Fas ligand gene expression is directly regulated by stress-inducible heat shock transcription factor-1.

Heat shock transcription factor-1 (HSF-1) is the primary stress responsive transcription factor that regulates expression of heat shock proteins (Hsps) in response to elevated temperature. We show that the transcriptional activity of HSF-1 can also directly mediate hyperthermia-induced Fas ligand (FasL) expression in activated T cells. We identify a conserved region within the human FasL promoter spanning from -276 to -236 upstream of the translational start site that contains two 15 bp non-identical adjacent HSF-1-binding sites or heat shock elements (HSEs) separated by 11 bp. Both the distal HSE (HSE1) (extending from -276 to -262) and the proximal HSE (HSE2) (spanning from -250 to -236) consist of two perfect and one imperfect nGAAn pentamers. We show the direct binding of HSF-1 to these elements and that mutation of these sites abrogates the ability of HSF-1 to bind and drive promoter activity. HSF-1 associates with these elements in a cooperative manner to mediate optimal promoter activity. We propose that the ability of HSF-1 to mediate stress-inducible expression of FasL extends its classical function as a regulator of Hsps to encompass a function for this transcription factor in the regulation of immune function and homeostasis.

Differential modulation of the TRAIL receptors and the CD95 receptor in colon carcinoma cell lines.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95 ligand (CD95L) are potent inducers of apoptosis in various tumour cell types. Death receptors DR4 and DR5 can induce and decoy receptors DcR1 and DcR2 can inhibit TRAIL-mediated apoptosis. The study aim was to investigate whether anticancer agents can modulate similarly TRAIL-receptor and CD95 membrane expression and TRAIL and CD95L sensitivity. Three colon carcinoma cell lines (Caco-2, Colo320 and SW948) were treated with 5-fluorouracil (5-FU), cisplatin or interferon-gamma. TRAIL-receptor and CD95 membrane expression was determined flow cytometrically. Sensitivity to TRAIL or CD95L agonistic anti-CD95 antibody was determined with cytotoxicity and apoptosis assays. SW948 showed highest TRAIL sensitivity. The protein synthesis inhibitor cycloheximide decreased FLICE-like inhibitory protein levels in all cell lines, and the TRAIL-resistant cell lines Caco-2 and Colo320 became sensitive for TRAIL. Exposure of the cell lines to 5-FU, cisplatin and interferon-gamma left TRAIL-receptor membrane expression and TRAIL sensitivity unaffected. CD95 membrane expression and anti-CD95 sensitivity was, however, modulated by the same drugs in all lines. Cisplatin and interferon-gamma raised CD95 membrane levels 6-8-fold, interferon-gamma also increased anti-CD95 sensitivity. These results indicate that the CD95 and TRAIL pathways use different mechanisms to respond to various anticancer agents. Induced CD95 membrane upregulation was associated with increased anti-CD95 sensitivity, whereas no upregulation of TRAIL-receptor membrane expression or TRAIL sensitisation could be established. For optimal use of TRAIL-mediated apoptosis for cancer therapy in certain tumours, downregulation of intracellular inhibiting factors may be required.

Modulation of death receptor pathways in oncology.

Chemotherapeutic efficacy is hampered by occurrence of drug resistance. Several mechanisms cause this phenomenon. A final common factor is the reduced capacity of resistant cells to go into apoptosis following treatment with DNA-damaging agents. It is therefore interesting to search for ways to facilitate this apoptotic process following use of chemotherapeutic drugs. The death receptor ligands tumor necrosis factor (TNF), FasL and TNF-related apoptosis-inducing ligand (TRAIL) might be interesting candidates as they are able to induce apoptosis by binding to their cell membrane receptors. Recombinant forms of these ligands potentiate chemotherapeutic drug effects in preclinical models. For the clinical application of TNF, FasL and TRAIL, it is of primary importance that their safety be guaranteed. RhTNF is the only ligand currently used in humans. However, systemic rhTNF has shown low antitumor activity and higher doses induce severe sepsis-like toxicity. Perfusion setting aimed at limb preservation with rhTNF plus melphalan is currently used in sarcoma patients. A number of options have been tested in the preclinical setting that might allow circumvention of TNF toxicity in the clinic. Systemic rhFasL administration in humans is not yet feasible because of observed severe liver toxicity in mice due to Fas-mediated apoptosis of hepatocytes. Measures to circumvent liver toxicity have not yet been exploited. Another option for using FasL in the clinic may be to identify an alternative route of administration. In the animal model, FasL appeared to be less toxic for the liver compared with anti-Fas antibodies when administered intraperitoneally. There are relatively nontoxic modulators of the Fas death pathway, such as interferon and nonsteroidal antiinflammatory drugs (NSAIDs), which might prove interesting in combination with chemotherapy. Finally, it may be possible to produce a modified FasL with a reduced toxicity profile. TRAIL, produced as soluble, zinc-stabilized rhTRAIL seems to be without preclinical toxicity. Agonistic DR4 and DR5 antibodies against their TRAIL death receptor are being studied as another potential clinical option to induce apoptosis. Due to the synergistic effect observed in the preclinical setting between death receptor ligands and other modulators of the death receptor pathways and chemotherapy, it may well be that this approach is especially of value in the clinic when combined with chemotherapy. Ideally, choices for specific (modified) death receptor ligands for the treatment of patients can be rationally made based on tumor characteristics.