p53-Mediated down-regulation of the human DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) via interaction with Sp1 transcription factor.

O(6)-Methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, reverses mutagenic and cytotoxic effects of O(6)-alkylguanine in DNA induced by chemotherapeutic N-alkyl N-nitrosourea and procarbazine type drugs by dealkylating the adduct. MGMT expression is down-regulated by wild-type p53 (WTp53) in human tumor cells. Here we report that p53 sequesters the Sp1 transcription factor to prevent its binding to the cognate cis elements in the MGMT promoter and thus inhibits MGMT expression. Sp1 overexpression abrogated the inhibitory effect of p53 on the MGMT promoter activity in a dose-dependent manner. Stable interaction of Sp1 with WTp53 was observed in HCT116 cells. Moreover, WTp53 overexpression reduced the binding of the nuclear extract to the Sp1 consensus sequence, even though recombinant p53 alone did not bind to the same sequence. Taken together, these results suggest that sequestration of Sp1 could be one of the mechanisms by which p53 negatively regulates MGMT expression, thus enhancing sensitivity of tumor cells to O(6)-alkylguanine generating drugs.

Interleukin-24 overcomes temozolomide resistance and enhances cell death by down-regulation of O6-methylguanine-DNA methyltransferase in human melanoma cells.

Melanoma is the most malignant of skin cancers, highly resistant to chemotherapy and radiotherapy. Temozolomide, a promising new derivative of dacarbazine, is currently being tested for treatment of metastatic melanoma. Resistance to alkylating agents such as temozolomide correlates with increased expression of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Interleukin-24 (IL-24; mda-7) is a tumor suppressor cytokine that selectively inhibits tumor cell growth by inducing apoptosis and cell cycle arrest in melanoma cell lines and solid tumors. This tumor-selective activity has been observed in multiple preclinical animal models and in clinical trials. In this study, we analyzed the ability of Ad-IL-24 and its protein product, IL-24, to overcome temozolomide resistance in human melanoma cells. We have shown that Ad-IL-24 via exogenous IL-24 protein induces combinatorial synergy of temozolomide-induced cell killing in temozolomide-resistant melanoma cells by inhibition of MGMT. Neutralizing antibodies against IL-24 or its receptors significantly blocked the apoptotic activity of IL-24 + MGMT treatment. We show that accumulation of functional p53 is essential for IL-24-induced down-regulation of MGMT. Using either MGMT small interfering RNA, p53 small interfering RNA, or a p53 dominant-negative mutant to block MGMT protein expression resulted in increased sensitization to temozolomide. However, MGMT blockade in combination with IL-24 + temozolomide resulted in loss of combinatorial synergy, indicating that MGMT expression is required for the reversal of temozolomide resistance in melanoma cells. This study shows that IL-24 can play a significant role in overcoming temozolomide resistance and that the clinical efficacy of temozolomide may be improved by using a biochemotherapy combination with IL-24.

Cancer targeting using tumor suppressor genes.

Conventional cancer treatments include cytotoxic chemotherapies and radiotherapy, which result in significant collateral toxicities. The goal for future cancer treatments is to leverage improved understanding of cancer biology mechanisms and thereby develop targeted drugs that display exquisite tumor selectivity and avoid iatrogenic damage. In this review, we discuss the potential of tumor suppressor genes for development of cancer-selective drugs using the tumor suppressor p53 as an archetype.

The double-stranded RNA-activated protein kinase mediates radiation resistance in mouse embryo fibroblasts through nuclear factor kappaB and Akt activation.

PURPOSE: Activation of the double-stranded RNA-activated protein kinase (PKR) leads to the induction of various pathways including the down-regulation of translation through phosphorylation of the eukaryotic translation initiation factor 2alpha (eIF-2alpha). There have been no reports to date about the role of PKR in radiation sensitivity. EXPERIMENTAL DESIGN: A clonogenic survival assay was used to investigate the sensitivity of PKR mouse embryo fibroblasts (MEF) to radiation therapy. 2-Aminopurine (2-AP), a chemical inhibitor of PKR, was used to inhibit PKR activation. Nuclear factor-kappaB (NF-kappaB) activation was assessed by electrophoretic mobility shift assay (EMSA). Expression of PKR and downstream targets was examined by Western blot analysis and immunofluorescence. RESULTS: Ionizing radiation leads to dose- and time-dependent increases in PKR expression and function that contributes to increased cellular radiation resistance as shown by clonogenic survival and terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) apoptosis assays. Specific inhibition of PKR with the chemical inhibitor 2-AP restores radiation sensitivity. Plasmid transfection of the PKR wild-type (wt) gene into PKR(-/-) MEFs leads to increased radiation resistance. The protective effect of PKR to radiation may be mediated in part through NF-kappaB and Akt because both NF-kappaB and Akt are activated after ionizing radiation in PKR+/+ but not PKR-/- cells. CONCLUSIONS: We suggest a novel role for PKR as a mediator of radiation resistance modulated in part through the protective effects of NF-kappaB and Akt activation. The modification of PKR activity may be a novel strategy in the future to overcome radiation resistance.

