Alternative splicing of p53 and p73: the novel p53 splice variant p53delta is an independent prognostic marker in ovarian cancer.

Similar to p73, the tumor suppressor gene p53 is subject to alternative splicing. Besides p53DeltaE6 and p53beta, we identified p53zeta, p53delta and p53varepsilon, arising from alternative splicing of exon 6 and intron 9, respectively. p53 splice variants were present in 18 of 34 ovarian cancer cell lines (52.9%) and 134 of 245 primary ovarian cancers (54.7%). p53delta expression was associated with impaired response to primary platinum-based chemotherapy (P=0.032). Also, p53delta expression constituted an independent prognostic marker for recurrence-free and overall survival (hazard ratio 1.854, 95% confidence interval 1.121-3.065, P=0.016; and hazard ratio 1.937, 95% confidence interval 1.177-3.186, P=0.009, respectively). p53beta expression was associated with adverse clinicopathologic markers, that is, serous and poorly differentiated cancers (P=0.002 and P=0.008, respectively), and correlated with worse recurrence-free survival in patients exhibiting functionally active p53 (P=0.049). DeltaN'p73 constituted the main N-terminally truncated p73 isoform and was preferentially found in ovarian cancer cell lines showing functionally active p53, supporting our hypothesis that N-terminally truncated p73 isoforms can alleviate the selection pressure for p53 mutations by the inhibition of p53 protein function.

DNA methylation of nuclear receptor genes--possible role in malignancy.

The members of the nuclear receptor superfamily are known to mediate a wide array of basic biological processes, such as regulation of cell growth and differentiation, and induction of apoptosis. In several human malignancies, this central control function of nuclear receptors is disturbed, which seems to play an important role in tumor development and progression. Many nuclear receptor genes have been reported to be downregulated in malignancies; however, only a few mutations, gene arrangements, deletions or similar genetic changes have been shown to occur in these tumors. During the last decade, increasing attention has been directed towards epigenetic mechanisms of gene regulation such as DNA methylation. Many nuclear receptor genes can be silenced through aberrant methylation in tumors; epigenetic silencing, therefore, represents an additional mechanism that modifies expression of key genes during carcinogenesis. This review will give insights into the role of DNA methylation in the silencing of nuclear receptor genes and its involvement in human malignancies.

Hyperandrogenism, postprandial hyperinsulinism and the risk of PCOS in a cross sectional study of women with epilepsy treated with valproate.

Among a sample of 43 women with epilepsy treated for at least 2 years with valproate (n=22) or other antiepileptic drugs (AEDs) (n=21), polycystic ovary syndrome (PCOS) was diagnosed in three women, two of them were treated with valproate. Although the rate of PCOS and of menstrual disturbances, weight body mass index (BMI) and waist to hip ratio as well as fasting blood glucose levels, fasting insulin, proinsulin and C-peptide values was similar in this small sample of women treated with valproate and other AEDs, valproate exposure was associated with higher androgen levels, higher postprandial (pp) insulin and proinsulin levels, as well as lower cholesterol and low density lipoprotein (LDL) cholesterol levels. The pronounced increase in pp insulin levels during VPA treatment may indicate an effect of the fatty acid derivate VPA on pancreatic islet cells.

New insights into p53 regulation and gene therapy for cancer.

Due to its critical involvement in cell cycle control and apoptotic signaling, the transcription factor p53 has become the most important tumor suppressor currently under investigation. TP53 is the most frequently mutated gene in human cancers and is thought to play a crucial role in malignant transformation. Therefore, p53 appears to be an appealing target for gene therapy. Adenoviral-based p53 gene transfection is now being introduced in large clinical trials. Viral cell entry was found to be the rate-limiting step of gene delivery and thus of therapeutic efficiency. Attachment of adenoviruses to the target cell surface is mediated through the coxsackie-adenovirus receptor, and internalization is achieved via interactions with integrins of the alpha v beta(3) and alpha v beta(5) class. The assumption that the restitution of the p53-dependent apoptotic pathway results in a higher responsiveness of solid tumors to cytostatic agents remains a major matter of debate. Combinations of p53-based gene therapy with other components involved in apoptosis, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/APO2L, or agents neutralizing tumor-promoting antiapoptotic signals, such as humanized anti-growth factor antibodies, should further improve the effectiveness of cancer treatment in the future.

