Role of GnRH-GnRH receptor signaling at the maternal-fetal interface.

OBJECTIVE: To investigate the expression and function of GnRH and GnRH receptor (GnRHR) subtypes at the maternal-fetal interface. DESIGN: In vitro experiments using freshly isolated human trophoblast cells, decidual stromal cells (DSCs), and immortalized cell lines. SETTING: University teaching hospital. PATIENT(S): Placenta-fetal membranes from term deliveries. INTERVENTION(S): Human trophoblast and DSCs were isolated, purified, and cultured. MAIN OUTCOME MEASURE(S): Expression of GnRH-I, GnRH-II, and GnRHR-I mRNA and protein in human trophoblast cell lines and tissues were evaluated by reverse-transcription polymerase chain reaction and Western blot. The effect of GnRH-I and -II on the production of select cytokines (hCG, interleukin [IL] 8, IL-6, matrix metalloproteinase 3, monocyte chemoattractant protein 1, vascular endothelial growth factor, soluble Fms-like tyrosine kinase 1, urokinase-type plasminogen activator, and plasminogen activator inhibitor 1) were measured by ELISA and normalized for protein content. RESULT(S): GnRH-I, GnRH-II, and GnRHR-I mRNA and protein were identified in trophoblasts and decidua. GnRH-I and -II stimulated hCG production by trophoblast and trophoblast-derived cell lines in a dose-dependent fashion (e.g., 2.8-fold, from 2.5 ± 0.5 to 7.0 ± 0.4 ng/mg protein per 24 h, for 1,000 nmol/L GnRH-I and 2.4-fold, from 2.5 ± 0.5 to 6.1 ± 0.6 ng/mg protein per 24 h, for 1,000 nmol/L GnRH-II) without affecting the production of other cytokines. CONCLUSION(S): Trophoblasts and decidua express GnRH-I, GnRH-II, and GnRHR-I mRNA and protein. GnRH-I and -II selectively stimulate hCG production by trophoblast cells without altering the production of select cytokines by trophoblasts or decidua. The role of GnRH-GnRHR signaling at the maternal-fetal interface therefore appears to be limited to the regulation of trophoblast hCG production.

Antitumor and normal cell protective effect of PKC412 in the athymic mouse model of ovarian cancer.

N-benzoyl-staurosporine (PKC412) is a selective inhibitor of protein kinase C, and it inhibits the growth of human cancer cells. In this study, we examined the antitumor effect of PKC412, given singly and in combination with paclitaxel, on tumor regression and chemotherapeutic side effects by assessing tumor burden and cytokine production responses in vivo. Twenty-six nude mice intraperitoneally inoculated with SKOV3 cells were treated differently in 4 treatment groups: PKC412 plus paclitaxel (n = 7), paclitaxel-only (n = 6), PKC412-only (n = 6), and controls (n = 7). At autopsy, we found that PKC412 itself slightly reduced the mass of tumor but did not fully inhibit tumor formation. The incidence of evident disease was decreased when PKC412 was combined with paclitaxel (43%). From the body weight of the tumor-bearing mice, we observed that PKC412 plus paclitaxel treated mice were less wasted than paclitaxel-only treated mice (18.1 g vs 22.4 g, p = 0.001). We measured intracellular TNFalpha, IFNgamma, IL-4, and IL-10 in stimulated mouse splenocytes using flow cytometry to determine if PKC412 inhibited cytokine production in T cells. TNFalpha, IFNgamma, and IL-10 production were all significantly inhibited in the paclitaxel-treated mice. The inhibitory effects on cytokine production by paclitaxel were compensated with PKC412 combination (p = 0.008, 0.035, 0.014, respectively). From this study, we deduce that PKC412 may have clinical applications in promoting tumor regression in ovarian cancer when combined with paclitaxel. Moreover, PKC412 is able to prevent weight loss and immunosuppression induced by paclitaxel because it rescues normal proliferating cells from cytotoxic effects.

Detection of p16(INK4A) in the Mixed Cell Populations of Normal Peripheral Blood Mononuclear Cells and Cervical Cancer Cell Lines.

