Cytochrome P2A13 and P1A1 gene polymorphisms are associated with the occurrence of uterine leiomyoma.

PROBLEM: To investigate the association between the occurrence of uterine leiomyoma and two SNPs of the CYP 2A13 and CYP 1A1 genes. METHOD OF STUDY: Prospective case control study with 132 women with clinically and surgically diagnosed uterine leiomyoma and 260 controls. Genotyping was performed by polymerase chain reaction (PCR) based amplification of CYP 2A13 and CYP 1A1 genes, and restriction fragment length polymorphism (RFLP) analysis. RESULTS: Comparing women with uterine leiomyoma and controls, we demonstrate statistical significant differences of allele frequency and genotype distribution for the CYP 1A1 polymorphism (P = 0.025 and P = 0.046, respectively). Furthermore, for the CYP 2A13 polymorphism we found a significant difference concerning allele frequency (P = 0.033). However, for the genotype distribution, only borderline significance was observed (P = 0.064). CONCLUSIONS: The CYP 2A13 and CYP 1A1 SNPs are associated with uterine leiomyoma in a Caucasian population and may contribute to the understanding of the pathogenic mechanisms of uterine leiomyoma.

hCG-dependent regulation of angiogenic factors in human granulosa lutein cells.

As prerequisite for development and maintenance of many diseases angiogenesis is of particular interest in medicine. Pathologic angiogenesis takes place in chronic arthritis, collagen diseases, arteriosclerosis, retinopathy associated with diabetes, and particularly in cancers. However, angiogenesis as a physiological process regularly occurs in the ovary. After ovulation the corpus luteum is formed by rapid vascularization of initially avascular granulosa lutein cell tissue. This process is regulated by gonadotropic hormones. In order to gain further insights in the regulatory mechanisms of angiogenesis in the ovary, we investigated these mechanisms in cell culture of human granulosa lutein cells. In particular, we determined the expression and production of several angiogenic factors including tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), Leptin, connective tissue growth factor (CTGF), meningioma-associated complimentary DNA (Mac25), basic fibroblast growth factor (bFGF), and Midkine. In addition, we showed that human chorionic gonadotropin (hCG) has distinct effects on their expression and production. hCG enhances the expression and production of TIMP-1, whereas it downregulates the expression of CTGF and Mac25. Furthermore it decreases the expression of Leptin. Our results provide evidence that hCG determines growth and development of the corpus luteum by mediating angiogenic pathways in human granulosa lutein cells. Hence we describe a further approach to understand the regulation of angiogenesis in the ovary.

Granulocyte colony-stimulating factor (G-CSF) receptor gene expression of ovarian carcinoma does not correlate with G-CSF caused cell proliferation.

BACKGROUND: Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a growth factor commonly used to avoid leukopenia after chemotherapy. Endogenous G-CSF is produced by macrophages and granulocytes that infiltrate tumors. It has been reported that rhG-CSF stimulates the proliferation of several cell lines as well as bladder carcinoma cells. Conversely, in some hematopoietic cell lines such as U-937, WEHI-3B, and K-562 no effect or in some cases a differentiation pattern was found. Moreover, the role of rhG-CSF on the proliferation of solid tumors is not well understood. METHODS: In this study, 10 ovarian carcinoma biopsies were characterized for the presence of G-CSF and G-CSF receptor by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analysis. Proliferation was analyzed by ATP viability assays. RESULTS: Performing RT-PCR, these biopsies and four ovarian carcinoma cell lines were analyzed for endogenous G-CSF production, which was found in some biopsies and in all cell lines. Despite the presence of the G-CSF receptor in all biopsies and cell lines, no proliferation was found after rhG-CSF incubation of the cell lines or the tumor samples for 3 and for 6 days, respectively. CONCLUSIONS: Summarizing the authors' in vitro studies, rhG-CSF does not affect the proliferation of ovarian carcinoma cells in vitro.

Evaluation of methods to detect p53 mutations in ovarian cancer.

OBJECTIVE: The p53 status is increasingly regarded as a marker predictive of response to particular cancer therapies, but for this approach it is self-evident that the p53 status must be determined correctly. METHODS: We have tested ovarian cancers with single-strand conformation polymorphism analysis (SSCP), immunohistochemical staining with DO-1 anti-p53 antibody (IHC), and yeast p53 functional assay (FASAY). RESULTS: These techniques commonly used to detect p53 mutations showed important differences in their sensitivity. Of 53 tumors tested with three indirect techniques, 27 (50%), 33 (62%) and 41 (77%) were positive by SSCP, IHC, and FASAY, respectively. In a subset of 32 tumors strongly suspected of containing mutations, 25 (78%), 26 (81%), 29 (91%) and 30 (94%) were positive by SSCP, immunostaining, DNA sequencing and yeast assay, respectively. CONCLUSIONS: Under comparable routine conditions, the FASAY reached the highest sensitivity. Since no single technique detected all mutations, we recommend the use of at least two different techniques in situations where the p53 status will affect patient management.

