Bifunctional DNA alkylator 1,3-bis(2-chloroethyl)-1-nitrosourea activates the ATR-Chk1 pathway independently of the mismatch repair pathway.

The presence of DNA damage initiates signaling through the ataxia-telangiectasia mutated kinase (ATM) and the ATM- and the Rad3-related kinase (ATR), which phosphorylate, thus activating, the checkpoint kinases (Chk) 1 and 2, which leads to cell cycle arrest. The bifunctional DNA alkylator 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is cytotoxic primarily by inducing DNA monoadducts and ultimately, interstrand cross-links, which block DNA replication. In this study, we investigated the activation of the ATR-Chk1 pathway in response to BCNU treatment and the dependence of this response on the DNA mismatch repair (MMR) capacity. Medulloblastoma cells were exposed to low and moderate doses of BCNU, and the effects on this DNA damage signaling pathway were examined. In response to BCNU, Chk1 was found to be phosphorylated at serine 345 and exhibited increased kinase activity. Caffeine and wortmannin, which are broad-spectrum inhibitors of ATM and ATR, reduced this phosphorylation. Cell cycle analysis further revealed an accumulation of cells in the S phase in response to BCNU, an effect that was attenuated by caffeine. Small interfering RNA knockdown of ATR also reduced Chk1 phosphorylation after exposure to BCNU. However, knockdown of ATM had no effect on the observed Chk1 phosphorylation, suggesting that ATR was primarily responsible for Chk1 activation. Analysis of Chk1 activation in cells deficient in MMR proteins MutLalpha or MutSalpha indicated that the DNA damage response induced by BCNU was independent of the MMR apparatus. This MMR-independent activation seems to be the result of DNA interstrand cross-link formation.

Glutathione S-transferase M1 and T1 genetic polymorphisms, alcohol consumption and breast cancer risk.

Alcohol consumption has been inconsistently associated with breast cancer risk. Recent studies suggest that genetic polymorphisms of glutathione S-transferases (GSTs) may modify this relation. To determine if breast cancer risk is associated with GSTM1 and GSTT1 genetic polymorphisms, and to evaluate the effect modification between GST genotypes and alcohol consumption in the risk of breast cancer, we conducted a case-control study in the state of Connecticut in the period 1998 and 2001. Cases were histologically confirmed, incident breast cancer patients in New Haven County, CT. Controls were randomly selected from women histologically confirmed to be without breast cancer. The study results show that, while GSTM1 genotypes were not associated with breast cancer risk, GSTT1-null genotype was associated with a significant 90% increased risk for postmenopausal women (OR=1.9, 95% CI 1.2-3.0). Analysis by GST genotypes and alcohol consumption shows that GSTM1A ever-drinking women had a 2.5-fold (OR=2.5, 95% CI 1.1-5.5) increased risk of breast cancer compared to the GSTM1A never-drinkers, and the risk increases with duration and daily amount of alcohol consumption. Postmenopausal women with GSTT1-null genotype, who consumed a lifetime of >250 kg of spirit-equivalents, had an almost seven-fold increased risk (OR=6.8, 95% CI 1.4-33.9), and drinking commencing at younger ages appears to carry a higher risk. An OR of 8.2 (95% CI 1.2-57.4) was observed for those with GSTM1A, and GSTT1-null genotypes who had consumed a lifetime of >250 kg of spirit-equivalents. In conclusion, alcohol consumption may increase breast cancer risk among those who carry susceptible GST genotypes.

Down-regulation of cathepsin B expression impairs the invasive and tumorigenic potential of human glioblastoma cells.

