High dose 17 beta-estradiol and the alpha-estrogen agonist PPT trigger apoptosis in human adrenal carcinoma cells but the beta-estrogen agonist DPN does not.

Previous studies have shown that high dose 17beta-estradiol (10 (-5) M) has a G2/M blocking effect in SW-13 human adrenal carcinoma cultures and strongly enhances apoptosis. To examine the differential effects of estrogen alpha and beta-receptors in this system, we incubated SW-13 cells with specific alpha- and beta-estrogen receptor agonists, PPT [4,4',4'-(propyl-[ (1)H]-pyrazole-1,3,5-triyl) trisphenol] and DPN [2,3-bis (4-hydroxyphenyl) propionitrile], respectively (each at 10 (-5) M). Flow cytometry was used to analyze the percentages of cells in various phases of the cell cycle [sub-G1 (apoptosis), G1, S, and G2/M] in each experimental condition. Exposure to 17 beta-estradiol for 48 hours increased apoptosis more than 5-fold (from 3.6+/-0.5 to 20+/-2.2% of cells; p<0.01). The alpha-estrogen agonist PPT had a similar effect, increasing apoptosis to 22+/-1.7% (p<0.01), but the beta-agonist DPN caused no change (3.6+/-0.5 vs. 3.9+/-0.8%). While estrogen and the alpha-estrogen agonist decrease apoptosis in this system, both of these compounds decreased the percentage of cells in G1 (from 59+/-1.4% for control to 34+/-2.3% for estrogen and 40+/-2.0% for PPT; p<0.01 for both agents relative to control); the beta-agonist again had no effect. Estrogen was also found to block the cell cycle in G2/M, increasing it from 15+/-0.4 to 21+/-1.0% of cells (p<0.01), but neither the alpha- nor beta-estrogen agonists had any effect at this point in the cell cycle, indicating that the influence of estrogen was not likely to be either alpha- or beta-receptor mediated. There was no apparent effect of any of these agents on DNA synthesis, as indicated by unchanged percentages of cells in S phase. These studies suggest that induction of apoptosis by estrogen in SW-13 human adrenal cortical carcinoma cultures is mediated by the alpha-receptor, but the G2/M blocking effect of estrogen is not likely to be related to either alpha or beta mechanisms.     

Effects of androgens and estrogens and catechol and methoxy-estrogen derivatives on mitogen-activated protein kinase (ERK(1,2)) activity in SW-13 human adrenal carcinoma cells.

We tested the effects of 17beta-estradiol as well as its catechol- and methoxy-derivatives, two androgens (DHEA and testosterone), a glucocorticoid (cortisol), a mineralocorticoid (aldosterone), and progesterone on the activity of ERK(1,2), a key component of the ERK/MAPK enzyme phosphorylation cascade, in SW-13 human adrenal carcinoma cells. After a 24-hour exposure SW-13 cells incubated with 10(-5) M concentrations of 17beta-estradiol, its 2-hydroxy or its 2-methoxy derivative, all had elevated ERK activities (196%, 159%, and 275%, respectively) relative to control cells (p < 0.01). Incubation with testosterone resulted in 162% of control ERK activity (p < 0.01), whereas incubation with the far weaker androgen DHEA or with cortisol, aldosterone, or progesterone had no significant effects. These findings suggest sex steroid-specific influences in the induction or activation of signal transduction pathways known to play a crucial role in cellular proliferation and differentiation.     

Expression of cyclin A,B1 and D1 after induction of cell cycle arrest in the Jurkat cell line exposed to doxorubicin

Jurkat human lymphoblastoid cells were incubated in increasing concentrations of doxorubicin (0.05, 0.1 and 0.15 μM) to induce cell death, and their expression of cyclin A, B1 and D1 was evaluated by flow cytometry (cell cycle progression, Annexin V assay, percentages and levels of each of the cyclins), transmission electron microscopy (ultrastructure) and confocal fluorescence microscopy (expression and intracellular localization of cyclins). After low‐dose doxorubicin treatment, Jurkat cells responded mainly by G2/M arrest, which was related to increased cyclin B1, A and D1 levels, a low level of apoptosis and/or mitotic catastrophe. The influence of doxorubicin on levels and/or localization of selected cyclins was confirmed, which may in turn contribute to the G2/M arrest induced by the drug.     

