The E3 ubiquitin ligase EDD is an adverse prognostic factor for serous epithelial ovarian cancer and modulates cisplatin resistance in vitro.

Despite a high initial response rate to first-line platinum/paclitaxel chemotherapy, most women with epithelial ovarian cancer relapse with recurrent disease that becomes refractory to further cytotoxic treatment. We have previously shown that the E3 ubiquitin ligase, EDD, a regulator of DNA damage responses, is amplified and overexpressed in serous ovarian carcinoma. Given that DNA damage pathways are linked to platinum resistance, the aim of this study was to determine if EDD expression was associated with disease recurrence and platinum sensitivity in serous ovarian cancer. High nuclear EDD expression, as determined by immunohistochemistry in a cohort of 151 women with serous ovarian carcinoma, was associated with an approximately two-fold increased risk of disease recurrence and death in patients who initially responded to first-line chemotherapy, independently of disease stage and suboptimal debulking. Although EDD expression was not directly correlated with relative cisplatin sensitivity of ovarian cancer cell lines, sensitivity to cisplatin was partially restored in platinum-resistant A2780-cp70 ovarian cancer cells following siRNA-mediated knockdown of EDD expression. These results identify EDD as a new independent prognostic marker for outcome in serous ovarian cancer, and suggest that pathways involving EDD, including DNA damage responses, may represent new therapeutic targets for chemoresistant ovarian cancer.

Estrogen inhibits GH signaling by suppressing GH-induced JAK2 phosphorylation, an effect mediated by SOCS-2.

Oral estrogen administration attenuates the metabolic action of growth hormone (GH) in humans. To investigate the mechanism involved, we studied the effects of estrogen on GH signaling through Janus kinase (JAK)2 and the signal transducers and activators of transcription (STATs) in HEK293 cells stably expressing the GH receptor (293GHR), HuH7 (hepatoma) and T-47D (breast cancer) cells. 293GHR cells were transiently transfected with an estrogen receptor-alpha expression plasmid and luciferase reporters with binding elements for STAT3 and STAT5 or the beta-casein promoter. GH stimulated the reporter activities by four- to sixfold. Cotreatment with 17beta-estradiol (E(2)) resulted in a dose-dependent reduction in the response of all three reporters to GH to a maximum of 49-66% of control at 100 nM (P < 0.05). No reduction was seen when E(2) was added 1-2 h after GH treatment. Similar inhibitory effects were observed in HuH7 and T-47D cells. E(2) suppressed GH-induced JAK2 phosphorylation, an effect attenuated by actinomycin D, suggesting a requirement for gene expression. Next, we investigated the role of the suppressors of cytokine signaling (SOCS) in E(2) inhibition. E(2) increased the mRNA abundance of SOCS-2 but not SOCS-1 and SOCS-3 in HEK293 cells. The inhibitory effect of E(2) was absent in cells lacking SOCS-2 but not in those lacking SOCS-1 and SOCS-3. In conclusion, estrogen inhibits GH signaling, an action mediated by SOCS-2. This paper provides evidence for regulatory interaction between a sex steroid and the GHJAKSTAT pathway, in which SOCS-2 plays a central mechanistic role.

Lycopene inhibition of cell cycle progression in breast and endometrial cancer cells is associated with reduction in cyclin D levels and retention of p27(Kip1) in the cyclin E-cdk2 complexes.

Numerous studies have demonstrated the anticancer activity of the tomato carotenoid, lycopene. However, the molecular mechanism of this action remains unknown. Lycopene inhibition of human breast and endometrial cancer cell growth is associated with inhibition of cell cycle progression at the G(1) phase. In this study we determined the lycopene-mediated changes in the cell cycle machinery. Cells synchronized in the G(1) phase by serum deprivation were treated with lycopene or vehicle and restimulated with 5% serum. Lycopene treatment decreased serum-induced phosphorylation of the retinoblastoma protein and related pocket proteins. This effect was associated with reduced cyclin-dependent kinase (cdk4 and cdk2) activities with no alterations in CDK protein levels. Lycopene caused a decrease in cyclin D1 and D3 levels whereas cyclin E levels did not change. The CDK inhibitor p21(Cip1/Waf1) abundance was reduced while p27(Kip1) levels were unaltered in comparison to control cells. Serum stimulation of control cells resulted in reduction in the p27 content in the cyclin E--cdk2 complex and its accumulation in the cyclin D1--cdk4 complex. This change in distribution was largely prevented by lycopene treatment. These results suggest that lycopene inhibits cell cycle progression via reduction of the cyclin D level and retention of p27 in cyclin E--cdk2, thus leading to inhibition of G(1) CDK activities.

c-Myc or cyclin D1 mimics estrogen effects on cyclin E-Cdk2 activation and cell cycle reentry.

