Jay Wimalasena

Microtubule Dysfunction Induced by Paclitaxel Initiates Apoptosis through Both c-Jun N-terminal Kinase (JNK)-dependent and -Independent Pathways in Ovarian Cancer Cells

The antineoplastic agent paclitaxel (TaxolTM), a microtubule stabilizing agent, is known to arrest cells at the G2/M phase of the cell cycle and induce apoptosis. We and others have recently demonstrated that paclitaxel also activates the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signal transduction pathway in various human cell types, however, no clear role has been established for JNK/SAPK in paclitaxel-induced apoptosis. To further examine the role of JNK/SAPK signaling cascades in apoptosis resulting from microtubular dysfunction induced by paclitaxel, we have coexpressed dominant negative (dn) mutants of signaling proteins of the JNK/SAPK pathway (Ras, ASK1, Rac, JNKK, and JNK) in human ovarian cancer cells with a selectable marker to analyze the apoptotic characteristics of cells expressing dn vectors following exposure to paclitaxel. Expression of these dn signaling proteins had no effect on Bcl-2 phosphorylation, yet inhibited apoptotic changes induced by paclitaxel up to 16 h after treatment. Coexpression of these dn signaling proteins had no protective effect after 48 h of paclitaxel treatment. Our data indicate that: (i) activated JNK/SAPK acts upstream of membrane changes and caspase-3 activation in paclitaxel-initiated apoptotic pathways, independently of cell cycle stage, (ii) activated JNK/SAPK is not responsible for paclitaxel-induced phosphorylation of Bcl-2, and (iii) apoptosis resulting from microtubule damage may comprise multiple mechanisms, including a JNK/SAPK-dependent early phase and a JNK/SAPK-independent late phase.

Estrogens and cell-cycle regulation in breast cancer

Clinical and experimental data have established that the leading cause of sporadic female breast cancer is exposure to estrogens, predominantly 17beta-estradiol. Recent advances in the understanding of cell-cycle control mechanisms have been applied to outline the molecular mechanisms through which estrogens regulate the cell cycle in cultured breast cancer cells, in particular, in MCF-7 cells. Here, we discuss how estrogens exert control over several key G1 phase cell-cycle regulators, namely cyclin D1, Myc, Cdk2, Cdk4, Cdk inhibitors and Cdc25A. Although the molecular mechanisms underlying estrogenic regulation of G1 phase regulators are far from clear, current evidence indicates that estrogens might regulate several key molecules required for S phase entry, this regulation being independent of cell-cycle transit per se.

Multifaceted Regulation of Cell Cycle Progression by Estrogen: Regulation of Cdk Inhibitors and Cdc25A Independent of Cyclin D1-Cdk4 Function

ABSTRACT Estrogens induce proliferation of estrogen receptor (ER)-positive MCF-7 breast cancer cells by stimulating G1/S transition associated with increased cyclin D1 expression, activation of cyclin-dependent kinases (Cdks), and phosphorylation of the retinoblastoma protein (pRb). We have utilized blockade of cyclin D1-Cdk4 complex formation through adenovirus-mediated expression of p16INK4a to demonstrate that estrogen regulates Cdk inhibitor expression and expression of the Cdk-activating phosphatase Cdc25A independent of cyclin D1-Cdk4 function and cell cycle progression. Expression of p16INK4a inhibited G1/S transition induced in MCF-7 cells by 17-β-estradiol (E2) with associated inhibition of both Cdk4- and Cdk2-associated kinase activities. Inhibition of Cdk2 activity was associated with delayed removal of Cdk-inhibitory activity in early G1 and decreased cyclin A expression. Cdk-inhibitory activity and expression of both p21Cip1 and p27Kip1 was decreased, however, in both control and p16INK4a-expressing cells 20 h after estrogen treatment. Expression of Cdc25A mRNA and protein was induced by E2 in control and p16INK4a-expressing MCF-7 cells; however, functional activity of Cdc25A was inhibited in cells expressing p16INK4a. Inhibition of Cdc25A activity in p16INK4a-expressing cells was associated with depressed Cdk2 activity and was reversed in vivo and in vitro by active Cdk2. Transfection of MCF-7 cells with a dominant-negative Cdk2 construct inhibited the E2-dependent activation of ectopic Cdc25A. Supporting a role for Cdc25A in estrogen action, antisenseCDC25A oligonucleotides inhibited estrogen-induced Cdk2 activation and DNA synthesis. In addition, inactive cyclin E-Cdk2 complexes from p16INK4a-expressing, estrogen-treated cells were activated in vitro by treatment with recombinant Cdc25A and in vivo in cells overexpressing Cdc25A. The results demonstrate that functional association of cyclin D1-Cdk4 complexes is required for Cdk2 activation in MCF-7 cells and that Cdk2 activity is, in turn, required for the in vivo activation of Cdc25A. These studies establish Cdc25A as a growth-promoting target of estrogen action and further indicate that estrogens independently regulate multiple components of the cell cycle machinery, including expression of p21Cip1 and p27Kip1.

Signal transduction through the ras/Erk pathway is essential for the mycoestrogen zearalenone-induced cell-cycle progression in MCF-7 cells

Zearalenone is a naturally occurring estrogenic contaminant of moldy feeds and is present in high concentrations in dairy products and cereals. Zearalenone was postulated to contribute to the overall estrogen load of women, but the mechanisms of its action are not known. We demonstrated that zearalenone could stimulate the growth of estrogen receptor–positive human breast carcinoma cell line MCF‐7. In addition, zearalenone functioned as an antiapoptotic agent by increasing the survival of MCF‐7 cell cultures undergoing apoptosis caused by serum withdrawal. Treatment of these cells with 100 nM zearalenone induced cell‐cycle transit after increases in the expression of c‐myc mRNA and cyclins D1, A, and B1 and downregulation of p27Kip‐1. G1/G2‐phase kinase activity and phosphorylation of the retinoblastoma gene product was also evident. Flow cytometric analysis demonstrated entry of cells into the S and G2/M phases of the cell cycle, and phosphorylation of histone H3 occurred 36 h after zearalenone treatment. Ectopic expression of a dominant‐negative p21ras completely abolished the zearalenone‐induced DNA synthesis in these cells, and the specific inhibitor PD98059 for mitogen/extracellular‐regulated protein kinase kinase arrested S‐phase entry induced by zearalenone. These data suggest that the mitogen‐activated protein kinase signaling cascade is required for zearalenones effects on cell‐cycle progression in MCF‐7 cells. Given the presence of this mycotoxin in cereals, milk, and meat, the possibility that zearalenone is a potential promoter of breast cancer tumorigenesis should be investigated further. Mol. Carcinog. 30:88–98, 2001.

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. 17β-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.

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.

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.

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.

Immunohistochemical Studies of the Adult Human Ovary: Possible Contribution of Immune and Epithelial Factors to Folliculogenesis

PROBLEM: Formation of primordial follicles in adult ovaries could be a cryptic process limited to relatively small areas of the ovarian cortex and occurring during a certain stage of the menstrual cycle. Such an event may require a specific milieu provided by factors involved in developmental processes, i.e., morphoregulatory molecules and macrophages.

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 ~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 ~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 ~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 ~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.