Changes in epidermal growth factor receptor expression and response to ligand associated with acquired tamoxifen resistance or oestrogen independence in the ZR-75-1 human breast cancer cell line.

We have examined the expression of receptors for epidermal growth factor (EGFR) by the ZR-75-1 human breast cancer cell line and tamoxifen resistant (ZR-75-9al 8 microM) and oestrogen independent/tamoxifen sensitive (ZR-PR-LT) variants. The parent line expressed a single class of high affinity binding sites (4,340 +/- 460 receptors/cell; Kd 0.23 +/- 0.04 nM). ZR-75-9al 8 microM cells, routinely maintained in medium containing 8 microM tamoxifen, were negative for oestrogen receptor (ER) and progesterone receptor (PGR) and expressed a markedly increased number of EGFR (14,723 +/- 2116 receptors/cell). Receptor affinity was unchanged. Time dependent reversal of the tamoxifen resistant phenotype was accompanied by a return to ER and PGR positivity and a fall in EGFR numbers to parent cell levels. In contrast ZR-PR-LT cells had a greatly reduced EGFR content (803 +/- 161 receptors/cell) accompanying elevated PGR numbers. Pre-treatment of these cells with suramin or mild acid stripping failed to expose receptors which may have been occupied by endogenously produced ligand. Increased proliferation of ZR-75-1 cells treated with EGFR (0.01-10 ng ml-1) was only observed in serum-free medium lacking insulin and oestradiol. Under these conditions untreated cells failed to proliferate. Both variant lines continued to proliferate in serum free medium in the absence or presence of insulin and oestradiol but failed to respond to exogenous EGF.     

Modulation by oestrogen and progestins/antiprogestins of alpha interferon receptor expression in human breast cancer cells

Human breast cancer ZR-75-1 cells expressed 1516 (105) (mean [S.D.]) interferon (IFN) receptors (IFNR) per cell with K_d of 0.61 (0.15) nmol/l. Oestrogen independent ZR-PR-LT and tamoxifen resistant ZR-75-9a1 8 μmol/l cells expressed similar numbers of IFNR. ZR-75-9a1 cells, which had been maintained in the absence of tamoxifen or known oestrogenic activity for 46 weeks, expressed a significantly higher number of IFNR (3170 [315]). Exposure of ZR-75-1 cells to 10⁻⁹ mol/l 17β-oestradiol (E2) led to a consistent reduction in IFNR numbers whilst 10⁻⁶ mol/l tamoxifen slightly increased IFNR expression. Since IFN increases oestrogen receptors in this cell line, IFN and E2 appear to have opposite effects on expression of each others' receptor. 10⁻⁹ mol/l medroxy progesterone acetate and mifepristone significantly increased IFNR numbers whilst ORG 2058 decreased IFNR expression and ZK 98.299 had no effect. Progestin/antiprogestin induced IFNR increase in this cell line correlated with down-regulation of progesterone receptor (PR). Thus an IFN/ER/PR axis may exist in ZR-75-1 cells and variants.     

Combined effects of 1,25-dihydroxyvitamin D3 and tamoxifen on the growth of MCF-7 and ZR-75-1 human breast cancer cells

Summary In the present study we assessed the effect of combined treatment with 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) and tamoxifen (TAM) on the growth of estrogen-responsive (MCF-7) and estrogen-dependent (ZR-75-1) human breast cancer cells. Both basal and 17-estradiol (17-E₂)-stimulated growth were studied. 1,25-(OH)₂D₃ (10^–10 – 10^–7 M) time- and dose-dependently inhibited basal growth of MCF-7 cells, with growth arrest at 10^–7 M. Also, 17-E₂-stimulated growth of MCF-7 and ZR-75-1 cells was inhibited by 1,25-(OH)₂D₃ in a time- and dose-dependent manner. TAM inhibited 17-E₂-stimulated growth of both cell lines and at high concentration (10^–6 M) it also inhibited basal growth of MCF-7 cells. 10^–6 M TAM together with 1,25-(OH)₂D₃ resulted in a further inhibition of basal (MCF-7 cells) as well as 17-E₂-stimulated proliferation (MCF-7 and ZR-75-1 cells) compared to the inhibition by these agents alone. TAM in combination with 10^–7 M 1,25-(OH)₂D₃ resulted in growth arrest of 17-E₂-stimulated growth of MCF-7 cells. The inhibition of basal and 17-E₂-stimulated growth of MCF-7 cells was additive at early time points (4 days), but less than additive at later time points (8–10 days). It was demonstrated that with co-treatment of MCF-7 cells an equipotent inhibition of basal growth could be reached with lower concentrations of 1,25-(OH)₂D₃, compared to treatment with 1,25-(OH)₂D₃ alone. Studies with low concentrations (< 10^–7 M) of TAM revealed a partial estrogenic effect, i.e. stimulation of MCF-7 proliferation in the absence of 17-E₂. This effect, which may resemble TAM-induced tumor flare, was completely prevented by co-treatment with a low concentration of 1,25-(OH)₂D₃ (10^–9 M). Together, these results demonstrate the potent inhibition of breast cancer cell proliferation by 1,25-(OH)₂D₃ combined with TAM and indicate a potential benefit of combining these agents for the treatment of breast cancer.     