Methylation and silencing of the retinoic acid receptor-beta2 gene in breast cancer.

BACKGROUND: A growing body of evidence supports the hypotheses that the retinoic acid receptor beta2 (RAR-beta2) gene is a tumor suppressor gene and that the chemopreventive effects of retinoids are due to induction of RAR-beta2. RAR-beta2 expression is reduced in many malignant tumors, and we examined whether methylation of RAR-beta2 could be responsible for this silencing. METHODS: RAR-beta2 expression was studied by reverse transcription-polymerase chain reaction (RT-PCR) analysis in eight breast cancer cell lines that were either treated with the demethylating agent 5-aza-2'-deoxycytidine and subsequently with all-trans-retinoic acid (ATRA) or left untreated. Sodium bisulfite genomic sequencing was used to determine the locations of 5-methylcytosines in the RAR-beta2 genes of three of these cell lines. In 16 breast cancer biopsy specimens and non-neoplastic breast tissue, methylation-specific PCR was used to determine the methylation status of RAR-beta2, and, in 13 of the specimens, RT-PCR analysis was used to detect RAR-beta2 expression. RESULTS: Cell lines SK-BR-3, T-47D, ZR-75-1, and MCF7 exhibited expression of RAR-beta2 only after demethylation and treatment with ATRA. The first exon expressed in the RAR-beta2 transcript was methylated in cell lines ZR-75-1 and SK-BR-3. Six breast cancer specimens showed methylation in the same region of the gene. No expression of RAR-beta2 was found in any grade III lesion. An inverse association between methylation and gene expression was found in all grade II lesions. The RAR-beta2 gene from non-neoplastic breast tissue was unmethylated and expressed. CONCLUSIONS: Methylation of the RAR-beta2 gene may be an initial step in breast carcinogenesis; treatment of cancer patients with demethylating agents followed by retinoic acid may offer a new therapeutic modality.

Expression of retinoic acid receptors in non-neoplastic epithelial disorders of the vulva and normal vulvar skin.

Retinoids and their nuclear retinoic receptors (RARs) are important modulators of epidermal cell proliferation and terminal differentiation. Aberrant expression of RARs in the epidermis has been found to be associated with altered differentiation capacity of keratinocytes. In this study, the expression of the various types of RARs (RAR-alpha, RAR-beta, and RAR-gamma) was investigated in surgical specimens from 17 patients with vulvar lichen sclerosus, 12 patients with vulvar squamous cell hyperplasia, and 11 specimens of normal vulvar skin by nonradioactive in situ hybridization. The results demonstrate that RAR-alpha expression is significantly decreased in lichen sclerosus (p < 0.0001) and squamous cell hyperplasia (p = 0.007) compared with normal vulvar skin. Furthermore, in normal vulvar skin RAR-alpha mRNA is mainly located in the suprabasal epidermal cell layers, whereas in lichen sclerosus RAR-alpha is expressed predominantly in the basal cell layers. In squamous cell hyperplasia RAR-alpha expression occurs in all cell layers. Compared with normal vulvar skin, RAR-gamma expression is higher in lichen sclerosus (p = 0.026), but no statistically significant differences are seen in squamous cell hyperplasia. These results suggest that partial loss and abnormal localization of RAR-alpha expression as well as increased RAR-gamma expression may play a role in the etiology of non-neoplastic epithelial disorders of the vulva.

Predictive value of uPA, PAI-1, HER-2 and VEGF in the serum of ovarian cancer patients.

BACKGROUND: Tissue levels of uPA, PAI-1, HER-2 and VEGF are known to have prognostic value in different malignant tumors. The prognostic potential of serum concentrations of these markers is less clear and was investigated. PATIENTS AND METHODS: The response to 2nd line chemotherapy was studied in 61 patients with recurrent ovarian cancer. Marker analyses were performed using specific and sensitive ELISA tests and the response to therapy was evaluated using multiple CA 125 determinations and including these values in a simple and comprehensive formula. RESULTS: Correlations were observed between VEGF and CA 125, HER-2 (inversely) as well as PAI-1, and between uPA and PAI-1. However, no marker showed a significant relation to the overall survival of patients, nor to treatment response. CONCLUSIONS: Serum levels of uPA, PAI-1, HER-2 and VEGF do not have enough predictive potential in recurrent ovarian cancer. Most likely additional sources contribute to the serum levels of the markers studied so that their levels are not only tumor specific.

