Cloning and sequencing of the pyrG encoding cytidine 5''-triphosphate synthetase from Mycobacterium bovis BCG

Treatment of estrogen receptor (ER)-negative MDA-MB-468 human breast cancer cells with 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced formation of a nuclear aryl hydrocarbon (Ah) receptor complex as determined by ligand-binding and gel electrophoretic mobility shift assays. TCDD also induced CYP1A1-dependent activity in MDA-MB-468 cells, which represents the first ER-negative Ah receptor-positive human breast cancer cell line that has been identified. Treatment of this cell line with TCDD and related compounds also caused a 50% inhibition of cell growth, which resembled the growth inhibitory effects previously reported for epidermal growth factor (EGF). However, EGF expression is minimal in this cell line and is not induced by TCDD; moreover, EGF and TCDD induced a different pattern of oncogene expression and apoptosis in MDA-MB-468 cells. In contrast, TCDD caused a rapid and sustained induction of transforming growth factor alpha (TGF alpha) gene expression and secreted protein (nearly 2-fold); moreover, the growth-inhibitory effects of TCDD could be blocked by antibodies to the EGF receptor. In a separate experiment, it was shown that TGF alpha also inhibited growth of MDA-MB-468 cells. The results of this study indicate that the mechanism of growth inhibition of MDA-MB-468 cells by TCDD is due to induction of TGF alpha, which is a potent antimitogen in this cell breast cancer line.     

EGF-related peptides are involved in the proliferation and survival of MDA-MB-468 human breast carcinoma cells

A majority of human breast carcinomas co-express the epidermal growth factor (EGF)-like peptides CRIPTO (CR), amphiregulin (AR) and transforming growth factor alpha (TGF-alpha). MDA-MB-468 breast carcinoma cells express CR, AR and TGFalpha, while SK-BR-3 cells express CR and TGF-alpha. Anti-sense phosphorothioate oligodeoxynucleotides (AS S-oligos) directed against either CR or TGF-alpha inhibit the proliferation of both cell lines. A 40-50% growth inhibition was observed at a 2-microM concentration of each AS S-oligo. Treatment with the AR AS S-oligo also resulted in a significant inhibition of MDA-MB-468 anchorage dependent growth (ADG). No significant growth inhibition was observed when MDA-MB-468 or SK-BR-3 cells were treated with a mis-sense S-oligo. The AS S-oligos inhibited the expression of AR, CR or TGF-alpha proteins and mRNAs, as assessed by immuno-cytochemistry and semi-quantitative RT-PCR. An additive growth-inhibitory effect was observed when MDA-MB-468 cells were treated with a combination of EGF-related AS S-oligos. Indeed, treatment of MDA-MB-468 cells with a combination of AR, CR and TGF-alpha AS S-oligos resulted in about 70% growth inhibition at a concentration of 0.7 microM each. Finally, treatment of MDA-MB-468 cells with a combination either of the 3 AS S-oligos or of an EGF receptor-blocking antibody (MAb 225) and either CR, AR or TGFalpha AS S-oligos resulted in a significant increase in DNA fragmentation. Our data suggest that the EGF-related peptides are involved in the proliferation and survival of breast carcinoma cells.     

Venous insufficiency in male workers with a standing profession. Part 1: epidemiology

