Telomerase prolongs the lifespan of normal human ovarian surface epithelial cells without inducing neoplastic phenotype.

OBJECTIVE: The aim of this study was to determine the effects of exogenous expression of the catalytic subunit of telomerase (hTERT) on the lifespan, growth characteristics, and tumorigenicity of normal human ovarian surface epithelial (OSE) cells. METHODS: Low-passage primary cultures of normal human OSE cells were transfected with hTERT and the resulting cell lines were characterized. RESULTS: The ectopic expression of hTERT stabilized the telomeres of the OSE cultures above 8 kb. The hTERT-transfected OSE cell lines grew beyond the normal lifespan seen in OSE cells and propagated in culture for more than 40 passages before senescing. Moreover, the hTERT-transfected cells demonstrated extensive proliferative capacity as evidenced by their ability to continuously grow even when seeded at low dilutions. The morphologic features and normal differentiation patterns seen in normal OSE cells were likewise retained by the hTERT-transfected cells. In addition, the cultures remained responsive to physiologic concentrations of epidermal growth factor and transforming growth factor-beta. Changes associated with neoplastic transformation like anchorage-independent growth, tumorigencity and karyotypic instability were not observed. CONCLUSIONS: We were able to show that the ectopic expression of hTERT in normal human OSE: 1) resulted in cultures with greater growth potential and longer lifespan and 2) did not induce a transformed phenotype previously seen in viral oncogene-transfected OSE cells. The established cell lines would not only provide sufficient material for comprehensive studies to investigate the normal physiology of OSE cells, but could also help in the understanding of the early steps of ovarian carcinogenesis.

An unexpected effect of glucocorticoids on stimulation of c-fms proto-oncogene expression in choriocarcinoma cells that express little glucocorticoid receptor.

OBJECTIVE: The purpose of this study was to determine the mechanism by which glucocorticoids stimulate c-fms proto-oncogene expression in JAR choriocarcinoma cells, which are reported to lack the glucocorticoid receptor. STUDY DESIGN: Glucocorticoid action on c-fms was tested with the use of ligand binding assays, Northern and Western blotting, immunohistochemistry, quantitative reverse transcriptase-polymerase chain reaction, and nuclear run-off experiments. RESULTS: Dexamethasone stimulated c-fms (EC(50)=1 nmol/L) in JAR cells in a specific manner. Both RU 486 and actinomycin D inhibited dexamethasone stimulation, which suggests receptor-mediated and transcriptionally regulated actions. Neither cytosol or whole cell binding assays nor immunohistochemistry detected glucocorticoid receptor in JAR cells. However, Southern blot analysis of reverse transcriptase-polymerase chain reaction products revealed levels of glucocorticoid receptor messenger RNA in JAR cells that were approximately 100-fold lower than in HeLa control cells. In all but 1 clone among several JAR clones that were tested, there was concordance between presence or absence of glucocorticoid receptor messenger RNA and glucocorticoid sensitivity. CONCLUSION: Some JAR cells contain low levels of glucocorticoid receptor, which mediate dexamethasone stimulation of c-fms expression. Such sensitivity to circulating glucocorticoids confers a survival advantage to these cells by stimulating the c-fms-related invasive behavior so characteristic of choriocarcinomas.

Apoptosis-based evaluation of chemosensitivity in ovarian cancer patients.

OBJECTIVE: Induction of apoptosis in target cells is a key mechanism by which chemotherapy induces cell killing. We have established an in vitro system for determining the chemosensitivity of epithelial ovarian cancer cells to carboplatin and paclitaxel (Taxol). Practical assays to predict the likelihood of individual tumor sensitivity are needed to facilitate the choice of adequate treatment. We sought to determine whether epithelial ovarian cancer cells (EOC) collected from the ascites fluid of patients known to be clinically chemosensitive or chemoresistant to carboplatin and paclitaxel would show a similar response to chemotherapeutic drugs after in vitro treatment. METHODS: Thirteen patients with stage III and IV ovarian cancer treated with carboplatin and paclitaxel were studied. Caspase-3 activation was used as a surrogate marker for activation of chemotherapy-induced programmed cell death. We compared the in vitro apoptotic response to the clinical response of the patients from whom the tumor cells were isolated. Clinical sensitivity was defined as no evidence of disease recurrence for 6 months after optimal debulking surgery and completion of chemotherapy. RESULTS: Of seven chemosensitive patients, five cell samples treated in vitro had increased caspase-3 activity in response to both carboplatin and paclitaxel. Five of six chemoresistant cases did not show caspase-3 activity in response to only one or to neither agent. CONCLUSION: Quantifiable markers of apoptosis such as caspase-3 activation have the potential to predict the clinical response to chemotherapy. Application of this assay in clinical laboratories could optimize the potential for efficient treatment and avoid the toxicities of ineffective drugs.

cDNA microarray analysis of invasive and tumorigenic phenotypes in a breast cancer model.