Clinical significance of serum and ascitic p53 autoantibodies in epithelial ovarian carcinoma.

BACKGROUND: Accumulation of mutated p53 in malignant cells can lead to the generation of anti-p53 autoantibodies in the serum and other body fluids of cancer patients. This retrospective study was performed to evaluate the prognostic significance of preoperative serum and ascitic anti-p53 antibodies in advanced ovarian carcinoma. METHODS: In 113 ovarian carcinoma patients who presented with significant amounts of ascites, anti-p53 autoantibodies were determined by a highly specific enzyme-linked immunosorbent assay of blood and ascites. Disease free and overall survival of study patients was estimated by the product limit method of Kaplan and Meier. Differences in survival were examined according to criteria of Mantel and Breslow. A multiple regression analysis based on the Cox proportional hazards model was used to determine the independence of prognostic variables. RESULTS: Serum and ascitic anti-p53 antibodies were found in 28 (25%) and 21 (19%) of the study patients, respectively. In univariate analysis, detection of anti-p53 antibodies in ascites but not in serum was found to be a sign of unfavorable disease free survival (P<0.003) and overall survival (P < 0.01). Multivariate analysis revealed that anti-p53 positivity in ascites retained independent significance only in the prediction of adverse progression free survival (P<0.01). CONCLUSIONS: The generation of a humoral immune response against p53 protein in the close tumor environment, as demonstrated by the occurrence of p53 autoantibodies in the ascitic fluid of ovarian carcinoma patients, is associated with poor disease free survival.

Studies on structure-activity relationships of retinoic acid receptor ligands by means of molecular modeling.

Vitamin A and its biologically active derivatives, retinal and retinoic acid, play an important role in vision, are required for reproduction, act as morphogenic agents during embryonic development, and regulate the growth and differentiation of a wide variety of cell types throughout the life of an organism. The biological action of retinoic acid and synthetic analogs, referred to as retinoids, is mediated by RAR alpha, RAR beta, or RAR gamma and/or by RXR alpha, RXR beta, or RXR gamma, all being nuclear receptors. Since retinoids exert profound effects on cell proliferation, differentiation, and apoptosis, these compounds seem to be promising agents for the treatment of cancer. Consequently, a large number of retinoids have been synthesized and examined to determine if they exert their biological activity according to retinoic acid receptor interaction. These screening methods are often expensive, time-consuming, and labor-intensive procedures. Since one can construct the pharmacophores of congeneric groups of drug molecules, molecular modeling techniques offer a new way to determine the binding abilities of different agents. We examined the structural properties of retinoids, which allow them to specifically bind to the different receptor subtypes. The thus-generated 3D pharmacophore models were used to predict the binding affinities of several retinoids to the retinoic receptor subtypes. Finally, the 3D models served as criteria for searching the Derwent World Drug Index for compounds that possess the features necessary for favorable ligand receptor interaction. The search resulted in a "hit list" containing 323 compounds, some of which are worth further investigation to determine if they act via retinoic acid receptor binding or not.

Peritoneal mesothelial cells as a significant source of ascitic immunostimulatory protein 90K.

The tumor associated antigen 90K is known to possess cytokine-like modulatory properties on the cellular immune system, whereby accessory cells are the primary target of this molecule. In 67 ovarian cancer patients presenting with significant amounts of ascites, immunostimulatory protein 90K was detected in all ascitic fluid samples examined. Furthermore, 90K levels correlated to ascitic s-IL-2R content. To elucidate the source of protein 90K in ascitic fluid; its in vitro release was investigated in primary cultured normal human peritoneal mesothelial cells (HPMC). Peritoneal mesothelium was found to produce five-fold more 90K than ovarian cancer cells. Release of protein 90K was significantly increased by treatment with IFN-gamma in both mesothelial and ovarian cancer cells. In contrast neither IL-1 beta nor TNF-alpha treatment consistently influenced the secretion of 90K in either cell type.

