Inhibition of Hsp90 function by ansamycins causes downregulation of cdc2 and cdc25c and G(2)/M arrest in glioblastoma cell lines.

Ansamycins exert their effects by binding heat shock protein 90 (Hsp90) and targeting important signalling molecules for degradation via the proteasome pathway. We wanted to study the effect of geldanamycin (GA) and its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) on glioblastoma cell lines. We show that these cells are growth inhibited by ansamycins by being arrested in G(2)/M and, subsequently, cells undergo apoptosis. The protein levels of cell division cycle 2 (cdc2) kinase and cell division cycle 25c (cdc25c) were downregulated upon GA and 17-AAG treatment and cdc2 kinase activity was inhibited. However, other proteins involved in the G(2)/M checkpoint were not affected. The cdc2 and cdc25c mRNA levels did not show significant differences upon ansamycin treatment, but the stability of cdc2 protein was reduced. The association of cdc2 and cdc25c with p50(cdc37), an Hsp90 co-chaperone, decreased, but the interaction of cdc2 and cdc25c with the Hsp70 co-chaperone increased after ansamycin treatment. Proteasome inhibitors were able to rescue the cdc2 downregulation, but not the cdc25c reduction. However, calpain inhibitors were able to rescue the cdc25c downregulation, suggesting that cdc25c is proteolysed by calpains in the presence of ansamycins, and not by the proteasome. We conclude that ansamycins downregulate cdc2 and cdc25c by two different mechanisms.

Tumour cells resistance in cancer therapy

Resistance to chemotherapeutic drugs presents a big caveat for cancer treatment. In this review we will describe the molecular mechanisms involved in chemoresistance, discussing the mechanisms of resistance related to tumour microenvironment, as well as their intracellular mechanisms. Chemoresistance can also appear as a consequence to treatments with new anticancer drugs. In this sense, we will exemplify this type of resistance discussing mechanisms of action of epidermal growth factor receptor (EGFR) inhibitors. We conclude that the main problem of chemoresistance is due to its pleiotropic and multifactorial nature(AU)

Molecular biology of exocrine pancreatic cancer.

Exocrine pancreatic cancer is one of the neoplasias with a worse prognosis, with conventional treatments having little impact on disease outcome. Research and genomic high-throughput technology is continuously expanding our knowledge of pancreas cancer biology. Characterization of genetic and epigenetic alterations in pancreatic tumors has allowed a better understanding of the progression model of the disease at the molecular level. The development of new therapeutic approaches with target- oriented agents is been tested in the preclinical and clinical settings. This review updates the current available data on pancreatic cancer molecular biology.

Regulation of estrogen receptor-alpha expression in MCF-7 cells by taxol.

Results presented in this study demonstrate that treatment of MCF-7 cells with taxol resulted in induction of estrogen receptor-alpha (ER alpha) gene transcription with a subsequent increase in ER alpha mRNA; this effect was promoter specific since taxol did not affect total transcription in MCF-7 cells and lacked an effect on transcription of the human acidic ribosomal phosphoprotein protein PO, progesterone receptor, and pS2 genes. In contrast to the increase in transcription of the ER alpha gene, taxol inhibited translation of the ER alpha mRNA. This effect is also transcript specific since taxol did not alter total protein synthesis and did not affect the concentration of progesterone receptor protein in the cell. The overall result of taxol treatment was to decrease the concentration of ER alpha protein in the MCF-7 cells. Evidence is presented that the effects of taxol on ER alpha gene transcription may be mediated through the induction of p53.

Regulation of estrogen receptor-alpha expression by the tumor suppressor gene p53 in MCF-7 cells.

The results presented here demonstrate that p53 upregulates estrogen receptor-alpha (ER alpha) expression in the human breast cancer cell line MCF-7. Two approaches were used to alter the activity of p53 in the cells. In the first approach, stable transfectants expressing an antisense p53 were established. In the stable clones, expression of antisense p53 resulted in a decrease in the expression of ER alpha protein. In the second approach, MCF-7 cells were transiently transfected with wild-type p53. Overexpression of p53 increased the amount of ER alpha. To determine whether the effects of p53 on the expression of ER alpha were due to changes in transcription, deletion mutants of the ER alpha promoter were used. This experimental approach demonstrated that p53 up-regulates ER alpha gene expression by increasing transcription of the gene through elements located upstream of promoter A. Transfection assays using p53 mutants further demonstrated that the p53-induced increase in ER alpha gene transcription was not dependent on the ability of p53 to bind to DNA but on its ability to interact with other proteins.

