LATS1 tumor suppressor regulates G2/M transition and apoptosis

The LATS1 gene is a mammalian member of the novel lats tumor suppressor family. Both lats mosaic flies and LATS1 deficient mice spontaneously develop tumors. Our previous studies have shown that inactivation of Drosophila lats leads to up-regulation of cyclin A in the fly, and the human LATS1 protein associates with CDC2 in early mitosis in HeLa cells, suggesting that the lats gene family may negatively regulate cell proliferation by modulating CDC2/Cyclin A activity. We demonstrate here that transduction of the human breast cancer cell MCF-7 with recombinant LATS1 adenovirus (Ad-LATS1), but not with EGFP adenovirus (Ad-EGFP), inhibits in vitro cell proliferation. Ectopic expression of LATS1 in MCF-7 cells specifically down-regulates Cyclin A and Cyclin B protein levels and dramatically reduces CDC2 kinase activity, leading to a G2/M blockade. Furthermore, Ad-LATS1 suppresses anchorage-independent growth of MCF-7 cells in soft agar and tumor formation in athymic nude mice. We also demonstrate that ectopic expression of LATS1 in MCF-7 cells and human lung cancer cell H460 up-regulates the level of BAX proteins and induces apoptosis. Finally, we show that LATS1 kinase activity is required for its ability to inhibit cell growth and induce apoptosis. The results indicate that the LATS1 tumor suppressor may play an important role in the control of human tumor development and that LATS1 suppresses tumorigenesis by negatively regulating cell proliferation and modulating cell survival.     

Double-stranded RNA-dependent protein kinase, PKR, down-regulates CDC2/cyclin B1 and induces apoptosis in non-transformed but not in v-mos transformed cells.

The interferon (IFN)-induced, double stranded RNA (dsRNA)-activated serine/threonine kinase, PKR, is a potent negative regulator of cell growth when overexpressed in yeast or mammalian cells. Paradoxically, while it can function as a tumor suppressor and inducer of apoptosis, it is overexpressed in a variety of human cancers. To resolve this enigma, we established cell-lines that overexpress PKR in non-transformed and in v-mos transformed CHO cells. Overexpression of PKR suppressed the proliferation of CHO cells by inducing a transient G0/G1 arrest, followed by a delayed G2/M arrest, which attenuated cell cycle progression. These effects were accompanied by early induction of p21/WAF-1 and delayed downregulation of CDC2 and cyclin B1. Induction of proapoptotic activity of the ectopic PKR paralleled the onset of G2/M arrest in CHO cells. However, while transiently inducing p21/WAF-1, PKR did not impose G2/M arrest or apoptosis in v-mos-transformed cells, nor was CDC2 or cyclin B1 down-regulated in those cells. These findings link the proapoptotic activity of PKR to the arrest of cell cycle at the G2/M phase. Consequently, the apoptotic activity of PKR could be counter-acted by an oncogene-like v-mos that overrides the G2/M arrest induced by PKR.     

Phenethyl isothiocyanate (PEITC) promotes G2/M phase arrest via p53 expression and induces apoptosis through caspase- and mitochondria-dependent signaling pathways in human prostate cancer DU 145 cells

Phenethyl isothiocyanate (PEITC), one of many compounds found in cruciferous vegetables, has been reported as a potential anticancer agent. In earlier studies, PEITC was shown to inhibit cell growth and induction of apoptosis in many cancer cell lines. However, no report has shown whether PEITC can induce apoptosis in human prostate cancer cells. Herein, we aimed to determine whether PEITC has anticancer activity in DU 145 human prostate cancer cells. As a result, we found that PEITC induced a dose-dependent decrease in cell viability through induction of cell apoptosis and cell cycle arrest in the G(2)/M phase of DU 145 cells. PEITC induced morphological changes and DNA damage in DU 145 cells. The induction of G(2)/M phase arrest was mediated by the increase of p53 and WEE1 and it reduced the level of CDC25C protein. The induction of apoptosis was mediated by the activation of caspase-8-, caspase-9- and caspase-3-depedent pathways. Results also showed that PEITC caused mitochondrial dysfunction, increasing the release of cytochrome c and Endo G from mitochondria, and led cell apoptosis through a mitochondria-dependent signaling pathway. This study showed that PEITC might exhibit anticancer activity and become a potent agent for human prostate cancer cells in the future.     

