Sensitization to cell death induced by soluble Fas ligand and agonistic antibodies with exogenous agents: A review

1.   Introduction

1.1.   Cell-death induction by soluble Fas ligand

The implementation of the cell-death induction against emerging malignant cells is one of the most important defensive actions for the maintenance of health in the human body. The failures in the controlled execution of cell-death processes via death ligand—death receptor systems including Fas ligand (FasL)—Fas receptor (FasR) result in the onset of many serious diseases, caused by either abnormal proliferation of malignant cells or excessive death of normal cells, such as cancers, various autoimmune diseases including rheumatoid arthritis, and type-I diabetes [1]–[3]. The apoptotic cell-death triggered by the binding of FasL to FasR is considered to play a central role for both the anti-pathogenic elimination of emerging harmful cells and the pathogenic destruction of beneficial cells [4]. Thus, from a medical viewpoint, if it is possible to devise an effective means for controlling the execution of Fas signaling system-mediated cell death at will, that will directly lead to the development of advanced therapeutic treatment strategy for such serious diseases.

Human Fas ligand extracellular domain (FasLECD) consists of 178 amino-acid residues located at the carboxyl-terminal side of whole human FasL protein molecule [5]. In human, the soluble, homo-trimeric extracellular-domain fragment called sFasL, comprised of the almost full region of FasLECD, is generated from cell-surface membrane-bound FasL (mFasL) by the processing with a cell-surface matrix-associated metalloproteinase, MMP7 (also called matrilysin), under many physiological and pathological circumstances [6],[7]. The relative cell-death inducing activity of human sFasL to that of mFasL was reported to be less than one thousandth [8], but sFasL still showed the significant activity against in vitro activated human peripheral blood T-cells [9]. The cytotoxic activity of human sFasL was greater than murine sFasL, and the difference was ascribed to the existence of a stalk region (Gln103–Pro136) at the N-terminal side of human FasLECD [10]. The cleaved bond in human mFasL by MMP7 was initially suggested to be the bond between Lys129 and Gln130 [11], but was corrected to that between Ser126 and Leu127 later [8].

Many diseases have been investigated in relation to the roles of sFasL during the onset processes of their pathological conditions. sFasL and mFasL can exert opposite biological functions with regard to the occurrence of diseases [12]. Because of the marginal cell-death inducing activity and the comparable affinity with mFasL toward Fas receptor extracellular domain (FasRECD), sFasL is generally considered to work as an antagonist of the mFasL-mediated cell-death induction [13]. Indeed, sFasL is known to stimulate the proliferation of fibroblast like synoviocytes of the patients suffered from rheumatoid arthritis by inhibiting the action of mFasL [14]. Also, the MMP7-mediated cleavage of mFasL to sFasL was pointed out to protect tumor cells from the cell-death induction by an anti-cancer drug, doxorubicin [7]. However, in contrast, many instances suggesting sFasL to play a role of the potent cell-death inducer in diseases have been reported, which include the host lymphocyte apoptosis by colon adenocarcinoma cells [15], the damage of ciliated epithelial cells in severe asthma patients [16], the acute kidney injury in cisplatin-treated mice [17], and also blister formation in pemphigus [18].

1.2.   Two alternative methods for triggering the cell-death via Fas signaling system by soluble agonistic proteins

It is possible to trigger the cell-death via Fas signaling system by the action of soluble agonistic proteins using two alternative methods. One way is the binding of the extracellular domain of the natural cognitive ligand, FasLECD, to FasRECD, and the other way is the binding of the specific monoclonal antibody (mAb) directed to FasRECD as the artificial substitute for FasLECD. The FasRECD-specific mAb are classified into two categories. One is cytotoxic, agonistic antibodies, represented by CH-11 (subclass IgM) and DX-2 (subclass IgG1), and the other is neutralizing, antagonistic antibodies, such as ZB4 [19]. Historically, the more frequently used reagents for triggering cell-death induction via the Fas signal transduction pathway were the FasR-mAbs in basic research, partly due to their better availability as a variety of commercial products at reasonable prices. It was revealed that the affinity strength was not always positively correlated to the potency of the cell-killing activity through the analysis of a series of IgG type monoclonal antibody [20].

From the viewpoint of therapeutic agents, it is possible to say that soluble proteins are more favorable than membrane proteins due to their uncomplicatedness in handling. However, as mentioned above, sFasL is functionally impaired in regard to the cell-killing activity as compared with mFasL. Therefore, if we intend to convert this soluble, but less potent as it is, protein molecule into effective cell-death inducing agents, employment of some engineering tactics would be required for obtaining the enough ability to induce the efficient death of target cells. One promising way is the application of the techniques for empowerment of sFasL molecule itself. This includes a further multimerization of self-trimeric FasLECD with the help of either additional domains for spontaneous oligomerization [21],[22] or crosslinking antibodies directed to the pre-attached peptide-tags in sFasL derivatives [23],[24]. Another potential approach for the enhancement of cell-death inducing activity will be the concentration of sFasL molecules around the surface of target cells by the use of binding domains to the target cells. Promising techniques have been developed for this purpose, where the affinity-providing protein domains toward target cells were engaged in. Methodologically, this can be accomplished by the conjugation to the N-terminal side of sFasL with antibody fragments, using either gene fusions with the single-chain Fv [25] or site-specific chemical modifications with Fab' fragments [26], as well as by construction of bispecific antibodies [27].

2.   Literature survey

The initial process of literature survey in this study was performed by collecting the candidate papers for the references extensively, by applying combinations of the selection key-words, “Fas”, “cell-death”, “sensitization”, “cisplatin/CDDP”, “5-fulorouracil/5-FU”, “doxorubicin/adriamycin”, “camptothecin”, “gemcitabine”, “interferon-gamma”, “interferon-alpha”, ”interferon-beta”, “tumor necrosis factor-alpha”, and “cytokines”, to the searching engines including PubMed, Web of Science and Google scholar, using the literature information retrieval system available. Final selection of appropriate literatures from the original hits was conducted based on a judging criteria for inclusion, which primarily depended on close relation to the scientific aim and scope of this survey and also the free-accessibility to whole paper contents for obtaining detailed experimental conditions as well. In Tables 1 to 3, the summarized results of the finally selected, relevant studies in terms of the target cells, the sensitization method, the cell-death induction method, and the primary findings in each study are listed in chronological order. The cell-death, directly caused by the administration of exogenous non-cytokine or cytokine agent independent of the soluble FasR agonists, was generally outside the scope of this review.

3.   Sensitization of Fas signaling system with exogenous non-cytokine agents

3.1.   Low molecular-weight anticancer drugs

It has been pointed out that malignant tumor cells escape from host's immune-surveillance system by suppressing cell-death arising from the immunocompetent mechanisms, including FasR-mediated apoptosis [4],[28]. The pharmaceutically important anti-proliferative agents, represented by anticancer drugs used for chemotherapies, are considered to primarily depend their therapeutic potentials on the cell-death inducing ability against diseased cells, where the Fas signaling system plays a key role in the implementation processes [29]. Therefore, it would be reasonable to expect cumulative therapeutic effects for the combination of an agonist against FasR with other substances or physical treatment. So far, a number of methods using exogenous non-cytokine agents, including clinical or non-clinical low-molecular weight compounds, non-cytokine macromolecules, virus or bacteria-derived factors, physical stresses, etc. have been examined for the sensitization to the cell-death inducing activity of sFasL or agonistic FasR-mAb.

Among them, numerous researchers' efforts have been especially devoted to seek the possibility that the clinically available low molecular-weight anticancer drugs, which are mainly considered to work as apoptosis inducers via intrinsic pathways, sensitize the tumor cells for making them more susceptible to Fas signaling system-mediated cell-death, known as a typical apoptotic event via an extrinsic pathway. A summary of the examination results concerning commonly used five representative clinical chemotherapeutics, namely cisplatin, doxorubicin (also called adriamycin), 5-fluorouracil (5-FU), camptothecin (including its derivatives) and gemcitabine, is presented in Table 1 [29]–[88]. As shown in Table 1, the level of sensitizing efficiency to Fas signaling system substantially depended on both the cell types and the nature of each sensitizing agent, which reflected the difference in the intracellular molecular-machineries required for the implementation of an efficient cell-death induction in individual target cells. The survey results suggested that the sensitization was frequently achieved by upregulation of either cell-surface FasR or intracellular pro-apoptotic proteins, such as initiator or effector caspases, Fas-associated death domain (FADD), oncogene B-cell lymphoma (Bcl)-extra-small protein (Bcl-X_S), Bcl-2-like protein 4 (Bax), or by downregulation of intracellular anti-apoptotic factors including Bcl-2, Bcl-extra-large protein (Bcl-X_L), Fas-associated death domain-like interleukin (IL)-1-converting enzyme inhibitory protein-long form (FLIP_L), as well as by simultaneous operation of these processes.

Table 1.  Sensitization of cell-death induction via Fas signaling system with five representative low molecular-weight clinical anti-cancer drugs.

Target cells: species, cell type (cell line/origin)	Sensitization method: type (mode), sensitizer (conc), treatment time	Cell-death induction method: inducer (name, subclass), conc, treatment time	Primary findings related to sensitization of cell-death induction via Fas signaling system	Refs.
hu, Ovarian cancer (A-2780, AD-10, C-30)	f-FR and f-CD (Pre and Co), CDDP (0.1–10 µg/ml) ± BHA (200 µM), 2–18 h (f-FR); 24 h (f-CD, Pre); 48 h (f-CD, Co)	FR-mAb (nd, IgM), 100–1000 ng/ml, Pre, 24 h; Co, 48 h	Up-FR and synergistic up-CD by either Pre or Co with CDDP. No inhibition in the synergism by coexistence of BHA.	[55]
hu, Colon cancer (HT-29, HCT-8R, HCT-116); leukemia (U937)	f-FR, and f-CD (Pre), CDDP (10 µM) ± ActD (5 µg/ml), 5FU (260 µM), DXR (10 µM) or CPT (0.3 µM), 3–6 h (f-FR, mRNA); 4 h (f-FR, protein and f-CD)	FR-mAb (CH-11, IgM), 5–100 ng/ml ± antagonistic FR-mAb (ZB-4, IgG1), 2 µg/ml or muFL-N2A, 0.5 U/ml (1 U: 1 µl of 100-fold conc), 72 h; PBL, E/T ratio: 25–100:1, 24 h	Up-FR with clinically relevant conc of CDDP, 5FU, DXR and CPT. Up-FR and up-CD with CDDP in agonistic FR-mAb-, sFL in muFL-N2A- or PBL-mediated cytotoxicity, blocked by ActD or antagonistic FR-mAb.	[30]
hu, Prostate cancer (PC-3, DU-145, LnCAP); mu, lymphoma (DT-140, LF−)	f-FR and f-CD (Pre and Co), CDDP (0.1–10 µg/ml), 18 h (f-FR); o/n (f-CD, Pre); 24 h (f-CD, Co)	muCTL, hybridoma (PMMI), TIL (kidney tumors), LAK (whole blood), E/T ratio: 0.3–30:1, 5–7 h; FR-mAb (CH-11, IgM), 100 ng/ml, 24 h	Up-FR in all cell lines except LF− and up-CD by CDDP treatment using PMMI cells or TIL/LAK cells in PC-3, DU-145, but not in LnCAP, associated with attenuation by coexistence of EGTA / MgCl2 and FR-mAb.	[61]
hu, Prostate cancer (PC-3, DU-145, LnCAP); ovarian cancer (AD-10)	f-FR and f-CD (Pre and Co), CDDP (1–10 µg/ml) or ADR (0.1–1 µg/ml), 18–24 h (f-FR and f-CD, Pre); 48 h (f-CD, Co)	FR-mAb (CH-11, IgM), 10–1000 ng/ml, Pre, 24 h; Co, 48 h; muCTL hybridoma (PMMI), E/T ratio: 0.3–10:1, Co, 7 h	Minimal effect on FR by CDDP treatment in all cell lines. Synergistic up-CD by either Pre with CDDP or Co with CDDP / ADR in PC-3 and DU-145, but much less in LnCAP.	[56]
hu, Glioma (T-98G, LN-229)	f-CD (Co), CPT (1 µM), 16–24 h	muFL-N2A, 100 U/ml, 16–24 h	Synergistic up-CD by Co with CPT and sFL, possibly mediated by enhanced Bax and decreased Bcl-2.	[48]
hu, Neuroblastoma (SHEP Tet-21/N)	f-FR and f-CD (Pre), DOX (100 ng/ml), 24 h	FR-mAb (nd, IgG3), 1 µg/ml, 24 h	Up-FR by treatment with DOX. Up-CD and up-FL by DOX Pre with cooperation by MycN oe.	[49]
hu, Colon cancer (HT-29, HCT-116); leukemia (U-937)	f-FL and f-FR, CDDP (10 µM) or DXR (10 µM), 4 h	None	No modification of cell-surface FL and up-reg of FR clustering with CDDP. Recruitment of FADD to FR by treatment with either drug.	[31]
hu, Colon cancer (HT-29, HCT-116, HCT-8R); myeloblastoma (U-3A, U-3A-STAT1)	f-FR, CDDP (10 µM) or DXR (10 µM), 72 h	None	Up-FR and up-reg of Bax in HT-29 cells by treatment with either drug. STAT1 independent up-reg of FADD, procasp-3 and -8.	[32]
hu, Prostate cancer (DU-145, LnCAP)	f-FL, f-FR and f-CD (Co), CPT (100 ng/ml) or CDDP (1.0 µg/ml),	FR-mAb (CH-11, IgM), 200 ng/ml, 24 h	No up-reg of FL and FR, and overcoming of CD resistance only in DU-145 cells by CPT treatment via activation of casp.	[62]
hu, Leukemia (CEM, REH, Nalm-6, KM-3, Nalm-6-RD)	f-FR and f-CD (Pre), DOX (10–500 ng/ml), 9–24 h (f-FR); 12 h (f-CD)	FR-mAb (nd, IgG3), 1 µg/ml plus Protein A (10 ng/ml), 1–48 h; LAK (peripheral blood), E/T ratio: 1:4, 24 h	Up-FR and up-CD in resistant pre-B-ALL cell lines by DOX Pre, associated with loss of Bcl-XL and up-reg of PARP cleavage.	[70]
hu, Breast cancer (MCF-7, EVSA-T)	f-FR, f-FL and f-CD (Co), DOX (100–500 µg/ml), 8–24 h (f-FR and f-FL); 48 h (f-CD)	FR-mAb (CH-11, IgM), 500 ng/ml, 48 h	Up-FR, but not up-FL, and up-CD by DOX Co in MCF-7 cells alone.	[66]
hu, Colon cancer (HT-29, HCT-8, HCT-116, VRC5/c1, RKO)	f-FR and f-CD (Co), 5FU (0.3–10 µM) plus LV (1 µM) ± dThd (20 µM), 48 h (f-FR); 72 h (f-CD)	FR-mAb (CH-11, IgM), 200 ng/ml, 72 h	Up-FR in wt p53 expressing cell lines with 5FU plus LV. Cl-dep reversal in up-CD by coexistence of dThd.	[33]
hu, Osteogenic sarcoma (MG-63)	f-FR and f-CD (Co), CDDP (0.5–5 µg/ml), 48 h	FR-mAb (CH-11, IgM), 100 ng/ml, 48 h	Up-CD by CDDP Co, associated with dn-reg of FLIP-L without up-FR.	[76]
hu, Head and neck cancer (HSC-2, HSC-3, HSC-4)	f-FR and f-FL, CDDP (5 µg/ml), 24 h	None	No up-FR, no up-FL, but up-reg of casp, in particular casp-9 by CDDP treatment.	[83]
hu, Non-small cell lung cancer (NCI-H-292, NCI-CorL-23, NCI-Colo-699)	f-FR and f-CD (Pre), GEM (0.005–0.5 µM), 24–72 h	FR-mAb (CH-11, IgM), 200 ng/ml, 3 h	Cl-dep and tm-dep up-FR and up-CD by GEM treatment. Synergistic effects on CD between GEM and FR-MAb in H-292 cells.	[41]
hu, Prostate cancer (DU-145)	f-CD (Co), CPT (100 ng/ml), 1–24 h	FR-mAb (CH-11, IgM), 200 ng/ml, 1–24 h	Tm-dep activation of SAPK / JNK activity for up-CD by CPT treatment, concomitant with c-Jun induction.	[63]
hu, Kidney cancer (ACHN, Caki-1, Caki-2, A-704, NC-65, fresh cells from patients' surgical specimens)	f-FR and f-CD (Pre and Co), ADR (0.1–10 µg/ml), 2–24 h	FR-mAb (CH-11, IgM), 0.01–1 µg/ml, 2–24 h	Up-FR and synergistic up-CD irrespective of cell types by ADR treatment.	[82]
hu, Breast cancer (MCF-7)	f-FR and f-CD (Co), DOX (10 ng/ml), 48 h	FR-mAb (CH-11, IgM), 500 ng/ml, 48 h	Marked up-FR, but insignificant up-CD by DOX treatment.	[67]
hu, Colon cancer with Val12-ras mutation (SW480, SW620)	f-FR and f-CD (Pre), 5FU (30 µg/ml), CPT-11 (1 µg/ml) or CDDP (0.1 µg/ml), 24 h	FR-mAb (CH-11, IgM), 500 ng/ml; anti-Val12-ras CTL, E/T ratio: 1.25–10:1, 18–20 h	Significant up-FR and up-CD by either inducer in SW480 cells, but much less FR and CD induction only with CTL in SW620 cells, after treatments with anti-cancer drugs.	[34]
hu, Melanoma (M-202, M-207, M-238, T-2); prostate cancer (DU-145, PC-3)	f-FR and f-CD (Pre), CDDP (0.1–10 ng/ml), 18 h	mu PMMI CTL or MART-1-specific CTL, E/T ratio: 1–30:1, 5–6 h	Up-FR and up-CD in M-202, T-2, DU-145 and PC-3, but M-207 and M-238 remained resistant to up-FR and up-CD, after CDDP treatment.	[64]
hu, Small cell lung cancer (POGB)	f-FR, DOX (200 ng/ml), 24 h	None	Up-FR similar to γ-ray treatment by DOX treatment, associated with marked up-reg of c-Myc.	[42]
hu, Glioblastoma (GBM)	f-FR and f-CD (Pre), topotecan (0.01 µg/ml), CPT-83 (0.1 µg/ml) or CDDP (0.3 µg/ml), 24 h	FR-mAb (nd), 1 µg/ml, 24–72 h	Up-FR by treatments with either CPT derivatives or CDDP, but synergistic up-CD with CPT derivatives alone.	[50]
hu, Cervical cancer (HeLa)	f-FR and f-FL, CDDP (0.25 µg/ml), 48–72 h	None	No up-reg in mRNAs of FR and FL by CDDP treatment.	[72]
mu, Thymoma (EL-4)	f-FR and f-CD (Pre and Co), DOX (0.001–200 µM) or CPT (0.001–200 µM), 0.5–20 h (f-FR); 24 h (f-CD, Co)	Crosslinked-sFL (2–200 ng/ml), 5 h (Pre); 24 h (Co)	Marked up-FR and up-CD by treatments with either DOX or CPT without any influence on FLIP-L.	[84]
hu, Liver/cervical cancer (Chang/HeLa)	f-FR and f-FL, ADR (0.5 µg/ml) ± Palgin (15.6 µg/ml), 2–10 h	None	Tm-dep up-FR and up-FL, associated with increase in casp-3 activity by combined treatment with ADR and an herb drug.	[73]
hu, Colon cancer (Caco-2, Colo-320, SW-948)	f-FR, 5FU (5–25 µM), CDDP, (5–25 µM), 24 h	None	Significant up-FR by CDDP treatment in Colo-320 alone.	[35]
hu, Oral squamous cell cancer (NA, HSC-4)	f-FR and f-CD (Pre), CDDP (1 µg/ml) and/or 5FU (2.5 µg/ml), 8–24 h	FR-mAb (CH-11, IgM), 100 ng/ml, 12 h	Marked up-FR and up-CD by CDDP and/or 5FU treatment with dn-reg of cFLIP. Increasing effect on CD by combined treatment.	[85]
hu, Colon cancer (HT-29)	f-CD (Pre), 5FU (100 µg/ml), CDDP (5 µg/ml) or DXR (2.5 µg/ml), 24 h	sFL, nd, 16 h	Marked up-CD by Pre with non-toxic conc of 5FU, CDDP or DXR.	[36]
hu, Ovarian cancer (2008, 2008-C13)	f-FR and f-FL, CDDP (20–40 µM) ± Z-VAD-fmk (50 µM), 6–24 h	None	Up-FL mRNA in CDDP-sensitive cells by the treatment, insensitive to coexistence of pan-casp inhibitor.	[57]
hu, Colon cancer (RKO, SW620)	f-FR and f-FL, CPT (5–40 nM) ± Ad-E2F-1 infection, 48–72 h	None	No change in FR and FL by CPT alone or a combined treatment with G1/S cell-cycle transition regulator.	[37]
ra, Cardiac myocytes (ventricles of 0-1 d-old Wister-Imamichi)	f-FR and f-CD (Pre), DOX (0.5–5 µM), 6–24 h	sFL, 10–1000 ng/ml, 12 h	Significant up-CD by DOX Pre, concomitant with tm-dep ROS-controlled FLIP-L dn-reg.	[88]
hu, Melanoma (UACC-903, DM-13, A-375)	f-FR and f-CD (Pre), 5FU (25 µg/ml), 48 h	FR-mAb (CH-11, IgM), 67–1800 ng/ml, 5–16 h; CD95L+-G209 specific-CTL, E/T ratio: 30–1:1, 6 h; ± FR-mAb (ZB4, IgG1), 2–10 g/ml	Significant up-FR and cl-dep up-CD by 5FU treatment. Almost complete block of FR-mAb-induced, but not CTL-induced, up-CD in the presence of antagonistic FR-mAb.	[81]
hu, Stromal-type neuroblastoma (SH-EP1, SK-N-AS)	f-FR, f-FL and f-CD (Co), DOX (0.5 µg/ml), 4–24 h	FR-mAb (CH-11, IgM), 1 µg/ml; sFL, 10 ng/ml, 24 h	Significant up-FR and up-CD by DOX Co, associated with tm-dep up-reg of casp-3, -8 and -9.	[51]
hu, Colon cancer (HT-29)	f-FR and f-CD (Co), CDDP (5 µg/ml), 4h (f-FR); 24 h (f-CD) ± nystatin (10 µg/ml) or imipramine (50 µM), 1 h pretreated	FR-mAb (CH-11, IgM), 100 ng/ml, 24 h	Marked clustering of FR in plasma membrane-raft and up-CD by CDDP treatment, associated with aSMase activation and ceramide production	[38]
hu, Epidemoid lung cancer (49-year-old man patient at stage T3N1M0)	f-FR and f-FL, in vivo: CDDP (10 mg/kg), 1–7 times; in vitro: CDDP (2 µg/ml), 24–48 h	None	Strong up-FR and up-FL in the beginning, followed by dn-reg in vivo, and dn-reg of FR mRNA in vitro by CDDP treatment.	[43]
hu, Testicular germ cell cancer (Tera)	f-FR and f-CD (Pre), CDDP (0.25–1.0 µM), 24 h	FR-mAb (7C11, IgM), 2 µg/ml, 20 h	Up-FR and up-CD by CDDP treatment without up-reg of p21 expression.	[86]
hu, Breast cancer (MCF-7, MCF-6/E6)	f-FR, CDDP (5 µM) ± Bcl2 specific AS-ODN (1 µM), 48 h	None	Up-FR by CDDP treatment irrespective of the presence of Bcl-2 specific AS-ODN or p53 activation status.	[68]
hu, Cervical cancer (HPV-18 positive and wt p53: HeLa; HPV-16 positive and wt p53: CaSki, SiHa; HPV negative and mut p53: C-33A)	f-FR, f-FL and f-CD (Pre), CDDP (0.5–5 µM), 24 h (f-FR); 2 h (f-CD)	FR-mAb (7C11, IgM), 50–1000 ng/ml, 24 h	Up-FR in all HPV positive cells, but not in C-33A, without major change in p53 levels by CDDP treatment. Strong up-CD only in HeLa and insignificant up-CD in other cells.	[74]
hu, Astrocytoma (U-373MG)	f-FR and f-CD (Pre), CDDP (25 µM), 2 h	FR-mAb (7C11, IgM), 200 ng/ml, 24 h	Up-FR and up-CD by CDDP treatment with requirement of active p73 protein.	[52]
hu, Glioblastoma (U-87, U-373, A-172, SNB-19)	f-FR, f-FL and f-CD (Co), CPT (1–100 µM), ADR (0.1–20 µM), 24 h	FR-mAb (CH-11, IgM), 50–1000 ng/ml, 24 h	No change in FR and FL by CPT alone treatment, but significant up-CD by Co with CPT or ADR regardless of p53 status.	[53]
hu, Colon cancer (HCT-116 with wt, null or mut p53, H630, RKO)	f-FR and f-CD (Pre), 5FU (5 µM), CPT-11 (5 µM), 24–48 h	FR-mAb (CH-11, IgM), 25–100 ng/ml, 12–48 h	p53 status independent up-FR by CPT-11, but not by 5FU treatment. Up-CD only in wt p53 cells by Pre with 5FU.	[39]
hu, Urinary bladder cancer (T-24, RT-4)	f-FR, f-FL and f-CD (Co), ADM (0.1–10 µg/ml), 24 h	FR-mAb (CH-11, IgM), 1–1000 ng/ml, 24 h	No up-FR, cl-dep change in FL and synergistic up-CD by ADM treatment.	[87]
mu, Lewis lung cancer (3LL)	f-FR, f-CD (Pre and Co), in vitro: DOX (0.03–3 µg/ml), 24–48 h; in vivo: DOX (1–4 mg/kg), 24 h	muFR-mAb (Jo2, IgM), 1 µg/ml, 48 h; splenic CTL, E/T ratio: 40–80:1, 20 h	Up-FR and ds-dep up-CD by DOX treatment in vitro. Up-FR mRNA with anti-tumor therapeutic effect on solid tumor in vivo.	[44]
hu, Ovarian cancer (OVCAR-3, advanced -stage patient's ascitic fluid)	f-FR and f-CD (Pre), CDDP (IC30 or 3 µM), 24 h	FR-mAb (CH-11, IgM), 300 ng/ml, 24 h	Up-FR and up-CD by CDDP treatment both with cell line sample and with the patient's ascitic sample.	[58]
hu, Osteosarcoma (U-2-OS, MG-63)	f-FR, CDDP (5 µM), 24–72 h	None	No significant change in FR and Bax expression by CDDP treatment in cells with either wt p53 or mut p53.	[77]
mu, Ovarian cancer (ID-8)	f-FR and f-CD (Pre), liposomal DOX (Doxil) (0.1–3 µg/ml), 6 h	muFR-mAb (Jo2, IgM), nd, plus Protein G, 2 µg/ml, 24 h; splenic CTL, E/T ratio: 10–20:1, 4h	Significant ds-dep up-FR, associated with MHC class1 up-reg and up-CD by liposomal DOX treatment.	[59]
hu, Lung cancer (ASTC-A-1)	f-FR and f-FL, CDDP (20 µM), 16 h ± ursolic acid (100 µM), 8 h preceded	None	Insignificant up-FR and up-FL by CDDP treatment alone or after preceding treatment with ursolic acid.	[45]
hu, Ovarian cancer (OVCAR-3)	f-FR, CDDP (1 µM) ± AT-101 (0.5 µM), 72 h	None	Up-reg of FR, Bax, Bad and dn-reg of Bcl-2 by CDDP treatment, augmented by combining with Mcl-1 inhibitor.	[60]
hu, Non-small cell lung cancer (H-292, COLO-699); immortalized normal bronchial (16-HBE)	f-FL and f-CD (Pre and Co), GEM (0.05 µM), 24–96 h	mAb (CH-11, IgM), 200 ng/ml, 3 h (Pre); LAK / MPL, E/T ratio: 5:1, 16 h (Co)	Significant up-FL mRNA level by GEM treatment in all cell lines. Up-reg of LAK / MPL-induced CD in H-292 cells by Pre with GEM.	[46]
hu, Colon cancer (Lovo)	f-FR and f-FL, CDDP (10 µM) ± andrographolide (10 µM), 24 h	None	Up-FR, up-FL and up-reg of FR-FL complex formation by andrographolide treatment, augmented by combination with CDDP.	[40]
hu, Non-small cell lung cancer (A-549, NCI-H-460)	f-FR, CDDP (22-26 µM), 24 h ± followed by gambogic acid (3.5–4 µM), 24 h	None	Augmented up-FR, up-reg of Bax and dn-reg of Bcl-2, but no effect on Bcl-XL by sequential treatments with CDDP and then with gambogic acid.	[47]
hu, Osteosarcoma (MG-63)	f-FR, DOX (1 µg/ml) ± MTE (50 mg/ml), 72 h	None	Up-FR by DOX treatment, augmented by combination with MTE.	[78]
hu, Monocytic leukemia (U-937)	f-FR, DOX (1 µM) ± MG132 (1 µM), 3 h	None	Up-reg of FR and Bax mRNAs and dn-reg Bcl-XL mRNA by combined treatment with DOX and MG132.	[71]
hu, Neuroblastoma (SK-N-AS)	f-FR and f-FL, CDDP (30 µM), 6–24 h ± TNF-α (100 ng/ml) followed, 24 h	None	Tm-dep up-reg of FL mRNA by CDDP treatment. Significant up-FR only after post-treatment with TNF-α.	[54]
hu, Cervical cancer (HeLa)	f-FL, CDDP (50-250 µg/ml) ± LPA (20 µM), 4 h	None	Ds-dep up-FL and up-reg of Bax and dn-reg of Bcl-2 by CDDP treatment, all negatively affected by LPA.	[75]
hu, Prostate cancer (DU-145)	f-FR, DOX (0.5 µM) ± sildenafil (10 µM), 48 h	None	Up-reg of surface localization of FR, by DOX treatment, enhanced with sildenafil. Dn-reg of increased FLIP-L and FLIP-S by DOX alone treatment after combination with sildenafil.	[65]
hu, Breast cancer (MCF-7); mu, Liver cancer (Hepa-1-6)	f-FR and f-FL, DOX (1 µM) ± MCD (5 mM) ± PFT-α (20 µM), 24 h ± p53 siRNA transfection, 18 h preceded	None	Up-FR, up-FL, up-reg of Bax, but dn-reg of Bcl-2, by DOX treatment. Up-FR and up-FL were augmented by combination with MCD, but attenuated with PFT-α or p53 siRNA transfection.	[69]
hu, Pancreatic cancer (SW-1990, Panc-1)	f-FR and f-CD (Pre), GEM (0.025–25 µM), 48 h	FR-mAb (CH-11, IgM), 200 ng/ml, 4 h; pancreatic cancer specific CTL, E/T ratio: 20–100:1 ± FR-mAb (ZB4), 500 ng/ml, 4 h	Ds-dep up-FR and up-CD with either pancreatic cancer specific CTL or agonistic FR-mAb by GEM treatment, attenuated by the presence of neutralizing FR-mAb.	[79]
hu, Pancreatic cancer (SUIT-020)	f-FR and f-CD (Pre and Co), GEM (1–10000 ng/ml), 24 h ± zVAD-fmk (50 µM) /NEC (50 µM) / SP600125 (10 µM), 30 m preceded	FR-mAb (nd, IgG3), 333 ng/ml, 30 m (Pre); 1.5–25 ng/ml, 24 h (Co)	Undetectable up-FR, but marked up-CD by GEM treatment, mediated through dn-reg of cFLIP and Mcl-1. Induction of apoptosis and necroptosis by combination of FL and GEM.	[80]
hu, Lung cancer (A-549); colon cancer (HCT-116); breast cancer (MCF-7)	f-FR and f-CD (Co), GEM (5–5000 nM) ± U0126 (1–10 µM) or SP600125 (1–10 µM), 24 h	Poly-His-tagged sFL, 50 ng/ml plus tag-crosslinking mAb, 3 µg/ml ± antagonistic FR-mAb 1-10 µg/ml, 24 h	Up-FR in all cells and up-CD in HCT-116 cells by GEM treatment at subtoxic doses. Reduction of up-FR by coexistence of JNK or ERK inhibitor.	[29]

