Expression levels of p53 and p73 isoforms in stage I and stage III ovarian cancer.

The p53 gene has been investigated for its role in epithelial ovarian cancer but data collected until now are contradictory. The evidence that p53 belongs with p63 and p73 to a family of transcription factors re-opened interest in this gene family. Here, we used quantitative real time RT-PCR to determine expression levels of TAp53, TAp73 and their N-terminal splice variants in a cohort of 169 ovarian cancer patients with stage I and stage III disease. The TAp73 levels in stage III biopsies differed by 100-fold depending on the p53 status and overall survival appears to be significantly related to DeltaNp73 expression. Kaplan-Meyer analyses did not suggest a correlation between overall survival and levels of TAp73, DeltaNp73 or the DeltaNp73/TAp73 ratio. In conclusion, these data suggest that at least in our patient cohort p53 and p73 expression levels are not correlated to malignant progression of ovarian cancer. They might, however, play a role in tumour initiation.

Cell cycle phase perturbations and apoptosis in tumour cells induced by aplidine.

Aplidine, dehydrodidemnin B, is a marine depsipeptide isolated from the Mediterranean tunicate Aplidium albicans currently in phase II clinical trial. In human Molt-4 leukaemia cells Aplidine was found to be cytotoxic at nanomolar concentrations and to induce both a G(1) arrest and a G(2) blockade. The drug-induced cell cycle perturbations and subsequent cell death do not appear to be related to macromolecular synthesis (protein, RNA, DNA) since the effects occur at concentrations (e.g. 10 nM) in which macromolecule synthesis was not markedly affected. Ten nM Aplidine for 1 h inhibited ornithine decarboxylase activity, with a subsequently strong decrease in putrescine levels. This finding has questionable relevance since addition of putrescine did not significantly reduce the cell cycle perturbations or the cytotoxicity of Aplidine. The cell cycle perturbations caused by Aplidine were also not due to an effect on the cyclin-dependent kinases. Although the mechanism of action of Aplidine is still unclear, the cell cycle phase perturbations and the rapid induction of apoptosis in Molt-4 cells appear to be due to a mechanism different from that of known anticancer drugs.

Trail activity in human ovarian cancer cells: potentiation of the action of cytotoxic drugs.

The ability of the TRAIL ligand to induce cell killing in three ovarian cancer cell lines was investigated using a glutathione-S-transferase (GST)-TRAIL fusion protein. One of the three lines was sensitive to TRAIL, which induced cell killing in a range of concentrations similar to those necessary to kill the TRAIL-sensitive leukaemic cell line Jurkat. The relative mRNA expression of the four TRAIL receptors did not explain the different sensitivities of the three ovarian cancer cell lines to TRAIL treatment. The TRAIL-sensitive IGROV-1 cell line expressed slightly lower levels of the anti-apoptotic protein FLIP than the two TRAIL-insensitive cell lines (A2780 and SKOV-3). Nevertheless, although TRAIL did not significantly reduce cell growth in the A2780 and SKOV-3 cells it did enhance the activity of paclitaxel and cisplatin (DDP), the two most widely used drugs for the treatment of ovarian cancer, increasing their ability to induce apoptosis. The use of TRAIL in combination with classical anticancer agents might thus boost the apoptotic response, improving the activity of DDP and paclitaxel in ovarian cancer.

Cell cycle-related phosphatases CDC25A and B expression correlates with survival in ovarian cancer patients.

Alterations in cell cycle regulating proteins are common in many cancer types. Recent data suggest a possible link between CDC25 phosphatases overexpression and malignancy. Our objective was to evaluate the expression of the three cell cycle-related phosphatases CDC25A, CDC25B and CDC25C in patients with ovarian cancer. All the patients had a minimal follow up of three years. CDC25A, CDC25B and CDC25C expression were investigated by immunohistochemistry in 106 patients with ovarian cancer. CDC25A and CDC25B were found expressed in almost all the samples analyzed, while CDC25C was undetectable in more than 80% of the patients. The low evaluable data on CDC25C expression, did not allow any association between the expression of this phosphatase and prognosis. The expression of CDC25A and CDC25B showed some evidences of association with a poor prognosis (p = 0.034 and p = 0.058 respectively). This relationship was independent of other factors such as tumor grade, histotype, stage and residual tumor after surgery. In the same patients the examined parameters (residual tumor, grade, stage and histotype) did show the expected relation with survival. The results indicate that high CDC25A and CDC25B expression is related to a worse prognosis in ovarian cancer patients. CDC25 phosphatases expression can be regarded as a possible prognostic factor for ovarian cancer and these proteins should be evaluated as potential molecular targets of novel drugs against this human neoplasm.

