Maryann B. Flick

Phenoxodiol - an isoflavone analog - induces apoptosis in chemoresistant ovarian cancer cells

Interference with the innate apoptotic activity is a hallmark of neoplastic transformation and tumor formation. In this study we characterize the cytotoxic effect of phenoxodiol, a synthetic anticancer drug analog of genestein, and demonstrate the mechanism of action by which phenoxodiol affects the components of the Fas apoptotic pathway on ovarian cancer cells. Primary ovarian cancer cells, isolated from ascitic fluids of ovarian cancer patients, resistant to conventional chemotherapy, undergo apoptosis following phenoxodiol treatment. This effect is dependent upon the activation of the caspase system, inhibiting XIAP, an inhibitor of apoptosis, and disrupting FLICE inhibitory protein (FLIP) expression through the Akt signal transduction pathway. We suggest that phenoxodiol is an efficient inducer of cell death in ovarian cancer cells and sensitizes the cancer cells to Fas-mediated apoptosis. We identified FLIP and XIAP signalling pathways as key factors regulating the survival of ovarian cancer cells. These findings demonstrate a novel nontoxic drug that controls FLIP/XIAP function and has the potential to eliminate tumor cells through Fas-mediated apoptosis.

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.

Recognition of activated CSF-1 receptor in breast carcinomas by a tyrosine 723 phosphospecific antibody

The macrophage colony stimulating factor receptor (CSF-1R), the product of the c-fms proto-oncogene, plays an important role in regulating the normal proliferation and differentiation of macrophages and trophoblasts. However, the abnormal expression of CSF-1R transcripts and protein by human breast carcinomas has been shown to correlate with advanced stage and poor prognosis. Ligand activated CSF-1R dimers transphosphorylate several tyrosines in their cytoplasmic domains which provide recognition sites for various effector proteins in multiple signal transduction pathways. In cells transformed by the c-fms oncogene, one of the major CSF-1R phosphotyrosines, pTyr723 is important for phenotypic expression of anchorage-independent growth and metastasis. In order to investigate the relationship between receptor activation/phosphorylation and cellular phenotypes in vitro and in vivo, we prepared a CSF-1R phosphorylation-state specific antibody raised against a specific phosphopeptide of CSF-1R, which included phosphorylated tyrosine 723. On immunoblots of lysates from cells expressing CSF-1R, this antibody recognizes phosphorylated CSF-1R in CSF-1 stimulated cells but not in unstimulated cells. As an immunohistochemical reagent, this antibody stained 52% of invasive human breast tumors (72% of CSF-1R positive cases) in a sample of 114 cases and 38% of carcinoma in situ. This data represents the first direct evidence of in vivo phosphorylation of CSF-1R in human breast carcinomas.

Apoptosis and Cutaneous T Cell Lymphoma

Abstract: The most successful therapies in the treatment of cutaneous T cell lymphoma (CTCL), which include localized X‐ray therapy, total skin electron beam therapy, total skin therapy with psoralen and UVA or other forms of UV light therapy, topical chemotherapy, and even topical steroids, all work by inducing T cell apoptosis. Yet, these same malignant T cells, which readily undergo apoptosis after these therapies, are far less likely to do so after therapy with systemic chemotherapeutic agents that are often curative in B cell lymphomas. Apoptosis in T cells is a normal phenotype in that only a small fraction of nonneoplastic T cells fail to undergo apoptosis. Although we still understand relatively little regarding the molecular biology of CTCL, it is clear that the neoplastic cell clone or clones that predominate in this disease have defects in normal apoptotic control mechanisms. These can be overridden in most cases by the strong proapoptotic signals triggered by ionizing radiation and other agents. Better understanding of the mechanisms by which the latter occurs should help us both improve our current therapies and devise new ones.

Expression of CSF-I and CSF-I receptor by normal lactating mammary epithelial cells.