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So far, the most and the second frequently employed anticancer drugs for the sensitization experiments were cisplatin and doxorubicin (adriamycin), respectively. The above mentioned low molecular-weight anti-cancer drugs have been examined with the malignant tumor cells derived from various kinds of organs, including colon [29]–[40], lung [29],[41]–[47], central nervous system [48]–[54], ovary [55]–[60], prostate [56],[61]–[65], breast [29],[66]–[69], blood cells [30],[31],[61],[70],[71], uterus [72]–[75], bone [76]–[78], pancreas [79],[80], skin [64],[81], kidney [82], head and neck [83], thymus [84], oral cavity [85], testis [86], bladder [87], and liver [69]. The sensitizing effects significantly depended on the kind of not only originated organs but also cell lines, and marked differences were observed even among the tumor cells originated from the same organs in some cases. For instances, upregulation of cell-death induction using the same FR-mAb were reported to be largely affected by the difference in cell lines of prostate cancer [56],[62] as well as that of colon cancer [34]. As for the intracellular factors which can critically influence the sensitization effects, the difference in either the mutation status or the expression level of other apoptosis execution-related proteins, such as p53 [33],[39],[53],[68],[77], p21 [86] and p73 [52], have been repeatedly investigated from the viewpoint regarding upregulation of the cell-death induction and that of cell-surface FasR expression. It is reasonable that the sensitization effects largely depended on the kind of anticancer drugs, since each group of anticancer drugs possesses its own specific interference mechanism different from others with proliferation of tumor cells. For example, the significant sensitization of cell-death induction was observed in the pretreatments with camptothecin, CPT-11 (a synthetic analog of camptothecin) or doxorubicin regardless of p53 mutation status of the target cells [39],[53], however the pretreatment with 5-fluorouracil was effective for the enhanced death of the cells with wild-type p53 alone [39]. Also, it is interesting to note that the pharmaceutical substances used in traditional medicines [47],[78] or that in treatment of non-tumor diseases [65] have been reported to augment the FasR expression in the target cells by combining with the anti-cancer drugs in some instances. With respect to non-tumor target cells, a large increase in the cell-death induction by sFasL treatment was observed for cardiac myocytes after the pretreatment with doxorubicin, accompanied by marked down-regulation of FLIP_L [88].

The other low molecular-weight clinical anticancer drugs already examined include daunorubicin [89], decitabine [90],[91], carboplatin [92], oxaliplatin [29], arsenic trioxide [93], bortezomib [94], mitoxantrone [95], and sorafenib [96]. In several cases, combinations of clinical anticancer drugs with other low-molecular weight pharmaceuticals, each of which exhibited limited efficiency in the sensitization as a sole agent, have been reported to show an improved efficacy by synergizing effects, resulting in a considerable level of cell-death induction associated with up-regulated FasR or FasL expression [40],[60],[91],[97].

3.2.   Other non-cytokine agents

Except for the clinical anti-cancer drugs, many low molecular-weight compounds including sodium butyrate [98],[99], enzyme inhibitors [100],[101], ceramides [23],[102], edelfosine [103],[104] and perillyl alcohol [105] have been examined for the sensitizing effect. Apart from the low molecular-weight agents, the components or the inactivated whole cells, derived from the virus and bacteria including human immunodeficiency virus [106], hepatitis C virus [107], Propionibacterium acnes [108] and Helicobacter pylori [109], have been also used for the sensitization experiments. The typical physical stress employed for the investigation was irradiation [110],[111].

4.   Sensitization of Fas signaling system with exogenous cytokine agents

4.1.   Interferon-γ (IFN-γ)

The number of studies on the sensitization of Fas signaling system-mediated cell-death induction using cytokine agents have fairly exceeded that employing non-cytokine agents described above. Among various clinically relevant cytokines responsible for the immune system, IFN-γ has been most extensively used exogenous agents for this purpose to date. This protein is the sole cytokine belonging to type-II interferon, and shows multifaceted important immune-modulatory biological functions including antiviral, cell-differentiative, and anti-proliferative activities via various mediators, in particular Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signal transduction pathway [112],[113]. Activation of STAT-1 signaling system directed by IFN-γ-primed signaling is known to be responsible for the enhancement of FasR and FasL as an important mechanism of tumor suppression [114]. IFN-γ is also suggested to be critically involved in tumor immunity in the human body by exerting pleiotropic overcoming of immunosuppression effects [115],[116].

Table 2 presents a summary of the survey results concerning the sensitization studies using exogenous agents containing IFN-γ as the component [117]–[283]. The concentration of exogenous IFN-γ, applied for the sensitization experiments, ranged from 0.1 to 10000 U/ml, however it fell within 10–1000 U/ml in most cases. Of note, nearly half of the studies listed in this Table were targeted to non-tumor cells. It was considered that one of the major reasons for the wide usage of IFN-γ by quite a few researchers of broad medical disciplines might come from the remarkable up-regulation effects on the surface FasR expression not only in various types of tumor cells but also in many types of other target cells, derived from patients with non-tumor diseases or healthy donor. An interesting feature about the investigations targeted to tumor cells using exogenous IFN-γ-containing agents is that a relatively large number of studies (approximately 40% of malignant tumor cases) have been conducted with colon cancer using a variety of cell-lines as the target cells [30],[35],[98],[117]–[146]. The primary organs of examined malignant tumors except for colon covered blood cells [117],[138],[147]–[162], central nervous system [54],[163]–[168], lung [169]–[174], breast [67],[174]–[177], uterus [74],[141],[144],[178]–[180], soft tissue [117],[124],[164],[181],[182], kidney [183]–[186], skin [187]–[189], bone [164],[190],[191], ovary [147],[192], stomach [109],[193],[194], liver [195], bile duct [196], pancreas [197], and prostate [198]. The samples of peripheral blood mononuclear cells from stage-IV cancer patients were also subjected to the analysis [133],[139].

As for non-tumor and benign tumor targets, many kinds of cells derived from human patients and animal models suffering from the diseases, including type-I diabetes [199],[200], insulinoma [201]–[204],[283], Graves' disease [205],[206], non-toxic and multi-nodular goiter [206],[267], Hashimoto's thyroiditis [206], epilepsy [207], asthma [208], Down syndrome [209], Sjögren's syndrome [210],[272], Simpson-Golabi-Behmel syndrome [211], Sézary syndrome [212], Leishmania major infection [213],[214], cardiac transplantation [215], keratoplasty [216], atherosclerosis [217],[218], peritoneal dialysis [219], glaucoma [220], and dry-eye [221], have been used for the studies. Also, normal or immortalized cells derived from healthy donors, including the samples of bone-marrow cells [222],[224]–[231]; mononuclear [223],[224], eosinophil [232],[233], basophil [235], monocyte [234], and erythroid progenitor [236] cells from blood; Kupffer [237], parenchymal [237], hematopoietic progenitor [238], and stellate [239] cells from liver; spleen cells [225]; small intestine lamina propria T-lymphocytes [240]; pancreas β-islet cells [200],[241],[242]; corpus luteum [243]–[247] and granulosa [248],[249] cells from ovary; placenta trophoblast cells [250],[251]; testis Sertoli cells [252]; mesangial [253],[254] and tubular [255] cells from kidney; microglia [256],[257], cortical astrocyte [258], and motoneuron [259] cells from central nervous system; keratinocyte [260],[261] and hair follicle [262] cells from skin; vasculature smooth muscle cells [153]; endothelial cells from umbilical vein [153],[263],[264], dermal microvasculature [263], and lung microvasculature [265]; epithelial cells from thyroid gland [266]–[270], salivary gland and duct [210],[271],[272], intestine [132],[273],[274], eye conjunctiva [275],[276], lung alveolus [265], small airway [277], bronchus [277], and uterus endometrium [278]; fibroblast and myofibroblast cells from lung [279], cardiac [280]; and uterus endometrial stromal cells [281],[282], have been employed for the sensitization experiments as the target cells.

In majority of the instances, the concomitant up-regulation of cell-death induced with sFasL or agonistic FR-mAb were observed for the cases that exhibited up-regulation of FasR expression at protein or mRNA level in cell line-, dose-, and treatment time-dependent manners. However, it should be pointed out that the increase in cell-surface expression of FasR by the sensitization with IFN-γ did not always directly correspond to the enhancement in death-induction level of the target cell. The existence of numerous, endogenous limiting-obstacles, which mainly consisted of up-regulation of anti-apoptotic factors and down-regulation of pro-apoptotic factors, has been suggested with respect to the demanding target cells. The representative former and the latter factors include enhanced expression or activation of Bcl-2, FLIP, STAT-3, and decoy receptor-3 (DcR3), and reduced expression or activation of caspase-1, -3, -8, and STAT-1, respectively. As a noteworthy example for the discrepancy between the responses to up-regulation of FasR expression and that to the augmentation of cell-death induction, the primary (SW-480) and the metastatic (SW-620) colon cell-lines harboring a mutation in Ras protein, originated from the same patient, exhibited significantly different responses to the sensitization under the identical experimental conditions [123],[127]–[129],[136]. In spite of the similar behavior in the up-regulation of cell-surface FasR, the metastatic cell-line emerged in vivo showed fairly delayed or largely diminished up-regulation in the cell-death induction by the treatment with either an agonistic FR-mAb or sFasL [123],[128], possibly due to the absence of cell-surface FasR capping [123] as well as the presence of intracellular expression of strong constitutive Bcl-2 [129] and IFN-γ inducible interferon consensus sequence-binding protein [136].

Further, it is important to note that the sensitization to the cell-death via Fas signaling system with IFN-γ was often synergistically or additively augmented by the combined treatment either with other exogenous cytokines such as tumor necrosis factor (TNF)-α [54],[122],[142],[163],[165],[180],[184],[190],[194],[203],[207],[211],[216],[219],[222],[231]–[233],[244],[245],[247],[248],[252],[255],[256],[258],[261],[267],[270],[273],[274],[276],[278],[279],[281],[282], IL-1α, -1β [180],[199],[201]–[204],[258],[261],[267]–[269],[282] and transforming growth factor (TGF)-β1, -β2 [163],[180],[216],[282], or with non-cytokine agents including 5-FU [139],[146], 5-aza-2′-deoxycytidine [173], cycloheximide [35],[129],[177], lipopolysaccharide [161],[255], all-trans retinoic acid [177], and mAb specific to CD40 [210],[265]. As an exceptional case, TGF-β2 has been reported to show substantial inhibitory effects against sensitization to the apoptosis of murine microglia cells by IFN-γ treatments in combination with or without TNF-α, where an intense induction of cellular FLIP expression was observed [257]. Also, in some instances, the up-regulation of cell-death was further enhanced by the coexistence of p44/p42 mitogen-activated protein-kinase kinase inhibitors [268], Helicobacter pylori extract [109], trichostatin A [182], tosyl lysine or phenylalanine chloromethyl ketone [144], and constitutive intracellular expression of interferon regulatory factor (IRF) [181]. In contrast, the event was significantly attenuated by exogenous addition of mAbs specific to human leukocyte antigen (HLA)-2 or intercellular adhesion molecule (ICAM) [127]; inhibitors to relevant caspases (general and caspase-1, -3, -7, -8, -9, -10) [141],[168],[201],[241], related kinases (phosphatidylinositol-3 kinase, protein kinase B, Janus kinase 2) [171],[215], intracellular FasR trafficking [215], and nitric monoxide synthase [192]; antisense-oligodeoxyribonucleotides (AS-ODN) to IRF-1 [185]; small interfering RNA (siRNA) targeted to activator subunits of immune-proteasome-8 [217], human apolipoprotein L6 [218] and autoantigens found in Sjögren's syndrome and systemic lupus erythematosus [179], as well as exogenously applied thyroid gland stimulating hormone [205],[266], IL-3, -5 [232], IL-4, -10 [206],[250],[274], granulocyte macrophage colony-stimulating factor (GM-CSF) [156],[232], phorbol 12-myristate 13-acetate [123], and dexamethasone [169],[216],[272].