Trichostatin A effects in different drug-resistant tumor cell lines

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Regulation of estrogen receptor-alpha gene expression by epidermal growth factor.

The role of epidermal growth factor (EGF) in the regulation of estrogen receptor-alpha (ER-alpha) gene expression in the human breast cancer cell line MCF-7 was investigated. Treatment of cells with 0.4 ng/ml EGF resulted in an approximately 60% decrease in ER-alpha protein concentration by 6 h and the amount of receptor remained suppressed for 24 h. Ligand binding assays demonstrated that the decrease in ER-alpha protein corresponded to a similar decrease (approximately 50%) in estradiol binding sites. Although EGF treatment resulted in a decrease in the number of binding sites, it had no effect on the binding affinity of ER-alpha. The dissociation constant of the estradiol-ER-alpha complex in the presence or absence of EGF was the same (K(d)=2.3x10(-)(10) M in control cells versus K(d)=1.98x10(-)(10) M in EGF-treated cells). The decrease in ER-alpha protein concentration paralleled a decrease in the steady-state amount of ER-alpha mRNA. By 9 h there was an approximately 60% decrease in ER-alpha mRNA. The amount of ER-alpha mRNA remained suppressed for 48 h. Transcription run-on experiments demonstrated that there was a decrease of approximately 70% in ER-alpha gene transcription upon EGF treatment, suggesting that the mechanism by which EGF regulates ER-alpha gene expression is transcriptional. In addition to regulating the amount of ER-alpha, EGF affected the activity of the receptor. At high concentrations, EGF induced progesterone receptor. Estradiol and high concentrations of EGF had an additive effect on progesterone receptor. In contrast to high concentrations, low concentrations of EGF had no effect on progesterone receptor and blocked estradiol induction. The effects of EGF on ER-alpha expression were inhibited by tyrophostins and wortmannin, suggesting that the effects of the growth factor are mediated by the EGF receptor and protein kinase B. When the cells were placed in serum-free medium and then treated with EGF, there was no effect on ER-alpha protein concentration or activity. However, increasing concentrations of serum restored the effects of EGF on ER-alpha, suggesting that an additional serum factor was required for the EGF-mediated effect on the decrease in ER-alpha protein concentration.

Role of insulin-like growth factor-I in regulating estrogen receptor-alpha gene expression.

The role of insulin-like growth factor-I (IGF-I) in regulating estrogen receptor-alpha (ER-alpha) gene expression and activity was investigated in the human breast cancer cell line MCF-7. Treatment of cells with 40 ng/ml IGF-I resulted in a 60% decrease in ER-alpha protein concentration by 3 h, and the amount of ER-alpha remained suppressed for 24 h. A multiple-dose ligand-binding assay demonstrated that the decrease in ER-alpha protein corresponded to a similar decrease of 50% in estradiol-binding sites with no effect on the binding affinity of ER-alpha. The dissociation constant of the estradiol-ER-alpha complex in the absence of IGF-I (K(d) = 3 x 10(-10) +/- 0.5 x 10(-10) M) was similar to the dissociation constant in the presence of IGF-I (K(d) = 6 x 10(-10) +/- 0.3 x 10(-10) M). The decrease in ER-alpha protein concentration was paralleled by an 80% decrease in the steady-state amount of ER-alpha mRNA by 3 h. The IGF-I induced decrease in ER-alpha mRNA was due to the inhibition of ER-alpha gene transcription. When an 128-base pair ER-alpha-promoter-CAT construct was transfected into MCF-7 cells, treatment with IGF-I resulted in a 40% decrease in CAT activity. In contrast to the effects on ER-alpha, treatment with IGF-I induced two endogenous estrogen-regulated genes, progesterone receptor and pS2, by 4- and twofold, respectively. The pure antiestrogen ICI-164, 384 blocked this induction, suggesting that ER-alpha mediates the effects of IGF-I. Transient co-transfections of wild-type ER-alpha and an estrogen response element-CAT reporter into COS-1 cells demonstrated that IGF-I increased reporter gene activity. This effect was also blocked by ICI 164,384. Protein kinase A and phosphatidylinositol 3-kinase inhibitors blocked the IGF-I effects on ER-alpha expression and activity, suggesting that these kinases may be involved in the cross-talk between the IGF-I and ER-alpha pathways.

Regulation of estrogen receptor-alpha gene expression by 1, 25-dihydroxyvitamin D in MCF-7 cells.

