c-Myc suppresses p21WAF1/CIP1 expression during estrogen signaling and antiestrogen resistance in human breast cancer cells

Estrogen rapidly induces expression of the proto-oncogene c-myc. c-Myc is required for estrogen-stimulated proliferation of breast cancer cells, and deregulated c-Myc expression has been implicated in antiestrogen resistance. In this report, we investigate the mechanism(s) by which c-Myc mediates estrogen-stimulated proliferation and contributes to cell cycle progression in the presence of antiestrogen. The MCF-7 cell line is a model of estrogen-dependent, antiestrogen-sensitive human breast cancer. Using stable MCF-7 derivatives with inducible c-Myc expression, we demonstrated that in antiestrogen-treated cells, the elevated mRNA and protein levels of p21(WAF1/CIP1), a cell cycle inhibitor, decreased upon either c-Myc induction or estrogen treatment. Expression of p21 blocked c-Myc-mediated cell cycle progression in the presence of antiestrogen, suggesting that the decrease in p21 is necessary for this process. Using RNA interference to suppress c-Myc expression, we further established that c-Myc is required for estrogen-mediated decreases in p21(WAF1/CIP1). Finally, we observed that neither c-Myc nor p21(WAF1/CIP1) is regulated by estrogen or antiestrogen in an antiestrogen-resistant MCF-7 derivative. The p21 levels in the antiestrogen-resistant cells increased when c-Myc expression was suppressed, suggesting that loss of p21 regulation was a consequence of constitutive c-Myc expression. Together, these studies implicate p21(WAF1/CIP1) as an important target of c-Myc in breast cancer cells and provide a link between estrogen, c-Myc, and the cell cycle machinery. They further suggest that aberrant c-Myc expression, which is frequently observed in human breast cancers, can contribute to antiestrogen resistance by altering p21(WAF1/CIP1) regulation.     

The cyclin-dependent kinase inhibitor p21CIP/WAF is a positive regulator of insulin-like growth factor I-induced cell proliferation in MCF-7 human breast cancer cells

To study the role of IGF-I receptor signaling on cell cycle events we utilized MCF-7 breast cancer cells. IGF-I at physiological concentrations increased the level of p21CIP/WAF mRNA after 4has well as protein after 8hby 10- and 6-fold, respectively, in MCF-7 cells. This IGF-1 effect was reduced by 50% in MCF-7-derived cells (SX13), which exhibit a 50% reduction in IGF-1R expression, demonstrating that IGF-1 receptor activation was involved in this process. Preincubation with the ERK1/2 inhibitor U0126 significantly reduced the IGF-1 effect on the amount of p21CIP/WAF protein in MCF-7 cells. These results were confirmed by the expression of a dominant negative construct for MEK-1 suggesting that the increase of the abundance of p21CIP/WAF in response to IGF-1 occurs via the ERK1/2 mitogen-activated protein kinase pathway. Using an antisense strategy, we demonstrated that abolition of p21CIP/WAF expression decreased by 2-fold the IGF-1 effect on cell proliferation in MCF-7. This latter result is explained by a delay in G1 to S cell cycle progression due partly to a reduction in the activation of some components of cell cycle including the induction of cyclin D1 expression in response to IGF-1. MCF-7 cells transiently overexpressing p21 showed increased basal and IGF-I-induced thymidine incorporation. Taken together, these results define p21CIP/WAF as a positive regulator in the cell proliferation induced by IGF-1 in MCF-7 cells.     

Helicobacter pylori releases a factor(s) inhibiting cell cycle progression of human gastric cell lines by affecting cyclin E/cdk2 kinase activity and Rb protein phosphorylation through enhanced p27(KIP1) protein expression

Helicobacter pylori, the main cause of chronic gastritis, plays a central role in the etiology of peptic ulcer disease and gastric cancer. In vitro studies have shown that H. pylori increases gastric epithelial cell turnover, thus increasing the risk for the development of neoplastic clones. The mechanisms by which H. pylori promotes perturbation of cell proliferation are not yet elucidated. To investigate whether products released by H. pylori in culture media interfere with cell cycle progression of human gastric epithelial cells, four cell lines (MKN 28, MKN 7, MKN 74, and AGS) were incubated in the presence of H. pylori broth culture filtrate. Cell cycle analysis showed that a H. pylori-released factor(s) significantly inhibited the G1- to S-phase progression of MKN 28 and MKN 7 cell lines, with a reversible, nonlethal mechanism, independent of the expression of VacA, CagA, and/or urease. The cell cycle inhibition occurred concomitantly with an increase in p27(KIP1) protein levels, a reduction in Rb protein phosphorylation on serine residues 807-811, and a significant decrease in cyclin E-associated cdk2 activity. In contrast, the cell cycle progression of MKN 74 and AGS cell lines was not affected by the H. pylori-released factor(s). In normal human fibroblasts, G1-phase cell accumulation was concomitant with the reduction in Rb protein phosphorylation; that, however, appeared to be dependent on p21(WAF1/CIP1) rather than on p27(KIP1) protein. A preliminary characterization showed that the molecular mass of the partially purified cell cycle inhibitory factor(s) was approximately 40 kDa. These results suggest that H. pylori releases a soluble factor(s) that may affect cell cycle progression of gastric epithelial cells through elevated levels of cdk inhibitor p27(KIP1). This factor(s) might act in vivo on noncolonized distant cells, the most proliferating cells of human gastric mucosa.     