Increases in abundance of cathepsin B transcript and protein correlate with increases in tumor grade and alterations in subcellular localization and activity of cathepsin B. The enzyme is able to degrade the components of the extracellular matrix (ECM) and activate other proteases capable of degrading ECM. To investigate the role played by this protease in the invasion of brain tumor cells, we transfected SNB19 human glioblastoma cells with a plasmid containing cathepsin B cDNA in antisense orientation. Control cells were transfected with vector alone. Clones expressing antisense cathepsin B cDNA exhibited significant reductions in cathepsin B mRNA, enzyme activity and protein compared to controls. Matrigel Invasion assay showed that the antisense-transfected cells had a markedly diminished invasiveness compared with controls. When tumor spheroids containing antisense transfected SNB19 cells expressing reduced cathepsin B were co-cultured with fetal rat brain aggregates, invasion of fetal rat brain aggregates was significantly reduced. Green Fluorescent Protein (GFP) expressing parental cells and antisense transfectants were generated for detection in mouse brain tissue without any post-chemical treatment. Intracerebral injection of SNB19 stable antisense transfectants resulted in reduced tumor formation in nude mice. These results strongly support a role for cathepsin B in the invasiveness of human glioblastoma cells and suggest cathepsin B antisense may prove useful in cancer therapy.

Enforced expression of wild-type p53 curtails the transcription of the O(6)-methylguanine-DNA methyltransferase gene in human tumor cells and enhances their sensitivity to alkylating agents.

We used isogenic human tumor cell lines to investigate the specific and direct effects of wild-type (wt) p53 on the expression of O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that confers tumor resistance to many anticancer alkylating agents. A p53-null, MGMT-proficient lung tumor cell line (H1299) was engineered to express wt p53 in a tetracycline-regulated system. High levels of p53 induction achieved by tetracycline withdrawal were accompanied by G(1) cell cycle arrest without significant apoptosis in this cell line. p53 accumulation resulted in a gradual and dramatic loss of MGMT mRNA, protein, and enzyme activity, whose levels were undetectable by day 3 of induction. The loss of MGMT protein was, however, not due to its degradation because the ubiquitin-promoted in vitro degradation of MGMT, which mediates the cellular disposal of the repair protein, was not altered by p53. Run-on transcription assays revealed a significant reduction in the rate of MGMT gene transcription. The negative regulation of MGMT expression by wt p53 was confirmed in two other human isogenic cell lines, namely, the GM47.23 glioblastoma, which contains a dexamethasone-inducible wt p53, and the H460 lung cancer cell line, in which wt p53 had been inactivated by the human papillomavirus E6 protein. Furthermore, a panel of four human tumor cell lines, including gliomas with wt p53 status, displayed markedly lower levels of MGMT gene transcripts than those having p53 mutations. Induction of wt p53 in these models led to a 3- and 2-fold increase in sensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea and temozolomide, respectively, which generate the MGMT-repairable O(6)-alkyl adducts in DNA. These results demonstrate that p53 is a negative regulator of MGMT gene expression and can create a MGMT-depleted state in human tumors similar to that achieved by O(6)-benzylguanine, a potent inhibitor of MGMT currently undergoing clinical trials. Thus, our study exposes an additional benefit associated with p53 gene therapy and provides a strong biochemical rationale for combining the MGMT-directed alkylators with p53 gene transfer to achieve improved antitumor efficacy.

Adenovirus-mediated antisense urokinase-type plasminogen activator receptor gene transfer reduces tumor cell invasion and metastasis in non-small cell lung cancer cell lines.

The urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in the proteolytic cascade involved in the metastasis of lung and other cancers. We report that the reduction in uPAR levels produced by an antisense strategy using an adenovirus construct (Ad-uPAR) in H1299 cells, an invasive human lung cancer cell line that produces high levels of uPAR, resulted in a decrease of uPAR levels to 80-90% of those seen in cells infected with mock or adenovirus (Ad)-cytomegalovirus vector controls. In addition, increasing the multiplicity of infection from 25 to 200 caused a corresponding decrease in the level of uPAR protein within 5 days of treatment, as shown by Western blot analysis. Furthermore, the in vitro translation of total RNA levels of Ad-uPAR-infected H1299 cells in a rabbit reticulocyte lysate system caused a 50-70% decrease in uPAR immunoprecipitate in Ad-uPAR-infected cells relative to the levels in cells of mock and vector controls. The Matrigel invasion assay showed the invasion of H1299 cells and A549 cells infected with Ad-uPAR to be decreased by 70% relative to mock- and vector-infected controls. Infection of tumor cells with Ad-uPAR before implantation significantly reduced the incidence of lung metastasis by 85% as compared with the control virus-infected cells injected into nude mice through the tail vein. Our collective results show that the uPAR system is a potential target of treatment for lung cancers.

