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.     

Nonlinear ionizing radiation-induced changes in eye lens cell proliferation,cyclin D1 expression and lens shape

Elevated cataract risk after radiation exposure was established soon after the discovery of X-rays in 1895. Today, increased cataract incidence among medical imaging practitioners and after nuclear incidents has highlighted how little is still understood about the biological responses of the lens to low-dose ionizing radiation (IR). Here, we show for the first time that in mice, lens epithelial cells (LECs) in the peripheral region repair DNA double strand breaks (DSB) after exposure to 20 and 100 mGy more slowly compared with circulating blood lymphocytes, as demonstrated by counts of γH2AX foci in cell nuclei. LECs in the central region repaired DSBs faster than either LECs in the lens periphery or lymphocytes. Although DSB markers (γH2AX, 53BP1 and RAD51) in both lens regions showed linear dose responses at the 1 h timepoint, nonlinear responses were observed in lenses for EdU (5-ethynyl-2′-deoxy-uridine) incorporation, cyclin D1 staining and cell density after 24 h at 100 and 250 mGy. After 10 months, the lens aspect ratio was also altered, an indicator of the consequences of the altered cell proliferation and cell density changes. A best-fit model demonstrated a dose-response peak at 500 mGy. These data identify specific nonlinear biological responses to low (less than 1000 mGy) dose IR-induced DNA damage in the lens epithelium.     

糖原合酶激酶3β以细胞周期蛋白D1依赖性方式引发人肺腺癌细胞A549细胞周期阻滞

对糖原合酶激酶3β（glycogen synthase kinase 3β,6SK3β）在细胞增殖中的作用研究,在不同细胞系和不同刺激因素作用下得出了不同结论,本文旨在探讨GSK3β在人肺腺癌细胞系A549细胞生长中的直接作用。A549细胞瞬时转染持续激活型S9A-GSK3β以及显性负突变型KM-GSK3β两种GSK3β突变型质粒,改变GSK3β活性。24 h后,分别进行细胞计数,流式细胞术及Western blot检测。结果显示,增强GSK3β活性可导致细胞数量下降,G．期细胞百分比升高。细胞周期蛋白D1表达水平被GSK3β下调。结果提示,GSK3β可能以细胞周期蛋白D1依赖性方式引发A549细胞的G,期阻滞,从而发挥生长抑制效应。     

Differentiation-inducing factor-1 induces cyclin D1 degradation through the phosphorylation of Thr286 in squamous cell carcinoma

Differentiation-inducing factors (DIFs) are morphogens which induce cell differentiation in Dictyostelium. We reported that DIF-1 and DIF-3 inhibit proliferation and induce differentiation in mammalian cells. In this study, we investigated the effect of DIF-1 on oral squamous cell carcinoma cell lines NA and SAS, well differentiated and poorly differentiated cell lines, respectively. Although DIF-1 did not induce the expression of cell differentiation makers in these cell lines, it inhibited the proliferation of NA and SAS in a dose-dependent manner by restricting the cell cycle in the G0/G1 phase. DIF-1 induced cyclin D1 degradation, but this effect was prevented by treatment with lithium chloride and SB216763, the inhibitors of glycogen synthase kinase-3beta (GSK-3beta). Depletion of endogenous GSK-3beta by RNA interference also attenuated the effect of DIF-1 on cyclin D1 degradation. Therefore, we investigated the effect of DIF-1 on GSK-3beta and found that DIF-1 dephosphorylated GSK-3beta on Ser9 and induced the nuclear translocation of GSK-3beta, suggesting that DIF-1 activated GSK-3beta. Then, we examined the effect of DIF-1 on cyclin D1 mutants (Thr286Ala, Thr288Ala, and Thr286/288Ala). We revealed that Thr286Ala and Thr286/288Ala mutants were highly resistant to DIF-1-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr286 was critical for cyclin D1 degradation induced by DIF-1. These results suggest that DIF-1 induces degradation of cyclin D1 through the GSK-3beta-mediated phosphorylation of Thr286.     

