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.     

Receptors for epidermal growth factor and insulin-like growth factor I and their relation to steroid receptors in human breast cancer

Levels of epidermal growth factor receptor (EGF-R) and insulin-like growth factor receptor (IGF-R) in breast cancer tissue were evaluated. The binding of growth factors was compared to the content of estrogen receptors (ER) and progesterone receptors (PgR). EGF-R correlated negatively to the ER and PgR (Kendall correlation, P less than 0.001), whereas the IGF-R correlated positively to ER and PgR (analysis of variance, P less than 0.001). In contrast, no correlation was found between EGF-R and IGF-R. IGF-R binding was higher in tumor tissues than in adjacent normal tissues (Wilcoxon rank test, P less than 0.001), whereas the EGF-R binding in normal tissue did not differ from that in cancer tissue. The degree of differentiation in ductal breast cancer correlated to EGF-R (chi 2 test, P = 0.018), but not to IGF-R. The bindings of both growth factors were the same in metastases and primary breast tumors. Our results show that EGF-R and IGF-R are present in normal breast tissue and breast cancer tissue. The growth factor receptors are related to steroid receptor content and their presence is associated with malignant transformation of breast cells and dedifferentiation of breast cancer.     

Receptors for epidermal growth factor and steroid hormones in human breast cancer

Epidermal growth factor (EGF) seems to play an important role in regulating the proliferation of human breast cancer. Fifty-five primary breast tumors and 7 lymph node metastases were simultaneously assayed for the presence of EGF receptors (EGFR), estrogen receptors (ER), and progesterone receptors (PR). Overall, 42% (23/55) of the tumors were EGFR positive. EGFR were more frequently present in ER- and PR-negative than in ER- and PR-positive tumors. In particular, a negative correlation between EGFR and PR (chi 2 = 6.8; p greater than 0.01) was observed. All metastatic tumors were EGFR negative, and in all cases but 1 the levels of EGFR were higher in metastatic than in primary tumors. Our results suggest the presence of a subclass of breast tumors, the growth of which is primarily regulated by EGF or EGF-like substances rather than by steroid hormones. In this group, not amenable to endocrine therapy, EGF receptors should represent a target for therapeutic intervention.     

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.     

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.     

HER-2 amplification, steroid receptors and epidermal growth factor receptor in primary breast cancer

Amplification of HER-2 oncogene was analysed in DNAs obtained from 291 primary human mammary carcinomas. 52/291 (18%) were found to contain amplified HER-2 oncogene. Moderate amplification (2- to 5-fold) was noted in 36/291 (12%). Thirteen tumors (4.5%) had a copy number of 5 to 10. A 10- to 20-fold and greater than 20-fold amplification was observed in 2 and 1 patient, respectively. Sample sizes allowed the determination of estrogen receptor (ER) and progesterone receptor (PgR) levels in 253/291 primary breast cancers. HER-2 gene amplification was noted in 14% of ER+ patients and in 28% of ER- patients, respectively (P = 0.02). Similarly a significantly greater number of PgR- primary mammary carcinoma exhibited an amplification of the HER-2 gene compared to PgR+ cases (22% vs. 16%, P = 0.01). Although statistically not significant, tumors with HER-2 gene amplification were found to have lower levels of ER and PgR. No association of HER-2 amplification with the androgen receptor and EGF receptor was observed. Present data combine to suggest that tumor progression is more stringently controlled by the oncogene upon loss of hormone dependency. Differences found in HER-2 amplification between steroid receptor positive and negative tumors could be helpful to define a specific subset of women to whom adjuvant therapy should be directed.     

