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.

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.

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.

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.

Diazepam binding inhibitor (DBI) increases after acute stress in rat.

Diazepam binding inhibitor (DBI) acts in brain by binding to GABAA/benzodiazepine receptors (GBR) and to mitochondrial benzodiazepine receptors (MBR). Because DBI acting at MBR, has been shown to be an effector of ACTH-induced steroidogenesis and stress is known to change the level of GBR and MBR, the model of acute noise stress in rats was used to study modifications of DBI and GRB or the content of MBR in various areas of the brain and adrenal gland. It was found that, in the brain of stressed rats, DBI and its processing products (ODN-like immunoreactivity), increased selectively in the hippocampus. This increase in the content of DBI was preceded and followed by a net decrease of GBR and an increase of MBR. Similarly, in adrenal cortex, the content of DBI and MBR increased during the first hour, following acute stress and this increase paralleled the increase in plasma corticosterone. These data suggest that DBI, acting on MBR may regulate steroidogenic function in stress.

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.

Hepatic protoporphyria is associated with a decrease in ligand binding for the mitochondrial benzodiazepine receptors in the liver.

Protoporphyrin IX (PP) and N-methylprotoporphyrin IX (N-MePP) added in vitro to liver membranes reduced dose-dependently the affinity of [3H]PK 11195 for the mitochondrial benzodiazepine receptors (MBRs), the latter being about 20 times more potent (Ki 4.5 and 0.25 microM). Preincubation of these two porphyrins with liver homogenates for 120 min at 4 degrees resulted in significant inhibition of [3H]PK 11195 binding even after repeated washings of the membranes due to the residual presence in the membranes of about 35 and 5% of PP and N-MePP, respectively. Thus, the hypothesis that an in vivo increase in the hepatic porphyrin content modifies the binding of the isoquinoline PK 11195 to the MBRs was investigated in an experimental model of protoporphyria. PP and N-MePP were allowed to accumulate in vivo through treatment with 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) (100 mg/kg i.p., once), and rats were killed 5 h after treatment when hepatic porphyrin accumulation was marked (10-fold increase), PP predominating. In the liver, treatment reduced the affinity (Kd) of [3H]PK 11195 for MBRs (from 3.56 to 15.37 nM, P < 0.01) and the maximum number of binding sites (Bmax) (55% decrease, P < 0.05); the affinity (Ki) of RO 5-4864 for [3H]PK 11195 binding sites was also reduced (from 23.9 to 72.99 nM, P < 0.05). No significant differences were found in the brain cortex. Liver and brain diazepam binding inhibitor levels and plasma corticosterone levels were unchanged. The reduction in [3H]PK 11195 binding to MBRs in the liver of DDC-treated rats thus appears to be attributable to a specific effect of the DDC-induced formation of the two protoporphyrins; this conclusion suggests that in hepatic protoporphyria processes modulated by MBRs may be altered.

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.

Comparative studies on the anorectic activity of d-fenfluramine in mice, rats, and guinea pigs.

The present study compares the anorectic activity of d-fenfluramine and its metabolite d-norfenfluramine in three animal species. d-Fenfluramine and d-norfenfluramine show anorectic activity at increasing doses (ED50) in rats, guinea pigs, and mice, d-norfenfluramine being more active than d-fenfluramine in all three species. Equiactive anorectic activities are reached with different brain levels of d-fenfluramine and d-norfenfluramine, guinea pigs being the most sensitive species, followed by rats then mice. The metabolite most probably plays a major role in the anorectic effect of d-fenfluramine in guinea pigs, contributes to the anorectic activity in rats, but adds little to the action of the parent drug in mice. The different sensitivity to d-fenfluramine and d-norfenfluramine in these three species does not appear to be explained by a number of biochemical parameters, including serotonin uptake or release, receptor subtypes, or 3H-d-fenfluramine binding and uptake.

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.