Cancer gene therapy using a novel secretable trimeric TRAIL.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, is a type II transmembrane cytokine molecule. Soluble TRAIL has been shown to induce apoptosis in a wide variety of cancer cells in vitro and to suppress tumor growth specifically without damaging normal cells and tissues in vivo. In our previous report, we have demonstrated that an artificial gene encoding the polypeptide composed of the three functional elements (a secretion signal, a trimerization domain and an apoptosis-inducing moiety of TRAIL gene sequence) expresses and secretes highly apoptotic trimeric TRAIL into the culture supernatant. Here, as an approach to TRAIL-based cancer gene therapy, we developed an adenoviral vector delivering the gene that encodes our secretable trimeric TRAIL (stTRAIL). This adenovirus (Ad-stTRAIL) potently induced apoptosis in vitro in cancer cell lines such as HeLa, MDA-MB-231, A549, HCT116 and U-87MG. In an animal xenograft tumor model bearing a human glioma cell line U-87MG, intratumoral delivery of Ad-stTRAIL dramatically suppressed tumor growth without showing detectable adverse side effects. Histological analysis revealed that Ad-stTRAIL suppresses tumor growth by inducing apoptotic cell death. Contrary to the known rapid clearance of systemically delivered TRAIL protein from the blood circulation, stTRAIL expressed by Ad-stTRAIL in tumor tissues persisted for more than 4 days. In a comparison of tumor suppressor activity between Ad-stTRAIL and Ad-flTRAIL (delivering the full-length TRAIL gene) after mixing infected cells with uninfected cells and implanting these mixed cells in nude mice, Ad-stTRAIL showed higher tumor suppressor activity than that of Ad-flTRAIL. Our data reveal that a gene therapy using Ad-stTRAIL has a promising potential to treat human cancers including gliomas.

Sodium nitroprusside enhances TRAIL-induced apoptosis via a mitochondria-dependent pathway in human colorectal carcinoma CX-1 cells.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo-2L) is a recently characterized member of the family of programmed cell death-inducing ligands that includes TNF-alpha and CD95L (FasL). It is well known that TRAIL binds to the death signaling receptors, DR4 and DR5, and initiates the TRAIL death pathway. Activation of this pathway, mediated through a caspase cascade, causes apoptosis. In this study, we hypothesized that oxidative stress facilitates TRAIL-induced apoptosis by promoting caspase activity through cytochrome c release from mitochondria. Human colorectal carcinoma CX-1 cells were treated with various concentrations of TRAIL (12.5-200 ng/ml) and/or sodium nitroprusside (SNP; 0.03-1 mM) for 12 h. SNP, a nitric oxide donor, which had little toxic effect by itself, enhanced TRAIL-induced cytotoxicity. For example, TRAIL-induced apoptosis (200 ng/ml) was increased by a factor of 2.5-fold in the presence of 1 mM SNP. The combined treatment also caused an increase in cytochrome c release, caspase-3 activity, and PARP cleavage. Overexpression of Bcl-2 completely blocked the SNP-promoting effects, but only moderately inhibited TRAIL-induced apoptosis. Similar results were observed in the presence of hydrogen peroxide or peroxynitrite. Taken together, the present studies suggest that SNP enhances TRAIL-induced cytotoxicity by facilitating the mitochondria-mediated caspase signal transduction pathway.

Signaling events triggered by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL): caspase-8 is required for TRAIL-induced apoptosis.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a TNF family member and potent apoptosis inducer. In contrast to TNF-alpha or Fas ligand, relatively little is known about the signaling events activated by TRAIL. In particular, the initial caspase(s) required for TRAIL-induced apoptosis remains to be determined Caspase-3-like protease but not caspase-1-like protease (YVADase) activity rapidly increased in HeLa cells in response to TRAIL treatment. The increase in protease activity correlated with the profile of apoptotic cell death that was inhibited by the pan-caspase inhibitor Z-VAD-fmk. In response to TRAIL, caspase-8, an initiator caspase in death receptor-mediated apoptosis, was activated within 1 h in association with Bid cleavage, cytochrome c release, caspase-3 activation, and DNA fragmentation factor 45 cleavage. Z-IETD-fmk, a caspase-8 inhibitor, completely blocked caspase-8 activation and resulted in inhibition of caspase-3 (a caspase-3-like protease) activation and apoptotic cell death. Overexpression of a caspase-8 dominant negative mutant inhibited apoptosis induced by TRAIL. Caspase-8-deficient Jurkat cells were resistant to both TRAIL and Fas-induced apoptosis, whereas wild-type Jurkat cells were susceptible to both TRAIL- and Fas-induced apoptosis. The caspase-8-reintro duced caspase-8-deficient Jurkat cells acquired normal susceptibility to both TRAIL and agonistic Fas antibody. Reverse transcription-PCR and sequence analyses have revealed that these caspase-8-deficient Jurkat cell express wild-type caspase-10. Therefore, our data indicate that caspase-8 is required for TRAIL-induced apoptosis and suggest that caspase-10 may play a minor role, if any, in TRAIL-induced apoptosis.

