Double level fractures of the femur treated with closed intramedullary nailing

Breast cancer patients receiving tamoxifen (Tam) are at an increased risk for developing endometrial carcinomas, possibly due to the partial estrogenic effect of Tam on endometrial cells. Progestational therapy has not routinely been included in Tam regimens. It was our aim to determine the presence of estrogen receptors (ERs) and progesterone receptors (PRs) in normal and abnormal endometria from postmenopausal women with breast cancer who were treated with Tam. Standard immunohistochemical staining of ERs and PRs was performed on paraffin sections from formalin-fixed uterine curettings or hysterectomy specimens from 40 patients who had received 20-40 mg of Tam daily for a minimum of 3 months. For comparison, normal endometria from 20 women who had not received Tam (11 premenopausal, 9 postmenopausal) were also studied for ER and PR expression. Staining was evaluated using semiquantitative immunoreactivity scores (IRS) ranging from 0 (negative) to 12 (strongly positive). In the group of patients receiving Tam, ERs and PRs were detected in the nuclei of glandular cells in 24/24 cases of endometrial atrophy (ER/PR-IRS, 2-12), in 8/8 endometrial polyps (ER-IRS, 6-12; PR-IRS, 4-12), in 4/4 adenomatous endometrial hyperplasias (ER-IRS, 3-8; PR-IRS, 1-12), and in 4/4 well-differentiated endometrioid adenocarcinomas (ER-IRS, 2-12; PR-IRS, 6-8). Of the 11 endometria from premenopausal patients who had not received Tam, 8 were ER+/PR+ (ER-IRS, 1-12; PR-IRS, 1-12), 1 was ER+/PR- (ER-IRS, 3; PR-IRS, 0), 1 was ER-/PR+ (ER-IRS, 0; PR-IRS, 2), and 1 was ER-/PR- (ER/PR-IRS, 0). Among 9 atrophic endometria from women not treated with Tam, 6 were ER+/PR+ (ER-IRS, 4-12; PR-IRS, 3-6), 1 was ER+/PR- (ER-IRS, 4; PR-IRS, 0), and 2 were ER-/PR- (ER/PR-IRS, 0). The consistent finding of ER and PR expression in endometria from postmenopausal women receiving Tam further supports the suspected estrogenic effect exerted by Tam on endometrial cells. Progestational therapy could be beneficial in the prevention of Tam-induced abnormal endometrial proliferations.     

Genetic data showing evolutionary links between Leishmania and Endotrypanum

We have established and characterized 3 new breast-cancer cell lines from pleural effusions of patients with advanced breast cancer. All 3 cell lines, designated IBEP-1, IBEP-2 and IBEP-3, showed typical ultrastructural characteristics of epithelial mammary tumor cells. Electron microscopy showed, among other characteristics, the presence of numerous microvilli, desmosomal junctions, intracytoplasmic duct-like vacuoles, well-developed endoplasmic reticulum and large nuclei. Immunohistochemical and biochemical studies revealed that the 3 cell lines expressed cytokeratin, epithelial membrane antigen, CEA and CA 15-3, but all showed negative immunoreaction for vimentin. On the other hand, other antigens (LEU-M1, GCDFP 15, c-erbB-2) were expressed by some of the cell lines, but in a variable manner. Ploidy studies confirmed the neoplastic origin of the cell lines. The doubling times were 68 hr for IBEP-1, 29 hr for IBEP-2 and 39 hr for IBEP-3. Only IBEP-2 cells expressed estrogen receptors (ER+), which were down-regulated after preincubation with E2, but they did not express progesterone receptors (PgR-). IBEP-1 and IBEP-3 cells were ER- but expressed PgR (PgR+). In these 2 cell lines, PgR were down-regulated after pre-incubation of the cells with progesterone (10(-8) M) for 24 hr. Estradiol (E2) increased the proliferation rate of IBEP-2 cells and progesterone increased the proliferation of IBEP-I and -3 cell lines. S.C. injection of the 3 IBEP cell lines into nude mice resulted in the growth of solid tumors between 11 and 16 weeks after inoculation. These cell lines could thus be new models for studying various aspects of the biology and the tumorigenicity of breast-cancer cells. A major interest of these new cell lines is that 2 of them were ER- and PgR+, which is an exceptional phenotypic feature. These 2 cell lines could be interesting models for studying the regulation of PgR and the effects of progestins and antiprogestins independently of the presence of ER.     