Allelic expression of p73 in human ovarian cancers.

BACKGROUND: The p73 gene is structurally related to the tumor suppressor gene p53. The role of p73 in tumor development is still unclear and no data on ovarian cancer are so far available. For this reason we have analyzed, in a panel of ovarian cancers, the allelic distribution and expression of p73. PATIENTS AND METHODS: Fifty-one samples from ovarian cancers and five human ovarian cancer cell lines growing in culture were analyzed. Allelic origin was analyzed by PCR after digestion with the restriction enzyme Sty I. Heterozygous, informative cases were selected for studies aimed at evaluating allelic expression of p73. RESULTS: We found an allelic distribution similar to that previously reported. LOH was found in two patients with ovarian cancer. In one case in which normal ovarian tissue was available biallelic expression of p73 was found. CONCLUSION: In comparisons of ovarian cancers and borderline tumors, no differences in allelic distribution and/or expression were found, suggesting that p73 does not play an important role in the pathogenesis and development of ovarian cancer.

CHK1 frameshift mutations in genetically unstable colorectal and endometrial cancers.

The protein encoded by the CHK1 gene plays an important role in the G2 checkpoint in mammalian cells. In its coding region it presents a sequence of nine consecutive adenines that are a potential site of mutations in tumors with microsatellite instability (MSI). We analyzed the presence of frameshift mutations in the CHK1 gene in human colon and endometrial cancer samples. In the same cancer samples genes known to be altered in these tumors (BAX, TGFBRII, and IGFIIR) were also analyzed. CHK1 frameshfit mutations were found in 1 out 10 colon cancers and 2 out of 7 endometrial cancers showing MSI. CHK1 alterations were associated with the presence of a high degree of MSI. No alterations were found in patients with tumors showing low frequency or lacking instability (microsatellite stable). The same was true for the other four genes analyzed. The insertion or deletion of one A in the poly A tract resulted in a truncated protein. Alterations of the CHK1 gene could represent an alternative way of cancer cells to escape from cell cycle control. Genes Chromosomes Cancer 26:176-180, 1999.

Microsatellite instability and frameshift mutations in genes involved in cell cycle progression or apoptosis in ovarian cancer.

The loss of mismatch repair enzymes increases the mutation rate in microsatellites and coding regions of the genome and appears to be involved in drug resistance. The replication error (RER+) phenotype, associated with microsatellite instability, has been widely described for both familial and sporadic colon cancers and for gastric and endometrial tumors. For ovarian cancer, the incidence of RER+ cases among sporadic tumors is still uncertain. We analyzed epithelial ovarian tumors and ovarian carcinoma cell lines for microsatellite instability and for mutations in the coding regions of different genes, including the recently discovered human CHK-1 gene, which has an important role in controlling cell cycle progression and whose coding region contains a poly(A)9 tract. Microsatellite instability and frameshift mutations in coding regions of BAX, TGFbetaRII, IGFIIR, E2F-4, ICE, and CHK-1 genes were analyzed in ovarian cancer samples and cell lines by polymerase chain reaction (PCR). Approximately 26% of patients showed microsatellite instability in two or more loci. BAT-26 locus showed no alteration in primary tumors. We detected a BAX mutation in one tumor sample and a TGFbetaRII mutation in one cell line. Our findings confirm the presence of the RER+ phenotype in sporadic ovarian cancer. The low rate of mutation in genes previously reported to be altered in colon and gastric cancer suggests that other not yet identified genes might be altered and could play a role in tumor progression and response to treatment in RER+ ovarian tumors.

Molecular characterisation of a panel of human ovarian carcinoma xenografts.

