BCL-2 family protein, BAD is down-regulated in breast cancer and inhibits cell invasion.

We have previously demonstrated that the anti-apoptotic protein BAD is expressed in normal human breast tissue and shown that BAD inhibits expression of cyclin D1 to delay cell-cycle progression in breast cancer cells. Herein, expression of proteins in breast tissues was studied by immunohistochemistry and results were analyzed statistically to obtain semi-quantitative data. Biochemical and functional changes in BAD-overexpressing MCF7 breast cancer cells were evaluated using PCR, reporter assays, western blotting, ELISA and extracellular matrix invasion assays. Compared to normal tissues, Grade II breast cancers expressed low total/phosphorylated forms of BAD in both cytoplasmic and nuclear compartments. BAD overexpression decreased the expression of ß-catenin, Sp1, and phosphorylation of STATs. BAD inhibited Ras/MEK/ERK and JNK signaling pathways, without affecting the p38 signaling pathway. Expression of the metastasis-related proteins, MMP10, VEGF, SNAIL, CXCR4, E-cadherin and TlMP2 was regulated by BAD with concomitant inhibition of extracellular matrix invasion. Inhibition of BAD by siRNA increased invasion and Akt/p-Akt levels. Clinical data and the results herein suggest that in addition to the effect on apoptosis, BAD conveys anti-metastatic effects and is a valuable prognostic marker in breast cancer.

hLH/hCG-receptor expression correlates with in vitro invasiveness in human primary endometrial cancer.

OBJECTIVE: Endometrial cancer (EC) is the most frequent cancer of the female genital tract. It has been hypothesized that those ECs that occur in the postmenopausal period, might be sensitive to elevated levels of luteinizing hormone/human chorionic gonadotropin (LH/hCG). Based on previous indications, we analyzed the functional expression of LH/hCG receptors (LH/hCG-R) in primary ECs. METHODS: We studied a cohort of primary ECs, in which both the LH/hCG-R mRNA and the LH/hCG-R protein were analyzed. Results were correlated with both clinical-pathological data and the effects of LH addition on cell invasion in vitro. RESULTS: The LH/hCG-R mRNA levels ranged from 4.67 e(-02) to 2.36 e(+03). The transcript was properly translated into a functional LH/hCG-R protein. The analysis of cell invasion in vitro in response to LH/hCG allowed us to divide the EC samples into two groups, one with a null or very low response (non-responders=NR) and the other with a significant response to LH (responders=R). The two groups had significantly different levels of LH/hCG-R mRNA expression: the NR group had a median value of 1.40 e(+)(00), while the R group of 7.42 e(+)(01) (p=0.043). CONCLUSION: In primary ECs a statistically significant correlation emerged between the levels of LH/hCG-R mRNA and the LH-induced cell invasion in vitro. These results suggest that therapies aimed at decreasing LH levels, through Gonadotropin Releasing Hormone (Gn-RH) analogues, could produce benefits in the treatment of recurrent or metastatic EC, especially in patients displaying high LH/hCG-R levels.

Activating transcription factor 3 and early growth response 1 are the novel targets of LY294002 in a phosphatidylinositol 3-kinase-independent pathway.

LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, has been widely used to study the function of PI3K in cellular responses. Based on its inhibitory effect on PI3K, LY294002 has been shown to exert antitumorigenic effect in vivo and in vitro. Here, we report that LY294002 alters early growth response 1 (EGR-1) phosphorylation and subsequently enhances activating transcription factor 3 (ATF3) expression independently of PI3K inhibition. This pathway may be, in part, responsible for the antitumorigenic effect of LY294002 in human colorectal cancer cells. ATF3 expression was increased by LY294002, followed by the induction of apoptosis in several colorectal cancer cell lines. This is consistent with results showing that the down-regulation of the ATF3 gene by small interfering RNA suppressed LY294002-induced apoptosis in HCT-116 cells. On the other hand, ATF3 expression was not affected by another PI3K inhibitor, wortmannin, as well as phosphatase and tensin homologue or dominant-negative Akt overexpression. We also found that LY294002 increases ATF3 promoter activity and the transactivation is partly mediated by a GC-rich sequence located in the promoter. EGR-1 binds to the ATF3 promoter as assessed by gel shift assay. Furthermore, phosphorylated EGR-1 was highly increased in LY294002-treated cells, indicating that EGR-1 phosphorylation induced by LY294002 may facilitate ATF3 transactivation. Our data suggest that EGR-1 acts as a mediator in LY294002-induced ATF3 expression via a PI3K-independent pathway. ATF3 and EGR-1 may provide a novel explanation for the antitumorigenic properties of LY294002 in human colorectal cancer cells.

