Responsive Breast Cancer Cell Lines Research Articles

Abstract LB335: Steroid receptor cross-talk in breast cancer

Abstract Breast cancer is the most frequently diagnosed cancer in women worldwide. Oestrogen Receptor-α (ERα), is expressed in two thirds of human breast cancers, and the majority of patients are sensitive to endocrine therapy, i.e. anti-oestrogens or aromatase inhibitors that aim to block oestrogen signalling. In addition to ERα, the other members of the steroid receptor family (androgen, AR; progesterone, PR; glucocorticoid, GR; mineralocorticoid receptors, MR) also appear to play an important role in tumour development and recurrence, but the role of this signalling in cancer progression has not been fully characterised. Therefore, this study aims to address the role of the steroid receptor family members in breast cancer and to assess the effect of downstream signalling upon prognosis. Investigation of receptor signalling cross-talk using reporter, proliferation and migration assays, demonstrated that ERα and the other steroid receptors inhibit each other’s activity in endocrine responsive breast cancer cell lines, MCF7 and T47D. Furthermore, treatment with antioestrogen reversed the suppressive effects of ER𝛼;;; on the activity of the androgen, glucocorticoid progesterone receptors. To characterize receptor crosstalk on a global scale, RNA-seq and ChIP-seq datasets were analysed. Analysis of the ChIP-seq data demonstrated that the PR, GR and AR reprogramming of ERα results in the loss of ERα canonical binding sites and the gain of novel response elements/binding sites. Motif enrichment analysis of the novel ERα binding sites suggested that the altered binding in response to AR, GR and PR signalling, promotes the recruitment of ERα to androgen, glucocorticoid and progesterone response elements, respectively. Gene ontology analysis also revealed that this change in ERα binding sites is correlated with alterations in genes linked to cell proliferation and growth. Moreover, transcriptome studies showed that cotreatment of cells with estradiol and glucocorticoid or mineralocorticoid resulted in the upregulation of apoptotic signalling pathways. These findings may provide an explanation for the observed downregulation of GR and MR expression in ERα positive breast cancer. Patient datasets were used to investigate the prognostic significance of the steroid receptor target genes of interest, more specifically, those deregulated as a result of cross-talk. Considering overall survival and disease specific survival, univariate and multivariate Cox models with Lasso Regression were used to identify prognostic gene signatures using RNA_seq datasets. Kaplan-Meier (KM) and ROC analyses were applied to evaluate risk models. Established genetic markers associated with MR, AR, GR and PR was used to stratify BC patients into low and high risk groups. Patients survival was found to be positively correlated with these signatures, demonstrates that these gene sets have prognostic value. Further studies are required to validate these preliminary results to understand the role of steroid receptor crosstalk in breast cancer formation, progression, and survival. Citation Format: Aygun Azadova, Greg Brooke, Antonio Marco. Steroid receptor cross-talk in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB335.

Investigation of Chemopreventive and Antiproliferative Potential of Dicliptera roxburghiana.

Context:Carcinogenesis causes much human misery. It is a process involving multistage alterations. Medicinal plants are candidates for beneficial anticancer agents.Objectives:Investigation of anticancer proficiencies of the plant Dicliptera roxburghiana.Material and methods:Crude extract and derived fractions were inspected for their inhibitory potential against nuclear factor KB (NFκB), nitric oxide synthase inhibition, aromatase inhibition and induction of quinone reductase 1 (QR 1). Antiproliferative activity was determined by using various cancer cell lines for example hormone responsive breast cancer cell line MCF-7, estrogen receptor negative breast cancer cell line MDA-MB-231, murine hepatoma cells Hepa 1c1c7, human neuroblastoma cells SK-N-SH and neuroblastoma cells MYCN-2.Results:Ethyl acetate and n-butanol fractions of D. roxburghiana were strongly active against NFκB with IC50 of 16.6 ± 1.3 and 8.4 ± 0.7 µg/ml respectively with 100% survival. Chloroform fraction of the plant exhibited an induction ratio of 2.4 ± 0.09 with CD value of 17.7 µg/ml. Regarding the nitrite assay, the n-hexane fraction exhibited significant inhibition of NO activity with IC50 of 17.8 ± 1.25 µg/ml. The n-butanol fraction exhibited strong antiproliferative activity against IcIc-7 cell lines with IC50 values of 13.6 ± 1.91 µg/ml; against MYCN-2 a cytotoxic effect developed with dose dependence, with IC50 of 12.6 ± 1.24 µg/ml. In antiproliferative activity against SK-N-SH cell lines, chloroform, ethyl acetate and n-butanol fractions were efficiently active with IC50 values of 11.2 ± 0.84, 14.6 ± 1.71 and 16.3 ± 1.57 respectively.Discussion and Conclusion:It was demonstrated that various fractions of D. roxburghiana displayed appreciable anticancer characteristics and could be a potent source for the development of anticancer leads.