Changes in radiation sensitivity and steroid receptor content induced by hormonal agents and ionizing radiation in breast cancer cells in vitro

Possible influences of tamoxifen and estradiol on in vitro radiation sensitivity and cellular receptor content after irradiation and/or tamoxifen treatment were studied in breast cancer cell lines; estrogen receptor (ER) and progesterone receptor (PgR) positive cell lines MCF-7 and MCF-7/TAM(R)-1 and the ER and PgR negative cell line MDA-MB-231. The tamoxifen resistant MCF-7/TAM(R)-1 cells were more resistant to ionizing radiation than the MCF-7 and MDA-MB-231 cells. Exposure to tamoxifen made the MCF-7 cells more radiation resistant, while estradiol made the MDA-MB-231 cells more radiation sensitive. A radiation dose of 6 Gy reduced the ER content in cytosol in both MCF-7 and MCF-7/TAM(R)-1 cells, but brought no alterations to the PgR content. In MCF-7/TAM(R)-1 cells tamoxifen exposure significantly increased the ER and reduced the PgR content, an effect not observed in the MCF-7 cells. To conclude, the present study indicates that irradiation and tamoxifen may modify the ER and PgR content in cytosol in breast cancer cells. Hormonal treatment may alter the radiation sensitivity, even in ER negative cells, suggesting that hormonal agents may act both via receptor and non-receptor binding mechanisms.     

The expression of thymidine phosphorylase/platelet-derived endothelial cell growth factor is correlated to angiogenesis in breast cancer

It has been shown that human thymidine phosphorylase (TP) is identical to platelet-derived endothelial cell growth factor and has angiogenic activity. In the present study, the expression of TP was examined in 139 mammary carcinomas and 35 benign mammary disorders using biochemical and immunohistochemical methods. Moreover, in order to evaluate the significance of TP expression in mammary carcinomas, the relationship between vascular density and various clinicopathological factors, including age and menopausal status of patients with a mammary carcinoma, were compared with the size, nodal status, expression of estrogen receptor (ER), progesterone receptor (PgR), c-erbB-2, p53 and TP of a mammary carcinoma. Thymidine phosphorylase expression increased in both the nuclei and cytoplasm of mammary carcinoma cells in comparison to mammary benign disorder cells. The number of microvessels in mammary carcinomas was generally correlated to the number of tumor cells with TP expression in cytoplasm. The number of cells with TP expression in cytoplasm was significantly large in tumors that measured 3-4 cm in diameter, compared with tumors measuring 1-2 and 5-6 cm in diameter. In mammary tumors of 1-4 cm diameter, TP expression and vessel density were significantly high in tumors negative for ER or positive for c-erbB2 and in tumors positive for TP or c-erbB2, respectively; whereas tumors of 5-6 cm in diameter were not modified by any clinicopathological factors. The results indicated that TP plays an important angiogenetic role in mammary carcinomas, especially tumors with a certain progression.     

Reported difficulties in access to quality care for children with asthma in the inner city

