Critical role for cyclin D2 in BCR/ABL-induced proliferation of hematopoietic cells.

Chronic myeloid leukemia is caused by the tyrosine kinase oncoprotein BCR/ABL. Using oligonucleotide arrays to assay mRNAs at different phases of the cell cycle in BCR/ABL-transformed cells, we found that cyclin D2 mRNA was constitutively expressed at high levels throughout the cell cycle, a pattern confirmed by immunoblotting of protein lysates. Bone marrow cells from cyclin D2-deficient strains of mice failed to proliferate in response to infection with a retrovirus carrying BCR/ABL and failed to generate transformed lymphoid cell lines in vitro. These results establish that BCR/ABL promotes cell cycle progression by altering expression of cyclin D2 and that cyclin D2 induction plays a critical role in proliferation of hematopoietic cells by BCR/ABL.     

The c-Rel transcription factor and B-cell proliferation: a deal with the devil

Activation of the Rel/NF-kappaB signal transduction pathway has been associated with a variety of animal and human malignancies. However, among the Rel/NF-kappaB family members, only c-Rel has been consistently shown to be able to malignantly transform cells in culture. In addition, c-rel has been activated by a retroviral promoter insertion in an avian B-cell lymphoma, and amplifications of REL (human c-rel) are frequently seen in Hodgkin's lymphomas and diffuse large B-cell lymphomas, and in some follicular and mediastinal B-cell lymphomas. Phenotypic analysis of c-rel knockout mice demonstrates that c-Rel has a normal role in B-cell proliferation and survival; moreover, c-Rel nuclear activity is required for B-cell development. Few mammalian model systems are available to study the role of c-Rel in oncogenesis, and it is still not clear what features of c-Rel endow it with its unique oncogenic activity among the Rel/NF-kappaB family. In any event, REL may provide an appropriate therapeutic target for certain human lymphoid cell malignancies.     

Decrease in susceptibility toward induction of apoptosis and alteration in G1 checkpoint function as determinants of resistance of human lung cancer cells against the antisignaling drug UCN-01 (7-Hydroxystaurosporine).

7-Hydroxystaurosporine (UCN-01) is a protein kinase inhibitor that is under development as an anticancer agent in the United States and Japan. Long-term exposure of human A549 non-small cell lung cancer cells to UCN-01 furnished cells (A549/UCN) with acquired resistance against UCN-01. In this study, the sensitivity of these cells toward the growth-arresting properties of certain conventional cytotoxic agents was explored. Cells were not cross-resistant against adriamycin, Taxol, staurosporine, and UCN-02, but they displayed 14- and 4.4-fold resistance against cisplatin and mitomycin C, respectively. Previous studies on the mechanism(s) of action of UCN-01 suggest that induction of apoptosis and G1 phase accumulation are important for its anticancer activity; therefore, we compared induction of apoptosis and cell cycle distribution caused by UCN-01 in wild-type A549 and A549/UCN cells using flow cytometry. UCN-01 (0.4 microM) induced apoptosis (62% terminal deoxynucleotidyl transferase-mediated nick end labeling-positive cells) in A549 cells, but not in A549/UCN cells. The percentages of cells that accumulated in G1 when exposed to UCN-01 (0.4 microM) were 22% in A549 cells and 67% in A549/UCN cells. These results suggest that acquired resistance of cancer cells against UCN-01 is characterized by attenuation of apoptosis induction associated with reinforcement of the G1 checkpoint and that apoptosis regulation is drastically altered in A549/UCN cells as compared with A549 cells. Cyclin-dependent kinase (CDK) inhibitor proteins p21 and p27 in A549/UCN cells were up-regulated, which was accompanied by overexpression of G1 cyclins D1 and E, but UCN-01 hardly affected levels of these proteins. In contrast, cyclin A, cyclin B1, retinoblastoma, and CDK2 proteins were apparently down-regulated, without changes in CDK4/6. UCN-01 hardly affected the expression level of cyclin B1 and induced dephosphorylation of retinoblastoma in both cell types. UCN-01 induced down-regulation of cyclin A level and CDK2 activity accompanied with its dephosphorylation in A549/UCN cells, but not in A549 cells. The antiapoptotic protein bcl-2 was apparently up-regulated in A549/UCN cells, however, bcl-xL, another antiapoptotic protein, was down-regulated, without changes in bak and bax. Taken together, these results are consistent with the notion that induction of apoptosis and block of cell cycle in G1 are important determinants of the sensitivity of cancer cells to UCN-01 and suggest that inhibition of CDK2 activity accompanied by its dephosphorylation and decrease of expression level of cyclin A might play an important role in the G1 phase accumulation induced by UCN-01.     