An important topic except for the cell-death inducing effects on tumor cells is the sensitization to the cells concerning reproduction of animals including human origin [243]–[251],[278],[281],[282]. Several types of the cells, including those originated from ovary corpus and placenta trophoblast, have been supplied for the examination. Although the influence of the pretreatments with exogenous IFN-γ depended on the cases, the sensitization efficiency to the up-regulation of cell-death or cell-surface FasR was often significantly augmented by the combination with the cytokines, especially TNF-α. [244],[247],[248],[278],[281],[282]. Another topical issue that has been targeted would be the sensitization effects on the cells of cardiovascular and lung alveolar systems [153],[215],[217],[263],[265]. In this case, the treatment efficiency tended to largely depend on the structural position of the cells, i.e. endothelial, smooth muscle or epithelial cells, and IFN-γ was mostly employed as the single cytokine agent, rather combined not with other cytokines but with non-cytokine agents, represented by low-molecular weight kinase inhibitors [215], so far.

Table 2.  Sensitization of cell-death induction via Fas signaling system with exogenous agents containing IFN-γ.

Target cells: species, cell type (cell line/origin)	Sensitization method: type (mode), sensitizer (conc), treatment time	Cell-death induction method: inducer (name, subclass), conc, treatment time	Main findings related to sensitization of cell-death induction via Fas signaling system	Refs.
hu, Lymphoma (U-937, Dauji), leukemia (HL-60, MOLT-4B), rhabdomyosarcoma (A-673), amnion-derived (FL), colon cancer (HT-29), foreskin fibroblast (FS-7, TM-11)	f-CD (Pre), IFN-γ (200 U/ml), 24 h	FR-mAb (nd, IgM), 10–100 ng/ml, 5–15 h	Up-CD in FR-mAb-primed cell-killing, indistinguishable from that primed with TNF-α, by IFN-γ Pre. Marked up-CD except for MOLT-4B and Daudi.	[117]
hu, Ovarian cancer (222, 222TR, SKOV-3, A-2780, AD-10, C-30), lymphoma (U-937, Raji)	f-CD (Pre), IFN-γ (50–150 U/ml), 24 h	FR-mAb (nd, nd), 0.1–10 µg/ml ± DTX, 10 pM–10 nM ± ADM, 1 µg/ml ± CDDP, 3 µg/ml, 24 h	Cl-dep up-CD of the target cells including multi-drug resistant cells by IFN-γ Pre, in synergy with DTX, ADM or CDDP.	[147]
hu, Lymphoma [U-937: uninfected (commercially obtained, cloned); HIV-infected (U1)]	f-CD (Co), IFN-γ (1000 U/ml), 72 h	FR-mAb (nd, IgM), 1 µg/ml, 72 h	Synergistic up-CD of several uninfected clones and HIV-infected cells by Co with IFN-γ and FR-mAb.	[148]
hu, Foreskin keratinocyte (non-lesional epidermis)	f-FR and f-CD (Pre), IFN-γ (10–10000 U/ml), 24 h	FR-mAb (CH-11, IgM), 1 µg/ml, 48 h	Marked up-FR and up-CD, associated with large increase in ICAM-1.	[260]
hu, Glioma (T-98G, LN-18, LN-215, LN-229, LN-308, LN-319, LN-405)	f-FR and f-CD (Pre), IFN-γ (100 U/ml) ± TNF-α (10 ng/ml) ± TGF-β2 (nd), 24 h	FR-mAb (CH-11, IgM), 0.1–1 µg/ml, 24 h	Cl-dep up-FR and up-CD by IFN-γ treatment, associated with synergistic effects of TNF-α or TGF-β2.	[163]
hu, Retinal pigment epithelium (keratoplasty donor eyes)	f-FR and f-CD (Pre), IFN-γ (100 U/ml) ± [TNF-α (10 ng/ml) or TGF-β2 (10 ng/ml)] ± DEX (10 µM), 24 h	FR-mAb (nd, IgM), 1 µg/ml, 16 h	Up-FR and synergistic up-CD by combined treatment with IFN-γ and TNF-α or TGF-β2, accompanied by significant dn-reg effect of DEX	[216]
hu, Multiple myeloma (RPMI-8226, ARH-77, KMM-1, U-266, Hs)	f-CD (Pre), IFN-γ (200 U/ml), 24 h	FR-mAb (nd, IgM), 100 ng/ml, 10 m-18 h	Cl-dep up-CD by IFN-γ Pre. No correlation between Bcl-2 expression and the sensitivity.	[149]
hu, Bone marrow hematogenic CD34+ progenitor (normal volunteers)	f-FR and f-CD (Pre), IFN-γ (1–10000 U/ml) ± TNF-α (10 U/ml) ± SCF (100 ng/ml), 48 h, (f-FR); 36 h (f-CD)	FR-mAb (CH-11, IgM), 1 µg/ml, 12 h	Ds-dep up-FR by IFN-γ treatment in synergy with TNF-α, but not with SCF. Insignificant effects on up-CD by IFN-γ Pre alone or in combination with TNF-α.	[222]
hu, Peripheral blood mononuclear (HIV-seronegative healthy donors)	f-FR, IFN-γ (1000 U/ml) ± TNF-α (1 µg/ml) ± VES (50 µg/ml), 48 h	None	No blocking effect of VES on up-FR by IFN-γ treatment alone or in combination with TNF-α.	[223]
hu, Primitive hematopoietic progenitor (CD34+CD38+/− fetal liver)	f-FR and f-CD (Co): selected combinations of IFN-γ, KL, GM-CSF (20 ng/ml each) and TNF-α (100 ng/ml), 3–5 d	FR-mAb (CH-11, IgM), 2 µg/ml, 3–5 d	Augmented up-FR by treatment with TNF-α and/or KL plus GM-CSF in the presence of IFN-γ. No up-CD, but reduction in hematopoietic potential.	[238]
hu, Cervical cancer cells (HeLa)	f-CD (Pre), IFN-γ (50 U/ml) ± pepstatin A (10−4 M), 20 h	FR-mAb (nd, IgG3), 50 ng/ml, 40 h	Suppression of up-CD by IFN-γ Pre with an inhibitor of cathepsin D.	[178]
hu, Leukemia (drug sensitive: K-562/WT, HL-60; multidrug-resistant: KG-1a, K-562/ADM)	f-FR and f-CD (Pre and Co), IFN-γ (100 U/ml), 24 h (f-FR and f-CD, Pre); IFN-γ (100 U/ml) ± DNR (10 ng/ml), VCR (3 ng/ml), CBDCA (1 mg/ml), ARC (1 ng/ml), DEX (4 ng/ml) or IR (1 Gy), 48 h (f-CD, Co)	FR-mAb (CH-11, IgM), 0.01–10 µg/ml, 48 h	Up-FR and up-CD by IFN-γ treatment alone and in combination with anticancer-drugs, IR or DEX in multidrug-resistant and sensitive cells, correlated with reduction of intracellular glutathione contents.	[150]
hu, Kidney mesangial (cadaver glomeruli)	f-FR and f-CD (Pre), IFN-γ (1000 U/ml), 48 h	FR-mAb (CH-11, IgM), 0.1–500 ng/ml, 24 h	Significant p-FR and up-CD by IFN-γ treatment.	[253]
hu, Colon cancer (HT-29, COLO-205)	f-CD (Pre), IFN-γ (100 U/ml), 72 h	FR-mAb (nd, IgG3), 500 ng/ml, 1–3 h	Up-CD by IFN-γ Pre, associated with de-adhesion from hyaluronate-coated plate and shedding of CD44.	[118]
hu, Thyrocytes (normal thyroid glands)	f-FR and f-CD (Pre), IFN-γ (500 U/ml) ± boTSH (10 mU/ml), 48 h	FR-mAb (nd, IgM), 1000 ng/ml, 18 h	Up-FR and up-CD by IFN-γ treatment, associated with significant inhibition by coexistence of TSH.	[266]
hu, Breast normal (MCF-10A), cancer (MCF-7, ZR75-1, T-47D, MB-231, MB-468, SKBr-3)	f-FR and f-CD (Pre), IFN-γ (20–2000 U/ml), 96 h	FR-mAb (CH-11, IgM), 1 µg/ml, 16 h	Up-CD in 4 of 5 resistant cell lines IFN-γ Pre, associated with up-reg of ICE. No or small up-FR in all cell-lines.	[175]
hu, Kidney cancer (OUR-10)	f-FR and f-CD (Pre), IFN-γ (nd) ± IFN-α (nd), 24 h	FR-mAb (CH-11, IgM), 330 ng/ml, 48 h	Up-FR and up-CD by treatment with IFN-γ, not augmented by coexistence of IFN-α.	[183]
mu, Pancreas islet-β (β-TC-1)	f-FR, muIFN-γ (105 U/l) ± muIL-1α (103–104 U/l), 6 h	None	Significant up-reg of FR mRNA by combined treatment with IFN-γ and IL-1α.	[283]
hu, Bone osteosarcoma (HOS/TE-85, MG-63, Saos-2)	f-FR and f-CD (Pre), IFN-γ (250–500 U/ml) ± TNF-α (250 U/ml), 24 h	FR-mAb (nd, nd), 1 µg/ml, 24 h	Synergistic up-FR and up-CD of resistant cells by combined treatment with IFN-γ and TNF-α.	[190]
hu, Thyroid gland epithelium (GD patients)	f-FR and f-CD (Pre), IFN-γ (500 U/ml) ± [healthy donors', GD patients' or IME patients' IgG (5mg/ml) or boTSH (5 mU/ml)], 48 h	FR-mAb (nd, IgM), 1000 ng/ml, 18 h	Up-FR and up-CD by IFN-γ treatment, associated with significant inhibition by coexistence of GD patients' IgG or TSH, but not of healthy donors' or IME patients' IgG.	[205]
hu, Colon cancer (HT-29)	f-FR, f-CD (Pre), IFN-γ (200 U/ml or 25 ng/ml) ± ActD (5 µg/ml), 3–6 h (f-FR, mRNA); 24–48 h (f-FR, protein and f-CD)	FR-mAb (CH-11, IgM), 5–100 ng/ml or muFL-N2A, 0.25–0.5 arbitrary U/ml ± antagonistic FR-mAb (ZB4, IgG1), 2 µg/ml, 72 h	More or equivalent up-FR and up-CD by treatment with IFN-γ, compared to clinical cytotoxic drugs. Blockade of the enhancing effects in the presence of ActD or antagonistic FR-mAb.	[30]
hu, Colon cancer (HT-29)	f-FR, f-CD (Pre), IFN-γ (200 U/ml), 16 h	FR-mAb (nd, nd), 10–50 ng/ml, 24 h	p53-state independent up-CD by IFN-γ Pre, linked with up-FR and up-reg of other many apoptosis-related gene products including Bak but not Bax.	[119]
mu, Ovary surface epithelium (OSE) [corpora lutea (CL) of pseudo-pregnant CD1 mice]	f-FR and f-CD (Pre), muIFN-γ (1–1000 U/ml), 24 h	muFR-mAb (Jo2, IgG2), 1 µg/ml, 8 h	Up-FR mRNA and marked up-CD by 10-1000 U/ml IFN-γ Pre. No effect on other cell types except OSE in CL culture.	[243]
hu, Bone marrow (mononuclear, CD34+) (posterior iliac crest of healthy volunteers)	f-FR and f-CD (Pre, colony-formation), IFN-γ (20–2000 U/ml), 48 h; (f-FR); 24 h (f-CD)	FR-mAb (CH-11, IgM), 1 µg/ml, 24 h	Ds-dep up-FR and up-CD by IFN-γ treatment, without significant change in IFN-γR expression.	[224]
hu, Stomach cancer (HM-02)	f-CD (Co), IFN-γ (10 ng/ml) plus TNF-α (20 ng/ml) ± H. p. extract (106 cfu/ml), 24 h	FR-mAb (nd, nd), 20 ng/ml, 24 h	Significant up-CD by IFN-γ plus TNF-α treatment, enhanced by the addition of H. p. extract.	[109]
hu, Lung cancer (A-549)	f-FR and f-CD (Pre), IFN-γ (250 U/ml) ± DEX (1mM), 24 h (f-FR); 12h (f-CD, Pre)	FR-mAb (nd, IgM), 100 ng/ml, 36–60 h	Up-FR and up-CD by IFN-γ treatment, associated with induction of ICE and IRF-1 expression. An inhibitory effect of DEX due to expression of CIAP-2.	[169]
hu, Colon cancer (HT-29, HCT-116, HCT-8)	f-FR and f-CD (Pre), IFN-γ (100 U/ml), 24 h	FR-mAb (CH-11, IgM), 50–200 ng/ml, 72 h	Up-FR in all cell lines and marked up-CD of HT-29 by IFN-γ treatment.	[120]
hu, Colon cancer (HT-29)	f-FR, IFN-γ (200 U/ml), 24 h	None	Up-FR by IFN-γ treatment.	[98]
mu, Bone marrow Mφ (infected with Leishmania major) (mice: wt, lpr-mut, gld-mut)	f-FR and f-CD (Pre), muIFN-γ (50 U/ml), 48 h	muFL-N2A, 20% (v/v), 4 h	Up-FR and up-CD in wt and gld-mut, but not in lpr-mut, Mφ infected with L. major by IFN-γ treatment.	[213]
hu, Colon cancer (COLO-205, HT-29)	f-FR and f-CD (Pre), IFN-γ (1–2000 U/ml), 24 h	muCTL cells (F3H, MD45), E/T ratio: 40:1–1:5, 16 h; sFL in COS cells supernatant, 1:2 dilution, 16 h	Marked up-FR and up-CD in COLO-205, but not in HT-29, by IFN-γ treatment, associated with enhanced cell-surface CEA expression.	[121]
hu, Colon cancer (COLO-201)	f-FR, f-FL and f-CD (Co), IFN-γ (3–100 U/ml) ± TNF-α (0.3 µg/ml), 3–5 d (f-FR and f-CD, Co); 3–12 h (f-FL)	FR-mAb (nd, nd), 100 ng/ml, 3d	Synergistic ds-dep up-FR and up-CD by combined treatment with IFN-γ and TNF-α, associated with dn-reg of Bcl-2 and up-reg of Bax.	[122]
hu, Blood eosinophils (peripheral blood of normal healthy volunteers)	f-FR and f-CD (Pre), IFN-γ ± TNF-α (1–100 ng/ml each) ± [IL-3 (0.0001–10 ng/ml), IL-5 or GM-CSF (10 ng/ml each)], 0.5–24 h	sFL in 293T cells (transfected with FL expression vector) supernatant, equal volume, 12 h	Tm-dep, ds-dep up-FR and significant up-CD by combined treatment with IFN-γ and TNF-α, associated with some inhibition against the up-FR by coexistence of IL-3, IL-5 and GM-CSF.	[232]
mu, Ovary granulosa (pregnant mare serum gonadotropin treated)	f-FR and f-CD (Pre or Pre plus Co), muIFN-γ (200 U/ml) ± muTNF-α or TNF-α (10 ng/ml), 24 h (Pre) ± following 24 h (Co)	muFR-Ab (Jo2, IgG2), 2 µg/ml ± muTNF-α, 10 ng/ml, 24 h ± CHX, 0.5 µg/ml, 2 h preceded	Synergistic up-reg of FR mRNA and up-CD by IFN-γ treatment combined with muTNF-α, but not with huTNF-α, in the presence or absence of CHX.	[248]
mu, Central nervous system microglia (primary)	f-FR and f-CD (Pre), muIFN-γ (100 U/ml) ± muTNF-α (10 ng/ml), 24–48 h	muFL-N2A, 20% (v/v), 20 h ± [FR-Fc (0.05–30 µg/ml) ± Protein A enhancer], 30 m preceded	Up-FR and synergistic up-CD by combined treatment with IFN-γ and TNF-α, associated with marked dn-reg of Bcl-XL expression and ds-dep inhibition by coexistence of FR-Fc.	[256]
hu, Multiple myeloma (IL-6 dependent: U-266-1970, U-1958; IL-6 independent: U-266-1984)	f-FR and f-CD (Pre), IFN-γ (1–1000 U/ml) + IL-6 (20–100 U/ml), 96 h	FR-mAb (CH-11, IgM), 100 ng/ml, 24 h	Significant ds-dep up-CD only in IL-6 dependent cells by IFN-γ treatment, without substantial change in FR.	[151]
hu, Colon cancer (COLO-205, SW-480, SW-620, HT-29)	f-FR and f-CD (Pre), IFN-γ (100 U/ml), 6–72 h ± PMA (5 ng/ml); SSP (35 µM) or Goe6983 (l µM), 0.5–1 h preceded	FR-mAb (nd, IgG3), 500 ng/ml, 3–24 h	Up-FR and up-CD by IFN-γ treatment, counteracted by presence of PMA. Delayed apoptosis of SW-620 as compared with SW-480, associated with the absence of FR capping.	[123]
hu, Colon cancer (HT-29), fibrosarcoma (U-3A, STAT-1 transfected U-3A)	f-FR, f-FL and f-CD (Pre), IFN-γ (0.1–500 U/ml), 3–48 h (f-FR and f-FL); 40 h (f-CD, Pre)	FR-mAb (CH-11, IgM), 500 ng/ml, 20 h	Ds- and tm-dependent, marked up-FR, modest up-FL and up-CD in HT-29 cells by IFN-γ treatment. Up-FR and up-FL in STAT-1 transfected U-3A, but not in STAT-1-deficient U-3A cells.	[124]
hu, Ovarian cancer (A-2780, AD-10)	f-FR and f-CD (Pre), IFN-γ (10–1000 U/ml) ± L-NMA (1.0 mM), 18–24 h	FR-mAb (CH-11, IgM), 10–1000 ng/ml, 24 h	Cl-dep, ds-dep up-FR and up-CD by IFN-γ treatment, associated with dn-reg effects by iNOS inhibitor co-treatment.	[192]
hu, Breast cancer (T-47D, MCF-7, BT-20)	f-FR and f-CD (Pre), IFN-γ (1000 U/ml), 24 h	Anti-ErbB-2 scFv transfected MD45 murine CTL cells, E/T ratio: 1–40:1, 16 h	Up-CD in two (T-47D, BT-20) of three ErbB-2 positive cells without up-FR, but not in MCF-7, by IFN-γ Pre.	[176]
hu, Myeloblastic leukemia (EoL-1)	f-FR and f-CD (Pre), IFN-γ (1000 U/ml), 72 h	FR-mAb (CH-11, IgM), 50–1000 ng/ml, 24 h	Up-FR and up-CD by IFN-γ treatment, but marked inhibition of TNF-α induced apoptosis.	[152]
hu, Stomach cancer (AGS)	f-FR, IFN-γ (12.5 ng/ml), 24 h	None	Up-FR comparable to H. p. infection by IFN-γ treatment.	[193]
ra, Liver Kupper Mφ (KC), parenchymal (PC) (male Wister rats)	f-FR, f-FL and f-CD (Co), raIFN-γ (100 U/ml) ± CsA (1 µM), 3–48 h (f-FR, f-FL); 24 h (f-CD, Co)	FR-mAb (CH-11, IgM), 10–100 ng/ml, 24 h	Up-FL in KC cells, up-FR in KC and PC cells, and up-CD in PC cells by IFN-γ treatment, all associated with a small inhibitory effect of CsA.	[237]
hu, Colon cancer (HT-29)	f-CD (Pre), IFN-γ (1000 U/ml), 24 h	FR-mAb (CH-11, IgM), 100 ng/ml, 17 h	Up-reg of α-1, 3 fucosyltransferase activity by IFN-γ treatment.	[125]
hu, Small intestinal epithelium (non-tumoral fatal crypt); colonic organ cultures	f-FR and f-CD (Co), INF-γ (1–1000 U/ml) ± TNF-α (0.01–10 ng/ml), 24–48 h	FR-mAb (CH-11, IgM), 1–500 ng/ml, 24–48 h	Synergistic up-FR and up-CD by combined treatment with IFN-γ and TNF-α, both in cell and organ cultures.	[273]
mu, Bone marrow (femurs and tibiae), spleen (C57BL/6 mice)	f-FR and f-CD (Co, colony formation), muIFN-γ (100 U/ml), 24 h	muFR-mAb (Jo2, IgG2), 5 µg/ml, 24 h	Up-FR and up-CD by IFN-γ treatment, evaluated by in vitro and in vivo colony-formation assays.	[225]
hu, Colon cancer (HT-29, Caco-2, HCT-8, HCT-116, GC3/c1, VRC5/c1)	f-FR, f-FL and f-CD (Co), IFN-γ (25–100 U/ml) ± 5-FU (3 µM) and LV (1 µM); IFN-γ (25–100 U/ml) ± anti-FR mAb (ZB4, IgG1) (100 ng/ml), 24–96 h	FR-mAb (CH-11, IgM), 10–200 ng/ml, 24–96 h	Cl-dep, tm-dep up-FR and up-FL, associated with some augmentation with 5-FU and LV, and up-CD, associated with inhibitory effect of antagonistic FR-mAb, by IFN-γ treatment.	[126]
hu, Melanoma (A-375, BLM, BS-1251, Colo-38, M-518, Me1Ju, Me1Juso, MeWo, SK-Mel-25, SK-Mel-28, W+); leukemia (Jurkat); lymphoma (Raji)	f-FR and f-CD (Co), IFN-γ (100–2500 U/ml), 48 h	FR-mAb (7C11, IgM), 50 ng/ml, 48 h	Cl-dep up-CD of melanoma cell-lines, associated with up-FR and/or up-reg of Bcl-XS, by IFN-γ treatment. No apparent correlation of up-FR degree with up-CD level.	[187]
mu, Pancreas islet (RAG2−/− NOD transgenic mice: wt and lpr-mut)	f-FR and f-CD (Co), muIFN-γ (250 U/ml) ± muIL-1α or muIL-1β (10–100 U/ml) ± Z-VAD-FMK (nd), 16 h	NOD mice-derived spleen CD4+-CTL, E/T ratio: 10:1, o/n; muFR-mAb (Jo2, IgG2), 1 µg/ml plus ActD, 30 µg/ml, 16 h	Up-FR and up-CD by treatment with IFN-γ, associated with enhancement by coexistence of IL-1α or IL-1β, and partial dn-reg with pan-casp inhibitor, in NOD mice islet cells with wt FR, but not in lpr-mut FR.	[199]
hu, Colon cancer with Val12-ras mutation (SW-480)	f-FR and f-CD (Pre), IFN-γ (250 U/ml), 24 h	Anti-Val12-ras CTL, E/T ratio: 1.25–10:1, nd; FR-mAb (CH-11, IgM), 1–1000 ng/ml, 24 h	Up-FR, associated with HLA-2 and ICAM-1 up-reg, up-CD induced with CTL or FR-mAb, and abrogation of CMA-inhibited CD, by IFN-γ treatment.	[127]
mu, Kidney cancer (Renca)	f-FR and f-CD (Pre), muIFN-γ (100 U/ml) ± muTNF-α (100 U ml), 3–24 h	muFR-mAb (Jo2, IgG2), 0.002–1 µg/ml plus mAb- crosslinking P815 cells, 1 × 105 cells/200 µl, 18 h	Some up-FR in the constitutively resistant cells by IFN-γ treatment, but significant up-CD only after combination with TNF-α.	[184]
hu, Colon cancer with Val12-ras mutation (SW-480, SW-620)	f-FR and f-CD (Pre), IFN-γ (250 U/ml), 18–24 h	Anti-Val12-ras CTL, E/T ratio: 2.5–20:1 ± CMA, 10 µM, 12 h; FR-mAb (CH-11, IgM), 1–2000 ng/ml or sFL, 0.1–100 ng/ml, 18 h	Significant up-FR and up-CD with CTL by IFN-γ treatment in both SW-480 and SW-620 cells, but associated with stronger CD-inhibition with CMA in SW-640 than in SW-480 cells. Marked up-CD by IFN-γ Pre, associated with either FR-mAb or sFL only in SW-480 cells.	[128]
hu, Eye conjunctival (Chang conjunctiva clone 1-5c-4)	f-CD (Pre), IFN-γ (30 U/ml), 24 h	FR-mAb (CH-11, IgM), 500 ng/ml, 1–24 h	Tm-dep up-CD by non-toxic dose of IFN-γ Pre, associated with up-reg of STAT-1.	[275]
mu, Brain neuron [euploid and Down syndrome model trisomy 16 (Ts16) fatal mice]	f-FR, muIFN-γ (67–600 U/sample), 48 h	None	Significant ds-dep up-FR in Ts16 cells with higher sensitivity, compared to euploid cells by IFN-γ treatment.	[209]
mu, Kidney cultured tubular epithelium (primary)	f-FR and f-CD (Pre plus Co), IFN-γ (300 U/ml), muTNF-α (30 ng/ml) and LPS (10 µg/ml), 48 h (f-FR); 24 h (f-CD, Pre), and then 24 h (f-CD, Co)	sFL, 1–100 ng/ml with 10-fold excess crosslinking antibody, 24 h	Up-FR and marked up-CD in the serum-deprived resistant cells by combined treatments with a mixture of IFN-γ, TNF-α and LPS.	[255]
hu, Peripheral blood eosinophils (normal non-atopic donors)	f-FR and f-CD (Pre), IFN-γ (1–100 ng/ml) ± TNF-α (1–100 ng/ml), 6–24 h	sFL, 100 ng/ml, 12 h	Synergistic up-FR and positive correlation of up-FR with up-CD in combined treatment with IFN-γ and TNF-α.	[233]
hu, Salivary duct epithelium (HSG)	f-FR and f-CD (Pre), IFN-γ (1–100 U/ml), 4–48 h (f-FR); 24 h (f-CD, Pre)	FR-mAb (DX-2, IgG1), 500 ng/ml, 3 h	Ds-dep, tm-dep up-FR and up-CD by INF-γ treatment.	[271]
hu, Colon cancer (HT-29, SW-620)	f-CD (Pre), IFN-γ (50–2000 U/ml), 16 h	FR-mAb (CH-11, IgM), 100 ng/ml ± CHX, 1 µg/ml, 24 h	Correlation of up-CD degree with constitutive expression level of Bcl-2. Necessity of CHX for maximal up-CD of SW-620 cells.	[129]
hu, Brain oligodendrocyte (adult intractable epilepsy patients)	f-FR and f-CD (Pre), IFN-γ (100 U/ml) ± TNF-α (100 U/ml), daily over 72 h	FR-mAb (nd, nd), 10 µg/ml, 24 h ± Z-VAD, 0.02 %, 1h preceded	Significant up-FR and up-CD by IFN-γ treatment, associated with augmentation by coexistence of TNF-α and inhibition with pan-casp inhibitor.	[207]
hu, Colon cancer (HT-29-D4)	f-CD (Pre), IFN-γ (40 ng/ml), 5 m pulse	FR-mAb (CH-11, IgM), 500 ng/ml ± des-(1-3)-IGF-1, 50 ng/ml, 24 h	Up-CD by IFN-γ Pre, associated with insignificant inhibition by coexistence of N-terminally truncated IGF-1.	[130]
mu, Testis seminiferous epithelium (Sertoli)	f-FR, f-FL and f-CD (Pre), muIFN-γ (500 U/ml) ± muTNF-α (20 ng/ml), 24 h	muFL-N2A, E/T ratio: 1–10:1, 7 h	Up-FR and up-CD by treatment with IFN-γ alone and synergistic up-reg of them after combination with TNF-α, without up-reg of FL mRNA.	[252]
hu, Breast cancer (MCF-7: wt p53, MDA-MB231: mut p53)	f-FR, f-FL and f-CD (Pre or Co), IFN-γ (10 ng/ml), 24–48 h (f-FR); 24–36 h (f-CD, Pre); 48–72 h (f-CD, Co)	FR-mAb (CH-11, IgM), 500 ng/ml, 24–72 h; sFL, 100 ng/ml plus 200 ng/ml sFL-crosslinker, 48 h	Up-FR without induction of FL mRNA, up-CD independent of p53 status by IFN-γ treatment, associated with casp-8 activation and prevented by intracellular Bcl-2 oe.	[67]