Genetic polymorphism at the glutathione S-transferase (GST) P1 locus is a breast cancer risk modifier.

The isolation of full-length cDNAs of naturally occurring GSTP1 gene variants, and the demonstration that these alleles are distributed in the normal population, have provided conclusive evidence that the human GSTP1 gene locus is polymorphic and that specific GSTP1 alleles may be associated with different risk for cancers or other diseases. Recent data have indicated that the different GSTP1 alleles encode proteins with different enzymatic activities against carcinogens. In this case-control study, we examined the effect of the GSTP1 genetic polymorphism and its interaction with other factors to determine breast cancer risk. GSTP1 and GSTM1 genotypes of 220 breast cancer patients and 196 controls, all residents of western France, were examined. Data on menopausal status and family cancer history were obtained from 195 patients and 147 controls. Exons 5 and 6 of the GSTP1 gene, which contain the polymorphic nucleotide transitions, were analyzed by DNA polymerase chain reaction-restriction fragment length polymorphism to distinguish between the GSTP1 alleles. In the control population, GSTP1 allelic frequencies were 64.3%, 26.0% and 9.7%, respectively, for GSTP1*A, GSTP1*B and GSTP1*C. In the breast cancer patients, the frequencies were 67.9% for GSTP1*A, 26.8% for GSTP1*B and 5.3% for GSTP1*C. In multivariate analysis, breast cancer risk increased by 7.7-fold (p < 0.001) in women with a family history of cancers and 2.18-fold (p = 0.026) in non-GSTP1*C individuals. GSTM1 genotypes did not emerge as risk factor. Our results show that in addition to well-known risk factors, in particular, a family history of cancer, GSTP1 allelopolymorphism is a significant modifier of breast cancer risk. The results also suggest a protective role against breast cancer for the GSTP1*C allele.

Effects of radiation on the levels of MMP-2, MMP-9 and TIMP-1 during morphogenic glial-endothelial cell interactions.

Radiation-induced damage to the central nervous system (CNS) is believed to target glial or endothelial cells or both, although the pathophysiology of the process is poorly understood. We therefore used a coculture system, in which glioblastoma SNB19 cells induced bovine retinal endothelial (BRE) cells to form capillary-like structures, to examine the role of ionizing radiation in modulating the production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase-1 (TIMP-1). In particular, we irradiated both BRE cells and cocultures of BRE and SNB19 cells with a single dose of X-rays and then estimated the levels of MMP-2, MMP-9 and TIMP-1. Gelatin zymography revealed a continuous increase in the levels of MMP-2 and MMP-9 during capillary-like structure formation. Of note, the levels of both MMP-2 and MMP-9 were markedly higher in irradiated cocultures at 72 hr after irradiation than in untreated cocultures. Northern blot analysis also demonstrated an increased expression of MMP-9 mRNA in the irradiated cocultures. In addition, TIMP-1 mRNA and protein levels increased up to 48 hr in both irradiated and nonirradiated BRE cells and in nonirradiated cocultures, but there was a significant decrease in the TIMP-1 mRNA and protein levels in irradiated cocultures. It takes about 72 hr for capillaries to form in nonirradiated cocultures, but these capillary networks fail to form in endothelial cells in irradiated cocultures. These findings establish that radiation differentially affects the production of MMP-2, MMP-9 and TIMP-1 during glial-endothelial morphogenesis and suggest mechanisms by which microvessels in the CNS respond to radiation.

DNA repair protein O6-alkylguanine-DNA alkyltransferase is phosphorylated by two distinct and novel protein kinases in human brain tumour cells.