Objective: Previous studies suggested a potential role for macrophage colony stimulating factor (CSF-1) in lactogenic differentiation of the breast. The aim of this study was to define the regulation of CSF-1 and its receptor (CSF-1R, the product of c-fms proto-oncogene) by lactogenic hormones in the breast in vivo during pregnancy and lactation and in vitro in organ culture and mammary epithelial cell lines. Methods: Immunohistochemical staining assays for the expression of CSF-1 and CSF-1R antigens were performjed on sections of breast biopsies from nonpregnant (n = 10), prepartum (n = 4), and postpartum lactating patients (n = 7_) and on sections of human mammary glands culture in the presence of the lctogenic hormones insulin, prolactin, and glucocorticoids. Northern blot analyses were used to study the regulation of CSF-1 and CSF-1R by these same lactogenic hormones in normal and neoplastic mammary epithelial cell lines in cell culture. Results: Normal, nonlactating mammary epithelium did not express CSF-1R and synthesized only low levels of CSF-1. During lactation, significant levels of both proteins could be observed in the epithelial cells that line actively lactating ducts and alveoli. Very similar increases in epithelial cell expression of CSF-1 and CSF-1R were observed in organ cultures of normal mammary gland biopsies exposed to prolactin, insulin, and glucocorticoids. Colony stimulating factor mRNA levels were increased by prolactin and/or insulin in a normal mammary epithelial cell line, while glucocorticoids had no apparent effect on CSF-1 mRNA levels. In contrast, we found that the levels of CSF-1R transcript are regulated primarily by glucocorticoids in breast carcinoma cells, while prolactin merely modulates the glucocorticoid effect. Conclusion: The observed increases in the expression of CSF-1 and its receptor during lactogenesis and the regulation of CSF-1/CSF-1R by lactogenic hormones suggests that this cytokine/receptor pair might play a regulatory role in the cellular events leading to the lactogenic differentiation of mammary epithelial cells.

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.

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. J. Cell. Biochem. 85: 10–23, 2002.

Correlation of tumor phenotype with c-fms proto-oncogene expression in an in vivo intraperitoneal model for experimental human breast cancer metastasis

Although proto-oncogene expression has been shown to correlate with clinical outcome in breast carcinoma, an experimental model has not been proposed to study this phenomenon in vivo. In addition, the ability to modulate this proto-oncogene in vivo to correlate with phenotypic behavior has not been determined. Utilizing an intraperitoneal model for metastatic spread with BT20 human breast carcinoma cells, clonally expanded cells expressing five fold higher c-fms protein were compared with parent BT20 cells as well as an underexpressing clone using intrasplenic injection following left flank cut-down in female nude and Severe combined immunodeficient (SCID) mice. Athymic BALB/c nude and SCID animals were observed for clinical evidence of tumorigenicity with necropsy performed at either 50 or 80 days unless compromised earlier. Immunohistochemistry (IHC) of the harvested tumors was performed to correlate c-fms expression from its original in vitro culture to the in vivo model. At day 50, differences in primary tumor take and spread to the pelvis were already evident favoring the c-fms over-expression group with IHC of these tumors revealing significantly higher intensity of staining for c-fms, (mean H score of 205 vs. 43 in the over-expression and parent groups, respectively). At day 80, tumor take and spread was comparable; however, tumor size in the over-expression group was significantly larger than the parent and under-expressing group in both the BALB/c and SCID experiments. Modulation of c-fms proto-oncogene expression was also achieved using the anti-glucocorticoid, RU-486, via oral administration to SCID mice with subsequent correlation to IHC staining. This model thus provides tumors of significant size and organ diversity which retain their phenotype early in tumorigenesis allowing an early endpoint to assess efficacy of novel treatments.

RU-486 Can Abolish Glucocorticoid-Induced Increases in CSF-1 Receptor Expression in Primary Human Breast Carcinoma Specimens

OBJECTIVE: We present the results of the application of organ culture techniques previously described in this journal to the study of steroid hormone responsiveness of primary breast carcinoma specimens. METHODS AND RESULTS: Nearly all breast carcinomas that express macrophage colony-stimulating factor-1R (CSF-IR) at tissue harvest (15 of 18) had levels of CSF-1R expression lowered after incubation in steroid-free media. The decrease in CSF-1R expression was reversed by treatment with glucocorticoids; this glucocorticoid-induced increase in CSF-1R expression can be blocked by mifepristone (RU-486), a competitive inhibitor of glucocorticoid action. CONCLUSION: These results demonstrate that steroid hormone responsiveness of primary breast carcinomas can be assayed in vitro, a result which can not only be employed to better predict the responsiveness of breast carcinomas to therapies with steroid hormone agonists and antagonists, but also suggests that the therapeutic utility of mifepristone in breast cancer deserves further study.