In a panel of 16 human ovarian tumours transplanted in nude mice, the expression of genes involved in cell cycle regulation and in response to drug treatment were characterised. In the 16 tumours analysed we could not detect overexpression of Erb-B2 oncogene while expression of MDR1 mRNA was not detected in 11/15 samples and was low in 4/15 tumours. Only three tumours had mutations in the p53 gene exons 5-8 and one of these mutations did not result in any amino acid alteration. The levels of mRNA for cyclins A, D1 and E were heterogeneous with some tumours expressing high levels and others not expressing them at all. The same was found for the cyclin dependent kinases (CDK) CDK2 and CDK4 and for CDK inhibitors p21/WAF1, p27/KIP1 and p16/CDKN2. Two genes belonging to the nucleotide excision repair, ERCC1 and ERCC3 were detectable in all the samples examined, as were the genes MGMT and MAG, also involved in DNA repair. The data indicate a heterogeneity in the expression of genes considered to be involved in the cellular responses to cytotoxic drug treatment and indicate the possibility of using these tumour models to test specifically molecules with a defined mechanism of action.

hMLH1 and hMSH2 expression and BAX frameshift mutations in ovarian cancer cell lines and tumors.

The expression of mismatch repair proteins hMSH2 and hMLH1 was investigated in human ovarian cancer cell lines and in biopsies of ovarian carcinomas obtained from 20 patients undergoing surgical operation. By Western blotting analysis hMSH2 protein was detected in all the tumor samples analyzed and in eight out of nine human ovarian cancer cell lines, while hMLH1 was undetectable in four out of 20 ovarian tumors and in five out of nine human ovarian cancer cell lines analyzed. The possible presence of frameshift mutations in the BAX gene, which contains a sequence of eight contiguous guanines in its third exon, was tested in all the samples. All the cell lines presented the normal alleles for the BAX gene while only in one of the tumor samples a heterozygous frameshift mutation was found. The frameshift mutation was associated to a low, almost undetectable, level of BAX protein which was instead present at much higher levels in all the other samples investigated. The results indicate that frameshift mutations in the BAX gene, possibly arising as a consequence of microsatellite instability (detectable in these tumors), is detectable in human ovarian cancer although quantitatively it does not appear to be a major determinant of the low apoptotic response to chemotherapy observed in ovarian cancer cells.

DNA-topoisomerase I activity and content in epithelial ovarian cancer.

BACKGROUND: The levels and activity of topoisomerase I were determined in 35 biopsies from patients with ovarian cancer. PATIENTS AND METHODS: The activity was defined by the ability to relax supercoiled DNA plasmid, and levels were determined by Western blotting and immunohistochemistry. RESULTS: We detected topoisomerase I activity in all samples, although at different levels. Enzymatic activity was the same in fresh and frozen tissues. Western blotting analysis detected topoisomerase I in 29 of 35 tumor samples but in the remaining six the levels were below the detection limit. We analyzed the distribution of topoisomerase I in tumor cells and normal infiltrating cells by immunohistochemistry. The enzyme was mainly associated with tumor cells although there were four samples in which tumor cells were negative but in which normal cells, mainly lymphocytes, yielded a positive result. CONCLUSIONS: The results indicate that topoisomerase I enzymatic activity is detectable in human ovarian tumors, varying among patients. No correlations were found between the levels of the enzyme and its activity. Because of the high heterogeneity observed, enzymatic activity assays should be combined with immunohistochemical evaluation of Topo I in the tumor and normal cells present in the tissue.

Expression of genes of potential importance in the response to chemotherapy and DNA repair in patients with ovarian cancer.

The expression of different genes potentially involved in DNA repair and in cell responses to chemotherapy was evaluated in 33 previously untreated ovarian cancer patients. In biopsies of the same patients the expression of repair genes O6-methylguanine DNA methyltransferase (MGMT), 3-methyladenine DNA glycosylase (MAG), ERCC1, MDR-1, DNA topoisomerase I, DNA topoisomerase IIalpha, and glutathione S-transferase-pi (GST-pi) was assessed by Northern blot analysis. No direct statistical correlation was found between the expression of these genes and the response to chemotherapy (mainly platinum-based with or without doxorubicin and cyclophosphamide). Univariate analysis showed a weak negative correlation (P = 0.037) between the expression of ERCC1 and mortality, whereas no statistically significant correlation was found for other parameters. The MDR-1 gene encoding for the P-glycoprotein P-170 was mostly undetectable in these patients (as assessed by Northern blotting), whereas relatively high levels of MAG and MGMT were found in the majority of patients. A statistically significant correlation was found between the expression of DNA topoisomerase I and the expression of either ERCC1 (P = 0.0026) or GST-pi (P = 0.0279).