Estrogen-induced progression through G1 phase of the cell cycle is preceded by increased expression of the G1-phase regulatory proteins c-Myc and cyclin D1. To investigate the potential contribution of these proteins to estrogen action, we derived clonal MCF-7 breast cancer cell lines in which c-Myc or cyclin D1 was expressed under the control of the metal-inducible metallothionein promoter. Inducible expression of either c-Myc or cyclin D1 was sufficient for S-phase entry in cells previously arrested in G1 phase by pretreatment with ICI 182780, a potent estrogen antagonist. c-Myc expression was not accompanied by increased cyclin D1 expression or Cdk4 activation, nor was cyclin D1 induction accompanied by increases in c-Myc. Expression of c-Myc or cyclin D1 was sufficient to activate cyclin E-Cdk2 by promoting the formation of high-molecular-weight complexes lacking the cyclin-dependent kinase inhibitor p21, as has been described, following estrogen treatment. Interestingly, this was accompanied by an association between active cyclin E-Cdk2 complexes and hyperphosphorylated p130, identifying a previously undefined role for p130 in estrogen action. These data provide evidence for distinct c-Myc and cyclin D1 pathways in estrogen-induced mitogenesis which converge on or prior to the formation of active cyclin E-Cdk2-p130 complexes and loss of inactive cyclin E-Cdk2-p21 complexes, indicating a physiologically relevant role for the cyclin E binding motifs shared by p130 and p21.

Identification of a human HECT family protein with homology to the Drosophila tumor suppressor gene hyperplastic discs.

Use of the differential display technique to isolate progestin-regulated genes in T-47D human breast cancer cells led to identification of a novel gene, EDD. The cDNA sequence contains a 2799 amino acid open reading frame sharing 40% identity with the predicted 2894 amino acid product of the Drosophila melanogaster tumor suppressor gene hyperplastic discs, while the carboxy-terminal 889 amino acids show 96% identity to a rat 100 kDa HECT domain protein. EDD mRNA was progestin-induced in T-47D cells and was highly abundant in testes and expressed at moderately high levels in other tissues, suggesting a broad role for EDD. Anti-EDD antibodies immunoprecipitated an approximately 300 kDa protein from T-47D cell lysates. HECT family proteins function as E3 ubiquitin-protein ligases, targeting specific proteins for ubiquitin-mediated proteolysis. EDD is likely to function as an E3 as in vitro translated protein bound ubiquitin reversibly through a conserved HECT domain cysteine residue. EDD was localized by FISH to chromosome 8q22, a locus disrupted in a variety of cancers. Given the homology between EDD and the hyperplastic discs protein, which is required for control of imaginal disc growth in Drosophila, EDD potentially has a role in regulation of cell proliferation or differentiation.

Estrogen and progestin regulation of cell cycle progression.

Estrogens and progesterone, acting via their specific nuclear receptors, are essential for normal mammary gland development and differentiated function. The molecular mechanisms through which these effects are mediated are not well defined, although significant recent progress has been made in linking steroid hormone action to cell cycle progression. This review summarizes data identifying c-myc and cyclin D1 as major downstream targets of both estrogen- and progestin-stimulated cell cycle progression in human breast cancer cells. Additionally, estrogen induces the formation of high specific activity forms of the cyclin E-Cdk2 enzyme complex lacking the cyclin-dependent kinase (CDK)3 inhibitor, p21. The delayed growth inhibitory effects of progestins, which are likely to be prerequisites for manifestation of their function in differentiation, also involve decreases in cyclin D1 and E gene expression and recruitment of CDK inhibitors into cyclin D1-Cdk4 and cyclin E-Cdk2 complexes. Thus estrogens and progestins affect CDK function not only by effects on cyclin abundance but also by regulating the recruitment of CDK inhibitors and, as yet undefined, additional components which determine the activity of the CDK complexes. These effects of estrogens and progestins are likely to be major contributors to their regulation of mammary epithelial cell proliferation and differentiation.