Estradiol abrogates apoptosis in breast cancer cells through inactivation of BAD: Ras-dependent nongenomic pathways requiring signaling through ERK and Akt.

Estrogens such as 17-beta estradiol (E(2)) play a critical role in sporadic breast cancer progression and decrease apoptosis in breast cancer cells. Our studies using estrogen receptor-positive MCF7 cells show that E(2) abrogates apoptosis possibly through phosphorylation/inactivation of the proapoptotic protein BAD, which was rapidly phosphorylated at S112 and S136. Inhibition of BAD protein expression with specific antisense oligonucleotides reduced the effectiveness of tumor necrosis factor-alpha, H(2)O(2), and serum starvation in causing apoptosis. Furthermore, the ability of E(2) to prevent tumor necrosis factor-alpha-induced apoptosis was blocked by overexpression of the BAD S112A/S136A mutant but not the wild-type BAD. BAD S112A/S136A, which lacks phosphorylation sites for p90(RSK1) and Akt, was not phosphorylated in response to E(2) in vitro(.) E(2) treatment rapidly activated phosphatidylinositol 3-kinase (PI-3K)/Akt and p90(RSK1) to an extent similar to insulin-like growth factor-1 treatment. In agreement with p90(RSK1) activation, E(2) also rapidly activated extracellular signal-regulated kinase, and this activity was down-regulated by chemical and biological inhibition of PI-3K suggestive of cross talk between signaling pathways responding to E(2). Dominant negative Ras blocked E(2)-induced BAD phosphorylation and the Raf-activator RasV12T35S induced BAD phosphorylation as well as enhanced E(2)-induced phosphorylation at S112. Chemical inhibition of PI-3K and mitogen-activated protein kinase kinase 1 inhibited E(2)-induced BAD phosphorylation at S112 and S136 and expression of dominant negative Ras-induced apoptosis in proliferating cells. Together, these data demonstrate a new nongenomic mechanism by which E(2) prevents apoptosis.

HES-1 inhibits 17beta-estradiol and heregulin-beta1-mediated upregulation of E2F-1.

We have previously shown that expression of the transcription factor HES-1 is required for the growth-inhibitory effect of all-trans retinoic acid on MCF-7 cells. In this study, we have used T47D cells with tetracyclin-regulated expression of wild-type or a dominant-negative form of HES-1. Expression of HES-1 in T47D cells inhibited G1/S-phase transition and activation of Cdk2 elicited by estrogen. Estrogen treatment of T47D cells caused increased expression of E2F-1, and this expression was inhibited by cotreatment with all-trans retinoic acid. We show that the effect is mediated through HES-1, which directly downregulates E2F-1 expression through a CACGAG-site within the E2F-1 promoter. Furthermore, proliferation caused by heregulin-beta1 treatment of T47D cells was inhibited by all-trans retinoic acid and this effect was mediated by HES-1. Interestingly, heregulin-beta1-mediated upregulation of E2F-1 expression was directly inhibited by HES-1 through the same CACGAG-site as seen with estrogen-stimulated induction. In addition, we found that two important downstream target genes of estrogen and heregulin-beta1 that are regulated through E2F-1, cyclin E and NPAT, were both regulated in a similar fashion by all-trans retinoic acid, and these effects were antagonized by dominant-negative HES-1. These findings establish that HES-1 inhibits both estrogen- and heregulin-beta1-stimulated growth of breast cancer cells, and further suggest that growth inhibition induced in these cells by all-trans retinoic acid occurs via HES-1-mediated downregulation of E2F-1 expression.

Placental expression of estrogen receptor beta and its hormone binding variant--comparison with estrogen receptor alpha and a role for estrogen receptors in asymmetric division and differentiation of estrogen-dependent cells.

