Jeanne L. Becker

Endometriosis in rhesus monkeys (Macaca mulatta) following chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The incidence of the reproductive disease endometriosis was determined in a colony of rhesus monkeys chronically exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) for a period of 4 years. Ten years after termination of dioxin treatment, the presence of endometriosis was documented by surgical laparoscopy and the severity of disease was assessed. The incidence of endometriosis was directly correlated with dioxin exposure and the severity of disease was dependent upon the dose administered (p < 0.001). Three of 7 animals exposed to 5 ppt dioxin (43%) and 5 of 7 animals exposed to 25 ppt dioxin (71%) had moderate to severe endometriosis. In contrast, the frequency of disease in the control group was 33%, similar to an overall prevalence of 30% in 304 rhesus monkeys housed at The Harlow Primate Center with no dioxin exposure. This 15-year study indicates that latent female reproductive abnormalities may be associated with dioxin exposure in the rhesus. Therefore, the effects of this toxin may be more diverse than previously recognized.

Three-dimensional culture of a mixed mullerian tumor of the ovary: Expression of in vivo characteristics

SummaryThe Rotating-Wall Vessel (RWV) is a novel in vitro cell culture system used to successfully culture a cell line derived from a heterologous mixed mullerian tumor cell of the ovary. Although the original tumor was comprised of both epithelial and mesodermal components, long-term culture in conventional flasks established a cell line from this tumor with homogenous epitheliallike growth characteristics (1). Cells from Passage 36 were seeded into a Rotating-Wall Vessel containing Cytodex-3 microcarrier beads. Scanning electron micrographs of tumor cells cultured for 32 d in the RWV showed the presence of heterogeneous cell populations organized into three-dimensional tissuelike architecture. Immunocytochemical analysis confirmed the cellular heterogeneity, as demonstrated by expression of both epithelial and mesenchymal antigens. Reverse transcription polymerase chain reaction amplification demonstrated the presence of mRNA for cellular oncogenes HER-2/neu, H-ras, K-ras, and tumor suppressor p53. Thus, there are two advantages to propagation of tissue in the RWV culture system: (a) tissue diversification representing populations present in the original tumor, and (b) the three-dimensional freedom to organize tissues morphologically akin to those observed in vivo. These data indicate that the RWV culture system is suitable for generating large quantities of ovarian tumor cells in vitro that are amenable to immunocytochemical, oncogenic, morphologic characteristics demonstrated in vivo.

Suppression of phagocytosis by dexamethasone in macrophage cultures: Inability of arachidonic acid, indomethacin, and nordihydroguaiaretic acid to reverse the inhibitory response mediated by a steroid-inducible factor

Glucocorticoid steroids inhibit phagocytosis and cell spreading in cultures of murine resident peritoneal macrophages. It is postulated that these suppressive responses are mediated by a steroid-induced substance elaborated from dexamethasone-treated macrophages. Accordingly, dialyzed medium from dexamethasone-treated cultures was analyzed for the presence of a factor that inhibits phagocytosis and cell spreading in macrophage cultures not exposed to the steroid. When previously untreated macrophages were supplied dialysates from steroid-treated cultures, cell spreading and the phagocytic capacity (i.e. percentages of phagocytes in macrophage populations and the ability of phagocytes to ingest heat-killed Saccharomyces cerevisiae particles) decreased dramatically between 2 and 24 h after exposure. A lesser transient inhibitory response was observed when dialysates from untreated cultures were used. Relative to these controls after 48 h, phagocytic capacity and cell spreading remained suppressed in cultures treated with dialysates from dexamethasone-treated cultures. The addition of arachidonate, in the absence and presence of cyclooxygenase and lipoxygenase inhibitors, did not affect the phagocytic capacity of control macrophages. Furthermore, the addition of these compounds, either alone or in combination, to dexamethasone-treated cultures did not modulate the steroid-induced suppression of phagocytosis. These results support the hypothesis that the inhibition of phagocytosis and cell spreading may be mediated by a dexamethasone-induced non-dialyzable factor. In addition, the inability of arachidonic acid and inhibitors of prostaglandin and leukotriene biosynthesis to reverse the steroid-induced suppression of phagocytosis implies that the inhibition of this important macrophage function is not associated with the failure of dexamethasone-treated macrophages to release these mediators.