Antisense estrogen receptor RNA expression increases epidermal growth factor receptor gene expression in breast cancer cells.

In human breast cancer, progression to a more malignant phenotype is often accompanied by decreased expression of estrogen receptor (ER) and increased expression of epidermal growth factor receptor (EGFR). Higher levels of this receptor tyrosine kinase are found in tumors lacking ER, and a quantitative, inverse relationship exists between the level of ER and EGFR mRNA in human breast cell lines. Antisense ER (ASER) RNA was used to evaluate the consequence of decreased ER expression in breast cancer cells, specifically to determine whether ER is involved in the regulation of EGFR gene expression. ER-positive MCF-7 human breast cancer cells were transfected with ASER, and clones constitutively expressing ASER RNA had decreased ER and up to a 3-fold increase in the expression of EGFR mRNA. To confirm that this observation was a direct consequence of ASER expression, a metal-inducible ASER expression construct was transfected into MCF-7 cells, and transfected clones were isolated and characterized. Northern analysis revealed an induction of ASER RNA within 1 h of the addition of zinc, which was followed by a 4-fold increase in EGFR mRNA levels, maximal at 6-12 h. The basal level of expression of the glucocorticoid receptor is also inversely related to that of ER among breast cancer cell lines, but neither constitutive nor inducible expression of ASER affected the expression of glucocorticoid receptor. These data support the hypothesis that the level of expression of ER specifically influences the expression of EGFR in human breast cancer cells and provides a potential link between loss of steroid sensitivity and the acquisition of autonomous growth.

Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

Estrogens induce cell proliferation in target tissues by stimulating progression through G1 phase of the cell cycle, but the underlying molecular targets remain undefined. To determine the role of the cyclin/cyclin-dependent kinase (CDK)/retinoblastoma protein (pRB) pathway in this response we treated MCF-7 breast cancer cells with the pure estrogen antagonist ICI 182780 to inhibit estrogen-induced gene expression and induce G1 phase arrest. Subsequent treatment with 17beta-estradiol resulted in the synchronous entry of cells into S phase commencing at 12 h. The proportion of cells in S phase reached a maximum of 60% at 21-24 h. Cells subsequently completed mitosis and entered a second semisynchronous round of replication. Entry into S phase was preceded by increased activity of both Cdk4 and cyclin E-Cdk2 and hyperphosphorylation of pRB, all within the first 3-6 h of estradiol treatment. The increase in Cdk4 activity was accompanied by increases in cyclin D1 mRNA and protein, indicating that an initiating event in the activation of Cdk4 was increased cyclin D1 gene expression. In contrast, the levels of Cdk2 and the CDK inhibitors p21 (WAF1/CIP1/SDI1) and p27 (KIP1) in total cell lysates and in cyclin E immunoprecipitates were unaltered at these early time points. However, an inhibitory activity was present in antiestrogen-pretreated cell lysates toward recombinant cyclin E-Cdk2 and was relieved by estradiol treatment. This activity was attributable predominantly to p21. These apparently conflicting data were resolved by performing gel filtration chromatography, which revealed that only a minority of cyclin E-Cdk2 complexes were active following estradiol treatment. Active complexes eluted at a higher molecular weight than inactive complexes, were relatively deficient in both p21 and p27, and contained Cdk2 with increased threonine 160 phosphorylation, consistent with a mechanism of activation of cyclin E-Cdk2 involving both reduced CDK inhibitor association and CDK-activating kinase-mediated phosphorylation of Cdk2. These results provide an explanation for the early activation of both cyclin D1-Cdk4 and cyclin E-Cdk2 complexes that accompany G1-S phase progression in response to estradiol.