Five furofuranone lignan glucosides from Terminalia citrina inhibit in vitro E2-enhanced breast cancer cell proliferation

Five new polyalkoxylated furofuranone lignan glucosides, terminalosides L–P (1–5), were isolated from EtOAc extracts of the leaves of Terminalia citrina, a Bangladeshi medicinal plant. The structures of the isolates were deduced primarily by NMR spectroscopy, and four of the isolates were found to contain rare tetraoxygenated aryl groups in their structures. The absolute configurations and conformations of the furofuranone ring were confirmed by ECD spectroscopy. All of the isolates were evaluated for their estrogenic and/or antiestrogenic properties using two estrogen responsive breast cancer cell lines, T47D and MCF-7. At a concentration of 10nM, terminaloside L (1) suppressed E2-enhanced T47D cell proliferation by 90%, while terminaloside M (2) showed 90% antiestrogenic activity against MCF-7 cells. Compared to 2, the antiestrogenic activity of terminaloside O (4) and P (5) was weak, possibly due to the different attachment positions of the sugar moiety that they share in common. This is the first report of furofuranone lignans from any Terminalia species, and also of their antiestrogenic activity.

Single-Cell States in the Estrogen Response of Breast Cancer Cell Lines

Estrogen responsive breast cancer cell lines have been extensively studied to characterize transcriptional patterns in hormone-responsive tumors. Nevertheless, due to current technological limitations, genome-wide studies have typically been limited to population averaged data. Here we obtain, for the first time, a characterization at the single-cell level of the states and expression signatures of a hormone-starved MCF-7 cell system responding to estrogen. To do so, we employ a recently proposed model that allows for dissecting single-cell states from time-course microarray data. We show that within 32 hours following stimulation, MCF-7 cells traverse, most likely, six states, with a faster early response followed by a progressive deceleration. We also derive the genome-wide transcriptional profiles of such single-cell states and their functional characterization. Our results support a scenario where estrogen promotes cell cycle progression by controlling multiple, sequential regulatory steps, whose single-cell events are here identified.

Abstract P5-05-07: Promestriene effects on estrogen-sensitive breast cancer cell proliferation in vitro

Abstract Introduction: Promestriene (3-propyl ethyl, 17B-methyl estradiol) is a synthetic estrogen analogue with potential as a topical treatment for vaginal atrophy caused by estrogen deprivation. Topical Promestriene is minimally absorbed with chronic use and has been reported to significantly improve the symptoms of vaginal atrophy. However, to be considered for use for these symptoms in estrogen responsive post-menopausal breast cancer survivors on an aromatase inhibitor, Promestriene would need to show none of the cyto-proliferative properties of estrogen. Methods: We conducted in vitro testing of estrogen responsive breast cancer cell line MCF-7, T-47D, and BT-474 for dose-dependent, estrogen-like functional responses to Promestriene in both estrogen-rich and estrogen-deprived conditions. As assay controls, cultures treated with Fulvestrant, Testosterone, Progesterone and Estradiol were analyzed in parallel. Analysis: Dose dependent Estrogen-like responses in the cell lines were measured as 1) proliferation using flow cytometric analysis of CSFE fluorescence, 2) estrogen receptor expression by flow cytometry, and 3) GREB 1 RNA expression using TaqMan real time PCR quantitative analysis. All cultures were grown to quiescence in RPMI medium supplemented with 10% fetal calf serum. To induce estrogen deprivation, half of the cultures were then held 3 days in media pre-treated and containing anti-estrogen specific antibodies to sequester any residual estrogen from the culture. Data for each culture treatment were analyzed using linear regression analysis and compared using multifactorial or pairwise analyses to determine the significance of estrogen-like responses relative to the estradiol and untreated controls. Results: In estrogen sufficient cultures, Promestriene exhibited no cell proliferative properties on MCF-7, BT-474, or T47-D tumor cells, even at very high concentrations. However, when estrogen in the media was sequestered and culture allowed to quiesce without estrogen receptor stimulation, low dose Promestriene effects on cell proliferation in MCF-7 estrogen responsive cells were not significantly different from low dose estradiol. Low dose Promestriene (2-10pg/ml), unlike comparable doses of estradiol, did not stimulate GREB1 expression in MCF-7 cells. In contrast, T-47D and BT-474 cell lines proliferated and increased GREB- expression in response Promestriene or Fulvestrant treatments. Conclusions: The potential estrogen-like properties of Promestriene to stimulate the growth of estrogen receptor responsive breast cancer cell lines, especially in estrogen-deprived conditions, suggest caution when prescribed for vaginal atrophy in post-menopausal breast cancer survivors on an aromatase inhibitor. Its ability to activate growth and gene expression in ER- breast cancer cells warrants further study. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-07.