We have previously reported that human breast carcinoma (HBC) cell lines expressing the mesenchymal intermediate filament protein vimentin (VIM+) are highly invasive in vitro, and highly metastatic in nude mice when compared to their VIM- counterparts. Since only VIM+ cell lines can be induced to activate matrix metalloproteinase-2 (MMP-2) upon stimulation with Concanavalin A (Con A), we have examined here membrane type 1 MMP (MT1-MMP), a cell surface activator of MMP-2. Northern analysis reveals baseline expression of MT1-MMP in five of the six VIM+ cell lines studied (MDA-MB-231, MDA-MB-435, BT-549, Hs578T, MCF-7(ADR)), each of which showed variable activation of exogenous MMP-2 after treatment with Con A. In contrast, the four VIM-, poorly invasive HBC cell lines studied (MCF-7, T47D, MDA-MB 468, ZR-75-1) lacked baseline MT1-MMP mRNA expression, and showed no induction of either MT1-MMP expression or MMP-2-activation with Con A. Such differential MT1-MMP expression was confirmed in vivo using in situ hybridization analysis of nude mouse tumor xenografts of representative cell lines. Western analysis of the MDA-MB-231 cells revealed baseline membrane expression of a 60 kDa species, which was strongly induced by Con A treatment along with a weaker band co-migrating with that from MT1-MMP-transfected COS-1 cells (63 kDa), presumably representing latent MT1-MMP. MT1-MMP immunofluorescence strongly decorated Con A-stimulated MDA-MB-231 cells in a manner consistent with membranous staining, but did not decorate the unstimulated MDA-MB-231 cells or MCF-7 cells under either condition. Collectively, the results suggest the constitutive production of active MT1-MMP which is unavailable for either MMP-2 activation or immuno-decoration until Con A treatment. Since VIM expression arises by virtue of the so-called epithelial to mesenchymal transition (EMT) in invasive embryonic epithelia, we propose that this represents a major metastasis mechanism in breast carcinomas. MT1-MMP on the surface of such 'fibroblastoid' carcinoma cells may mediate a paracrine loop for the utilization of stromally produced MMP-2, and contribute to the poorer survival associated with VIM+ breast carcinomas.     

Possible involvement of calpain in the growth of estrogen receptor positive breast cancer cells

Calpain (Ca2(+)-activated neutral protease, EC 3.4.22.17) has been reported to hydrolyze the estrogen receptor (ER). However, there has been no report available regarding the role of calpain in the growth of breast cancer cells. To investigate the role of calpain in the growth of various breast cancer cell lines, we employed a synthetic peptide, calpeptin, which is a cell permeable specific inhibitor of calpain. Calpeptin inhibited the cell growth of ER positive breast cancer cells, such as MCF-7, T-47D, and ZR-75-1 in a dose dependent manner in the presence of E2. However, the growth of ER negative breast cancer cells, MDA-MB-231, was not inhibited by calpeptin. It is suggested that calpain plays an important role in the growth of ER positive breast cancer cells.     

Expression of cytosolic sulfotransferases in normal mammary epithelial cells and breast cancer cell lines

Breast cancers require the presence of estrogens for the maintenance of growth at some time in the course of their development, as does normal breast tissue. Sulfation is an important process in the metabolism and inactivation of steroids, including estrogens, because the addition of the charged sulfonate group prevents the binding of the steroid to its receptor. Also, many of the therapeutic and chemopreventive agents used in the treatment of breast cancer are substrates for the sulfotransferases (STs). The activity and expression of four cytosolic STs, which are the human phenol-sulfating and monoamine-sulfating forms of phenol ST (PST), dehydroepiandrosterone ST, and estrogen ST (hEST), were assayed in normal breast cells and in breast cancer cell lines. ST activities and immunoreactivities were assayed in the estrogen receptor-positive human breast cancer cell lines ZR-75-1, T-47D and MCF-7; in the estrogen receptor-negative breast cancer cell lines BT-20, MDA-MB-468, and MDA-MB-231; and in normal human mammary epithelial cells. The PSTs were the most highly expressed ST activities in the breast cancer cell lines, although the levels of activity varied significantly. ZR-75-1 and BT-20 cells possessed the highest levels of activity of the human phenol-sulfating form of PST. The breast cancer cell lines showed only trace levels of dehydroepiandrosterone ST and hEST activities. In contrast, hEST was the only ST detectable in human mammary epithelial cells. Understanding the regulation of ST activity in these breast cancer and normal breast cell lines will improve our knowledge of the role of sulfation in breast cancer and provide a model with which to study the mechanism of action of estrogens in mammary cells.     

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)     

Tumor necrosis factor alpha enhances secretion of transforming growth factor beta2 in MCF-7 breast cancer cells