Transforming growth factor-beta3 promotes mesenchymal cell proliferation and angiogenesis mediated by the enhancement of cyclin D1, Flk-1, and CD31 gene expression during CL/Fr mouse lip fusion

BACKGROUND: Cleft lip with or without cleft palate is the most common congenital anomaly in the craniofacial region. Knowledge of the molecular mechanisms behind normal lip fusion can contribute to better intervention and improved functional clinical outcome. Transforming growth factor-beta3 (TGF-beta3) has been implicated in lip morphogenesis. Therefore, we hypothesized that TGF-beta3 functions during lip fusion through regulation of angiogenesis and mesenchymal cell cycle progression during early developmental stages. METHODS: To test this hypothesis we used the CL/Fraser mouse model, which has a high incidence of cleft lip. Lips isolated from embryonic day (ED) 11.5 mouse embryos were allowed to develop in serum-free organ cultures in the presence or absence of TGF-beta3. The lips that developed in these cultures fused in 2 days. RESULTS: During normal development, we detected positive immunoreactions for TGF-beta3 at the site of fusion. We also detected mesenchymal cells that were immunopositive for Flk-1 and CD31, which are markers for endothelial cell precursors. Exogenous TGF-beta3 accelerated lip fusion in culture. This enhancement was associated with an increase in the number of capillary blood vessels in the lips cultured in the presence of TGF-beta3, in comparison with controls. In tandem, TGF-beta3 increased the level of expression of both Flk-1 and CD31. Our data suggest that an elevated level of TGF-beta3 may promote angiogenesis in developing lips that is mediated by increased Flk-1 and CD31 expression. We also detected increased cyclin D1 expression (a marker for cell proliferation) in the presence of TGF-beta3, which suggests that TGF-beta3 promoted cell proliferation. CONCLUSIONS: TGF-beta3 promoted cell proliferation and angiogenesis in lip mesenchymal tissues. These events led to enhanced lip fusion in the presence of TGF-beta3.     

Differential expression of D-type G1 cyclins during mouse development and liver regeneration in vivo

D-type Gl cyclins are the primary cell cycle regulators of G1/S transition in eukaryotic cells, and are differentially expressed in a variety of cell lines in vitro. Little is known, however, about the expression patterns of D-type G1 cyclins in normal mouse in vivo. Thus, in the present study, tissue-specific expressions of cyclin D1 and D3 genes were examined in several tissues derived from adult male mice, and stage-specific expression of cyclin genes was studied in brain, liver, and kidney of developing mice from embryonic day 13 to postnatal day 11. Cell cycle-dependent expression of cyclins was also examined in regenerating livers following partial hepatectomy. Our results indicate that (l) cyclins Dl and D3 are expressed in a tissue-specific manner, with cyclin Dl being highly expressed in kidney and D3 in thymus; (2) cyclin D3 mRNA is abundantly expressed in young proliferating tissues and is gradually reduced during development, whereas cyclin Dl mRNA fluctuates during development; and (3) compensatory regeneration of liver induces cyclin Dl gene expression 12 hr after partial hepatectomy, and cyclin D3 gene expression from 36 to 42 hr (at the time of G1/S transition). In conclusion, this study indicates that cyclin D1 and D3 genes are differentially expressed in vivo in a tissue-specific, developmental stage-dependent, and cell cycle-dependent manner. © 1996 Wiley-Liss, Inc.     

Effects of the vitamin D3 analog 1α,25-dihydroxyvitamin D3-3β-bromoacetate on rat osteosarcoma cells: Comparison with 1α,25-dihydroxyvitamin D3

The actions of the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1α,25-(OH)₂D₃ in many cell types including osteoblastic cells. We have compared the effects of 1α,25-(OH)₂D₃ and a novel 1α,25-(OH)₂D₃ bromoester analog (1,25-(OH)₂-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 × 10⁻⁸ M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)₂-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE, intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 × 10⁻⁸ M, both 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)₂-BE are similar to those of 1α,25-(OH)₂D₃, and since 1,25-(OH)₂-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors. © 1996 Wiley-Liss, Inc.     

Colocalization of the cyclin B homologue P56 and β-tubulin during the cell cycle in a unicellular eucaryote dinoflagellate

We provide evidence for an unusual behavior of the cyclin B homologue, p56, in the dinoflagellate Crypthecodinium cohnii. p56, of which we previously demonstrated the presence in this original eukaryotic protist, is present all along the cell cycle progression, and is exclusively cytoplasmic as revealed after immunofluorescence labeling with anti-p56 Ab and counterstaining with Dapi. It was never found in the nucleus as is the case in higher eukaryotic cells. During motosis, p56 was essentially associated with the mitotic apparatus: centrosomes and mitotic spindle, as shown after double immunofluorescence labeling with anti p56 and anti β-tubulin Ab. Using high pressure freeze fixation, we clearly detected in transmission electron microscopy (TEM) the localization of p56 cyclin B homologue and β-tubulin: single immunogold labeling demonstrated that p56 is localized along the whole cell cortex, along the cleavage furrow of anaphase to cytokinesis cells and into cytoplasmic channels passing throughout the mitotic nucleus where is located the mitotic spindle. Double immunogold labeling realized with anti-p56 and anti-β-tubulin antibodies confirm that p56 antigens colocalize with β-tubulin in many sites. The significance of the exclusively cytoplasmic localization of the cyclin B homologue is discussed.     