Quanititation of gene expression by means of HPLC analysis of RT-PCR products.

A method for the quantitative analysis of specific mRNA species by reverse transcription-polymerase chain reaction (RT-PCR) and subsequent detection of products by means of ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) on alkylated micropellicular polystyrene-divinylbenzene particles has been developed. For RT-PCR, we used the EZrTth RNA PCR kit (Perkin Elmer). This method allows reverse transcription and amplification of specific target mRNA in a single reaction tube. RT-PCR products were analyzed qualitatively and quantitatively by means of IP-RP-HPLC and UV detection at 254 nm. The enzymatic amplification combined with chromatographic separation and UV detection permitted high precision (and intra assay CV < 10%), with good practicability (two pipetting steps only). A total RNA preparation of a tissue that highly expressed the sequence of interest and that was stored in multiple aliquots, was diluted to give a standard curve. This external standard curve was used to define when samples have to be diluted, i.e., when the signal is in the plateau phase of amplification. The validity of the method is demonstrated with the example of human retinoic acid receptor mRNA.

Retinoic acid modulates prolactin receptor expression and prolactin-induced STAT-5 activation in breast cancer cells in vitro.

Two recent papers demonstrate that prolactin plays an important role in the induction and progression of mammary tumours. Retinoids have been shown to be potent inhibitors of breast carcinogenesis. We studied expression of prolactin receptor mRNA in human breast cancer cell lines MCF-7, SKBR-3, T47D and BT-20 treated with and without retinoids using Northern blot and a quantitative polymerase chain reaction (PCR) method. In all cell lines, all-trans- and 9-cis-retinoic acid, as well as the retinoic acid receptor gamma (RAR-gamma) selective agonists CD2325 and CD437 (1 microM), were able to down-regulate prolactin receptor. After 1 h, a significant reduction was detectable and maximal effect was achieved after 24 h of treatment. Pretreatment with retinoic acid also reduced the prolactin-/prolactin receptor-dependent signal transduction and activation of transcription 5 (STAT-5) activation in T47D cells. Cycloheximide failed to abrogate the retinoic acid-induced decline in prolactin receptor mRNA levels, indicating that this effect was not dependent upon continuing protein synthesis. Similarly, no change in the stability of prolactin receptor mRNA was observed during 12 h of retinoic acid treatment. In conclusion, our results demonstrate that retinoids are able to inhibit the expression of prolactin receptor message, which encodes an important growth factor receptor in breast cancer cells. This action could be responsible for the anti-tumour effects of retinoids.

Peritoneum and tissues of the female reproductive tract as physiological sources of CA-125.

The origin of physiological CA-125 serum levels, which in normally menstruating women were shown to depend on their actual menstrual cycle phase, has not yet been completely elucidated. It is furthermore conceivable that physiological CA-125 sources may contribute to serum elevations in the various pathologies associated with increased circulating CA-125. The present review deals with menstrual cycle-dependent expression of CA-125 in normal tissues of the female reproductive tract in relation to the actual circulating CA-125 levels together with in vivo data concerning the inductive effect of medroxyprogesterone acetate on circulating CA-125 studied in 24 postmenopausal women. Furthermore, in vitro results on constitutive, steroid hormone- and cytokine-modulated CA-125 shedding from human peritoneal mesothelial and ovarian surface epithelial cells are summarized.

Childbirth as a biological model for stress? Associations with endocrine and obstetric factors.