We studied the effect of tumor necrosis factor alpha (TNF-alpha) on transforming growth factor beta (TGF-beta) secretion by human breast cell lines to further characterize the antitumor effects of TNF-alpha. We found that TNF-alpha increased the secretion of TGF-beta in two established breast cancer cell lines (MCF-7 and ZR-75-1) but not in two immortalized human mammary epithelial cell lines (184B5 and MCF-10A). In MCF-7 cells, TNF-alpha increased the secretion of total TGF-beta 6.1-fold within 72 h in a dose-dependent manner. The secretion of both latent and active forms of TGF-beta was increased, and their ratio altered from 25:1 to 12:1 in the medium. TNF-alpha converted the secretory pattern of TGF-beta by MCF-7 cells from the heterodimeric form TGF-beta1.2 to the homodimeric form TGF-beta2. Immunoblot analysis under nonreducing conditions identified four molecular mass species of TGF-beta secreted in the culture media of untreated MCF-7 cells (238, 210, 40-55, and 25 kDa). Under reducing conditions, three molecular mass species of TGF-beta were identified: 88, 44, and 12 kDa. Gel filtration analysis demonstrated that the secreted TGF-beta within the range of 12-88 kDa was biologically active. TNF-alpha treatment did not alter the size of molecular mass species secreted by MCF-7 cells and did not change steady-state levels of mRNA for TGF-beta1 or TGF-beta2. These findings indicate that TNF-alpha may regulate quantitatively and qualitatively TGF-beta secretion by human breast cancer cells in vitro. The diverse biological activities of TGF-beta may also allow TNF-alpha to regulate the growth and metabolism of human mammary epithelial cells and/or stromal cells in a paracrine manner.     

Identification of HTLV-1-specific CTL directed against synthetic and naturally processed peptides in HLA-B*3501 transgenic mice

Most human breast tumors start as estrogen-dependent, but during the course of the disease become refractory to hormone therapy. The transition of breast tumors from estrogen dependent to independent behavior may be regulated by autocrine and/or paracrine growth factor(s) that are independent of the estrogen receptor (ER). We have investigated the role(s) of NDF (neu-differentiation factor) in the biology of estrogen positive breast cancer cells by using MCF-7 cells as a model system. Treatment of MCF-7 cells with human recombinant NDF-beta 2 (NDF) inhibited the ER expression by 70% and this was associated with growth stimulation in an estrogen-independent manner. To explore the mechanism(s) of action of NDF in MCF-7 cells, we examined the expression of NDF-inducible gene products. We report here that NDF stimulated the levels of expression of a 46 kD protein (p46) (in addition to few minor proteins) in ER positive breast cancer cells including MCF-7, T-47-D, and ZR-75-R cells but not in ER negative breast cancer cells including MDA-231, SK-BR-3, and MDA-468 cells. This effect of NDF was due to induction in the rate of synthesis of new p46. The observed NDF-mediated induction of p46 expression was specific as there was no such effect by epidermal growth factor or 17-beta-estradiol, and inclusion of actinomycin D partially inhibited the p46 induction elicited by NDF. NDF-inducible stimulation of p46 expression was an early event (2-6 h) which preceded the period of down-regulation of ER expression by NDF. These results support the existence of NDF-responsive specific cellular pathway(s) that may regulate ER, and these interactions could play a role(s) in hormone-independence of ER positive breast cancer cells.     

Antiestrogenic effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on 17 beta-estradiol-induced pS2 expression

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exhibits a broad spectrum of antiestrogenic activities in rodents and mammalian cells in culture. The effects of TCDD on 17 beta-estradiol (E2)-induction of pS2, a prognostic marker for breast cancer, were investigated in MCF-7, ZR-75, HeLa, and Hepa 1c1c7 wild-type and mutant cells. These effects were compared to the suppressive activities of the congener, 2,8-dichlorodibenzo-p-dioxin, and the established antiestrogens, ICI 164,384 and tamoxifen, in order to determine the relative potency of TCDD and to distinguish the mechanism of action of Ah receptor-mediated antiestrogens. Treatment of MCF-7 cells with 10 nM TCDD decreased E2-induced secreted pS2 protein levels by 50% and the induction of the transiently transfected -1100 to -86 pS2 promoter-regulated reporter gene (pS2-LUC) by 57%. Comparable effects on PS2-LUC activity were observed in HeLa and ZR-75 cells. In contrast, TCDD had minimal effects on pS2ERE(-405 to -393)-LUC induction, whereas treatment with 10 nM ICI 164,384 caused a 60% decrease in luciferase activity. In Hepa 1c1c7 wild-type and clone 1 (C1) mutant cells, TCDD also reduced E2 induction of pS2-LUC activity but had little effect in clone 4 (C4) or clone 12 (C12) mutant cells. However, suppression was reestablished following transfection of the human Ah receptor nuclear translocator (ARNT) complementary DNA expression vector into C4 cells and the mouse Ah receptor (AhR) complementary DNA expression vector into C12 cells. Induction of pS2-LUC activity by the ligand-dependent and -independent chimeric estrogen receptors (HE15, HE19, ERcVP16, and ERGR) were also used to examine the role of E2 metabolism and the mechanism of TCDD-mediated antiestrogenic activity. Induction by HE15 and ERcVP16 was suppressed by 57 and 74%, respectively, following treatment with TCDD, whereas ICI 164,384 was significantly less effective (38 and 20%, respectively). These results demonstrate a role for the Ah receptor in TCDD-mediated suppression of E2-induced pS2 expression. Data is presented demonstrating that the effect requires sequences within the pS2 promoter other than the estrogen response element and is independent of E2 oxidative metabolism.     