High progesterone receptor concentration in a variant of the ZR-75-1 human breast cancer cell line adapted to growth in oestrogen free conditions

Culture of ZR-75-1 human breast cancer cells for 5 days in the absence of oestrogens (phenol red-free medium supplemented with dextran coated charcoal stripped 5% fetal calf serum) resulted in a slowing of growth rate and loss of progesterone receptors. Oestradiol at 10(-9) M markedly stimulated growth and progesterone receptor synthesis over a 5-day period. While medroxyprogesterone acetate (10(-10) to 10(-6) M) inhibited growth of ZR-75-1 cells growing in complete medium, in the short-term absence of oestrogens low concentrations were growth stimulatory. Cells deprived of oestrogens for 5 days retained sensitivity to growth inhibition by 4-hydroxy tamoxifen. ZR-75-1 cells were also adapted to growth in the absence of oestrogens over a 5-month period. These cells (ZR-PR-LT) failed to express binding sites characteristic of the type 1 oestrogen receptor but progesterone receptor expression was at a level normally associated with oestrogen induction. Adapted cells were growth inhibited by oestradiol, 4-hydroxy tamoxifen and medroxyprogesterone acetate, but despite elevated progesterone receptor expression the progestin was only marginally more inhibitory than in the parent line. Our data indicate a poor quantitative relationship between response to progestins in vitro and progesterone receptor concentration and support previous findings that acquisition of an oestrogen independent phenotype does not necessarily result in resistance to anti-oestrogens.     

Bag1 proteins regulate growth and survival of ZR-75-1 human breast cancer cells

Bag1 proteins bind heat shock protein M(r) 70,000 (Hsp 70) family molecular chaperones and regulate diverse pathways involved in cell proliferation, apoptosis, and stress responses. Four isoforms of Bag1 can be produced from a single gene in humans, including a nuclear-targeted long version (Bag1L)and a shorter cytosolic isoform (Bag1). Because overexpression of Bag1and Bag1L has been reported in breast cancers, we explored the effects of Bag1 and Bag1L on the growth of ZR-75-1 human breast cancer cells cultured in vitro and in tumor xenograft models using immunocompromised mice. Cells stably transfected with expression plasmids encoding either Bag1 or Bag1L displayed comparable rates of growth in cultures containing 10% serum, compared with control-transfected ZR-75-1 cells. In contrast, ZR-75-1 cells stably expressing mutants of Bag1 or Bag1L, which lack the COOH-terminal domain (DeltaC) required for heat shock protein M(r) 70,000 binding, displayed retarded growth rates. When cultured without serum, the viability of control-transfected, as well as Bag1DeltaC- and Bag1LDeltaC-expressing, cells declined with time, whereas Bag1- and Bag1L-overexpressing ZR-75-1 cells survived for over a week in culture. Caspase protease activation induced by serum deprivation was also prevented by stable expression of either Bag1 or Bag1L in ZR-75-1 cells. In addition, sensitivity to anchorage dependence was restored partially in ZR-75-1 cells expressing dominant-negative Bag1DeltaC and Bag1LDeltaC. In tumor xenograft studies involving injection of ZR-75-1 cells into mammary fat pads of female nu/nu mice, ZR-75-1 cells expressing Bag1 or Bag1L formed 1.4-1.6-fold larger tumors compared with control-transfected cells, whereas tumors formed by Bag1DeltaC- and Bag1LDeltaC-expressing cells grew very slowly and reached sizes < one-third of tumors generated by Neo-control ZR-75-1 cells. Altogether, these findings demonstrate that Bag1 and Bag1L provoke similar changes in breast cancer cell growth and survival and suggest that interference with Bag1 or Bag1L function might be a useful strategy for opposing breast cancer.     