We showed recently that human O(6)-alkylguanine-DNA alkyltransferase (AGT), an important target for improving cancer chemotherapy, is a phosphoprotein and that phosphorylation inhibits its activity [Srivenugopal, Mullapudi, Shou, Hazra and Ali-Osman (2000) Cancer Res. 60, 282-287]. In the present study we characterized the cellular kinases that phosphorylate AGT in the human medulloblastoma cell line HBT228. Crude cell extracts used Mg(2+) more efficiently than Mn(2+) for phosphorylating human recombinant AGT (rAGT) protein. Both [gamma-(32)P]ATP and [gamma-(32)P]GTP served as phosphate donors, with the former being twice as efficient. Specific components known to activate protein kinase A, protein kinase C and calmodulin-dependent kinases did not stimulate the phosphorylation of rAGT. Phosphoaminoacid analysis after reaction in vitro with ATP or GTP showed that AGT was modified at the same amino acids (serine, threonine and tyrosine) as in intact HBT228 cells. Although some of these properties pointed to casein kinase II as a candidate enzyme, known inhibitors and activators of casein kinase II did not affect rAGT phosphorylation. Fractionation of the cell extracts on poly(Glu/Tyr)-Sepharose resulted in the adsorption of an AGT kinase that modified the tyrosine residues and the exclusion of a fraction that phosphorylated AGT on serine and threonine residues. In-gel kinase assays after SDS/PAGE and non-denaturing PAGE revealed the presence of two AGT kinases of 75 and 130 kDa in HBT228 cells. The partly purified tyrosine kinase, identified as the 130 kDa enzyme by the same assays, was strongly inhibited by tyrphostin 25 but not by genestein. The tyrosine kinase used ATP or GTP to phosphorylate the AGT protein; this reaction inhibited the DNA repair activity of AGT. Evidence that the kinases might physically associate with AGT in cells was also provided. These results demonstrate that two novel cellular protein kinases, a tyrosine kinase and a serine/threonine kinase, both capable of using GTP as a donor, phosphorylate the AGT protein and affect its function. The new kinases might serve as potential targets for strengthening the biochemical modulation of AGT in human tumours.

Glutathione S-transferase GSTP1 and cyclin D1 genotypes: association with numbers of basal cell carcinomas in a patient subgroup at high-risk of multiple tumours.

We previously described associations between basal cell carcinoma (BCC) numbers and allelic variants at loci that mediate host response to ultraviolet radiation (UV). These associations were largely exerted in cases with the multiple presentation phenotype (MPP). This phenotype describes patients who present at their first or a later presentation with a cluster of BCC (2-10 new BCC). Remaining BCC cases have the single presentation phenotype (SPP) and may develop more than one BCC but only have single new lesions at any presentation. We proposed that the MPP cases comprise a high-risk group as they suffer significantly more lesions than SPP cases. We are attempting to determine, in the total BCC case group and subgroups, how many genes influence BCC numbers and their relative importance. In this study, we assessed the influence of two further candidates, glutathione S-transferase GSTP1 and cyclin D1 (CCND1), on tumour numbers in a total group of 457 patients comprising MPP and SPP cases. The relative importance of these genes in comparison with occupational UV exposure and host response (skin type) was also considered. We found that the frequencies of GSTP1 genotypes based on the Ile105 and Val105-expressing alleles and CCND1 AA, AG, GG genotypes were similar in MPP and SPP cases and that there were no significant associations between GSTP1 or CCND1 genotypes and BCC numbers in the total or SPP groups. However, in the MPP cases, GSTP1 Val105/Val105 was associated with more tumours (P = 0.05, reference GSTP1 Ile105/Ile105). Inclusion of skin type and indoor/outdoor occupation in the negative binomial regression models did not alter the associations of these genotypes with tumour numbers. DNA from 258 cases was analysed to identify GSTP1*A (Ile105-Ala114), GSTP1*B (Val105-Ala114), GSTP1*C (Val105-Val114) and GSTP1*D (Ile105-Val114). In SPP cases, there was no association between BCC numbers and GSTP1 BB, though the association with GSTP1 BC approached significance (P = 0.09). In MPP cases, GSTP1 BC was associated with BCC numbers (P = 0.03). We also found that the interaction term, GSTP1 Val105/Val105 with CCND1 AA, was associated with BCC numbers in the total (P = 0.001) and MPP (P = 0.006) but not SPP (P = 0.68) groups. In a stepwise model including GSTP1 Val105/Val105, CCND1 AA and their interaction terms as well as GSTM1, GSTT1 and CYP2D6 genotypes, skin type 1 and gender, the combination of genotypes was the best predictor of BCC numbers. These data suggest that study of further genes involved in cell-cycle control and protection from oxidative stress will be useful, particularly in high-risk subgroups.

