Overexpression of cyclin D1 enhances taxol induced mitotic death in MCF7 cells

Treatment of MCF7 human breast cancer cells with taxol induces G2/M arrest followed by mitotic death. A moderate overexpression of ectopic cyclin D1 accelerated these G2/M associated events and resulted in a reduced clonogenic survival upon taxol treatment. Taxol treatment resulted in elevated expression of p53 and of p21, which was more pronounced and persistent in cyclin D1 overexpressing cells. Overexpression of cyclin D1 altered sensitivity to taxol by modulating exit from mitosis, which is controlled by p21. These results indicate that overexpression of cyclin D1 sensitizes MCF7 cells to treatment with taxol.     

Study of the G2/M cell cycle checkpoint in irradiated mammary epithelial cells overexpressing Cul-4A gene

PURPOSE: Members of the cullin gene family are known to be involved in cell cycle control. One of the cullin genes, Cul-4A, is amplified and overexpressed in breast cancer cells. This study investigates the effect of Cul-4A overexpression upon G2/M cell cycle checkpoint after DNA damage induced by either ionizing or nonionizing radiation. METHODS AND MATERIALS: The normal mammary epithelial cell line MCF10A was stably transfected with full-length Cul-4A cDNA. Independent clones of MCF10A cells that overexpress Cul-4A proteins were selected and treated with either 8 Gy of ionizing radiation or 7 J/M(2) of UV radiation. The profile of cell cycle progression and the accumulation of several cell cycle proteins were analyzed. RESULTS: We found that overexpression of Cul-4A in MCF10A cells abrogated the G2/M cell cycle checkpoint in response to DNA damage induced by ionizing irradiation, but not to DNA damage induced by nonionizing radiation. Analysis of cell cycle proteins showed that after ionizing irradiation, p53 accumulated in the mock-transfected MCF10A cells, but not in the Cul-4A transfectants. CONCLUSION: Our results suggest a role for Cul-4A in tumorigenesis and/or tumor progression, possibly through disruption of cell cycle control.     

Radiation and the Apo2L/TRAIL apoptotic pathway preferentially inhibit the colonization of premalignant human breast cells overexpressing cyclin D1

The role of cyclin D1 overexpression in human breast premalignancy was investigated using immortal, nontumorigenic MCF-10A cells. Previous work documented that cyclin D1 overexpression promoted in vitro anchorage-independent colonization. We now report that the colonization of MCF-10A cyclin D1 transfectants was preferentially inhibited by gamma-radiation and specific classes of apoptosis inducers [Apo-2 ligand (Apo-2L), but not tumor necrosis factor alpha]. Antibody inhibition studies and semiquantitative PCR indicated that radiation inhibition of colonization was partially mediated via the Apo2L/TRAIL pathway. The apoptotic removal of cyclin D1-overexpressing, colonization-competent premalignant breast cells by Apo2L/TRAIL or other biologicals may represent a novel approach to the prevention of breast cancer.     

HuR contributes to cyclin E1 deregulation in MCF-7 breast cancer cells

Many cancers overexpress cyclin E1 and its tumor-specific low molecular weight (LMW) isoforms. However, the mechanism of cyclin E1 deregulation in cancers is still not well understood. We show here that the mRNA-binding protein HuR increases cyclin E1 mRNA stability in MCF-7 breast carcinoma cells. Thus, mRNA stabilization may be a key event in the deregulation of cyclin E1 in MCF-7 cells. Compared with MCF10A immortalized breast epithelial cells, MCF-7 cells overexpress full-length cyclin E1 and its LMW isoforms and exhibit increased cyclin E1 mRNA stability. Increased mRNA stability is associated with a stable adenylation state and an increased ratio of cytoplasmic versus nuclear HuR. UV cross-link competition and UV cross-link immunoprecipitation assays verified that HuR specifically bound to the cyclin E1 3'-untranslated region. Knockdown of HuR with small interfering RNA (siRNA) in MCF-7 cells decreased cyclin E1 mRNA half-life (t(1/2)) and its protein level: a 22% decrease for the full-length isoforms and 80% decrease for the LMW isoforms. HuR siRNA also delayed G(1)-S phase transition and inhibited MCF-7 cell proliferation, which was partially recovered by overexpression of a LMW isoform of cyclin E1. Overexpression of HuR in MCF10A cells increased cyclin E1 mRNA t(1/2) and its protein level. Taken together, our data show that HuR critically contributes to cyclin E1 overexpression and its growth-promoting function, at least in part by increasing cyclin E1 mRNA stability, which provides a new mechanism of cyclin E1 deregulation in breast cancer.     

