Time-dependent supplementation of vitamin E influences leptin expression in the aortic layers of rats fed atherogenic diet.

An excessive consumption of a diet rich in saturated fatty acids is a key factor in pathogenesis of cardiovascular diseases which are strictly connected with leptin imbalance in the vessels. However, whether vitamin E supplementation would influence leptin expression in aortic layers is still unknown. For 3 or 6 weeks male Wistar rats were fed a high-fat (20% w/w) diet with lard as dietary fat source with or without vitamin E supplementation (50 mg/100 g of diet). The 6-week intake of an atherogenic diet increased total cholesterol (TC) and high density lipoprotein cholesterol (HDL) plasma levels simultaneously lowering TC/HDL ratio (ANOVA p≤0.0001 for all three parameters). After longer period of feeding it was stated that leptin expression in all three aortic layers was enhanced (ANOVA p≤0.0001 for endothelium, tunica media and adventitia, respectively) with decreased leptin plasma concentration (ANOVA p≤0.0001). After both periods of feeding vitamin E supplementation caused an increase in plasma HDL content and a decrease of TC/HDL ratio. In the 3-week experiment vitamin E addition caused a decrease in leptin plasma levels (Fisher's test, 3L versus 3LE: p≤0.002) and an increase in leptin expression in all three aortic layers (Fisher's test, 3L versus 3LE p≤0.005, p≤0.01 and p≤0.05 respectively for endothelium, tunica media and adventitia). The contradictory results were observed in the 6-week experiment in which vitamin supplementation decreased leptin expression in the aortic endothelium (Fisher's test, 6L versus 6LE: p≤0.001) with lack of changes in the other two layers of the aorta and plasma. The study showed that vitamin E supplementation influenced leptin expression in aortic layers in rats fed atherogenic diet differently depending on the length of feeding period. It may suggest that vitamin E consumption plays an important role in the control of leptin status in the endothelium.

Ovarian small cell carcinoma of hypercalcemic type - evidence of germline origin and SMARCA4 gene inactivation. a pilot study.

Ovarian tumors from two patients, compatible by histological and immunohistochemical criteria with small cell carcinoma of hypercalcemic type (SCCHT) (WT1+, EMA dispersed+, synaptophysin+ or dispersed+), were extensively sampled in order to find clues to their histogenesis. Subsequently, small foci of immature teratoma were found in both of them (in 1/122 and in 3/80 tumor sections). In one case, microfoci of yolk sac tumor were also present within the teratoma area as well as in the background of the small cell tumor population - in the primary tumor and in omental metastasis. We found a resemblance of the microscopic patterns of SCCHT and atypical teratoid/rhabdoid tumor (AT/RT) of the central nervous system, and this prompted us to evaluate INI-1 and SMARCA4 immunohistochemical expression, because their alternative loss is regarded as a molecular hallmark of AT/RT. INI-1 expression was retained, while that of SMARCA4 was lost. We therefore analyzed tumor DNA by PCR amplification and sequencing for mutations in the SMARCA4 gene (NG_011556.1), which were identified in both tumors (c.2184_2206del; nonsense c.3277C>T - both in one tumor; nonsense c.3760G>T in another tumor). These data suggest that SCCHT is most likely an embryonal tumor originating from immature teratoma and related to malignant rhabdoid tumor. Further analyses are necessary to determine whether the tumors diagnosed as SCCHT constitute a homogeneous group or represent more than one entity.

The SNARE-associated component SNAPIN binds PUMILIO2 and NANOS1 proteins in human male germ cells.

It has been reported that a highly conserved human protein PUMILIO2 forms a complex with NANOS1 in human male germ cells, as does the Drosophila ancestor Pumilio, which binds Nanos to regulate translation of specific mRNAs. Here, we found that PUMILIO2 interacts also with SNAPIN, a modulator of SNARE complex assembly, which is involved in vesicle trafficking. We demonstrated that SNAPIN interacts additionally with NANOS1 protein. This is the first report demonstrating that the N-terminal region of NANOS1 is necessary for protein binding. In human testis, SNAPIN co-localizes with PUMILIO2 and NANOS1 in prenatal and also in spermatogenic germ cells of the adult. We describe for the first time the expression of SNAPIN in germ cells which raises possibility that SNAPIN plays an extra role in mammals which is germ cell specific. The presence of a coiled-coil domain responsible for protein-protein interaction could enable SNAPIN to be an adaptor of PUMILIO2 and NANOS1, binding other factors to regulate translation in the development of the human germ cells.

PTEN mutation, expression and LOH at its locus in ovarian carcinomas. Relation to TP53, K-RAS and BRCA1 mutations.