Cellular adaptation to drug exposure: evolution of the drug-resistant phenotype

The efficacy of all chemotherapeutic agents is limited by the occurrence of drug resistance. For etoposide (VP-16), increased expression of MDR-1 or MRP and alterations in topoisomerase IIalpha have been shown to confer tolerance. To further understand resistance to VP-16, three sublines, designated MCF-7-VP17, ZR-75B-VP13, and MDA-MB-231-VP7, were initially isolated as single clones from parental cells by exposure to VP-16. Subsequently, a population of cells from each subline was exposed to 3-fold higher drug concentrations, allowing stable sublines to be established at higher extracellular drug concentrations. Characterization of the resistant sublines demonstrates the adaptation that occurs with advancing drug concentrations during in vitro selections. Reduced topoisomerase II mRNA and protein levels were observed in the initial isolates. This reduction was accompanied by a decrease in topoisomerase II activity and cellular growth rate and was associated with 6-314-fold resistance to topoisomerase II poisons. With advancing resistance, MRP expression increased and VP-16 accumulation decreased. This adaptation allowed for partial restoration of topoisomerase II activity as a result of increased expression (MCF-7-VP17 and ZR-75B-VP13) or hyperphosphorylation (MDA-MB-231-VP7), with a resultant increase in growth rate. In MDA-MB-231-VP7 cells, hyperphosphorylation coincided with increased casein kinase II mRNA and protein levels, suggesting a role for this kinase in the acquired hyperphosphorylation. In this cell line, hyperphosphorylation mediated the increased activity despite a fall in topoisomerase IIalpha protein levels secondary to an acquired 600-bp deletion in one topoisomerase IIalpha allele, which resulted in reduced protein levels. In all three sublines, high levels of resistance were attained as a result of synergism between the reduced topoisomerase IIalpha levels and MRP overexpression. These studies demonstrate how cellular adaptation to increasing drug pressure occurs and how more than one mechanism can contribute to the resistant phenotype when increasing selecting pressure is applied. Reduced expression of topoisomerase II is sufficient to confer substantial resistance early in the selection process, with synergy from MRP overexpression helping to confer high levels of resistance.     

Cytotoxic effects of adenovirus-mediated wild-type p53 protein expression in normal and tumor mammary epithelial cells

To evaluate the effects of the wild-type p53 expression in normal and tumor cells, we have constructed a recombinant adenovirus vector (E1 minus) expressing human wild-type p53 cDNA (AdWTp53). Infection of normal and tumor cells of lung and mammary epithelial origin with AdWTp53 resulted in high levels of wild-type p53 expression. Production of p53 protein following infection was dependent on the dose of AdWTp53 with maximum amounts of p53 produced following infection with 50 plaque-forming units/cell. AdWTp53 infection inhibited the growth of all human cell lines studied. However, tumor cells that were null for p53 prior to infection (H-358 and MDA-MB-157) and tumor cells that expressed mutant endogenous p53 protein (MDA-MB-231 and MDA-MB-453) were more sensitive to AdWTp53 cytotoxicity than cells that contained the wild-type p53 (MCF-7, MCF-10, 184B5, and normal mammary epithelial cells). All cells exhibited WAF1/Cip1 mRNA and protein induction following AdWTp53 infection. AdWTp53-induced cytotoxicity of human tumor cell lines expressing mutant p53 was mediated by apoptosis as revealed by nucleosomal DNA fragmentation analysis. No detectable nucleosomal DNA fragmentation was observed following AdWTp53 infection of human cells expressing wild-type p53. These data suggest that endogenous p53 status is a determinant of AdWTp53-mediated cell killing of human tumor cells.     