The role of caspase-8 in resistance to cancer chemotherapy.

Toxicity of chemotherapeutic agents against cancer cells is mediated through the initiation of programmed cell death. Apoptosis is an evolutionarily conserved cascade of intracellular proteolytic events propagated by a family of cysteine proteases called caspases. Many receptor- and non-receptor-mediated death signals induce apoptosis via activation of caspase-8 (FLICE/MACH). Mechanisms of tumor resistance to cytotoxic drugs through decreased apoptosis may occur by altered expression of caspase-8, upregulation of caspase-8 inhibitors like FLIP (FLICE-like Inhibitory Protein), or sequestration of caspase-8 by Bcl-2. Modulation of caspase-8 and apoptosis may be a therapeutic strategy for sensitization of drug-resistant malignancies to radiation or combination chemotherapy.

Cysteine 230 modulates tumor necrosis factor-related apoptosis-inducing ligand activity.

Biologically active tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein is known to form a homotrimer in solution. Unexpectedly, the recombinant active human TRAIL protein purified from bacteria produced two bands (a Mr 21,000 monomer derived from the disruption of the trimer in SDS gels and a Mr 42,000 dimer) on nonreducing SDS gels. The treatment of this TRAIL protein with DTT, a reducing agent, abolished formation of the Mr 42,000 band, suggesting that the Mr 42,000 band was the result of intermolecular disulfide bridge formation. Inspection of the amino acid sequence of human TRAIL protein identified a unique cysteine residue at position 230, and subsequent site-directed mutagenesis revealed that this amino acid residue is responsible for the appearance of the Mr 42,000 dimer. The binding analysis using the TRAIL protein and a TRAIL receptor (death receptor 5) revealed that both the dimer and the trimer bind to death receptor 5 with similar affinity. Interestingly, mutation of cysteine 230 to glycine completely abolished the apoptotic activity of TRAIL protein. The disruption of the dimer in the mixture of TRAIL dimer and trimer increased the apoptotic activity slightly, suggesting that the dimer has less apoptotic activity than the trimer. Therefore, our data indicate that cysteine 230 is not only required for TRAIL function but also modulates the apoptotic activity of TRAIL by forming an intermolecular disulfide bridge.

Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to induce apoptosis in various tumor cells but not in nontransformed, normal cells. Preclinical studies in mice and nonhuman primates have shown that administration of TRAIL can induce apoptosis in human tumors, but that no cytotoxicity to normal organs or tissues is found. The susceptibility of tumor cells to TRAIL and an apparent lack of activity in normal cells has lead to a proposal to use TRAIL in cancer therapy. Here, we assessed the sensitivity of hepatocytes from rat, mouse, rhesus monkey and human livers to TRAIL-induced apoptosis. TRAIL induced apoptosis in normal human hepatocytes in culture but not in hepatocytes isolated from the other species. Human hepatocytes showed characteristic features of apoptosis, including cytoplasmic shrinkage, the activation of caspases and DNA fragmentation. Apoptosis and cell death in human hepatocytes was massive and rapid, occurring in more than 60% of the cells exposed to TRAIL within 10 hours. These results indicate that there are species differences in sensitivity to TRAIL, and that substantial liver toxicity might result if TRAIL were used in human cancer therapy.

Transcriptional activation of the hepatocyte growth factor receptor (c-met) gene by its ligand (hepatocyte growth factor) is mediated through AP-1.

Hepatocyte Growth Factor (HGF) exerts its biological effects via binding and activating a transmembrane protein tyrosine kinase receptor known as c-Met. Previous studies from our laboratory demonstrated that c-met gene expression is inducible by its own ligand (HGF). However, the molecular mechanism(s) involved in this process are unknown. The present study was carried out to address this question. Transfection of various c-met-CAT promoter constructs into the mouse hepatocellular carcinoma cell line Hepa 1-6 in combination with electrophoretic mobility shift assays (EMSA) identified the responsive element as an activated protein-1 (AP-1) binding site (TGAGTCA) within the c-met core promoter region at position -158 to -152. The c-met AP-1 element binds specifically to AP-1 protein as verified by supershift assays. EMSA studies and mutational analyses of the promoter region also revealed that the members of the Sp family of transcription factors (Sp-1 and Sp-3) bind to the c-met Sp-1 element (located at position -124) which is adjacent to the AP-1 site. We show that Sp binding dampens binding of AP-1 to its cognate site in the c-met promoter region. Stimulation of Hepa 1-6 cells with HGF resulted in a rapid and dramatic enhancement of the AP-1 binding activity as well as an overall increase in the level of AP-1 protein. Cotransfection of AP-1 expression vectors (c-Fos plus c-Jun) with c-met promoter constructs resulted in stimulation of c-met promoter activity. We found that transactivation of the c-met promoter by AP-1 can be blocked by Curcumin, an inhibitor of AP-1. Moreover, we found that the induction of the endogenous c-met gene by HGF is inhibited by the addition of Curcumin. The results demonstrate that the HGF-induced transcription of the c-met gene by HGF is, at least in part, due to activation of the AP-1 pathway.

