Immunomodulatory and transcriptional effects of progesterone through progesterone A and B receptors in Hec50co poorly differentiated endometrial cancer cells.

OBJECTIVE: Derivatives of progesterone, progestins, are used to treat endometrial cancer; however, the pathways activated by the hormone have not been fully investigated. Progesterone acts through two receptor isoforms, progesterone receptors A and B (PRA and PRB), transcription factors that control the expression of downstream genes leading to endometrial differentiation. The purpose of this study was to perform an expression analysis to identify the mechanisms underlying progesterone's growth suppressive and immunomodulatory effects in endometrial cancer. METHODS: To study the molecular effects of progesterone, PRs were introduced into Hec50co cells. Expression array analyses followed by confirmatory semiquantitive reverse-transcriptase polymerase chain reaction (RT-PCR) experiments were performed. RESULTS: Expression analysis demonstrated a significant effect of progesterone after 12 hours of treatment on a number of genes, including cell signaling, DNA remodeling, apoptotic, tumor-suppressor, and transcription factors. Of particular interest was the consistent modulation of cytokines, which generally predicted for a powerful anti-inflammatory effect of progesterone through PR. Specifically, pro-inflammatory genes such as TNFalpha, IL-1beta, and MCP-1/MCAF-1 were down-regulated and anti-inflammatory genes such as TRAP1 and SMAD4 were induced. CONCLUSION: We have discovered that progesterone has a modulatory effect on inflammation and many other important cellular functions. These effects likely underlie the inhibitory effects of progesterone on tumor growth and invasion.

Experimental intrauterine infection with Prevotella bivia in New Zealand White rabbits.

OBJECTIVE: The purpose of this study was to develop a model of chronic intrauterine and fetal infection with Prevotella bivia, an anaerobe of the lower genital tract that is associated often with bacterial vaginosis. STUDY DESIGN: Thirty timed pregnant New Zealand White rabbits on gestational day 21 were inoculated with P bivia or saline solution in a planned ratio of 4:1 (24 P bivia: 6 saline solution). Rabbits were inoculated 6 cm transcervically with 10(5) to 10(8) colony-forming units/uterine horn of P bivia or with saline solution. Necropsy was scheduled on days 4, 6, or 7 after inoculation. Cultures were collected from blood, uterus, amniotic fluid and fetal brain, lung, and heart. Tissues from placenta, uterus, fetal brain, and lung were evaluated with the histologic inflammation score, with a range of 0 to 13. Amniotic fluid was assayed for tumor necrosis factor-alpha by bioassay. Animals with contamination by other organisms were excluded. Categoric data were evaluated with the use of the Fisher exact test, and continuous data were evaluated with the use of the Wilcoxon rank sum. RESULTS: After the exclusion of 8 animals because of contamination with other organisms, 22 animals were evaluated. Of 3 rabbits with an inoculum of 10(8) P bivia colony-forming units/horn, 2 animals (67%) had fever within 24 hours. These results were not compatible with chronic, subclinical infection. Therefore, 14 does had inocula of 10(5-6) P bivia colony-forming units/horn, with necropsy planned at day 4 (n=5 animals), day 6 (n=3 animals), and day 7 (n=6 animals), and 5 animals were inoculated with saline solution. Animals that had been inoculated with P bivia were significantly more likely to have a positive culture than were those animals that were inoculated with saline solution (64% vs 0%; P<.04). Preterm delivery without fever occurred in 21% of does (3/14 does) that were inoculated with P bivia overall and in 33% of the does (3/9 does) that were followed for 6 to 7 days. No saline-solution inoculated animal had preterm birth. There was an increase in amniotic fluid tumor necrosis factor-alpha levels over time in the P bivia group (P=.12). Histologic inflammation scores were not significantly different between P bivia and saline solution groups. CONCLUSION: Inoculation with P bivia at 10(5-6) colony-forming units/horn leads to chronic intrauterine and fetal infection that are accompanied by preterm birth in up to 33% of cases. This model may serve to explore the mechanism of preterm birth that is induced by chronic infection with genital tract anaerobes.

The Six1 homeoprotein stimulates tumorigenesis by reactivation of cyclin A1.