Changes in BRCA1 and BRCA2 expression produced by chemotherapeutic agents in human breast cancer cells

We have investigated the effects of chemotherapeutic agents such as adriamycin (ADR), camptothecin (CPT), mitomycin-C (MYC-C) and methotrexate (MTX) on the regulation of expression of the tumor susceptibility genes (BRCA1 and BRCA2), and the association of cell cycle progression in human breast cancer and normal breast epithelial cells. Results revealed that the mRNA and protein expression levels of BRCA1/2 were reduced by the treatment of chemotherapeutic agents used in the breast cancer cell lines tested, with ADR being the most effective. The regulation of the cell cycle was dose-dependent and low doses of ADR (1.5 microM) induced G2/M phase arrest whereas a late S phase arrest was observed with a higher dose of ADR (15 microM) in both breast cancer cells (MCF-7 and MDA-MB-231) tested. In addition, a negative correlation was observed between BRCA1/2 mRNA and expressions of the proteins with the cell cycle alterations being regulated by chemotherapeutic agents.     

乳腺癌中Rho A蛋白表达及其与Cyclin D1和P21WAF1/CIP1蛋白表达的相关性

目的探讨Rho A蛋白在人乳腺癌中的表达情况,Rho A蛋白与临床病理因素的关系,及其与细胞周期蛋白Cyclin D1,细胞周期抑制蛋白 P21 WAF1/CIP1表达的相关性.方法应用免疫组化S-P法,检测64例乳腺癌组织及20例正常乳腺组织中Rho A蛋白、Cyclin D1和P21 WAF1/CIP1蛋白的表达情况.结果 (1)Rho A、 Cyclin D1和P21 WAF1/CIP1蛋白在正常乳腺组织中的表达率分别为5.00%、25.00%、15.00%,在乳腺癌组织中的表达率分别为73.44%、59.38%、48.44%,三者在乳腺癌组织中的阳性表达分别与正常乳腺组织相比,均差异有显著性意义(P< 0.01).(2)Rho A蛋白表达与病理组织分级,淋巴结转移相关(P< 0.05),与患者年龄、肿瘤大小及临床分期无关.(3)RhoA蛋白与P21 WAF1/CIP1蛋白表达呈负相关(χ2=4.548,P<0.05),与Cyclin D1蛋白表达无关.结论乳腺癌患者RhoA蛋白过表达与预后不良有关.RhoA蛋白通过下调P21 WAF1/CIP1蛋白参与细胞周期调节,进而与乳腺癌发展及侵袭转移相关.     

Low doses of EPO activate MAP kinases but not JAK2-STAT5 in rat vascular smooth muscle cells.

Previous reports have shown a direct effect of erythropoietin (Epo) on vascular smooth muscle cells (VSMCs). Our aim was to assess expression of the Epo receptor (EpoR) on VSMCs and to study the activation of two major signaling cascades activated by Epo, namely JAK2/STAT5 and MAPK pathways. All experiments were performed in parallel using the Epo-responsive UT7 cell line. From semiquantitative RT-PCR experiments, VSMCs were estimated to express approximately 30-fold less EpoR mRNA than UT7 cells. Epo-induced phosphorylation of proteins involved in the EpoR/JAK2/STAT5 cascade could not be detected in VSMCs, even using pharmacological doses of Epo (250 IU/ml). In contrast, a strong activation of MAP kinase pathway was detected with as low as 10 IU/ml Epo. We suggest that MAPK activation reflects a physiologically relevant effect of Epo on VSMCs that may be correlated to cell proliferation.     