During human pregnancy, the production of 17-beta-estradiol (E2) rises steadily to eighty fold at term, and placenta has been found to specifically bind estrogens. We have recently demonstrated the expression of estrogen receptor alpha (ER-alpha) protein in human placenta and its localization in villous cytotrophoblast (CT), vascular pericytes, and amniotic fibroblasts. In vitro, E2 stimulated development of large syncytiotrophoblast (ST) aggregates. In the present study we utilized ER-beta affinity purified polyclonal (N19:sc6820) and ER-alpha monoclonal (clone h-151) antibodies. Western blot analysis revealed a single approximately 52 kDa ER-beta band in chorionic villi (CV) protein extracts. In CV, strong cytoplasmic ER-beta immunoreactivity was confined to ST. Dual color immunohistochemistry revealed asymmetric segregation of ER-alpha in dividing villous CT cells. Prior to separation, the cell nuclei more distant from ST exhibited high ER-alpha, while cell nuclei associated with ST showed diminution of ER-alpha and appearance of ER-beta. In trophoblast cultures, development of ST aggregates was associated with diminution of ER-alpha and appearance of ER-beta immunoreactivity. ER-beta was also detected in endothelial cells, amniotic epithelial cells and fibroblasts, extravillous trophoblast (nuclear and cytoplasmic) and decidual cells (cytoplasmic only). In addition, CFK-E12 (E12) and CWK-F12 (F12) monoclonal antibodies, which recognize approximately 64 kDa ER-beta with hormone binding domain, showed nuclear-specific reactivity with villous ST, extravillous trophoblast, and amniotic epithelium and fibroblasts. Western blot analysis indicated abundant expression of a approximately 64 kDa ER-beta variant in trophoblast cultures, significantly higher when compared to the chorionic villi and freshly isolated trophoblast cell protein extracts. This is the first report on ER-beta expression in human placenta and cultured trophoblast. Our data indicate that during trophoblast differentiation, the ER-alpha is associated with a less, and ER-beta with the more differentiated state. Enhanced expression of approximately 64 kDa ER-beta variant in trophoblast cultures suggests a unique role of ER-beta hormone binding domain in the regulation of trophoblast differentiation. Our data also indicate that asymmetric segregation of ER-alpha may play a role in asymmetric division of estrogen-dependent cells.

Expression and localization of estrogen receptor-alpha protein in normal and abnormal term placentae and stimulation of trophoblast differentiation by estradiol.

Estrogens play an important role in the regulation of placental function, and 17-beta-estradiol (E2) production rises eighty fold during human pregnancy. Although term placenta has been found to specifically bind estrogens, cellular localization of estrogen receptor alpha (ER-alpha) in trophoblast remains unclear. We used western blot analysis and immunohistochemistry with h-151 and ID5 monoclonal antibodies to determine the expression and cellular localization of ER-alpha protein in human placentae and cultured trophoblast cells. Western blot analysis revealed a ~65 kDa ER-alpha band in MCF-7 breast carcinoma cells (positive control). A similar band was detected in five normal term placentae exhibiting strong expression of Thy-1 differentiation protein in the villous core. However, five other term placentae, which exhibited low or no Thy-1 expression (abnormal placentae), exhibited virtually no ER-alpha expression. In normal placentae, nuclear ER-alpha expression was confined to villous cytotrophoblast cells (CT), but syncytiotrophoblast (ST) and extravillous trophoblast cells were unstained. In abnormal placentae no CT expressing ER-alpha were detected. Normal and abnormal placentae also showed ER-alpha expression in villous vascular pericytes and amniotic (but not villous) fibroblasts; no staining was detected in amniotic epithelial cells or decidual cells. All cultured trophoblast cells derived from the same normal and abnormal placentae showed distinct ER-alpha expression in western blots, and the ER-alpha expression was confined to the differentiating CT, but not to the mature ST. Trophoblast cells from six additional placentae were cultured in normal medium with phenol red (a weak estrogen) as above (PhR+), or plated in phenol red-free medium (PhR-) without or with mid-pregnancy levels of E2 (20 nM). Culture in PhR- medium without E2 caused retardation of syncytium formation and PhR-medium with E2 caused acceleration of syncytium formation compared to cultures in PhR+ medium. These data indicate that the considerable increase in estrogen production during pregnancy may play a role, via the ER-alpha, in the stimulation of CT differentiation and promote function in normal placentae. This mechanism, however, may not operate in abnormal placentae, which show a lack of ER-alpha expression.