BCR signals target p27(Kip1) and cyclin D2 via the PI3-K signalling pathway to mediate cell cycle arrest and apoptosis of WEHI 231 B cells.

Cross-linking of the B cell antigen receptor (BCR) on immature WEHI 231 B cells results in G1 cell cycle arrest and apoptosis. Here we investigated the molecular mechanisms that are necessary and sufficient for these changes to occur. We show that BCR stimulation of WEHI 231 cells results in down-regulation of cyclin D2 and up-regulation of p27(Kip1), which are associated with pocket protein hypophosphorylation and E2F inactivation. Ectopic expression of p27(Kip1) by TAT-fusion protein or retroviral transduction is sufficient to cause G1 cell cycle arrest, followed by apoptosis. In contrast, over-expression of cyclin D2 overcomes the cell cycle arrest and apoptosis induced by anti-IgM, indicating that down-regulation of cyclin D2 is necessary for the cell cycle arrest and apoptosis activated by BCR stimulation. Thus, cyclin D2 and p27(Kip1) have opposing roles in these pathways and our data also suggest that cyclin D2 functions upstream of p27(Kip1) and the pRB pathway and therefore plays an essential part in integrating the signals from BCR with the cell cycle machinery. We next investigated which signal transduction pathways triggered by the BCR regulate cell proliferation and apoptosis via cyclin D2 and p27(Kip1). Inhibition of PI3-K signalling by LY294002 down-regulated cyclin D2 and up-regulated p27(Kip1) expression at both protein and RNA levels, mimicking the effects of BCR-stimulation. Furthermore, ectopic expression of a constitutively active form of AKT blocked the cell cycle arrest and apoptosis triggered by anti-IgM and also abrogated down-regulation of cyclin D2 and up-regulation of p27(Kip1) expression induced by BCR-engagement. These results indicate that BCR activation targets p27(Kip1) and cyclin D2 to mediate cell cycle arrest and apoptosis and that down-regulation of PI3-K/AKT activity post BCR stimulation is necessary for these to occur.     

E2F3a在U251胶质瘤细胞中的功能研究

背景与目的：E2F3a是一种重要的转录激活因子，在多种肿瘤组织中均呈现表达上调。E2F3a在恶性化程度较高的神经胶质瘤中表达较高，目前它在胶质瘤细胞中的功能尚不清楚。本文的目的是研究E2F3。对U251胶质瘤细胞周期和凋亡的影响。方法：通过脂质体介导质粒转染在U251细胞中过表达E2F3a：用Western blotting检测细胞内E2F3a蛋白质的表达水平：用PT-核DNA染色结合流式细胞术分析细胞周期分布：用Annexin V—PE和7-AAD染色结合流式细胞术分析细胞凋亡比例；用实时荧光定量PCR方法测定细胞内A2、B1、D1和E细胞周期素mRNA的相对表达水平。结果：与空载体转染对照组相比．E2F3a过表达可将位于S期的细胞比例提高28％（P〈0．01），将位于G0/G1期和G2/M的细胞比例分别降低15％和13％（P〈0．01）。E2F3a过表达对细胞凋亡无明显影响。进一步研究发现，E2F3a可显著上调细胞周期素D1和E的mRNA表达．而不影响周期素A2和B1的mRNA表达。结论：E2F3a是胶质瘤细胞增殖的促进因子，它可通过上调细胞周期素D1和E的表达加速细胞由G，期进入S期。     