Hormonal factors have been implicated in the development of both female and male breast cancers (MBC). However, MBCs are rare and seem to have different biological behavior than those of females. The aim of this study was to evaluate proliferative activity and to establish an association with steroid hormone receptor concentration and clinicopathological parameters in MBC. Proliferative activity was assessed in 18 MBC by mitotic figure counts and immunohistochemical evaluation of MIB-1 and proliferating cell nuclear antigen (PCNA). Estrogen (ER), progesterone (PR) and androgen (AR) receptors were evaluated in serial section from the same tumor by immunohistochemistry. PCNA (range 17-73%; mean, 51.6%) and MIB-1 (range 18.5-58%; mean 38.4%) were positive correlated with the mitotic rate. High proliferative activity assessed either by mitotic index or MIB-1 expression was associated with more poorly differentiated tumors. Sixty one percent (11/18) of the tumors were ER+, 72% (13/18) PR+ and 38.5% (5/13) AR+. Proliferative activity in tumors displaying ER+/PR+ phenotype showed a tendency to be higher than in ER-/PR- tumors. This difference was statistically significant when MIB-1 expression was used as proliferation marker. An association between AR concentration and age at diagnosis was found; in the AR negative group (8/13) mean age at diagnosis was 54.4 +/- 7.3 which was significantly lower than the age of patients with AR+ tumors, 63.2 +/- 11.1 (5/13). Results presented here show that decreased androgen action (AR-) within the breast might contribute to an earlier development of MBC. Besides that, the presence of ER and PR in carcinoma cells is considered to provide a growth advantage as shown by the positive association between the phenotype (ER+/PR+) and high proliferative activity. These results add information for a better understanding of hormonal control of MBC growth and development.     

A paracrine role for the epithelial progesterone receptor in mammary gland development

Recently generated progesterone receptor (PR)-negative (PR-/-) mice provide an excellent model for dissecting the role of progesterone in the development of the mammary gland during puberty and pregnancy. However, the full extent of the mammary gland defect in these mice caused by the absence of the PR cannot be assessed, because PR-/- mice do not exhibit estrous cycles and fail to become pregnant. To circumvent this difficulty, we have transplanted PR-/- breasts into wild-type mice, and we have demonstrated that the development of the mammary gland in the absence of the PR is arrested at the stage of the simple ductal system found in the young virgin mouse. Mammary transplants lacking the PR in the stromal compartment give rise to normal alveolar growth, whereas transplants containing PR-/- epithelium conserve the abnormal phenotype. Chimeric epithelia in which PR-/- cells are in close vicinity to PR wild-type cells go through complete alveolar development to which the PR-/- cells contribute. Together, these results indicate that progesterone acts by a paracrine mechanism on a subset of mammary epithelial cells to allow for alveolar growth and that expression of the PR is not required in all the cells of the mammary epithelium in order for alveolar development to proceed normally.     

Interleukin 6 acts as a paracrine growth factor in human mammary carcinoma cell lines

The effect of interleukin 6 (IL-6) on normal and human mammary carcinoma epithelial cells was studied. IL-6 inhibited the growth of estrogen receptor-positive [ER(+)] breast cancer cell lines, which underwent apoptosis with prolonged treatment. In contrast, ER(-) breast cancer cell lines were resistant to IL-6-mediated growth inhibition. By examining the components of the IL-6 receptor (IL-6R) system, we found that ER(+) breast cancer cells expressed predominantly soluble IL-6Ralpha, whereas the ER(-) breast cancer cells expressed primarily the transmembrane form of the IL-6R, gp130. In addition, detectable levels of IL-6 were secreted into the medium by ER(-) but not ER(+) breast cancer cells. Furthermore, the supernatant obtained from IL-6-secreting, ER(-) cells suppressed the growth of IL-6-sensitive, ER(+) breast cancer cells in a paracrine fashion. Although IL-6 is secreted by ER(-) breast cancer cells, this cytokine does not seem to stimulate the proliferation of these cells in an autocrine fashion. These studies indicate that IL-6 can regulate the growth of normal and transformed human mammary epithelial cells differentially, and that IL-6 secretion by some ER(-) breast cancer cells can function as a paracrine growth factor, suppressing the growth of ER(+) breast cancer cells in vitro.     