Changes of ovarian interstitial cell hormone receptors and behavior of resident mesenchymal cells in developing and adult rats with steroid-induced sterility.

In the present paper, we report that injection of testosterone propionate (500 microg) during the critical window of rat development (postnatal day 5) induces temporary appearance of aged interstitial cells in developing ovaries (days 7 and 10). Aged interstitial cells showed large size (> or = 12 microm), enhanced androgen receptor (AR) and low estrogen (ER) and luteinizing hormone receptor (LHR) expression. Although normal mature interstitial cells (large size and strong ER and LHR expression) appeared later (day 14), and ovaries of androgenized rats were similar to normal ovaries between days 14 and 35, ovaries of adult androgenized females showed only aged and no mature interstitial cells. Androgenization on day 10 caused the development of aged interstitial cells on day 14, but adult ovaries were normal. Long lasting postnatal estrogenization (estradiol dipropionate for four postnatal weeks) caused in developing and adult ovaries a lack of interstitial cell development beyond the immature state. Immature interstitial cells were characterized by a small size (< or = 7 microm) and a lack of AR, ER and LHR expression. Because the critical window for steroid-induced sterility coincides with the termination of immune adaptation, we also investigated distribution of mesenchymal cells (Thy-1 mast cells and pericytes, ED1 monocyte-derived cells, CD8 T cells, and cells expressing OX-62 of dendritic cells) in developing and adult ovaries. Developing ovaries of normal, androgenized and estrogenized females were populated by similar mesenchymal cells, regardless of differences in the state of differentiation of interstitial cells. However, mesenchymal cells in adult ovaries showed distinct behavior. In normal adult ovaries, differentiation of mature interstitial cells was accompanied by differentiation of mesenchymal cells. Aged interstitial cells in ovaries of androgenized rats showed precipitous degeneration of resident mesenchymal cells. Immature interstitial cells in ovaries of estrogenized rats showed a lack of differentiation of resident mesenchymal cells. These observations indicate that an alteration of interstitial cell differentiation during immune adaptation toward the aged phenotype results in precipitous degeneration of resident mesenchymal cells and premature aging of ovaries in adult rats, and alteration toward immature phenotype results in a lack of differentiation of mesenchymal cells and permanent immaturity of ovaries in adult females.

Cyclin D1 degradation and p21 induction contribute to growth inhibition of colorectal cancer cells induced by epigallocatechin-3-gallate.

PURPOSE: The public has paid attention to green tea due to its health benefits. Epigallocatechin-3-gallate (EGCG), the major component of green tea, is well documented to induce apoptosis and cell cycle arrest in cancer cells by targeting multiple signal transduction pathways. However, the detailed mechanism(s) of action needs to be determined. METHODS: Cell growth was evaluated by MTT assay, cell cycle analysis, and caspase 3/7 activity. Protein expression was analyzed through Western blotting. Reverse transcription polymerase chain reaction was used for examining mRNA expression of p21 and cyclin D1. The promoter activity of p21 was assessed by the luciferase reporter system. RESULTS: We identified cyclin D1 and p21 as molecular targets of EGCG in human colorectal cancer cells. We observed that cyclin D1 was down-regulated, while p21 expression was up-regulated by EGCG in dose- and time-dependent manners. Furthermore, we found EGCG decreased cyclin D1 protein stability, therefore triggering ubiquitin-dependent proteasomal degradation. Meanwhile, EGCG increased p21 promoter activity, resulting in up-regulation of p21 mRNA and protein, which was likely dependent on extracellular-signal-regulated kinase (ERK), inhibitor of nuclear factor kappa-B kinase (IKK) and phosphoinositide 3-kinase (PI3 K). CONCLUSION: The data presented here details a novel mechanism by which EGCG inhibits cell growth of colorectal cancer cells. Namely, EGCG-induced cyclin D1 degradation and p21 transcriptional activation partially contribute to growth suppression in these cells.

