Multiple steroid metabolic pathways in ZR-75-1 human breast cancer cells

In order to characterize the main enzymatic systems involved in androgen and estrogen formation as well as metabolism in ZR-75-1 human breast cancer cells, incubation of intact cells was performed for 12 or 24 h at 37 degrees C with tritiated estradiol (E2), estrone (E1), androst-5-ene-3 beta, 17 beta-diol (5-ene-diol), dehydroepiandrosterone (DHEA), testosterone (T), androstenedione (4-ene-dione), dihydrotestosterone (DHT) or androsterone (ADT). The extra- and intracellular steroids were extracted, separated into free steroids, sulfates and non-polar derivatives (FAE) and identified by HPLC coupled to a Berthold radioactivity monitor. Following incubation with E2, 5-ene-diol or T, E1, DHEA and 4-ene-dione were the main products, respectively, thus indicating high levels of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). When 4-ene-dione was used, on the other hand, a high level of transformation into 5 alpha-androstane-3,17-dione (A-dione), Epi-ADT and ADT was found, thus indicating the presence of high levels of 5 alpha-reductase as well as 3 alpha- and 3 beta-hydroxysteroid dehydrogenase. Moreover, some T was formed, due to oxidation by 17 beta-HSD. No estrogen was detected with the androgen precursors T or 4-ene-dione, thus indicating the absence of significant aromatase activity. Moreover, significant amounts of sulfates and non-polar derivatives were found with all the above-mentioned substrates. The present study shows that ZR-75-1 human breast cancer cells possess most of the enzymatic systems involved in androgen and estrogen formation and metabolism, thus offering an excellent model for studies of the control of sex steroid formation and action in breast cancer tissue.     

Overexpression of PTEN induces cell growth arrest and apoptosis in human breast cancer ZR-75-1 cells

Phosphatase and tensin homolog （PTEN） is a tumor suppressor gene located at human chromosome 10q23, might play an important role in cell proliferation, cell cycle and apoptosis of cancer cells. In this study, the eukaryotic expression vectors pBP-wt-PTEN （containing a wild-type PTEN gene） and pBP-G129R-PTEN （containing a mutant PTEN gene） were used to transfect breast cancer ZR-75-1 cells. After transfection, ZR-75-1 cells expressing PTEN were obtained and tested. The blue exclusion assay showed the growth rate of the cells transfected with pBP-wt-PTEN was significantly lower than that of the control cells transfected with pBP-G129R-PTEN. Analysis of the cell cycle by flow cytometry showed that the progression from the G1 to the S phase was arrested in cells expressing wild-type PTEN. Some typical morphological changes of apoptosis were also observed in cells transfected with pBP-wt-PTEN, but not in those transfected with pBP-G 129R-PTEN. This study shows that overexpression of PTEN in ZR-75-1 cells leads to cell growth arrest and apoptosis.     

Inhibitory effect of estrogens on GCDFP-15 mRNA levels and secretion in ZR-75-1 human breast cancer cells

In order to better understand the mechanisms responsible for the antagonism between steroids in human breast cancer cells, we have studied the effect of 17 beta-estradiol (E2), dihydrotestosterone (DHT), and dexamethasone (DEX) alone or in combination on the expression of the breast gross cystic disease fluid protein-15 (GCDFP-15) in ZR-75-1 cells. Incubation with E2 markedly decreased basal GCDFP-15 mRNA levels accompanied by a parallel inhibition of the secretion of this tumor marker, the estrogenic effect being exerted at a half-maximal concentration of about 44 pM E2. The inhibitory effect of E2 on GCDFP-15 expression was competitively reversed by the antiestrogen LY156758. In addition, 1 nM E2 inhibited the marked stimulation induced by 1 nM DHT or 300 nM DEX on GCDFP-15 mRNA accumulation and on the secretion of the glycoprotein. However, at the concentration used, E2 reversed by only 65% the stimulation achieved by the combination of DHT and DEX on GCDFP-15 mRNA levels. It is of interest to mention that the effect of DHT, DEX, and E2 on GCDFP-15 expression is opposite to the respective effect of each steroid on ZR-75-1 cell proliferation. The present data on the regulation of GCDFP-15 mRNA demonstrate an estrogen-induced inhibition of mRNA levels under physiological conditions, thus offering a unique opportunity to study the mechanisms involved in the down-regulation of gene expression by estrogens and to achieve a better understanding of the antagonism between estrogens, androgens, glucocorticoids, and progestins in breast cancer cells. Furthermore, GCDFP-15 could well be a good marker for monitoring the response to androgens and antiestrogens during the course of breast cancer therapy.     