Identification of PRG1, a novel progestin-responsive gene with sequence homology to 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

To define early molecular targets of progestin action, the differential display technique was used to identify genes with altered levels of expression in T-47D breast cancer cells treated with the synthetic progestin ORG 2058 for 3 h. PRG1 was first isolated as a 200-bp cDNA clone and its progestin regulation confirmed by Northern analysis. Cloning of the complete coding region of PRG1 revealed that it shared a high degree of amino acid sequence identity with isoforms of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from several tissues and species. Expression of PRG1 mRNA was observed in several normal breast epithelial and breast cancer cell lines and in a variety of human tissues, with highest expression in the breast, aorta, and brain. In T-47D cells, PRG1 mRNA was rapidly and transiently induced by progestins, expression peaking between 2 and 4 h and returning to control levels by 12 h. Progestin-induced increases in PRG1 mRNA were inhibited by the progestin antagonist RU 486 and occurred via the progesterone receptor. Progestin induction of PRG1 mRNA was also inhibited by actinomycin D but not by cycloheximide. PRG1 is therefore a novel human gene that is directly regulated by progestins via the progesterone receptor.

D-penicillamine causes free radical-dependent inactivation of activator protein-1 DNA binding.

D-Penicillamine (beta, beta-dimethyl cysteine) is an antirheumatic thiol drug with a poorly understood mechanism of action. On the basis that gold(I) thiolates and D-penicillamine are both capable of forming stable bonds with endogenous thiols, we sought a common target of action. Cysteine residues in the basic DNA binding domains of Jun and Fos, members of the activator protein-1 (AP-1) transcription factor family, have been identified as likely targets for the therapeutic action of antirheumatic gold(I) thiolates. The current study demonstrates that AP-1 DNA binding is inhibited by D-penicillamine in the presence of Fenton reagents (Fe2+/EDTA and H2O2) but not with either agent alone. The effect is biphasic, with maximum inhibition in the concentration range of approximately 100-250 microM. Cysteine has qualitatively similar properties, although the effect is less pronounced. In contrast, glutathione and thiomalate do not inhibit AP-1 DNA binding, even in the presence of Fenton reagents. Mutant proteins were used to identify the cysteine residues within the DNA binding domains of Jun and Fos that are essential for the inhibitory action of D-penicillamine. The results suggest that D-penicillamine is distinguished from other thiols by its formation of sulfur-containing radicals capable of inhibiting AP-1 DNA binding by a mechanism involving the cysteine residues of Jun and Fos.

Cyclins and breast cancer.

Recent advances in the understanding of cell cycle control by cyclins and cyclin-dependent kinases provide a basis for delineating the molecular mechanisms of proliferation control by steroids and the development and progression of hormone-dependent cancers. Cyclin D1 is necessary, rate-limiting and sufficient for G1 progression in breast cancer cells and regulation of cyclin D1 expression or function is an early response to steroid and steroid antagonist regulation of proliferation. The cyclin D1 gene is amplified in approximately 15%, and its product overexpressed in 40-50%, of primary breast carcinomas. The strong evidence that cyclin D1 plays a major role in cell cycle control in breast epithelial cells suggests that its deregulated expression may have effects on disease progression and phenotype including sensitivity to endocrine therapies.

Antiestrogen inhibition of cell cycle progression in breast cancer cells in associated with inhibition of cyclin-dependent kinase activity and decreased retinoblastoma protein phosphorylation.

To define the mechanisms by which antiestrogens inhibit breast cancer cell proliferation, the effects of the antiestrogen ICI 182780 on G1 cyclins and their cyclin-dependent kinase (CDK) partners were investigated in MCF-7 cells. Inhibition of entry into S phase became evident 9 h after treatment, with the proportion of cells in S phase reaching a minimum by 24 h. ICI 182780 increased the proportion of the hypophosphorylated, growth inhibitory form of the retinoblastoma protein (pRB). This change began at 4-6 h, preceding effects on S phase. This suggests that there are early effects on the activities of CDKs that target pRB that are not merely a consequence of changes in cell cycle progression. The kinase activity of Cdk2 decreased to low levels at 18-24 h when changes in S phase and pRB phosphorylation were well advanced. An earlier effect was seen on kinase activity associated with immunoprecipitated cyclin D1, which was reduced approximately 40% by 12 h, with further decreases at 18-24 h. Cdk2 and Cdk4 protein levels remained constant over 24 h. Cyclin D1 messenger RNA and protein were down-regulated by ICI 182780 from 2 h, with levels halved at 8 h. ICI 182780 also increased the expression of the CDK inhibitors p27KIP1 and p21WAF1/CIP1 at later times. These observations are compatible with the hypothesis that antiestrogens block entry of cells into S phase and inhibit cell proliferation as the consequence of an early decline in pRB phosphorylation contributed to by reduced cyclin D1/Cdk4 activity. At later times, increased CDK inhibitor abundance may act to repress Cdk2 and Cdk4 activities, causing additional reductions in pRB phosphorylation, thus maintaining the antiestrogen blockade of cell cycle progression.