Homeobox genes constitute a large family of transcription factors that are essential during normal development and are often dysregulated in cancer. However, the molecular mechanisms by which homeobox genes influence cancer remain largely unknown. Here we show that the tissue-restricted cyclin A1 is a transcriptional target of the Six1 homeoprotein. Both genes are expressed in the embryonic but not the terminally differentiated mammary gland, and Six1-knockout mice show a dramatic reduction of cyclin A1 in the embryonic mammary gland. In addition, both genes are reexpressed in breast cancers. Six1 overexpression increases cyclin A1 mRNA levels and activity, cell proliferation, and tumor volume, whereas Six1 down-regulation decreases cyclin A1 mRNA levels and proliferation. Overexpression of Six1 in wild-type mouse embryonic fibroblasts, but not in knockout variants lacking the cyclin A1 gene, induces cell proliferation. Furthermore, inhibition of cyclin A1 in Six1-overexpressing mammary carcinoma cells decreases proliferation. Together these results demonstrate that cyclin A1 is required for the proliferative effect of Six1. We conclude that Six1 overexpression reinstates an embryonic pathway of proliferation in breast cancer by up-regulating cyclin A1.

Inhibition of JNK reduces G2/M transit independent of p53, leading to endoreduplication, decreased proliferation, and apoptosis in breast cancer cells.

c-Jun N-terminal kinase (JNK) is activated by diverse cell stimuli, including stress, growth factors, and cytokines. Traditionally, activation of JNK by stress treatment is thought to induce cell death. However, our recent data indicate that JNK's ability to sensitize cells to apoptosis may be, in part, cell cycle dependent. Here, we show that the majority of both paclitaxel- and UV-induced apoptosis can be inhibited by the pharmacological JNK inhibitor, SP600125, in MCF-7 cells. However, inhibition of JNK does little to reverse doxorubicin-induced apoptosis in MCF-7 cells or doxorubicin- and UV-mediated death in MDA MB-231 cells. SP treatment causes G2/M arrest of three breast cancer cell lines and results in the endoreduplication (cellular DNA content >4N) of MCF-7 and MDA MB-231 cells. These effects on cell cycle and apoptosis are not significantly altered by the inhibition of p53, indicating that JNK is functioning independently of p53. Lastly, inhibition of JNK using both SP and antisense oligonucleotides targeted to JNK1 and JNK2 reduced proliferation of all three breast cancer cell lines. Taken together, these results suggest that the activation of JNK is important for the induction of apoptosis following stresses that function at different cell cycle phases, and that basal JNK activity is necessary to promote proliferation and maintain diploidy in breast cancer cells.

Risk factors for cesarean delivery at presentation of nulliparous patients in labor.

OBJECTIVE: To identify risk factors that place a term nulliparous patient in labor at risk for cesarean delivery. METHODS: This was a case-control, chart review study of 325 nulliparous patients presenting in labor at term with singleton vertex fetuses with either cesarean (patients) or vaginal (controls) delivery. Dichotomous variables were analyzed by chi(2) or Fisher exact tests; continuous variables were assessed by the Wilcoxon two-sample test. Multiple logistic regression was used to identify independent risk factors for cesarean delivery, and a model for predicting risk was built and evaluated. RESULTS: In univariate analysis, 22 variables were significantly different between patients and controls. Of 11 that were known within 2 hours of admission, five (change in cervical dilatation, maternal weight, gestational age, fetal station at 2 hours, and preeclampsia) remained independently significant in a multiple logistic regression model for cesarean delivery. The multiple regression model could divide our study population into quintiles in which the lowest risk group had a 5% incidence and the highest risk group had an 88% incidence of cesarean delivery. CONCLUSION: It may be possible to offer early cesarean delivery to patients at highest risk, reducing the potential morbidity of long labor or failed operative vaginal delivery followed by a later cesarean delivery.

Progesterone inhibits human endometrial cancer cell growth and invasiveness: down-regulation of cellular adhesion molecules through progesterone B receptors.