Establishment and characterization of six new human endometrial adenocarcinoma cell lines

The endometrial carcinoma cell lines EC-MZ-1, 2, 3, 5, 9, and 11 were established between 1986 and 1990. Four cell cultures were started from endometrial tissue, one from ascites, and one from a lymph node metastasis. Lines have to date been subcultured up to 180 times and the doubling time varies between 26 hr and 3 weeks. Immunocytochemically the coexpression of cytokeratin (predominantly simple-epithelial cytokeratin polypeptides) and vimentin intermediate filaments was detectable in all cell lines, but three lines (EC-MZ-5, 9, 11) expressed vimentin only at low level. By transmission electron microscopy the tumor cells exhibited features of epithelial differentiation. After subcutaneous transplantation into nude mice three lines (EC-MZ-1, 2, 5) produced slow-growing tumors. The histological classification of these tumors ranged from moderately differentiated adenocarcinoma to undifferentiated carcinoma and closely corresponded to the original tumor. Even after long-term in vitro culture, without any addition of estrogens to the culture medium, the moderately differentiated receptor-positive cell line (EC-MZ-2) retained its morphological differentiation. The cells were propagated without estrogens in the culture medium. The estrogen and progesterone receptor levels of cultured cells were determined. Three lines (EC-MZ-1, 2, 3) were positive for the progesterone receptor in low passage number only, the other cell lines were negative for both receptors. The transplantable lines were investigated for hormonal receptor expression in ovariectomized nude mice. In the moderately differentiated cell line (EC-MZ-2) we observed an enhanced expression of the estrogen receptor under optimal stimulation of the nude mouse with estradiol benzoate. There was no effect on the expression of the progesterone receptor.     

Angiostatin-mediated suppression of cancer metastases by primary neoplasms engineered to produce granulocyte/macrophage colony-stimulating factor

We determined whether tumor cells consistently generating granulocyte/macrophage colony- stimulating factor (GM-CSF) can recruit and activate macrophages to generate angiostatin and, hence, inhibit the growth of distant metastasis. Two murine melanoma lines, B16-F10 (syngeneic to C57BL/6 mice) and K-1735 (syngeneic to C3H/HeN mice), were engineered to produce GM-CSF. High GM-CSF (>1 ng/10(6) cells)- and low GM-CSF (<10 pg/10(6) cells)-producing clones were identified. Parental, low, and high GM-CSF-producing cells were injected subcutaneously into syngeneic and into nude mice. Parental and low-producing cells produced rapidly growing tumors, whereas the high-producing cells produced slow-growing tumors. Macrophage density inversely correlated with tumorigenicity and directly correlated with steady state levels of macrophage metalloelastase (MME) mRNA. B16 and K-1735 subcutaneous (s.c.) tumors producing high levels of GM-CSF significantly suppressed lung metastasis of 3LL, UV-2237 fibrosarcoma, K-1735 M2, and B16-F10 cells, but parental or low-producing tumors did not. The level of angiostatin in the serum directly correlated with the production of GM-CSF by the s.c. tumors. Macrophages incubated with medium conditioned by GM-CSF- producing B16 or K-1735 cells had higher MME activity and generated fourfold more angiostatin than control counterparts. These data provide direct evidence that GM-CSF released from a primary tumor can upregulate angiostatin production and suppress growth of metastases.     

Methylation of estrogen and progesterone receptor gene 5' CpG islands correlates with lack of estrogen and progesterone receptor gene expression in breast tumors

Hormonal factors have a profound influence on the development, treatment, and outcome of breast cancer. The absence of steroid hormone receptors is highly correlated with resistance to antihormonal treatments. Work in cultured human breast cancer cell lines has shown that the absence of estrogen receptor (ER) gene expression in ER- cells is associated with extensive methylation of the ER gene 5' CpG island, and treatment with agents that demethylate the ER gene CpG island results in the production of functional ER protein. The current study shows that CpG islands in the 5' region of the ER and progesterone receptor (PR) genes are methylated in a significant fraction of primary human breast cancer tissues. The ER CpG island is methylated at the methylation-sensitive NotI restriction site in 9 of 39 (25%) of primary ER- breast cancers but remains unmethylated in 53 ER+ breast cancers and 9 normal breast specimens. Three methylation-sensitive restriction sites in the PR gene CpG island are not methylated in normal breast specimens and PR+ human breast cancers but are hypermethylated in 40% of PR- human breast tumors. These data demonstrate that methylation of the ER and PR gene CpG islands is associated with the lack of ER and PR gene expression in a significant fraction of human breast cancers.     