Curcumin selectively induces apoptosis in deregulated cyclin D1-expressed cells at G2 phase of cell cycle in a p53-dependent manner

Curcumin (diferuloylmethane) is known to induce apoptosis in tumor cells. In asynchronous cultures, with time-lapse video-micrography in combination with quantitative fluorescence microscopy, we have demonstrated that curcumin induces apoptosis at G(2) phase of cell cycle in deregulated cyclin D1-expressed mammary epithelial carcinoma cells, leaving its normal counterpart unaffected. In our search toward delineating the molecular mechanisms behind such differential activities of curcumin, we found that it selectively increases p53 expression at G(2) phase of carcinoma cells and releases cytochrome c from mitochondria, which is an essential requirement for apoptosis. Further experiments using p53-null as well as dominant-negative and wild-type p53-transfected cells have established that curcumin induces apoptosis in carcinoma cells via a p53-dependent pathway. On the other hand, curcumin reversibly inhibits normal mammary epithelial cell cycle progression by down-regulating cyclin D1 expression and blocking its association with Cdk4/Cdk6 as well as by inhibiting phosphorylation and inactivation of retinoblastoma protein. In addition, curcumin significantly up-regulates cell cycle inhibitory protein (p21Waf-1) in normal cells and arrests them in G(0) phase of cell cycle. Therefore, these cells escape from curcumin-induced apoptosis at G(2) phase. Interestingly, these processes remain unaffected by curcumin in carcinoma cells where cyclin D1 expression is high. Similarly, in ectopically overexpressed system, curcumin cannot down-regulate cyclin D1 and thus block cell cycle progression. Hence, these cells progress into G(2) phase and undergo apoptosis. These observations together suggest that curcumin may have a possible therapeutic potential in breast cancer patients.     

孕激素诱导cyclin G1在小鼠子宫内膜上皮细胞的表达及其对细胞增殖的影响

本文在体外培养条件下研究卵巢激素诱导小鼠子宫内膜上皮细胞cyclin G1的表达及细胞增殖和细胞周期进程的变化,以探讨孕激素依赖的细胞周期调控因子cyclin G1对子宫内膜上皮细胞增殖的负调控作用.原代培养小鼠子宫内膜上皮细胞,待其生长汇合后分为4组:对照组(C组)、雌激素组(E组)、孕激素组(P组)、雌、孕激素共同作用组(EP组).加入相应激素作用24 h后,用细胞免疫化学方法检测各组细胞cyclin G1的表达水平:四甲基偶氮唑蓝(MTT)比色法检测各组细胞活力,间接观察子宫内膜上皮细胞的增殖情况;用流式细胞仪检测分布在细胞周期各时相的子宫内膜上皮细胞所占百分数.细胞免疫化学结果显示,cyclin G1在C组和E组子宫内膜上皮细胞上无明显表达,而在P组和EP组子宫内膜上皮细胞中表达明显,且定位于细胞核内.MTT法结果显示,与C组相比,E组细胞活力明显增高,而P组和EP组的细胞活力均明显下降,表明雌激素能促进子宫内膜上皮细胞增殖,而孕激素则具有抑制子宫内膜上皮细胞增殖的作用.流式细胞术检测显示,与C组相比,E组中处于S期的子宫内膜上皮细胞百分数增多;P组与EP组中处于S期的子宫内膜上皮细胞百分数明显减少,而处于G1期的细胞百分数和G2/M期的细胞百分数则明显增加.上述结果提示,孕激素依赖的cyclin G1可能通过阻滞细胞周期进程来参与孕激素对子宫内膜上皮细胞增殖的负调控作用.     

Effects of 2-methoxyestradiol on proliferation, apoptosis and gene expression of cyclin B1 and c-Myc in esophageal carcinoma EC9706 cells

2-Methoxyestradiol (2-ME) is an endogenous metabolite of 17β-estradiol. In this study, we determined the antitumour activities of 2-ME on the well-differentiated EC9706 esophageal carcinoma cells in vitro. 2-ME had a strong antiproliferative effect on EC9706 cells and caused an increase in the population of apoptotic cells, detected by flow cytometry. A significant number of cells were blocked in the G(2)/M phase of the cell cycle. 2-ME-treated cells demonstrated an increase in cyclin B1 and c-Myc protein levels, as well as an increase in the percentage of G(2)/M phase. Their up-regulation may be involved in 2-ME-induced apoptosis and G(2)/M cell cycle arrest of the EC9706 cells, and it precedes the onset of apoptosis.     