PI-3K/Akt signal pathway plays a crucial role in arsenite-induced cell proliferation of human keratinocytes through induction of cyclin D1

Exposure of arsenite can induce hyperproliferation of skin cells, which is believed to play important roles in arsenite-induced carcinogenesis by affecting both promotion and progression stages. However, the signal pathways and target genes activated by arsenite exposure responsible for the proliferation remain to be defined. In the present study, we found that: (1) exposure of human keratinocytic HaCat cells to arsenite caused an increase in cell proliferation, which was significantly inhibited by pretreatment of wortmannin, a specific chemical inhibitor of PI-3K/Akt signal pathway; (2) arsenite exposure was also able to activate PI-3K/Akt signal pathway, which thereby induced the elevation of cyclin D1 expression level in both HaCat cells and human primary keratinocytes based on that inhibition of PI-3K/Akt pathway by either pretreatment of wortmannin or the transfection of their dominant mutants, significantly inhibited cyclin D1 expression upon arsenite exposure; (3) PI-3K/Akt pathway is implicated in arsenite-induced proliferation of HaCat cells through the induction of cyclin D1 because either knockdown of cyclin D1 by its siRNA or inhibition of PI-3K/Akt signal pathway by their dominant mutants markedly impaired the proliferation of HaCat cells induced by arsenite exposure. Taken together, we provide the direct evidence that PI-3K/Akt pathway plays a role in the regulation of cell proliferation through the induction of cyclin D1 in human keratinocytes upon arsenite treatment. Given the importance of aberrant cell proliferation in cell transformation, we propose that the activation of PI-3K/Akt pathway and cyclin D1 induction may be the important mediators of human skin carcinogenic effect of arsenite.     

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.     

Expression of galectin-1 mRNA in the mouse uterus is under the control of ovarian steroids during blastocyst implantation

Galectin-1 is a member of β-galactoside-binding lectins expressed in a variety of mammalian tissues. We report here that galectin-1 mRNA is abundantly expressed in the mouse reproductive organs such as the uterus and ovary. Uterine expression of galectin-1 mRNA is specifically regulated in the embryonic implantation process. Its expression increased at a high level on the fifth day post coitum (dpc 5) when embryos hatched into the endometrial epithelial cells. In the absence of embryos, however, galectin-1 expression in the mouse uterus decreased on dpc 5. In the delayed implantation mice, galectin-1 mRNA level was augmented by the termination of the delay of implantation. Ovarian steroids progesterone and estrogen differentially regulated galectin-1 mRNA level in uterine tissues. Treatment with RU486, a progesterone receptor antagonist, blocked progesterone-induced galectin-1 mRNA level in uterine tissues of ovariectomized mouse. ICI182780, a pure estrogen receptor antagonist, clearly blocked the estrogen effect. Taken together, galectin-1 gene expression in the uterine tissues was regulated by ovarian steroids and this regulation correlated with the implantation process. Mol. Reprod. Dev. 48:261–266, 1997. © 1997 Wiley-Liss, Inc.     

GSK-3beta mediates in the progesterone inhibition of estrogen induced cyclin D2 nuclear localization and cell proliferation in cyclin D1-/- mouse uterine epithelium

We report that glycogen synthase kinase (GSK)-3beta is phosphorylated at ser9 and inactivated in uterine epithelial cells from E(2)-treated cyclin D1 null mutant mice. Simultaneous administration of P(4) together with E(2) blocked this effect. Pharmacological inhibition of GSK-3beta activity in mice treated with P(4)E(2) reversed the nuclear exclusion of cyclin D2 in the uterine epithelial cells and this caused phosphorylation of Rb protein and progression of cells towards S-phase. Our results indicate that GSK-3beta is a major target of E(2) and P(4) in regulation of cyclin D2 localization in the mouse uterine epithelium.     

Molecular cloning, gene localization, and structure of human cyclin B3

Here we describe the molecular cloning of human cyclin B3, its localization, and its structure. It is localized in the subcentromeric region of the X chromosome, still not completely sequenced by the Human Genome Project. This cyclin B3 is unusually large for a mitotic cyclin. Its mRNAs were found in all tissues and were particularly abundant in testis. At least three splice variants were found in the ORF and three variants in the 5'UTR.     