Ectopic expression of epidermal growth factor receptors induces hormone independence in ZR-75-1 human breast cancer cells.

Epidermal growth factor (EGF) receptor is inversely related to expression of estrogen receptor (ER) and progesterone receptor in primary breast tumors and is a negative predictor for response to endocrine therapy. To investigate a possible causal role of EGF receptor expression in breast cancer progression to hormone independence, we have created an experimental cell system. Epidermal growth factor receptor complementary DNA was introduced in estrogen-dependent ZR-75-1 breast cancer cells, and the resulting ZR/HERc cells exhibited a mitogenic response to epidermal growth factor, thus bypassing estrogen dependence. This EGF-induced proliferation could not be inhibited by antiestrogens. In addition, we noted changes in cell morphology and keratin expression of EGF-stimulated ZR/HERc cells, suggestive of an altered differentiation state. Furthermore, intolerance of functional ER and EGF receptor signal transduction pathways in ZR/HERc cells was observed during simultaneous activation, which possibly explains the inverse relationship of ER and EGF receptor expression in primary tumors. In contrast to the parental cells, ZR/HERc cells rapidly progressed to a stable ER-negative phenotype when cultured in the presence of the antiestrogen hydroxy-tamoxifen. These results suggest a possible role for EGF receptor in progression of breast cancer to hormone independence.     

Antiproliferative effects of the somatostatin analogue octreotide (SMS 201-995) on ZR-75-1 human breast cancer cells in vivo and in vitro.

The somatostatin analogue octreotide (SMS 201-995) inhibits secretion and growth of certain tumor cells, and current efforts are directed toward the elucidation of its mode of antiproliferative action. In this study, the effect of octreotide on the growth of ZR-75-1 human breast cancer cells has been characterized in immunodeficient nude mice and in cell culture. These results have been related to the expression of somatostatin receptors in vivo and in vitro. Continuous infusion of 10 micrograms/kg/h of octreotide yielded plasma levels of 5.7 ng/ml and elicited highly significant growth inhibitory effects on solid ZR-75-1 breast tumors in nude mice. After 2 and 4 weeks of treatment, tumor volumes in the octreotide group were 39.1 and 36.7% of those of control animals treated with vehicle, respectively. Autoradiographic studies demonstrated that 8 of 12 ZR-75-1 tumors studied were somatostatin receptor positive. When ZR-75-1 tumor cells were exposed in vitro to nanomolar concentrations of octreotide, a dose-dependent inhibition of cell growth was observed in the presence of 5% fetal calf serum or under serum-free conditions using epidermal growth factor, insulin-like growth factor type I, or insulin as growth stimulus. In parallel receptor-binding experiments, ZR-75-1 cells were shown to express specific high-affinity somatostatin receptors (Kd value = 0.9 nM, Bmax = 6000 sites/cell). From these experiments, we conclude that octreotide is a powerful inhibitor of ZR-75-1 tumor cell growth in nude mice and in culture. This inhibitory action of octreotide and the presence of somatostatin receptors on ZR-75-1 tumor cells in vitro and in vivo suggest a direct, somatostatin receptor-mediated effect of octreotide.     

Characterisation of a tamoxifen-resistant variant of the ZR-75-1 human breast cancer cell line (ZR-75-9a1) and ability of the resistant phenotype

A 6-month exposure of ZR-75-1 human breast cancer cells to tamoxifen (1 microM rising to 2 microM). resulted in a fall in oestrogen receptor (ER) levels from 225 fmol mg protein-1 to 56 fmol mg protein-1 while progesterone receptor (PGR) concentration fell from 63 fmol mg protein-1 to undetectable levels. Sensitivity to the anti-proliferative effects of tamoxifen was unchanged. A further 6 months' exposure to 4 microM tamoxifen resulted in loss of detectable ER and PGR and development of resistance to tamoxifen. Resistant cells, designated ZR-75-9a1, displayed morphological changes consistent with the acquisition of a less well differentiated phenotype. Flow cytometric studies demonstrated that the cell cycle distribution pattern of the resistant variant growing in the presence of 8 microM tamoxifen was identical to that of the untreated parent line, which showed marked accumulation of cells in G0/G1 when exposed to 8 microM tamoxifen. The resistant phenotype was not stable if cells were transferred to complete drug-free medium, but remained stable for at least 3 months in the presence of medium lacking oestrogenic activity. ZR-75-9a1 cells differ from previously reported tamoxifen-resistant variants of the MCF-7 line which retain ER and may prove a valuable model for the study of the development and stability of tamoxifen resistance in human breast cancer.     