OBJECTIVE: We aimed to evaluate frequency of PTEN mutation, LOH and expression in ovarian tumors. In search for a molecular pathway, we confronted PTEN gene mutations with TP53, K-RAS and BRCA1 gene status in the same tumors. We also evaluated clinical significance of PTEN expression in a subgroup of patients uniformly treated with platinum-based regimens. METHODS: Molecular analysis was performed on 105 ovarian tumors (100 carcinomas) with the use of the SSCP and sequencing. Seventy-six tumors were analyzed for LOH at 10q23 locus with the use of six polymorphic markers. Immunohistochemical PTEN expression was done on paraffin-embedded material. Multivariate and univariate analysis was performed with the STATA program. RESULTS: PTEN mutations occurred in 5/100 (5%) of all carcinomas and in 3/15 (20%) of endometrioid carcinomas (EC). Low-grade EC that developed in borderline tumors had PTEN and/or K-RAS mutation (4/5, 80%), while high-grade EC had TP53 mutations only. There was a reverse association between PTEN and TP53 mutations (P = 0.005). LOH at PTEN locus was found in 60% of endometrioid and in 28% of serous and clear cell carcinomas. PTEN expression did not associate with PTEN mutations or LOH. Strong PTEN expression diminished risk of death in a TP53 positive group only (HR = 0.35, P = 0.029). CONCLUSION: Our results suggest that PTEN mutations may play a role in a development of low-grade endometrioid tumors. PTEN haploinsufficiency caused by LOH or epigenetic events may possibly contribute to development of other histological types and may be an adverse prognostic factor.

P21WAF1, P27KIP1, TP53 and C-MYC analysis in 204 ovarian carcinomas treated with platinum-based regimens.

BACKGROUND: The prognostic and predictive value of cell cycle regulatory proteins in ovarian cancer has not been established. We evaluated the clinical and biological significance of P21(WAF1), P27(KIP1), C-MYC, TP53 and Ki67 expressions in ovarian cancer patients. MATERIALS AND METHODS: Immunohistochemical analysis was performed on 204 ovarian carcinomas of International Federation of Gynecology and Obstetrics (FIGO) stage IIB to IV treated with platinum-based chemotherapy. Multivariate analysis with Cox and logistic regression models was performed in the whole group, and in the TP53-negative and TP53-positive subgroups. RESULTS: High P21(WAF1) labeling index (LI) was an independent positive predictor of platinum-sensitive response (P = 0.02). Overall survival was positively influenced by P21(WAF1) LI (P = 0.02) or by P21(WAF1) plus P27(KIP1) LI (P = 0.004) in the TP53-negative group only. Ki67 LI showed borderline association with disease-free survival (P = 0.05). Growth fraction was negatively associated with P21(WAF1) and P27(KIP1) indices in the TP53-negative group (P = 0.023 and 0.008, respectively), and these associations were borderline or lost in the TP53-positive group. Endometrioid and clear cell carcinomas differed from other carcinomas by having a low incidence of TP53 accumulation, a high incidence of C-MYC overexpression (70%) and a low median Ki67 LI (all with P <0.001). CONCLUSIONS: We have shown an independent predictive value of P21(WAF1) LI in ovarian carcinoma patients. The prognostic value of P21(WAF1) and P21(WAF1) plus P27(KIP1) LI was determined by TP53 status. A high frequency of C-MYC overexpression in endometrioid and clear cell carcinomas may suggest its role in the development of these tumor types.

Evaluation of clinical significance of TP53, BCL-2, BAX and MEK1 expression in 229 ovarian carcinomas treated with platinum-based regimen.

In cell line studies, BCL-2, BAX, as well as novel MEK1 protein levels have strong influence on ovarian cancer response to cisplatin-based chemotherapy. However, such associations have not been demonstrated clinically. We evaluated prognostic/predictive significance of these proteins with regard to TP53 status. Immunohistochemical analysis was performed on 229 ovarian carcinomas FIGO stage IIB-IV treated with platinum-based chemotherapy; the results were analysed by the Cox and logistic regression models. Clinical parameters (residual tumour size, patient age, FIGO stage) were the only indicators of overall survival (OS) and the strongest predictors of complete remission (CR). On the other hand, BAX expression was the strongest (P=0.005) or the only (in FIGO IIIC, P=0.02) prognostic indicator of disease-free survival (DFS) in the TP53(+) group. TP53(+) and TP53(-) ovarian carcinomas differed in clinical and molecular prognostic and predictive factors. Another novel finding is that CR was negatively influenced by high BAX expression in all patients group (P=0.047) and by BCL2 expression in the TP53(-) group (P=0.05). High MEK1 expression was associated with endometrioid and clear cell carcinomas (P=0.049); its loss was found with advancing FIGO stage (P=0.002). Our results suggest that binomial TP53 status divides ovarian carcinomas into two biologically distinct groups. BAX expression is an important factor of DFS in the TP53(+) group. BCL-2 and BAX, but not MEK1 expressions have predictive value in ovarian cancer patients treated with platinum-based chemotherapy.