Luteinizing hormone receptor mediates neuronal pregnenolone production via up-regulation of steroidogenic acute regulatory protein expression.

The functional consequences of luteinizing hormone/human chorionic gonadotropin signaling via neuronal luteinizing hormone/human chorionic gonadotropin receptors expressed throughout the brain remain unclear. A primary function of luteinizing hormone (LH) in the gonads is the stimulation of sex steroid production. As LH can cross the blood-brain barrier, present in cerebrospinal fluid and is expressed by neuronal cells, we tested whether LH might also modulate steroid synthesis in the brain. Treatment of differentiated rat primary hippocampal neurons and human M17 neuroblastoma cells with LH (100 mIU/mL) resulted in a twofold increase in pregnenolone secretion in both cell types, suggesting an increase in P450scc-mediated cleavage of cholesterol to pregnenolone and its secretion from neurons. To explore how LH might regulate the synthesis of pregnenolone, the precursor for steroid synthesis, we treated rat primary hippocampal neurons with LH (0, 10 and 100 mIU/mL) and measured changes in the expression of LH receptor and steroidogenic acute regulatory protein (StAR). LH induced a rapid (within 30 min) increase in the expression of StAR, but induced a dose-dependent decrease in LH receptor expression. Consistent with these results, the suppression of serum LH in young rats treated with leuprolide acetate for 4 months down-regulated StAR expression, but increased LH receptor expression in the brain. Taken together, these results indicate that LH induces neuronal pregnenolone production by modulating the expression of the LH receptor, increasing mitochondrial cholesterol transport and increasing P450scc-mediated cleavage of cholesterol for pregnenolone synthesis and secretion.

Breast cancer cell proliferation is inhibited by BAD: regulation of cyclin D1.

Recent investigations suggest that functions of the proapoptotic BCL2 family members, including BAD, are not limited to regulation of apoptosis. Here we demonstrate that BAD inhibits G(1) to S phase transition in MCF7 breast cancer cells independent of apoptosis. BAD overexpression inhibited G(1) transit and cell growth as well as cyclin D1 expression. Inhibition of cyclin D1 expression was mediated through inhibition of transcription activated by AP1. Chromatin immunoprecipitation assays indicated that BAD is localized at the 12-O-tetradecanoylphorbol-13-acetate-response element (TRE) and cAMP-response element (CRE) in the cyclin D1 promoter. This was shown to reflect direct binding interactions of BAD with c-Jun, and this interaction inhibited the activity of AP1 complexes at TRE. BAD did not interact with phosphorylated forms of c-Jun. Our data suggest that inhibitory TRE/CRE-c-Jun-BAD complexes are present at the cyclin D1 promoter in quiescent cells. Estrogen stimulation displaced BAD from TRE/CRE elements in MCF7 cells, whereas BAD overexpression inhibited estrogen-induced cyclin D1 synthesis and cell proliferation. Inhibition of endogenous BAD in MCF7 cells markedly increased the proliferative fraction and DNA synthesis, activated Cdks, and increased cyclin D1 protein levels. This action of BAD required serine residues Ser(75) and Ser(99). Both phosphorylated and unphosphorylated forms of BAD localized to the nuclei of human breast epithelial cells. Thus, we demonstrate a novel role for BAD in cell cycle regulation dependent upon its phosphorylation state and independent of the BAD/BCL2 interaction and apoptosis.

Oogenesis in adult mammals, including humans: a review.