The fms oncogene encodes the macrophage colony-stimulating factor receptor (CSF1R), a transmembrane tyrosine kinase receptor, which is abnormally expressed in breast cancer. Transfection of wild-type CSF1R into HC11 mammary epithelial cells (HC11-CSF1R) renders the transfectants capable of in vitro local invasion and in vivo tumorigenesis. Transfection with CSF1R mutated to express phe at the tyr-721 autophosphorylation site (HC11-CSF1R-721) creates a phenotype that lacks metastastic competence but maintains local invasiveness. Conversely, HC11 cells transfected with CSF1R mutated at tyr-807 (HC11-CSF1R-807) retain their metastatic competence, but are not locally invasive. Our aims were to determine which genes were differentially expressed with transfection of HC11 with wild-type CSF1R, and to determine the effect of mutation at the autophosphorylation sites on gene expression, using 4.6 K cDNA microarrays. Complementary DNA from HC11, HC11-CSF1R-721 and HC11-CSF1R-807 were each hybridized together with HC11-CSF1R on individual arrays. A principal component spectral method combined with prenormalization procedures was used for sample clustering. Differentially expressed genes were identified by the analysis of variance. Confirmation by Northern blotting was performed for MAP kinase phosphatase-1, WDNM1 (extracellular proteinase inhibitor), Trop 2 (tumor-associated calcium signal transducer-2), procollagen type IV alpha, secretory leukoprotease inhibitor, prenylated snare protein Ykt6, ceruloplasmin and chaperonin 10. Many of these genes have not previously been associated with tumor invasion and metastasis. We have successfully identified genes that can be linked to the invasive phenotypes or to tumorigenesis. These genes provide a basis for further studies of metastatic progression and local invasiveness, and can be evaluated as therapeutic targets.

Macrophage colony-stimulating factor-1 receptor expression is associated with poor outcome in breast cancer by large cohort tissue microarray analysis.

PURPOSE: Macrophage colony-stimulating factor-1 receptor (CSF-1R) is a transmembrane tyrosine kinase receptor, which is abnormally expressed in invasive breast cancer. Small cohort studies have demonstrated that increased expression of CSF-1R is associated with ipsilateral breast cancer recurrence. Correlation with survival has not been reported. Our aim was to further evaluate the role of CSF-1R in breast cancer, by studying the expression of CSF-1R in a large cohort of clinical specimens. EXPERIMENTAL DESIGN: Tissue microarrays containing 301 node-negative and 280 node-positive cases were used. Immunohistochemical staining was performed and correlated with overall survival, nodal status, and other clinicopathological data. RESULTS: CSF-1R expression was strongly associated with nodal status. Of the node-negative cases, 114 (38.9%) stained positive for CSF-1R, whereas 189 (67.5%) of the node-positive cases expressed CSF-1R (P < 0.0001). CSF-1R expression is also associated with larger tumor size (P = 0.02). Positive staining was strongly associated with decreased survival (P = 0.0003). Among node-negative patients, CSF-1R expression was associated with decreased overall survival (P = 0.045), whereas among node-positive patients, it was not (P = 0.47). In multivariate analysis, CSF-1R was not independent of nodal status as a predictor of survival. CONCLUSIONS: CSF-1R expression is a strong predictor of poor outcome in nonmetastatic breast cancer. It is significantly more frequently expressed in patients with nodal involvement. Among the node-negative patients, it has a stronger association with survival than among the node-positive patients. Our findings support other preclinical findings that CSF-1R may be involved in local invasion and metastasis. Thus, this receptor may be an effective target for therapeutic agents.

Expression of colony-stimulating factor 1 receptor during prostate development and prostate cancer progression.

Colony-stimulating factor-1 receptor (CSF-1R) is the major regulator of macrophage development and is associated with epithelial cancers of the breast and ovary. Immunohistochemistry analysis of murine prostate development demonstrated epithelial expression of CSF-1R during the protrusion of prostatic buds from the urogenital sinus, during the prepubertal and androgen-driven proliferative expansion and branching of the gland, with a decline in older animals. Models of murine prostate cancer showed CSF-1R expression in areas of carcinoma- and tumor-associated macrophages. Several human prostate cancer cell lines and primary cultures of human prostate epithelial cells had low but detectable levels of CSF-1R. Human prostatectomy samples showed low or undetectable levels of receptor in normal glands or benign prostatic hypertrophy specimens. Staining was strongest in areas of prostatic intraepithelial neoplasia or carcinoma of Gleason histological grade 3 or 4. The activated form of the receptor reactive with antibodies specific for phosphotyrosine modified peptide sequences was observed in samples of metastatic prostate cancer. Immunohistochemistry showed strong expression of CSF-1R by macrophage lineage cells, including villous macrophages and the syncytiotrophoblast layer of placenta, Kupper cells in the liver, and histiocytes infiltrating near prostate cancers. These observations correlate CSF-1R expression with changes in the growth and development of the normal and neoplastic prostate.