CDK4 inhibition and doxorubicin mediate breast cancer cell apoptosis through Smad3 and survivin

Cyclin D1/CDK4 activity is upregulated in up to 50% of breast cancers and CDK4-mediated phosphorylation negatively regulates the TGFβ superfamily member Smad3. We sought to determine if CDK4 inhibition and doxorubicin chemotherapy could impact Smad3-mediated cell/colony growth and apoptosis in breast cancer cells. Parental and cyclin D1-overexpressing MCF7 cells were treated with CDK4 inhibitor, doxorubicin, or combination therapy and cell proliferation, apoptosis, colony formation, and expression of apoptotic proteins were evaluated using an MTS assay, TUNEL staining, 3D Matrigel assay, and apoptosis array/immunoblotting. Study cells were also transduced with WT Smad3 or a Smad3 construct resistant to CDK4 phosphorylation (5M) and colony formation and expression of apoptotic proteins were assessed. Treatment with CDK4 inhibitor/doxorubicin combination therapy, or transduction with 5M Smad3, resulted in a similar decrease in colony formation. Treating cyclin D overexpressing breast cancer cells with combination therapy also resulted in the greatest increase in apoptosis, resulted in decreased expression of anti-apoptotic proteins survivin and XIAP, and impacted subcellular localization of pro-apoptotic Smac/DIABLO. Additionally, transduction of 5M Smad3 and doxorubicin treatment resulted in the greatest change in apoptotic protein expression. Collectively, this work showed the impact of CDK4 inhibitor-mediated, Smad3-regulated tumor suppression, which was augmented in doxorubicin-treated cyclin D-overexpressing study cells.     

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。     

Tissue-specific consequences of cyclin D1 overexpression in prostate cancer progression

The cyclin D1 oncogene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the Rb protein and promotes progression through G(1) to S phase of the cell cycle. Several prostate cancer cell lines and a subset of primary prostate cancer samples have increased cyclin D1 protein expression. However, the relationship between cyclin D1 expression and prostate tumor progression has yet to be clearly characterized. This study examined the effects of manipulating cyclin D1 expression in either human prostatic epithelial or stromal cells using a tissue recombination model. The data showed that overexpression of cyclin D1 in the initiated BPH-1 cell line increased cell proliferation rate but did not elicit tumorigenicity in vivo. However, overexpression of cyclin D1 in normal prostate fibroblasts (NPF) that were subsequently recombined with BPH-1 did induce malignant transformation of the epithelial cells. The present study also showed that recombination of BPH-1 + cyclin D1-overexpressing fibroblasts (NPF(cyclin D1)) resulted in permanent malignant transformation of epithelial cells (BPH-1(NPF-cyclin D1) cells) similar to that seen with carcinoma-associated fibroblasts (CAF). Microarray analysis showed that the expression profiles between CAFs and NPF(cyclin D1) cells were highly concordant including cyclin D1 up-regulation. These data indicated that the tumor-promoting activity of cyclin D1 may be tissue specific.     

Overexpression of cyclin D1 promotes tumor cell growth and confers resistance to cisplatin-mediated apoptosis in an elastase-myc transgene-expressing pancreatic tumor cell line.

PURPOSE: Elevated cyclin D1 in human pancreatic cancer correlates with poor prognosis. Because pancreatic cancer is invariably resistant to chemotherapy, the goal of this study was to examine whether the drug resistance of pancreatic cancer cells is in part attributed to cyclin D1 overexpression. EXPERIMENTAL DESIGN: Stable overexpression and small interfering RNA (siRNA)--mediated knockdown of cyclin D1 were done in the newly established Ela-myc pancreatic tumor cell line. Cisplatin sensitivity of control, overexpressing, and siRNA-transfected cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, clonogenic, and apoptotic assays [DNA fragmentation, sub-G1, and poly(ADP-ribose) polymerase cleavage analysis]. The role of nuclear factor-kappaB and apoptotic proteins in cyclin D1-mediated chemoresistance was examined by EMSA and Western blotting, respectively. RESULTS: Overexpression of cyclin D1 in Ela-myc pancreatic tumor cells promoted cell proliferation and anchorage-independent growth. Moreover, cyclin D1-overexpressing cells exhibited significantly reduced chemosensitivity and a higher survival rate upon cisplatin treatment, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic assays, respectively. Although overexpression of cyclin D1 rendered cells more resistant to cisplatin-induced apoptosis, siRNA-directed suppression of cyclin D1 expression resulted in enhanced susceptibility to cisplatin-mediated apoptosis. The attenuation of cisplatin-induced cell death in cyclin D1-overexpressing cells was correlated with the up-regulation of nuclear factor-kappaB activity and maintenance of bcl-2 and bcl-xl protein levels. CONCLUSIONS: These results suggest that overexpression of cyclin D1 can contribute to chemoresistance of pancreatic cancer cells because of the dual roles of cyclin D1 in promoting cell proliferation and in inhibiting drug-induced apoptosis.     