Inhibition of Myc-dependent apoptosis by eukaryotic translation initiation factor 4E requires cyclin D1

Ectopically expressed eukaryotic translation initiation factor 4E (eIF4E) stimulates cell proliferation, suppresses apoptosis in growth factor restricted cells, and induces malignant transformation in primary rodent fibroblasts when coexpressed with protooncogene myc. We report here that eIF4E rescued rat embryo fibroblasts ectopically expressing c-Myc (REF/Myc) from genotoxic and non-genotoxic cytostatic drugs and identify cyclin D1 as a downstream effector in the antiapoptotic mechanism. In clones of REF/Myc ectopically expressing eIF4E, resistance to apoptosis paralleled steady state levels of cyclin D1. Stable expression of cyclin D1 in REF/Myc inhibited apoptosis in response to a broad range of cell cycle specific cytostatic agents. Partial loss-of-cyclin D1 function in REF/Myc ectopically expressing eIF4E (REF/Myc/4E) significantly increased chemosensitivity; either soluble antisense cyclin D1 oligomers or transfection with a dominant negative cyclin D1 mutant that prevents translocation of cyclin D-dependent kinases to the nucleus, significantly blunted the antiapoptotic effect of eIF4E. These data directly link eIF4E rescue from cytostatic drugs to cyclin D1. Since overexpression of eIF4E and cyclin D1 is observed in many aggressive forms of chemoresistant cancers, these findings provide insight into possible mechanisms responsible for this biological behavior.     

BCR targets cyclin D2 via Btk and the p85alpha subunit of PI3-K to induce cell cycle progression in primary mouse B cells

The p85alpha subunit of PI3-K and Btk are two crucial components of the B-cell receptor (BCR) signalling pathway. In the present study, we showed that primary splenic B cells from p85alpha null and xid (Btk-deficient) mice fail to induce cyclin D2 expression and enter early G1, but not S phase of the cell cycle in response to BCR engagement. Furthermore, these Btk or p85alpha null B cells displayed increased cell death compared with wild type following BCR engagement. These findings are further confirmed by studies showing that specific pharmacological inhibitors of Btk (LFM-A13), PI3-K (LY294002 and Wortmannin) and PLCgamma (U73122) also block cyclin D2 expression and S phase entry following BCR stimulation, as well as triggering apoptosis. Collectively, these data provide evidence for the concept that the B-cell signalosome (p85alpha, Btk, BLNK and PLCgamma) is involved in regulating cyclin D2 expression in response to BCR engagement. PKC and intracellular calcium are two major downstream effectors of the B-cell signalosome and can be activated by PMA and ionomycin, respectively. In small resting (G0) B cells, costimulation with PMA and ionomycin, but not PMA or ionomycin alone, induces cyclin D2 expression and cell-cycle progression. Consistent with this, we also showed that the BCR-mediated cyclin D2 induction could be abolished by pretreatment of resting B cells with specific inhibitors of capacitative Ca(2+) entry (SK&F 96365) or PKC (G?6850). Our present results lead us to propose a model in which the B-cell signalosome targets cyclin D2 via the Ca(2+) and PKC-dependent signalling cascades to mediate cell-cycle progression in response to BCR engagement.     

Cyclin A1, the alternative A-type cyclin, contributes to G1/S cell cycle progression in somatic cells

Cyclin A1 is an alternative A-type cyclin that is essential for spermatogenesis, but it is also expressed in hematopoietic progenitor cells and in acute myeloid leukemia. Its functions during cell cycle progression of somatic cells are incompletely understood. Here, we have analysed the cell cycle functions of cyclin A1 in transformed and nontransformed cells. Murine embryonic fibroblasts derived from cyclin A1-deficient mice were significantly impaired in their proliferative capacity. In accordance, cyclin A1-/- cells accumulated in G1 and G2/M phase while the percentage of S phase cells decreased. Also, lectin stimulated splenic lymphocytes from cyclin A1-/- mice proliferated slower than their wild-type counterparts. Forced cyclin A1 overexpression in NIH3T3 cells and in U937 leukemic cells either by transient transfection or by retroviral infection enhanced S phase entry. Consequently, siRNA mediated silencing of cyclin A1 in highly cyclin A1 expressing ML1 leukemic cells significantly slowed S phase entry, decreased proliferation and inhibited colony formation. Taken together, these analyses demonstrate that cyclin A1 contributes to G1 to S cell cycle progression in somatic cells. Cyclin A1 overexpression enhances S phase entry consistent with an oncogenic function. Finally, cyclin A1 might be a therapeutic target since its silencing inhibited leukemia cell growth.     