OBJECTIVE: The aims of this investigation were to measure corticotropin-releasing hormone (CRH), corticotropin (ACTH) and cortisol before, during and after delivery searching for an endocrine intercorrelation of the hypothalamic-pituitary-adrenal (HPA) axis and to correlate these findings with obstetrical variables. METHODS: Blood was sampled from 50 women with singleton pregnancies at term without uterine contractions, during delivery (after full cervical dilatation) and on the 4th postnatal day. Hormones were measured by radioimmunoassay (RIA). The correlation between obstetric variables, sociodemographic and endocrine data were evaluated using the Spearman rank coefficient. Group comparisons for continuous variables were calculated using the Mann-Whitney U test and Kruskal-Wallis test. RESULTS: Maternal plasma ACTH and cortisol increased significantly during labor, declining toward the 4th postnatal day (p < 0.001) and showing a significant intercorrelation (p < 0.01). Compared to women without uterine contractions CRH rose during labor (p < 0.05) and decreased rapidly to the 4th postnatal day (p < 0.001). No correlations between CRH and ACTH or cortisol were observed. None of the obstetrical variables (parity, newborn's weight, duration of delivery) revealed any significant correlation with ACTH. Analgetic medication (pethidine hydrochloride) was not able to influence the endocrine response to labor stress. CONCLUSIONS: Stressful experience during childbirth has an impact on endocrine response. However, this is not fully evident along the HPA axis in a simple biological model with monocausal dependencies. This 'biological stress model' is not sensitive enough to detect different childbirth conditions and the hormones in the maternal compartment have partially fetal (placental) origin.

Ascitic interleukin-12 is an independent prognostic factor in ovarian cancer.

PURPOSE: The clinical impact of endogenous cytokines supplied with deterministic properties in the generation of either T helper (Th)1 -type or Th2-type immune response was investigated in patients with ovarian cancer. Whereas interleukin (IL)- 12 initiates the differentiation of naive Th0 cells toward Th1 phenotype, IL-4 and IL-10 mediate the development of Th2-type immunity. PATIENTS AND METHODS: Cytokines were determined before treatment by means of enzyme-linked immunosorbent assay (ELISA) in ascites fluid and serum of 76 patients with ovarian cancer. Cytokine levels were compared with each other and with standard clinicopathologic parameters. A stepwise logistic regression was calculated to rule out interdependence in the associations of the various variables. Survival analyses were performed with the Kaplan-Meier method and differences in survival were examined according to Mantel and Breslow. Cox proportional hazards analysis was used to identify independent prognostic factors. RESULTS: Whereas IL-10 and IL-12 were detectable in all ascites-fluid samples, IL-4 was measurable in only 43% of the specimens. With the exception of neopterin, macrophage colony-stimulating factor (M-CSF), and IL-4, determined cytokine levels were significantly elevated in ascites fluid compared with serum (P < .01). In univariate analyses, high ascitic-fluid concentrations of either neopterin, tumor necrosis factor-alpha (TNF-alpha), or IL-12 were associated with poor disease-free (P < .005) and overall (P < .01) survival. Multivariate Cox regression analysis showed ascitic-fluid IL-12 levels to be the only immunologic variable that retained independent prognostic significance (P < .03 for disease-free and P < .01 for overall survival), together with residual disease, Fédération Internationale de Gynécologie et d'Obstétrique (FIGO)-stage, and patient age. CONCLUSION: In ovarian cancer, high ascitic-fluid IL-12 levels, which may indicate a local Th1-generated immune response, are associated with disease progression.

Regulation of CA 125 expression in cultured human carcinoma cells.

CA 125 shedding is not a constitutive and stable process but may be affected by cell cycle and cell proliferation as well as by various growth factors and cytokines. Interferons, interleukin-1 beta, tumor necrosis factor-alpha and transforming growth factor-alpha have been shown to induce while glucocorticoids and transforming growth factor-beta have been shown to suppress the release of the tumor marker CA 125 from ovarian carcinoma cells. Several endogenous as well as exogenous factors may affect CA 125 biosynthesis; however, a major question remains whether this observed modulation of CA 125 expression in vitro is of clinical importance.

CD44 splice variant expression in normal and malignant uterine cervical epithelium.