DNA methyltransferase levels and altered CpG methylation in the total genome and in the GSTP1 gene in human glioma cells transfected with sense and antisense DNA methyltransferase cDNA.

This study examines the efficacy of using plasmid expression vectors containing sense and antisense DNA MTase cDNA to both up- and downregulate intracellular DNA MTase levels in human glioma cells. The effects of the changes in MTase levels on global genomic DNA methylation and on the methylation status of CpG dinucleotides in the GSTP1 gene were determined in a glioma cell line that overexpresses the GSTP1 gene. In cells transfected with sense DNA MTase cDNA, MTase gene transcripts increased to a maximum of 2. 5-fold at 24 h, while MTase activity increased to a maximum of 3. 6-fold at 48 h. The effects of antisense MTase cDNA transfections were less pronounced, and levels of MTase gene transcripts and enzyme activity in transfectants were decreased to only, approximately, one-half the levels of controls. The alterations in DNA MTase expression were associated with corresponding changes in the level of global DNA methylation and in the methylation of the GSTP1 gene in the cells, however, with no detectable morphological or cytotoxic effects on the cells. No significant changes in GSTP1 gene expression were detected after the transfections, presumably because of the high levels of basal GSTP1 expression in the cells. Consequently, the p16 gene, known to be repressed transcriptionally by DNA methylation, was examined for the functional effects of the altered MTase levels. The results showed a 2-fold decrease in p16 gene transcripts with the sense MTase transfectants, while in the MTase antisense-transfected cells p16 transcript levels increased by 30%. Together, these results demonstrate the feasibility of using both sense and antisense DNA MTase expression vectors to regulate DNA MTase levels in glioma cells and that, over relatively short periods of time, the alterations in MTase activities are not deleterious to the cells. The system provides a model with which the role of DNA methylation in critical genes and DNA sequences can be investigated in glioma cells.

Protein phosphorylation is a regulatory mechanism for O6-alkylguanine-DNA alkyltransferase in human brain tumor cells.

The biochemical regulation of human O6-alkylguanine-DNA alkyltransferase (AGT), which determines the susceptibility of normal tissues to methylating carcinogens and resistance of tumor cells to many alkylating agents, is poorly understood. We investigated the regulation of AGT by protein phosphorylation in a human medulloblastoma cell line. Incubation of cell extracts with [gamma-32P]ATP resulted in Mg(2+)-dependent phosphorylation of the endogenous AGT. Immunoprecipitation after exposure of the cells to 32P-labeled inorganic phosphate showed that AGT exists as a phosphoprotein under physiological conditions. Western analysis and chemical stability studies showed the AGT protein to be phosphorylated at tyrosine, threonine, and serine residues. Purified protein kinase A (PKA), casein kinase II (CK II), and protein kinase C (PKC) phosphorylated the recombinant AGT protein with a stoichiometry of 0.15, 0.28, and 0.44 (mol phosphate incorporated/mol protein), respectively. Residual phosphorylation of the endogenous AGT by the PKs present in cell homogenates and phosphorylation of the recombinant AGT by purified serine/threonine kinases, PKA, PKC, and CK II reduced AGT activity by 30-65%. Conversely, dephosphorylation of cell extracts by alkaline phosphatases stimulated AGT activity. We also identified consensus phosphorylation motifs for many cellular kinases, including PKA and CK II in the AGT protein. These data provide the first and conclusive evidence of AGT phosphorylation and suggest that reversible phosphorylation may control the activity of this therapeutically important DNA repair protein in human normal and cancer cells.