Direct interaction with and activation of p53 by SMAR1 retards cell-cycle progression at G2/M phase and delays tumor growth in mice

The tumor-suppressor p53 is a multifunctional protein mainly responsible for maintaining genomic integrity. p53 induces its tumor-suppressor activity by either causing cell-cycle arrest (G(1)/S or G(2)/M) or inducing cells to undergo apoptosis. This function of wild-type p53 as "guardian of the genome" is presumably achieved by forming molecular complexes with different DNA targets as well as by interacting with a number of cellular proteins, e.g., Mdm2, Gadd45, p21, 14-3-3sigma, Bax and Apaf-1. Upon activation, p53 activates p21, which in turn controls the cell cycle by regulating G(1) or G(2) checkpoints. Here, we report SMAR1 as one such p53-interacting protein that is involved in delaying tumor progression in vivo as well as in regulating the cell cycle. SMAR1 is a newly identified MARBP involved in chromatin-mediated gene regulation. The SMAR1 gene encodes at least 2 alternatively spliced variants: SMAR1(L) (the full-length form) and SMAR1(S) (the shorter form). We report that expression of SMAR1(S), but not of SMAR1(L), mRNA was decreased in most of the human cell lines examined, suggesting selective silencing of SMAR1(S). Overexpression of SMAR1(S) in mouse melanoma cells (B16F1) and their subsequent injection in C57BL/6 mice delays tumor growth. Exogenous SMAR1(S) causes significant retardation of B16F1 cells in the G(2)/M phase of the cell cycle compared to SMAR1(L). SMAR1(S) activates p53-mediated reporter gene expression in mouse melanoma cells, breast cancer cells (MCF-7) and p53 null cells (K562), followed by activation of its downstream effector, p21. We further demonstrate that SMAR1 physically interacts and colocalizes with p53. These data together suggest that SMAR1 is the only known MARBP that delays tumor progression via direct activation and interaction with tumor-suppressor p53.     

Induction of apoptosis and G2/M cell cycle arrest by DCC.

The Deleted in Colorectal Cancer gene (DCC) encodes a cell surface receptor that belongs to the Ig superfamily. Inactivation of the DCC gene has been implicated in human tumor progression. However, little is known about the biological function of the DCC protein. In the present study, we demonstrated that expression of DCC activated caspase-3 and programmed cell death, or induced G2/M cell cycle arrest in tumor cells. In some cell lines, apoptosis was evident within 24 h of DCC expression. Timing of the appearance of apoptotic cells coincided with that of the cleavage of poly (ADP-ribose) polymerase, a substrate of caspase-3. Expression of the apoptosis inhibitory gene Bcl-2 was not able to abrogate the DCC-induced apoptosis. In the G2/M cycle arrest cells, cdk1 activity was inhibited. Our results suggest that the DCC protein may transduce signals resulting in activation of caspases or inhibition of Cdk1. These data provide a possible mechanism by which DCC suppresses tumorigenesis.     

Induction of cell cycle arrest and apoptosis in human breast cancer cells by quercetin

Quercetin, a widely distributed bioflavonoid, has been shown to induce growth inhibition in certain cancer cell types. In the present study we have pursued the mechanism of growth inhibition in MCF-7 human breast cancer cells. Quercetin treatment resulted in the accumulation of cells specifically at G2/M phase of the cell cycle. Mitotic index measured by MPM2 staining clearly showed that cells were transiently accumulated in M phase, 24 h after treatment. The transient M phase accumulation was accompanied by a transient increase in the levels of cyclin B1 and Cdc2 kinase activity. However, 24 h or longer treatment caused a marked accumulation of cells in G2 instead of M phase. Levels of cyclin B1 and cyclin B1-associated Cdc2 kinase activity were also decreased. We also found that quercetin markedly increased Cdk-inhibitor p21CIP1/WAF1 protein level after treatment for 48 h or longer, and the induction of p21CIP1/WAF1 increased its association with Cdc2-cyclin B1 complex, however, up-regulation of p53 by quercetin was not observed. Quercetin also induced significant apoptosis in MCF-7 cells in addition to cell cycle arrest, and the induction of apoptosis was markedly blocked by antisense p21CIP1/WAF1 expression. The present data, therefore, demonstrate that a flavonoid quercetin induces growth inhibition in the human breast carcinoma cell line MCF-7 through at least two different mechanisms; by inhibiting cell cycle progression through transient M phase accumulation and subsequent G2 arrest, and by inducing apoptosis.     