Abstract P5-05-01: IGFBP-7 Reduces Growth of Xenografted Breast Tumors in Mice and Inhibits Breast Cancer Cell Proliferation and Migration through the MEK-ERK Pathway

Abstract To identify genes correlated with breast tumorigenesis and eventual metastasis, gene expression profiles were generated using 28K whole genome microarrays. Gene expression profiles of primary breast tumors that grew and metastasized in the NOD/SCID mice were compared with other primary breast tumors that had different growth and metastatic potentials. Five hundred and eighty two genes were significantly differentially expressed by our statistical comparison criteria using the GeneSpring GX 7.3.1 software suite. In the metastatic set eight genes were selectively overexpressed (YB1, MMP7, MMP9, RAB5A, RABGDIB, EPHRB3, WNT2B, CSF1R) and 12 were selectively underexpressed (IGFBP7, GATA3, CST5, CDK6, SERBP1, MGP, TGF1L4, ESE3, ELF3, EDNRB, HECTD1, TINP1). In the present study we focused on the IGFBP-7 gene product which was found to be inversely correlated with disease progression in breast cancer. To further investigate the role of IGFBP-7 in breast tumor suppression, it was overexpressed in the triple negative MDA-MB-468 human breast cancer line. Ectopic overexpression of IGFBP-7 clearly reduced the growth of the MDA-MB-468/IGFBP-7 cells compared to the parental MDA-MB-468 cells. Ectopic overexpression or addition of rIGFBP-7 to breast cancer cell lines in vitro clearly reduced the growth of several breast cancer cell lines and resulted in phenotypic changes characterized by cell aggregation. Investigation of downstream signalling pathways affected by IGFBP-7 revealed that addition of IGFBP-7 to a triple negative breast cancer cell line not only inhibited phosphorylation of ERK-1/2 but also increased expression of the cyclin-dependent kinase inhibitor 1, p21. Similar IGFBP-7 treatment of an ER+ breast cancer line resulted not only in inhibition of ERK-1/2 phosphorylation, but also AKT phosphorylation, suggesting that IGFBP-7 may affect multiple pathways in hormone responsive breast cancer cell lines. IGFBP-7 protein is processed into a shorter form by matriptase. Breast cancer cell lines which were unable to process IGFBP-7 into the shorter form were unaffected by IGFBP-7 mediated growth inhibition, suggesting that cleaved IGFBP-7 may be required for growth inhibitory effects. When injected subcutaneously into NOD/SCID mice, the increased expression of IGFBP-7 in the MDA-MB-468/IGFBP-7 cells reduced the rate of tumor growth in comparison to the parental MDA-MB-468 cells. Furthermore, injection of rIGFBP-7 protein at the tumor site into breast cancer xenografted NOD/SCID mice also resulted in significant tumor growth reduction. These results suggest that the growth of breast cancer could be prevented by the forced expression of IGFBP-7 protein. Work in progress will further uncover the mechanism of IGFBP-7-mediated inhibition on signalling pathways, as well as differentiate the impact of both full length and cleaved IGFBP-7 protein on breast tumor inhibition. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-01.