Wide spectrum of steroids serving as substrates for the formation of lipoidal derivatives in ZR-75-1 human breast cancer cells

Recently, several natural steroids have been found to be esterified to long-chain fatty acids (FAE) in various mammalian tissues. The purpose of the present study was to determine the ability of a series of 3H-labeled steroids to serve as substrates for the formation and accumulation of such non-polar derivatives in intact cells, using the hormone-responsive ZR-75-1 human breast cancer cell line as model. All 14 steroids tested were found to be converted, directly or following further metabolism, to lipoidal ester derivatives. The percentage of intracellular steroids recovered as FAE derivatives was usually substantial (14-90%), especially in the case of C-19 steroids (75-90%). The composition of the lipoidal steroid fractions recovered from the labeled cell extracts was characterized by chromatographic comparison with synthetic steroid FAEs and by saponification of the steroid FAEs and identification of the released steroidal moieties. Following metabolism, most steroid substrates were converted into multiple lipoidal esters. Furthermore, 5 alpha-androstane-3 alpha, 17 beta-diol, 5 alpha-androstane-3 beta, 17 beta-diol, as well as androst-5-ene-3 beta, 17 beta-diol formed lipoidal diesters in addition to the monoester form. The high level of intracellular steroid FAE accumulation reported in this study suggests that these yet poorly known steroid derivatives may play important functions in the regulation of steroid hormone metabolism and action.     

Effects of folate receptor expression following stable transfection into wild type and methotrexate transport-deficient ZR-75-1 human breast cancer cells.

Two biochemically distinct systems, the high affinity folate receptor and the lower affinity reduced-folate carrier, have each been implicated in mediating the transport of folates and antifolates into cells. Previous studies from our laboratory have shown that methotrexate accumulation into wild type (WT) ZR-75-1 human breast cancer cells involves a system with characteristics of the reduced-folate carrier, that this system is deficient in methotrexate resistant (MTXR) ZR-75-1 cells in which methotrexate transport is undetectable and that neither breast cancer cell line expresses folate receptors. In this report we examined the possible interaction of the reduced-folate carrier with folate receptors by stably transfecting both WT ZR-75-1 and MTXR ZR-75-1 cells with an expression vector containing a folate receptor cDNA. Clones of stably transfected MTXR ZR-75-1 and WT ZR-75-1 cells expressing comparable levels of folate receptors were studied and compared to the nontransfected cell lines. Although nontransfected WT and MTXR ZR-75-1 cell lines require concentrations > or = 100 nM folic acid for growth, the expression of folate receptors in transfected WT and MTXR ZR-75-1 cells permitted the growth of both cell lines in low concentrations (1 nM) of folic acid. While the defect in the reduced-folate carrier system in MTXR ZR-75-1 cells inhibits their growth in medium containing low concentrations of folinic acid (< or = 1 microM), MTXR ZR-75-1 cells expressing folate receptors display uninhibited growth in 1 nM folinic acid. The accumulation of folic acid, folinic acid, and methotrexate is enhanced in folate receptor-transfected WT ZR-75-1 cells and MTXR ZR-75-1 cells. Furthermore, the accumulation of folates and antifolate was similar in both transfected WT and MTXR ZR-75-1 cell lines that expressed folate receptors. This suggests that alterations in the reduced-folate carrier do not affect folate receptor function. We also examined the effect of folate receptor expression on the sensitivity of WT and MTXR ZR-75-1 cells to methotrexate and to the lipophillic antifolate trimetrexate. Increased folate receptor expression decreased the sensitivity of WT ZR-75-1 cells toward the antifolate trimetrexate, presumably through increased uptake of reduced folates. Although the expression of the folate receptor enhanced the growth of both cell lines in low folate concentrations, it did not affect the sensitivity of either WT or MTXR ZR-75-1 cells to methotrexate.     