MiR-23a-3p promoted G1/S cell cycle transition by targeting protocadherin17 in hepatocellular carcinoma

MiR-23a-3p has been shown to promote liver cancer cell growth and metastasis and regulate that of chemosensitivity. Protocadherin17 (PCDH17) is a tumor suppressor gene and plays an essential part in cell cycle of hepatocellular carcinoma (HCC). This study aimed at evaluating the effects of miR-23a-3p and PCDH17 on HCC cell cycle and underlining the mechanism. The level of miR-23a-3p was up-regulated, while PCDH17 level was down-regulated in HCC tissues compared to adjacent tissues. For the in vitro studies, high expression of miR-23a-3p down-regulated PCDH17 level; increased cell viability; promoted G1/S cell cycle transition; up-regulated cyclin D1, cyclin E, CDK2, CDK4, p-p27, and p-RB levels; and down-regulated the expression of p27. Dual luciferase reporter assay suggested PCDH17 was a target gene of miR-23a-3p. MiR-23a-3p inhibitor and PCDH17 siRNA led to an increase in cell viability and the number of cells in the S phase and up-regulated cyclin D1 and cyclin E levels, compared with miR-23a-3p inhibitor and NC siRNA group. For the in vivo experiments, high expression of miR-23a-3p promoted tumor growth and reduced PCDH17 level in the cytoplasm. These results indicated that high expression of miR-23a-3p might promote G1/S cell cycle transition by targeting PCDH17 in HCC cells. The miR-23a-3p could be considered as a molecular target for HCC detection.

     

Evidence for post-transcriptional regulation of cyclin B1 mRNA in the cell cycle and following irradiation in HeLa cells.

Cyclin B1 mRNA expression varies markedly through the cell cycle with its peak in G2/M and lowest level in G1. Cyclin B1 mRNA levels are also transiently reduced in HeLa cells after gamma-irradiation, coincident with the radiation-induced G2 block. In order to understand the mechanisms underlying these variations, we have measured cyclin B1 mRNA stability in HeLa cells during different phases of the cell cycle. The half-life of the mRNA measured after actinomycin D administration is 1.1-1.8 h in both early and late G1, 8 h in S and 13 h in G2/M. We therefore conclude that altered RNA stability is important in modulating cyclin B1 mRNA levels through the HeLa cell cycle. Furthermore, 3 h after irradiation of HeLa cells in S phase with 10 Gy, the half-life of cyclin B1 mRNA is reduced to 5 h; it is further reduced to 2-3 h at 14 h after irradiation. Thus, decreased stability contributes to the reduction in cyclin B1 mRNA following irradiation.     

WWOX suppresses prostate cancer cell progression through cyclin D1-mediated cell cycle arrest in the G1 phase

WW domain-containing oxidoreductase (WWOX) has been reported to be a tumor suppressor in multiple cancers, including prostate cancer. WWOX can induce apoptotic responses to inhibit tumor progression, and the other mechanisms of WWOX in tumor suppression have also been reported recently. In this study, we found significant down-regulation of WWOX in prostate cancer specimens and prostate cancer cell lines compared with the normal controls. In addition, an ectopically increased WWOX expression repressed tumor progression both in vitro and in vivo. Interestingly, overexpression of WWOX in 22Rv1 cells led to cell cycle arrest in the G1 phase but did not affect sub-G1 in flow cytometry. GFP-WWOX overexpressed 22Rv1 cells were shown to inhibit cell cycle progression into mitosis under nocodazole treatment in flow cytometry, immunoblotting and GFP fluorescence. Further, cyclin D1 but not apoptosis correlated genes were down-regulated by WWOX both in vitro and in vivo. Restoration of cyclin D1 in the WWOX-overexpressed 22Rv1 cells could abolish the WWOX-mediated tumor repression. In addition, WWOX impair c-Jun-mediated cyclin D1 promoter activity. These results suggest that WWOX inhibits prostate cancer progression through negatively regulating cyclin D1 in cell cycle lead to G1 arrest. In summary, our data reveal a novel mechanism of WWOX in tumor suppression.     