Effects of an acute exposure to lindane (gamma-hexachlorocyclohexane) on brain and liver carbohydrate metabolism of rainbow trout

The ZR-75-1 ER positive breast cancer cell line, xenografted in female nude mice, has been used to determine the effect of tamoxifen on cell proliferation (as measured by mitosis) and cell death (as evidenced by apoptosis and necrosis). After 2 days treatment, there was a significant rise in apoptosis (p < 0.05), whereas a fall in mitosis was not apparent until 7 days (p < 0.05). Furthermore there was an increase in the apoptotic:mitotic ratio on day 7 (p < 0.05). These changes antedated tumour regression, which did not reach not significance until day 14. Tamoxifen did not increase necrosis (which significantly decreased in treated tumours once they had regressed (p < 0.01). In contrast tamoxifen treatment of xenografted MDA-MB-231 ER-negative breast cancer cells produced no significant effects on growth, apoptosis, or mitosis. This study presents clear evidence for tamoxifen inducing apoptosis in ZR-75-1 xenografts (but not MDA-MB-231 tumours). Since changes in apoptosis and mitosis antedate tumour regression, their assessment may provide the potential by which to predict tumour response to tamoxifen therapy.     

Activation of tissue-factor gene expression in breast carcinoma cells by stimulation of the RAF-ERK signaling pathway

Tissue factor (TF) is a cell-surface glycoprotein responsible for initiating the extrinsic pathway of coagulation. The overexpression of TF in human malignancy has been correlated with the angiogenic phenotype, poor prognosis, and thromboembolic complications. The mechanisms underlying constitutive expression of TF in cancer cells are poorly defined. We cloned TF cDNA on the basis of its strong expression in metastatic MDA-MB-231 breast carcinoma cells in contrast to its weak expression in non-metastatic MCF-7 cells. Transient transfection analysis showed that TF promoter activity in MCF-7 cells could be stimulated by expression of a membrane-targeted raf kinase (raf-CAAX). raf-induced activity was dependent on the presence of an AP-1/NF-kappaB motif in the TF promoter and was inhibited by dominant-negative mutants of jun and by I-kappaB alpha. MDA-MB-231 cells were found to contain higher levels of ERK1/2 kinase activity than did MCF-7 cells. Electrophoretic mobility shift assays showed that MDA-MB-231 nuclear proteins bound strongly to an oligonucleotide corresponding to the AP-1/NF-kappaB sequence, whereas MCF-7 nuclear extracts showed weak binding to this element. Finally, we showed that TF mRNA levels in MDA-MB-231 cells declined after addition of the mitogen-activated protein kinase kinase inhibitor PD98059. Our data showed that activation of the raf-ERK pathway led to activation of TF expression in breast carcinoma cells and suggested that constitutive activation of this pathway leads to high TF expression in MDA-MB-231 cells.     

Haloperidol and apomorphine differentially affect neuropeptidase activity

The effects of lipid peroxidation and the antioxidant vitamin E contained in LDL isolated from control plasma (LDL--) and from plasma preincubated with 0.5 mmol/ml alpha-tocopherol (LDL+) on the proliferation of estrogen-receptor positive (ER+ : ZR-75, T-47-D, MCF-7) and negative (ER--: HBL-100, MDA-MB-231) human breast cancer cells were studied. Human skin fibroblasts served as controls. Incubation of plasma with 0.5 mmol/ml alpha-tocopherol resulted in a 3-fold increase of its content and a significant reduction in lipid hydroperoxides and conjugated dienes in LDL. Incubation of fibroblasts or ER+ tumor cells with LDL- or LDL+ had an effect on neither cell proliferation nor on the cellular levels of peroxidation products as compared to control incubations in the absence of LDL. In ER- cells, however, LDL+ stimulated the proliferation, whereas LDL- yielded a cytotoxic effect. Moreover, LDL- supplementation resulted in an increase in the content of hydroperoxides and conjugated dienes. LDL+ supplemented cells exhibited hydroperoxide levels in these tumor cells comparable to the basal levels measured in the absence of LDL. Our data suggested that peroxidation products in LDL are cytotoxic to estrogen-receptor negative breast tumor cells and vitamin E counteracts this effect.