A novel retinoic acid receptor-selective retinoid, ALRT1550, has potent antitumor activity against human oral squamous carcinoma xenografts in nude mice

We have identified a novel retinoid, ALRT1550, that potently and selectively activates retinoic acid receptors (RARs). ALRT1550 binds RARs with Kd values of approximately equal to 1-4 nM, and retinoid X receptors with low affinities (Kd approximately equal to 270-556 nM). We studied the effects of ALRT1550 on cellular proliferation in squamous carcinoma cells. ALRT1550 inhibited in vitro proliferation of UMSCC-22B cells in a concentration-dependent manner with an IC50 value of 0.22 +/- 0.1 (SE) nM. 9-cis-Retinoic acid (ALRT1057), a pan agonist retinoid that activates RARs and retinoid X receptors, inhibited proliferation with an IC50 value of 81 +/- 29 nM. In vivo, as tumor xenografts in nude mice, UMSCC-22B formed well-differentiated squamous carcinomas, and oral administration (daily, 5 days/week) of ALRT1550, begun 3 days after implanting tumor cells, inhibited tumor growth by up to 89% in a dose-dependent manner over the range of 3-75 micrograms/kg. ALRT1550 (30 micrograms/kg) also inhibited growth of established tumors by 72 +/- 3% when tumors were allowed to grow to approximately equal to 100 mm3 before dosing began. In comparison, 9-cis retinoic acid at 30 mg/kg inhibited growth of established tumors by 73 +/- 5%. Interestingly, retinoids did not appear to alter tumor morphologies in UMSCC-22B tumors. Notably, ALRT1550 produced a therapeutic index of approximately equal to 17 in this model, indicating a separation between doses that inhibited tumor growth and that induced symptoms of hypervitaminosis A. In summary, ALRT1550 potently inhibits cellular proliferation in vitro and in vivo in this squamous cell carcinoma tumor model. These data support additional study of ALRT1550 for its potential for improving anticancer therapy in human clinical trials.     

Effects and metabolism of steroid hormones in human neuroblastoma cells

The development of the central nervous system is influenced by sex steroids and by their metabolites. However, little information on the possible effects of steroid hormones on neuroblastoma cells is available. Human neuroblastoma cell lines have been used as a model of human neuroblasts in vitro to study the metabolism of steroid hormones; in addition, the effects of steroids and steroid antagonists on neuroblastoma cell growth have also been investigated. The results obtained show that SH-SY5Y human neuroblastoma cells may actively metabolize testosterone and progesterone to their respective 5 alpha-reduced metabolites and that differentiation of neuroblastoma cells is paralleled by a significant increase in expression of the type-1 5 alpha-reductase and of the formation of steroid metabolites. All these data are suggestive of a potential role of steroid 5 alpha-reduced metabolites in the biology of neuroblastoma cells. Studies performed to analyze the role of steroid hormones on neuroblastoma cell proliferation show that progesterone at low doses may induce minor stimulation, and at higher doses, a toxic effect on the neuroblastoma cell line SK-N-SH is seen. Moreover, the antiprogestin 17 beta-hydroxy-11 beta-(4-dimethylamino-phenyl-1)-17-(prop-1-ynyl)estra-4,9-dien+ ++-3-one (RU486) decreases the proliferation of these cells in a dose-dependent manner. The effect of RU486 is not antagonized by either progesterone or dexamethasone, a result that seems to exclude the action of RU486 via classic intracellular steroid hormone receptors.     