Progestin inhibition of estrogen-dependent proliferation in ZR-75-1 human breast cancer cells: Antagonism by insulin

The effect of R5020 [17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione], a synthetic progestin, was studied in the hormone-responsive ZR-75-1 human breast cancer cell line. Following a 12-day incubation with increasing concentrations of R5020, the mitogenic effect of 17-estradiol (E₂,1 nM) was partially (60–80%) antagonized by the progestin, with a half-maximal effective concentration measured at about 30 pM. This effect of R5020 was completely reversed by the addition of physiological concentrations of bovine insulin, as well as by the potent antiprogestin RU486 [17-hydroxy-11-(4-dimethylaminophenyl)-17-(1-propynyl)-4,9-estradien-3-one], but not by the antiandrogen hydroxyflutamide (,,-trifluoro-2-methyl-4-nitro-m-lactotoluidide). Moreover, the effect of R5020 required the presence of estrogens, thus further indicating a progesterone receptor (PgR)-mediated effect. Low (<100 nM) concentrations of R5020 increased the specific binding of [¹²⁵I]-insulin up to 2- to 2.5-fold in intact ZR-75-1 cells, an effect which was reversed by RU486. The effect was rapid, being nearly maximal after 24 h of incubation with R5020. The PgR-mediated effect of R5020 on cell proliferation was abolished by the addition of a pure steroidal antiestrogen. The present results suggest a physiological role for progestins in increasing the responsiveness to insulin, which could, in turn, reverse the antiproliferative effect of progestins on estrogen action and thus decrease the efficacy of progestins in the treatment of breast cancer.     

Relationship of coregulator and oestrogen receptor isoform expression to de novo tamoxifen resistance in human breast cancer

This study addresses the hypothesis that altered expression of oestrogen receptor-beta and/or altered relative expression of coactivators and corepressors of oestrogen receptors are associated with and may be mechanisms of de novo tamoxifen resistance in oestrogen receptor positive breast cancer. All cases were oestrogen receptor +, node negative, primary breast tumours from patients who later had no disease progression (tamoxifen sensitive) or whose disease progressed while on tamoxifen (tamoxifen resistant). Using an antibody to oestrogen receptor-beta that detects multiple forms of this protein (total) but not an antibody that detects only full-length oestrogen receptor-beta 1, it was found that high total oestrogen receptor beta protein expressors were more frequently observed in tamoxifen sensitive tumours than resistant tumours (Fisher's exact test, P=0.046). However, no significant differences in the relative expression of oestrogen receptor beta2, oestrogen receptor beta5 and full-length oestrogen receptor beta1 RNA in the tamoxifen sensitive and resistant groups were found. Also, when the relative expression of two known coactivators, steroid receptor RNA activator and amplified in breast cancer 1 RNA to the known corepressor, repressor of oestrogen receptor activity RNA, was examined, no significant differences between the tamoxifen sensitive and resistant groups were found. Altogether, there is little evidence for altered coregulators expression in breast tumours that are de novo tamoxifen resistant. However, our data provide preliminary evidence that the expression of oestrogen receptor beta protein isoforms may differ in primary tumours of breast cancer patients who prove to have differential sensitivity to tamoxifen therapy.     

Differential effects of retinoids on nitric oxide production by promonocytic U937 cells and ZR-75-1 human breast cancer cells

We demonstrated the presence of inducible and endothelial nitric oxide synthases in histiocytic lymphoma U937 cells by staining with anti-iNOS and anti-eNOS antibodies. We also investigated the effects of retinol and retinoic acid on nitric oxide production by both U937 cells and ZR-75-1 human breast cancer cells. U937 cells which had been treated with either retinol or retinoic acid (10-10-10-6 M) exhibited no significant difference in nitric oxide secretion into conditioned medium. Conversely, for ZR-75-1 cells, both retinol and retinoic acid (10-10-10-6 M) caused a significant (p<0.001) increase in the amount of nitrite secreted. Our results indicate that retinoid induced growth inhibition of breast cancer cells is associated with an increase in NO production, however, an increase in NO synthesis does not mediate retinoid induced differentiation of U937 cells.     