WWOX suppresses prostate cancer cell progression through cyclin D1-mediated cell cycle arrest in the G1 phase

WW domain-containing oxidoreductase (WWOX) has been reported to be a tumor suppressor in multiple cancers, including prostate cancer. WWOX can induce apoptotic responses to inhibit tumor progression, and the other mechanisms of WWOX in tumor suppression have also been reported recently. In this study, we found significant down-regulation of WWOX in prostate cancer specimens and prostate cancer cell lines compared with the normal controls. In addition, an ectopically increased WWOX expression repressed tumor progression both in vitro and in vivo. Interestingly, overexpression of WWOX in 22Rv1 cells led to cell cycle arrest in the G1 phase but did not affect sub-G1 in flow cytometry. GFP-WWOX overexpressed 22Rv1 cells were shown to inhibit cell cycle progression into mitosis under nocodazole treatment in flow cytometry, immunoblotting and GFP fluorescence. Further, cyclin D1 but not apoptosis correlated genes were down-regulated by WWOX both in vitro and in vivo. Restoration of cyclin D1 in the WWOX-overexpressed 22Rv1 cells could abolish the WWOX-mediated tumor repression. In addition, WWOX impair c-Jun-mediated cyclin D1 promoter activity. These results suggest that WWOX inhibits prostate cancer progression through negatively regulating cyclin D1 in cell cycle lead to G1 arrest. In summary, our data reveal a novel mechanism of WWOX in tumor suppression.     

Oxidative stress-induced cyclin D1 depletion and its role in cell cycle processing

Background

Cyclin D1 is immediately down-regulated in response to reactive oxygen species (ROS) and implicated in the induction of cell cycle arrest in G2 phase by an unknown mechanism. Either treatment with a protease inhibitor alone or expression of protease-resistant cyclin D1 T286A resulted in only a partial relief from the ROS-induced cell cycle arrest, indicating the presence of an additional control mechanism.

Methods

Cells were exposed to hydrogen peroxide (H₂O₂), and analyzed to assess the changes in cyclin D1 level and its effects on cell cycle processing by kinase assay, de novo synthesis, gene silencing, and polysomal analysis, etc.

Results

Exposure of cells to excessive H₂O₂ induced ubiquitin-dependent proteasomal degradation of cyclin D1, which was subsequently followed by translational repression. This dual control mechanism was found to contribute to the induction of cell cycle arrest in G2 phase under oxidative stress. Silencing of an eIF2α kinase PERK significantly retarded cyclin D1 depletion, and contributed largely to rescuing cells from G2 arrest. Also the cyclin D1 level was found to be correlated with Chk1 activity.

Conlclusions

In addition to an immediate removal of the pre-existing cyclin D1 under oxidative stress, the following translational repression appear to be required for ensuring full depletion of cyclin D1 and cell cycle arrest. Oxidative stress-induced cyclin D1 depletion is linked to the regulation of G2/M transit via the Chk1–Cdc2 DNA damage checkpoint pathway.

General significance

The control of cyclin D1 is a gate keeping program to protect cells from severe oxidative damages.     

Progesterone inhibits estrogen-induced cyclin D1 and cdk4 nuclear translocation, cyclin E- and cyclin A-cdk2 kinase activation, and cell proliferation in uterine epithelial cells in mice

The response of the uterine epithelium to female sex steroid hormones provides an excellent model to study cell proliferation in vivo since both stimulation and inhibition of cell proliferation can be studied. Thus, when administered to ovariectomized adult mice 17beta-estradiol (E2) stimulates a synchronized wave of DNA synthesis and cell division in the epithelial cells, while pretreatment with progesterone (P4) completely inhibits this E2-induced cell proliferation. Using a simple method to isolate the uterine epithelium with high purity, we have shown that E2 treatment induces a relocalization of cyclin D1 and, to a lesser extent, cdk4 from the cytoplasm into the nucleus and results in the orderly activation of cyclin E- and cyclin A-cdk2 kinases and hyperphosphorylation of pRb and p107. P4 pretreatment did not alter overall levels of cyclin D1, cdk4, or cdk6 nor their associated kinase activities but instead inhibited the E2-induced nuclear localization of cyclin D1 to below the control level and, to a lesser extent, nuclear cdk4 levels, with a consequent inhibition of pRb and p107 phosphorylation. In addition, it abrogated E2-induced cyclin E-cdk2 activation by dephosphorylation of cdk2, followed by inhibition of cyclin A expression and consequently of cyclin A-cdk2 kinase activity and further inhibition of phosphorylation of pRb and p107. P4 is used therapeutically to oppose the effect of E2 during hormone replacement therapy and in the treatment of uterine adenocarcinoma. This study showing a novel mechanism of cell cycle inhibition by P4 may provide the basis for the development of new antiestrogens.     