Coupling of heterotrimeric Gi proteins to the erythropoietin receptor

To identify new proteins involved in erythropoietin (Epo) signal transduction, we purified the entire set of proteins reactive with anti-phosphotyrosine antibodies from Epo-stimulated UT7 cells. Antisera generated against these proteins were used to screen a lambdaEXlox expression library. One of the isolated cDNAs encodes Gbeta2, the beta2 subunit of heterotrimeric GTP-binding proteins. Gbeta and Galpha(i) coprecipitated with the Epo receptor (EpoR) in extracts from human and murine cell lines and from normal human erythroid progenitor cells. In addition, in vitro Gbeta associated with a fusion protein containing the intracellular domain of the EpoR. Using EpoR mutants, we found that the distal part of the EpoR (between amino acids 459-479) was required for Gi binding. Epo activation of these cells induced the release of the Gi protein from the EpoR. Moreover in isolated cell membranes, Epo treatment inhibited ADP-ribosylation of Gi and increased the binding of GTP. Our results show that heterotrimeric Gi proteins associate with the C-terminal end of the EpoR. Receptor activation leads to the activation and dissociation of Gi from the receptor, suggesting a functional role of Gi protein in Epo signal transduction.     

Identification and characterization of mechanism of action of P61-E7, a novel phosphine catalysis-based inhibitor of geranylgeranyltransferase-I

Small molecule inhibitors of protein geranylgeranyltransferase-I (GGTase-I) provide a promising type of anticancer drugs. Here, we first report the identification of a novel tetrahydropyridine scaffold compound, P61-E7, and define effects of this compound on pancreatic cancer cells. P61-E7 was identified from a library of allenoate-derived compounds made through phosphine-catalyzed annulation reactions. P61-E7 inhibits protein geranylgeranylation and blocks membrane association of geranylgeranylated proteins. P61-E7 is effective at inhibiting both cell proliferation and cell cycle progression, and it induces high p21(CIP1/WAF1) level in human cancer cells. P61-E7 also increases p27(Kip1) protein level and inhibits phosphorylation of p27(Kip1) on Thr187. We also report that P61-E7 treatment of Panc-1 cells causes cell rounding, disrupts actin cytoskeleton organization, abolishes focal adhesion assembly and inhibits anchorage independent growth. Because the cellular effects observed pointed to the involvement of RhoA, a geranylgeranylated small GTPase protein shown to influence a number of cellular processes including actin stress fiber organization, cell adhesion and cell proliferation, we have evaluated the significance of the inhibition of RhoA geranylgeranylation on the cellular effects of inhibitors of GGTase-I (GGTIs). Stable expression of farnesylated RhoA mutant (RhoA-F) results in partial resistance to the anti-proliferative effect of P61-E7 and prevents induction of p21(CIP1/WAF1) and p27(Kip1) by P61-E7 in Panc-1 cells. Moreover, stable expression of RhoA-F rescues Panc-1 cells from cell rounding and inhibition of focal adhesion formation caused by P61-E7. Taken together, these findings suggest that P61-E7 is a promising GGTI compound and that RhoA is an important target of P61-E7 in Panc-1 pancreatic cancer cells.     

Induction of cell cycle arrest and apoptosis in HT-29 human colon cancer cells by the dietary compound luteolin

Luteolin is 3',4',5,7-tetrahydroxyflavone found in celery, green pepper, and perilla leaf that inhibits tumorigenesis in animal models. We examined luteolin-mediated regulation of cell cycle progression and apoptosis in the HT-29 human colon cancer cell line. Luteolin decreased DNA synthesis and viable HT-29 cell numbers in a concentration-dependent manner. It inhibited cyclin-dependent kinase (CDK)4 and CDK2 activity, resulting in G1 arrest with a concomitant decrease of phosphorylation of retinoblastoma protein. Activities of CDK4 and CDK2 decreased within 2 h after luteolin treatment, with a 38% decrease in CDK2 activity (P < 0.05) observed in cells treated with 40 micromol/l luteolin. Luteolin inhibited CDK2 activity in a cell-free system, suggesting that it directly inhibits CDK2. Cyclin D1 levels decreased after luteolin treatment, although no changes in expression of cyclin A, cyclin E, CDK4, or CDK2 were detected. Luteolin also promoted G2/M arrest at 24 h posttreatment by downregulating cyclin B1 expression and inhibiting cell division cycle (CDC)2 activity. Luteolin promoted apoptosis with increased activation of caspases 3, 7, and 9 and enhanced poly(ADP-ribose) polymerase cleavage and decreased expression of p21(CIP1/WAF1), survivin, Mcl-1, Bcl-x(L), and Mdm-2. Decreased expression of these key antiapoptotic proteins could contribute to the increase in p53-independent apoptosis that was observed in HT-29 cells. We demonstrate that luteolin promotes both cell cycle arrest and apoptosis in the HT-29 colon cancer cell line, providing insight about the mechanisms underlying its antitumorigenic activities.     