抑癌基因PTEN对乳腺癌ZR-75-1细胞增殖和转移的抑制作用

背景与目的:研究表明,抑癌基因PTEN不仅能抑制肿瘤细胞的增殖,还能抑制其转移,但其机理还不甚明了.本文研究抑癌基因PTEN对人乳腺癌ZR-75-1细胞增殖和转移的作用.方法:以脂质体介导法分别将野生型PTEN质粒和磷酸酶缺陷的PTEN质粒转染人乳腺癌ZR-75-1细胞,用MTT法测定细胞增殖抑制率:转染后用嘌呤霉素筛选阳性克隆.用Western blot法检测细胞中PTEN蛋白的表达.通过细胞-基质粘附实验和人工重组基底膜侵袭实验,检测细胞粘附抑制率与侵袭抑制率.结果:野生型PTEN质粒转染的ZR-75-1细胞增殖明显被抑制,并伴有部分细胞凋亡;该细胞与未经转染的和磷酸酶缺陷的PTEN质粒转染的ZR-75-1细胞比较.细胞增殖抑制率差异均有统计学意义(42.7% vs.0%及2.7%,P＜0.01),细胞增殖抑制效应随细胞培养时间与质粒浓度的增加而增强.而磷酸酶缺陷的PTEN质粒转染的与未经质粒转染的ZR-75-1细胞比较,细胞增殖抑制率差异无统计学意义(2.7%vs.0%,P＞0.05).在两种PTEN质粒转染的ZR-75-1细胞中PTEN蛋白均明显表达,其中转染野生型PTEN质粒的细胞的粘附抑制率与侵袭抑制率分别达65.7%和70.4%,而转染磷酸酶缺陷的PTEN质粒的ZR-75-1细胞的粘附抑制率与侵袭抑制率分别只有8.8%和6.9%(P＜0.05).结论:具有双特异磷酸酶活性的野生型PTEN基因对ZR-75-1细胞的增殖和转移有一定的抑制作用.     

Growth-independent induction of spermidine transport by IL-4 and IL-13 in ZR-75-1 human breast cancer cells

Polyamine transport is strongly induced by insulin and estradiol (E₂) in ZR-75-1 human breast cancer cells. Because signal transduction mechanisms of insulin and interleukin-4 (IL-4) partly overlap, we have compared the ability of these agents as well as that of interleukin-13 (IL-13), a cytokine that often mimics IL-4, to modulate spermidine transport in these cells. In the presence of E₂, insulin increased DNA content and the rate of [³H]spermidine uptake by 2.1- and 3.7-fold, respectively, after an 8-day incubation, whereas the sole addition of IL-4 caused a quantitatively similar induction of [³H]spermidine uptake while leaving cell growth unaffected. No comparable induction of spermidine transport was observed with interleukins-1α and -6, and the effect of IL-4 was not additive to that elicited by insulin plus E₂. IL-4 and IL-13 stimulated [³H]spermidine uptake to a comparable extent, with half-maximal effects observed at 80 and 400 pg/ml, respectively. Interferon-γ inhibited IL-4- and IL-13-dependent spermidine uptake to a much greater extent than basal or insulin-induced transport of the polyamine. IL-4 and IL-13 increased the V_max and K_m of [³H]spermidine uptake by about 4- and 2.5-fold, respectively. Na⁺-dependent amino acid uptake was increased by insulin but not by IL-4 or IL-13, indicating that the cytokines do not induce a general increase in membrane transport activity. IL-4 and IL-13 did not interfere with feedback inhibition of polyamine uptake, and only modestly decreased polyamine content after prolonged incubation, suggesting that these cytokines stimulate spermidine uptake by increasing total transport capacity rather than by repressing an endogenous inhibitor. © 1996 Wiley-Liss, Inc.     

