mad—overexpression down regulates the malignant growth and p53 mediated apoptosis in human hepatocellular carcinoma BEL—7404 cells

Mad protein has been shown as an antagonist of cMyc protein in some cell lines.The effect of Mad protein to the malignant phenotype of human hepatoma BEL-7404 cell line was investigated experimentally.An eukarryotic vector pCDNA Ⅲ containing full ORF fragment of mad cDNA was transfected into targeted cells.Under G418 selection,stable Mad-overexpressed cells were cloned.Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells.DNA synthesis,cell proliferation and anchorage-independent growth in soft-agar of the madtransfected cells were partially inhibited in comparison to control cells.Flos cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase,resulting in the retardation of cell proliferation.RT-PCR detected a marked inhibition of the expression of cdc25A,an important regulator gene of G0/G1 to S phase in cell cycle.It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-74040M1 cells in the absence of serume.Thus,Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL-7404 cells.     

Induction of apoptosis and expression of cell cycle regulatory proteins in response to a phytosphingosine derivative in HaCaT human keratinocyte cells

Ceramide, a compound derived from sphingomyelin, a sphingolipid precursor, affects cell functions such as growth, differentiation, cell division and apoptosis. We have shown that the phytosphingosine derivative, tetra-acetyl phytosphingosine (TAPS), inhibits the growth of HaCaT cells mainly by inducing apoptosis. In this study, we investigated its effect on the cell cycle and on cell cycle regulatory proteins. We showed by flow cytometry and staining for BrdU and phosphorylated histone H3 that the cells accumulated in S phase and arrested in G2 phase and did not divide before undergoing apoptosis. The level of the pro-apoptotic regulator Bax peaked after 6 h and then returned to normal, whereas the level of the anti-apoptotic regulator Bcl-xL, which is presumably induced in order to inhibit apoptosis, started to increase at 6 h, and remained high for 24 h. Phosphorylation of Cdc2 on Tyr-15 greatly increased while p21 rose to a plateau at 8 h. Levels of p53 and Mad2 proteins were unchanged. Our observations suggest that TAPS induces apoptosis of the HaCaT cells at least in part via transient G2 arrest.     

Diarsenic and tetraarsenic oxide inhibit cell cycle progression and bFGF- and VEGF-induced proliferation of human endothelial cells

Arsenic trioxide (As2O3, diarsenic oxide) has recently been reported to induce apoptosis and inhibit the proliferation of various human cancer cells derived from solid tumors as well as hematopoietic malignancies. In this study, the in vitro effects of As2O3 and tetraasrsenic oxide (As4O6) on cell cycle regulation and basic fibroblast growth factor (bFGF)- or vascular endothelial growth factor (VEGF)-stimulated cell proliferation of human umbilical vein endothelial cells (HUVEC) were investigated. Significant dose-dependent inhibition of cell proliferation was observed when HUVEC were treated with either arsenical compound for 48 h, and flow cytometric analysis revealed that these two arsenical compounds induced cell cycle arrest at the G1 and G2/M phases--the increases in cell population at the G1 and G2/M phase were dominantly observed in As2O3- and As4O6-treated cells, respectively. In both arsenical compounds-treated cells, the protein levels of cyclin A and CDC25C were significantly reduced in a dose-dependent manner, concomitant to the reduced activities of CDK2- and CDC2-associated kinase. In G1-synchronized HUVEC, the arsenical compounds prevented the cell cycle progression from G1 to S phase, which was stimulated by bFGF or VEGF, through the inhibition of growth factor-dependent signaling. These results suggest that arsenical compounds inhibit the proliferation of HUVEC via G1 and G2/M phase arrest of the cell cycle. In addition, these inhibitory effects on bFGF- or VEGF-stimulated cell proliferation suggest antiangiogenic potential of these arsenical compounds.     