This report describes an investigation of the role of 1, 25-dihydroxyvitamin D (VD(3)) in the regulation of estrogen receptor-alpha (ER) in the ER-positive breast cancer cell line, MCF-7. Treatment of cells with 10 nM VD(3) resulted in a 50% decline in the concentration of ER protein at 24 h. Scatchard analysis showed a corresponding decrease in the number of estradiol binding sites and no alteration in the binding affinity of estradiol for the ER (K(d) = 0.08 nM in VD(3)-treated cells compared with K(d) = 0.07 nM in control cells). Vitamin D treatment also caused a 50% decrease in the steady state amount of ER mRNA, which was maximal by 18 h. In vitro transcription run-on experiments demonstrated a decrease of approximately 60% in transcription of the estrogen receptor gene. Transient transfections using an ER promoter-CAT construct also demonstrated a 40% decrease in CAT activity after VD(3) treatment. Sequence analysis identified a potential vitamin D response element (nVDRE) within the ER promoter. When this element was mutated, the ability of VD(3) to block transcription from the ER promoter was lost. When the nVDRE was placed upstream of a heterologous promoter, nVDRE-SV40-CAT, treatment with VD(3) resulted in a 50% decrease in CAT activity. Interestingly, co-transfection of either the ER promoter-CAT or the nVDRE-SV40-CAT construct and a vitamin D receptor expression vector into COS-1 or CV-1 cells showed an approximately 4-fold increase in CAT activity after VD(3) treatment. Taken together these data suggest that VD(3) inhibition of ER gene transcription is mediated through a nVDRE in the ER promoter. Inhibition appears to be cell specific.

Estradiol regulates estrogen receptor mRNA stability.

Previous studies suggest that post-transcriptional events play an important role in estrogen-induced loss of estrogen receptor expression. The present study shows that treatment of MCF-7 cells with estradiol resulted in a six-fold decrease in estrogen receptor mRNA half-life from 4 h in control cells to 40 min in estradiol treated cells. To determine the role of protein synthesis in the regulation of estrogen receptor mRNA stability, several translational inhibitors were utilized. Pactamycin and puromycin, which prevent ribosome association with mRNA, inhibited the effect of estradiol on receptor mRNA stability, whereas cycloheximide, which has no effect on ribosome association with mRNA, had no effect on estradiol regulation of estrogen receptor mRNA stability. In control cells, the total cellular content of estrogen receptor mRNA was associated with high molecular weight polyribosomes. Treatment with estradiol resulted in a 70% decrease in estrogen receptor mRNA associated with polyribosomes but had no effect on the polyribosome distribution of estrogen receptor mRNA. In an in vitro degradation assay, polyribosomes isolated from estradiol-treated cells degraded ER mRNA faster than polyribosomes isolated from control cells. The nuclease activity associated with the polysome fraction appeared to be Mg2+ independent and inhibited by RNasin. Freeze-thawing and heating at 90 degrees C for 10 min resulted in the loss of nuclease activity. These studies suggest that an estrogen-regulated nuclease activity associated with ribosomes alters the stability of estrogen receptor mRNA.

The role of transforming growth factor-beta in the regulation of estrogen receptor expression in the MCF-7 breast cancer cell line.

The role of transforming growth factor-beta1 (TGFbeta1) in the regulation of estrogen receptor (ER) expression in MCF-7 cells was investigated. After treatment of the cells with 100 pM TGFbeta1, ER protein declined by about 30% at 6 h from a concentration of 413.5 fmol/mg protein in control cells to 289.5 fmol/mg protein in treated cells. The concentration of receptor remained suppressed for 24 h. Scatchard analysis demonstrated that the decrease in ER protein corresponded to a decrease in estradiol-binding sites, with no effect on the binding affinity of the ER. The dissociation constant of the estradiol-ER complex was 0.117 nM in TGFbeta1-treated cells compared to 0.155 nM in control cells. Treatment with TGFbeta1 did not influence the half-life of the ER. In TGFbeta1-treated cells, as well as in control cells, the half-life of the receptor was approximately 4 h. In contrast to the effect on ER concentration, TGFbeta1 treatment resulted in a greater decrease in the steady state level of ER messenger RNA (approximately 75%) at 6 h. By 24 h, a small recovery in the amount of messenger RNA was observed. Transcription run-on experiments demonstrated a decrease of approximately 70% in the level of ER gene transcription at 3 h. Transient transfections using an ER promoter-chloramphenicol acetyltransferase construct demonstrated that after TGFbeta1 treatment, chloramphenicol acetyltransferase activity decreased by 50%, suggesting that TGFbeta1 inhibition of the ER gene transcription is mediated through the ER promoter. Although treatment with TGFbeta1 decreased the ER concentration, the growth factor had no effect on the activity of ER, as measured by its effects on estradiol induction of progesterone receptor and pS2, suggesting that TGFbeta1 does not inhibit proliferation of MCF-7 cells by blocking ER activity.