Hypoxia induces proteasome-dependent degradation of estrogen receptor alpha in ZR-75 breast cancer cells

Regulation of estrogen receptor alpha (ERalpha) plays an important role in hormone responsiveness and growth of ER-positive breast cancer cells and tumors. ZR-75 breast cancer cells were grown under conditions of normoxia (21% O(2)) or hypoxia (1% O(2) or cobaltous chloride), and hypoxia significantly increased hypoxia-inducible factor 1alpha protein within 3 h after treatment, whereas ERalpha protein levels were dramatically decreased within 6-12 h, and this response was blocked by the proteasome inhibitor MG-132. In contrast, hypoxia induced only minimal decreases in cellular Sp1 protein and did not affect ERalpha mRNA; however, hypoxic conditions decreased basal and 17beta-estradiol-induced pS2 gene expression (mRNA levels) and estrogen response element-dependent reporter gene activity in ZR-75 cells. Although 17beta-estradiol and hypoxia induce proteasome-dependent degradation of ERalpha, their effects on transactivation are different, and this may have implications for clinical treatment of mammary tumors.     

Role of extracellular electrolytes in the activation of ribosomal protein S6 kinase by epidermal growth factor, insulin-like growth factor 1, and insulin in ZR-75-1 cells

Activation of ribosomal protein S6 kinase by epidermal growth factor (EGF), insulin, and insulin-like growth factor 1 (IGF1) was studied in the human mammary tumor cell line ZR-75-1 in isotonic buffers. In contrast to growth factor-dependent S6 phosphorylation which is strongly dependent on extracellular pH (Chambard, J. C., and J. Pouyssegur. 1986. Exp. Cell Res. 164:282-294.) preincubation of cells in buffers with different pH values ranging from 7.5 to 6.5 had no effect on basal or EGF-stimulated S6 kinase activity. Replacement of extracellular Na+ with choline or replacement of extracellular Ca++ with EGTA also did not inhibit stimulation of S6 kinase by EGF. When intracellular Ca++ was buffered with the permeable Ca++ chelator quin2, EGF stimulation was reduced 50%. A similar inhibition of the EGF response was observed when cells were incubated in buffers with high K+ concentrations or in the presence of the K+ ionophore valinomycin. Insulin and IGF1 stimulation of S6 kinase were also inhibited by high K+ concentrations and by buffering intracellular Ca++. In contrast to the responses to EGF, insulin- and IGF1-activation of S6 kinase was enhanced when glucose was present and depended on the presence of bicarbonate in the medium. The results indicate that ionic signals generated by growth factors and insulin, such as increases in intracellular pH or Na+, do not seem to be involved in the activation of S6 kinase. However, effects of growth factors or insulin on membrane potential and/or K+ fluxes and redistribution of intracellular Ca++ may play a role in the activation process. Furthermore, the mechanism of insulin activation of S6 kinase is distinct from the growth factors by its dependency on extracellular bicarbonate.     