槲皮素对人乳腺癌裸鼠移植瘤细胞周期的影响

 目的　研究槲皮素对人乳腺癌细胞株MCF 7裸鼠移植瘤细胞周期的影响。方法　 2 4只MCF 7细胞株移植成功的裸鼠分为对照组、槲皮素组、阿霉素组及联合用药组 ,每组 6只。用流式细胞仪分析细胞周期分布 ,用免疫组化测定cyclinD1表达水平。结果　G0 /G1期占细胞周期比例对照组明显低于槲皮素组 ,阿霉素组及联合用药组 (P <0 .0 1) ,S期占细胞周期比例对照组明显高于槲皮素组 ,阿霉素组及联合用药组 (P <0 .0 1) ,cyclinD1表达水平对照组明显高于槲皮素组、阿霉素组及联合用药组 (P <0 .0 1)。结论　槲皮素可作用于MCF 7移植瘤的G1/S节点 ,可抑制移植瘤细胞增殖及cyclinD1表达 ,延缓肿瘤生长。     

Constitutive overexpression of cyclin D1 but not cyclin E confers acute resistance to antiestrogens in T-47D breast cancer cells

Cyclin D1 and cyclin E are overexpressed in approximately 45% and 30% of breast cancers, respectively, and adverse associations with patient outcome have been reported. The potential roles of cyclin D1 and cyclin E expression as markers of therapeutic responsiveness to the pure steroidal antiestrogen ICI 182780 were investigated using T-47D breast cancer cell lines constitutively overexpressing cyclin D1 or cyclin E. Measurement of S phase fraction, phosphorylation states of the retinoblastoma protein, and cyclin E-cyclin-dependent kinase (Cdk) 2 activity demonstrated that overexpression of cyclin D1 decreased sensitivity to antiestrogen inhibition at 24 and 48 h. Overexpression of cyclin E produced a less pronounced early cell cycle effect indicating only partial resistance to antiestrogen inhibition in the short-term. In ICI 182780-treated cyclin D1-overexpressing cells, sufficient Cdk activity was retained to allow retinoblastoma protein phosphorylation and cell proliferation, despite an increase in the association of p21 and p27 with cyclin D1-Cdk4/6 and cyclin E-Cdk2 complexes. After longer-term (>7 days) treatment, antiestrogens inhibited colony growth in cyclin D1- or cyclin E-overexpressing breast cancer cells, but with an approximately 2-2.5-fold decrease in dose sensitivity. This was associated with a fall in cyclin D1 levels, a reduction in the half-life of cyclin D1 protein and a decline in cyclin E-Cdk2 activity in cyclin D1-overexpressing cells, and the maintenance of cyclin E-p27 association in the cyclin E-overexpressing cells. These data confirm that cyclin D1 expression and cyclin E-p27 association play important roles in antiestrogen action, and suggest that cyclin D1 or cyclin E overexpression has subtle effects on antiestrogen sensitivity. Additional studies to elucidate the contribution of alterations in cyclin D1 stability to antiestrogen action and to assess the relationship between antiestrogen sensitivity and expression of cyclin D1, cyclin E, or p27 in a clinical setting are required.     