Altered gene expression in drug-resistant human breast cancer cells.

It is increasingly recognized that drug-resistant cells undergo transitions not directly linked to "classical" drug resistance. We examined the expression of growth factors, growth factor receptors, and the estrogen receptor in 17 drug-resistant and 2 revertant human breast cancer sublines to provide an understanding of the phenotypic changes that occur and how these changes could affect the biology of the cell. These sublines were derived from five parental human breast cancer cell lines (MCF-7, ZR75B, T47D, MDA-MB-231, and MDA-MB-453). The expression of estrogen receptor was absent or decreased in 6 of the 15 resistant MCF-7, ZR75B, and T47D sublines. Increases of as much as 49-fold compared to parental levels were observed in transforming growth factor alpha, epidermal growth factor receptor, c-erbB2, and/or c-erbB3 mRNA expression in 14 of the 17 resistant sublines. Altered amphiregulin and insulin-like growth factor-I receptor expression was observed in nine and four drug-resistant sublines, respectively. No major alterations were observed in epidermal growth factor and c-erbB4 expression. Few alterations were observed in two sublines derived from estrogen receptor-negative cells. Higher levels of phosphotyrosine residues were detected in a subset of the resistant sublines, indicating an increased tyrosine kinase activity in these cells. Interestingly, decreased growth rates were observed in all of the sublines, despite up-regulated growth factor-related gene expression. Taken together, these data suggest that loss of estrogen receptor, increased expression of growth factor pathway genes, and decreased growth rate regularly occur in drug-resistant breast cancer cells. Although we do not know whether the altered expression of growth factor pathway genes is linked as a cause or a consequence of the reduced growth rate, it is well established that decreased growth rate confers drug resistance. These phenotypic changes in drug-resistant human breast cancer cells could serve to initiate, support, or extend the drug resistance.

Regulation of estrogen receptor concentration and activity by an erbB/HER ligand in breast carcinoma cell lines.

Expression of the erbB-2 oncogene in breast cancer patients correlates with poor prognosis and failure of hormonal therapy. In this study, the effects of a putative erbB/HER ligand, gp30, on estrogen receptor (ER) concentration and activity was investigated in the estrogen receptor positive human breast cancer cells, BT474 and MCF-7, which express either high or low levels of erbB-2 and erbB-4, respectively. Treatment of cells with gp30 resulted in a decrease in the steady-state level of estrogen receptor protein by approximately 70-80%. The effect of gp30 on the concentration of ER was independent of serum in the media and was not inhibited by an epidermal growth factor receptor blocking antibody. In addition to the effect on ER protein, gp30 decreased the steady-state level of ER messenger RNA. Transcription run on experiments demonstrated that the decrease in ER expression was mediated by a decrease in ER gene transcription. The effect of gp30 on estrogen receptor activity was also investigated in this study. Treatment of cells with gp30 blocked estradiol induction of progesterone receptor. Inhibition was observed at the level of progesterone receptor protein, messenger RNA, and gene transcription. gp30 also blocked estradiol induction of pS2 gene transcription. In addition to its effects on progesterone receptor and pS2, gp30 blocked activation of an estrogen response element in a transient transfection assay and inhibited ER binding to its response element in a DNA mobility shift assay, suggesting a direct effect on the estrogen receptor. The effects of gp30 on estrogen receptor concentration and activity were independent of the level of erbB-2 and erbB-4 in the cell. These data show that gp30 regulates the concentration of ER and modulates ER activity.

Regulation of retinoblastoma gene expression in hormone-dependent breast cancer.