Aspirin effects on endometrial cancer cell growth

Objective To find whether aspirin (acetylsalicylic acid, ASA) inhibits the growth of endometrial cancer cells in vitro in a way similar to that in colorectal cancer cells and to investigate the mechanisms by which aspirin might lead to growth inhibition. Methods Ishikawa human endometrial tumor cells were grown in the presence of ASA (1–5 mM) for 96 hours. Controls were treated with vehicle (absolute ethanol). Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay. Apoptosis was determined by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling assay. Analysis of cell-cycle distribution and bcl-2 expression was assessed by flow cytometry. Results Acetylsalicylic acid induced a dose-dependent inhibition of Ishikawa cells in vitro. The percentage of growth inhibition was 21–88% at concentrations of 1–5 mM. It also induced apoptosis and reduced bcl-2 expression in Ishikawa cells in a dose-dependent manner. Control cells and cells treated with the lowest concentration of ASA exhibited 2% apoptosis and more than 60% of the population expressed bcl-2. Apoptosis levels increased as levels of ASA increased from 2 to 5 mM (7–58%) with a concommitant decrease in bcl-2 expression from 46% at 2 mM to 2% at 5 mM. Acetylsalicylic acid concentrations of 3 mM or greater induced a shift from the resting phase (G0/G1) to S phase of the cell cycle. Conclusion Acetylsalicylic acid inhibited Ishikawa cell growth in vitro in a dose-dependent manner. Apoptosis is one of the mechanisms involved in the response, which can be mediated in part by downregulation of bcl-2.

ENDOMETRIOSIS IS ASSOCIATED WITH ALTERATIONS IN THE RELATIVE ABUNDANCE OF PROTEINS AND IL-10 IN THE PERITONEAL FLUID

A growing body of evidence suggests that endometriosis modulates the microenvironment of peritoneal cavity. Therefore, in this study, we compared the protein profile of peritoneal fluids from normal fertile women with those from patients with infertility, and patients with mild to severe endometriosis. Two-dimensional gel electrophoresis of peritoneal fluids from normal subjects exhibited a distinct and reproducible pattern of proteins in the size ranges of approximately 35 to 80 kD and pI close to 4.5 to 6.6. Infertility without evidence of endometriosis was not associated with changes in the relative abundance of proteins present in the peritoneal fluid. However, mild endometriosis was associated with a mild reduction in the amount of several peritoneal protein spots with the approximate molecular weights of 35-40 kD and pI close to 5.7-6.0. These changes became markedly apparent in the peritoneal fluid of patients who suffered from the severe form of this disease. Severe endometriosis was also associated with appearance of protein spots in the gels that were not detectable in the peritoneal fluids of normal subjects. Consistent with these data, enzyme-linked immunosorbent assay showed that moderate to severe endometriosis was associated with markedly elevated levels of IL-10 in the peritoneal fluid. Reverse transcription followed by polymerase chain reaction amplification using primers specific to IL-10 confirmed presence of IL-10 mRNA in cells derived from peritoneal fluids. These findings show that endometriosis is associated with disturbed secretion of proteins into the peritoneal cavity and with an elevated level of IL-10 in the peritoneal fluid. The studies also show cells resident in peritoneum as a major source of IL-10.

CYTOGENETIC, MORPHOLOGIC AND ONCOGENE ANALYSIS OF A CELL LINE DERIVED FROM A HETEROLOGOUS MIXED MULLERIAN TUMOR OF THE OVARY

SummaryA cell line was established from a mixed mullerian tumor of the ovary and designated LN1. Histopathologic analysis of the fresh tumor specimen demonstrated a highly aneuploid heterologous tumor comprised of undifferentiated mesodermal components with carcinomatous cells present as a smaller population. Long-term in vitro culture resulted in the establishment of a cell line that exhibits an epithelial-like morphology and expresses epithelial antigens cytokeratin, epithelial membrane antigen, and carcinoma antigen TAG-72. These cells also express mesenchymal intermediate filaments, vimentin, and desmin. Karyotypic analysis revealed a basic triploid pattern with multiple chromosomal abnormalities, most notably an isochromosome of the short arm of five present in three copies. Analysis of oncogene expression revealed that LN1 cells constitutively express mRNA for c-ras, c-erbB2, and p53. The expression of mRNA for cellular oncogenes correlated with the presence of corresponding oncoproteins, p21H-ras, p21K-ras, and p185erbB2 and mutant p53 protein. In summary, coexpression of epithelial and mesenchymal antigens by LN1 cells lends support to the hypothesis that epithelial and mesenchymal elements comprising mixed mullerian tumors of the ovary are derived from a common stem cell precursor. Furthermore, this cell line represents a functional in vitro model to evaluate the biologic activities of these unusual and highly aggressive ovarian malignancies.