Obligatory requirement of sulfation for P-selectin binding to human salivary gland carcinoma Acc-M cells and breast carcinoma ZR-75-30 cells

Stimulated endothelial cells and activated platelets express P-selectin, which reacts with P-selectin glycoprotein ligand-1 (PSGL-1) for leukocyte rolling on the stimulated endothelial cells and heterotypic aggregation of the activated platelets on leukocytes. P-selectin also binds to several cancer cells in vitro and promotes the growth and metastasis of human colon carcinoma in vivo. The P-selectin/PSGL-1 interaction requires tyrosine sulfation. However, it is unknown whether sulfation is necessary for P-selectin binding to somatic cancer cells. In this study, we show that P-selectin mediated adhesion of Acc-M cells, a cell line derived from a human adenoid cystic carcinoma of salivary gland. These cells had a moderate expression of heparan sulfate-like proteoglycans, but had no detectable expressions of PSGL-1, CD24, Lewis(x), and sialyl Lewis(x). Treatment with sodium chlorate (a sulfation biosynthesis inhibitor), but not 4-methylumbelliferyl-beta-D-xyloside (a proteoglycan biosynthesis inhibitor) or heparinases, reduced adhesion of these cells to P-selectin. Sodium chlorate also inhibited the P-selectin precipitation of the 160-, 54-, and 36-kDa molecules from the cell surface of Acc-M cells. Furthermore, P-selectin could bind to human breast carcinoma ZR-75-30 cells in a sulfation-dependent manner. Our results thus indicate that sulfation is essential for adhesion of nonblood-borne, epithelial-like human cancer cells to P-selectin.     

Overexpression of insulin-like growth factor II (IGFII) in ZR-75-1 human breast cancer cells: higher threshold levels of receptor (IGFIR) are required for a proliferative response than for effects on specific gene expression

Previous transfection experiments using a zinc-inducible expression vector have shown that overexpression of insulin-like growth factor II (IGFII) in MCF7 human breast cancer cells can reduce dependence on oestrogen for cell growth in vitro (DALY RJ, HARRIS WH, WANG DY, DARBRE PD. (1991) Cell Growth Differentiation 2, 457-464.). Parallel transfections now performed into another oestrogen-dependent human breast cancer cell line (ZR-75-1) yielded three clones of transfected ZR-75-1 cells that produced levels of zinc-inducible IGFII mRNA and secreted mature IGFII protein similar to those found in the transfected MCF7 cells. However, unlike in MCF7 cells, no resulting effects were found on cell growth in the ZR-75-1 clones, even though the ZR-75-1 clones possessed receptors capable of binding 125I-IGFI and showed a growth response to exogenously added IGFII. Medium conditioned by the ZR-75-1 clones could stimulate growth of untransfected MCF7 cells, indicating that the secreted IGFII protein was bioactive. Furthermore, zinc-induced IGFII was capable of increasing both pS2 mRNA levels and CAT activity from a transiently transfected AP1-CAT gene in the ZR-75-1 clones. Constitutive co-overexpression of the protein processing enzyme PC2 resulted in reduced levels of large forms of zinc-inducible IGFII, but zinc treatment still produced no effect on cell growth rate. Finally, however, constitutive co-overexpression of the type I IGF receptor (IGFIR) did result in zinc-inducible increased basal cell growth and reduced dependence on oestrogen for cell growth. These results demonstrate that while overexpression of IGFII per se was sufficient to deregulate MCF7 cell growth, the ZR-75-1 cells are limited in their proliferative response by their intrinsic receptor levels. However, although the proliferative response was limited, molecular responses (expression of pS2 and AP1-CAT) were not limited, indicating that different cellular responses can have different threshold receptor level requirements.     