PURPOSE: Human papilloma viruses (HPVs) play a central role in the pathogenesis of neoplastic lesions of the uterine cervix. The viral oncoprotein HPV E6 degrades the p53 protein, and the HPV E7 protein inactivates pRB and increases the expression of the CDK inhibitor, p16(INK4A). We investigated the usefulness of p16(INK4A) as a biologic marker for the cervical dysplastic and neoplastic cells. MATERIALS AND METHODS: We examined the expression of p16(INK4A) and cytokeratin in a mixed population of normal peripheral blood mononuclear cells (PBMC) and the cervical cancer cell lines (HeLa, SiHa, and CasKi) using flow cytometry. RESULTS: The DNA indices of the HeLa, SiHa and CasKi cell lines were 1.89, 1.53 and 1.75, respectively, indicating that these cells are aneuploid cells. Furthermore, the positive rate of p16(INK4A) expression was 86.7% for the HeLa mixed population, 85.6% for the SiHa mixed population, and 92.2% for the CasKi mixed population. According to the FL3A vs FL3W histogram, electrical gating of the HeLa, SiHa and CasKi mixed populations showed the expression levels of both cytokeratin and p16(INK4A) to be identical, at 86.6%, 84.8% and 85.0%, respectively. These findings revealed that almost all cells selected through electrical gating were cervical cancer cells originating from the epithelium and which expressed cytokeratin and p16(INK4A). On the other hand, when each mixed population was electrically gated for normal PBMC, we found that the PBMCs expressed neither cytokeratin nor p16(INK4A). CONCLUSION: Using flow cytometry, we observed the enhanced expression of p16(INK4A) in cervical cancer cell lines. These. RESULTS: suggest the usefulness of p16(INK4A) for the selective detection of cervical dysplastic and cancer cells in the liquid-based samples, which are taken from the cervices and contaminated with blood and stromal cells.

Multiparametric flow cytometric analysis in a breast cancer cell line (MCF-7).

OBJECTIVE: To investigate whether multiparameter flow cytometric analysis of solid tumor specimens, including gynecologic tumors, which were stained triply with phycoerythrin (PE), fluorescein isothiocyanate (FITC) and propidium iodide (PI), can be performed simultaneously without interference from normal diploid cell populations and spectral overlap on a standard flow cytometer. METHODS: MCF-7 breast cancer cell lines and heterogeneous cell populations mixed with MCF-7 cells and human peripheral blood lymphocytes (PBL) were fixed with 1% paraformaldehyde and permeabilized with 100% methanol. Cytokeratin and several proliferation-associated cellular antigens (proliferating cell nuclear antigen (PCNA), p53, c-erbB/2 and c-myc) were labeled with PE and FITC, which was followed by DNA staining using PI. These labeled cells were measured on a standard FACScan flow cytometer equipped with a 488 nm single laser. RESULTS: The coefficient of variation (CV) of the G0G1 peak of MCF-7 cells was 4.3 and the cell cycle phase fractions of G0G1, S and G2M were 44.9, 45.9 and 9.2%, respectively. Fluorescein isothiocyanate, PE and PI fluorescences were detected without interference. The MCF-7 cells expressed cytokeratin, PCNA, p53, c-erbB/2 and c-myc antigen. In the heterogeneous population of MCF-7 cells mixed with PBL, two cellular populations were clearly separated into diploid PBL and aneuploid MCF-7 cells without interference. The CV of G0G1 peak of PBL was 2.3 and the G0G1, S and G2M phase fractions were 85.5, 2.7 and 11.8%, respectively. The DNA index of MCF-7 cells was 1.7, which indicated that the MCF-7 cell line was composed of tumor cells with aneuploid DNA. The CV of the G0G1 peak of the MCF-7 cells was 4.2, and the cell cycle phase fractions were 47.5% for G0G1, 42.3% for S, and 10.2% for G2M. The MCF-7 cells expressed cytokeratin, but the PBL did not. CONCLUSIONS: Multiparameter flow cytometer analysis was useful to determine DNA ploidy status, phase fraction of the cell cycle and expression of cellular antigens and selective cytokeratin expression allowed epithelial originated tumor cells to be differentiated from normal stromal cells. This analysis could be performed without interference of spectral overlaps of fluorochromes using software-based algorithmic compensation of spectral overlaps. Thus, this method offers new possibilities for multiparameter flow cytometric analysis and its use should be extended to future studies of the diagnosis, treatment and prediction of prognosis of the neoplasm.