The origin of oocytes and primary follicles in ovaries of adult mammalian females has been a matter of dispute for over 100 yr. The prevailing belief that all oocytes in adult mammalian females must persist from the fetal period of life seems to be a uniquely retrogressive reproductive mechanism requiring humans to preserve their gametes from the fetal period for several decades. The utilization of modern techniques during last 10 yr clearly demonstrates that mammalian primordial germ cells originate from somatic cell precursors. This indicates that if somatic cells are precursors of germ cells, then somatic mutations can be passed on to progeny. Mitotically active germline stem cells have been described earlier in ovaries of adult prosimian primates and recently have been reported to also be present in the ovaries of adult mice. We have earlier shown that in adult human females, mesenchymal cells in the ovarian tunica albuginea undergo a mesenchymal-epithelial transition into ovarian surface epithelium cells, which differentiate sequentially into primitive granulosa and germ cells. Recently, we have reported that these structures assemble in the deeper ovarian cortex and form new follicles to replace earlier primary follicles undergoing atresia (follicular renewal). Our current observations also indicate that follicular renewal exists in rat ovaries, and human oocytes can differentiate from ovarian surface epithelium in fetal ovaries in vivo and from adult ovaries in vitro. These reports challenge the established dogma regarding the fetal origin of eggs and primary follicles in adult mammalian ovaries. Our data indicate that the pool of primary follicles in adult human ovaries does not represent a static but a dynamic population of differentiating and regressing structures. Yet, the follicular renewal may cease at a certain age, and this may predetermine the onset of the natural menopause or premature ovarian failure. A lack of follicular renewal in aging ovaries may cause an accumulation of spontaneously arising or environmentally induced genetic alterations of oocytes, and that may be why aging females have a much higher chance of having oocytes with more mutations in persisting primary follicles.

Estrogen receptor beta inhibits 17beta-estradiol-stimulated proliferation of the breast cancer cell line T47D.

Estrogen receptor (ER) beta counteracts the activity of ERalpha in many systems. In agreement with this, we show in this study that induced expression of ERbeta in the breast cancer cell line T47D reduces 17beta-estradiol-stimulated proliferation when expression of ERbeta mRNA equals that of ERalpha. Induction of ERbeta reduces growth of exponentially proliferating cells with a concomitant decrease in components of the cell cycle associated with proliferation, namely cyclin E, Cdc25A (a key regulator of Cdk2), p45(Skp2) (a key regulator of p27(Kip1) proteolysis), and an increase in the Cdk inhibitor p27(Kip1). We also observed a reduced Cdk2 activity. These findings suggest a possible role for ERbeta in breast cancer and imply that ERbeta-specific ligands may reduce proliferation of ER-positive breast cancer cells through actions on the G(1) phase cell-cycle machinery.

Removal of Cdk inhibitors through both sequestration and downregulation in zearalenone-treated MCF-7 breast cancer cells.

Treatment of MCF 7 cells with the fungal estrogen zearalenone induced cyclin E-associated kinase activity transiently within 9-12 h; total cyclin-dependent kinase (Cdk) 2 activity was elevated for 24 h and beyond. This increased cyclin E/Cdk2 activity was associated with sequestration of the Cdk inhibitor p27 Cdk inhibitor 1B (p27(KIP1)) by newly formed cyclin D1/Cdk4 complexes and with downregulation of p27(KIP1) expression. The activation of cyclin A/Cdk2 activity corresponded with virtual elimination of p27(KIP1). The activity of cyclin E/Cdk2 complexes from zearalenone-treated lysates was inhibited in vitro by recombinant p27(KIP1), and this inhibition was relieved by the addition of recombinant cyclin D1/Cdk4 complexes. Thus, sequestration of p27(KIP1) by cyclin D1/Cdk4 resulted in activation of Cdk2 in vitro. Cdk inhibitory activity in lysates of zearalenone-treated cells was depleted by anti-p27(KIP1) and anti-Cdc2 interacting protein (p21(CIP1)) antibodies. Overexpression of the Cdk4/6-specific Cdk inhibitor of Cdk4 p16(INK4A) was associated with increased association of p27(KIP1) with Cdk2, concomitant with disruption of D cyclin/Cdk4 complexes. The proteasome inhibitor 2-leu-leu-leu-H aldehyde (MG-132) was relatively ineffective in inhibiting the initial, sequestration-dependent activation of cyclin E/Cdk2 yet was as effective as p16(INK4A) in inhibiting activation of cyclin A/Cdk2 later in G(1). Downregulation of p27(KIP1) proceeded in p16(INK4A)-expressing cells after zearalenone treatment, and G(1) arrest afforded by p16(INK4A) expression was reversible upon prolonged treatment with zearalenone. Zearalenone treatment of MCF-7 cells elicited expression of F-box protein S phase kinase-associated protein 2 (p45(SKP2)), a substrate-specific component of the ubiquitin-ligase complex that targets p27(KIP1) for degradation in the proteasome. These studies suggest that both sequestration of Cdk inhibitors by cyclin D1/Cdk4 complexes and downregulation of p27(KIP1) play major roles in the induction of Cdk2 activity and S phase entry elicited by estrogens in MCF-7 cells.