Extensive esterification of adrenal C19-delta 5-sex steroids to long-chain fatty acids in the ZR-75-1 human breast cancer cell line

Estrogen-sensitive human breast cancer cells (ZR-75-1) were incubated with the 3H-labeled adrenal C19-delta 5-steroids dehydroepiandrosterone (DHEA) and its fully estrogenic derivative, androst-5-ene-3 beta,17 beta-diol (delta 5-diol) for various time intervals. When fractionated by solvent partition, Sephadex LH-20 column chromatography and silica gel TLC, the labeled cell components were largely present (40-75%) in three highly nonpolar, lipoidal fractions. Mild alkaline hydrolysis of these lipoidal derivatives yielded either free 3H-labeled DHEA or delta 5-diol. The three lipoidal fractions cochromatographed with the synthetic DHEA 3 beta-esters, delta 5-diol 3 beta (or 17 beta)-monoesters and delta 5-diol 3 beta,17 beta-diesters of long-chain fatty acids. DHEA and delta 5-diol were mainly esterified to saturated and mono-unsaturated fatty acids. For delta 5-diol, the preferred site of esterification of the fatty acids is the 3 beta-position while some esterification also takes place at the 17 beta-position. Time course studies show that ZR-75-1 cells accumulate delta 5-diol mostly (greater than 95%) as fatty acid mono- and diesters while DHEA is converted to delta 5-diol essentially as the esterified form. Furthermore, while free C19-delta 5-steroids rapidly diffuse out of the cells after removal of the precursor [3H]delta 5-diol, the fatty acid ester derivatives are progressively hydrolyzed, and DHEA and delta 5-diol thus formed are then sulfurylated prior to their release into the culture medium. The latter process however is rate-limited, since new steady-state levels of free steroids and fatty acid esters are rapidly reached and maintained for extended periods of time after removal of precursor, thus maintaining minimal concentrations of intracellular steroids. The rapid rate and large extent of esterification of DHEA and delta 5-diol to long-chain fatty acids in breast cancer cells indicate that this reaction could constitute an important regulatory step in the estrogenic action of DHEA and delta 5-diol in these cells.     

Purification and characterization of a novel growth factor from human breast cancer cells.

We have purified and characterized a novel 30-kDa glycoprotein (gp30) with TGF alpha-like properties secreted from the estrogen receptor negative breast cancer cell line MDA-MB-231. This factor was immunoprecipitated by an anti-TGF alpha polyclonal antibody and also had TGF alpha-like biological activity, as assayed by EGF radioreceptor assay and anchorage-independent assays. In addition, the novel growth factor stimulated phosphorylation of the EGF receptor and erbB-2 receptor. However, the novel growth factor, unlike EGF and TGF alpha, bound to heparin-Sepharose. Purification of gp30 was obtained to apparent homogeneity by heparin affinity chromatography and subsequent reversed-phase chromatography. Tunicamycin treatment in vivo or N-glycanase deglycosylation in vitro revealed a putative precursor of approximately 22 kDa molecular mass in contrast to the "normal" 16-kDa precursor species for TGF alpha. In vitro translation of total mRNA from MDA-MB-231 cells confirmed the size of the putative precursor. Biochemical characterization of gp30 was begun by V8 protease digestion of the deglycosylated polypeptide and the translated products. Peptide mapping of V8-digested, immunoprecipitated material suggests that the amino acid sequence of this unique protein is distinct from mature TGF alpha and not the result of a posttranslational modification of the precursor. We conclude that this TGF alpha-like (gp30) polypeptide is a novel growth factor with agonistic activity for both EGF and erbB-2 receptors.     

Insulin-stimulated cell growth in insulin receptor substrate-1–deficient ZR-75-1 cells is mediated by a phosphatidylinositol-3-kinase–independent pathway