Androgens inhibit basal and estrogen-induced cell proliferation in the ZR-75-1 human breast cancer cell line

This study describes the inhibitory effect of 5 alpha-dihydrotestosterone (5 alpha-DHT) and its precursors testosterone (T) and androst-4-ene-3,17-dione (delta 4-DIONE) on the growth of the estrogen-sensitive human breast cancer cell line ZR-75-1. In the absence of estrogens, cell proliferation measured after a 12-day incubation period was 50-60% inhibited by maximal concentrations of 5 alpha-DHT, T, or delta 4-DIONE with half-maximal effects (IC50 values) observed at 0.10, 0.15 and 15 nM, respectively. This growth inhibition by androgens was due to an increase in generation time and a lowering of the saturation density of cell cultures. The antiestrogen LY156758 (300 nM) induced 25-30% inhibition of basal cell growth, its effect being additive to that of 5 alpha-DHT. The mitogenic effect of 1 nM estradiol (E2) was completely inhibited by increasing concentrations of 5 alpha-DHT with a potency (IC50 = 0.10 nM) similar to that measured when the androgen was used alone. E2 had a more rapid effect on cell proliferation than 5 alpha-DHT, the latter requiring at least 5 to 6 days to exert significant growth inhibition. As found in the absence of estrogens, maximal inhibition of cell proliferation in the presence of E2 was achieved by the combination of the antiestrogen and 5 alpha-DHT. Supraphysiological concentrations of E2 (up to 1 microM) were needed to completely reverse the growth inhibitory effect of a submaximal concentration of 5 alpha-DHT (1 nM). The antiproliferative effect of androgens was competitively reversed by the antiandrogen hydroxyflutamide, thus indicating an androgen receptor-mediated mechanism. The present data suggest the potential benefits of an androgen-antiestrogen combination therapy in the endocrine management of breast cancer.     

Androgen and glucocorticoid receptor-mediated inhibition of cell proliferation by medroxyprogesterone acetate in ZR-75-1 human breast cancer cells

Medroxyprogesterone acetate (MPA) is a synthetic progestin, currently used in the adjuvant treatment of advanced breast cancer, which induces remission rates (30–40%) comparable to those obtained with other types of endocrine therapies. Since, in addition to its progestin-like action, MPA exhibits androgen- and glucocorticoid-like activities in other tissues, the present study was designed to assess the relative contribution of the different steroid receptor systems in the direct action of MPA on breast cancer cell growth, using the ZR-75-1 human mammary carcinoma cell line as anin vitro model.Unlike pure progestins, MPA potently inhibited the proliferation of ZR-75-1 cells in a concentrationdependent manner either in the presence or in the absence of estrogens, and the addition of insulin had only marginal effects on its growth-inhibitory activity. On the other hand, both hydroxyflutamide (OHF, a non-steroidal monospecific antiandrogen) and RU486 (a potent antiglucocorticoid and antiprogestin also endowed with antiandrogenic activity) competitively reversed MPA antiproliferative effects. MPA further decreased the growth of ZR-75-1 cells co-incubated with maximally inhibitory concentrations of either 5-dihydrotestosterone (DHT) or dexamethasone (DEX), although at about 300-fold higher MPA concentrations with DHT-treated than with DEX-treated ZR-75-1 cells, thus demonstrating a highly predominant androgenic effect. However, MPA had no effect on the growth of ZR-75-1 cells co-incubated with DHT and DEX simultaneously, thus supporting the predominant role of androgen and glucocorticoid receptors in MPA action. A 12-day preincubation of ZR-75-1 cells with increasing concentrations of MPA (10^–12 to 3 × 10^–6M) decreased the specific uptake of [³H]estradiol (E₂) by intact cell monolayers to the same extent as 10 nM DHT, an effect which was competitively blocked by the addition of OHF (3 µM). MPA action on ZR-75-1 cell growth also significantly differed from that of progestins in being additive to the inhibition of E₂-stimulated growth by the steroidal antiestrogen ICI164384.The present data indicate that the main action of MPA on ZR-75-1 human breast cancer cell growth is due to its androgen receptor-mediated inhibitory action, while its glucocorticoid-like activity could play an additional role at high concentrations.     