Vitamin E succinate in combination with mda-7 results in enhanced human ovarian tumor cell killing through modulation of extrinsic and intrinsic apoptotic pathways.

Adenovirus-mediated mda-7 (Ad-mda7) gene transfer has been shown to induce apoptosis in various human cancer cells while sparing normal cells. Vitamin E succinate (VES) is also known to exhibit antitumor activity against a number of human cancer cell lines. We hypothesized that a combination of the two agents would produce an enhanced antitumor effect in MDAH2774 human ovarian cancer cells. Treatment of MDAH2774 cells with Ad-mda7 plus VES resulted in enhanced antitumor activity that involved the activation of two apoptotic pathways. Activation of the extrinsic pathway was demonstrated by increased cell-surface Fas expression and cleavage of Bid and caspase-8. Activation of the intrinsic pathway was demonstrated by disruption of mitochondrial potential; and activation of downstream capase-9 and caspase-3 via cytochrome C release. In contrast, the combination of Ad-mda7 plus VES did not show any antitumor activity against normal fibroblasts, indicating selective tumor cell killing. Our in vitro results provide a basis for further preclinical testing of Ad-mda7 plus VES as a potential cancer treatment strategy.

Human interleukin 24 (MDA-7/IL-24) protein kills breast cancer cells via the IL-20 receptor and is antagonized by IL-10.

The melanoma differentiation-associated gene-7 (mda-7/IL-24) is a unique member of the interleukin 10 (IL-10) family of cytokines, with ubiquitous tumor cell pro-apoptotic activity. Recent data have shown that IL-24 is secreted as a glycosylated protein and functions as a pro-Th1 cytokine and as a potent anti-angiogenic molecule. In this study, we analyzed the activity of Ad-mda7 and its protein product, secreted IL-24, against human breast cancer cells. We show that Ad-mda7 transduction of human breast cancer cells results in G(2)/M phase cell cycle arrest and apoptotic cell death, which correlates with secretion of IL-24 protein. Neutralizing antibody against IL-24 significantly inhibited Ad-mda7 cytotoxicity. IL-24 and IL-10 both engage their cognate receptors on breast cancer cells resulting in phosphorylation and activation of STAT3, however, IL-10 receptor binding failed to induce cell killing, indicating that tumor cell killing by IL-24 is independent of STAT3 phosphorylation. Treatment with exogenous IL-24 induced apoptosis in breast cancer cells and this effect was abolished by addition of anti-IL-24 antibody or anti-IL-20R1, indicating that bystander cell killing is mediated via IL-24 binding to the IL-20R1/IL-20R2 heterodimeric receptor complex. Co-administration of the related cytokine IL-10 inhibited killing mediated by IL-24 and concomitantly inhibited IL-24 mediated up-regulation of the tumor suppressor proteins, p53 and p27(Kip1). In summary, we have defined a tumor-selective cytotoxic bystander role for secreted IL-24 protein and identified a novel receptor-mediated death pathway in breast cancer cells, wherein the related cytokines IL-24 and IL-10 exhibit antagonistic activity.

Mitotic arrest, apoptosis, and sensitization to chemotherapy of melanomas by methionine deprivation stress.