Estrogens down-regulate p27Kip1 in breast cancer cells through Skp2 and through nuclear export mediated by the ERK pathway.

The cyclin-dependent kinase (CDK) inhibitor p27Kip1 plays a key role in growth and development of the mammary epithelium and in breast cancer. p27Kip1 levels are regulated through ubiquitin/proteasome-mediated proteolysis, promoted by CDK2 and the F box protein Skp2 at the G1/S transition, and independent of Skp2 in mid-G1. We investigated the respective roles of Skp2 and subcellular localization of p27Kip1 in down-regulation of p27Kip1 induced in MCF-7 cells by estrogens. 17beta-Estradiol treatment increased Skp2 expression in MCF-7 cells; however, this increase was prevented by G1 blockade mediated by p16Ink4a or the CDK inhibitor roscovitine, whereas down-regulation of p27Kip1 was maintained. Exogenous Skp2 prevented growth arrest of MCF-7 cells by antiestrogen, coinciding with decreased p27Kip1 expression. Under conditions of G1 blockade, p27Kip1 was stabilized by inhibition of CRM1-dependent nuclear export with leptomycin B or by mutation of p27Kip1 (Ser10 --> Ala; S10A) interfering with CRM1/p27Kip1 interaction. Antisense Skp2 oligonucleotides and a dominant-interfering Cul-1(1-452) mutant prevented down-regulation of p27Kip1S10A, whereas Skp2 overexpression elicited its destruction in mitogen-deprived cells. Active mediators of the extracellular signal-regulated kinase (ERK) pathway including Raf-1caax induced cytoplasmic localization of p27Kip1 in antiestrogen-treated cells and prevented accumulation of p27Kip1 in these cells independent of Skp2 expression and coinciding with ERK activation. Genetic or chemical blockade of the ERK pathway prevented down-regulation and cytoplasmic localization of p27Kip1 in response to estrogen. Our studies indicate that estrogens elicit down-regulation of p27Kip1 in MCF-7 cells through Skp2-dependent and -independent mechanisms that depend upon subcellular localization of p27Kip1 and require the participation of mediators of the Ras/Raf-1/ERK signaling pathway.

Variability of placental expression of cyclin E low molecular weight variants.

Cyclin E, a G(1) cyclin serving to activate cyclin-dependent kinase 2, is the only cyclin gene for which alternative splicing leading to structurally different proteins has been described. Different cyclin E proteins are present in tumor tissues but absent from normal (steady) tissues. Cyclin E contributes to the regulation of cell proliferation and ongoing differentiation and aging. Because trophoblast has invasive properties and differentiates into syncytium and placental aging may develop at term, we examined cyclin E protein variants in human placenta. Placental samples were collected from 27 deliveries between 33 and 41 wk and were compared with ovarian cancer (positive control). Both placental and tumor tissues showed seven cyclin E low molecular weight (LMW) bands migrating between 50 and 36 kDa. Placental expression of cyclin E showed certain variability among cases. Lowest cyclin E expression was detected in normal placentas (strong expression of Thy-1 differentiation protein in villous core and low dilatation of villous blood sinusoids). Abnormal placentas (significant depletion of Thy-1 and more or less pronounced dilatation of sinusoids) showed significant increase either of all (early stages of placental aging) or only certain cyclin E proteins (advanced aging). Our studies indicate that a similar spectrum of cyclin E protein variants is expressed in the placental and tumor tissues. Low cyclin E expression in normal placentas suggests a steady state. Overexpression of all cyclin E proteins may indicate an activation of cellular proliferation and differentiation to compensate for developing placental insufficiency. However, an enhanced expression of some cyclin E LMW proteins only might reflect an association of cyclin E isoforms with placental aging or an inefficient placental adaptation.