2-Methoxyestradiol-induced caspase-3 activation and apoptosis occurs through G(2)/M arrest dependent and independent pathways in gastric carcinoma cells

BACKGROUND: 2-Methoxyestradiol (2-Me), one of the estrogen metabolites, has recently been found to possess anti-angiogenesis activity in vivo. Many chemotherapeutic agents, such as taxol, docetaxel, and vinblastine, interact with microtubules and then induce apoptosis. It has been suggested that 2-Me acts on microtubules and results in G(2)/M-cycle arrest of tumor cells. Whether 2-Me induces apoptosis in gastric carcinoma cell lines is not known. Moreover, reactive oxygen species (ROS) produced by 2-Me may be involved in cytotoxicity of tumor cells. Thus, another objective of this study was to evaluate the relation between cell cycle arrest, ROS formation, and caspase activity levels after 2-Me treatment in gastric carcinoma cells. METHODS: It was determined whether 2-Me directly induced apoptosis in two gastric carcinoma cell lines (SC-M1 and NUGC-3) through caspase-3 and caspase-8 activation and, eventually, induced DNA fragmentation. To clarify the effect of 2-Me-induced G(2)/M arrest in apoptosis, calcium ionophore, A23187, and thapsigargin were used to modulate 2-Me-induced cell cycle responses. Moreover, the role of 2-Me-induced ROS formation in the cell cycle responses also was evaluated. RESULTS: It was found that 2-Me treatment resulted in G(2)/M-cycle arrest, caspase-8 and caspase-3 activation, and DNA fragmentation. In addition, the 2-Me induced, concomitant increases of peroxide and superoxide anions were correlated with G(2)/M-cycle arrest. Treatment with calcium ionophore A23187 and thapsigargin partially reversed the 2-Me-induced G(2)/M-cycle arrest, with a concomitant decrease in both peroxide and superoxide levels. Moreover, A23187 blocked the 2-Me-induced caspase-3 activation, whereas thapsigargin had no effect. Treatment with calcium channel blockers did not affect 2-Me-induced cell cycle arrest or caspase-3 activation. CONCLUSIONS: These results suggest that the 2-Me-induced apoptosis of gastric carcinoma cells by DNA fragmentation accompanied caspase activation. Elevation of free radicals was associated with G(2)/M-cycle arrest. The induction of G(2)/M-cycle arrest is not a prerequisite for caspase activation.     

Differential suppression of human cervical cancer cell growth by adenovirus delivery of p53 in vitro: arrest phase of cell cycle is dependent on cell line.

It has been reported that overexpression of wild-type p53 protein induces suppression of tumor cell growth in vivo and in vitro. In this study, we further evaluated the differential effects of p53 delivered in an adenovirus vector on the cell growth, apoptosis and cell cycle progression in cervical cancer cell lines. We constructed a recombinant adenovirus expressing p53 and then delivered this into cervical carcinoma cell lines (CaSki, SiHa, and HeLa, HeLaS3) along with adenovirus expressing beta-galactosidase as a negative control. Adenovirus-delivered p53 overexpression resulted in a more significant suppression of cell growth in HPV 18-infected cells (HeLa and HeLaS3) and a lesser suppression in HPV 16-infected cells (CaSki and SiHa). However, no suppression was observed in cells infected with a negative control virus. p53 overexpression also induced apoptosis and cell cycle arrest, as determined by annexin V and propidium iodide staining. In particular, the cell cycle was arrested in the G(2)/M phase in CaSki cells. In contrast, cell cycles were arrested in the G(1) phase in HeLa cells, suggesting that the arrest phase is dependent upon the cervical cancer cell line. Taken together, these data support the idea that overexpressed p53 protein plays a differential role in suppressing cervical cancer cell growth through apoptosis and cell cycle arrest in either G(1) or G(2)/M phase, depending on the cancer cell line.     