Progesterone is a critical steroid hormone that controls cell proliferation and differentiation in the female reproductive tract. Progesterone acts through two nuclear receptor isoforms, progesterone receptors A and B (PRA and PRB, respectively), each with unique cellular effects. Loss of PRB has recently been linked to the development of poorly differentiated endometrial tumors, a lethal form of cancer. To study the molecular effects of progesterone, progesterone receptors were introduced into Hec50co endometrial cancer cells by adenoviral vectors encoding either PRA or PRB. Progesterone induced the cyclin-dependent kinase inhibitors p21 and p27, thereby significantly reducing the percentage of proliferating cells. Cancer cell invasion was also markedly inhibited as measured by Matrigel invasion studies. Similarly, a differentiated, secretory phenotype was induced by progesterone in cells expressing PRB. However, replicative senescence was induced by progesterone only in cells expressing PRA. Expression array analysis followed by confirmatory semiquantitative reverse transcription-PCR experiments demonstrated a significant progesterone-dependent inhibition of expression of a cadre of cellular adhesion molecules, including fibronectin, integrin alpha3, integrin beta1, integrin beta3, and cadherin 6. The level of down-regulation of adhesion molecule expression was significantly greater in the presence of the B isoform, demonstrating that progesterone acts principally through B receptors to inhibit cancer cell invasiveness modulated by adhesion molecules.

Chronic intrauterine infection and inflammation in the preterm rabbit, despite antibiotic therapy.

OBJECTIVE: In a pregnant rabbit model using intracervical inoculation of Escherichia coli with delayed antibiotic therapy, we investigated the rate of positive cultures and histologic inflammation of maternal and fetal compartments and the concentration of tumor necrosis factor-alpha in the amniotic fluid for up to 5 days. STUDY DESIGN: New Zealand White rabbits at 70% gestation were inoculated intracervically with 10(3) - 10(4) colony-forming units of E coli per uterine horn. At varying intervals after inoculation (0.5 - 4.0 hours), antibiotic therapy was initiated with ampicillin-sulbactam. Primary outcomes were positive cultures and histologic inflammation score. Tumor necrosis factor-alpha levels in the amniotic fluid were determined by bioassay. RESULTS: A total of 60 animals were inoculated with E coli. At the endpoint, uterine cultures were positive more commonly than in the fetus or amniotic fluid (41.8% vs 27.5% vs 17.3%, respectively), which was consistent with an ascending pathway of infection. Inflammation scores were similar in uterus and placenta but lower in fetal lung and absent in fetal brain (2.8 vs 3.1 vs 0.84 vs 0.0, respectively). Comparing the durations of delay in antibiotic administration, we found a significant increase in positive uterine cultures and a significant increase in histologic inflammation score with increasing delay. The proportion of dead pups within a litter was significantly associated with the log of the tumor necrosis factor-alpha concentration in amniotic fluid and the degree of histologic inflammation in the uterus, but not with amniotic fluid or other culture positivity. CONCLUSION: The administration of therapeutic doses of antibiotic does not consistently eradicate bacteria from the rabbit uterus nor, more importantly, from the fetus and the amniotic fluid. Obtaining a negative amniotic fluid culture does not exclude either infection in the decidua or the fetus or histologic inflammation with tumor necrosis factor-alpha elaboration.

Differential gene regulation by the two progesterone receptor isoforms in human breast cancer cells.

The PR-A and PR-B isoforms of progesterone receptors (PR) have different physiological functions, and their ratio varies widely in breast cancers. To determine whether the two PR regulate different genes, we used human breast cancer cell lines engineered to express one or the other isoform. Cells were treated with progesterone in triplicate, time-separated experiments, allowing statistical analyses of microarray gene expression data. Of 94 progesterone-regulated genes, 65 are uniquely regulated by PR-B, 4 uniquely by PR-A, and only 25 by both. Almost half the genes encode proteins that are membrane-bound or involved in membrane-initiated signaling. We also find an important set of progesterone-regulated genes involved in mammary gland development and/or implicated in breast cancer. This first, large scale study of PR gene regulation has important implications for the measurement of PR in breast cancers and for the many clinical uses of synthetic progestins. It suggests that it is important to distinguish between the two isoforms in breast cancers and that isoform-specific genes can be used to screen for ligands that selectively modulate the activity of PR-A or PR-B. Additionally, use of natural target genes, rather than "consensus" response elements, for transcription studies should improve our understanding of steroid hormone action.