MicroRNA-218 Inhibits Cell Cycle Progression and Promotes Apoptosis in Colon Cancer by Downregulating BMI1 Polycomb Ring Finger Oncogene

Deregulated miRNAs participate in colorectal carcinogenesis. In this study, miR-218 was found to be downregulated in human colorectal cancer (CRC) by miRNA profile assay. miR-218 was silenced or downregulated in all five colon cancer cells (Caco2, HT29, SW620, HCT116 and LoVo) relative to normal colon tissues. miR-218 expression was significantly lower in 46 CRC tumor tissues compared with their adjacent normal tissues (P < 0.001). Potential target genes of miR-218 were predicted and BMI1 polycomb ring finger oncogene (BMI-1), a polycomb ring finger oncogene, was identified as one of the potential targets. Upregulation of BMI-1 was detected in CRC tumors compared with adjacent normal tissues (P < 0.001) and in all five colon cancer cell lines. Transfection of miR-218 in colon cancer cell lines (HCT116, HT29) significantly reduced luciferase activity of the wild-type construct of BMI-1 3′ untranslated region (3′UTR) (P < 0.001), whereas this effect was not seen in the construct with mutant BMI-1 3′UTR, indicating a direct and specific interaction of miR-218 with BMI-1. Ectopic expression of miR-218 in HCT116 and HT29 cells suppressed BMI-1 mRNA and protein expression. In addition, miR-218 suppressed protein expression of BMI-1 downstream targets of cyclin-dependent kinase 4, a cell cycle regulator, while upregulating protein expression of p53. We further revealed that miR-218 induced apoptosis (P < 0.01), inhibited cell proliferation (P < 0.05) and promoted cell cycle arrest in the G2 phase (P < 0.01). In conclusion, miR-218 plays a pivotal role in CRC development through inhibiting cell proliferation and cycle progression and promoting apoptosis by downregulating BMI-1.     

Cell Death of MCF-7 Human Breast Cancer Cells Induced by EGFR Activation in the Absence of Other Growth Factors

The Epidermal Growth Factor (EGF) Receptor (EGFR) plays an important role in the growth and progression of breast cancer. Overexpression of EGFR or the high activity of EGFR signal pathway has been related with increases in cell proliferation and a poor prognosis in patients with breast cancer. Several human breast cancer cell lines depend on estrogen for their proliferation. EGF may bypass the requirement of estrogen for the proliferation of breast cancer cells. To evaluate this hypothesis, MCF-7 breast cancer cells were stimulated with EGF and the effects on cell proliferation, signal pathways, and cell cycle progression were determined. The results demonstrate that EGF stimulation in the absence of others growth factors induced a modest effect on cell proliferation and the induction of a cellular arrest in the G1 phase of the cell cycle. Although phosphorylation of AKT and ERK proteins were detected, this phosphorylation was insufficient to support of cell cycle progression. Cellular arrest in G1 phase was accompanied by an increase in p21CIP1 protein, down regulation of the BCL-2 protein, induction of caspase-8, and ARHI/NOEY2 an imprinted tumor suppressor gene. These results indicate that EGFR activation by itself is not sufficient for the proliferation of breast cancer cells and suggest the existence of a mechanism that induces apoptosis upon EGFR activation.     

c‐FLIP is involved in erythropoietin‐mediated protection of erythroid‐differentiated cells from TNF‐α‐induced apoptosis

The TNF‐α (tumour necrosis factor) affects a wide range of biological activities, such as cell proliferation and apoptosis. Cell life or death responses to this cytokine might depend on cell conditions. This study focused on the modulation of factors that would affect the sensitivity of erythroid‐differentiated cells to TNF‐α. Hemin‐differentiated K562 cells showed higher sensitivity to TNF‐induced apoptosis than undifferentiated cells. At the same time, hemin‐induced erythroid differentiation reduced c‐FLIP (cellular FLICE‐inhibitory protein) expression. However, this negative effect was prevented by prior treatment with Epo (erythropoietin), which allowed the cell line to maintain c‐FLIP levels. On the other hand, erythroid‐differentiated UT‐7 cells – dependent on Epo for survival – showed resistance to TNF‐α pro‐apoptotic action. Only after the inhibition of PI3K (phosphatidylinositol‐3 kinase)‐mediated pathways, which was accompanied by negative c‐FLIP modulation and increased erythroid differentiation, were UT‐7 cells sensitive to TNF‐α‐triggered apoptosis. In summary, erythroid differentiation might deregulate the balance between growth promotion and death signals induced by TNF‐α, depending on cell type and environmental conditions. The role of c‐FLIP seemed to be critical in the protection of erythroid‐differentiated cells from apoptosis or in the determination of their sensitivity to TNF‐mediated programmed cell death. Epo, which for the first time was found to be involved in the prevention of c‐FLIP down‐regulation, proved to have an anti‐apoptotic effect against the pro‐inflammatory factor. The identification of signals related to cell life/death switching would have significant implications in the control of proliferative diseases and would contribute to the understanding of mechanisms underlying the anaemia associated with inflammatory processes.