Protective effect of pregnancy for development of uterine leiomyoma.

Many factors that can modulate the risk of developing uterine leiomyoma have been identified, including parity. Epidemiological data on decreased risk of developing this disease has been subject to different interpretations regarding whether pregnancy per se is protective or, as leiomyomas are a major cause of infertility, women that develop these tumors are less fertile and thus have lower pregnancy rates. We have utilized an animal model genetically predisposed to uterine leiomyoma to investigate the potential protective effect of pregnancy on the risk of developing this disease. Female Eker rats that carry a mutation in the tuberous sclerosis 2 (Tsc-2) tumor suppressor gene develop uterine leiomyoma with a frequency of 65% when nulliparous. These animals were bred with intact or vasectomized males and tumor incidence determined after a single pregnancy (to confirm fertility) or multiple pregnancies over the lifetime of the animals. Females with multiple litters displayed a dramatic shift in tumor incidence and presentation. Tumor incidence decreased from 71% in single litter females to 10% in females that had multiple litters (average: five litters/animal). Interestingly, females bred with vasectomized males also exhibited a reduced tumor incidence of 41%, suggesting that the hormonal changes associated with early stages of pregnancy that occur in pseudopregnant females may have contributed to the protective effect of pregnancy.

Lessons from pregnancy and parturition: uterine leiomyomas result from discordant differentiation and dedifferentiation responses in smooth muscle cells.

Leiomyomas, benign smooth muscle tumors of the uterus, are the most common gynecological neoplasm in women. Studies with human tissues and primary cultures have revealed little about the development of leiomyomas, although several genes have been shown to be differentially expressed in leiomyomas compared to matched normal myometrium. We propose that uterine smooth muscle tumor cells mimic a differentiated myometrial cell of pregnancy, and are associated with a hypersensitivity to sex steroid hormones, preventing the cells from responding to normal apoptotic or dedifferentiation signals which would return the cells to a nongravid phenotype. Support of this hypothesis is derived from experimental studies in female Eker rats which develop uterine leiomyoma with many similarities to the human disease. Members of the steroid receptor superfamily as well as the binding partners and co-regulators necessary for transactivation and gene transcription, may be involved in the altered pathway of cellular differentiation and regulation observed in uterine leiomyomas.

Pregnancy, parturition, and prostaglandins: defining uterine leiomyomas.

Leiomyomas, benign smooth muscle tumors of the uterus, are the most common gynecologic neoplasm in women. Studies with surgically resected human tissues and primary cultures have revealed that several genes are differentially expressed in leiomyomas compared to matched normal myometrium. An estrogen-driven pattern of gene expression in leiomyomas, similar to that seen in normal myometrium during pregnancy and parturition, is associated with a persistent inappropriate response of neoplastic myometrial smooth muscle cells to ovarian hormones. This is possibly due to aberrant expression levels or signaling via estrogen and progesterone receptors. We propose the hypothesis that uterine leiomyomas mimic a differentiated myometrial cell at pregnancy and exhibit a hypersensitivity to sex steroid hormones that prevents the cells from responding to normal apoptotic or dedifferentiation signals and from returning to a nongravid phenotype. Support of this hypothesis is derived from experimental studies in female Eker rats that develop uterine leiomyomas with many similarities to the human disease. Our hypothesis accounts for the benign nature of these tumors and their high incidence in women during the reproductive years. By identifying the factors that participate in parturition and involution of the pregnant myometrium, we may better define uterine leiomyomas and thus identify novel targets for therapeutic strategies to treat these tumors.

Growth inhibition in G(1) and altered expression of cyclin D1 and p27(kip-1 )after forced connexin expression in lung and liver carcinoma cells.

Gap junctional intercellular communication (GJIC) and connexin expression are frequently decreased in neoplasia and may contribute to defective growth control and loss of differentiated functions. GJIC, in E9 mouse lung carcinoma cells and WB-aB1 neoplastic rat liver epithelial cells, was elevated by forced expression of the gap junction proteins, connexin43 (Cx43) and connexin32 (Cx32), respectively. Transfection of Cx43 into E9 cells increased fluorescent dye-coupling in the transfected clones, E9-2 and E9-3, to levels comparable to the nontransformed sibling cell line, E10, from which E9 cells originated. Transduction of Cx32 into WB-aB1 cells also increased dye-coupling in the clone, WB-a/32-10, to a level that was comparable to the nontransformed sibling cell line, WB-F344. The cell cycle distribution was also affected as a result of forced connexin expression. The percentage of cells in G(1)-phase increased and the percentage in S-phase decreased in E9-2 and WB-a/32-10 cells as compared to E9 and WB-aB1 cells. Concomitantly, these cells exhibited changes in G(1)-phase cell cycle regulators. E9-2 and WB-a/32-10 cells expressed significantly less cyclin D1 and more p27(kip-1) protein than E9 and WB-aB1 cells. Other growth-related properties (expression of platelet-derived growth factor receptor-beta, epidermal growth factor receptor, protein kinase C-alpha, protein kinase A regulatory subunit-Ialpha, and production of nitric oxide in response to a cocktail of pro-inflammatory cytokines) were minimally altered or unaffected. Thus, enhancement of connexin expression and GJIC in neoplastic mouse lung and rat liver epithelial cells restored G(1) growth control. This was associated with decreased expression of cyclin D1 and increased expression of p27(kip-1), but not with changes in other growth-related functions.