hu, Umbilical vein endothelial (HUVEC), vascular smooth muscle (hVSMC) (internal mammary artery), leukemia (Jurkat E6-1)	f-FR and f-CD (Co), IFN-γ (100 ng/ml), 24 h (f-FR); 48 h (f-CD, Co)	Ad-muFL, multiplicity of infection: 300, 48 h	Marked up-FR in HUVEC cells, but not in hVSMC and Jurkat cells, and significant up-CD in hVSMC cells, but not in HUVEC cells, after Ad-muFL infection, by IFN-γ treatment.	[153]
hu, Thyroid gland follicular [nontoxic goiter (NTG), Hashimoto's thyroiditis (HT) and Grave's disease (GD) patients]	f-CD (Pre), IFN-γ (1000 U/ml) ± [IL-4 (20 ng/ml) or IL-10 (40 ng/ml)], 24 h	FR-mAb (CH-11, IgM), 200 ng/ml, 24 h	Small up-CD by IFN-γ Pre in HT and NTG cells. Enhanced Bcl-XL and cFLIP in GD cells. Abolition of the sensitizing effect by coexistence of IL-4 or IL-10.	[206]
hu, Stomach cancer (Kato-III, AGS-NY-2, N-87)	f-FR and f-CD (Pre), IFN-γ (100 U/ml) ± H. p. (300 bacteria/epithelial cell) ± TNF-α (40 ng/ml), 24–48 h (f-FR), 6–48 h (f-CD)	FR-mAb (CH-11, IgM), 50–100 ng/ml, 12 h	Synergistic up-FR by IFN-γ treatment in combination with H. p. and TNF-α. Up-FR and up-CD in all cell lines by IFN-γ Pre alone.	[194]
mu, Central nervous system microglia (primary)	f-FR, muIFN-γ (100 U/ml) ± muTNF-α (10 ng/ml) ± TGF-β2 (20 ng/ml), 24 h	None	Up-FR by IFN-γ ± TNF-α treatment, associated with significant reduction by coexistence of TGF-β2 in the case of treatment with IFN-γ alone.	[257]
hu, Salivary gland epithelium cells (non-neoplastic primary derived from SS patients and control individuals)	f-FR and f-FL, IFN-γ (500 U/ml), 48 h ± DEX (500 nM), 60 h	None	Significant up-FR and up-FL by IFN-γ treatment, associated with complete inhibition by coexistence of DEX.	[272]
hu, Lung cancer (A-549)	f-CD (Pre), IFN-γ (40 ng/ml), 6 h	FR-mAb (CH-11, IgM), 100 ng/ml, 24 h	Up-CD by IFN-γ Pre, associated with significant up-reg casp-1 and -3.	[170]
hu, Colon cancer (HT-29)	f-CD (Pre), IFN-γ (10 ng/ml), 16 h	FR-mAb (CH-11, IgM), 5–200 ng/ml or TNF-α, 0.05–2.0 ng/ml, 24 h	Up-CD by IFN-γ Pre via FR-mAb stimulation similar to that via TNF-α stimulation, but through different NF-κB activation mechanism in IκB-α degradation.	[131]
hu, Liver normal (Chang liver), cancer (SK-HEP-1, HEP-G2, -3B, -G2.2.15, PLC/PRF/5, SNU-182, -354, -387, -398, -423, -449, -475)	f-FR and f-CD (Pre), IFN-γ (250 U/ml), 24 h (f-FR); 36 h (f-FR and f-CD)	FR-mAb (CH-11, IgM), 250 ng/ml, 36 h	Insignificant up-CD in most cell lines except three (SNU-354, 387 and 423) by IFN-γ Pre. Significant up-FR in the strongly sensitized cell lines.	[195]
hu, Lymphoma (U-937), myeloid leukemia (HL-60, THP-1)	f-CD (Pre), IFN-γ (10 U/ml), 24 h	FR-mAb (CH-11, IgM), 20 ng/ml, 24–36 h	Up-CD by IFN-γ Pre, associated with dn-reg of Bcl-2 and up-reg of casp-8.	[154]
hu, Bile duct cancer (Sk-ChA-1)	In vitro: f-FR and f-CD (Pre), IFN-γ (250 U/ml), 12–18 h; in vivo (5 × 106 tumor cells-injected athymic 6–8 w age female mice): 5 × 104 U/d/mouse, (3 daily injections in 4 d) × 3 in 2 w	FR-mAb (CH-11, IgM), 250 ng/ml, 15–20 h	Up-FR and up-CD in either FR level-low or -high subpopulation cells in vitro by IFN-γ treatment, associated with up-reg of casp-3, -4, -8, -7 -10a and Bak. Decreased tumorigenicity of FR level-low cells in nude mice.	[196]
bo, Ovary corpus luteum (midluteal stage of estrous cycle)	f-FR and f-CD (Pre plus Co), boIFN-γ (50 ng/ml) ± TNF-α (50 ng/ml), 24 h, (f-FR; f-CD, Pre) followed by 24 h (f-CD, Co)	sFL, 100 ng/ml, 24 h	Significant up-reg of FR mRNA and up-CD by IFN-γ treatment, associated with augmentation by coexistence of TNF-α.	[244]
hu, Placenta trophoblast (A-3, HTR/8)	f-FR, f-FL and f-CD (Pre and Co), IFN-γ (5–10 ng/ml) ± IL-10 (10 ng/ml), 12–24 h (f-FR, f-FL); 24 h (f-CD)	FR-mAb (nd, nd), 20–500 ng/ml, 24 h	Up-FR without up-FL, and up-CD by IFN-γ treatment, associated with inhibition against up-CD by coexistence of IL-10.	[250]
hu, Lung cancer (A-549)	f-FR and f-CD (Pre), IFN-γ (250 U/ml) ± IL-1β (50 pg/ml), 24 h ± LY294002 (10 µM), 30 m preceded	FR-mAb (CH-11, IgM), 10–100 ng/ml, 4–24 h	Up-FR and up-CD by IFN-γ treatment. Sequence-dependent opposing effects to IFN-γ regarding up-CD by IL-1β treatment, associated with reversal by Pre with PI3K inhibitor.	[171]
hu, Leukemia (B-lineage: SEM, RS-4;11, REH, Nalm-6; T-lineage: Jurkat, CEM), lymphoma (Raji, Ramos)	f-FR and f-CD (Pre), IFN-γ (500 U/ml) ± [castanospermine (1.06 mM), 1-deoxymannojirimycin (2 mM) or swainsonine (57.7 µM)], 48 h	FR-mAb (CH-11, IgM), 200 ng/ml, 48 h	Up-reg of high molecular-weight form FR in B-lineage cell lines, except Nalm-6, by IFN-γ treatment. Change in CD-sensitivity, but not in up-FR, by treatments with N-linked glycosylation inhibitors.	[155]
hu, Neuroblastoma (CHP-126, KAN, SH-SY5Y, Kelly), medulloblastoma (D283Med), Ewing's sarcoma (CADO-ES-1, STA-ET-2.1, 6647, RDES), fibrosarcoma [2fTGH, U-3A, U-3A-STAT-1]	f-FR and f-CD (Co), IFN-γ (1000 U/ml) ± [Z-VAD-FMK (50 µM) or Z-IETD-FMK (50 µM)], 24–72 h	FR-mAb (nd, IgG3), 0.1 µg/ml, 24–72 h	Up-FR and up-reg of casp-8 in various neuroblastoma, Ewing's sarcoma and medulloblastoma cells. Blockade of up-CD by Co with pan-casp or casp-8 specific inhibitor, or by deficiency in STAT-1 protein.	[164]
hu, Lung cancer (A-549)	f-CD (Co), IFN-γ (500 U/ml) ± [Z-VAD-FMK (20 µM) or IETD-FMK (50 µM)], 1–3 d (Co)	FR-mAb (nd, nd), 200 ng/ml, 1–3 d (Co)	Up-CD by IFN-γ Co via up-reg of casp-8, and selective increase in casp-7 and -8, by treatment with IFN-γ alone.	[172]
hu, Umbilical vein endothelium (HUVEC), dermal microvasculature endothelium (HDMEC)	f-FR and f-CD (Pre), IFN-γ (4–300 ng/ml), 3–22 h (f-FR); IFN-γ (100 ng/ml), 20 h (f-CD, Pre)	CTL (preincubated with PMA, 10ng/ml plus ionomycin, 0.5 µM, 1 h), E/T ratio: 15:1, 5 h	Ds-dep, tm-dep up-FR in both types of normal endothelial cells. Up-CD of HUVEC cells by IFN-γ Pre.	[263]
hu, Normal intestine crypt epithelium (primary, colon and jejunum) (HIPEC); colon cancer (HT-29), leukemia (Jurkat)	f-FR and f-CD (Pre), IFN-γ (200 U/ml) ± TNF-α (10 ng/ml), 18–20 h	FR-mAb (CH-11, IgM), 100–300 ng/ml, 1–24 h ± Z-IETD-FMK, Z-LEHD-FMK or Z-DEVD-FMK (5 µM each), 15 m preceded	Insignificant up-FR, but significant up-CD of HIPEC cells by IFN-γ Pre, to the level of Jurkat cells with delay, associated with considerable degree of reduction by coexistence of casp-3, -8 and -9 inhibitors.	[132]
hu, Thyroid gland epithelium (primary, normal and multinodular goiter patients' thyroid tissues)	f-CD (Pre), IFN-γ (100 U/ml) ± TNF-α (50 ng/ml) ± IL-1β (50 U/ml), 4 d	FR-mAb (CH-11, IgM), 1 µg/ml, 20 h	Up-CD in goitrous cells, much less than normal cells, by Pre with IFN-γ in combination with IL-1β and/or TNF-α.	[267]
hu, Colon cancer (HT-29, in vitro), peripheral mononuclear blood (PBMC, in vivo) (stage-IV cancer patients)	f-FR, in vitro: IFN-γ (1-10 U/ml), 2 h; in vivo: IFN-γ (25–100 µg/m2), administered at days 1 and 3	None	Tm-dep up-FR with the maximum of 3-fold in HT-29 in vitro, and 2.5-fold in CD15+ PBMC from cancer patients in vivo, by IFN-γ treatment.	[133]
hu, Colon cancer (HT-29)	f-CD (Co), IFN-γ (80 U/ml, for HT-29 cells only), ± Z-VAD-FMK (50 µM)	FR-mAb (CH-11, IgM), 0.2–200 ng/ml, 3–4 d	Up-CD by IFN-γ Co in HT-29, not blocked by coexistence of pan-casp inhibitor.	[134]
mu, Bone marrow Mφ (Leishmania major infected)	f-CD (Co), IFN-γ (10 ng/ml), 48 h	sFL, 100 ng/ml, 48 h	Requirement of Co with IFN-γ and sFL for leishmanicidal activity in the infected bone marrow Mφ.	[214]
hu, Blood erythroid colony-forming (normal donors)	f-CD (Co), IFN-γ (400 U/ml) ± SCF (100 ng/ml), 24-120 h	sFL, 10–50 ng/ml, 24–120 h	Protective effect of SCF from up-CD by IFN-γ Co via inhibition of casp-3 and -8 activation and up-reg of FLIP.	[236]
hu, Colon cancer (HT-29, HCT-116, HCT-116 p53-/-)	f-FR, IFN-γ (100 U/ml) ± ZD9331 (50–100 nM), 24 h	None	p53-state dependent enhancing effect of thymidylate synthase inhibitor on up-FR by IFN-γ treatment.	[135]
hu, Small cell lung cancer (SCLC) (N-417)	f-FR and f-CD (Co), IFN-γ (2500 U/ml) ± 5dAzaC (1 µM), 72 h	muFL-N2A, 10%, 72 h	Enhanced up-FR and up-CD in SCLC cells by IFN-γ treatment after combination with 5dAzaC.	[173]
hu, Acute myeloid leukemia (GM-CSF-dependent) (AML-193)	f-FR and f-CD (Co), IFN-γ (1000 U/ml), 24–40 h, after pre-cultivation with GM-CSF (20 ng/ml), at least 2 w	FR-mAb (FAS18, IgG2a), 5 µg/ml, 24–40 h	No difference in FR and 2.5-fold median up-CD by IFN-γ treatment in the absence of GM-CSF, associated with selective reduction of G1 cell-cycle state.	[156]
hu, Colon cancer [SW-480, SW-620, both original and IFN-consensus binding protein (ICSBP)-gene transfected]	f-FR and f-CD (Pre), IFN-γ (250 U/ml), 4–24 h	FR-mAb (CH-11, IgM), 1 µg/ml, 18–24 h ± casp-1 inhibitor (Z-YVAD-FMK or Z-LEVD-FMK), 20 µM, 30 m preceded	Marked up-FR in both primary and metastatic cells by IFN-γ treatment. Significant up-CD by IFN-γ Pre in SW-480 cells and ICSBP-gene transfected SW-620 cells.	[136]
hu, Colon cancer [SW-480 (parent), SW-480sell (metastatic subline selected in vitro), SW-480spl (metastatic subline obtained in vivo, SW-620 (primary metastatic tumor)]	f-FR and f-CD (Pre), IFN-γ (250 U/ml), 18–24 h	FR-mAb (CH-11, IgM), 1 µg/ml, 18–24 h	Resemblance of SW-480sell and SW-480spl cells to SW-620 cells regarding CD resistance in spite of significant up-FR by IFN-γ treatment and morphology, associated with identical genome-wide gene expression profiles.	[137]
hu, Peritoneum mesothelial (effluents from peritoneal dialysis patients)	f-FR and f-CD (Pre), IFN-γ (300 U/ml) ± TNF-α (5000 U/ml), 24 h	sFL (aggregated), 10–100 ng/ml, 24 h	Up-FR, associated with additive effect of TNF-α, and significant up-CD, by IFN-γ treatment.	[219]
hu and mu, Prostate cancer (hu: LNCaP, PC-3; mu: RM-1)	f-CD (Pre), IFN-γ (100 U/ml) or muIFN-γ (25–200 U/ml), 24 h	FR-mAb (nd, nd) or muFR-mAb (nd, nd), 10 µg/ml each, 1 d; sFL in 30x conc. TC268 cell culture medium, 1:1 (v/v), 18 h; Ad-muFL, moi: 3.125-25, 1h exposure plus 24 h incubation	Synergistic up-CD in all prostate cancer cells, irrespective of the induction via either exogenously provided FR-mAb or sFL, as well as via transfection with Ad-muFL, by IFN-γ Pre.	[198]
hu, Kidney cancer (ACHN)	f-CD (Pre plus Co), IFN-γ (400 U/ml) ± [DEVD-CHO, YVAD-CHO (10 mM each), IRF-1 AS-ODN (2.5–20 µM) or random ODN (10 µM)], 31 h (Pre) followed by 17 h (Co)	FR-mAb (CH-11, IgM), 800 ng/ml, 17 h	Up-CD by IFN-γ treatment without up-FR, inhibited strongly either by casp-3/7 inhibitor or by IRF-1 AS-ODN, weakly by casp-1 inhibitor, but not by random ODN.	[185]
hu, Pancreas cancer (AsPC-1, BxPC-3, Capan-2, CFPAC-1, HPAC, MIAPaCa-2, PANC-1)	f-FR and f-CD (Pre plus Co), IFN-γ (40 U/ml), 48 h (f-FR); 24 h (f-CD, Pre) followed by 48 h (f-CD, Co)	FR-mAb (CH-11, IgM) or sFL, 1–100 ng/ml, 24–72 h; mFL-transfected murine L5178 lymphoma cells (mFL), E/T ratio: 10:1, 48 h	Up-CD in 5 of 7 cell lines induced with FR-mAb, 4 in 7 cell lines with mFL, by IFN-γ treatment without up-FR. Significant correlation of mFL-, but not FR-mAb-, mediated growth inhibition with endogenous DcR3 level.	[197]
hu, Colon cancer (Caco-2, Colo-320, SW-948)	f-FR and f-CD (Pre), IFN-γ (1–1000 U/ml), 24 h ± CHX (5 µg/ml), 1 h preceded to FR-mAb addition	FR-mAb (7C11, IgM), 50–1000 ng/ml, 24 h	Marked up-FR and up-CD by IFN-γ treatment, augmented by coexistence of CHX, in SW-948 cells, less in Colo-320 cells, but not significant in Caco-2 cells.	[35]
hu, Spontaneously transformed umbilical vein endothelium (ECV-340)	f-FR and f-CD (Pre), IFN-γ (100 U/ml), 6–48 h (f-FR); 12 h (f-CD, Pre)	sFL, 50–5000 ng/ml or FR-mAb (CH-11, IgM), 50–500 ng/ml, 20 h	Tm-dep up-reg of FR mRNA and ds-dep up-CD, associated with enhancement of IL-8 and MCP-1 secretion.	[264]
hu, Placenta villus trophoblasts and explanted tissues (woman with uncomplicated pregnancies)	f-FR, f-FL and f-CD (Co, explant tissues), IFN-γ (0.1–50 ng/ml), 24 h ± FR-Fc (2 ng/ml), 1 h preceded	Endogenous FR-mediated CD inducer in explanted tissues	No up-FL, insignificant up-FR, and up-CD by IFN-γ treatment, associated with suppression by FR-Fc Pre.	[251]
mu, Pancreas β-cells insulinoma (NIT-1) (autoimmune diabetes-prone NOD mice)	f-FR and f-CD (Co), IFN-γ (1000 U/ml) plus IL-1β (100 U/ml) ± Z-VAD, 100 µM, 24 h	Super-FL, 100 ng/ml, 24 h	Significant up-FR and up-CD by IFN-γ treatment, associated with up-reg of casp-3 activity and prevention with pan-casp inhibitor.	[201]
hu, Oligodendroglioma (HOG, MO-3.13)	f-FR, f-FL and f-CD (Pre), IFN-γ (1–500 ng/ml) ± TNF-α (25–100 ng/ml), 24 h	sFL, 1–10 ng/ml, 24–72 h	Significant up-FR and up-CD by IFN-γ treatment, associated with up-reg of MHC-I expression and augmented up-FR in combination with TNF-α.	[165]
hu, Astrocytoma (CRT-J, U-373-MG, U-87-MG, U-251-MG, CH-235-MG)	f-FR and f-CD (Pre), IFN-γ (20–1000 U/ml), 0–72 h	FR-mAb (CH-11, IgM), 100–1000 ng/ml, 24 h	Up-FR in all cell lines, associated with cl-dep up-reg of casp-1, but up-CD by IFN-γ treatment, limited to CRT-J, U251-MG and CH235-MG.	[166]
mu, Brain cortical astrocytes (primary) (1 to 2 d old mice)	f-FR and f-CD (Pre), muIFN-γ (5 U/ml) and/or muTNF-α (10 ng/ml) and/or muIL-1β (10 ng/ml) ± SB203580 or SB202190 (0.1–20 µM each), 0.5–24 h ± W1400 (10–50 µM) or indomethacin (10 µM), prior addition	muFR-mAb (Jo2, IgG2), 1–104 µg/ml, sFL, 10−2–102 ng/ml plus enhancer, 1 µg/ml or muFL-N2A, 2%, 24 h	Tm-dep up-FR mRNA and up-CD in resistant cells by Pre with complete cytokines (IFN-γ, TNF-α and IL-1β) mix, accompanied by ds-dep inhibition of CD with p38 mitogen activated protein kinase inhibitor, but neither with iNOS inhibitor or COX-2 inhibitor.	[258]
hu, Proximal lung epithelium [normal bronchus (NHBE), small airway (DLEC)]	f-FR and f-CD (Pre and Co), IFN-γ (100 U/ml), 18 h	sFL, 25–500 ng/ml, 18h (Pre); last 16 h (Co)	No up-CD in DLEC cells, but significant up-CD in NHBE cells, without significant up-FR, by IFN-γ Pre. No up-CD in both types of cells by IFN-γ Co.	[277]
hu, Colon cancer (HT-29, DLD-1), monocyte leukemia (THP-1)	f-CD (Pre plus Co), IFN-γ (10 pM), o/n-9 d (Pre), followed by 24 h (Co)	FR-mAb (APO-1-3, IgG3), 60–600 pM, 24 h; sFL, 3.1 nM, 24 h	Significant up-CD in HT-29 and DLD-1, but not THP-1, by Pre with 10 pM IFN-γ o/n. Decrease in CD response by prolonged IFN-γ Pre.	[138]
hu, Melanoma [HeLa, cell death inhibiting RNA (CDIR) oe or control vector transfected]	f-CD (Pre), IFN-γ (200 U/ml), 24 h	FR-mAb (CH-11, IgM), 100 ng/ml, 10–30 h	Inhibition of up-CD caused by IFN-γ Pre in HeLa cells, transfected with CDIR oe vector.	[188]
hu, Kidney mesangial (glomeruli from normal portion of renal tissue)	f-FL, IFN-γ (1000 U/ml) ± lactacystin (20 µM), 24 h	None	Significant up-FL by IFN-γ treatment, associated with complete reversal by coexistence of NFκB blocker.	[254]
hu, Colon cancer (HT-29, in vitro); peripheral blood mononuclear (PBMC, in vivo) (stage-IV cancer patients)	f-FR, in vitro: IFN-γ (1–10 U/ml), 6.5 h ± 5FU (38.5 µM) and LV (1 µM), 2 h preceded; in vivo: IFN-γ (10–100 µg/m2), at days 1, 3 and 5 of a 28 d cycle with LV (200 mg/m2) and 5FU (370 mg/m2), at days 1–5 daily	None	Up-FR in PBMC from cancer patients in vivo and HT-29 in vitro by treatment with clinically achievable IFN-γ conc. Positive correlation of up-FR with the area under conc–time curve and time above 33.3 pg/ml in several compartments of PBMC.	[139]
ra, Colon cancer (CC-531s)	f-CD (Pre), IFN-γ (100 U/ml), 24 h	sFL, 1–100 ng/ml ± crosslinking antibody, 1 µg/ml, 18 h; LSEC cells, E/T ratio: 2.5–10:1 ± anti-FL mAb, 20 µg/ml, NMA, 1 mM or DEX, 10 µg/ml, 18 h	Up-CD induced with either sFL or co-cultured LSEC cells by IFN-γ Pre. No effect of anti-FL mAb and suppression effects on CD with nitric monoxide inhibitors	[140]
hu, Breast cancer (MCF-7)	f-FR and f-CD (Pre), IFN-γ (10–1000 U/ml) ± AT (0.1–1000 nM) ± CHX (20 µM), 6–72 h (f-FR); 48 h (f-CD, Pre)	FR-mAb (CH-11, IgM), 500 ng/ml ± CHX, 5 µM, 12–48 h	Ds-dep, tm-dep up-FR and modest up-CD by synergistic treatment with IFN-γ and AT, associated with up-reg of STAT-1, TR1 and PKR. Enhanced up-CD in the presence of CHX.	[177]
hu, Multiple myeloma (U-266-1970, U-1958; both IL-6 dependent)	f-FR, f-FL and f-CD (Pre), IL-6 (20 U/mL) ± IFN-γ (1000 U/ml) and, 0.5–96 h	FR-mAb (CH-11, IgM), 100 ng/ml, 6–24 h	Significant up-FR and up-CD by IFN-γ treatment without large change in Bcl-2 family members' expression, associated with enhancement of STAT-1 activation and attenuation of STAT-3 activation.	[157]
Target cells: species, cell type (cell line/origin)	Sensitization method: type (mode), sensitizer (conc), treatment time	Cell-death induction method: inducer (name, subclass), conc, treatment time	Main findings related to sensitization of cell-death induction via Fas signaling system	Refs.
hu, Cervical cancer (HPV-18 positive and wt p53: HeLa; HPV-16 positive and wt p53: CaSki, SiHa; HPV negative and mut p53: C-33A)	f-FR, f-FL and f-CD (Pre), IFN-γ (10–100 U/ml), 24 h (f-FR); 2 h (Pre)	FR-mAb (7C11, IgM), 50–1000 ng/ml, 24 h	Up-FR in all HPV positive cells, but not in C-33A, without major change in p53 levels by IFN-γ treatment. Strong up-CD only in HeLa and insignificant up-CD in other cells.	[74]
hu, Thyroid epithelium (primary, contralateral lobes of normal thyroid tissues)	f-FR and f-CD (Pre), IFN-γ (100 U/ml) plus IL-1β (50 U/ml), 4 d	FR-mAb (CH-11, IgM), 0.1–1 µg/ml, 1 h - o/n ± U0126, 10–50 µM, 30 m preceded	Significant up-FR and up-CD by IFN-γ treatment, associated with significant up-reg of many pro-apoptotic molecules, including Bid. Enhanced up-CD by coexistence of a p44/p42 MAPK kinase inhibitor.	[268]
hu, Salivary gland epithelium (healthy donors, Sjögren's syndrome patients)	f-FR and f-CD (Pre), IFN-γ (1000 U/ml), 72 h	FR-mAb (CH-11, IgM), 1 µg/ml ± anti-CD40 mAb, 1 µg/ml, 24 h	Up-FR and marked up-CD by IFN-γ treatment, only under cooperated stimulation with anti-CD40 mAb.	[210]
hu, Cervical cancer (KB: original, Bcl-2, FLIP-L or FLIP-S gene-transfected); Colon cancer (HT-29)	f-FR and f-CD (Pre), IFN-γ (20 ng/ml), 24 h	[FLAG-tagged sFL, 0.01–1000 ng/ml + Anti-FLAG mAb (M2, IgG1), 0.5–1 µg/ml, or Fc-sFL, 500 ng/ml] ± Z-VAD-FMK, 20 µM, 30 m-18 h	Up-FR and up-CD in both KB and HT-29 cells by IFN-γ treatment, associated with up-reg of IκBα, IL-6 and IL-8 expression. Inhibition of up-CD by coexistence of pan-casp inhibitor or FLIP-L/S oe, and less by Bcl-2 oe.	[141]
hu, Neuroblastoma (Paju)	f-FR and f-CD (Pre), IFN-γ (100 ng/ml), 16 h (f-FR); 24 h (f-CD, Pre)	FR-mAb (CH-11, IgM), 3 µg/ml, 24 h	Up-FR, but insignificant up-CD, by IFN-γ treatment, associated with neurite outgrowth.	[167]
mu, Small intestine epithelium (immortalized by SV-40 large T-gene transfection) (MODE-K, IEC-4.1)	f-FR and f-CD (Pre), [IFN-γ ± TNF-α (2–10 ng/ml each)] ± IL-10 (5–50 ng/ml), 24–48 h	muFR-mAb (Jo2, IgG2), 1 µg/ml, 24 h	Marked up-FR and up-CD by synergistic treatment with IFN-γ and TNF-α, associated with suppression by coexistence of IL-10.	[274]
hu, Skin pre-adipocyte and adipocyte (primary, SGBS patients)	f-FR and f-CD (Co), IFN-γ (1000 U/ml) ± TNF-α (10 nM), 6–72 h	FR-mAb (nd, IgG3), 1 µg/ml, 6–72 h	Tm-dep synergistic up-FR and up-CD by combined treatment with IFN-γ and TNF-α, associated with increased DISC formation.	[211]
hu, Lung fibroblast (fatal, MRC-5; primary, undifferentiated from normal lung tissue), myofibroblast (both, differentiated with TGF-β)	f-FR and f-CD (Pre), IFN-γ (50 U/ml) ± TNF-α (20 ng/ml), 36–48 h	FR-mAb (CH-11, IgM), 250 ng/ml, 4–12 h	Marked up-FR and up-CD, associated with efficient DISC formation, by synergistic combined treatment with IFN-γ and TNF-α, but not by IFN-γ treatment alone.	[279]