Nitric oxide prevents p21 degradation with the ubiquitin-proteasome pathway in vascular smooth muscle cells.

PURPOSE: We have shown that gene transfer of the inducible nitric oxide synthase (iNOS) gene to injured arteries inhibits the development of intimal hyperplasia. One mechanism by which nitric oxide (NO) may inhibit this process is through the upregulation of the cyclin-dependent kinase inhibitor p21, which induces a G0/G1 cell cycle arrest, leading to an inhibition of vascular smooth muscle cell (VSMC) proliferation. Because NO induced such a dramatic upregulation of p21 and because p21 is a universal inhibitor of the cell cycle, this study aimed to determine how NO upregulates p21 protein expression in VSMCs. METHODS: p21 messenger RNA (mRNA) levels in rat aortic smooth muscle cells (RASMCs) were determined by Northern blot analysis after treatment with S-nitroso-N-acetylpenicillamine (SNAP) or after adenoviral iNOS gene transfer. p21 protein levels in RASMCs in similar conditions were determined by Western blot analysis. Levels of ubiquinated p21 in these same treatment groups were assessed by immunoprecipitation of p21 from RASMCs, followed by western blot analysis for ubiquitin. Protein tyrosine and protein serine/threonine phosphatase activity after treatment with SNAP, plus or minus the phosphatase inhibitors calyculin A or cantharidin, were measured with (32)P-labeled myelin basic protein as a substrate. RESULTS: NO exposure by the NO-donor SNAP or iNOS gene transfer induced a dose- and time-dependent increase in p21 protein expression in RASMCs. p21 mRNA levels were significantly increased after SNAP treatment only at the 6-hour point, but were not increased at 24 hours. In contrast, protein levels were increased from 6 to 24 hours, and transcriptional inhibitors did not inhibit this increase in protein synthesis. The increase in p21 protein expression induced by NO was associated with less of the ubiquinated form of p21 at both early and late points. Furthermore, NO induced an increase in both protein tyrosine and protein serine/threonine phosphatase activity. Inhibition of these phosphatases with calyculin A or cantharidin prevented the upregulation of p21 protein expression by NO. CONCLUSION: These data indicate that one mechanism by which NO upregulates p21 protein expression is through the prevention of p21 protein degradation by the ubiquitin-proteasome pathway in association with increased protein tyrosine and serine/threonine phosphatase activity.

Regulation of the c-met proto-oncogene promoter by p53.

In the present study, we have investigated the possible involvement of p53 in the transcriptional regulation of the c-met gene. Cotransfection of various c-met promoter reporter vectors with p53 expression plasmids demonstrated that only wild-type p53 but not tumor-derived mutant forms of p53 resulted in a significant enhancement of c-met promoter activity. Functional assays revealed that the p53 responsive element in the c-met promoter region is located at position -278 to -216 and confers p53 responsiveness not only in the context of the c-met promoter but also in the context of a heterologous promoter. Electrophoretic mobility shift assays using purified recombinant p53 protein showed that the p53 binding element identified within the c-met promoter specifically binds to p53 protein. Induction of p53 by UV irradiation in RKO cells that express wild-type p53 increased the level of the endogenous c-met gene product and p21(WAF1/CIP1), a known target of p53 regulation. On the other hand, in RKO cells in which the function of p53 is impaired either by stable transfection of a dominant negative form of p53 or by HPV-E6 viral protein, no induction of the endogenous c-met gene or p21(WAF1/CIP1) was noted by UV irradiation. These results suggest that the c-met gene is also a target of p53 gene regulation.

A caspase-9 variant missing the catalytic site is an endogenous inhibitor of apoptosis.