Galectin-3 mediates genistein-induced G(2)/M arrest and inhibits apoptosis

Many recent studies have focused on potential chemopreventive activities of dietary genistein, a natural isoflavonoid compound found in soy products. Genistein has been implicated in anticancer activities, including differentiation, apoptosis, inhibition of cell growth and inhibition of angiogenesis. In previous studies, genistein was shown to induce apoptosis and cell cycle arrest at G(2)/M in several cancer cell lines in vitro, which is associated with induction of p21(WAF1/CIP1), a universal inhibitor of cyclin-dependent kinases. At present, the molecular basis for diverse genistein-mediated cellular responses is largely unknown. In the present study, we investigated whether galectin-3, an anti-apoptotic gene product, regulates genistein-mediated cellular responses. We show that genistein effectively induces apoptosis without detectable cell cycle arrest in BT549, a human breast epithelial cell line which does not express galectin-3 at a detectable level. In galectin-3 transfected BT549 cells, genistein induced cell cycle arrest at the G(2)/M phase without apoptosis induction. Interestingly, genistein induces p21(WAF1/CIP1) expression in galectin-3-expressing BT549 cells, but not in control BT549 cells undergoing apoptosis. Collectively, the results of the present study suggest that galectin-3, at least in part, is a critical determinant for genistein-mediated cell cycle arrest and apoptosis, and genistein induction of p21(WAF1/CIP1) is associated with cell cycle arrest, but not required for apoptosis induction.     

CDC20在子宫内膜癌中的表达及其对RL95-2细胞增殖和凋亡的影响

目的：评估细胞分裂周期蛋白20（CDC20）在子宫内膜癌（EC）中的表达，探讨其对EC细胞RL95-2周期和凋亡的影响及可能的机制。方法：从癌症基因组图谱（TCGA）数据库获取EC 的mRNA 表达矩阵以及患者的临床信息，通过R 语言分析CDC20 mRNA的差异表达情况及其与肿瘤分期的相关性，qPCR及WB法检测CDC20在RL95-2细胞中的表达；向RL95-2细胞转染sh-CDC20以敲减CDC20的表达，采用CCK-8法和流式细胞术检测敲减CDC20对RL95-2细胞增殖活力、细胞周期分布和凋亡的影响，WB法分析对Mcl-1/p-Chk1信号活性的影响；建立RL95-2细胞裸鼠移植瘤模型，评估敲减CDC20 对肿瘤生长的抑制作用及对移植瘤组织中Mcl-1/p-Chk1信号轴和细胞凋亡的影响。结果：CDC20在EC组织及RL95-2细胞中呈高表达（均P<0.01），且CDC20的高表达与EC 的分期有关联。敲减CDC20可显著降低RL95-2 细胞增殖活力（P<0.01），阻滞细胞周期于G1期（P<0.01），促进细胞凋亡（P<0.01），抑制细胞中Mcl-1和p-Chk1的表达（P<0.05 或P<0.01）。敲减CDC20 可显著抑制RL95-2细胞裸鼠移植瘤的生长（P<0.01），降低移植瘤组织内Mcl-1 和p-Chk1 的表达（P<0.01），促进移植瘤细胞凋亡（P<0.01）。结论：CDC20在EC组织中呈高表达且与肿瘤分期有关联，敲减CDC20 能够抑制RL95-2细胞及其裸鼠移植瘤的生长而促进凋亡，这可能与Mcl-1/p-Chk1信号轴有关。     

蛋白酶体抑制剂MG132诱导HL-60细胞凋亡前G2/M期阻滞及机制

背景和目的:蛋白酶体(proteasome)抑制剂能够诱导多种肿瘤细胞凋亡,是一种潜在的有应用前景的抗肿瘤剂.本研究旨在探讨蛋白酶体抑制剂MGl32(Z-Leu-Leu-Leu-CHO)诱导白血病细胞HL-60凋亡和C2/M期阻滞的机制.方法:采用荧光显微镜观察、流式细胞术和免疫印迹研究测定MG132诱导HL-60细胞凋亡和周期阻滞及机制.结果:2μmol/L的MG132能够有效地诱导HL-60细胞凋亡,用药后24 h就显现有细胞凋亡;在MG132诱导HL-60细胞凋亡出现之前有一个明显的G2/M期阻滞,加MG132后12 h时G2/M期时相百分比为63.42±2.02;24 h时加MG132组细胞凋亡为16.67±1.48,与对照组G2/M期时相百分比为7.29±3.01及细胞凋亡为0相比,两者之间有显著性差别(P＜0.01);咖啡因CAF能够减少MG132诱导HL-60细胞出现的G2/M期阻滞,同时也减少凋亡细胞的比例;细胞周期检查点的负调控因子p21waf/cip1蛋白在加MG132处理后3 h有明显的表达,但并未能检测到p53和p27蛋白.结论:MG132诱导HL-60细胞凋亡之前有一个明显的G2/M期阻滞,p21蛋白表达明显上调提示:是p21waf/cip1而不是p53或其同源蛋白参与了其中的调控.     