mu, Mastocytoma (P-815), lymphoma (LS-102.9); wc, hepatocytes (WCM-260)	f-FL and f-CD (Pre), wcIFN-γ (1.5–1500 U/ml) ± muFR-mAb (Jo2, IgG2) (nd), 18 h	Wc hepatocytes (IFN-γ pretreated WCM-260), E/T ratio: 1.5–12:1, 18 h	Significant up-FL and up-reg of MHC-I expression in wc hepatocytes by IFN-γ Pre. Ds-dep up-CD of mu cells by treatment with IFN-γ-sensitized wc hepatocytes, associated with inhibition by muFR-mAb Pre.	[158]
hu, Oral mucosa melanoma (MMN9)	f-CD (Co), IFN-γ (1000 U/ml), 24 h	FR-mAb (mHFE7A, IgG), 0.1 µg/ml, 4 h, then with cross-linking antibody, 1.0 µg/ml, 24 h,	Weaker Up-CD by IFN-γ Co using an IgG subclass FR-mAb than that using an IgM subclass FR-mAb as the agonistic for CD-induction.	[189]
hu, Aorta vascular smooth muscle (cardiac transplantation patients)	f-FR and f-CD (Pre), IFN-γ (500 U/ml), 24 h ± LY2940002 (10 µM), BFA (5 µg/ml), ActD (1 µg/ml), tyrphostin AG490 (10 µM) or SH5 (20 µM), 30 m preceded	mFL, 1:2000, 6–24 h	Up-reg of cell-surface FR without increase in total FR and up-CD by IFN-γ treatment, associated with abrogation by coexistence of inhibitors to intracellular trafficking, PI3K, Akt, and Jak2.	[215]
mu, Colon cancer (C-15-4.3: MC-38 transfected with hu CEA gene)	f-FR and f-CD (Pre), muIFN-γ (1000 U/ml) ± muTNF-α (250 U/ml), o/n	CEA-mimicking epitope (3H1)-specific CTL, E/T ratio: 50:1 ± [CMA (500 nM), anti-FL mAb or anti-TRAIL mAb (10 µg/ml each)], 8 h	Marked up-FR and up-CD by synergistic treatment with IFN-γ and TNF-α, associated with inhibition by additional combination of CMA, anti-FL mAb and anti-TRAIL mAb.	[142]
hu, Uterus endometrial stroma (ESC: undifferentiated, decidualized) (benign hysterectomy tissue from pre-menopausal woman)	f-FR and f-CD (Pre), IFN-γ (50 ng/ml) ± TNF-α (25 ng/ml) ± hCG (1 U/ml), 24–48 h	FR-mAb (CH-11, IgM), 100 ng/ml, 24 h	Up-FR and up-CD irrespective of differentiation state by combined Pre with IFN-γ and TNF-α without strong influence by coexistence of hCG, associated with up-reg of FLIP mRNA.	[281]
hu, Thyroid gland epithelium (primary, contralateral lobes of normal thyroid tissues)	f-CD (Pre), IFN-γ (100 U/ml) plus IL-1β (50 U/ml), 3 d ± Z-VAD, Inhibitor for casp- (IC)1, IC3, IC4, IC8, IC10, SB203580 or MG132 (singly or in pairs), 60 m preceded	FR-mAb (CH-11, IgM), 1 µg/ml, o/n	Up-CD by IFN-γ plus IL-1β Pre, associated with significant prevention with inhibitors of pan-casp or specific to casp-3/7, -10 and p38 MAPK kinase, but not with proteasome inhibitor.	[269]
hu, Eye subconjunctival Tenon fibroblast (biopsy specimen from glaucoma patients)	f-FR, IFN-γ (100 U/ml) ± IFN-α (5000 U/ml), 48 h	None	Significant up-FR by IFN-γ treatment, associated with augmentation by coexistence of IFN-α and up-reg of sensitivity to MMC-mediated CD.	[220]
mu, Soft tissue sarcoma (CMS-4: parental; transfectant with empty-control, K79E-mut or R289E-mut IRF-8 gene)	f-FR and f-CD (Pre), IFN-γ (100 U/ml), o/n-24 h	sFL, 200 ng/ml, 24 h	Up-FR and marked up-CD by IFN-γ treatment in wt IRF-8 expressing cells, associated with enhanced STAT-1 activation and significant inhibition by oe of functionally-impaired mut IRF-8.	[181]
hu, Peripheral blood CD4+ leukocyte [healthy donors (HD) and Sézary syndrome (SzS) patients]	f-FR and f-CD (Pre), IFN-γ (100 U/ml), o/n	Super-FL, 100 ng/ml, 6–8 h	Significant up-FR and up-CD in both CD4+ leukocytes from HD and SzS patients by IFN-γ treatment.	[212]
mu, Thyroid gland epithelium [primary, parental (Tg−) and FLIP transgenic (Tg+) mice]	f-FR, f-FL and f-CD (Pre), IFN-γ (100 U/ml) and TNF-α (50 U/ml), 4 d	muFR-mAb (Jo2, IgG2), 1 µg/ml, o/n	Up-reg of FR and FL mRNAs in both FLIP Tg+ and Tg− cells and up-CD only in Tg− cells by combined treatment with IFN-γ and TNF-α.	[270]
hu, Astroglioma (ADF, LN-229, U-373)	f-FR, f-FL and f-CD (Pre), IFN-γ (500 U/ml) ± lactacystin (1.25–10 µM), 24–72 h	FR-mAb (CH-11, IgM), 500 ng/ml, 24 h ± casp inhibitor (Z-VAD-, IETD-, AEVD- or LEHD-FMK) (50 µM each), 2 h preceded	Tm-dep up-FR, up-reg of sFL release, associated with up-reg of Bax, Bak expression. Marked up-CD by IFN-γ treatment, associated with ds-dep up-reg by a proteasome-specific inhibitor, but with attenuation by coexistence of casp-specific inhibitors.	[168]
hu, Heart vascular fibrous cap (atherosclerotic lesion)	f-FR and f-CD (Pre), IFN-γ (5 ng/ml), 20–24 h ± siRNA (non-targeted and targeted to PSME-1, PSME-8 or PSME-1 and -8 mix) (total 50 nM), 24–48 h preceded	FR-mAb (CH-11, IgM), 50–100 ng/ml, 24 h	Up-FR without block by siRNA Pre and up-CD by IFN-γ treatment, associated with inhibition by Bcl-XL oe or Pre with siRNA targeted to PSME-8 component for immunoproteasome.	[217]
mu, Convoluted-type T-cell lymphoma (ST4: wild-type and LI255-256 to AA mutation in IFN-γ receptor 2)	f-FR and f-FL, IFN-γ (1000 U/ml), 24 h	None	Marginal up-FR in both types of cells by IFN-γ treatment. Significant up-FL only in cells expressing mut IFN-γ receptor, associated with more persisted STAT-1 phosphorylation.	[159]
eq, Ovary corpus luteum (midluteral stage of estrous cycle)	f-CD (Co), IFN-γ plus TNF-α (10 ng/ml each), 24 h	sFL, 10 ng/ml, 24 h	Modest up-CD by Co with IFN-γ and TNF-α, associated with up-reg in PARP 1 activity, P4 production, and dn-reg of PGF2α production.	[245]
bo, Ovary corpus luteum (midluteral stage of estrous cycle)	f-CD (Co), boIFN-γ (2.5 nM) plus TNF-α (2.9 nM), 48 h	sFL, 2.9 nM, 48 h	Insignificant change in CD and cFLIP levels by treatment with IFN-γ plus TNF-α, after combination with sFL.	[246]
hu, Lung cancer (A-549); breast cancer (MCF-7) (parental and Rel A-kd with both cells)	f-FR, IFN-γ (50 ng/ml) plus TNF-α (20 ng/ml) ± anti-IFN-γRα mAb (nd) plus anti-TNF-α mAb (nd), 24 h	None	Up-FR by IFN-γ treatment, associated with reduction by either blocking IFN-γ and TNF-α or kd of main member of NF-κB family.	[174]
mu, Bone marrow lymph-node (inguinal, axillary and lateral axillary from normal B6 mice)	f-FR, muIFN-γ (20-60 ng/ml), 18–72 h	None	Up-FR in total and residual cells after body γ-ray irradiation by IFN-γ treatment, accompanied by up-reg expression and co-localization of FR with MHC-class II.	[226]
hu, Colon cancer (HT-29)	f-FR and f-CD (Co), in vitro: IFN-γ (250 U/ml) or Let-7 inhibitors (50 nM), 24 h; in vivo: IFN-γ (1000 U) or Let-7 inhibitors (20 µg), intra-tumoral injections at days 4, 6 and 8 in 18 d period	FR-mAb (CH-11, IgM), 1 µg/ml (in vitro), 24 h; 20 µg/injection (in vivo)	Up-FR by IFN-γ treatment and its enhancement in the presence of FR-mAb. Enhancing effect of inhibitor against micro RNA targeting to FR mRNA, similar to IFN-γ, on up-CD both in vitro and in vivo.	[143]
ra, Pancreas insulinoma (INS-1E: uninfected, infected with Ad expressing Bcl-6 or control luciferase)	f-FR, raIFN-γ (100 U/ml) plus IL-1β (10 U/ml), 6–24 h	None	Marked up-reg of FR mRNA by combined-cytokines treatment in either uninfected or control Ad-infected cells, but significant attenuation in Bcl-6-expressing Ad-infected cells.	[202]
mu, Bone marrow (tibiae and femurs of 8-w-old mice)	f-FR and f-FL, muIFN-γ (100 ng/ml) plus M-CSF (100 ng/ml), 4 d	None	Up-FL in non-adherent cells, but no significant up-FR, irrespective of cell-adherence by IFN-γ treatment.	[227]
hu, Bone osteosarcoma (HOS, U-2OS)	f-FR and f-CD (Pre and Co), IFN-γ (500 U/ml), 48 h (f-FR and f-CD, Pre for CH-11 treatment; f-CD, Co for γδ-T cells treatment)	FR-mAb (CH-11, IgM), 100 ng/ml, 24 h; γδ-T cells ± [CMA (15 nM) or anti-FL mAb (10 µg/ml)], 30 m preceded, E/T ratio: 1.5–12:1, 4–18 h	Significant up-FR and up-CD by IFN-γ treatment, associated with reduction of CD after IFN-γ Pre induced with γδ-T cells Co by coexistence of FR-mediated pathway inhibitor, but weaker effects than perforin-mediated pathway inhibitor.	[191]
hu, Pancreas islet β [βLox5: wild type, mitochondria (mt)-depleted mutant (ρ0), cybrid of ρ0 with platelet cells holding normal mt DNA]	f-FR and f-CD (Co), IFN-γ (250–1000 U/ml), o/n (f-FR); IFN-γ (1000 U/ml) ± Z-VAD-FMK (50 µM × 2), 48 h (f-CD, Co)	FR-mAb (CH-11, IgM), 500 ng/ml, 48 h	Significant up-FR and up-CD in wt cells, and up-CD in ρ0 and cybrid cells by IFN-γ Co, associated with complete suppression by coexistence of pan-casp inhibitor.	[241]
mu, Bone marrow mesenchymal stem (bone cavity of tibiae and femurs)	f-FR, IFN-γ (50 ng/ml) ± TNF-α (20 ng/ml), 24 h ± Fas siRNA treatment (nd), nd	None	Up-FR by combined treatment with IFN-γ and TNF-α, associated with suppression by Fas siRNA treatment. Significant up-FR, but insignificant activation of casp-3 and -8, by treatment with IFN-γ alone.	[228]
mu, Pancreas islets (wild-type and Bid deficient NOD mice)	f-CD (Co), IFN-γ (100 U/ml) plus IL-1β (10 U/ml) ± TNF-α (1000 U/ml), 3–5 d	sFL, 100 nM, 3–5 d	Significant protection of Bid-deficient islet cells from sFL-induced CD under Co with IFN-γ plus IL-1β ± TNF-α.	[200]
mu, Eye conjunctiva epithelium (dry-eye model mice; wild-type and IFN-γ gene-kd)	f-FR, in vivo: muIFN-γ (10000 U/eye/injection), before, at 2nd and 4th d in 5 d desiccating stress period	None	More enhanced up-FR mRNA level in wt mice as compared with IFN-γ-gene- kd mice by IFN-γ treatment, associated with up-reg of casp-3, -8, -9 activities.	[221]
hu, Blood vessel smooth muscle cells-like myofibroblast (carotid artery atherosclerotic lesion)	f-CD (Pre), IFN-γ (50 U/ml), 8 h ± ApoL6 siRNA (10 nM), o/n preceded	FR-mAb (CH-11, IgM), 10–25 ng/ml, 20 h	Significant up-CD by IFN-γ Pre, associated with partial suppression by Pre with ApoL6 siRNA.	[218]
ra, Pancreas islet-β (primary); insulinoma (INS-1E); (either transfected with CHOP-kd or control siRNA)	f-FR, raIFN-γ (100 U/ml) plus [IL-1β (10 U/ml) or TNF-α (1000 U/ml)], 8 h (INS-1E); raIFN-γ (1000 U/ml) plus [IL-1β (50 U/ml) or TNF-α (1000 U/ml)], 24 h (primary)	None	Up-reg of FR-mRNA by combined-cytokines treatment. Suppression by siRNA-kd of CHOP, associated with decrease in NF-kB activity and in degradation of Bcl-2 and Mcl-1.	[203]
mu, Soft tissue sarcoma (CMS-4: wild-type, mutant IRF-8-expressing or IRF-8-deficient via shRNA transfection)	f-CD (Pre), IFN-γ (200 U/ml), 24 h ± TSA (20–100 nM), 4 h preceded	sFL, 100 ng/ml, 24 h	Up-CD by IFN-γ Pre, associated with augmentation by coexistence of TSA and attenuation by either deficiency or impaired function of IRF-8 using shRNA transfection.	[182]
hu, Multiple myeloma (U-266-1970, U-266-1970-STAT-1C)	f-CD (Pre), IFN-γ (1000 U/ml), 96 h	FR-mAb (CH-11, IgM), 100 ng/ml, 24 h	Insignificant enhancement in up-CD by IFN-γ Pre after stable expression of a constitutively active mut STAT-1 alone.	[160]
hu, Cervical cancer (HeLa: Ro52 siRNA or control siRNA transfected)	f-CD (Pre), IFN-γ (50 ng/ml), 24h	FR-mAb (nd, nd), 250 ng/ml, 3 h	Up-CD by IFN-γ Pre, associated with significant suppression after Ro52-siRNA transfection.	[179]
mu and hu, lung alveolar wall (normal lung tissue from healthy mice) (mu, in vivo); normal microvascular endothelium (HPMVEC) and alveolar epithelium (HPAEpiC) (hu, in vitro)	f-FR and f-CD (Co), in vivo: [muIFN-γ (100 µg) ± anti-muCD40 mAb (30 µg)] per intratracheal injection, every 3 d × 8; in vitro: IFN-γ (1000 U/ml) ± anti-CD40 mAb (0.2–20 µg/ml), 12 h	muFR-mAb (Jo2, IgG2), in vivo: 0.1 µg/g/injection, every 3 d × 8; in vitro: FR-mAb (nd, nd), 2.0 µg/ml, 12 h	Up-FR and up-CD specific to HPMVEC by treatment with IFN-γ alone either in vivo or in vitro. More pronounced up-CD in HPMVEC than in HPAEpiC by IFN-γ treatment alone or combination with anti-CD40 mAb.	[265]
mu, Airway CD4+ T lymphocytes (lung tissue and peri-bronchial lymph nodes of OVA-sensitized asthma model mice); spleen CD4+ T lymphocytes (naïve)	f-FR and f-FL, in vivo: IFN-γ (10–1000 U/mouse/d), 6 d, 5 m preceding each OVA inhalation ± anti-FL mAb (MFL-3, IgG) (100 µg/mouse), at day 2; in vitro: IFN-γ (0.1–10 U/ml) ± anti-FL mAb (MFL-3, IgG) (10 µg/ml), 6–24 h	None	Ds-dep dn-reg of cell-surface FR and FL in OVA-sensitized airway T-cells in vivo, and up-reg of cell-surface FL in naïve and OVA-treated spleen T-cells in vitro, by IFN-γ treatment. Both occurred without significant effect of anti-FL mAb.	[208]
hu, Eye conjunctiva epithelium (IOBA-NHC) (normal, spontaneously immortalized)	f-FR and f-FL, IFN-γ (500 U/ml) ± TNF-α (25 ng/ml), 24-48 h ± CsA (10 µM), 24 h-pre-, co- or 24 h-post-treatment	None	Up-FR and up-FL by combined treatment with IFN-γ and TNF-α, but only up-FR with IFN-γ alone, associated with treatment procedures-dependent reduction by coexistence of CsA.	[276]
ra, Pancreas islet β (RINm5F; wild-type and plasmid vector for SOCS1-oe transfected)	f-FR, IFN-γ (1000 U/ml) ± IL-1β (50 U/ml) ± TNF-α (1000 U/ml)	None	No alteration in FR mRNA level by treatments with IFN-γ alone or in combination with other cytokines, irrespective of SOSC1 oe.	[242]
hu, Epidermal keratinocyte (HEK) (primary from normal neonatal foreskin)	f-FL, IFN-γ (20 ng/ml) ± TNF-α (75 ng/ml) ± IL-1β (500 pg/ml), 24–72 h ± NMA (3 mM), 2 h preceded	None	Significant up-FL mRNA by combined-cytokines treatment containing IFN-γ, accompanied by up-reg of NO production with suppression by coexistence of iNOS inhibitor.	[261]
bo, Ovary corpus luteum (midluteal stage of estrous cycle)	f-FR and f-FL, IFN-γ (50 ng/ml) plus TNF-α (50 ng/ml) ± LPA (435 ng/ml), 6–12 h	None	Up-FR and up-FL by combined treatment with IFN-γ and TNF-α, associated with suppression by coexistence of LPA and up-reg of Bax, but not of Bcl-2, expression.	[247]
hu, Blood monocytes, Mφ (buffy coats of healthy donors)	f-FR, IFN-γ (10 ng/ml) ± IL-10 (50 ng/ml) + DEX (100 nM), 3 d (for monocytes); 8 d (for Mφ, IL-10 and DEX added on day 5)	None	Up-FR by IFN-γ treatment, associated with significant augmentation by coexistence of IL-10 or DEX and reduction of MerTK expression.	[234]
bo, Uterus endometrium epithelial and stromal (intercaruncular endometrial tissues form healthy Holstein cows, 2–5 d after ovulation)	f-FR and f-CD (Pre plus Co or Co alone), boIFN-γ (50 ng/ml) plus TNF-α (100 ng/ml), 24 h (f-FR, Co); 24 h plus 24 h (Pre plus Co)	sFL, 50–100 ng/ml, 24 h	Marked up-reg of FR mRNA and up-CD by Pre with IFN-γ and TNF-α in both types of cells. More prominent CD in stromal cells than in epithelial cells by the combined treatment.	[278]
hu, Uterus endometrial epithelium cancer (RL95-2: wild-type, Sdc-1-kd)	f-FR and f-CD (Pre), IFN-γ, IL-1β (10 ng/ml each), TNF-α (5 ng/ml) plus TGF-β1 (0.5 ng/ml), 24 h	FR-mAb (EOS9.1, IgM), 5 µg/ml, 7 h	Up-FR and up-CD in both types of cells by combined-cytokines Pre plus FR-mAb treatment, associated with up-reg of Bad, FADD and XIAP.	[180]
mu, Blood Mφ-like leukemia (RAW264)	f-CD (Pre), IFN-γ (10 ng/ml) plus LPS (1 µg/ml), 24 h	mu auricular chondrocytes (wt, gld mut), E/T ratio: 1.25–10:1, 4 h	Ds-dep more up-CD by Pre with IFN-γ plus LPS induced with wt effector cells than that with gld mut cells.	[161]
hu, Kidney cortex / proximal tubule epithelium (HK-2) (transformed with HPV-16)	f-CD (Co), IFN-γ (50 ng/ml) ± TNF-α (25 ng/ml), 48 h	FR-mAb (CH-11, IgM), 0.5 µg/ml, 48 h	No up-CD by Co with either IFN-γ alone or combination of IFN-γ and TNF-α.	[186]
mu, Small intestine lamina propria CD4+ T-lymphocytes (naïve and peanuts-derived allergen sensitized 6-8-w-old mice)	f-FL, IFN-γ (20 ng/ml), nd	None	Significant up-FL by treatment with IFN-γ alone in cells from either naïve or allergen-sensitized mice, associated with increased unmethylation of FL gene.	[240]
hu, Uterus immortalized endometrial stromal (St-T1: wild-type, Sdc-1-kd: both non-differentiated, decidualized)	f-FR and f-CD (Pre), IFN-γ (10 ng/ml) ± [IL-1β (10 ng/ml), TNF-α (5 ng/ml) plus TGF-β1 (0.5 ng/ml)], 24 h	FR-mAb (EOS9.1, IgM), 5 µg/ml, 24 h	Up-FR and significant up-CD by treatment with all combined-cytokines containing IFN-γ, associated with sdc-1-kd or differentiation state dependent difference in up-reg degree.	[282]
mu, Bone marrow hematopoietic stem and progenitor (KSL, KL, KLCD150+, KLCD150+ CD48−) (tibiae and femurs)	f-FR and f-CD (Co), in vitro: muIFN-γ (25–50 ng/ml) plus [IL-3, IL-6, SCF and Flt-3 L (10 ng/ml each)], 24 h - 6 d; in vivo: IFN-γ (10 ng/mouse), 20 h - 2 d	muFR-mAb (nd, nd), 0.5–2.0 µg/ml, 24 h	Significant up-FR in all types of cells by treatments with IFN-γ either in vitro or in vivo, and reduced recovery of total KSL or BM cells by Co with IFN-γ and FR-mAb.	[229]
hu, Neuroblastoma [SH-SY5Y, SK-N-BE (2)]	f-FR and f-CD (Pre), IFN-γ (100 ng/ml), 24 h ± TNF-α (100 ng/ml), 24 h followed	Fc-sFL, 100 ng/ml, 24 h	Significant up-FR and up-CD in both cells by IFN-γ treatment, associated with enhanced CD in highly resistant cells by additional Pre with TNF-α.	[54]
hu, Cervical cancer (HeLa), colon cancer (HT-29)	f-FR and f-CD (Pre), IFN-γ (100–1000 U/ml), 2–24 h ± [TLCK, TPCK or EI (10–100 µM)], 30 m preceded	FR-mAb (nd, nd), 5–1000 ng/ml, 2–4 d	Significant up-FR and up-CD by IFN-γ treatment, associated with augmentation by Pre with TLCK or TPCK, but not with EI.	[144]
hu, Blood basophils (venous EDTA-blood of healthy non-selected donors)	f-CD (Pre), IFN-γ (1000 U/ml), 24–48 h	Super-FL, 5–100 ng/ml, 24 h	No up-CD by IFN-γ Pre, in spite of STAT-1 activation.	[235]
ra, Ovary granulosa [diethylstilbestrol-primed (relatively undifferentiated); eCG-primed (differentiated)]	f-FR and f-CD (Pre plus Co), raIFN-γ (10–1000 U/ml), 18 h (Pre) plus 6 h (Co)	muFR-mAb (Jo2, IgG2), 1 µg/ml, 6 h	Up-reg of FR mRNA, requiring a higher IFN-γ dose in differentiated cells, and more prominent up-CD in relatively undifferentiated cells, by Pre with IFN-γ.	[249]
mu, Bone marrow mesenchymal stem (bone cavity of tibiae and femurs of pathogen-free mice)	f-FR, IFN-γ (200 ng/ml) ± IL-17 (200 ng/ml), 24–72 h	None	Tm-dep synergistic up-reg of FR mRNA by combined treatment with IFN-γ and IL-17, associated with STAT-1 and casp-8 activation.	[230]
mu, Skin hair follicle (alopecia areata mice)	f-FR, [IFN-γ (50 ng/ml) plus TNF-α (25 ng/ml)] ± SOCS3 (nd), 48 h	None	Marked suppression of up-FR mRNA by IFN-γ plus TNF-α treatment in the presence of SOCS3, associated with concomitant dn-reg of MHC-I mRNA.	[262]
hu, Liver hepatic stellate (hTERT-immortalized)	f-FR, IFN-γ (100 ng/ml) ± 1MT (0.5 mM), 3–72 h	None	Tm-dep up-FR by IFN-γ treatment, associated with up-reg of STAT-1, IDO-1, -2 and dn-reg of α-SMA as well as up-reg of IFN-γRβ1 in the presence of 1MT.	[239]
hu, Leukemia (K-562)	f-FR and f-FL, IFN-γ (10–1000 U/ml), 12–48 h	None	Ds-dep, tm-dep up-FR and up-FL by IFN-γ treatment.	[162]
hu, Colon cancer (HT-29)	f-CD (Pre), IFN-γ (0.1–100 U/ml), 24–72 h	SSCC of sFL with fluorescein or avidin, 0.001–1000 ng/ml, 4–72 h	Ds-dep and tm-dep up-CD of resistant colon cancer cells by Pre with IFN-γ.	[145]
ra, Pancreas islet-β insulinoma (INS-1E)	f-FR, raIFN-γ (1000 U/ml) plus IL-1β (10 U/ml), 48 h ± PRL (0.5 µg/ml), 24 h preceded	None	Marked up-FR by combined treatment with IFN-γ plus IL-1β, associated with significant attenuation, concomitant with dn-reg Iκ-Bα, MCP-1, A20 and Cxcl-10 expression, by Pre with PRL.	[204]
mu, Spinal cord motoneuron (wt mice)	f-CD (Pre), muIFN-γ (10 ng/ml), 24 h	CD8+ T-lymphocyte cells (wt, E/T ratio: 10:1, 72 h	Insignificant up-CD in co-culture together with wt T-cells by Pre with IFN-γ.	[259]
hu, Cardiac myofibroblast (normal adult ventricles)	f-FR and f-FL, IFN-γ (100 ng/ml) ± 1MT (0.5 mM), 3–72 h	None	Tm-dep up-FR and up-FL by treatment with IFN-γ, associated with significant augmentation by coexistence of IDO inhibitor.	[280]
hu, Colon cancer (HT-29)	f-CD (Pre), IFN-γ (10–1000 U/ml) ± 5FU (25–100 U/ml), 24 h	SSCC of sFL with biotin, 100 ng/ml, 72 h	Enhanced up-CD by IFN-γ Pre in combination with 5FU.	[146]
mu, Mesenchymal stem / stromal (tibia and femur bone marrow from wt, FR−/− lpr-mut, iNOS-deficient mice)	f-FR and f-CD (Co), [IFN-γ (10 ng/ml) ± TNF-α (10 ng/ml)] ± SNAP (20 µM), 3–48 h	muFR-mAb (Jo2, IgG2), nd, 48 h	Tm-dep significant up-reg of FR mRNA and CD-induction in wt cells, less in iNOS-deficient cells and no in FR−/− lpr-mut cells, by synergistic treatment with IFN-γ and TNF-α.	[231]