Roles of cyclin D1 and related genes in growth inhibition, senescence and apoptosis

It is now apparent that apoptosis is closely linked to the control of cell cycle progression. During the G1 to S progression, cyclin D1, p53, and the cyclin dependent kinase inhibitors p21^WAF1 and p27^kip1 can play roles in induction of apoptosis. During the G2 and M phases, premature activation of Cdk1 can cause cells to enter mitotic catastrophe, which results in apoptosis. In this review we focus on factors acting during G1 and S, particularly cyclin D1, and their effects on cell growth, senescence and apoptosis. We emphasize that cyclin D1 can have diverse effects on cells depending on its level of expression, the specific cell type, the cell context and other factors. Possible mechanisms by which cyclin D1 exerts these diverse effects, via cyclin dependent kinase-dependent and -independent pathways, are discussed.     

Flow cytometry study of human cyclin B1 and cyclin E expression in leukemic cell lines: cell cycle kinetics and cell localization

Experiments by flow cytometry (FCM) after nuclei isolation have never been done to investigate cyclins. We have conducted different experiments by FCM using whole cells and isolated nuclei to study the immunolocalization and kinetic patterns of cyclin B1 and cyclin E in various leukemic cell lines. During asynchronous growth, all whole cells had a scheduled, cell cycle phase-restricted expression of cyclin B1. By using a washless immunostaining of unfixed nuclei, cyclin B1 was detected in all cell cycle phases, including G1, although to a lesser extent than in G2/M, suggesting that in whole cells the cyclin B1 epitope is masked and accessible only in isolated nuclei. When the cells were synchronized at the G1/S boundary by thymidine or in the G1 phase by sodium n-butyrate, an identical accumulation of cyclin B1 was observed. As for cyclin E, its expression was higher with thymidine treatment than with sodium n-butyrate, particularly in nuclei. The elevated cyclin B1 level in the cells arrested at the G1/S boundary may reflect the increased half-life of this protein stabilized as the result of cyclin E overexpression. However, our FCM data also support the notion that accumulation of human cyclin B1 in leukemic cell lines begins during the G1 phase of the cell cycle, probably in the nucleus. The detection of cyclin B1 by Western blot in cells sorted in the G1 phase of the cell cycle confirms this finding. It is possible, therefore, that tumor transformation or leukemic phenotype may invariably be associated with altered cyclin B1 expression.     

Alterations in radiation induced cell cycle perturbations by 2-deoxy-D-glucose in human tumor cell lines

In the present studies, effects of glucose analogue, 2-deoxy-D-glucose (2-DG) on radiation-induced cell cycle perturbations were investigated in human tumor cell lines. In unirradiated cells, the levels of cyclin B1 in G2 phase were significantly higher in both the glioma cell lines as compared to squamous carcinoma cells. Upon irradiation with Co60 gamma-rays (2 Gy), the cyclin B1 levels were reduced in U87 cells, while no significant changes could be observed in other cell lines, which correlated well with the transient G2 delay observed under these conditions by the BrdU pulse chase measurements. 2-DG (5 mM, 2 hr) induced accumulation of cells in the G2 phase and a time-dependent increase in the levels of cyclin B1 in both the glioma cell lines, while significant changes could not be observed in any of the squamous carcinoma cell lines. 2-DG enhanced the cyclin B1 level further in all the cell lines following irradiation, albeit to different extents. Interestingly, an increase in the unscheduled expression of B1 levels in G1 phase 48 hr after irradiation was observed in all the cell lines investigated. 2-DG also increased the levels of cyclin D1 at 24 hr in BMG-1 cell line. These observations imply that 2-DG-induced alterations in the cell cycle progression are partly responsible for its radiomodifying effects.     