Synthesis and in vitro antitumor activity of some amino-deoxy 7-hexofuranosylpyrrolo[2,3-d]pyrimidines

7-(6-amino-6-deoxy-beta-D-glucofuranosyl)-5-cyanopyrrolo[2,3 -d]pyrimidine (22) and 7-(3-amino-methyl-3-deoxy-beta-D-allofuranosyl)-5- cyanopyrrolo[2,3-d]pyrimidine (28) were synthesized by sequentially coupling silylated 4-amino-6-bromo-5- cyanopyrrolo[2,3-d]pyrimidine with the corresponding protected sugars 9 and 17, followed by deblocking and catalytic hydrogenation. Conversion of the 5-nitrile in 22 and 28 into a carboxamide gave the corresponding sangivamycin derivatives 23 and 29. Whereas 5'-aminomethyl nucleosides 22 and 23 inhibited the growth of four different human tumor cell lines at microM concentrations, the 3'-aminomethyl analogs 28 and 29 were much less active against these cells.     

Proprotein-processing endoprotease furin controls growth of pancreatic beta-cells

We have previously reported that in the well-differentiated beta-cell line MIN6 cells, the beta-cell-specific differentiated characteristics, such as insulin content, expression of prohormone convertases PC2 and PC3, and glucose-regulated insulin secretion, diminished when the proprotein-processing endoprotease furin was highly expressed. Since furin converts many growth-related protein precursors to their bioactive forms, we compared the four pancreatic islet cell lines RINm5F, betaTC3, betaHC9, and MIN6 with respect to cell growth rate, furin expression, endoprotease activity, and insulin content. RINm5F cells exhibited the strongest furin expression, higher furin-type endoprotease activity, and the fastest cell growth, but had the least insulin content. In contrast, MIN6 cells exhibited only a weak furin expression, little furin-type endoprotease activity, and the slowest cell growth, but had the highest insulin content. To test whether furin-expressing cells secrete growth-promoting factors cleaved by furin, we prepared conditioned media from RINm5F and furin cDNA-introduced MIN6 (MIN6-F) cells. The conditioned media from RINm5F and MIN6-F induced increased DNA synthesis and promoted the growth of normal MIN6 cells, compared with the medium from the empty vector-introduced MIN6-0 cells. We then examined the effect of the protease inhibitors alpha1-antitrypsin and its variants by infecting their vaccinia recombinants to the four cell lines. All conditioned media from each cell line expressing the furin-specific alpha1-antitrypsin variant exhibited the least DNA synthetic capacity on normal MIN6 cells. Furthermore, all three sublines of MIN6-F grew faster than MIN6-0 and MIN6. Thus, we suggest that the islet cells with higher furin expression may induce increased production of growth factors, which result in an increase in cell growth, through an autocrine/paracrine mechanism.     

A clinico-radiological correlation of breast diseases during lactation and the significance of unilateral failure of lactation

OBJECTIVES: The Ishikawa endometrial cancer cell line is hormonally responsive, expressing estrogen and progesterone receptors (ER, PR) when grown in traditional monolayer culture. The purpose of this paper is to demonstrate a three-dimensional spheroid culture system for cancer cells. We used this system to determine the response of the Ishikawa cell line to estradiol-17 beta (E), tamoxifen (T), megestrol acetate (MA), and progesterone (P). METHODS: Ishikawa cells were incubated in polyurethane culture bags using phenol red-free media containing ethanol (0.1%, controls), E (1 mumol, or 1 nmol), T (1 mumol, or 10 nmol), MA (1 mumol, or 10 nmol), or P (1 mumol). Cellular morphology was assessed by hematoxylin and eosin staining, and expression of estrogen and progesterone receptors was determined immunohistochemically using an immunoperoxidase technique. RESULTS: Cells in control cultures demonstrated minimal organization and lacked hormone receptors. In contrast, cells exposed to either E or T displayed significant glandular formation, with multicellular, microvilli-rich, columnar epithelia exhibiting polarized nuclear arrangements. Within 4 weeks, E- and T-treated cultures showed upregulated nuclear staining for PR, with little ER present. Cells treated with MA or P showed less glandular organization but expressed ER with PR downregulation. CONCLUSIONS: These data support the use of this novel three-dimensional culture system to study the modulation of tumor cell biologic activity in response to hormonal agents. Future applications of this model include examining in vitro responsiveness of cancer cell lines to additional biologic agents and chemotherapeutic regimens.     