Selective suppression of matrix metalloproteinase-9 in human glioblastoma cells by antisense gene transfer impairs glioblastoma cell invasion.

Increased expression of matrix metalloproteinases (MMPs) has been associated with human glioblastoma tumor progression. In this study, we sought to down-regulate MMP-9 expression by stably transfecting a high-grade glioblastoma cell line with a plasmid vector capable of expressing an antisense transcript complementary to a 528-bp segment at the 5' end of human MMP-9 cDNA. Stable transfectants were obtained through selection with G418. Of the clones transfected with vector, sense, and antisense constructs, Northern blotting, Western blotting, and gelatin zymography showed that MMP-9 expression was significantly reduced only in the antisense-transfected cells. A Matrigel invasion assay revealed marked reductions in invasiveness for the antisense clones relative to the parental, vector, and sense clones. Cocultures of tumor spheroids and fetal rat brain aggregates showed that the antisense-transfected stable clones showed no invasion of the rat brain aggregates; in contrast, 90% of the parental, vector, and sense clones invaded the rat brain aggregates. Intracerebral injection of antisense stable transfectants in nude mice produced no tumors or very small tumors, but intracerebral injection of parental or vector clones did produce tumors. These results suggest that MMP-9 expression is essential for the invasiveness of glioblastoma cells.

Modulation of endothelial cell morphogenesis in vitro by MMP-9 during glial-endothelial cell interactions.

The purpose of this study was to investigate the roles of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the formation of capillary structures by human brain microvascular endothelial cells cocultured with SNB19 glioblastoma cells. Unstimulated cocultures did not form capillaries and produce MMP-9 but stimulation with the protein kinase C (PKC) activator 4-phorbol-12-myristate 13-acetate (PMA) produced MMP-9 and capillary networks. Addition of recombinant MMP-9 increased capillary formation. Anti-MMP-9 antibodies, TIMP-1, the synthetic MMPs inhibitor Batimastat (BB-94), and the PKC inhibitor calphostin-C all reduced MMP-9 activity and capillary network formation in these cocultures. Cytochalasin-D in the presence of PMA suppressed MMP-9 expression and capillary formation, but colchicine-B had no such effect. Finally, PMA-induced MMP-9 expression and capillary formation were inhibited by the MEKK-specific inhibitor PD98059. These results suggest that MMP-9 is important in endothelial cell morphogenesis and the formation of capillaries in glial/endothelial cocultures in vitro.

Regulation of MMP-9 (type IV collagenase) production and invasiveness in gliomas by the extracellular signal-regulated kinase and jun amino-terminal kinase signaling cascades.

Our previous studies have shown that MMP-9 levels are significantly elevated during the progression of human gliomas. In the current study, we examined the role of JNK- and ERK-dependent signaling modules in the regulation of MMP-9 production and the invasive behavior of the human glioblastoma cell line SNB19, in which JNK/ERK1 is constitutively activated. SNB19 cells that were transfected with dominant-negative JNK, MEKK, and ERK1 expression vectors showed reduced MMP-9 promoter activity. In addition, conditioned medium collected from SNB19 cells transfected with these expression vectors showed diminished MMP-9 activity in the presence of phorbol myristate acetate, as determined by gelatin zymography. The cotransfection of SNB19 cells with kinase-deficient c-raf also diminished MMP-9 promoter activity. Further, in the presence of a specific inhibitor of MEKK (PD098059), the Matrigel invasion assay showed the invasiveness of dominant-negative SNB19 cells transfected with dominant-negative JNK1 or ERK1 to be remarkably reduced. In conclusion, our studies showed for the first time that MMP-9 production and the invasive behavior of SNB 19 cells are regulated by JNK- and ERK-dependent signaling modules and that interfering with either of the pathways reduces invasiveness.

Amplification of telomeric DNA directly correlates with metastatic potential of human and murine cancers of various histological origin.