Nuclear type II [3H]estradiol binding site ligands: inhibition of ER-positive and ER-negative cell proliferation and c-Myc and cyclin D1 gene expression

These studies assessed the effects of 3,4-dihydroxybenzalacetone (ZN-1) and 1-(3,4-dihydroxyphenyl)-2-propanol (ZN-2) on MCF-7 cell proliferation. The compounds blocked [3H]estradiol binding to nuclear type II sites, but did not compete for [3H]estradiol binding to recombinant ERalpha or ERbeta. ZN-1 and ZN-2 inhibited the proliferation of ERalpha and ERbeta positive (MCF-7) and negative (MCF-10A) breast cells, further ruling out direct binding to ER in the mechanism of action of these compounds. Pre-loading type II sites with ZN-1 or ZN-2 reduced [3H]estradiol exchange, strongly suggesting the drugs were binding covalently. ZN-1 treatment resulted in complete occupancy of type II sites and sustained (9 days) inhibition of MCF-7 cell proliferation following its removal from the tissue culture medium. This cell growth inhibition was not due to non-specific toxicity, as the numbers of viable, attached cells per dish (determined by trypan blue dye exclusion) remained constant throughout this 9-day period and eventually reversed by day 19. ZN-2 effects on cell proliferation reversed more rapidly following discontinuation of treatment, a response consistent with the inability of the compound to totally block type II binding. Both ZN-1 and ZN-2 blocked estradiol stimulation of c-Myc and cyclin D1 gene expression in MCF-7 cells, two events that are clearly coupled to cell cycle progression. We suspect this may occur through ZN-1 or ZN-2 modification of nucleosome function and/or chromatin remodeling since nuclear type II sites are localized to a complex of histones H3 and H4 (Shoulars et. al, J Steroid Biochem. Mol. Biol. 96: 19-30, 2005).     

Ras signals principally via Erk in G1 but cooperates with PI3K/Akt for Cyclin D induction and S-phase entry

Numerous studies exploring oncogenic Ras or manipulating physiological Ras signalling have established an irrefutable role for Ras as driver of cell cycle progression. Despite this wealth of information the precise signalling timeline and effectors engaged by Ras, particularly during G1, remain obscure as approaches for Ras inhibition are slow-acting and ill-suited for charting discrete Ras signalling episodes along the cell cycle. We have developed an approach based on the inducible recruitment of a Ras-GAP that enforces endogenous Ras inhibition within minutes. Applying this strategy to inhibit Ras stepwise in synchronous cell populations revealed that Ras signaling was required well into G1 for Cyclin D induction, pocket protein phosphorylation and S-phase entry, irrespective of whether cells emerged from quiescence or G2/M. Unexpectedly, Erk, and not PI3K/Akt or Ral was activated by Ras at mid-G1, albeit PI3K/Akt signalling was a necessary companion of Ras/Erk for sustaining cyclin-D levels and G1/S transition. Our findings chart mitogenic signaling by endogenous Ras during G1 and identify limited effector engagement restricted to Raf/MEK/Erk as a cogent distinction from oncogenic Ras signalling.     

Differential regulation of cyclin D1 and D3 expression in the control of astrocyte proliferation induced by endothelin-1

We have previously shown that the mitogenic effect of endothelin-1 (ET-1) in primary astrocytes is dependent on activation of both extracellular signal-regulated kinase (ERK)- and cytoskeleton (CSK)-dependent pathways. In this study, we evaluated the contribution of each of these pathways to the expression and activation of proteins mediating cell cycle progression. Our results suggest that ET-1-induced expression of cyclins D1 and D3 is dependent on the ERK- and CSK-dependent pathways, respectively; moreover, a decrease in the levels of the cyclin-dependent kinase inhibitor (CKI) p27 was observed as a consequence of ERK activation. Expression of both cyclins D1 and D3 together with a decrease in the p27 levels are essential for retinoblastoma protein (pRB) phosphorylation and cyclin A expression. Furthermore, the molecular events responsible for cell-cell contact inhibition of astrocyte proliferation were found to be independent of the mitogenic pathways leading to D-type cyclin expression. Cell growth arrest in confluent astrocytes was found to be correlated with increased expression of CKI p21, resulting in inhibition of D-type cyclin-associated pRB phosphorylation and cyclin A expression. Taken together, these results indicate that cyclins D1 and D3, which constitute the key mediators of the proliferative response of primary astrocytes to ET-1, are regulated by distinct signaling pathways.