Absence of deletions but frequent loss of expression of p16INK4 in human ovarian tumours.

The cyclin-dependent kinase inhibitor p16 gene (P16, MTS1, CDKN2) has been shown to be altered by deletion or point mutation in some human tumours and cancer cell lines, suggesting that it works as a tumour suppressor. We analysed p16 gene mutation and p16 protein expression in 42 primary ovarian carcinomas and in five human ovarian cancer cell lines. Polymerase chain reaction (PCR) amplifications of exons 1 and 2 of the gene showed no deletion or gross rearrangement in the p16 gene. The lack of deletion was further demonstrated by Southern blot analysis. Looking for point mutations, we used single-strand confirmation polymorphism (SSCP) analysis and, in half of the tumours, we sequenced both strands of exons 1 and 2. No mutations were detected. In 11 out of 42 patients (26%), however, we detected no protein expression by Western blot analysis, suggesting that decreased expression of p16 rather than deletion of the gene can occur in a significant percentage of human ovarian cancers. In the same experiment CDK4 protein was found homogeneously expressed in all the tumour specimens and in the five cell lines. The lack of expression of p16 was not due to hypermethylation of the gene assessed by digestion of genomic DNAs with a methylation sensitive enzyme, suggesting that other mechanisms, not yet identified, are involved in the decreased expression of the p16 gene in human ovarian tumours.

Glutathione S-transferase activity and glutathione content in human bladder carcinoma associated with schistosomiasis: comparison with uninvolved surrounding tissues.

Glutathione (GSH), glutathione S-transferase (GST) activity and GSTpi expression were measured in 10 human bladder tumors and adjacent uninvolved specimens from Egyptian patients with a history of schistosomal infection. GSH was higher in the tumor than in surrounding uninvolved tissue (not significant). Total GST activity per mg tissue protein and GSTpi expression were higher in tumor tissues (P < 0.05) than in uninvolved tissues. There was a positive correlation between GST activity and GSH content and between total GST activity and GSTpi expression in both tumor and uninvolved tissues.

Interferon-beta can induce progesterone receptors in human endometrial adenocarcinoma.

BACKGROUND: The induction of estrogen and progesterone receptors (ER and PGR) has been reported in breast and endometrial cancer cells exposed to human fibroblast interferon-beta (hIFN-beta). Clinical verification of this finding might provide the rationale for new therapeutic approaches. This study was designed to evaluate whether clinical treatment with high doses of hIFN-beta induced ER and PGR in patients with endometrial adenocarcinoma. METHODS: Two biopsies were obtained, 1 before and 1 after hIFN-beta treatment (3 x 10(6) i.m. every other day for 3 weeks) from 36 patients with endometrial adenocarcinoma. ER and PGR were determined with standard procedures using radiolabeled ligands. RESULTS: hIFN-beta treatment did not affect the proportion of ER-positive (i.e., >15 fmol/mg protein) or PGR-positive (i.e., >20 fmol/mg protein) cases. However, in patients with detectable ER and PGR at baseline, hIFN-beta raised the levels. Using a 35% difference before and after therapy as a cut-off, 72 and 79% of cases had increases in ER and PGR, respectively. The difference was highly significant for PGR. CONCLUSIONS: In patients with endometrial adenocarcinoma with undetectable ER or PGR, hIFN-beta did not induce the expression of these receptors. When the receptors were present they were upregulated by hIFN-beta. Whether this increase in receptor levels, particularly PGR, has therapeutic applications remains to be established.

Flow cytometric detection of glutathione S-transferase isoenzymes by quantitative immunofluorescence under nonsaturating conditions.