Telomeres, repeated DNA sequences (T2AG3)n that guard the ends of chromosomes, serve as a checkpoint for cell-cycle progression and regulate cell senescence and apoptosis. Loss of the telomeric repeats promotes genomic instability, which is the hallmark of most cancer cells. Whether this loss differs among tumor cells with malignant potential is unknown and was the goal of this study. An all-human telomeric DNA probe was used to perform fluorescence in situ hybridization (FISH) and the telomeric signals in interphase nuclei were quantitated using a computer software package. Southern blot analysis was carried out to measure terminal restriction fragment length (TRFL) in multiple cancer cell lines, including nonmetastatic and metastatic human breast, lung, prostate, colon, brain, and renal carcinomas, as well as human and murine melanoma clones and somatic cell hybrids. The metastatic capability of all cell lines, clones and somatic cell hybrids was evaluated subsequent to orthotopic implantation into nude mice. FISH preparations with telomeric DNA probes showed that the mean percent telomeric area in the metastatic nuclei was significantly greater than their nonmetastatic counterparts and Southern blotting in selected samples confirmed our findings. These data suggest that amplification of telomeres is directly correlated with invasive and metastatic potential of murine or human tumor cells.

Structure of the human allelic glutathione S-transferase-pi gene variant, hGSTP1 C, cloned from a glioblastoma multiforme cell line.

We recently reported the cloning of full-length cDNAs corresponding to mRNAs of three GST-pi genes, hGSTP1*A, hGSTP1*B and hGSTP1*C, as well as, the isolation of the full-length hGSTP1*C, of the human glutathione S-transferase-pi (GST-pi) gene that is characterized by a A-->G transition at +1404 in exon 5 and a C-->T transition at +2294 in exon 6. Although the promoter of the isolated gene was identical to that of the previously described GST-pi gene isolated from the MCF 7 and the HPB-ALL cell lines, both of which were hGSTP1*A, a number of structural differences were observed, including, nucleotide transitions, transversions, deletions and insertions, some of which created new restriction enzyme cleavage sites. A guanine insertion in the insulin response element, IRE, in intron 1 created an additional site for 5'-cytosine methylation. Seven repeat retinoic acid response element (RARE) consensus half sites, A(G)GG(T)TC(G)A at +1521 to +1644 were identified in the cloned hGSTP1*C. Five of the RARE half-sites had the minimal spacer nucleotide requirement for functionality and DNA mobility shift analysis with different pairs of the RARE half-sites and supershift studies using antibodies against RAR-beta showed significant binding of nuclear protein complexes from RA-treated cells to these RAREs. GST-pi gene expression was increased significantly in cells transfected with the GST-pi gene and treated with all-trans RA. These results contrast with those in a previous report in which RA was shown to suppress the GST-pi promoter, and indicate a complex mechanism of RA-mediated GST-pi gene regulation in tumor cells.

Translational inhibition of messenger RNA of the human pi class glutathione S-transferase by antisense oligodeoxyribonucleotides.

In this study, a T7 plasmid expression vector containing the cDNA of a variant human GST-pi gene, hGSTP1*C, was used to examine the translational inhibition of the GST-pi mRNA with antisense deoxyribonucleotides (AS-ONs), and to investigate the dependency of the inhibition on ribonuclease (RNAse) H, AS-ON and target mRNA sequence specificity and AS-ON back bone modification. Translational inhibition of hGSTP1*C mRNA showed significant AS-ON concentration-dependency and was both target mRNA and AS-ON sequence specific. Fully modified phosphoromonthioate AS-ONs were less inhibitory than their partial phosphoromonthioate analogs; unmodified AS-ONs were inactive. RNAse H enhanced the translational inhibition by AS-ON specific to the translation initiation region mRNA, and was associated with cleavage of the target mRNA at the site of AS-ON:mRNA hybridization. AS-ONs directed to the A-->G and C-->T transitions, unique to hGSTP1*C, were more RNAse H-dependent than AS-ONs directed against the translation initiation site, indicating a greater involvement of RNAse H-dependent mRNA cleavage in the mechanism of translational inhibition by AS-ON at the polymorphic site. These data suggest that AS-ONs provide a potentially effective means of specific down-regulation of the human GST-pi gene, and demonstrate that the sites of GST-pi gene allelo-polymorphism can be targeted to translationally down-regulate the different GST-pi gene variants, specifically and differentially targeted.