稳定表达Cyclin E细胞系的建立及Cyclin E高表达对人乳腺癌MCF 7细胞生长及细胞周期的影响

 目的 观察cyclin E 的高表达对乳腺癌细胞MCF 7 生长及周期的影响。方法 构建cyclin EcDNA真核表达载体并采用lipofectAMINE 转染方法将其导入MCF 7 细胞,获得稳定表达cyclin E的细胞系。通过对细胞生长曲线绘制、3H TdR测定及细胞周期分布等的分析,观察其对细胞生长、增殖的影响。结果 cyclin E的高表达可以使细胞的生长速度加快（ 约为对照1-52 倍） 及DNA 掺入增加（4-2 倍）,G1 S移行加速;pRB的磷酸化形式的增多（3 倍）.结论 cyclin E的高表达可明显影响MCF 7 细胞的生长、增殖,并可能通过pRB 磷酸化,影响细胞周期G1 S期移行而实现其对生长的调节。     

Cyclin D1 induction of cellular migration requires p27(KIP1)

The cyclin D1 gene is amplified and overexpressed in human breast cancer, functioning as a collaborative oncogene. As the regulatory subunit of a holoenzyme phosphorylating Rb, cyclin D1 promotes cell cycle progression and a noncatalytic function has been described to sequester the cyclin-dependent kinase inhibitor protein p27. Cyclin D1 overexpression correlates with tumor metastasis and cyclin D1-deficient fibroblasts are defective in migration. The genetic mechanism by which cyclin D1 promotes migration and movement is poorly understood. Herein, cyclin D1 promoted cellular migration and cytokinesis of mammary epithelial cells. Cyclin D1 enhanced cellular migratory velocity. The induction of migration by cyclin D1 was abolished by mutation of K112 or deletion of NH(2)-terminal residues 46 to 90. These mutations of cyclin D1 abrogated physical interaction with p27(KIP1). Cyclin D1(-/-) cells were p27(KIP1) deficient and the defect in migration was rescued by p27(KIP1) reintroduction. Conversely, the cyclin D1 rescue of cyclin D1(-/-) cellular migration was reversed by p27(KIP1) small interfering RNA. Cyclin D1 regulated p27(KIP1) abundance at the posttranslational level, inhibiting the Skp2 promoter, Skp2 abundance, and induced p27(KIP1) phosphorylation at Ser(10). Together, these studies show cyclin D1 promotes mammary epithelial cell migration. p27(KIP1) is required for cyclin D1-mediated cellular migration.     

Direct trans-activation of the human cyclin D2 gene by the oncogene product Tax of human T-cell leukemia virus type I

Cyclins are one of the pivotal determinants regulating cell cycle progression. We previously reported that the trans-activator Tax of human T-cell leukemia virus type I (HTLV-I) induces endogenous cyclin D2 expression along with cell cycle progression in a resting human T-cell line, Kit 225, suggesting a role of cyclin D2 in Tax-mediated cell cycle progression. The cyclin D2 gene has a typical E2F binding element, raising the possibility that induction of cyclin D2 expression is a consequence of cell cycle progression. In this study, we examined the role and molecular mechanism of induction of the endogenous human cyclin D2 gene by Tax. Introduction of p19(INK4d), a cyclin dependent kinase (CDK) inhibitor of the INK4 family specific for D-type CDK, inhibited Tax-mediated activation of E2F, indicating requirement of D-type CDK in Tax-mediated activation of E2F. Previously indicated E2F binding element and two NF-kappaB-like binding elements in the 1.6 kbp cyclin D2 promoter fragment had little, if any, effect on responsiveness to Tax. We found that trans-activation of the cyclin D2 promoter by Tax was mainly mediated by a newly identified NF-kappaB-like element with auxiliary contribution of a CRE-like element residing in sequences downstream of -444 which were by themselves sufficient for trans-activation by Tax. These results indicate that Tax directly trans-activates the cyclin D2 gene, resulting in growth promotion and perhaps leukemogenesis through activation of D-type CDK.     