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4.2.   Other cytokine agents

In addition to the combined treatments with IFN-γ, many exogenous cytokine agents independent of IFN-γ have been applied for the sensitization studies. The summarized results of them are listed in Table 3 [54],[122],[124],[132],[138],[142],[151],[153],[156],[157],[158],[161],[163],[165],[171],[181],[183],[184],[186],[187],[190],[194],[199],[205],[207],[211],[212],[216],[219],[220],[224],[227],[228],[230]–[235],[238],[240],[244],[248],[250]–[252],[254],[256]–[258],[261],[266],[267],[271]–[274],[276],[277],[279],[281]–[310]. As compared to the agents containing IFN-γ, the number of the studies employing the other cytokine agents lacking IFN-γ as a component is still rather limited. Among them, TNF-α and IFN-α, -β are the representative molecules, which have been frequently used for the sensitization experiments to date. TNF-α is a protein that belongs to the same TNF ligand-superfamily as is the case for FasL. However, unlike FasL, TNF-α is known to exert primarily not only pro-apoptotic but also anti- or non-apoptotic biological activities, such as proliferation, angiogenesis and metastasis, on the target cells, due to the presence of a pair of receptors, TNFR-I and TNFR-II. These receptors are responsible for the mediation of several distinct signaling pathways including nuclear factor-kappa B (NF-κB) and c-Jun N-terminal kinase (JNK) signaling pathways, which can give negative feedbacks against the apoptosis pathway [311]. On the other hand, IFN-α and IFN-β belong to the Type-I interferon family, which regulates many cellular functions by activating signaling processes, including the JAK-STAT pathway that closely crosstalk to each other with apoptosis-priming cytokines, represented by IFN-γ [112],[312]. Thus, the sensitization effects caused by TNF-α or IFN-α, -β to Fas signaling system can be less straightforward in transmitting the signals received at each responsible receptor than the cases primed with IFN-γ, because of the presence of rather complicated regulation mechanisms arising from many crosstalk between each signaling system and the IFN-γ signaling system. Nevertheless, in majority of the cases, the up-regulation of cell-death induction concomitant with the enhancement of cell-surface FasR expression was also observed in the treatment with the exogenous agents, containing TNF-α or IFN-α, -β, but lacking IFN-γ, which accompanied intracellular pro-apoptotic events, such as the activation of caspases-3, -7, -8, or STAT-1; increase in poly (ADP-ribose) polymerase cleavage; and decrease in STAT-3 activation. The sensitization degrees were often significantly correlated with the intracellular expression levels of FLIP, Bcl-2, Bcl-X_L, Bax, Bcl-2 homologous antagonist / killer protein (Bak), Bcl-2 like protein 11 (Bim) and Bcl-2 homology domain 3 interacting-domain death agonist (Bid). In the treatments with TNF-α-containing agents, IL-1β or TGF-β exhibited the enhancing [216],[258] and suppressing [257],[298] action to TNF-α in multiple studies. Also, the inhibition of NF-κB signaling pathway reversed the up-regulation effects on cell-surface FasR and cell-death of the target cells, caused by TNF-α treatment [54],[292],[310]. The TNF-α treatment combined with ribavirin synergistically up-regulated the cell-death of hepatoma [293]. As for IFN-α and IFN-β, the combined treatments with other low molecular weight compounds, such as epigallocatchin-gallate [304] or methotrexate [305], and post-treatment with temozolomide [307], have been identified as the enhancing factors for the sensitization in some cases.

Apart from TNF-α and IFN-α, -β, a variety of other cytokines and hormones, including IL-1α, -1β [163],[171],[199],[205],[251],[254],[258],[261],[266],[267],[271]–[273],[277],[282],[283],[287],[309], IL-2 [183],[271],[287],[303], IL-3 [238],[235], IL-12 [285],[294], IL-17, -18 [230],[234],[240],[289],[294], other ILs [161],[163],[232],[234],[250],[271],[274],[287],[298], TGF-β1, -β2 [163],[186],[257],[282],[298], TNF-β [163], TNF related apoptosis-inducing ligand (TRAIL) [302], macrophage inflammatory protein (MIP)-1α, -1β, -2, IFN-γ-inducible protein-10 (IP-10), keratinocyte-derived chemokine (KC) [161], kit ligand (KL) [238], GM-CSF [132],[232],[238],[271],[298], granulocyte colony-stimulating factor (G-CSF) [161], macrophage colony-stimulating factor (M-CSF) [163], platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF) [124], and thyroid stimulating hormone (TSH) [205],[266], have been examined for sensitization to target cells as the exogenous agents individually. Among them, IL-1α and IL-1β constitute the most frequently used cytokines after TNF-α and IFN-α, -β. IL-1 are known to exert pleiotropic biological functions including the augmentation of IFN-γ-mediated cytolytic activity of natural killer cells [112],[313] and anti-tumor activity of macrophages [314], where Fas signaling system is expected to have profound connections with them. However, the actual actions of IL-1 in the sensitization to FasR-mediated cell-death largely depended on the cases, and even significant down-regulation of FasL expression was observed in an instance [272]. This may be closely related to the mechanism that the signal transduction by IL-1 is implemented through the modulation of NF-κB [315], which is also a major transcription factor for cell-proliferation, and to the phenomenon that IL-1β was released by the action of FasL during the events in inflammation [316]. Further extensive studies in relation to Fas signaling system would be required for making discussion about general mechanistic characteristics of the sensitization to cell-death via FasR-mediated signaling by exogenous treatments with other cytokine agents.

Table 3.  Sensitization of cell-death induction via Fas signaling system with exogenous cytokine agents independent of IFN-γ.