Inhibition of AP-1 binding and transcription by gold and selenium involving conserved cysteine residues in Jun and Fos.

Gold(I) salts and selenite, which have diverse therapeutic and biological effects, are noted for their reactivity with thiols. Since the binding of Jun-Jun and Jun-Fos dimers to the AP-1 DNA binding site is regulated in vitro by a redox process involving conserved cysteine residues, we hypothesized that some of the biological actions of gold and selenium are mediated via these residues. In electrophoretic mobility-shift analyses, AP-1 DNA binding was inhibited by gold(I) thiolates and selenite, with 50% inhibition occurring at approximately 5 microM and 1 microM, respectively. Thiomalic acid had no effect in the absence of gold(I), and other metal ions inhibited at higher concentrations, in a rank order correlating with their thiol binding affinities. Cysteine-to-serine mutants demonstrated that these effects of gold(I) and selenite require Cys272 and Cys154 in the DNA-binding domains of Jun and Fos, respectively. Gold(I) thiolates and selenite did not inhibit nonspecific protein binding to the AP-1 site and were at least an order of magnitude less potent as inhibitors of sequence-specific binding to the AP-2, TFIID, or NF1 sites compared with the AP-1 site. In addition, 10 microM gold(I) or 10 microM selenite inhibited expression of an AP-1-dependent reporter gene, but not an AP-2-dependent reporter gene. These data suggest a mechanism regulating transcription factor activity by inorganic ions which may contribute to the known antiarthritic action of gold and cancer chemoprevention by selenium.

Expression and regulation of cyclin genes in breast cancer.

Cyclins, the regulatory subunits of cyclin-dependent kinases, control passage through key check-points within the cell cycle. Since dysregulated expression and function of cyclins can lead to loss of normal growth control some of these genes are oncogenes. We have studied cyclin gene expression, regulation and function in breast cancers. Induction of cyclin D1 is an early event in mitogenic stimulation of breast cancer cells by growth factors and steroids. Furthermore, inhibition of cyclin D1 expression is an early response to growth inhibition by antioestrogens. Ectopic expression of cyclin D1 in T-47D breast cancer cells demonstrated that cyclin D1 is rate-limiting for progression through G1 phase and is sufficient for growth arrested cells to complete the cell cycle. Since this gene is frequently overexpressed in human breast cancers it may contribute to the development and progression of some breast carcinomas.

Steroidal regulation of cell cycle progression.

Sex steroid hormones and their antagonists have well-defined mitogenic and growth-inhibitory effects on target cells including cancer cells. These effects are mediated by cell cycle phase-specific actions, implying that steroids control rates of cell cycle progression by regulating the expression of key cell cycle regulatory genes. An emerging model of cell cycle control involves transcriptional induction of cyclin genes and consequent activation of cyclin-dependent kinases, which initiate cellular events necessary to complete checkpoints within the cell cycle. Our recent studies have focused on the roles of G1 cyclins, particularly cyclin D1, in the control of cell cycle progression in human breast cancer cells. These studies show that cyclin D1 induction is an early response to mitogenic stimulation by oestrogens and progestins, is rate-limiting for G1 progression and is sufficient for completion of the cell cycle in cells arrested in early G1 phase by serum deprivation. Furthermore, inhibition of cyclin D1 expression is an early response to growth-inhibitory anti-oestrogens. These results suggest that cyclin D1 is a target for regulation of cell cycle progression by sex steroids and their antagonists.

Overexpression of estrogen receptor in HTB 96 human osteosarcoma cells results in estrogen-induced growth inhibition and receptor cross talk.