Methionine deprivation stress (MDS) eliminates mitotic activity in melanoma cells regardless of stage, grade, or TP53 status, whereas it has a negligible effect on normal skin fibroblasts. In most cases, apoptosis accounts for the elimination of up to 90% of tumor cells from the culture within 72 hours after MDS, leaving a scattered population of multinucleated resistant cells. Loss of mitosis in tumor cells is associated with marked reduction of cyclin-dependent kinase (CDK) 1 transcription and/or loss of its active form (CDK1-P-Thr(161)), which is coincident with up-regulation of CDKN1A, CDKN1B, and CDKN1C (p21, p27, and p57). Expression of the proapoptotic LITAF, IFNGR, EREG, TNFSF/TNFRSF10 and TNFRSF12, FAS, and RNASEL is primarily up-regulated/induced in cells destined to undergo apoptosis. Loss of Aurora kinase B and BIRC5, which are required for histone H3 phosphorylation, is associated with the accumulation of surviving multinucleated cells. Nevertheless, noncycling survivors of MDS are sensitized to temozolomide, carmustin, and cisplatin to a much greater extent than normal skin fibroblasts possibly because of the suppression of MGMT/TOP1/POLB, MGMT/RAD52/RAD54, and cMET/RADD52, respectively. Sensitivity to these and additional genotoxic agents and radiation may also be acquired due to loss of cMET/OGG1, reduced glutathione reductase levels, and a G(2)-phase block that is a crucial step in the damage response associated with enhancement of drug toxicity. Although the genes controlling mitotic arrest and/or apoptosis in response to low extracellular methionine levels are unknown, it is likely that such control is exerted via the induction/up-regulation of tumor suppressors/growth inhibitor genes, such as TGFB, PTEN, GAS1, EGR3, BTG3, MDA7, and the proteoglycans (LUM, BGN, and DCN), as well as the down-regulation/loss of function of prosurvival genes, such as NFkappaB, MYC, and ERBB2. Although MDS targets several common genes in tumors, mutational variability among melanomas may decide which metabolic and signal transduction pathways will be activated or shutdown.

Role for the double-stranded RNA-activated protein kinase PKR in Ad-TNF-alpha gene therapy in esophageal cancer.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) is a cytokine with direct antitumor activity. Clinical trials with TNF-alpha have been limited because of the severe side effects of systemic administration. Gene therapy with an adenoviral vector allows delivery of high local doses of TNF-alpha. Activation of protein kinase R (PKR) has been implicated as a general transducer of apoptosis in response to a variety of different stimuli including TNF-alpha. We, therefore, evaluated the role of PKR in Ad-TNF-alpha-induced apoptosis in esophageal cancer cells. METHODS: A tetracycline-responsive adenoviral vector was used to transfect the TNF-alpha gene (Ad-TNF-alpha) into human esophageal cancer cell lines Bic1, Seg1 and TT, as well as in transformed PKR(+/+) and PKR(-/-) early-passage mouse embryo fibroblasts. Ad-luciferase, Ad-Bak, and mock infection with phosphate buffered saline solution were used as controls. Gene expression was determined by Western blot analysis. Apoptosis was detected by propidium iodide staining and fluorescence-activated cell sorter analysis. RESULTS: Overexpression of TNF-alpha in the lysate was evident in all cell lines treated with Ad-TNF-alpha. Treatment with Ad-TNF-alpha was associated with PKR upregulation and induction of apoptosis. Inhibition of TNF-alpha expression by tetracycline resulted in downregulation of PKR and decreased apoptosis. Transduction of PKR(+/+) and PKR(-/-) mouse embryo fibroblasts with Ad-TNF-alpha demonstrated that Ad-TNF-alpha-induced apoptosis was mediated in part through a PKR-dependent process. CONCLUSIONS: These results suggest that Ad-TNF-alpha-mediated apoptosis in esophageal cancer cell lines is dependent in part on PKR upregulation. Strategies to enhance PKR upregulation may allow increased Ad-TNF-alpha antitumoral activity in the treatment of esophageal cancer.

mda-7/IL24 kills pancreatic cancer cells by inhibition of the Wnt/PI3K signaling pathways: identification of IL-20 receptor-mediated bystander activity against pancreatic cancer.

The melanoma differentiation-associated gene (mda-7; approved gene symbol IL24) is a tumor suppressor gene whose protein expression in normal cells is restricted to the immune system and to melanocytes. Recent studies have shown that mda-7 gene transfer inhibits cell growth and induces apoptosis in melanoma, lung cancer, breast cancer, and other tumor types through activation of various intracellular signaling pathways. In the current study, we demonstrate that Ad-mda7 transduction of human pancreatic cancer cells results in G2/M cell cycle arrest and cell killing. Cytotoxicity is mediated via apoptosis in a time- and dose-dependent manner. Tumor cell killing correlates with regulation of proteins involved in the Wnt and PI3K pathways: beta-catenin, APC, GSK-3, JNK, and PTEN. Additionally, we identify bystander cell killing activated by exposure of pancreatic tumor cells to secreted human MDA-7 protein. In pancreatic tumor cells, exogenous MDA-7 protein activates STAT3 and kills cells via engagement of IL-20 receptors. The specificity of bystander killing is demonstrated using neutralizing anti-MDA-7 antibodies and anti-receptor antibodies, which inhibit the apoptotic effects. In sum, we show that Ad-mda7 is able to induce growth inhibition and apoptosis in pancreatic cancer cells via inhibition of the Wnt/PI3K pathways and identify a novel bystander mechanism of MDA-7 killing in pancreatic cancer that functions via IL-20 receptors.