It is likely that endogenous inhibitors of the apical caspases such as caspase-9 exist to prevent undesirable activation of caspase cascades. A naturally occurring variant of caspase-9 named caspase-9S was cloned from human liver. Caspase-9S is missing most of the large subunit of caspase-9, including the catalytic site, but has the intact prodomain and small subunit. Caspase-9S did not show apoptotic activity in transfection analysis. Overexpression of caspase-9S inhibited apoptosis induced by caspase-9, indicating that caspase-9S is an endogenous dominant-negative of caspase-9. Moreover, caspase-9S inhibited apoptosis induced by tumor necrosis factor(TNF)-alpha, TNF factor-related apoptosis-inducing ligand (TRAIL), Bax, or Fas-associated death domain-containing protein (FADD) as well as the combination of Apaf-1 and caspase-9. In vitro binding assays demonstrated that caspase-9S binds to Apaf-1 and blocks the binding of caspase-9 to Apaf-1. Coexpression of caspase-9 and caspase-9S mRNA was identified in various cell lines. Thus, caspase-9S acts as a dominant-negative inhibitor of caspase-9 activation, at least in part, by blocking Apaf-1-caspase-9 interaction.

Nitric oxide suppression of apoptosis occurs in association with an inhibition of Bcl-2 cleavage and cytochrome c release.

It is now known that caspase-3-like protease activation can promote Bcl-2 cleavage and mitochondrial cytochrome c release and that these events can lead to further downstream caspase activation. NO has been proposed as a potent, endogenous inhibitor of caspase-3-like protease activity. Experiments were carried out to determine whether NO could interrupt Bcl-2 cleavage or cytochrome c release by the inhibition of caspase activity linking these events. NO inhibited the capacity of purified caspase-3 to cleave recombinant Bcl-2. Both Bcl-2 cleavage and cytochrome c release were inhibited in tumor necrosis factor alpha- and actinomycin D-treated MCF-7 cells exposed to NO donors. The NO-mediated inhibition of Bcl-2 cleavage and cytochrome c release occurred in association with an inhibition of apoptosis and caspase-3-like activation. Thus, NO suppresses a key step in the positive feedback amplification of apoptotic signaling by preventing Bcl-2 cleavage and cytochrome c release.

Structural and functional characterization of the mouse c-met proto-oncogene (hepatocyte growth factor receptor) promoter.

The c-met gene encoding Hepatocyte Growth Factor Receptor is predominantly expressed in epithelial cell types and overexpressed in a variety of human and mouse neoplastic tissues and cell lines. To understand the molecular mechanisms of the transcriptional regulation of this gene, we have cloned and functionally characterized the mouse c-met promoter region. Transient transfection analysis using a series of 5'-end deletion met-CAT chimeric constructs in epithelial (C-33A) and fibroblast (NIH3T3) cell lines demonstrated that the c-met promoter acts in a cell-type specific manner. These experiments also localized functionally important regulatory regions at -1390 to -279, relative to the transcription start site, which exert repressive activity, and at -278 to -77 which exhibit enhancing effects on c-met promoter activity. Further analysis by electrophoretic mobility shift assays using specific competitors and antibodies identified Sp1 protein binding to two cognate response elements at -221 and -124 within the enhancer region. Cotransfection experiments revealed that Sp1 stimulated promoter activity of the met-CAT constructs containing the two Sp1 binding sites. These results demonstrate that Sp1 is actively involved in the transcriptional regulation of the c-met promoter.

Co-expression and regulation of Met and Ron proto-oncogenes in human hepatocellular carcinoma tissues and cell lines.

Met and ron proto-oncogenes encode the cell surface receptors for hepatocyte growth factor (HGF) and hepatocyte growth factor-like (HLP) protein, respectively, and induce mitogenesis, motogenesis, morphogenesis, and metastatic activity in various cell types. Overexpression of met in human carcinoma has been reported by several groups including ours; however, the mechanisms that control met gene expression are thus far unclear. The present study focuses on the expression and regulation of the Met and Ron receptors in human hepatocellular carcinoma (HCC). We report here that abnormal expression of met and ron proteins occurs in some cases of human HCC. Using several HCC cell lines as a model system, we show that HGF, as well as other cytokines, such as epidermal growth factor (EGF), interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha), induce met and ron expression. Using several chimeric constructs consisting of various lengths of the met promoter region fused to the reporter gene of chloramphenicol acetyl transferase (CAT), and by performing transient transfection of these constructs into HepG2 cells, we show that induction of met gene expression by HGF and other cytokines is, at least in part, through up-regulation of met gene promoter activity. The DNA region conferring responsiveness to cytokine induction was located within 0.2 kb of the met core promoter. Interestingly, EGF did not stimulate met promoter activity in any of the met-CAT chimeric constructs. These results provide evidence that met and ron are modulated in the liver by a similar cytokine network. In the case of met expression, the 0.2-kb region in the met gene promoter may play an important role in mediating its gene induction in response to HGF and other cytokines. Our results also suggest that unregulated expression of met and ron may be associated with pathological conditions, such as HCC, in the liver.