CD44 expression was investigated immunohistochemically on paraffin sections obtained from 88 uterine cervical cancers and 31 normal cervices, using monoclonal antibodies against CD44 variant epitopes v4, v5, v6, v7/8, v9 and the standard form of the CD44 protein. Normal epithelium showed expression of all CD44 splice variants, at least in traces, and it was located predominantly in basal and parabasal cells. In cervical carcinomas CD44 expression was widely heterogeneous. CD44 variant v9 staining was moderate or strong in 86% of the tumors, and it was significantly correlated with CD44 v6 staining. Also a significant correlation between expression of CD44 v4 and v6 occurred. Highly significant correlations between CD44 expression of variants v4 and v6 and tumor stage as well as patients age were found. In addition, these variants were more frequently expressed in squamous cell carcinomas than in adenocarcinomas. However, in contrast to the recently reported data, we were not able to confirm the hypothesis that CD44 v6 represents a prognostic indicator in cervical cancer. In FIGO stages III and IV, patients with CD44 variant v4 positive tumors had a significantly longer disease-free and overall survival than patients with CD44 variant v4 negative tumors. In conclusion, our data indicate that CD44 v6 tissue expression cannot be considered as a prognostic factor in cervical cancer. Regarding the unexpected outcome of patients with CD44 v4 positive tumors, further investigations are needed to elucidate the exact clinical value of this variant isoform.

Loss of retinoic acid receptor beta expression in breast cancer and morphologically normal adjacent tissue but not in the normal breast tissue distant from the cancer.

Retinoids and their receptors [retinoic acid receptors (RARs) and retinoid X receptors] play an important role in maintaining the balance between proliferation and apoptosis. Recently, Deng et al. [Science (Washington DC), 274: 2057-2059, 1996] reported a loss of heterozygosity on chromosome 3p24 in breast cancer specimens and the morphologically normal appearing adjacent tissue. The 3p24 locus includes, among other genes, the region coding for RAR-beta. This study was designed to determine whether there are abnormalities in the expression of retinoid receptors in surgical specimens of patients with breast cancer. In 14 patients, transcripts of nuclear retinoid receptors were detected by in situ hybridization in formalin-fixed, paraffin-embedded specimens by means of digoxigenin-labeled riboprobes specific for RAR-alpha, -beta and -gamma. We found RAR-alpha expressed in all specimens, whereas RAR-gamma was expressed in 100% of normal breast tissue but in only 11 of 14 tumorous lesions. RAR-beta was found in all cases of normal breast tissue localized distant from the tumor, but in 13 of 14 cases it was completely absent in the tumor and the morphologically normal appearing tissue adjacent to the tumor. One possibility to explain the suppression of RAR-beta is a mutation in the promoter region. Sequencing the DNA extracted from paraffin-embedded tumor tissue of the corresponding breast cancer specimens, we were not able to detect any mutation in the retinoic acid-responsive element. Our results clearly indicate a crucial role of RAR-beta in the carcinogenesis of breast cancer.

Paclitaxel- and docetaxel-dependent activation of CA-125 expression in human ovarian carcinoma cells.

Taxanes represent a new class of antineoplastic agents that are being evaluated in several malignant tumors; they have been shown to induce a high remission rate and to prolong survival in ovarian cancer patients. However, CA-125 has been suggested to be an unreliable marker for monitoring response to paclitaxel therapy. Therefore, we were interested in whether taxanes may directly modulate CA-125 expression. Human ovarian carcinoma cell lines OVCAR-3, HOC-7, SKOV-6, 2780, 2774, and HTB-77 were treated with paclitaxel or docetaxel. Secreted, surface-associated, and cytosolic CA-125 were estimated by means of a sandwich solid-phase RIA or by immuno-flow cytometry. In addition to in vitro antiproliferative activity, paclitaxel and docetaxel augmented the expression of the tumor marker CA-125 in the three ovarian carcinoma cell lines, OVCAR-3, HOC-7, and SKOV-6, constitutively expressing this tumor marker. The three CA-125-negative cell lines, 2780, 2774, and HTB-77, did not respond to taxane treatment by expressing this tumor marker, although their proliferation was markedly inhibited. The taxane-mediated induction of CA-125 was found to be dependent on intact protein and RNA biosynthesis. However, CA-125 concentration was increased in the supernatant medium only and not on cell surface or cytosol. Our results demonstrate an in vitro activation of ovarian carcinoma cells in terms of CA-125 secretion by taxanes. This may explain the CA-125 fluctuations observed in vivo under paclitaxel treatment and may indicate that CA-125 is not a reliable tumor marker during taxane chemotherapy.