Sequential Design for Microarray Experiments

This article features online supplementary material.A critical aspect in the design of microarray studies is the determination of the sample size necessary to declare genes differentially expressed across different experimental conditions. In this article, we propose a sequential approach where the decision to stop the experiment depends on the accumulated microarray data. The study could stop whenever sufficient data have been accumulated to identify gene expression changes across several experimental conditions. The gene expression response is modeled by a robust linear regression model. We then construct a sequential confidence interval for the intercept of this model, which represents the median gene expression at a given experimental condition. We derive the stopping rule of the experiment for both continuous and discrete sequential approaches and give the asymptotic properties of the stopping variable. We demonstrate the desirable properties of our sequential approach, both theoretically and numerically. In our application to a study of hormone responsive breast cancer cell lines, we estimated the stopping variable for the sample size determination to be smaller than the actual sample size available to conduct the experiment. This means that we can obtain an accurate assessment of differential gene expression without compromising the cost and size of the study. Altogether, we anticipate that this approach could have an important contribution to microarray studies by improving the usual experimental designs and methods of analysis.

Sodium arsenite induces ROS generation, DNA oxidative damage, HO-1 and c-Myc proteins, NF-κB activation and cell proliferation in human breast cancer MCF-7 cells

Epidemiological evidence has associated exposure to arsenic (As) in drinking water with an increased incidence of human cancers in the skin, bladder, liver, kidney and lung. Sodium arsenite mimics the effects of estradiol and induces cell proliferation in the estrogen responsive breast cancer cell line MCF-7. Therefore, our aim was to further explore the ability of sodium arsenite to induce MCF-7 epithelial breast cell proliferation and some of its underlying mechanisms by studying ROS production, c-Myc and HO-1 protein levels, 8-OHdG formation and NF-κB activation. Low arsenite concentrations (0.5–5 μM) induced ROS production and ROS-related depolarization of the mitochondrial membrane suggesting that mitochondria played an important role in the oxidative effects of As. ROS-mediated DNA damage as measured by the presence of 8-OHdG DNA-adducts in their nuclei, IκB phosphorylation, NF-κB activation and increases in c-Myc and HO-1 protein levels were also observed, suggesting that these factors play a relevant role in the arsenite induced MCF-7 cell recruitment into the S-phase of the cell cycle and cell proliferation observed. In conclusion, arsenite activates several pathways involved in MCF-7 cell proliferation suggesting that arsenite exposure may pose a risk for breast cancer in human exposed populations notwithstanding that most studies to date have not yet implicated this metalloid as a cofactor in the etiology of this disease.

Delineation of molecular mechanisms of sensitivity to lapatinib in breast cancer cell lines using global gene expression profiles

Lapatinib (GW572016) is a small-molecule dual inhibitor of epidermal growth factor receptor (ErbB1) and ErbB2 receptor kinase activities currently in phase III clinical trials. We used phosphoprotein and microarray analyses to carry out targeted pathway studies of phosphorylation and gene expression changes in human breast cancer cell lines in the presence or absence of lapatinib. Studies were done in four breast cancer cell lines, two of which were responsive and two of which were nonresponsive to lapatinib. Responsive cell lines, BT474 and SKBr3, constitutively overexpress ErbB2 and show an IC(50) of 25 or 32 nmol/L for lapatinib, respectively. In contrast, nonresponsive MDA-MB-468 and T47D cells expressed a low basal level of ErbB2 and showed IC(50) values in the micromolar range. Cells responsive to lapatinib exhibited strong differential effects on multiple genes in the AKT pathway. After 12 h of exposure to 1.0 micromol/L of lapatinib, AKT1, MAPK9, HSPCA, IRAK1, and CCND1 transcripts were down-regulated 7- to 25-fold in responsive BT474 and SKBr3 cells. In contrast, lapatinib weakly down-regulated the AKT pathway in nonresponsive breast cancer cell lines (<5-fold down-regulation of most genes in the pathway). Furthermore, the proapoptotic gene FOXO3A, which is negatively regulated by AKT, was up-regulated 7- and 25-fold in lapatinib-responsive SKBr3 and BT474 cells, respectively. Phosphorylated Akt and Akt-mediated phosphorylation of FOXO3A also decreased in responsive breast cancer cell lines exposed to lapatinib. Gene expression profiling also revealed that lapatinib stimulated the expression of estrogen and progesterone receptors and modulated the expression of genes involved in cell cycle control, glycolysis, and fatty acid metabolism. In BT474 and T47D cells, which expressed moderate basal levels of the estrogen and progesterone receptors, 1.0 micromol/L of lapatinib induced expression by 7- to 11-fold. These data provide insight into the mechanism of action of lapatinib in breast cancer cells.