Prognostic significance of insulin-like growth factor 1 receptors in human breast cancer

Overall survival (OS) and relapse free survival (RFS) were studied in 297 patients according to the presence of insulin-like growth factor 1 receptors (IGF1-R). All the patients were surgically treated for locoregional disease in the same institution from January 1986. The median duration of follow-up was 40 months. RFS was better in patients with IGF1-R in their tumors as assessed by actuarial survival (P = 0.014) as well as Cox analysis (P = 0.016). OS was better in IGF1-R positive tumors studied by actuarial (P = 0.007) as well as Cox analysis (P = 0.010). By Cox analysis the other prognostic factors on RFS were estrogen receptor (P = 0.002), progesterone receptor (P = 0.002), axillary node metastases (P = 0.032), histoprognostic grading (GHP) according to the standard of Scarff and Bloom (P = 0.004), and tumor diameter (P = 0.019). The other prognostic factors on OS (Cox analysis) were estrogen receptor (P = 0.001), axillary node metastases (P = 0.010), GHP (P = 0.009), progesterone receptor (P = 0.012), and tumor diameter (P = 0.007). When combining IGF1-R, GHP, and axillary node metastases, it appeared that IGF1-R, GHP, and axillary node metastases had independent prognostic significance. In this prospective study IGF1-R had a prognostic significance on RFS as well as on OS studied by actuarial as well as Cox analysis.     

Type I insulin-like growth factor receptor function in breast cancer

Experimental evidence suggests an important role of the type I IGF receptor (IGF-IR) in breast cancer development. Breast tumors and breast cancer cell lines express the IGF-IR. IGF-IR levels are higher in cancer cells than in normal breast tissue or in benign mammary tumors. The ligands of the IGF-IR are potent mitogens promoting monolayer and anchorage-independent growth of breast cancer cells. Interference with IGF-IR activation, expression, or signaling inhibits growth and induces apoptosis in breast cancer cells. In addition, recent studies established the involvement of the IGF-IR in the regulation of breast cancer cell motility and adhesion. We have demonstrated that in MCF-7 cells, overexpression of the IGF-IR promotes E-cadherin-dependent cell aggregation, which is associated with enhanced cell proliferation and prolonged survival in three-dimensional culture.The expression or function of the IGF-IR in breast cancer cells is modulated by different humoral factors, such as estrogen, progesterone, IGF-II, and interleukin-1. The IGF-IR and the estrogen receptor (ER) are usually co-expressed and the two signaling systems are engaged in a complex functional cross-talk controlling cell proliferation.Despite the convincing experimental evidence, the role of the IGF-IR in breast cancer etiology, especially in metastatic progression, is still not clear. The view emerging from cellular and animal studies is that abnormally high levels of IGF-IRs may contribute to the increase of tumor mass and/or aid tumor recurrence, by promoting proliferation, cell survival, and cell-cell interactions. However, in breast cancer, except for the well established correlation with ER status, the associations of the IGF-IR with other prognostic parameters are still insufficiently documented.     

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.     

Significance of epidermal growth factor receptor expression in breast cancer

Recent interest of many investigators is focused on epidermal growth factor receptor (EGFR) family, because of their potential role in the pathogenesis and progression of breast cancer. Paraffin tumor sections were collected retrospectively from 181 breast cancer patients diagnosed between 2002 and 2003. Immunohistochemical staining with ErbB-1, ErbB-2, ErbB-3, and ErbB-4 monoclonal antibodies was performed. The ErbB expression was correlated with the other clinicopathological variables. Overexpression of ErbB-1, ErbB-2, ErbB-3, and ErbB-4 was observed in 20.6, 18.2, 14.3, and 5.7% cases, respectively. Overexpression of ErbB-1 and ErbB-2 was associated with poor prognostic features and decreased 5-year disease-free survival. The patients with co-overexpression of ErbB-1 and ErbB-2 had a shorter DFS, although this difference was not statistically significant. ErbB-1 overexpression may indicate a subset of patients with a poor disease prognosis. Assays for ErbB-1 and ErbB-2 may be more useful than a single assay in predicting prognosis of a breast cancer patient.     

Immunohistochemical expression of epidermal growth factor receptor in breast cancer

Background  

Molecular-targeting drugs able to treat breast cancer expressing epidermal growth factor receptor (EGFR) would be clinically valuable. The aim of the current study was to determine the further significance of immunohistochemical expression of EGFR in breast cancer.     