CycD1, a putative G1 cyclin from Antirrhinum majus, accelerates the cell cycle in cultured tobacco BY-2 cells by enhancing both G1/S entry and progression through S and G2 phases

A putative G1 cyclin gene, Antma;CycD1;1 (CycD1), from Antirrhinum majus is known to be expressed throughout the cell cycle in the meristem and other actively proliferating cells. To test its role in cell cycle progression, we examined the effect of CycD1 expression in the tobacco (Nicotiana tabacum) cell suspension culture BY-2. Green fluorescent protein:CycD1 is located in the nucleus throughout interphase. Using epitope-tagged CycD1, we show that it interacts in vivo with CDKA, a cyclin dependent protein kinase that acts at both the G1/S and the G2/M boundaries. We examined the effect of induced expression at different stages of the cell cycle. Expression in G0 cells accelerated entry into both S-phase and mitosis, whereas expression during S-phase accelerated entry into mitosis. Consistent with acceleration of both transitions, the CycD1-associated cyclin dependent kinase can phosphorylate both histone H1 and Rb proteins. The expression of cyclinD1 led to the early activation of total CDK activity, consistent with accelerated cell cycle progression. Continuous expression of CycD1 led to moderate increases in growth rate. Therefore, in contrast with animal D cyclins, CycD1 can promote both G0/G1/S and S/G2/M progression. This indicates that D cyclin function may have diverged between plants and animals.     

Comparative effects of chemotherapeutic agents on the growth and survival of human adrenal carcinoma cells in culture.

Adrenocortical carcinoma is an uncommon malignancy that is usually fatal within a short time after diagnosis. We have investigated the effects on the growth and survival of SW-13 human adrenal carcinoma cells in culture of some currently used and some potentially new agents in the treatment of adrenal cancer. Established drugs tested were mitotane, cisplatin, etoposide, 5-fluorouracil, and suramin. Other agents studied included adenine arabinofuranoside, cytosine arabinofuranoside, 2-methoxyestradiol, and paclitaxel. The most potent chemotherapeutic agents in this system were paclitaxel and 2-methoxyestradiol, with EC (50) of 1.8x10 (-8) and 3.3x10 (-7) M, respectively. Cytosine arabinofuranoside and cisplatin both had the same EC (50) of 7.0x10 (-7) M, and etoposide 1.1x10 (-6) M. All the other agents tested required much higher doses for effect, including mitotane, the current most commonly used chemotherapy for adrenal cancer, with an EC (50) of 3.3x10 (-4) M. These data suggest that paclitaxel, 2-methoxyestradiol, and cytosine arabinofuranoside should be further evaluated for their potential in the chemotherapy of adrenal carcinoma.     

Estrogen activates cyclin-dependent kinases 4 and 6 through induction of cyclin D in rat primary osteoblasts

Estrogen plays important roles in maintaining bone density and protecting against osteoporosis, but the underlying mechanisms of estrogen action via estrogen receptors (ERs) in bone remain to be clarified. In the present study, we isolated primary osteoblasts derived from transgenic rats harboring a dominant negative ER mutant, rat ERalpha (1-535) cDNA, and from their wild-type littermates. We observed that the rate of cell growth of osteoblasts from the transgenic rats was reduced compared to that of wild-type osteoblasts. Utilizing cDNA microarray analysis, we found that mRNA level of cyclin D2 was lower in the osteoblasts from the transgenic rats. D-type cyclins including cyclin D1, cyclin D2, and cyclin D3 are cell cycle regulators that promote progression through the early-to-mid G1 phase of the cell cycle. The protein levels of D-type cyclins including cyclin D2 and cyclin D3 but not cyclin D1 were elevated in wild-type osteoblasts with 17beta-estradiol treatment, resulting in the activation of cyclin-dependent kinases 4 and 6 (Cdk4/6) activities and the promotion of cell growth. Moreover, an anti-estrogen ICI 182,780 abolished the induction of the expression of D-type cyclins by 17beta-estradiol. Our findings indicate that estrogen and its receptors enhance Cdk4/6 activities through the induction of D-type cyclins, leading to the growth promotion of osteoblasts.