Studies have shown an increased risk for breast cancer in the mothers of children suffering from retinoblastoma and osteosarcoma, suggesting a role for the retinoblastoma susceptibility (Rb) gene product in breast cancer. We now show that estradiol decreases the expression of Rb at the level of protein and messenger RNA (mRNA) in estrogen-dependent breast cancer cell lines. Treatment of MCF-7 cells with 10(-9) M estradiol for 48 h resulted in a 70% decrease in the level of Rb protein. Ribonuclease protection assays showed a 50% decrease in the steady state levels of Rb mRNA by 12 h and a 70% decrease in Rb mRNA by 24 h. Treatment with estradiol had no effect on the rate of Rb gene transcription or on Rb mRNA stability, but resulted in an increase in the steady state level of Rb mRNA in the nucleus. The effect of estradiol was inhibited by 10(-7) M 4-hydroxytamoxifen. In the absence of estradiol, the antiestrogens 4-hydroxytamoxifen and ICI 164,384 increased Rb mRNA by 50% over that in estrogen-depleted conditions. Estradiol regulation of Rb mRNA also occurred in other estrogen-dependent breast cancer cell lines. Insulin-like growth factor I, insulin, progestins, and epidermal growth factor had no effect on Rb expression. In summary, these results show that estradiol specifically regulates the expression of the Rb susceptibility gene product in hormone-dependent breast cancer by a posttranscriptional mechanism that occurs in the nucleus. The results from this study suggest that the negative regulation of Rb expression by estradiol, rather than Rb loss or mutation, may play an important role in breast carcinogenesis.

Bidirectional interactions between the estrogen receptor and the cerbB-2 signaling pathways: heregulin inhibits estrogenic effects in breast cancer cells.

The responsiveness of estrogen receptor (ER)-positive breast cancer to endocrine therapy is frequently reduced in cells over-expressing c-erbB-2. Stimulation of ER suppresses c-erbB-2, indicating that estrogen controls the activity of c-erbB-2. Heregulin (HRG) has been described to bind to c-erbB-3/c-erbB-4 and to stimulate c-erbB-2. Here we describe the effects of HRG on cell growth and on ER and c-erbB-2 expression in breast cancer cell lines containing distinct levels of c-erbB-2 and ER (BT-474: c-erbB-2 , ER+; MDA-MB-361: c-erbB-2++, ER++; MCF-7: c-erbB-2+, ER ). Proliferation of estrogen-stimulated, c-erbB-2 and ER-positive cells is inhibited by HRG in a dose-dependent manner. In addition, HRG dose-dependently inhibits ER expression. Estrogen, however, inhibits c-erbB-2. Estrogen-mediated down-regulation of c-erbB-2 is most pronounced in MCF-7 but weaker in BT-474. In the latter cells HRG efficiently blocks the estrogenic effect on c-erbB-2. In MCF-7 cells, however, the inhibition of c-erbB-2 cannot be completely reverted by HRG. This modulation occurs in all 3 cell lines at protein, RNA and transcriptional levels, suggesting that the activity of the c-erbB-2 promoter, which contains an estrogen-responsive region, is affected by HRG. The intensity of the mutual inhibition between the HRG/c-erbB-2 and the estrogen/ER system depends on the relative levels of ER and c-erbB-2 expression in the respective cell lines.

Effects of 12-O-tetradecanoylphorbol-13-acetate on estrogen receptor activity in MCF-7 cells.

The effects of long term treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) on estrogen receptor (ER) expression in the human breast cancer cell line, MCF-7, were studied. This study demonstrates that treatment of cells with the phorbol ester blocked estrogen receptor activity. Treatment of cells with 100 nM TPA resulted in an 80% decrease in the level of ER protein and a parallel decrease in ER mRNA and binding capacity. Following removal of TPA from the medium, the level of ER protein and mRNA returned to control values; however, the receptor failed to bind estradiol. These cells also failed to induce progesterone receptor in response to estradiol. In addition, TPA treatment blocked transcription from an estrogen response element in transient transfection assays and inhibited ER binding to its response element in a DNA mobility shift assay. The estrogen receptor in treated cells was recognized by two monoclonal anti-ER antibodies and was not quantitatively different from ER in control cells. RNase protection analysis failed to detect any qualitative changes in the ER mRNA transcript. Mixing experiments suggest that TPA induces/activates a factor which interacts with the ER to block binding of estradiol. The effects of TPA on ER levels and binding capacity were concentration-dependent. Low concentrations of TPA inhibited estradiol binding without a decrease in the level of protein, whereas higher concentrations were required to decrease the level of ER protein. The effects of TPA appear to be mediated by activation of protein kinase C since the protein kinase C inhibitors, H-7 and bryostatin, block the effects of TPA on estradiol induction of progesterone receptor. TPA treatment had no effect on the level or binding capacity of the glucocorticoid receptor, indicating that the effects are not universal for steroid receptors. These data demonstrate that activation of the protein kinase C signal transduction pathway modulates the estrogen receptor pathway. The long term effect of protein kinase C activation is to inhibit estrogen receptor function through induction/activation of a factor which interacts with the receptor.

