Steroid receptors in epithelial ovarian carcinoma: relation to clinical parameters and survival.

Estrogen receptor (ER) and progestin receptor were measured in samples of tumors obtained at first laparotomy from 97 previously untreated patients suffering with a primary ovarian epithelial tumor, for whom a 3-year follow-up was available. The presence or absence of steroid receptors (threshold arbitrarily fixed at 10 fmol/mg of cytoplasmic protein) was determined by the dextran coated charcoal method and related to a number of patient characteristics such as the residual disease (cutoff, 2 cm), histological type, International Federation of Gynecologists and Obstetricians grade and stage, and age. Results were analyzed by univariate and multivariate methods. (a) The tumor ER positivity was associated with better survival; progestin receptor showed a similar trend but did not reach statistical significance. (b) After stratification for residual tumor the association ER positivity/better survival was still statistically significant in the subset of patients with residual tumor greater than 2 cm. (c) When the median survival times were considered it became apparent that progestin receptor absence nullified the effect associated with positive ER. (d) Multivariate analysis confirmed that among the variables considered the main determinants of prognosis were the size of the residual tumor, serous histological type, and positive ER.

Progress report on the anorexia induced by drugs believed to mimic some of the effects of serotonin on the central nervous system.

Some agents that increase serotoninergic transmission in the brain show anorectic activity at doses that do not interfere with the behavior of rats and other animal species. These agents reduce food intake by a mechanism that clearly differs from that involved in the anorectic activity of d-amphetamine. d-Fenfluramine, fluoxetine, and sertraline are three drugs that have already been tested and are used in man. These compounds accumulate in the brain and are metabolized through N-dealkylation. They affect the uptake and release of serotonin at different concentrations, with mechanisms that do not completely overlap. There is pharmacological evidence that d-fenfluramine and sertraline exert their anorectic activity by enhancing the stimulation of 5-HT1nonA receptors whereas fluoxetine seems to affect at anorectic doses both serotoninergic and dopaminergic systems. The role of serotonin in controlling food intake will be discussed, and the effects of agents that reduce serotoninergic transmission will also be considered.

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.

The effects of single and repeated anorectic doses of 5-hydroxytryptamine uptake inhibitors on indole levels in rat brain.

1. The effects of acute and repeated equiactive anorectic doses (ED50) of recently marketed 5-hydroxytryptamine (5-HT) uptake inhibitors on the content of brain indoles were compared in rats in relation to the brain regional concentrations of unchanged drug and its known active metabolite. 2. Single intraperitoneal (i.p.) doses of the anorectic ED50 of fluoxetine (35 mumol kg-1), fluvoxamine (60 mumol kg-1), paroxetine (20 mumol kg-1) and sertraline (49 mumol kg-1) slightly reduced brain 5-hydroxyindoleacetic acid (5-HIAA), with regional differences, this being compatible with 5-HT uptake blockade. Only fluvoxamine and sertraline significantly enhanced the content of 5-HT in the cortex. 3. The regional sensitivity to the acute effect of a given drug was not related to any preferential drug distribution, as these compounds distributed almost uniformly in the brain areas considered (cortex, striatum and hippocampus). 4. Repeating the same doses twice daily, i.p. for 14 days, however gave a different picture, fluvoxamine having little or no effect on the content of indoles and fluoxetine, paroxetine and sertraline lowering both 5-HT and 5-HIAA in all the brain regions compared to pair-fed control animals, 1 h after the last dose. 5. One week later only fluoxetine-treated animals still had reduced brain 5-HT, this probably being related to the accumulation of its main metabolite norfluoxetine in rat brain after chronic dosing. 6. Further studies on the relationship between the long-term neurochemical changes and anorectic activity are required but it appears from these results that anorectic drugs with similar acute effects on 5-HT uptake may differ in their long-term effects on 5-HT mechanisms.

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.

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.

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.

Accumulation and metabolism of uneven fatty acids present in single cell protein.

Liquipron and Toprina, obtained by growing yeasts (Candida maltosa and Candida lipolytica) on n-hydrocarbons, were investigated to ascertain the biological significance and possible toxicological implications of their high content of uneven fatty acids (UFA). It was confirmed that the extent to which UFA accumulate in adipose tissue of rats fed the 2 products reflects only partially their UFA contents. The presence of UFA in rat tissues does not appear to alter intermediate metabolism. The capacity of liver mitochondria ot oxidize palmitic acid was similar in control and in Liquipron-treated rats. Palmitic acid and heptadecanoic acid did not compete for oxidation when mixed at concentrations which reflect their presence in the tissues of animals fed high levels of Liquipron.

Anorectic activity of fluoxetine and norfluoxetine in mice, rats and guinea-pigs.

The present study aimed to establish the role of the metabolite norfluoxetine in the anorectic activity of fluoxetine, and to relate the anorectic doses (ED50) to the brain concentrations of the parent drug and its metabolite. Fluoxetine showed anorectic activity at increasing intraperitoneal doses (ED50 = 39.1, 34.7 and 21.7 mumol kg-1 in mouse, rat and guinea-pig, respectively) and norfluoxetine was slightly more active (24.3, 22.9 and 19.1 mumol kg-1, respectively) in all three species. In terms of maximum concentration (Cmax) and area under the curve (AUC) within the experimental period (0-90 min), brain concentrations varied widely and were poorly related to the dose; guinea-pig appeared to be much more sensitive to fluoxetine than was mouse or rat. Administered norfluoxetine was present in the brain of the three species in approximately the same order as fluoxetine, i.e. lower in guinea-pig than in mouse or rat. The Cmax and AUC of norfluoxetine after fluoxetine administration was 50-60% of the values after an equiactive dose of norfluoxetine in mouse and guinea-pig, and more than 80% in rat.

Factors affecting plasma free fatty acids rise during hemodialysis.

We studied the mechanisms responsible for causing acute changes in plasma lipids during hemodialysis. Dialysis decreased plasma triglycerides to the same extent as when heparin was given without dialysis. Cholesterol increased in proportion to hemoconcentration. Plasma free fatty acids (FFA) levels were also increased, but more so than with heparin alone. Glucose and acetate did not play a role, nor did carnitine loss, and hemofiltration elicited similar effects. The rise in plasma FFA is therefore likely to be caused by other as yet unknown mechanism.

Estrogen and progesterone receptors in leiomyomas and normal uterine tissues during reproductive life.

Estrogen and progesterone receptors (ER, PR) were measured in leiomyomas and normal uterine tissues. Estrogen receptors concentration was higher in endometrium than in leiomyomas, lowest in normal myometrium. In the case of progesterone receptors, the concentrations in endometrium and leiomyomas were similar whereas that of myometrium was lower. ER and PR concentration were similar in leiomyomas of the uterine fundus, body and isthmus and steroid receptor content in the inner parts of large myomas was the same as in the outer parts. ER and PR concentrations in tumor-bearing myometrium were not different from those in myometrium of a control group.

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.

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.

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.

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.