Human peritoneal macrophage and T lymphocyte populations in mild and severe endometriosis

PROBLEM: The aim of this study was to characterize the phenotype of peritoneal lymphocyte and macrophage populations in mild versus severe endometriosis.

Aspirin-induced inhibition of ovarian tumor cell growth.

OBJECTIVE To determine if aspirin inhibits the growth of ovarian tumor cells in vitro and to investigate possible mechanisms involved in inhibition. METHODS OVCAR‐3 ovarian tumor cells were grown in monolayer cultures and then harvested for use in proliferation assays. The cells were then treated with vehicle (1% absolute ethanol), 1–5‐mmol/L aspirin, 1‐μg/mL of anti HER‐2/neu monoclonal antibody, or 20‐ng/mL heregulin, the ligand for the HER‐2/neu receptor either alone or in combination. Cellular proliferation was determined spectrophotometrically by the reduction of tetrazolium dye. Expression of Her‐2/neu was assessed by flow cytometry. RESULTS Aspirin induced inhibition of OVCAR‐3 tumor cell growth in a dose‐dependent fashion ranging from little to no inhibitory response in cultures treated with 1‐mmol/L aspirin to 68% in those treated with 5‐mmol/L aspirin. Expression of HER‐2/neu was likewise reduced in a dose‐dependent manner from 87% expression in control cells to 16% in those treated with 5‐mmol/L aspirin. Addition of heregulin alone resulted in 23% proliferation over the control. The combination of heregulin plus 2‐mmol/L aspirin caused 66% inhibition of tumor cell growth, whereas the blocking of the HER‐2/neu receptor with the monoclonal antibody resulted in an even greater inhibitory response of 82%. CONCLUSION OVCAR‐3 tumor cell growth is inhibited by aspirin, and suppression appears to be potentiated by blocking the HER‐2/neu receptor.

Suppression of yeast ingestion by dexamethasone in macrophage cultures: evidence for a steroid-induced phagocytosis inhibitory protein.

The mechanism by which glucocorticoid steroids suppress yeast phagocytosis in cultures of resident and thioglycollate-elicited murine peritoneal macrophages was examined. Time course and dose-response studies demonstrated that the phagocytic capacity of resident macrophages was suppressed by dexamethasone to the same extent in both newly established cultures and cultures that were incubated for several days. In contrast, relative to newly established cultures of elicited cells that were treated with the drug, elicited macrophages that were incubated for at least 1 day prior to exposure to dexamethasone, exhibited enhanced sensitivity to the action of the steroid. Steroid-induced phagocytic inhibitory responses were blocked by the metabolic inhibitors cycloheximide and actinomycin D. The suppression of phagocytosis by dexamethasone was mediated by a factor, present in the cellular homogenates of steroid-treated macrophages, that was partially purified by Sephadex G-25 chromatography. Since the phagocytic inhibitory activity in these homogenates was destroyed following exposure to heat and trypsin, the factor has been named phagocytosis inhibitory protein (PIP). The antiphagocytic activity of PIP was neutralized by treatment with RM23, a monoclonal antibody directed against lipocortin. The results support the hypothesis that the suppression of yeast ingestion is mediated by the action of PIP, which is induced in dexamethasone-treated macrophage cultures. Moreover, PIP appears to belong to the lipocortin family of phospholipase inhibitory proteins.

Dexamethasone action inhibits the release of arachidonic acid from phosphatidylcholine during the suppression of yeast phagocytosis in macrophage cultures.

Dexamethasone suppresses phagocytosis of heat killed Saccharomyces cerevisiae in cultures of murine peritoneal macrophages. Recent observations suggest that dexamethasone induces a phagocytic inhibitory protein that suppresses yeast ingestion by inhibiting macrophage phospholipase A2 activity. The present investigation, therefore, examined whether macrophage lipid metabolism is modulated by dexamethasone. Control and steroid treated macrophages were allowed to incorporate radiolabeled arachidonate and were incubated subsequently in the absence and presence of yeast. Following ingestion by control macrophages, arachidonate from phosphatidylcholine was readily cleaved to free fatty acid and transferred to the neutral lipid fraction. In contrast, arachidonate release was inhibited in dexamethasone treated macrophages. These results suggest that the suppression of yeast phagocytosis by dexamethasone action may be associated with the inhibition of phospholipase A2 activity.