Target cells: species, cell type (cell line/origin)	Sensitization method: type (mode), sensitizer (conc), treatment time	Cell-death induction method: inducer (name, subclass), conc, treatment time	Main findings related to sensitization of cell-death induction via Fas signaling system	Refs.
hu, Glioma (T-98G, LN-18, LN-215, LN-229, LN-308, LN-319, LN-405)	f-FR and f-CD (Pre), TNF-α and/or TGF-β2, IL-10, IL-13 (10 ng/ml each), TNF-β, IL-8 (20 ng/ml each), IL-1β (2.5 ng/ml), IL-4 (5 ng/ml), IL-6 (100 U/ml), or M-CSF (10 U/ml), 24 h	FR-mAb (CH-11, IgM), 0.1–1 µg/ml, 24 h	Cl-dep up-FR and up-CD by treatment with TNF-α, TGF-β2, IL-1β or IL-8, and up-CD of only T-98G cells by Pre with IL-10 or -13. Correlation of CD sensitivity with cell-surface FR expression level after TNF-α treatment.	[163]
hu, Retinal pigment epithelium (keratoplasty donor eyes)	f-FR and f-CD (Pre), TNF-α (10 ng/ml) ± TGF-β2 (10 ng/ml) ± DEX (10 µM), 24 h	FR-mAb (nd, IgM), 1 µg/ml, 16 h	Up-FR by treatment with TNF-α alone, Synergistic up-CD by combination of TNF-α with TGF-β2, associated with inhibition by coexistence of DEX.	[216]
mu, Pancreas islet (primary, β-TC-1)	f-FR and f-CD (Pre), muIL-1α (102–105 U/l) ± NMA 0.2–1.0 mM) or minus L-arginine, 1–12 h	muFR-mAb (RK8, nd), 0.5–5 mg/l, 12–72 h	Tm-dep up-reg of FR and FR-mRNA, and ds-dep up-CD by treatment with IL-1α alone, without inhibition by coexistence of iNOS inhibitor and dependence on L-arginine.	[283]
hu, Primitive hematopoietic progenitor (CD34+CD38+/− fetal liver)	f-FR, f-CD (Co), selected combinations of KL, IL-3, GM-CSF (20 ng/ml each) or TNF-α (100 ng/ml), 3–5 d	FR-mAb (CH-11, IgM), 2 µg/ml, 3–5 d	Up-FR by treatment with KL plus GM-CSF ± TNF-α. No up-CD, but reduction in hematopoietic potential by treatment with TNF-α alone.	[238]
hu, Kidney cancer (OUR-10)	f-FR and f-CD (Pre), IFN-α (nd) or IL-2 (nd), 24 h	FR-mAb (CH-11, IgM), 330 ng/ml, 48 h	Insignificant up-FR or up-CD by treatment with IFN-α or IL-2 alone.	[183]
hu, Thyrocytes (normal thyroid gland)	f-FR and f-CD (Pre), IL-1β (10 U/ml) and/or boTSH (0.1–20 mU/ml), 24–72 h	FR-mAb (nd, IgM), 1000 ng/ml, 18 h	Tm-dep, ds-dep dn-reg of FR by treatment with TSH alone. Up-FR and up-CD by IL-1β treatment, associated with significant inhibition by coexistence of TSH.	[266]
hu, Thyroid gland epithelium (GD patients)	f-FR and f-CD (Pre), IL-1β (10 U/ml) ± [healthy donors', GD patients' or IME patients' IgG (5mg/ml) or boTSH (5 mU/ml)], 48 h	FR-mAb (nd, IgM), 1000 ng/ml, 18 h	Up-FR and up-CD by IL-1β treatment, associated with significant inhibition by coexistence of GD patients' IgG or TSH, but not of healthy donors' or IME patients' IgG.	[205]
hu, Osteosarcoma (HOS/TE-85, MG-63, Saos-2)	f-FR, TNF-α (250–500 U/ml), 24–48 h	None	Significant up-FR by treatment with TNF-α alone, associated with much weaker up-reg of soluble FR.	[190]
hu, Bone marrow (mononuclear, CD34+) (posterior iliac crest of healthy volunteers)	f-FR, f-CD (Pre, colony formation) TNF-α (2–20 ng/ml), 48 h (f-FR); 24 h (f-CD)	FR-mAb (CH-11, IgM), 1 µg/ml, 24 h	Ds-dep up-FR and marked up-CD by TNF-α treatment, without significant change in TNFR-I and -II expression.	[224]
hu, Brain glioma (LN-229, LN-308, T-98G)	f-FR and f-CD (Pre), IFN-α (100–2500 U/ml), 48 h	muFL-N2A, 125-250 U/ml (1 U: half-maximal CD activity against LN-18 cell line), 24 h	No modulation of FR, but ds-dep significant up-CD, unaffected by ectopic expression of Bcl-2 or FR.	[284]
hu, Blood eosinophils (peripheral blood of normal healthy volunteers)	f-FR and f-CD (Pre), TNF-α (1–100 ng/ml), IL-3, IL-5 or GM-CSF (10 ng/ml each), 2–24 h	sFL in 293T cells (transfected with FL expression vector) supernatant, equal volume, 12 h	Small up-FR by TNF-α treatment alone. No up-reg of FR mRNA, but augmentation of early apoptosis by treatment with IL-3, IL-5 or GM-CSF.	[232]
mu, Ovary granulosa (pregnant mare serum gonadotropin treated)	f-FR and f-CD (Pre or Pre plus Co), TNF-α or muTNF-α (10 ng/ml), 24 h (Pre) ± following 24 h (Co)	muFR-Ab (Jo2, IgG2), 2 µg/ml, 24 h ± CHX, 0.5 mg/ml, 2 h preceded	Up-reg of FR mRNA by muTNF-α treatment, but not by huTNF-α treatment. Insignificant up-CD by Pre with muTNF-α alone.	[248]
mu, Central nervous system microglia (primary, BV-2)	f-FR and f-CD (Pre), muTNF-α (0.01–100 ng/ml), 24–48 h	muFL-N2A, 20% (v/v), 20 h	Up-FR and ds-dep up-CD by treatment with TNF-α alone, associated with marked dn-reg of Bcl-XL expression and up-reg of FLIP mRNA.	[256]
hu, Multiple myeloma (IL-6 dependent: U-266-1970, U-1958; IL-6-independent: U-266-1984)	f-FR and f-CD (Pre), IFN-α (1–1000 U/ml) ± IL-6 (20-100 U/ml), 96 h	FR-mAb (CH-11, IgM), 100 ng/ml, 24 h	Significant ds-dep up-CD by IFN-α treatment, irrespective of IL-6 dependency, without substantial change in FR.	[151]
hu, Colon cancer (HT-29), fibrosarcoma (U-3A, STAT-1 transfected U-3A)	f-FR, f-FL and f-CD (Pre), TNF-α (1000 U/ml), PDGF, EGF or FGF (20 ng/ml each), 3–48 h (f-FR and f-FL); 40 h (f-CD, Pre)	FR-mAb (CH-11, IgM), 500 ng/ml, 20 h	Up-FR by TNF-α or FGF, up-FL by TNF-α, and up-CD by TNF-α, PDGF, or EGF treatment in HT-29 cells, and No up-FR and no up-FL by TNF-α treatment, in U-3A or STAT-1 U-3A cells.	[124]
mu, Spleen (CD4+) , (received in GVHD mice)	f-FR, in vivo: IL-12 (4900 IU at day 0, 2400 IU at days 3, 5 and 7 of BM transplantation by injection)	None	Up-FR in lethally irradiated GVHD mice after BM transplantation, by IL-12 treatment.	[285]
hu, Colon cancer (COLO-201)	f-FR, f-FL and f-CD (Co), TNF-α (0.01–0.3 µg/ml), 3–5 d (f-FR); 3 d (Co); 3–12 h (f-FL)	FR-mAb (nd, nd), 100 ng/ml, 3d	Up-FR and up-CD by TNF-α treatment alone in ds-dep, tm-dep manner, associated with dn-reg of Bcl-2 and up-reg of Bax expression.	[122]
hu, Melanoma (A-375, BLM, BS-1251, Colo-38, M-518, Me1Ju, Me1Juso, MeWo, SK-Mel-25, SK-Mel-28, W+)	f-CD (Co), IFN-α-2b (500 U/ml), 48 h	FR-mAb (7C11, IgM), 50 ng/ml, 48 h	Insignificant up-CD in any melanoma cell-line by treatment with IFN-α alone.	[187]
hu, Small intestinal epithelium (non-tumoral fatal crypt); colonic organ cultures	f-FR and f-CD (Co), TNF-α (1–100 ng/ml) or IL-1β (0.1–100 ng/ml), 24–48 h	FR-mAb (CH-11, IgM), 1–500 ng/ml, 24–48 h	Ds-dep significant up-FR in cell culture, slight up-FR in organ culture, and modest up-CD, by treatment with TNF-α alone, but not with IL-1β alone.	[273]
hu, Synovial (osteoarthritis patients' tissue)	f-FR and f-CD (Pre), TNF-α (5–10 ng/ml), 2–5 d	FR-mAb (CH-11, IgM), 1 µg/ml ± Z-IETD-FMK, Ac-DEVD-CHO or Ac-YVED-CHO, 5–500 µM, 24 h	Insignificant up-FR and tm-dep up-CD by TNF-α treatment, associated with inhibition by coexistence of casp-3, 8 inhibitors.	[286]
mu, Pancreas islet (RAG2−/− NOD transgenic mice: wt and lpr-mut)	f-FR and f-CD (Co), muIL-1α or muIL-1β (10–100 U/ml each), 16 h-o/n	NOD mice-derived spleen CD4+-CTL, E/T ratio: 10:1, o/n; muFR-mAb (Jo2, IgG2), 1 µg/ml plus ActD, 30 µg/ml, 16 h	Significant up-FR and up-CD by treatment with IL-1α, and less with IL-1β, in NOD mice islet cells possessing wt FR, but not in cells possessing lpr-mut FR.	[199]
mu, Kidney cancer (Renca)	f-FR and f-CD (Pre), muTNF-α (100 U ml), 3–24 h	muFR-mAb (Jo2, IgG2), 0.002–1 µg/ml plus mAb- crosslinking P815 cells, 1 × 105 cells/200 µl, 18 h	Some up-FR, but no up-CD, in the constitutively resistant cells by treatments with TNF-α alone.	[184]
hu, Peripheral blood eosinophils (normal non-atopic donors)	f-FR, TNF-α (1–100 ng/ml), 6–24 h	None	Small up-FR by treatment with TNF-α alone.	[233]
hu, Salivary duct epithelium (HSG)	f-FR and f-CD (Pre), TNF-α (1–100 U/ml), IL-1β, IL-2 (0.001–0.1 U/ml), IL-4 (3–30 U/ml) or GM-CSF (0.01–1 mg/ml), 6–48 h (f-FR); 24 h (f-CD, Pre)	FR-mAb (DX-2, IgG1), 500 ng/ml, 3 h	Ds-dep up-FR and up-CD by TNF-α treatment, but no significant up-FR by treatment with other cytokines.	[271]
hu, Brain oligodendrocyte (adult intractable epilepsy patients)	f-CD (Pre), TNF-α (100 U/ml), daily over 72 h	FR-mAb (nd, nd), 10 µg/ml, 24 h	Insignificant up-CD by Pre with TNF-α alone.	[207]
hu, Umbilical vein endothelial (HUVEC), vascular smooth (hVSMC) (internal mammary artery), leukemia (Jurkat E6-1)	f-FR and f-CD (Co), TNF-α (25 ng/ml), 24 h (f-FR); 48 h (f-CD, Co)	Ad-muFL, multiplicity of infection: 300, 48 h	Modest up-FR in HUVEC, but not in hVSMC and Jurkat cells, by TNF-α treatment. Modest up-CD in hVSMC, but no up-CD in HUVEC after Ad-muFL treatment	[153]
ra, Non-transformed gastric mucosal (RGM-1)	f-FR, muIL-1β (1 ng/ml), raIL-2 (20 U/ml), IL-8 (50 ng/ml) or TNF-α (300 U/ml), 2–24 h	None	Up-reg of FR mRNA by treatment with IL-1β or TNF-α, but not with other cytokines.	[287]
hu, Stomach cancer (Kato-III)	f-FR, TNF-α (40 ng/ml), 48 h	None	Significant up-FR by TNF-α treatment alone.	[194]
mu, Testis seminiferous epithelium (Sertoli)	f-FR, f-FL and f-CD (Pre), muTNF-α (0.5–250 ng/ml), 24 h	muFL-N2A, E/T ratio: 1–10:1, 7 h	Ds-dep up-FR and up-CD by TNF-α treatment alone, associated with up-reg of soluble FR, and without up-reg of FL mRNA.	[252]
hu, Prostate cancer (LNCap)	f-FR, f-FL and f-CD (Co), TNF-α (0.4–40 ng/ml) ± TLCK (20-50 µM), 6–48 h ± Z-VAD (50 µM), 1 h preceded	FR-mAb (CH-11, IgM), 0.4–4 µg/ml ± γ-ray IR, 8–20 Gy, 20–24 h	Up-FR and ds-dep synergistic f-CD by TNF-α treatment combined with IR, associated with complete inhibition by coexistence of pan-casp inhibitor.	[288]
mu, Central nervous system microglia (primary, BV-2)	f-FR, f-CD (Pre), muTNF-α (10 ng/ml) ± TGF-β2, muIL-4 or muIL-10 (20 ng/ml), 24 h; TGF-β2 (0.02–100 ng/ml) 1–24 h	muFL-N2A, 0.5–20% (v/v), 1–24 h	Marked up-FR and up-CD by TNF-α treatment, associated with significant protection from CD by coexistence of TGF-β2 via up-reg of FLIP expression.	[257]
mu, Spleen T-lymphocyte (acute GVHD model by bone-marrow transplantation; donors: wt, lpr-mut, IFN-γ-kd mice)	f-FR and f-CD (Co), in vivo: muIL-18 (1 µg i.p. injection/d/mouse), each d at day -2 to 2 (day 0: transplantation day)	Endogenous FR-mediated CD inducer in recipient mouse	Up-FR and up-CD by IL-18 treatment, resulting protection from GVHD in wt donor, but not in functional FR-deficient donor and IFN-γ-kd donor.	[289]
hu, Salivary gland epithelium cells (non-neoplastic primary derived from SS patients and control individuals)	f-FR and f-FL, TNF-α, IFN-α (500 U/ml each) or IL-1β (5 ng/ml), 48 h	None	Significant up-FR and up-FL by treatment with TNF-α, but not with IFN-α, and dn-reg of FL with IL-1β.	[272]
mu, T-cell hybridoma (DO11.10); CD8−/− spleen (primary, CD4+ 85%-enriched)	f-CD (Pre), TNF-α (1-100 ng/ml), 6 h	muFR-mAb (Jo2, IgG2), immobilized with 5 µg/ml of anti-goat Ab, 16 h; FLAG-tagged sFL (100 ng/ml) plus anti-FLAG mAb (2 µg/ml), 24 h	Ds-dep up-CD by TNF-α treatment in both types of cells without significant up-FR, accompanied by dn-reg of FLIP and up-reg of Bax mRNA expression.	[290]
hu, Peripheral blood activated T-lymphocyte (relapsing- remitting multiple sclerosis patients)	f-FR, in vivo: IFN-β-1a, 22 µg × 3/w, 44 µg × 3/w, 30 µg × 1/w or 2.5–6.5 y (mean: 4.8 y)	None	Insignificant change in FR and Bcl-2, and dn-reg of surviving, by long-term IFN-β treatment.	[291]
bo, Ovary corpus luteum (midluteal stage of estrous cycle)	f-FR and f-CD (Pre plus Co), TNF-α (50 ng/ml), 24 h, (f-FR; f-CD, Pre) followed by 24 h (f-CD, Co)	sFL, 100 ng/ml, 24 h	Insignificant up-reg of FR mRNA and up-CD by treatment with TNF-α alone.	[244]
hu, Placenta trophoblast (A-3, HTR/8)	f-FR, f-FL and f-CD (Pre and Co), TNF-α, IL-6 or IL-10 (10 ng/ml each), 1–24 h (f-FR, f-FL); 24 h (f-CD)	FR-mAb (nd, nd), 20–500 ng/ml, 24 h	Tm-dep up-FR by TNF-α treatment. Proliferation by IL-6 treatment after FR-mAb stimulation. Ds-dep, tm-dep up-FL by IL-10 treatment, associated with up-reg of FLIP expression.	[250]
hu, Lung cancer (A-549)	f-FR and f-CD (Pre), IL-1β (50 pg/ml), 24 h	FR-mAb (CH-11, IgM), 10–100 ng/ml, 4–24 h	No up-FR and inhibition of CD by treatment with IL-1β alone.	[171]
hu, Normal intestine crypt epithelium (primary, colon and jejunum) (HIPEC)	f-FR and f-CD (Pre), TNF-α (10 ng/ml) or GM-CSF (20 ng/ml), 18–20 h	FR-mAb (CH-11, IgM), 100 ng/ml, 18–24 h	Insignificant up-FR and no up-CD of HIPEC cells by Pre with either TNF-α or GM-CSF alone, not due to absence of each receptor expression.	[132]
hu, Thyroid gland epithelium (primary, normal and multinodular goiter patients' thyroid tissues)	f-CD (Pre), TNF-α (50 ng/ml) and/or IL-1β (50 U/ml), 4 d	FR-mAb (CH-11, IgM), 1 µg/ml, 20 h	Up-CD in goitrous cells by Pre with TNF-α and/or IL-1β, in much less extent than normal cells.	[267]
hu, Acute myeloid leukemia (GM-CSF-dependent) (AML-193)	f-FR and f-CD (Co), IFN-α-2b (500 U/ml), 24–40 h, after pre-cultivation with GM-CSF (20 ng/ml), at least 2 w	FR-mAb (FAS18, IgG2a), 5 µg/ml, 24–40 h	No difference in FR and 2.5-fold median up-CD by IFN-α treatment in the absence of GM-CSF, associated with selective reduction of G1 cell-cycle state.	[156]
hu, Peritoneum mesothelial (effluents from peritoneal dialysis patients)	f-FR and f-CD (Pre), TNF-α (500–5000 U/ml), 24 h	sFL (aggregated), 10–100 ng/ml, 24 h	Insignificant up-FR and significant up-CD only by treatment with high conc of TNF-α.	[219]
hu, Placenta villus trophoblasts and explanted tissues (woman with uncomplicated pregnancies)	f-FR, f-FL and f-CD (Co, explant tissues), TNF-α or IL-1β (0.1–50 ng/ml each), 24 h ± FR-Fc (2 ng/ml), 1 h preceded	Endogenous FR-mediated CD inducer in explanted tissues	No up-FL, insignificant up-FR and up-CD by treatment with either TNF-α or IL-1β, associated with inhibition by Pre with FR-Fc in the case of TNF-α treatment alone.	[251]
hu, Follicular dendritic cell-like (HK from 2 different donors' tonsils)	f-FR and f-CD (Pre), TNF-α (20 ng/ml), 24 h ± ALLN (20 µM), 30 m preceded	sFL containing culture supernatant of CHO-K1-sFL cells, 10% (v/v), ± CHX (2 µg/ml), 16 h	Up-FR and up-CD by TNF-α treatment, associated with up-reg of CD by coexistence of CHX as well as diminished up-FR by inclusion of NF-κB inhibitor.	[292]
hu, Hepatoma (HepG-2)	f-CD (Pre), IFN-α (5–100 kU/ml) ± ribavirin (250–100 µM), 24 h	FR-mAb (nd, nd), 0.25 µg/ml, 12 h	Ds-dep synergistic up-CD by combined Pre with IFN-α and ribavirin, accompanied by up-reg of casp-3, -7, -8 activation and PARP cleavage.	[293]
hu, Kidney mesangial (glomeruli from normal portion of renal tissue)	f-FL, IL-1β (20 ng/ml) ± lactacystin (20 µM), 24 h	None	Up-FL by treatment with IL-1β than that with IFN-γ, associated with strong NF-κB induction and complete inhibition with NF-κB blocker.	[254]
hu, Oligodendroglioma (HOG, MO-3.13)	f-FR, f-FL and f-CD (Pre), TNF-α (1–100 ng/ml), 24 h	sFL, 1 ng/ml, 48 h	Cl-dep significant up-FR and up-CD by treatment with TNF-α alone, associated with up-reg of MHC-I expression.	[165]
mu, Basophil precursor-enriched splenocytes (wt, depleted of mature cells, c-kit− fraction); Lymphocytic leukemia (L1210: non-transfected, FR-transfected)	f-FR and f-CD (Pre), in vitro: muIL-12 (10 ng/ml) and/or muIL-18 (100 ng/ml), 24 h; in vivo: muIL-12 (0.2 µg/mouse) plus muIL-18 (0.25 µg/mouse), single i.v. injection, 2 h	Splenocytes (total and NK1.1+) cells, E/T ratio: 0.75–12 (in vitro Pre cells); 3–24 (in vivo Pre cells), 18 h	Up-FR in immature basophil precursor splenocytes, and up-reg of FR-mediated-CD via splenocytes in leukemia cells, by IL-12 and IL-18 treatment with synergistic effects.	[294]
hu, Proximal lung epithelium [normal bronchus (NHBE), small airway (DLEC)]	f-FR and f-CD (Pre and Co), TNF-α or IL-1β (10 ng/ml each), 18 h	sFL, 25–500 ng/ml, 18h (Pre); last 16 h (Co)	No up-FR in both types of cells by either cytokine. Up-CD in NHBE by Pre with TNF-α or IL-1β, but in DLEC only by Pre with IL-1β.	[277]
mu, Brain cortical astrocytes (primary) (1 to 2 d old mice)	f-CD (Pre), muTNF-α (10 ng/ml) and/or muIL-1β (10 ng/ml) ± SB203580 or SB202190 (0.1–4 µM each), 0.5–24 h	muFR-mAb (Jo2, IgG2), 1–104 µg/ml, 24 h	Up-CD in resistant cells by synergistic Pre with TNF-α and IL-1β, associated with ds-dep inhibition of CD by coexistence of p38 mitogen activated protein kinase inhibitor.	[258]
hu, Colon cancer (HT-29, DLD-1); leukemia (THP-1, Jurkat)	f-CD (Pre plus Co), TNF-α (100 pM), 1–7 d (Pre), followed by 24 h (Co)	FR-mAb (APO-1-3, IgG3), 180 pM or sFL, 3.1 nM, 24 h	Up-CD in HT-29 cells, but not in DLD-1, THP-1 or Jurkat cells, by o/n Pre with TNF-α, associated with decrease in CD response by prolonged Pre.	[138]
hu, T-lymphocyte (naïve CD4+ from neonates)	f-FR and f-CD (Pre), IFN-α2 (1 nM), 20 h ± additional 48–72 h	FR-mAb (CH-11, IgM), 50 ng/ml, 16 h	Significant up-FR, and up-CD, as judged by casp-3, -8 activation, by Pre with IFN-α, associated with up-reg of Bcl-2 expression and dn-reg of active Bax expression.	[295]
hu, Renal cell cancer (SK-RC-44, SK-RC-07)	f-FR and f-CD (Pre plus Co), IFN-α (300–3000 U/ml) ± G3139 (300 nM), 48 h (f-FR and f-CD, Pre) plus 24 h (f-CD, Co)	FR-mAb (CH-11, IgM), 12.5–100 ng/ml, 8–24 h ± antagonistic FR-mAb (ZB4, IgG1), 1 µg/ml, 1 h preceded	Significant up-FR and up-CD by Pre with IFN-α, associated with enhancement by coexistence of Bcl-2 AS-ODN, but abolition by Pre with antagonistic mAb.	[296]
hu, Multiple myeloma (U-266-1970, U-1958; both IL-6 dependent; primary, BM from newly diagnosed or relapsed MM patients)	f-FR, F-FL and f-CD (Pre), IL-6 (20 U/mL) ± IFN-α-2b (1000 U/ml), 0.5–96 h	FR-mAb (CH-11, IgM), 100 ng/ml, 6–24 h	Up-FR and up-CD by IFN-α treatment, associated with enhancement of STAT-1 activation, but attenuation of STAT-3 activation.	[157]
mu, Bone marrow osteoclast progenitor (RAW-264.7); differentiated osteoclast (from RAW-264.7)	f-FR and f-CD (Pre), RANKL (1.1–3.3 nM) plus M-CSF (10 ng/ml), 6 d	muFR-mAb (Jo2, IgG2), 1 µg/ml, 24 h	Ds-dep, tm-dep up-reg of FR and CD in osteoclast progenitor, and ds-dep dn-reg of FR and CD in differentiated osteoclast, by treatment with RANKL	[297]
hu, Follicular dendritic cell-like (HK)	f-FR and f-CD (Pre), TNF-α (20 ng/ml) and/or TGF-β1 (10 ng/ml); IL-4 (20 ng/ml), GM-CSF (50 ng/ml) or LT-α (10 ng/ml), 6–48 h	sFL in culture supernatant of CHO-K1-sFL cells, 10% (v/v) plus CHX, 2 mg/ml, 18 h	Significant up-FR and up-CD by Pre with TNF-α, associated with tm-dep counteraction and dn-reg of casp-8 expression by coexistence of TGF-β.	[298]
hu, Synovial fibroblasts (RA-SF, synovial tissues from rheumatoid arthritis patients)	f-FR and f-CD (Pre), TNF-α (10–100 ng/ml) ± [Ad-TIMP-3 transduction (100–1000 pfu) or TIMP-3 (10–1000 ng/ml)], 24 h	sFL, 100 ng/ml, 16 h	Up-FR and ds-dep up-CD in Ad-TIMP-3 transduced cells, but not in cells co-treated with exogenous TIMP-3, by TNF-α treatment.	[299]
hu, Multiple myeloma (U-266-1970, IL-6 dependent)	f-CD (Pre), IFN-α (1000 U/ml) plus IL-6 (20 U/mL), 24 h	FR-mAb (CH-11, IgM), 100 ng/ml, 24 h	Up-CD by Pre with IFN-α, associated with reduction of telomerase activity repressed at mRNA level.	[300]
mu, Small intestine epithelium (immortalized by SV-40 large T-gene transfection) (MODE-K, IEC-4.1)	f-FR and f-CD (Pre), TNF-α (2–10 ng/ml) or IL-10 (5–50 ng/ml), 24 h	muFR-mAb (Jo2, IgG2), 1 µg/ml, 24–48 h	Significant up-FR and up-CD by treatment with TNF-α alone, but dn-reg of CD by Pre with IL-10 alone.	[274]
hu, Prostate cancer (PC-3)	f-FR and f-CD (Pre), TNF-α (1–100 U/ml) ± sTNF-RI (0.3 µg/ml), 12–24 h	FR-mAb (CH-11, IgM), 30 ng/ml, 12 h	Dn-reg of FR and CD by TNF-α treatment, associated with partial reversal by co-existence of sTNF-RI.	[301]
hu, Skin pre-adipocyte and adipocyte (primary, SGBS patients)	f-FR and f-CD (Co), TNF-α (10 nM), 6–72 h	FR-mAb (nd, IgG3), 1 µg/ml, 6–72 h	Tm-dep up-FR and up-CD by treatment with TNF-α alone, associated with increased DISC formation.	[211]
mu, Liver hepatocytes (primary from wt and Bim-deficient mice)	f-CD (Pre), TRAIL (30 ng/ml), nd	muFL-N2A, E/T ratio: 10–90:1, nd; Fc-sFL, 0.15–5 ng/ml, nd	Up-CD by TRAIL treatment, associated with activation of JNK and Bim.	[302]
hu, Lung fibroblast (fetal, MRC-5; primary, undifferentiated from normal lung tissue), myofibroblast (both, differentiated with TGF-β)	f-FR and f-CD (Pre), TNF-α (0.01–20 ng/ml), 36–48 h	FR-mAb (CH-11, IgM), 250 ng/ml, 4–12 h	Up-FR and ds-dep up-CD, irrespective of differentiation state, by treatment with TNF-α alone, associated with efficient DISC formation.	[279]
wc, Hepatocytes (WCM-260)	f-FL, wcTNF-α (35 U/ml), 18 h	None	Insignificant up-FL mRNA, but associated with increased MHC-I expression, by Pre with TNF-α.	[158]
mu, Colon cancer (C-15-4.3: MC-38 transfected with hu CEA gene)	f-FR, muTNF-α (250 U/ml), o/n	None	Significant up-FR by treatment with TNF-α alone.	[142]
mu, Soft tissue sarcoma (CMS-4: parental; transfectant with empty-control, K79E-mut or R289E-mut IRF-8 gene)	f-CD (Pre), TNF-α (100 U/ml), o/n	sFL, 200 ng/ml, 24 h	Up-CD by TNF-α treatment alone in wt IRF-8 expressing cells, associated with significant inhibition by oe of functionally-impaired mut IRF-8.	[181]
hu, Eye subconjunctival Tenon fibroblast (biopsy specimen from glaucoma patients)	f-FR, IFN-α-2b (5000 U/ml), 48 h	None	Slight up-FR by IFN-α treatment alone, accompanied by up-reg of sensitivity to MMC-mediated CD.	[220]
hu, Uterus endometrial stroma (ESC: undifferentiated, decidualized) (benign hysterectomy tissue from pre-menopausal woman)	f-FR and f-CD (Pre), TNF-α (25 ng/ml) ± hCG (1 U/ml), 24–48 h	FR-mAb (CH-11, IgM), 100 ng/ml, 24 h	Small effect on both FR and CD by TNF-α treatment alone, without significant influence by coexistence of hCG.	[281]
hu, Periphral blood mononuclear [healthy donors, T-large-granular lymphocyte (LGL) patients]	f-CD (Pre), IL-2 (500 U/ml) ± anti-CD3 mAb (0.01–10 µg/ml), 24 h	FR-mAb (Apo-1, IgG3), 500–1000 ng/ml, 45 m-24 h	Up-CD only in LGL patients' cells, associated with enhancement of DISC formation, and up-reg c-FLIP-L/S only in healthy donors' cells, by Pre with IL-2.	[303]
hu, Peripheral blood CD4+ leukocyte [healthy donors (HD) and Sézary syndrome (SzS) patients]	f-FR and f-CD (Pre), IFN-α (100 U/ml), o/n	Super-FL, 100 ng/ml, 6–8 h	Significant up-FR and up-CD in either CD4+ leukocytes from HD or SzS patients by IFN-α treatment.	[212]
hu, Melanoma (Hs-294T)	f-FR and f-CD (Pre), IFN-α-2b (1000 U/ml) ± EGCG (10 µg/ml), 48 h	FLAG-tagged sFL (50 ng/ml) plus anti-FLAG mAb (1 µg/ml), 16 h	Up-FR and up-CD by treatment with IFN-α ± EGCG, associated with marked decrease in NF-κB expression.	[304]
hu, Cutaneous T-cell lymphoma (HH, SZ-4, MyLa, Hut-78); SzS blood tumor (SS1)	f-FR and f-CD (Pre), IFN-α (100 U/ml) ± MTX (10 µM), nd	FLAG-tagged sFL (50 ng/ml) plus anti-FLAG mAb (1 µg/ml), 16 h	Up-FR and up-CD by IFN-α treatment, associated with enhancement by coexistence of MTX.	[305]
mu, Bone marrow (tibiae and femurs of 8-w-old mice)	f-FR and f-FL, muTNF-α (100 ng/ml) plus M-CSF (100 ng/ml), 4 d	None	Up-FR in adherent cells, but no significant up-FL, irrespective of cell-adherence.	[227]
mu, Bone marrow mesenchymal stem (tibiae and femurs)	f-FR, TNF-α (20 ng/ml), 24 h	None	Insignificant up-FR and casp-3 activation by treatment with TNF-α alone.	[228]
mu, Hepatocyte (in vitro: primary from wt, Bid−/− mut, XIAP −/− mut, FR−/− mut, FL gld-mut; in vivo: wt mice of 8-12-w-old mice)	f-CD (Pre), in vitro: TNF-α (25 ng/ml), 0.2–48 h ± SP600125 (25 µM), preceded ± Bim siRNA treatment (10 nM); in vivo: TNF-α (40 µg/kg), 2 h	in vitro: muFL-N2A, 50 ng/ml, 6 h; in vivo: muFR-mAb (Jo2, IgG2), 80 µg/kg, 5 h	Tm-dep significant up-CD, both in vitro and in vivo, by TNF-α treatment, associated with abolition by loss of FR or Bid, but not of XIAP or mut of FL, and with suppression by Pre with JNK inhibitor or Bim siRNA.	[306]
hu, Melanoma (wt p53: D05, A375: both parent and transfected with DN-FADD gene; mut p53: D14)	f-CD (Pre plus Co), IFN-β-1a (300 U/ml), 24 h (Pre) ± TMZ (50 µM), 72 h (Pre) plus 24 h (Co) followed	FR-mAb (APO-1-3, IgG3), 25 ng/ml plus Protein A, 25 ng/ml, 24 h	Up-CD, irrespective of p53 state, by Pre with IFN-β, associated with enhancement by TMZ treatment and reduction by DN-FADD transfection.	[307]
hu, Peripheral blood T-lymphocyte (CD4+, CD8+) (healthy donors, at least 1 y HIV-I positive patients)	f-FR and f-CD (Pre), IFN-α or IFN-β (1000 U/ml each) plus plate-bound anti-CD3 mAb (OKT-3, IgG2a) (0.1 µg/ml), 3 d	Plate-bound FR-mAb (CH-11, IgM), 5 µg/ml, 14 h	Up-FR and up-CD in healthy donors' cells and up-CD in HIV patients' cells by treatment with type-I IFNs, associated with up-reg of Bak expression alone among many Bcl-2 family members.	[308]
hu, Eye conjunctiva epithelium (IOBA-NHC) (normal, spontaneously immortalized)	f-FR and f-FL, TNF-α (25 ng/ml), 24–48 h	None	Insignificant up-FR or up-FL by treatment with TNF-α alone.	[276]
hu, Epidermal keratinocyte (HEK) (primary from normal neonatal foreskin)	f-FL, TNF-α (75 ng/ml) and/or IL-1β (500 pg/ml), 24–72 h	None	Significant up-reg of FL mRNA by treatment with TNF-α and/or IL-1β, associated with modest up-reg of NO production.	[261]
hu, Blood monocytes, Mφ (buffy coats of healthy donors)	f-FR, [IL-4 (20 ng/ml) or IL-17 (100 ng/ml)] ± IL-10 (50 ng/ml) ± DEX (100 nM), 3 d (for monocytes); 8 d (for Mφ, IL-10 and DEX added on day 5)	None	Up-FR by treatment with IL-10, but not with IL-4 or IL-17, associated with significant augmentation by coexistence of DEX and inverse correlation between FR and MerTK.	[234]
mu, Hepatocyte (primary from wt, Bid−/− mut, Bim−/− mut, or XIAP−/− mut 8-14-w-old mice)	f-CD (Pre), IL-1β (1–20 ng/ml) or TNF-α (25 ng/ml), 12–24 h ± Q-VD-Oph (25 µM), 30 m preceded	muSFL-N2A, 50 ng/ml, 6 h	Protection from CD by Pre with IL-1β, without influence from deficiency in Bid or Bim, associated with further dn-reg of CD by coexistence of pan-casp inhibitor.	[309]
hu, Auricular chondrocytes (remnant cartridge of microtia patients)	f-FL mRNA, G-CSF, IL-6, IP-10, KC, MIP-1α, -1β, -2 (0.1–10 × ED50 each), ± AG490 (nd), 5 d	None	Enhanced FL mRNA by G-CSF, IL-6, KC, MIP-1α /-1β treatment, associated with dn-reg by coexistence of JAK-2 inhibitor.	[161]
hu, Kidney cortex / proximal tubule epithelium (HK-2) (transformed with HPV-16)	f-CD (Co), TNF-α (25 ng/ml) or TGF-β1 (1 ng/ml), 48 h	FR-mAb (CH-11, IgM), 0.5 µg/ml, 48 h	No up-CD by Co with either TNF-α or TGF-β1 alone.	[186]
mu, Small intestine lamina propria CD4+ T-lymphocytes (naïve and peanuts-derived allergen sensitized 6-8-w-old mice)	f-FL, IL-18 (100 ng/ml) ± anti-CD3/CD24 mAb (2 µg/ml) ± anti-IFN-γ mAb (200 ng/ml), 3 d	None	Insignificant up-FL by treatment with IL-18 alone in both types of cells, but associated with increase in FL by coexistence of anti-CD3 / CD24 mAb and its complete reversal with anti-IFN-γ mAb.	[240]
hu, Neuroblastoma [MYCN amplified: LAI-5S, SK-N-BE (2), IMR-32, Tet-21-N; MYCN non-amplified: SK-N-AS, SK-N-SH, CHLA-90, SH-SY5Y]	f-FR and f-CD (Pre and Co), TNF-α (100 ng/ml), 0.5–24 h ± prior treatment with BAY11-7082 (10-25 µM)	Fc-sFL, 1–100 ng/ml, 24 h	Cl-dep, tm-dep up-FR and up-CD by TNF-α treatment, associated with full abrogation of up-FR by coexistence of NF-κB inhibitor and positive correlation of up-CD with up-FR.	[54]
hu, Uterus immortalized endometrial stromal (St-T1: wt, Sdc-1-kd: both non-differentiated, decidualized)	f-CD (Pre), IL-1β (10 ng/ml), TNF-α (5 ng/ml) or TGF-β1 (0.5 ng/ml), 24 h	FR-mAb (EOS9.1, IgM), 5 µg/ml, 24 h	Greater up-CD in Sdc-1-kd-cells than wt cells, irrespective of differentiation state, by treatment with individual cytokine alone.	[282]
hu, Blood basophils (venous EDTA-blood of healthy non-selected donors)	f-CD (Pre), IFN-α (1000 U/ml) and/or IL-3 (50 ng/ml), 24–48 h	Super-FL, 5–100 ng/ml, 24 h	Insignificant up-CD by Pre with IFN-α, associated with enhanced Bcl-2 cleavage and protection from CD by coexistence of IL-3	[235]
mu, Bone marrow mesenchymal stem (bone cavity of tibiae and femurs of pathogen-free mice)	f-FR, IL-17 (200 ng/ml), 24–72 h	None	Tm-dep small up-reg of FR mRNA by treatment with IL-17 alone.	[230]
mu, hepatoma (Hepa-1-6, 3T3); hepatocytes (primary from 14-w-old mice, AML-12); embryo fibroblasts (3T3); hu, Hepatoma (HepG-2)	f-FR and f-CD (Pre), in vitro: TNF-α (20 ng/ml), 2–14 h; in vivo: TNF-α (200 µg/kg), 14 h; either ± NF-κB p65-subunit shRNA treatment	Fc-sFL, 20 ng/ml, 1–5 h	Marked up-FR and up-CD in all types of cells except HepG-2 by either in vitro or in vivo treatment with TNF-α, associated with inhibition by NF-κB p65-subunit shRNA treatment.	[310]
mu, Mesenchymal stem / stromal (tibia and femur bone marrow from iNOS-deficient mice)	f-CD (Co), TNF-α (10 ng/ml) plus SNAP (20 µM), 48 h	muFR-mAb (Jo2, IgG2), nd, 48 h	Insignificant up-CD in iNOS-deficient cells by treatment with TNF-α alone, in the presence of NO donor.	[231]