In many human breast cancers and cultured cell lines, insulin receptor expression is elevated, and insulin, via its own insulin receptor, can stimulate cell growth. It has recently been demonstrated that the enzyme phosphatidylinositol-3-kinase (PI3-K) mediates various aspects of insulin receptor signaling including cell growth. In order to understand the mechanisms for insulin-stimulated cell growth in human breast cancer, we measured insulin-stimulable PI3-K activity in a non-transformed breast epithelial cell line, MCF-10A, and in two malignantly transformed cell lines, ZR-75-1 and MDA-MB157. All three cell lines express comparable amounts of insulin receptors whose tyrosine autophosphorylation is increased by insulin, and in these cell lines insulin stimulates growth. In MDA-MB157 and MCF-10A cells, insulin stimulated PI3-K activity three- to fourfold. In ZR-75-1 cells, however, insulin did not stimulate PI3-K activity. In ZR-75-1 cells PI3-K protein was present, and its activity was stimulated by epidermal growth factor, suggesting that there might be a defect in insulin receptor signaling upstream of PI3-K and downstream of the insulin receptor. Next, we studied insulin receptor substrate-1 (IRS-1), a major endogenous substrate for the insulin receptor which, when tyrosine is phosphorylated by the insulin receptor, interacts with and activates PI3-K. In ZR-75-1 cells, there were reduced levels of protein for IRS-1. In these cells, both Shc tyrosine phosphorylation and mitogen-activated protein kinase (MAP-K) activity were increased by the insulin receptor (indicating that the p21^ras pathway may account for insulin-stimulated cell growth in ZR-75-1 cells). The PI3-K inhibitor LY294002 (50 μM) reduced insulin-stimulated growth in MCF-10A and MDA-MB157 cell lines, whereas it did not modify insulin effect on ZR-75-1 cell growth. The MAP-K/Erk (MEK) inhibitor PD98059 (50 μM) consistently reduced insulin-dependent growth in all three cell lines. Taken together, these data suggest that in breast cancer cells insulin may stimulate cell growth via PI3-K–dependent or–independent pathways. J. Cell. Biochem. 70:268–280, 1998. © 1998 Wiley-Liss, Inc.     

Secretion of human epidermal growth factor by Corynebacterium glutamicum

AIMS: To examine the secretion of human epidermal growth factor (hEGF) by Corynebacterium glutamicum. METHODS AND RESULTS: We recently showed that a novel protein-secretion system in C. glutamicum could produce Streptomyces mobaraensis transglutaminase. In the present study, the industrially important protein hEGF was secreted into the culture medium in a fully active form by C. glutamicum and accumulated at a rate of up to 156 mg l(-1) day(-1). CONCLUSIONS: These results demonstrated that the hEGF protein could be secreted in an active form by C. glutamicum. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data confirmed that the pharmaceutically important human protein hEGF could be efficiently secreted in an active form by the C. glutamicum protein-expression system. Moreover, we demonstrated that this bacterium has potential as a host for the industrial-scale production of human proteins.     

Characterization of insulin-like growth factor binding proteins from human breast cancer cells

The insulin-like growth factor binding proteins (IGF-BPs) are structurally and immunologically distinct from the IGF type 1 or type 2 receptors and are characterized by two major forms: a large, GH-dependent BP found in human plasma (Mr = 150 k) and a small GH-independent BP (Mr = 28-42 k) present in human plasma, amniotic fluid, and HEP G2 cells. Using affinity cross-linking techniques, we have identified several binding proteins secreted by human breast cancer cell lines (Hs578T, MDA-231, T-47D, and MCF-7). Under nonreducing conditions these proteins migrated at an apparent Mr = 35, 28, 27, and 24 k, while reducing conditions revealed bands of apparent Mr = 35, 32, 27, and 24 k. Competitive binding studies in T-47D-conditioned media demonstrated that these BPs bound more IGF-II than IGF-I, and that IGF-II potently inhibited binding of either IGF-I or -II. Immunological studies using a polyclonal antibody against the HEP G2 small BP revealed no immunoreactive BP in conditioned media from MCF-7 and T-47D and only slight immunoreactivity in conditioned media from Hs578T and MDA 231. Analysis by Northern blot, using a probe from the cDNA sequence of the HEP G2 BP, demonstrated that Hs578T and MDA-231 cell lines contained small amounts of the 1.65 kilobase mRNA characteristic of the HEP G2 BP, while MCF-7 and T-47D tested negative.(ABSTRACT TRUNCATED AT 250 WORDS)