Bystander activity of Ad-mda7: human MDA-7 protein kills melanoma cells via an IL-20 receptor-dependent but STAT3-independent mechanism.

The melanoma differentiation-associated gene-7 (mda-7/IL24) is a unique member of the IL-10 family of cytokines, with ubiquitous tumor cell proapoptotic activity. Transduction of tumor or normal cells with the mda-7 gene results in secretion of glycosylated MDA-7 protein. Recent data indicate that secreted MDA-7 protein functions as a pro-Th1 cytokine and as a potent antiangiogenic molecule. MDA-7 protein binds two distinct type II cytokine heterodimeric receptor complexes, IL-20R1/IL-20R2 (type 1 IL-20R) and IL-22R1/IL-20R2 (type 2 IL-20R). In this study we analyzed the activity of glycosylated secreted MDA-7 against human melanoma cells. MDA-7 protein induces phosphorylation and nuclear translocation of STAT3 in melanoma cells via both type 1 and type 2 IL-20R. MDA-7 induces dose-dependent cell death in melanoma tumor cells. MDA-7 receptor engagement results in up-regulation of BAX and subsequent apoptosis induction; this effect is mediated by STAT3-independent signaling. Additional IL-10 family members (IL-10, -19, -20, and -22) also activate STAT3; however, these ligands do not activate death pathways in melanoma. In normal cells, MDA-7 can bind to its cognate receptors and induce phosphorylation of STAT3, without cytotoxic sequelae. This study defines a tumor-selective cytotoxic bystander role for secreted MDA-7 protein and identifies a novel receptor-mediated, STAT3-independent, and PKR-independent death pathway.

The p53-induced oncogenic phosphatase PPM1D interacts with uracil DNA glycosylase and suppresses base excision repair.

The wild-type p53-induced phosphatase PPM1D (or Wip1) is a serine/threonine phosphatase that is transcriptionally upregulated by p53 following ultraviolet and ionizing radiation. PPM1D is an oncogene in transformation assays and is amplified or overexpressed in several human tumor types. Here, we demonstrate that PPM1D interacts with the nuclear isoform of uracil DNA glycosylase, UNG2, and suppresses base excision repair (BER). Point mutations that inactivate PPM1D phosphatase activity abrogate BER suppression, indicating that dephosphorylation by PPM1D is important for BER inhibition. We have identified UNG2 phosphorylation sites at threonines 6 and 126 that exhibit enhanced phosphorylation following UV irradiation. The UV-induced phosphorylated forms of UNG2 are more active than nonphosphorylated forms in mediating uracil-associated DNA cleavage. PPM1D dephosphorylation of UNG2 at phosphothreonine 6 is associated with reduced UNG2 activity. Thus, PPM1D may inhibit BER by dephosphorylating UNG2 to facilitate its inactivation after completion of DNA repair.

Multifaceted resistance of gliomas to temozolomide.

PURPOSE AND EXPERIMENTAL DESIGN: The contributions of O6-methylguanine-DNA-methyltransferase(MGMT), p53 status, mismatch repair, and apoptotic response to the resistance of glial tumors to temozolomide (TMZ) were tested using seven established human glial tumor cell lines in culture and xenografts in athymic mice. RESULTS: Resistance to TMZ was only marginally dependent on MGMT activity, because subtoxic doses of TMZ easily eliminated MGMT reserves for at least 18 h after treatment. Resistance to TMZ varied most notably with the p53 status of the tumor. Tumors with wild-type (wt) p53 and a functional p53 response to DNA damage (SWB40 and SWB61) were most sensitive. The p21-related cell cycle arrest was intimately linked to TMZ toxicity because tumors with wt p53 but lacking a robust increase in p21 protein level (D-54) were resistant to TMZ. In contrast, tumors with a dysfunctional p53 cycle and a weak cell cycle response to DNA damage (SWB39 and SWB77) were extremely unresponsive to treatment even with the aid of MGMT inactivators. Notable exceptions to the above were observed with the p53 mutated tumors SWB33 and SWB95, which were arrested by TMZ in G1-S and consequently underwent apoptosis despite their failure to express p21. CONCLUSIONS: By testing a limited number of glial tumors in cell culture and also as xenografts, we have shown that mobilization of the p53 in response to TMZ damage is likely to induce a cell cycle arrest and apoptosis in glial tumors. Additional pathways linking cell cycle arrest and apoptosis contribute to the efficacy of TMZ against p53 mutated glial tumors. The unusual resistance of tumors, of which the cell cycle was not arrested in response to TMZ treatment, was associated with allelic losses during regrowth of treated tumors. Nevertheless such resistance was not related to dysfunctional mismatch repair.