Overexpression of the low molecular weight cyclin E in transgenic mice induces metastatic mammary carcinomas through the disruption of the ARF-p53 pathway

In tumor cells, cyclin E deregulation results in the appearance of five low molecular weight (LMW) isoforms. When overexpressed in breast cancer cells, these forms of cyclin E induce genomic instability, resistance to inhibition by p21 and p27, and resistance to antiestrogen therapy. Additionally, the LMW forms of cyclin E strongly correlate with decreased survival in patients with breast cancer. However, the oncologic role of the LMW forms of cyclin E in breast cancer tumorigenesis is yet to be determined. To this end, we generated transgenic mice expressing full-length cyclin E alone (M46A), full-length and the EL4 isoforms (EL1/EL4), or the EL2/3 isoforms of cyclin E (T1) under the control of the mouse mammary tumor virus promoter. Compared with full-length cyclin E, LMW cyclin E overexpression induces delayed mammary growth during the pubertal phase and abnormal cell morphology during lactation. Both primary mammary tumor formation and metastasis were markedly enhanced in LMW cyclin E transgenic mice. LMW cyclin E overexpression in mammary epithelial cells of mice is sufficient by itself to induce mammary adenocarcinomas in 34 of 124 (27%) animals compared with 7 of 67 (10.4%) mice expressing only the full-length cyclin E (P < 0.05). In addition, metastasis was seen in 25% of LMW cyclin E tumor-bearing animals compared with only 8.3% of tumors in the full-length cyclin E background (P < 0.05). Moreover, LMW cyclin E overexpression selects for inactivation of p53 by loss of heterozygosity and spontaneous and frequent inactivation of ARF. Therefore, LMW cyclin E overexpression strongly selects for spontaneous inactivation of the ARF-p53 pathway in vivo, canceling its protective checkpoint function and accelerating progression to malignancy.     

Murine embryonic fibroblasts lacking TC-PTP display delayed G1 phase through defective NF-kappaB activation

Previous results suggested a potential role for T-cell protein tyrosine phosphatase (TC-PTP) in cell proliferation. However, no conclusive data has supported such a function in the modulation of this process. In order to clarify this issue, we isolated TC-PTP-/- murine embryonic fibroblasts (MEFs) as well as cell lines to characterize the role of TC-PTP in the control of cell proliferation and cell cycle. Both TC-PTP-/- primary MEFs and cell lines proliferate slower than TC-PTP+/+ cells. We also demonstrated that TC-PTP-/- cells have a slow progression through the G1 phase of the cell cycle. Further characterization of the G1 defect indicates that the kinetics of cyclin D1 induction was delayed and that p27(KIP1) remains at higher levels for an extended period of time. Moreover, cells lacking TC-PTP showed a delayed activation of CDK2. This slow progression through the early G1-phase resulted in decreased phosphorylation of the RB protein and subsequent delay into the S phase transition. In contrast, no further defects were detected in other phases of the cell cycle. Survey of the potential signaling pathways leading to this delayed cyclin D1 expression indicated that NF-kappaB activation was compromised and that IKKbeta activity was also reduced following PDGF stimulation. Reintroduction of wild-type TC-PTP into the TC-PTP-/- cells rescued the defective proliferation, cyclin D1 expression, NF-kappaB activation as well as IkappaB phosphorylation. Together, these results confirm that TC-PTP plays a positive role in the progression of early G1 phase of the cell cycle through the NF-kappaB pathway.