DNA damage in peripheral blood mononuclear cells correlates with response to biochemotherapy in melanoma.

The combination of cisplatin-based chemotherapy with interleukin-2 (IL-2) and interferon, referred to as biochemotherapy, has shown encouraging results in patients with advanced melanoma. Toxicity is high, however and no objective parameters exist to distinguish between patients who are likely to respond and those who are not. The purpose of this pilot study was to determine whether in vitro cisplatin-induced damage to the glutathione S-transferase-pi (GST-pi) gene in peripheral blood mononuclear cells (PBMCs) before therapy correlated with the histological response in melanoma patients with local-regional metastases who received concurrent biochemotherapy before definitive surgery. Before therapy, PBMCs from 16 patients were exposed to cisplatin at concentrations of 25, 50 or 100 microM for 3 h and the extent of damage to the GST-pi gene was quantitated by polymerase chain reaction (PCR). Patients were subsequently treated on a biochemotherapy regimen consisting of cisplatin 20 mg/m2 intravenously (i.v.) on days 1-4, vinblastine 1.5 mg/m2 i.v. on days 1-4, dacarbazine 800 mg/m2 i.v. on day 1, IL-2 9 MIU/m2 per day i.v. by continuous infusion on days 1-4 (total of 96 h), and interferon alpha2a 5 MU/m2 subcutaneously on days 1-5. The 16 patients were categorized into two groups: major responders (n = 7) and non-major responders (n = 9). Although we observed a wide interpatient variation, a statistically significant correlation existed between the histological response and the degree of DNA damage caused in the PBMCs at all three cisplatin concentrations tested (P = 0.024 for 25 microM; P = 0.036 for 50 microM; P = 0.007 for 100 microM). Our pilot study suggests that determination of in vitro cisplatin-induced DNA damage using a gene-specific PCR assay may be useful in predicting the histological response to biochemotherapy.

Nitric oxide and neopterin levels and clinical response in stage III melanoma patients receiving concurrent biochemotherapy.

The combination of cisplatin-based chemotherapy with interleukin-2 (IL-2) and interferon-alpha (IFN-alpha), referred to as biochemotherapy, has produced overall response rates of greater than 50% in advanced melanoma patients, with durable complete responses in the range of 5-10%. The mechanism of action of biochemotherapy is unknown. Preclinical work suggests synergistic interactions between the cytotoxic agents, especially cisplatin, and the biological agents in killing melanoma cells. Immune effector cells activated by the components of the biochemotherapy may also be involved, as direct cytotoxic effectors and/or as sources of secondary cytokines, which can induce nitric oxide (NO) production in a wide variety of cell types. In addition, high levels of neopterin, a marker of monocyte/macrophage activation, have been found in patients undergoing immunotherapy or biochemotherapy for melanoma. Based on these data, we hypothesized that the degree of elevation of serum NO metabolic products and neopterin during treatment would correlate with the response to biochemotherapy in melanoma patients. Blood samples were obtained before and during preoperative biochemotherapy with cisplatin, vinblastine, dacarbazine, IL-2 and IFN-alpha in 45 melanoma patients with locoregionally advanced disease. NO was measured as nitrite after enzymatic reduction, using the colorimetric assay of Griess, and neopterin was measured by radioimmunoassay. Our results demonstrate a higher day 5 nitrite level (of borderline statistical significance, P = 0.057) in major responders to the therapy than in those who did not achieve a major response, while there was no difference in the elevation in neopterin level during therapy between major and non-major responders. These results suggest that induction of NO during biochemotherapy may be playing a role in the mechanism of action of this therapy, while the role of monocyte/macrophage activation is still in question.