Acute inhibition of spontaneous uterine contractions by an estrogenic polychlorinated biphenyl is associated with disruption of gap junctional communication.

An estrogenic polychlorinated biphenyl, 4-hydroxy-2',4', 6'-trichlorobiphenyl (4-OH-TCB), inhibits oscillatory uterine contractions immediately. Because increased gap junction formation is associated with the development of synchronized uterine contractions at term, we examined whether the inhibitory effect of 4-OH-TCB on spontaneous oscillatory contractions was due to the disruption of gap junctional communication. The effect of 4-OH-TCB on gap junctional communication was determined by intercellular Lucifer yellow dye transfer in primary cultures of myometrial myocytes isolated from midgestation rats. Intercellular dye transfer was inhibited by 4-OH-TCB or 17beta-estradiol in a concentration-dependent manner. The inhibitory effect of 4-OH-TCB on intercellular dye transfer was reversed by tetraethylammonium (TEA). To examine effects on uterine contraction, longitudinal uterine strips were excised from midgestation rats and placed in muscle baths for isometric force measurement. Spontaneous uterine oscillation was suppressed by 4-OH-TCB or 17beta-estradiol. The inhibitory effects of 4-OH-TCB and 17beta-estradiol on spontaneous oscillations were counteracted by TEA but were not affected by a calcium ionophore (A23187) or a calcium-dependent potassium channel blocker (apamin). These results suggest that the acute inhibition of spontaneous oscillatory contractions by an estrogenic polychlorinated biphenyl may result from the disruption of intercellular communication.

Role of gap junctions in lung neoplasia.

Reduced gap junctional intercellular communication (GJIC) has been noted in many types of neoplastic cells and may contribute to the neoplastic phenotype. This study assessed GJIC (by fluorescent dye-coupling) and gap junction protein (connexin) expression in mouse and human lung carcinoma cell lines and investigated whether reduced GJIC was involved in their neoplastic phenotype. Dye-coupling and connexin43 (Cx43) expression were much lower in most of the carcinoma lines (16 of 22) compared to nontransformed lung epithelial cells. Other connexins were not detected. A poorly communicating mouse lung carcinoma cell line (E9) was transfected with Cx43 or transduced with Cx32 and several stable clones were isolated that had 2- to 4-fold increased dye coupling. When evaluated for growth in vitro, the population doubling times were increased and the saturation densities were decreased in the clones. When assessed for tumorigenicity, the parental E9 cells formed tumors with a 100% incidence (6/6 mice), whereas the clones varied in tumorigenic response (0-88% incidence). The best communicating clone (E9-2) was not tumorigenic. The highly communicating Cx32 clone, E9/32-9, gave a tumor incidence of 88%. These results suggest that restoration of GJIC by forced connexin expression can reduce the growth and tumorigenicity of lung carcinoma cells in a connexin-specific manner.

Frequent reduction of gap junctional intercellular communication and connexin43 expression in human and mouse lung carcinoma cells.

The reduced gap junctional intercellular communication (GJIC) and gap junction protein (connexin) expression that have been noted in many neoplastic cell types may contribute to the neoplastic phenotype. We assessed GJIC (by fluorescent dye micro-injection) and connexin expression (by Northern blotting, Western blotting and immunohistochemistry) in five mouse and 17 human lung carcinoma cell lines; both measures were lower in neoplastic cells compared to non-transformed lung epithelial cells. Other connexins were not detected in these cells. Co-culture experiments indicated that carcinoma cell lines able to transfer dye among themselves (homologous GJIC) had little capacity for dye-coupling with non-transformed cells (heterologous GJIC). Southern blot analyses indicated that reductions in GJIC and connexin43 expression were not due to deletions or rearrangements of this gene, but were more likely accounted for by transcriptional down-regulation and/or post-transcriptional factors. No correlations between GJIC and known oncogene and tumor suppressor gene alterations in the human lung carcinoma cells were apparent, suggesting that other mechanisms down-regulate GJIC in these cells. Since the neoplastic cell lines exhibited low GJIC (either homologous or heterologous), this characteristic may be involved in expression of the neoplastic phenotype.

Role of protein kinase C in the deficient gap junctional intercellular communication of K-ras-transformed murine lung epithelial cells.

BACKGROUND: Protein kinase C (PKC) activity is increased and gap junctional intercellular communication (GJIC) is decreased frequently in Ras-transformed cells. We investigated the roles of Ras and PKC in the deficient gap junctional intercellular communication (GJIC) of K-ras-transformed E9 mouse lung carcinoma cells. METHODS: GJIC was measured by fluorescent dye microinjection. Ras activity was blocked with lovastatin or a K-ras antisense oligonucleotide. PKC activity was inhibited with GF 109203X or apigenin or was downregulated by overnight treatment with 12-O-tetradecanoylphorbol-13-acetate. The content and phosphorylation of the gap junction protein, connexin43 (Cx43), was assessed by Western blot. RESULTS: E9 cell GJIC was increased two-three fold by lovastatin, the K-ras antisense oligonucleotide, and PKC inhibition/downregulation. Cx43 content and phosphorylation were unchanged, however. CONCLUSIONS: Oncogenic Ras blocks GJIC in E9 cells through a PKC-dependent mechanism, but this does not directly involve Cx43 expression or phosphorylation.