Lipoxygenase inhibitors prevent lung carcinogenesis and inhibit non-small cell lung cancer growth

The effects of lipoxygenase inhibitors were investigated using human lung cancer cell lines and A/J mice. By RT-PCR, 5-, 12-, and 15-lipoxygenase mRNA was detected in NSCLC cells. NDGA inhibited 5-LO activity in adenocarcinoma cell line NCI-H1264. Using an MTT assay, NDGA, MK591 and AA861 inhibited the growth of NSCLC cell lines tested with IC50 values of 3, 2, and 7 microM, respectively. Using a clonogenic assay, 10 microM NDGA significantly reduced NSCLC colony number. NDGA significantly slowed NSCLC xenograft growth in nude mice. When the tumors were excised and analyzed, nude mice treated with NDGA had significantly more apoptotic figures than did untreated tumors. A/J mice treated with urethane developed adenomas after 4 months and NDGA administration significantly reduced lung adenoma number. These data indicate that lipoxygenase inhibitors inhibit lung cancer growth and prevent lung carcinogenesis.     

A model to describe how a point mutation of the estrogen receptor alters the structure-function relationship of antiestrogens

The antiestrogen tamoxifen [(Z)-1(p-beta-dimethylamino-ethoxyphenyl)-1,2- diphenylbut-1-ene] is an effective anticancer agent for the treatment of hormone responsive breast cancer. Previous studies have demonstrated that a point mutation in the estrogen receptor (ER) resulted in an alteration of the pharmacology of 4-hydroxytamoxifen, the active metabolite of tamoxifen (Jiang et al, Mol Endocrinol 6:2167-2174, 1992). We have extended our studies to evaluate the effect of a point mutation, a Val substitution for Gly at amino acid 400 in the ligand binding domain of ER, on the pharmacology of other antiestrogens in ER stable transfectants derived from the ER-negative breast cancer cell line MDA-MB-231 CL10A. The compounds were tested with or without estradiol-17 beta (E2) for their effects on cell growth in cells expressing the wild type ER (S30) or the mutant ER (ML alpha 2H) or in control antisense ER transfectant AS23 which does not express ER protein. MCF-7 cells, which express the wild type ER, were also used as a control. The growth of AS23 cells was not affected by any of the compounds at a concentration of 1 microM. E2 stimulated the growth of MCF-7 cells but inhibited the growth of ER transfectants S30 and ML alpha 2H. The ML alpha 2H cells were about 10 to 100-fold less sensitive to E2 and antiestrogens than S30 and MCF-7 cells. Keoxifene, an antiestrogen with a high affinity for the ER, maintained antiestrogenic activities in both ER transfectants and MCF-7 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

A role for caveolin in transport of cholesterol from endoplasmic reticulum to plasma membrane

Caveolin is a 22-kDa membrane protein found associated with a coat material decorating the inner membrane surface of caveolae. A remarkable feature of this protein is its ability to migrate from caveolae directly to the endoplasmic reticulum (ER) when membrane cholesterol is oxidized. We now present evidence caveolin is involved in transporting newly synthesized cholesterol from the ER directly to caveolae. MA104 cells and normal human fibroblasts transported new cholesterol to caveolae with a half-time of approximately 10 min. The cholesterol then rapidly flowed from caveolae to non-caveolae membrane. Cholesterol moved out of caveolae even when the supply of fresh cholesterol from the ER was interrupted. Treatment of cells with 10 microg/ml progesterone blocked cholesterol movement from ER to caveolae. Simultaneously, caveolin accumulated in the lumen of the ER, suggesting cholesterol transport is linked to caveolin movement. Caveolae fractions from cells expressing caveolin were enriched in cholesterol 3-4-fold, while the same fractions from cells lacking caveolin were not enriched. Cholesterol transport to the cell surface was nearly 4 times more rapid in cells expressing caveolin than in matched cells lacking caveolin.