The glutathione (GSH)-glutathione S-transferase (GST) detoxification system is an important element in cellular defence against injurious agents and anticancer drugs. GST isoenzymes may represent biochemical markers of neoplastic transformation, and, possibly, drug resistance is associated with altered GST-isoenzyme levels. The ability to measure GST-isoenzymes in cell populations would be useful for several biological and clinical applications. We have developed an immunofluorescence flow cytometric method for the simultaneous detection of different GST-isoenzymes and of DNA in fixed cells. Due to the impossibility of working under saturating conditions for the anti-GST antibody, a normalizing procedure was developed to permit quantitative analysis of single cells labelled with the anti-GST antibody at high dilution. A theoretical model and experimental data supported the use of this procedure. The method proposed is general and could be applied to other antibodies in order to obtain quantitative data outside saturating conditions. The method was challenged in different applications in order to compare it with other classical techniques. First, we characterized sublines resistant to different anticancer drugs with respect to variations of GST isotypes. In a second application, we studied the intercellular heterogeneity of GST content in mouse renal cells. In addition, GST was determined in aneuploid cells from solid tumor biopsies by separation from diploid cells on the basis of DNA content. Finally, GST distribution during cell-cycle progression was studied in two different cell lines by the biparametric analysis of GST/DNA.

Different vimentin expression in two clones derived from a human colocarcinoma cell line (LoVo) showing different sensitivity to doxorubicin.

We selected two clones, isolated from the human colocarcinoma cell line LoVo, showing a sensitivity to doxorubicin similar to (LoVo clone 5) or three times lower than (LoVo clone 7) the parental cell line. Since vimentin was atypically expressed in a human breast carcinoma cell line made resistant to doxorubicin, we looked at vimentin expression in these two clones with spontaneously different sensitivity to the drug. For comparison we used the parental cell line LoVo WT and LoVo/DX made resistant pharmacologically. mRNA for vimentin was undetectable by Northern blot analysis in LoVo WT and in LoVo clone 5, while expression of this gene was high in LoVo clone 7 and in LoVo/DX. This increase in mRNA levels was not related to an amplification of DNA, as suggested by Southern blot analysis. Immunofluorescence and immunocytochemistry findings confirmed, at protein level, the mRNA data. In LoVo clones 5 and 7, there were respectively 8.6% and 71% vimentin-positive cells, although the two clones showed similar expression of multidrug resistance gene 1 (mdr-1) and accumulated intracellular doxorubicin at similar levels. Similarly, drug efflux was the same for both clones. Our results show for the first time that cells resistant to doxorubicin express vimentin independently of the mdr glycoprotein. However when cells from clone 5 were transfected with human vimentin cDNA, they did not become resistant, indicating that vimentin can be considered as a marker of resistance in these cells but does not give rise to a resistant phenotype by itself.

Acute noise stress reduces [3H]5-hydroxytryptamine uptake in rat brain synaptosomes: protective effects of buspirone and tianeptine.

Acute noise stress decreased [3H]5-hydroxytryptamine ([3H]5-HT) uptake in synaptosomes from rat hypothalamus, hippocampus and cerebral cortex. The decrease was due to the maximum rate of [3H]5-HT uptake, which peaked 30 min after stress and partly returned to resting values within 4 h, with no changes in affinity (Km values). No changes in [3H]paroxetine binding and basal [3H]5-HT release were found in stressed rats. Tianeptine, given at the dose of 10 mg/kg 1 h before stress, counteracted the noise-induced decrease of 5-HT uptake, since it increased [3H]5-HT uptake in both resting and stressed animals, but did not prevent the rise in plasma corticosterone of stressed rats. Buspirone pretreatment had no effect on [3H]5-HT uptake in resting rats but prevented the noise-induced decrease in [3H]-HT uptake. Diazepam did not modify either the basal or the noise-induced reduction in [3H]5-HT uptake. The evidence that treatments reducing extrasynaptic 5-HT, by increasing its reuptake (tianeptine) or reducing its release (buspirone) in innervated regions are able to modify the stress-induced decrease in 5-HT uptake, further confirms the importance of serotonin in the mechanisms mediating neurochemical responses to stress.