Resveratrol modulates gene expression associated with apoptosis, proliferation and cell cycle in cells with mutated human c-Ha-Ras, but does not alter c-Ha-Ras mRNA or protein expression

An accumulating body of evidence suggests that resveratrol can inhibit carcinogenesis through antiproliferative and apoptotic effects. One proposed mechanism for this is the modulation of genes, for example, Ras and p53, frequently associated with human cancer. To test the effect of resveratrol on gene expression, we used the WR-21 cell line because it contains a mutated human c-Ha-ras gene. Cells at > or =70% confluency were incubated with media alone or with increasing concentrations of trans-resveratrol (0.1-1000 microM) for 24 h. Resveratrol (30-100 microM) decreased cellular proliferation by 80% (bromodeoxyuridine incorporation) and increased apoptosis by 60% (TUNEL). Cells were then treated with media alone or with 50-microM resveratrol for 24 h. RNA was isolated for nylon-based macroarray analyses and protein for immunoblotting. Resveratrol increased (+) and decreased (-) gene expression associated with apoptosis (Birc5+, Cash+, Mcl-1+, Mdm2+, Rpa-like+), cellular proliferation (Ctsd+, Mdm2+, Egr1+, ODC+) and cell cycle (cyclin D+, cyclin g+, Gadd45a-, Mad2l-, Mdm2+). Resveratrol consistently increased by > or =6-fold Mdm2 expression and other downstream p53 effectors, but not p53 itself at 24 h. Subsequent cell cycle analysis indicated a significant accumulation of cells in G2/M, and a decrease in G1/G0 suggesting a G2/M blockade. Further RT-PCR and Western blot analyses indicated no differential changes in Ras mRNA expression or p21(ras) protein levels, respectively. These results suggest that resveratrol potently inhibits cellular proliferation, increases apoptosis, alters cell cycle dynamics and modulates associated gene expression. Furthermore, these effects appear mediated, in part, by p53 without direct modulation of mutant c-Ha-ras expression.     

Pro-apoptotic effect and cytotoxicity of genistein and genistin in human ovarian cancer SK-OV-3 cells

We investigated the effects of genistein and genistin on proliferation and apoptosis of human ovarian SK-OV-3 cells and explored the mechanism for these effects. SK-OV-3 cells were treated with genistein and genistin at various concentrations (ranging from 1 to 100 muM) either alone or in combination for 24 and 48 h. Cell proliferation was estimated using an MTT assay, and cell cycle arrest was evaluated using FACS. Caspase-3 activity and annexin-based cell cycle analysis were used as measures of apoptosis. In addition, genistein- and genistin-induced cytotoxicity was determined by measuring release of LDH. Genistein treatment for 24 or 48 h substantially inhibited SK-OV-3 cell proliferation in a dose-dependent manner, and genistin treatment for 48 h also inhibited cell proliferation. Genistein caused cell cycle arrest at G2/M phase in dose- and time-dependent manner, and genistin caused cell cycle arrest not only at G2/M phase but also at G1 phase. Genistein markedly induced apoptosis and significantly increased LDH release, whereas genistin did not affect LDH release. Moreover, exposure to both genistein and genistin in combination for 48 h induced apoptosis without increasing LDH release. Genistein and genistin inhibit cell proliferation by disrupting the cell cycle, which is strongly associated with the arrest induction of either G1 or G2/M phase and may induce apoptosis. Based on our findings, we speculate that both genistein and genistin may prove useful as anticancer drugs and that the combination of genistein and genistin may have further anticancer activity.     

A Critical Function of Mad2l2 in Primordial Germ Cell Development of Mice

The development of primordial germ cells (PGCs) involves several waves of epigenetic reprogramming. A major step is following specification and involves the transition from the stably suppressive histone modification H3K9me2 to the more flexible, still repressive H3K27me3, while PGCs are arrested in G2 phase of their cycle. The significance and underlying molecular mechanism of this transition were so far unknown. Here, we generated mutant mice for the Mad2l2 (Mad2B, Rev7) gene product, and found that they are infertile in both males and females. We demonstrated that Mad2l2 is essential for PGC, but not somatic development. PGCs were specified normally in Mad2l2^−/− embryos, but became eliminated by apoptosis during the subsequent phase of epigenetic reprogramming. A majority of knockout PGCs failed to arrest in the G2 phase, and did not switch from a H3K9me2 to a H3K27me3 configuration. By the analysis of transfected fibroblasts we found that the interaction of Mad2l2 with the histone methyltransferases G9a and GLP lead to a downregulation of H3K9me2. The inhibitory binding of Mad2l2 to Cyclin dependent kinase 1 (Cdk1) could arrest the cell cycle in the G2 phase, and also allowed another histone methyltransferase, Ezh2, to upregulate H3K27me3. Together, these results demonstrate the potential of Mad2l2 in the regulation of both cell cycle and the epigenetic status. The function of Mad2l2 is essential in PGCs, and thus of high relevance for fertility.     