G-CSF receptor expression in ovarian cancer.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is clinically used to overcome neutropenic periods during chemotherapy. In vitro studies using cell lines as a model system have recently suggested that G-CSF can promote ovarian cancer growth. The objective of this work is to determine whether tumor cells express G-CSF-receptors (G-CSFR). A set of ovarian tumor biopsies and ovarian cancer cell lines was analyzed by RT-PCR, immunohistochemistry and immunofluorescence. The presence of a 276 bp-amplicon (exon 8-10) obtained by RT-PCR showed that 12 out of 16 ovarian tumor biopsies and two out of four ovarian cancer cell lines expressed G-CSFR-mRNA. G-CSFR-protein was detected in tumor cells of the 12 biopsies that also contained G-CSFR-mRNA. A second 409 bp-amplicon (exon 17) obtained by RT-PCR from the variable C-terminal cytoplasmic region of G-CSFR could be amplified only in four out of 16 biopsies and in none of the ovarian cancer cell lines studied. The results presented here indicate that G-CSFR is frequently expressed in ovarian cancer cells. Moreover, the failure of RT-PCR amplification of the 409 bp-amplicon in samples that express G-CSFR-mRNA suggests that C-terminal truncated receptor forms are also expressed.

[An immunohistochemical study of the steroid hormone receptors in endometrial hyperplasia]. / Studiul imunohistochimic al receptorilor hormonilor steroizi în hiperplaziile endometriale.

AIM: Evaluation of the estrogen and progesterone receptors (ER and PR) in different degrees of endometrial hyperplasias. METHODS: 10 cases of each degree of endometrial hyperplasia (simple, complex and atypical) were analyzed using the avidin-biotin-peroxidase technique and monoclonal antibodies applied to formalin-fixed, paraffin-embedded tissue. RESULTS: We found similar, high level of both ER and PR in simple and complex hyperplasias and a significant decrease of these in atypical hyperplasia. CONCLUSIONS: Endometrial hyperplasias develop in a setting of estrogen excess. This explains the high level of endometrial receptors and the response to progesterone in typical hyperplasia. In atypical hyperplasia, the decrease of steroid receptors results in a low sensibility of this lesion to progestative therapy, but there are cases with high receptor levels which could have a chance for hormonal therapy.

[p53 and steroid receptors support double endometrial carcinogenesis]. / p53 si receptorii steroizi sustin o dubla carcinogeneza endometriala.

AIM: This study investigated the p53 positivity correlated with the receptor status, in different grades and stages of endometrial carcinoma. METHODS: The study included 88 endometrioid-type carcinomas and 5 non-endometrioid-type carcinomas. Paraffin sections were used for the ordinary histological diagnosis and for immunohistochemical diagnosis by avidin-biotin-peroxidase technique. RESULTS: p53 positivity was identified in 10 endometrioid-type carcinomas (11.4%) and 3 non-endometrioid type carcinomas (60%). Most of the p53 positive cases were receptor negative and observed in advanced stages and histological grades. No associated endometrial hyperplasia was p53 positive, while a positive intraepithelial non-endometrioid carcinoma was identified. CONCLUSIONS: p53 positivity is most frequently identified in non-endometrioid type, receptor negative adenocarcinomas, even in a precursor lesion as it is intraepithelial carcinoma, while in endometrioid, receptor positive carcinomas it appear in late stages of development, never being identified in precursor lesions.

Expression of interleukin-6 and interleukin-6 receptors in human granulosa lutein cells.

Cytokines are important regulators of reproductive functions. Significant amounts of interleukin-6 (IL-6) have been detected in the serum and ascites of patients with ovarian hyperstimulation syndrome (OHSS). These findings suggest the involvement of IL-6 as a mediator in the pathogenesis of OHSS. This study was performed to analyse IL-6 and IL-6 receptor (IL-6-R) expression in human granulosa lutein cells (GC). GC were cultured after isolation from follicular fluid. IL-6 concentrations in follicular fluid and serum from individual patients and GC supernatants were measured by enzyme-linked immunosorbent assay. We found detectable concentrations of IL-6 in serum and follicular fluid of all patients. Expression of IL-6 in GC was shown immunocytochemically. IL-6 mRNA was detected in GC by in-situ hybridization. Gene expression for IL-6 and IL-6-R in GC was demonstrated using reverse transcription-polymerase chain reaction. IL-6 significantly inhibited human chorionic gonadotrophin (HCG)-induced progesterone secretion of GC. The results of our study suggest that IL-6 is expressed in HGC and that this cytokine is able to modulate GC function via its specific receptor. This is the first report that describes the precence of IL-6-R in human granulosa lutein cells.

Immunohistochemical localization of thromboxane synthase in human intrauterine tissues.

Uterine tissues are known to be able to synthesize thromboxane A2 (TXA2), but there is little information about the nature of cells actually responsible for its production. In this study human placenta, fetal membranes, umbilical cord and pregnant myometrium were investigated immunohistochemically. The avidin-biotin method for a monoclonal antibody against human thromboxane synthase (Tü 300) was applied on frozen tissue sections. In placenta, fetal membranes and umbilical cord, staining was positive for Hofbauer cells and fibroblasts. Further, in sections of placenta, capillary endothelium showed antigenicity for TX synthase. Leiomyocytes in the umbilical cord vessels contained the enzyme as well. Preparations of pregnant myometrium were shown to express TX synthase in leiomyocytes, endothelial cells and connective tissue cells. Amnion, trophoblast and decidua did not possess antigenicity for this enzyme. Since TXA2 plays an important role for the regulation of vascular tone and aggregation of platelets and may stimulate myometrial contractions during parturition, the abundance of TX synthase in pregnancy-specific tissues confirms previous in vivo and in vitro observations. Further, TXA2 synthesized by Hofbauer cells may be involved in immunological reactions during pregnancy, and the number and level of activation of Hofbauer cells may be closely related to the initiation of labour. Thromboxane production by the endothelium lining the fetal vessels points to its regulatory role for the blood flow in the fetoplacental unit.