Significance of epidermal growth factor receptor expression in breast cancer

Colorectal cancer is one of the most common cancers worldwide. The anticancer effect of Wolfberry (Lycium barbarum) polysaccharide (LBP) on colon cancer cells is largely unknown. To investigate the growth effect of LBP on human colon cancer cell and its possible mechanisms, human colon cancer SW480 and Caco-2 cells were treated with 100–1,000 mg/l LBP for 1–8 days. Cell growth was measured by MTT assay and crystal violet assay. Distribution of the cell cycle was analyzed by flow cytometry. Western blotting was used to indicate changes in the level of cyclins and cyclin-dependent kinases (CDKs). LBP treatment inhibited both colon cancer cell lines in a dose-dependent manner. At concentrations from 400 to 1,000 mg/l, LBP significantly inhibited the growth of SW480 cells (400 mg/l, P < 0.01; 800 and 1,000 mg/l, P < 0.001); while at concentrations from 200 to 1,000 mg/l, LBP significantly inhibited the growth of Caco-2 cells (200 mg/l, P < 0.05; 400–1,000 mg/l, P < 0.001). Crystal violet assay showed that LBP had a long-term anti-proliferative effect. More importantly, cells were arrested at the G0/G1 phase. The changes in cell-cycle-associated protein, cyclins, and CDKs were consistent with the changes in cell-cycle distribution. This is one of the first studies to focus on LBP-induced interruption of the cell cycle in human colon carcinoma cells. The results suggest that LBP is a candidate anticancer agent.     

Significance of epidermal growth factor receptor expression in breast cancer

Recent interest of many investigators is focused on epidermal growth factor receptor (EGFR) family, because of their potential role in the pathogenesis and progression of breast cancer. Paraffin tumor sections were collected retrospectively from 181 breast cancer patients diagnosed between 2002 and 2003. Immunohistochemical staining with ErbB-1, ErbB-2, ErbB-3, and ErbB-4 monoclonal antibodies was performed. The ErbB expression was correlated with the other clinicopathological variables. Overexpression of ErbB-1, ErbB-2, ErbB-3, and ErbB-4 was observed in 20.6, 18.2, 14.3, and 5.7% cases, respectively. Overexpression of ErbB-1 and ErbB-2 was associated with poor prognostic features and decreased 5-year disease-free survival. The patients with co-overexpression of ErbB-1 and ErbB-2 had a shorter DFS, although this difference was not statistically significant. ErbB-1 overexpression may indicate a subset of patients with a poor disease prognosis. Assays for ErbB-1 and ErbB-2 may be more useful than a single assay in predicting prognosis of a breast cancer patient.

     

Characterization of insulin-like growth factor 1 receptors (IGF1-R) in human breast cancer cell lines

The binding characteristics of IGF1 on membranes prepared from 5 human breast cancer cell lines were investigated in detail. The presence of one class of high affinity IGF1 binding sites was demonstrated (BT-20: n = 230 fmol/mg protein, Ka = 0.7 nM-1; MCF-7: n = 124 fmol/mg protein, Ka = 1 nM-1; T-47D: n = 61 fmol/mg protein, Ka = 1.1 nM-1; HBL-100: n = 18 fmol/mg protein, Ka = 3.2 nM-1; MDA-MB-231: n = 7 fmol/mg protein, Ka = 2.8 nM-1). Chemical cross-linking of 125I IGF1 to breast cancer cell membranes then sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed one major band of relative molecular weight 130000. The specificity of these receptors was studied: native or recombinant IGF1 had the same potency to inhibit 125I IGF1 binding; IGF2 was able to compete for binding, whereas insulin competed with a potency lower than 1/100 that of IGF1. These characteristics of IGF1 binding sites in breast cancer cell membranes correspond to the previously described binding unit of type I IGF receptors (IGF1-R). Finally we determined that for a routinely used standard assay, it was necessary to incubate for 5 h at 4 degrees C a high amount of membrane protein (400 micrograms) and 200,000 cpm of tracer. Considering the known effect of IGF1 on breast cancer cell multiplication, it is tempting to suggest that this factor might play a major role in the growth of breast cancer: the measurement of IGF1-R, using this standardized method, will give an assessment of these tumors IGF1 sensitivity; it can be performed on the membrane fraction obtained when preparing cytosol for steroid receptor assay.     