Nijmegen breakage syndrome gene (NBS1) alterations and its protein (nibrin) expression in human ovarian tumours.

We looked for NBS1 gene (602667) alterations and changes in nibrin expression in 162 human gynaecological tumours, mostly ovarian. Exons 6-8 and 10 of the NBS1 gene were evaluated by the SSCP and direct sequencing method. Nibrin expression was detected immunohistochemically with the use of the p95NBS1 (Ab-1) antibody. The 657del5 mutation (Slavic mutation) was found in two of 117 carcinomas studied (1.7%) - in both cases it was present in the germline; one of these tumours showed loss of heterozygosity (LOH) for the 657del5 mutation and loss of nibrin expression. We have found three types of novel germline intron variants: (1) two concomitant transitions (G to A) at bases 14009 and 14256; (2) C to T transition at base 13998; (3) G to C transversion at base 20035. Among the carcinomas studied, the intron variants were associated with a clear cell histological type (p = 0.004). Our results may suggest that NBS1 gene alterations contribute to the development of rare ovarian carcinomas. LOH for 657del5 in tumour tissue may support the hypothesis that the NBS1 gene functions as a tumour suppressor.

Spontaneous apoptosis in ovarian carcinomas: a positive association with p53 gene mutation is dependent on growth fraction.

Changes in cell survival contribute to tumour development, influence tumour biology and its response to chemotherapy. p53 gene alterations should negatively affect apoptosis by impaired p53-dependent apoptotic response. We looked for associations between spontaneous apoptosis, p53 gene mutation, p53 protein accumulation, growth fraction, bcl-2 expression and histological parameters in 64 ovarian, four tubal and three peritoneal carcinomas. Apoptotic cells were detected with the TUNEL method. p53 gene variants were detected by the single-strand conformation polymorphism and were sequenced directly. P53, Ki-67 and bcl-2 protein expressions were detected immunohistochemically. A weighed multiple logistic regression model was applied. Apoptotic index (AI) ranged 0.02-0.18 (mean 0.11); proliferation index (PI) ranged 3-90% (mean 54%). p53 gene mutations were present in 51, p53 protein accumulation in 46, and diffuse bcl-2 expression in 29 of 71 tumours. The AI was positively associated with the presence of p53 gene mutation (P = 0.011). However, the PI included into the analysis did positively influence the AI (P = 0.02) and diminished the association with p53 gene mutation (P = 0.082). The AI was negatively associated with good histological differentiation (P = 0.0006), the serous tumour type (P = 0.002), and diffuse bcl-2 expression (P = 0.025). Strong bcl-2 expression was associated with endometrioid tumour type (P = 0.002). FIGO stage and p53 protein accumulation were the only parameters that influenced overall survival time. Thus, our results suggest that histological tumour type and grade are major determinants of spontaneous apoptosis in ovarian carcinomas; p53 alterations do not adversely but rather positively affect spontaneous apoptosis by increasing growth fraction. This, in turn, suggests p53-independency of spontaneous apoptosis in ovarian carcinomas.

Steroidogenic activity in surface epithelium of the human ovary.

The steroidogenic activity of the ovarian surface epithelium in various steps of biosynthesis of the sex steroids was investigated on 105 specimen of the ovaries obtained during laparotomy in the Department of Obstetrics and Gynecology of Postgraduate Medical School. The slices of the ovary were incubated according to the method elaborated by Levy et al. The studies were performed during the follicular and luteal phase of the menstrual phase. For the determination of the activity of delta 5-3-beta-hydroxysteroid dehydrogenase pregnenolone and dehydroepiandrosterone were used. For the determination of 17 beta-hydroxysteroid dehydrogenase androstenedione and testosterone were applied. The results obtained indicate, that the cells of the human surface epithelium have the enzyme system necessary for the different stages of steroidogenesis. However the results obtained do not prove that steroidogenic precursors are synthesized de novo in cells of the surface epithelium.

Influence of embryonic and adult testis on the differentiation of embryonic ovary in the mouse.

The development of 11 1/2-13 1/2-day embryonic mouse ovaries subjected to the influence of adult and embryonic testes was investigated. The environment of adult testis caused severe restriction of ovarian growth, but did not produce any effects which might be considered as masculinization. The presence of embryonic testis was distinctly unfavourable to the embryonic ovary, resulting in restriction of the growth of the latter and degeneration of oocytes. Reversal of the course of differentiation of genetically female germ cells has never been observed.