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5.   Conclusions and perspectives

The present survey clarified that many non-cytokines represented by clinical anticancer drugs and cytokines such as interferon-γ are effective in sensitizing the death of a wide range of target cells through FasR-mediated signaling, as the exogenous agents. The cell-death induction via death receptors such as FasR has been repeatedly suggested to become a possible alternative strategy to the conventional chemo- and radio-therapies for the treatment of various tumors [317],[318]. An artificially planned implementation of the death of a wide range of target cells, including various types of malignant tumor cells, could be attained by treatments with soluble cell-death inducers. Soluble FasL and agonistic FasR-mAb are the promising candidates for this purpose. However, despite of their great potentials, the development of the treatment strategy using these agonistic proteins in clinical medicine remains still on the way. There are two important problems that have to be overcome for making these proteins more powerful as the cytotoxic pharmaceuticals. One is the impaired cell-death inducing activity as compared to the natural mFasL protein, existing on the surface of immune cells. Hence, the development of effective sensitization methods is essential for the efficient cell-death induction using the soluble agonists. Once effectively potentiated with any appropriate method, the lesser cell-death inducing activity of the soluble agonists can turn out to be a merit, since this property is beneficial for the avoidance of unwanted, potential adverse effects, which might be caused by the too strong action of membrane-bound agonists. Also, the treatment with soluble agonists is in principle free of the possible problems derived from the cell-killing processes through the direct contact between the immune cells and the target cells, represented by tumor cells' counter-attack against infiltrating lymphocytes [319]. Moreover, soluble proteins will be manufactured in a large amount much more easily at reasonable costs, as compared with either water-insoluble membrane proteins or delicate immune cells, since the former is generally more stable and easier to handle than the latter.

As also suggested from the literature survey conducted in this study, it is obviously important to pay attentions to the possible induction of toxicity caused by the death of the cells constituting normal or non-targeted organs, particularly the liver [108],[320], which may be substantially augmented by the exogenous agents. The toxicity induction can raise a serious problem, especially in the cases that we plan to apply the soluble FasR-agonists for the systemic, therapeutic treatments depending on the selective death execution against malignant cells in the future. Nevertheless, it is fair to speculate that the greater activity of mFasL in the surface membrane of the immune cells as compared to sFasL at least partly derives from the coexistence of other molecules, which specifically recognizes the target cells. Hence, it is reasonable to expect that the increase in the specificity of the soluble FasR agonists to the target cells using protein-engineering technologies will significantly contribute to the reduction of this unwanted adverse effect. Further, at present, it is still difficult to draw a complete mechanistic picture of each experimental result in all the studies surveyed in this paper, however the basic researches striving for the understanding of detailed mechanisms would also continue to shed useful, informative lights on the solution of critical problems in the practical applications. The specific issues, which have already been investigated, include the connection of the apoptosis system via extrinsic FasR-mediated pathway to that via intrinsic mitochondrial pathway involving Bid [321], the stimulation of FasR by singlet oxygen [322], the interaction of the activated Fas signaling system with the activation of NADPH oxidase [323],[324], and also that with the enhancement of inducible nitric oxide synthase [325].

Figure 1.  A strategy for translating soluble agonists at Fas receptor into clinical protein pharmaceuticals.

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In Figure 1, a possible strategy for the translation of the soluble agonists at FasR into practical protein pharmaceuticals is shown. In addition to finding of the solution using the sensitization with exogenous agents, further in-depth characterization studies aimed at overcoming the resistance problems in the demanding targets, which are often mediated by intracellular anti-apoptotic factors represented by Bcl-2 and Bcl-X_L [326] as well as the functional modulation caused by post-translational modifications of FasR molecule [327], are also essential for the development of advanced strategies in the enhancement of cell-death induction. As shown in Tables 2 and 3, owing to the recent progress in the synthetic technology of oligonucleotides, multiple versatile tools to interfere with specific genes involved in the counteractions against the effective cell-death, such as AS-ODN [185],[296], siRNA [179],[203],[217],[218],[228], and short hairpin RNA (shRNA) [182], have already been employed for the sensitization studies in combination with exogenous cytokine agents. However, at the moment, it seems that they have been mainly used as the evaluation aid for analytical purposes. Further technical advancements in the treatment efficacy would be required for safe clinical applications of these tools. Additional finding of the unexplored compounds including repositioning of the existing drugs may also significantly contribute to the effective sensitization in the future.

As mentioned above, apart from the enhancement of cell-death inducing activity, another critical problem to be solved with regard to the medical applications of sFasL or agonistic FR-mAb is the lack of specificity. Thus, multiple protein engineering techniques have been devised to empower the targeting ability by conjugations of affinity-providing domains, represented by single-chain antibody fragment, to date [5],[25]. The coexistence of a decoy receptor can be a serious obstacle to efficient implementation of the death against the target cells. Thus, the achievement of affinity reduction for a decoy receptor, DcR3, relative to the genuine receptor, FasR, is an another important targetable issue worth to be tackled in finding the good solution, since FasR and DcR3 share a comparable affinity for sFasL to each other in spite of considerably weak homology (17%) in the primary structures [328],[329]. The sensitization of cell-death induction using exogenous agents in combination with increase in the targeting specificity using protein engineering techniques, which can be assisted by suitable preventions against the endogenous resistance in refractory cells using co-treatments with function-masking molecules, is proposed to be a valuable approach for improving the efficacy of the soluble agonistic proteins reviewed in this paper as the therapeutic substances. It is hoped that this strategy will ultimately contribute toward translating them into novel pharmaceuticals for curative treatments in clinical medicine.