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.     

Increased epidermal growth factor receptor gene expression by gamma-interferon in a human breast carcinoma cell line

The interferons are a group of naturally occurring proteins that inhibit the growth of tumours in vivo and many transformed cell lines in vitro. The mechanisms of action of interferon, however, remain unclear. The IFN induced inhibition of growth of many epithelial cancer cell lines is associated with changes in Epidermal Growth Factor Receptor (EGFR) binding or expression. Therefore, we examined the effect of IFN treatment on the expression of EGFR in a human breast carcinoma cell line, MDA 468. We have found the IFN-gamma inhibited, in a dose dependent fashion, the growth of MDA 468 cells. IFN decreased cell surface binding of 125I-EGF to EGFR by changing receptor number rather than affinity. However, total cellular receptor protein, as measured by immunoprecipitation with monoclonal antibodies, was increased in IFN-treated cells. The half-life of the metabolically labelled receptor was unchanged by treatment with IFN. Increased amounts of EGFR mRNA were observed in MDA 468 cells treated with IFN-gamma for 3 days. The levels of mRNA increased with time in culture, reaching a peak of four times control values after 5 days of treatment. This effect was observable with as little as 10 U ml-1 of IFN-gamma. Treatment of the cells with Actinomycin D to inhibit new RNA synthesis suggested that the stability of EGFR mRNA was not enhanced in IFN-gamma treated cells. The increase in receptor mRNA induced by IFN was not inhibited by cycloheximide. These data suggest IFN-gamma can increase expression of EGFR mRNA and protein in MDA 468 cells. Increased expression of EGFR mRNA and protein by IFN-gamma is associated with inhibition of cell growth.     

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.     

Regulation of epidermal growth factor receptor by progestins and glucocorticoids in human breast cancer cell lines

Human breast cancer cells secrete a number of autocrine peptides which modulate their proliferation rates. The known effects of steroid hormones on breast cancer cell proliferation may be mediated in part by altering the production of these growth factors and/or their interactions with cellular receptor sites. Receptors for epidermal growth factor (EGF), which also bind the autocrine growth factor, alpha-transforming growth factor, are present on a number of breast cancer cell lines and it has previously been shown that T-47D and MCF-7 cells respond to progestins with an increase in the concentration of EGF receptors (EGF-R). In the present study we examined the effects of both progestins and glucocorticoids on EGF binding in 10 human breast cell lines. Five of these lines were progesterone receptor positive and all lines expressed the glucocorticoid receptor (GR). All cell lines were initially incubated for 24 hr with increasing concentrations of the synthetic progestin, medroxyprogesterone acetate (MPA), and the level of specifically bound EGF was determined. An increase in specific binding of EGF was confirmed in two PR-positive lines but, in addition, increases in EGF binding were observed in 4 PR-negative cell lines. In these last lines the synthetic glucocorticoid, dexamethasone, was a more potent inducer of EGF binding than MPA, a known glucocorticoid agonist, while the high-affinity PR ligand, ORG 2058, was without effect. Furthermore, MPA competed with dexamethasone for binding to GR in these cell lines, supporting the view that the induction of EGF binding by MPA in these cells was mediated via the GR. This conclusion was further supported by studies in which addition of the glucocorticoid and progestin antagonist, RU 486, inhibited the effect of ORG 2058 in two cell lines and completely abrogated the effect of dexamethasone in two other lines. Detailed binding studies revealed that the increase in EGF binding was accompanied by an increase in the concentration of EGF-R. This effect was observed when EGF binding was assayed at either 0 degree or 37 degrees C. Further studies demonstrated that the increases in EGF binding following ORG 2058 and dexamethasone treatment were accompanied by increases in EGF-R mRNA levels. Our data illustrate that the binding of EGF by some human breast cancer cells can be regulated by both progestins and glucocorticoids acting via their respective receptors and inducing increases in EGF-R mRNA levels.     

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.