Exploration of low-dose estrogen effects: identification of No Observed Transcriptional Effect Level (NOTEL).

Identifying a minimal dose capable of eliciting a biological response is a fundamental issue in a number of scientific fields, including: drug development, signal transduction research, and environmental toxicology. Frequently, proliferation, viability, and other assays based on the cellular response to a treatment are used to assess the threshold dose for minimal activity. Here we propose a novel approach for identifying the effects of low dose treatments and pinpointing the threshold dose. Using microarrays, we examined the transcriptional response of a hormone responsive breast cancer cell line (MCF-7) stimulated with various concentrations of estrogen. Previous studies have focused on transcriptional responses to physiologically relevant concentrations of estrogen. However, relatively few studies have examined the transcriptional effects of concentrations below normal physiologic levels. These doses may not stimulate the expression of any genes or, alternatively, may regulate a different subset of genes that had not been previously characterized as estrogen responsive. We used gene expression profiling, coupled with a detailed analysis of replicates, to measure estrogen effects on many transcriptional targets and found that only physiologically relevant doses of estrogen (1 x 10(-10) M and higher) were capable of inducing a transcriptional response. This study demonstrates the utility of gene expression profiling as a means to identify concentrations that do not elicit a change in gene expression, or simply a No Observed Transcriptional Effect Level (NOTEL). The identification of a NOTEL for a given compound may be beneficial in several different scientific disciplines. For example, in the development of therapeutic drugs, a NOTEL could be used to identify doses of pharmaceutical compounds that are no longer effective at modulating the expression of biomarkers of efficacy.

The cooked food derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine is a potent oestrogen: a mechanistic basis for its tissue-specific carcinogenicity.

The cooked meat carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces tumours of the breast, colon and prostate in rats. Here we show that in addition to its well-established genotoxicity, which can be detected at concentrations >10(-6) M, PhIP is also oestrogenic. In COS-1 cells transiently transfected with an oestrogen-responsive reporter gene, PhIP (10(-10)-10(-6) M) mediated transcription through oestrogen receptor (ER) alpha, but not ER-beta, and inhibition by the pure ER antagonist ICI 182 780 demonstrated a requirement for a functional ER. In contrast, the structurally related food-derived carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) failed to induce reporter gene transcription. Additionally, we show that in a hormonally responsive breast cancer cell line (MCF-7 cells), PhIP induced transcriptional activation using endogenously expressed ER. Examination of the genotoxic potential of PhIP using a model mammalian cell mutation assay (hprt(-) locus) demonstrated that the genetic toxicology of PhIP was readily detectable, but separate, in terms of effective concentration, from its oestrogenic activity. To determine whether the oestrogenicity of PhIP could mediate oestrogen-dependent responses such as cell growth, we examined the growth of hormonally responsive cells (MCF-7 cells). We show that PhIP can stimulate cell proliferation and, again, this was dependent upon a functional ER. Using ligand blotting, we further show that PhIP can stimulate the expression of progesterone receptor (PR-A and PR-B) and c-MYC and activate the MAPK signal transduction pathway. These responses were similar to that produced by oestradiol, in terms of temporal aspects, potency and a requirement for a functional ER. Each of these dose-dependent mitogenic responses occurred at concentrations of PhIP ( approximately 10(-9)-10(-11)M) that are likely to be equivalent to systemic human exposure via consumption of cooked meat. Thus PhIP can induce cellular responses that encompass altered gene expression and mitogenesis. We suggest that the combination of genetic toxicology and oestrogen-like promotion of genomic and cellular events provide a mechanism for the tissue-specific tumorigenicity of this compound.

Estrogen receptor-alpha and C-ERBB-4 expression in breast carcinomas.