Total skin electron radiation in the management of mycosis fungoides: Consensus of the European Organization for Research and Treatment of Cancer (EORTC) Cutaneous Lymphoma Project Group.

Radiotherapy has been successfully implemented in the treatment of mycosis fungoides (MF) for almost a century. With the development of the modern linear accelerator, it has become possible to treat extended areas of the skin with accelerated electrons. Total skin electron beam radiation (TSEB) has been in use for several decades, and a number of technical modifications have been made with the goals of optimizing dose distribution and improving clinical outcome. Emerging evidence from recent studies suggests an association between TSEB techniques and efficacy in the treatment of MF. Based on this evidence, the European Organization for Research and Treatment of Cancer Cutaneous Lymphoma Project Group, in association with experts from radiotherapy centers in North America, has reached a consensus on acceptable methods and clinical indications for TSEB in the treatment of MF. The aims of this report are to enhance accessibility of this highly efficacious treatment modality to patients with MF and to provide a point of reference for further clinical research.

Regulation of Fas ligand expression by estrogen in normal ovary.

OBJECTIVE: The clinical significance of the Fas/Fas ligand (FasL) system in hormone-sensitive carcinomas such as breast and ovary has been reported. However, only a few studies have investigated the potential hormonal regulation of its expression. In this study, we evaluated the expression of FasL in normal ovarian tissue during the normal female reproductive cycle with the goal of identifying potential hormones that can regulate FasL expression. METHODS: We used Western blot analysis to examine the expression of FasL in the rat ovary throughout the natural estrous cycle. We employed Western blot and reverse transcriptase-polymerase chain reaction to study hormonal regulation of FasL in human ovarian epithelial cells and normal ovarian tissues. RESULTS: FasL protein expression levels change in the ovary during the female reproductive cycle. FasL protein appeared intensively in estrus, declined sharply in metestrous, further decreased to a very low level in diestrus, and was absent in proestrus. Because the protein expression pattern of FasL in the cycling ovary was similar to the estrogen receptor beta expression pattern, we examined the effect of estrogen on the level of FasL protein and found that estrogen indeed upregulates the expression of FasL protein and mRNA levels in ovarian epithelial cells as well as in normal ovarian tissues. Furthermore, we showed that the estrogen-induced increase in the FasL protein and mRNA levels could be abolished by 4-hydroxytamoxifen, which suggests that the observed increase in FasL expression was mediated by estrogen receptor. CONCLUSION: Our findings support the hypothesis that the expression of FasL in normal ovary is hormonally sensitive and could have a key role in the physiology of normal ovarian tissue.

Hormonal regulation of the c-fms proto-oncogene in breast cancer cells is mediated by a composite glucocorticoid response element.

We have previously reported that glucocorticoids markedly increase and anti-glucocorticoids (such as RU-486) block c-fms RNA and protein expression in some breast cancer cell lines, but not in others, and that this increase is the consequence of increased transcription from the first, epithelial cell-specific promoter of the c-fms gene (encoding CSF-1R, macrophage colony-stimulating factor receptor). Employing DNaseI protection and electrophoretic mobility shift assays (EMSA), we now demonstrate that DNA-transcription factor protein complexes are formed on the c-fms first promoter at a composite regulatory element containing overlapping binding sites for AP-1 proteins, bHLH factors, and the glucocorticoid receptor (GR). Competition studies indicate that transcription factor proteins bind the AP-1 site and the GR element (GRE) and both GR and AP-1 proteins are involved in DNA-protein complex formation. The complexes differ in quantity and glucocorticoid inducibility in the different breast cancer cell lines studied depending on whether the promoter responds to glucocorticoid stimulation. Transient transfection of promoter/reporter gene constructs resulted in reduced basal transcription activity of this promoter and lack of glucocorticoid stimulation when the AP-1 site was mutated. We conclude that AP-1 proteins, GR and associated co-factors regulate transcription from the c-fms first promoter and that differences in recruitment of the various components are responsible for cell specific repression and activation of this gene in breast carcinoma cell lines.