Estrogenic effects on the proliferation and differentiated cellular functions of bone cells have been described in vivo and in vitro. In particular, stimulatory effects on the growth rate of osteoblasts have been observed, although these are generally small. In an attempt to produce a more sensitive model for the study of estrogen action in bone, HTB 96 human osteoblast-like osteosarcoma cells, which lack endogenous estrogen receptor (ER), were stably transfected with an expression vector coding for the human ER gene. Several HTB 96 sublines expressing ER protein, detected by ligand binding and immunoassay, were isolated. The ability of 17 beta-estradiol (E2) to induce chloramphenicol acetyltransferase (CAT) activity from a cotransfected reporter vector containing the CAT gene linked to the Xenopus vitellogenin A2 gene estrogen response element demonstrated that the expressed ER was functional. ER continued to be expressed over a 30 week culture period. E2 but not other steroids significantly reduced growth rates and produced an altered morphology in HTB 96 sublines expressing higher levels of ER. The antiestrogen 4-hydroxytamoxifen partially reversed the E2 effect on growth rate. Transient transfection of cells expressing ER with a vector containing the CAT gene linked to the mouse mammary tumor virus long terminal repeat sequence, which contains response elements for the glucocorticoid receptor but not the ER, showed that E2 was able to inhibit CAT induction by dexamethasone. This result suggest that in ER-transfected HTB 9 cells the effects of E2 may result not from direct activation of endogenous genes but instead by transcriptional interference.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiestrogen regulation of cell cycle progression and cyclin D1 gene expression in MCF-7 human breast cancer cells.

The molecular mechanisms by which antiestrogens inhibit breast cancer cell proliferation are not well understood. Using cultured breast cancer cell lines, we studied the effects of antiestrogens on proliferation and cell cycle progression and used this information to select candidate cell cycle regulatory genes that are potential targets for antiestrogens. Under estrogen- and serum-free conditions antiestrogens inhibited proliferation of MCF-7 cells stimulated with insulin. Cells were blocked at a point in G1 phase. These effects are comparable with those in serum- and estrogen-containing medium and were also seen to a lesser degree in nude mice bearing MCF-7 tumors. Similar observations with other peptide mitogens suggest that the process inhibited by antiestrogens is common to estrogen and growth factor activated pathways. Other studies have identified G1 cyclins as potential targets for growth factor and steroid hormone/steroid antagonist regulation of breast epithelial cell proliferation. In MCF-7 cells growing in the presence of fetal calf serum, cyclin D1 mRNA was rapidly down-regulated by steroidal and nonsteroidal antiestrogens by an apparently estrogen receptor mediated mechanism. Cyclin D1 gene expression was maximally inhibited before effects on entry into S phase and inhibition was therefore not merely a consequence of changes in cell cycle progression. Together with data on the effects of antiestrogens in serum-free conditions [1], these results suggest down-regulation of cyclin D1 by antiestrogens may be a general phenomenon in estrogen receptor-positive breast cancer cells, independent of culture conditions and class of antiestrogen. These observations are compatible with the hypothesis that reductions in cyclin D1 levels may mediate in part the action of antiestrogens in blocking entry of cells into S phase.

Cyclin gene expression and growth control in normal and neoplastic human breast epithelium.

Recent advances in defining the molecular mechanisms of cell cycle control in eukaryotes provide a basis for better understanding the hormonal control of cell proliferation in normal and neoplastic breast epithelium. It is now clear that a number of critical steps in cell cycle progression are controlled by families of serine/threonine kinases, the cdks. These kinases are activated by interactions with various cyclin gene products which form the regulatory subunits of the kinase complexes. Several families of cyclins control cell cycle progression in G1 phase, cyclins C, D and E, or in S, G2 and mitosis, cyclins A and B. Recent studies have defined the expression and regulation of cyclin genes in normal breast epithelial cells and in breast cancer cell lines. Following growth arrest of T-47D breast cancer cells by serum deprivation restimulation with insulin results in sequential induction of cyclin genes. Cyclin D1 mRNA increases within 1 h of mitogenic stimulation and is followed by increased expression of cyclins D3 and E in G1 phase, cyclin A in late G1/early S phase and cyclin B1 in G2. Similar results were observed following epidermal growth factor stimulation of normal breast epithelial cells. Other hormones--oestrogens and progestins--and growth factors--insulin-like growth factor-I and basic fibroblast growth factor--with actions in G1 were also investigated for their effects on G1 cyclin gene expression. In all cases there was an excellent correlation between the induction of cyclin D1 mRNA and subsequent entry into S phase. Furthermore, growth inhibition by antioestrogens and concurrent G1 arrest were preceded by an acute decrease in cyclin D1 gene expression. These observations suggest a likely role for cyclin D1 in mediating many of the known hormonal effects on cell proliferation in breast epithelial cells.