The presence of estrogen receptors (ERs) in breast carcinomas is important for clinical response to endocrine therapy. However, the cellular mechanisms following ER activation are not fully understood. It has been indicated that expression of the ER is associated with the expression of c-erbB-4. To address this question, 103 breast carcinoma samples were studied using reverse transcriptase polymerase chain reaction (RT-PCR) analysis after application of a microselection method for RNA isolation. Total RNA for RT-PCR was isolated from 20-microm-thick frozen sections, which were made from microselected areas. Paraffin blocks from 98 of these 103 tumors were also immunohistochemically examined. Significant associations between ER-alpha and c-erbB-4 mRNA and protein expressions were found in the present study with both methods. One-fourth of the tumors did not express ER-alpha (22%, 24%, and 26% with chemical binding, immunohistochemistry, and RT-PCR, respectively). About one-half of the ER-alpha negative tumors did not express c-erbB-4 on both mRNA and protein levels (48% with RT-PCR and 46% with immunohistochemistry, P=0.001 for both methods). The endocrine therapy responsive breast cancer cell lines MCF-7 and T47-D were positive for both ER-alpha and c-erbB-4 expression, while the endocrine therapy nonresponsive breast cancer cell lines MDA-MD-231 and SK-BR-3 were not. Thus, we confirm the association between the expression of ER-alpha and c-erbB-4 mRNA and protein in breast carcinomas, indicating a role for c-erbB-4 in estrogen signal transduction.

Influence of 1 alpha,25-dihydroxyvitamin D-3 on growth regulation through epidermal growth factor receptor in human breast cancer cell lines

We investigated the relationship between epidermal growth factor (EGF) dependent cell growth and antiproliferative effects of 1 alpha,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) in hormone responsive breast cancer cell lines in vitro. MCF-7 breast cancer cells and GMC-M, which is a serum-independent, hormone receptor-positive subtype derived from MCF-7, were used in this study. EGF stimulated the growth Of both cell lines, and 1,25(OH)(2)D-3 inhibited the EGF-stimulated cell growth in a dose dependent fashion. But treatment with 1,25(OH)(2)D-3 did not change the EGF receptor (EGFR) level significantly in either cell line. GMC-M had a higher level of EGFR and was more sensitive to EGF than MCF-7. These results suggest that other mechanisms of action, which are different from EGFR modulation, concern with the growth inhibitory effect of 1,25(OH)(2)D-3, and that 1,25(OH)(2)D-3 will be a new effective treatment for breast cancer irrespective of EGFR.

Dexamethasone mediated stabilization of insulin receptor mRNA.

To determine the mechanism of glucocorticoid mediated enhancement of insulin receptor (IR) gene expression, we cotransfected a glucocorticoid receptor expression vector and a plasmid containing a reporter gene driven by an MMTV or IR promoter into COS 7 cells. Dexamethasone (Dex) increased MMTV promoter activity by 100% but had no effect on IR promoter activity. In the glucocorticoid responsive breast cancer cell line, MCF-7, Dex increased IR mRNA by 60%, and increased the IR mRNA half-life from approximately 6hrs to > 24 hrs. No glucocorticoid responsive element could be located in the insulin receptor 3' untranslated region. Glucocorticoids stabilize IR mRNA.

Oestrogen receptor expression is decreased by oestrogen in MCF-7 cells but increased by oestrogen in three other oestrogen responsive breast cancer cell lines

Oestrogen receptor expression is decreased by oestrogen in MCF-7 cells but increased by oestrogen in three other oestrogen responsive breast cancer cell lines

Oestrogen regulates cathepsin D mRNA levels in oestrogen responsive human breast cancer cells.

A cDNA library was constructed from the mRNA of the ZR-75 oestrogen responsive human breast cancer cell line and screened for oestrogen regulated mRNA sequences. Of the recombinants isolated, 30 contained cDNA that corresponded to a single mRNA species of 2.1 kb that was induced between 10 and 15 fold by oestradiol treatment. The sequence of the entire open reading frame and 3' non-coding region of the mRNA was determined and shown to encode the aspartyl protease cathepsin D. The induction of cathepsin D mRNA is specific for oestrogen and is maximal at 3 X 10(-10) M. Cathepsin D mRNA levels were increased by oestrogen in 3 oestrogen responsive breast cancer cell lines. Cathepsin D mRNA was expressed but not regulated in an oestrogen receptor negative breast tumour cell line, BT 20, and in 2 other malignant cell lines, Hela and A431.