Sequence dependence of Alimta (LY231514, MTA) combined with doxorubicin in ZR-75-1 human breast carcinoma cells

Alimta is a new-generation antifolate with inhibitory activity against multiple enzymes, including thymidylate synthase, glycinamide ribonucleotide formyltransferase and dihydrofolate reductase. Alimta is undergoing broad phase II evaluation as a single agent, and preliminary results show responses in several tumor types, including breast carcinoma. Doxorubicin is often used in combination chemotherapy of breast cancer. Because the two drugs have mechanisms of action that might be complementary, we investigated a possible synergism between doxorubicin and Alimta on growth inhibition of ZR-75-1 human breast carcinoma cells. Cytostatic activity was evaluated using semi-automated MTT assays, and drug interactions were determined using CalcuSyn (Chou/Hayball) multiple drug effect analyses. The cells were exposed to Alimta or doxorubicin as single agents and combinations for 24 hours starting at the time of plating or for 72 hours starting 24 hours after plating with a total culture time of 96 hours. Preincubation with Alimta for 24 hours followed by exposure to doxorubicin for 72 hours resulted in highly synergistic activity, whereas the opposite sequence or simultaneous exposure produced mainly an additive response. DNA flow cytometry studies indicated that Alimta causes a build-up of cells near the G1/S interface after 24 hours of incubation. The data suggest that, to obtain maximal antitumor activity, Alimta should precede doxorubicin when the drugs are given in combination chemotherapy protocols.     

Estrogen regulation of vascular endothelial growth factor gene expression in ZR-75 breast cancer cells through interaction of estrogen receptor alpha and SP proteins

Vascular endothelial growth factor (VEGF) is expressed in multiple hormone-dependent cancer cells/tumors. Treatment of ZR-75 breast cancer cells with 17beta-estradiol (E2) induced a greater than fourfold increase of VEGF mRNA levels. ZR-75 breast cancer cells were transfected with pVEGF1, a construct containing a -2018 to +50 VEGF promoter insert, and E2 induced reporter gene (luciferase) activity. Deletion and mutation analysis of the VEGF gene promoter identified a GC-rich region (-66 to -47) which was required for E2-induced transactivation of pVEGF5, a construct containing the minimal promoter (-66 to +54) that exhibited E2-responsiveness. Interactions of nuclear proteins from ZR-75 cells with the proximal GC-rich region of the VEGF gene promoter were investigated by electrophoretic mobility shift and chromatin immunoprecipitation assays. The results demonstrate that both Sp1 and Sp3 proteins bound the GC-rich motif (-66 to -47), and estrogen receptor alpha (ERalpha) interactions were confirmed by chromatin immunoprecipitation. Moreover, E2-dependent activation of constructs containing proximal and distal GC/GT-rich regions of the VEGF promoter was inhibited in ZR-75 cells transfected with small inhibitory RNAs for Sp1 and Sp3. These results were consistent with a mechanism of hormone activation of VEGF through ERalpha/Sp1 and ERalpha/Sp3 interactions with GC-rich motifs.     

Recombinant human interferon-alpha 2a increases hormone receptor level of a human breast carcinoma xenograft in nude mice and enhances the anti-proliferative activity of tamoxifen.

The effect of recombinant human interferon-alpha 2a (rhIFN-alpha 2a) on the hormone receptor level and antitumor activity of tamoxifen (TAM) was investigated in nude mice using ZR-75-1, an estrogen receptor (ER)-positive, and progesterone receptor (PgR)-negative human breast carcinoma xenograft. ER levels (maximum binding sites) of tumors treated with rhIFN-alpha 2a at a dose of 6 x 10(5) U/mouse/day for 1 or 3 wk were not significantly different from the control, whereas those with rhIFN-alpha 2a at a dose of 6 x 10(4) U/mouse/day for 1 or 3 wk were higher than the control (3.9- to 4.4-fold) with a significant difference at P < 0.01. The increase of ER by rhIFN-alpha 2a was investigated using a sucrose density gradient method. The peak was only seen at 8S in both rhIFN-alpha 2a-treated tumor and control ER, and the sedimentation patterns were almost the same, suggesting that both ERs were essentially equivalent. On the other hand, PgR of all the treated groups could be detected, while that of the control group was undetectable. The antitumor effect of the combination treatment of rhIFN-alpha 2a and TAM was compared with those of single treatments. While rhIFN-alpha 2a at a dose of 6 x 10(5) U/mouse/day and TAM did not show a combination effect, rhIFN-alpha 2a at a dose of 6 x 10(4) U/mouse/day and TAM showed a synergistic combination effect, and ER was decreased to the threshold of detection by the combination treatment. These findings indicated that a low dose of rhIFN-alpha 2a increased the ER levels of ER-positive human breast cancer in vivo as well as in vitro and enhanced the anti-proliferative effect of TAM, and the newly synthesized ER was essentially the same as the original ER.