The role of peroxiredoxin II in radiation-resistant MCF-7 breast cancer cells

Although several signaling pathways have been suggested to be involved in the cellular response to ionizing radiation, the molecular basis of tumor resistance to radiation remains elusive. We have developed a unique model system based upon the MCF-7 human breast cancer cell line that became resistant to radiation treatment (MCF+FIR30) after exposure to chronic ionizing radiation. By proteomics analysis, we found that peroxiredoxin II (PrxII), a member of a family of peroxidases, is up-regulated in the radiation-derived MCF+FIR3 cells but not in the MCF+FIS4 cells that are relatively sensitive to radiation. Both MCF+FIR3 and MCF+FIS4 cell lines are from MCF+FIR30 populations. Furthermore, the resistance to ionizing radiation can be partially reversed by silencing the expression of PrxII by PrxII/small interfering RNA treatment of MCF+FIR3 resistant cells, suggesting that PrxII is not the sole factor responsible for the resistant phenotype. The relevance of this mechanism was further confirmed by the increased radioresistance in PrxII-overexpressing MCF+FIS4 cells when compared with vector control cells. The up-regulation of the PrxII protein in radioresistant cancer cells suggested that human peroxiredoxin plays an important role in eliminating the generation of reactive oxygen species by ionizing radiation. The present finding, together with the observation that PrxII is also up-regulated in response to ionizing radiation in other cell systems, strengthens the hypothesis that the PrxII antioxidant protein is involved in the cellular response to ionizing radiation and functions to reduce the intracellular reactive oxygen species levels, resulting in increased resistance of breast cancer cells to ionizing radiation.     

Cold‐inducible RNA‐binding protein contributes to human antigen R and cyclin E1 deregulation in breast cancer

The cell cycle regulator cyclin E1 is aberrantly expressed in a variety of human cancers. In breast cancer, elevated cyclin E1 correlates with poor outcome, as do high cytoplasmic levels of the stress‐induced RNA‐binding protein human antigen R (HuR). We showed previously that increased cytoplasmic HuR elevates cyclin E1 in MCF‐7 breast cancer cells by stabilizing its mRNA. We show here that cold‐inducible RNA‐binding protein (CIRP) co‐regulates cyclin E1 with HuR in breast cancer cells. CIRP had been shown to interact with HuR in Xenopus laevis oocytes and to be decreased in endometrial cancer. To investigate if human CIRP and HuR co‐regulate cyclin E1, HuR and CIRP levels were altered in MCF‐7 cells and effects on cyclin E1 assessed. Altering HuR expression resulted in a reciprocal change in CIRP expression, while altering CIRP expression resulted in corresponding changes in HuR and cyclin E1 expression. CIRP and HuR co‐precipitated in the presence of RNA and CIRP enhanced HuR binding to the cyclin E1 mRNA and increased cyclin E1 mRNA stability. CIRP co‐localized with HuR predominantly in the nucleus, but also in discrete cytoplasmic foci identified as stress granules (SGs). CIRP overexpression increased the number of HuR‐containing SGs, while its knockdown decreased them. Our results suggest that CIRP positively regulates HuR, ultimately resulting in increased protein synthesis of at least one of its targets. © 2009 Wiley‐Liss, Inc.     

Differential regulation of cdk2 and cyclin D1 in irradiated human glioma cells

We have utilized a series of glioma cell lines to study the effects of ionizing radiation on the regulation of proteins that contribute to cell cycle progression. While no alterations of cyclin E or cdk4 were detected, a high percentage of glioma cell lines exhibited constitutive overexpression of cdk2 protein and aberrant patterns of cyclin D1 protein. The fraction of glioma cells expressing cdk2 was similar to that observed in normal astrocytes, but individual glioma cells overexpressed cdk2. In response to ionizing radiation, both cyclin D1 and cdk2 accumulated in control cells but not in gliomas with overexpressed cdk2 or aberrant cyclin D1. These novel findings provide the first evidence of altered cyclin-cdk regulation in gliomas in response to ionizing radiation.     

Induction of cytostasis in mammary carcinoma cells treated with the anticancer agent perillyl alcohol.

The monoterpene perillyl alcohol (POH) has preventive and therapeutic effects in a wide variety of pre-clinical tumor models, including those for breast cancers, and is currently being tested in human phase I clinical trials. POH causes both cytostasis and apoptosis in rat mammary carcinomas. In vitro, POH inhibits cellular proliferation in a variety of mammalian cell lines. Here we investigated the mechanisms underlying cytostasis by studying the effects of POH on the cell cycle in vitro using the murine mammary transformed cell line TM6. In TM6 cells, POH causes an early G(1) cell-cycle block and slows the G(2)-M transition. An increase in pRB in its hypophosphorylated state is associated with the early G(1) block caused by POH. POH treatment inhibits two important targets in the cells during the G(1)-S transition: cyclin D1- and cyclin E-associated kinase. POH treatment leads to a reduction in cyclin D1 RNA and protein levels and prevents the formation of active cyclin D1-associated kinase complexes in synchronous cells during the exit of G(0) and entry into the cell cycle. In addition, POH treatment induces an increased association of p21(WAF1) with cyclin E-Cdk2 complexes, and inhibits the activating phosphorylation of Cdk2. All these effects of POH may contribute to the inhibition of the transition out of the G(1) phase of the cell cycle.