转录因子Foxo3a高表达对小鼠T淋巴瘤EL-4细胞周期和凋亡的影响

为了研究转录因子Foxo3a高表达对小鼠T淋巴瘤EL-4细胞周期和凋亡的影响,采用电穿孔法将真核表达载体pEGFP-N1/Foxo3a转染小鼠T淋巴瘤细胞系EL-4细胞,并通过聚合酶链式反应和免疫印迹法分别检测Foxo3a mRNA及蛋白表达。转录因子Foxo3a高表达后,采用细胞计数法绘制其细胞生长曲线;采用荧光显微镜法及流式细胞仪定性和定量观察典型EL-4细胞凋亡形态特征、细胞凋亡百分率及细胞周期变化情况。结果表明,转录因子Foxo3a真核表达质粒pEGFP-N1/Foxo3a经酶切鉴定及测序检测序列正确。转染pEGFP-N1/Foxo3a的小鼠EL-4细胞表达Foxo3a mRNA和蛋白水平显著升高。Foxo3a高表达明显抑制EL-4细胞的增殖能力,并使EL-4细胞发生明显G2期阻滞(P<0.001)。Foxo3a基因高表达后,荧光显微镜可以观察到典型凋亡的细胞形态。同时,EL-4细胞凋亡百分率显著升高(P<0.01)。结果提示,Foxo3a高表达可以有效抑制小鼠T淋巴瘤细胞体外细胞增殖,使细胞周期G2时相阻滞,并具有诱导细胞凋亡的作用。     

Mitochondrial ribosomal protein L41 mediates serum starvation-induced cell-cycle arrest through an increase of p21(WAF1/CIP1)

Ribosomal proteins not only act as components of the translation apparatus but also regulate cell proliferation and apoptosis. A previous study reported that MRPL41 plays an important role in p53-dependent apoptosis. It also showed that MRPL41 arrests the cell cycle by stabilizing p27(Kip1) in the absence of p53. This study found that MRPL41 mediates the p21(WAF1/CIP1)-mediated G1 arrest in response to serum starvation. The cells were released from serum starvation-induced G1 arrest via the siRNA-mediated blocking of MRPL41 expression. Overall, these results suggest that MRPL41 arrests the cell cycle by increasing the p21(WAF1/CIP1) and p27(Kip1) levels under the growth inhibitory conditions.     

Expression and DNA-binding activity of MYCN/Max and Mnt/Max during induced differentiation of human neuroblastoma cells

Amplification of MYCN is one of the most important prognostic markers for neuroblastoma and is correlated with rapid tumor progression and poor prognosis. MYCN belongs to the Myc/Max/Mad/Mnt network of proteins that regulate proliferation, apoptosis, and differentiation. It is well established that MYCN is downregulated during induced differentiation of neuroblastoma cells carrying an amplified MYCN gene, but very little is known about other components of the network, i.e., the Max, Mad, and Mnt proteins, during this process. In this study we show that Mad and Mnt expression was only modestly regulated in differentiating SK-N-BE(2) neuroblastoma cells, while MYCN was rapidly downregulated. This downregulation was reflected in a decreased MYCN/Max DNA-binding activity while the Mnt/Max binding did not change during differentiation. In parallel experiments we also analyzed the Myc/Max/Mad expression and DNA binding capacity during induced differentiation in the MYCN single copy neuroblastoma cell line SH-SY5Y. In this cell line only modest changes in expression of the components of the MYCN/Max/Mad/Mnt network was detected, but since the cell line expresses relatively low levels of MYCN and c-Myc, these changes might be of functional significance. Cell cycle analyses of SK-N-BE(2) demonstrated an increase in the G1-phase fraction after RA-treatment. These data show that the decreased MYCN expression and MYCN DNA-binding is correlated with retarded cell cycle progression. Furthermore, when Mad1 or Mnt was overexpressed in SK-N-BE(2) cells they retained the capacity to differentiate, underscoring the notion that MYCN downregulation, and not changes in Mad/Mnt expression, is essential for neuroblastoma cell differentiation.