Estrogen and phorbol esters regulate amphiregulin expression by two separate mechanisms in human breast cancer cell lines.

The actions of 17 beta-estradiol (E2) and protein kinase C (PKC) appear to converge in the regulation of expression of certain growth modulatory genes, such as the growth factor amphiregulin (AR). AR is known to modulate cell growth by binding to the epidermal growth factor receptor. In the current report we established the mechanisms of the PKC-activating phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) and the steroid hormone E2 on the induction of AR expression in human breast carcinoma cell lines. TPA (100 nM) and E2 (1 nM) induce AR messenger RNA (mRNA) expression by 6- to 8-fold and 3- to 6-fold, respectively, in a time- and dose-dependent manner. In addition, immunoreactive AR protein is induced by both TPA and E2 by 6- to 8-fold and 2- to 4-fold, respectively. The PKC-modulating drugs, bryostatin and H-7, and antiestrogens (ICI 164,384 and 4-hydroxytamoxifen) interfere with AR induction by TPA and estrogen, respectively. The effects of TPA and E2 on the induction of AR mRNA were both closely associated with enhanced transcription of the AR gene. However, TPA had an additional effect at the posttranscriptional level by stabilizing the AR mRNA. The protein synthesis inhibitor, cycloheximide, prevented AR induction by TPA, suggesting that a component of the TPA induction of AR is indirect and dependent upon protein synthesis. Conversely, the E2 induction of AR transcription was found to be a direct response, independent of protein synthesis. The results presented herein thus demonstrate that TPA and E2 are able to stimulate AR gene transcription by two separate mechanisms.

Effect of cadmium on estrogen receptor levels and estrogen-induced responses in human breast cancer cells.

The effects of cadmium on estrogen receptor and other estrogen-regulated genes in the human breast cancer cell line MCF-7 were studied. Treatment of MCF-7 cells with 1 microM cadmium decreased the level of estrogen receptor 58%. Cadmium induced a parallel decrease in estrogen receptor mRNA (62%). Progesterone receptor levels increased 3.2-fold after cadmium treatment. This induction was blocked by the anti-estrogen ICI-164,384. Progesterone receptor mRNA was also increased by cadmium, as well as cathepsin D mRNA. An in vitro nuclear transcription run-on assay showed that cadmium increased the transcription of the progesterone receptor and pS2 genes and decreased transcription of the estrogen receptor gene. These are not general effects of heavy metals, as zinc, 25 and 100 microM, did not affect progesterone receptor protein and mRNA levels. Cadmium stimulated pS2 and progesterone receptor mRNAs in a clone of MDA-MB-231 cells transfected with the human estrogen receptor, but had no effect in MDA-MB-231 cells transfected with antisense estrogen receptor. Cadmium also stimulated an estrogen response element in transient transfection experiments. These data suggest that the effects of cadmium are mediated by the estrogen receptor independent of estradiol. In addition to its effect on gene expression, cadmium induced the growth of MCF-7 cells 5.6-fold.

Increased epidermal growth factor receptor in an estrogen-responsive, adriamycin-resistant MCF-7 cell line.

We examined the expression of the estrogen and epidermal growth factor (EGF) receptors in a drug-resistant subline of MCF-7 cells in order to study potential alterations in hormone dependence or in the growth factor pathway that could be related to the development of drug resistance in human breast cancer. The drug-resistant subline was derived from MCF-7 cells by selection with Adriamycin in the presence of the P-glycoprotein antagonist, verapamil, to prevent acquisition of the classical multidrug resistance phenotype. The Adriamycin-resistant cells retain estrogen-binding, estrogen-responsive monolayer growth, and estrogen-dependent tumorigenesis. Estrogen-binding studies demonstrate 1.4 x 10(6) sites per cell with unaltered affinity when compared to parental MCF-7 cells, which have 2.7 x 10(5) sites per cell. An increase in expression of EGF receptor, eight to 12-fold, occurred early in the selection for drug resistance, and appears to be unrelated to verapamil exposure, since cells maintained in Adriamycin without verapamil also have increased EGF receptor expression. Partially drug-sensitive revertants carried a verapamil, but out of Adriamycin, demonstrate a decline in EGF receptor expression. We postulate that activation of growth factor pathways in drug-resistant cells may enhance mechanisms of drug resistance, or provide mitogenic stimuli for cells to recover after damage by drug exposure.