tetra-O-methylnordihydroguaiaretic acid inhibits melanoma in vivo

tetra-O-methylnordihydroguaiaretic acid is a derivative of a naturally-occurring lignan, nordihydroguaiaretic acid, that has previously been shown to inhibit various cancer types in vitro and in vivo. Additionally, nordihydroguaiaretic acid has been shown to have nephrotoxic effects in the rat. Here we show that tetra-O-methylnordihydroguaiaretic acid inhibits the growth of a number of tumor cell lines in vitro by inducing apoptosis in a non-schedule-dependent manner. Further, this compound inhibits the synthesis of DNA by melanoma cells and causes cell cycle arrest in G0/G1 and G2/M phases of the cell cycle. tetra-O-Methylnordihydroguaiaretic acid also inhibits the growth of both murine and human melanomas and human colon cancer in vivo without apparent hepatic or renal toxicity.     

Gambogic acid-induced G2/M phase cell-cycle arrest via disturbing CDK7-mediated phosphorylation of CDC2/p34 in human gastric carcinoma BGC-823 cells

Molecular mechanisms of cell-cycle arrest caused by gambogic acid (GA), a natural product isolated from the gamboge resin of Garcinia hanburryi tree, have been investigated using BGC-823 human gastric carcinoma cells as a model. Based on our 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazoliumbromide (MTT) assay and flow cytometric analysis, treatment of BGC-823 cells with growth suppressive concentrations of GA caused an irreversible arrest in the G(2)/M phase of the cell cycle. Western blot analysis demonstrated that GA-induced cell-cycle arrest in BGC-823 cells was associated with a significant decrease in CDC2/p34 synthesis, which led to the accumulation of phosphorylated-Tyr(15) (inactive) form of CDC2/p34. Real-time PCR, western blot and kinase activity assays revealed that GA-induced reduction of CDC2/p34 expression was mediated through the inhibition of cyclin-dependent kinase (CDK)-activating kinase (CDK7/cyclin H) activity. In addition, GA-treated cells were shown to have a low level of CDK7 kinase-phosphorylated-Thr(161) CDC2/p34 (active). Taken together, our results suggested that the inhibited proliferation of GA-treated BGC-823 cells was associated with the decreased production of CDK7 mRNA and protein, which in turn, resulted in the reduction of CDK7 kinase activity. The reduced CDK7 kinase activity is responsible for the inactivation of CDC2/p34 kinase and the irreversible G(2)/M phase cell-cycle arrest of human gastric carcinoma BGC-823 cells.     

Lats2, a putative tumor suppressor,inhibits G1/S transition

Lats2 is a new member of the Lats tumor suppressor family. The human LATS2 gene is located at chromosome 13q11-12, which has been shown to be a hot spot (67%) for LOH in nonsmall cell lung cancer. In order to understand the function of LATS2 in the control of tumor development, we ectopically expressed mouse Lats2 via retroviral infection in NIH3T3/v-ras cells to examine whether Lats2 plays a role in suppressing tumor development and regulating cell proliferation. We have found that ectopic expression of Lats2 in NIH3T3/v-ras cells suppresses development of tumors in athymic nude mice and inhibits proliferation of NIH3T3/v-ras cells in an in vitro assay. Cell cycle profile analysis demonstrated that ectopic expression of Lats2 inhibited the G1/S transition. Further mechanistic studies revealed that cyclin E/CDK2 kinase activity was downregulated in Lats2-transduced NIH3T3/v-ras cells, while other cell cycle regulators controlling the G1/S transition were not affected. We have also shown that LATS2 kinase activity and two LATS conserved domains (LCDs) are required for Lats2 to suppress tumorigenicity and to inhibit cell growth. In addition, the LATS2 protein is cytoplasmic during interphase in NIH3T3 cells, while it becomes localized to the mitotic apparatus during mitosis. Finally, we propose a model in which a combination of mammalian Lats2 and Lats1 control cell proliferation by negatively regulating different cell cycle check points.