Polymorphisms of p53 codon 72 and MDM2 promoter 309 and the risk of endometrial cancer

Genetic polymorphisms of p53 and its negative regulator murine double minute 2 homolog (MDM2) have been shown to be closely associated with tumorigenesis in a variety of human cancers. In the present study, single nucleotide polymorphism (SNP) at p53 codon 72 and MDM2 promoter 309 was examined for germline DNA samples from 102 endometrial cancer cases and 95 controls using polymerase chain reaction-based fragment analysis. There were no significant differences in the genotype and allele prevalence between control subjects and endometrial cancer patients for p53 codon 72. The GG genotype frequency of MDM2-SNP309 was statistically higher in endometrial cancer patients than that in normal healthy women when compared with the TG genotype ( P = 0.0088). However, no statistically significant differences were found between the TT and TG or GG genotype frequencies and allele prevalence. Interestingly, the combination of the homozygous Arg/Arg genotype of p53 codon 72 and homozygous GG genotype of MDM2 SNP309 polymorphisms was significantly associated with the risk of endometrial cancer (odds ratio = 3.28, 95% confidence interval = 1.13 to 9.53, P = 0.0212). The homozygous variants of wild p53 codon 72 and mutant MDM2 promoter 309 may cooperatively increase the risk of endometrial cancer in a Japanese population.     

8-氯腺苷诱导的组蛋白H3K79双甲基化促进NBS1和p21的基因转录激活

组蛋白H3第79位赖氨酸甲基化（H3K79me）修饰有单甲基、双甲基及三甲基3种形式,是常染色质的标志.然而,对于组蛋白H3K79三种甲基化各自在基因转录、DNA损伤修复中所起的作用尚不十分清楚.本研究以8-氯腺苷（8-Cl-Ado）为DNA双链断裂（DNA double-stranded breaks,DSB）诱导剂,采用Western 印迹,在人肺癌细胞H1299检测出了DNA修复分子NBS1、细胞周期检验点相关分子p21,并发现H3K79me1、H3K79me2和H3K79me3三种甲基化修饰的组蛋白明显增加;染色质免疫共沉淀结合实时定量PCR实验显示,只H3K79me2与DNA损伤检验点分子p21、DNA修复分子NBS1的启动子区域相结合,说明H3K79双甲基化修饰与这些基因的转录激活有关.结果提示,在8-氯腺苷引起 DSB时,是H3K79me2、而不是H3K79me1和H3K79me3参与NBS1和p21基因转录激活时的染色质重塑.8-氯腺苷诱导H3K79双甲基化增强、促进H3K79me2所在染色质区域的NBS1和p21基因转录激活可能是8-Cl-Ado抑制肿瘤细胞生长作用机制之一.     

D-glucosamine inhibits proliferation of human cancer cells through inhibition of p70S6K

Although D-glucosamine has been reported as an inhibitor of tumor growth both in vivo and in vitro, the mechanism for the anticancer effect of D-glucosamine is still unclear. Since there are several reports suggesting D-glucosamine inhibits protein synthesis, we examined whether D-glucosamine affects p70S6K activity, an important signaling molecule involved in protein translation. In the present study, we found D-glucosamine inhibited the activity of p70S6K and the proliferation of DU145 prostate cancer cells and MDA-MB-231 breast cancer cells. D-glucosamine decreased phosphorylation of p70S6K, and its downstream substrates RPS6, and eIF-4B, but not mTOR and 4EBP1 in DU145 cells, suggesting that D-glucosamine induced inhibition of p70S6K is not through the inhibition of mTOR. In addition, D-glucosamine enhanced the growth inhibitory effects of rapamycin, a specific inhibitor of mTOR. These findings suggest that D-glucosamine can inhibit growth of cancer cells through dephosphorylation of p70S6K.     

Chlorophyll reduction in the seed of Brassica napus with a glutamate 1-semialdehyde aminotransferase antisense gene*

Chlorophyll reduction in the seed of Brassica can be achieved by downregulating its synthesis. To reduce chlorophyll synthesis, we have used a cDNA clone of Brassica napus encoding glutamate 1-semialdehyde aminotransferase (GSA-AT) to make an antisense construct for gene manipulation. Antisense glutamate 1-semialdehyde aminotransferase gene (Gsa) expression, directed by a Brassica napin promoter, was targeted specifically to the embryo of the developing seed. Transformants expressing antisense Gsa showed varying degrees of inhibition resulting in a range of chlorophyll reduction in the seeds. Seed growth and development were not affected by reduction of chlorophyll. Seeds from selfed transgenic plants germinated with high efficiency and growth of seedlings was vigorous. Seedlings from T₂ transgenic lines segregated into three distinctive phenotypes: dark green, light green and yellow, indicating the dominant inheritance of Gsa antisense gene. These transgenic lines have provided useful materials for the development of a low chlorophyll seed variety of B. napus.     

Functional inhibition of the p75 receptor using a small interfering RNA

The neurotrophin receptor p75(NTR) mediates a wide variety of biological effects. Consistent with the function in controlling the survival and neurite formation, p75(NTR) is expressed during the developmental stages of the nervous system. Importantly, p75(NTR) is re-expressed in various pathological conditions and is suggested to contribute to the inhibition of neuronal regeneration and the death of the neurons. Here we develop a tool to knock down the expression of p75(NTR) by employing a small interfering RNA (siRNA). The siRNA for p75(NTR) effectively reduces the expression of endogenous p75(NTR) both in Schwann cells and dorsal root ganglion neurons in vitro. NGF-induced cell death in Schwann cells and the neurite retraction in DRG neurons induced by myelin-associated glycoprotein are attenuated by the siRNA. Inhibition of p75(NTR) in specific pathological conditions by the siRNA may provide a potential therapeutic agent.     

Requirement of c-Jun NH2-terminal kinase activation in interferon-alpha-induced apoptosis through upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Daudi B lymphoma cells

Interferon alpha (IFN-alpha) inhibits growth, at least in part, through induction of apoptosis. However, the molecular mechanisms underlying IFN-alpha-induced apoptosis are not completely understood. In the present study, we found that IFN-alpha induced a sustained activation of c-Jun N-terminal kinase 1 (JNK1), but not extracellular kinases (ERKs), in Daudi B lymphoma cells, as assessed by Western blotting using phospho-specific antibodies. Several lines of evidence support the notion that the IFN-alpha-induced activation of JNK is responsible for IFN-alpha-induced apoptosis, at least in part, through upregulation of TNF-related apoptosis-inducing ligand (TRAIL). First, pretreatment of Daudi cells with a JNK inhibitor reduced IFN-alpha-induced upregulation of TRAIL and loss of mitochondrial membrane potential (DeltaPsim) and annexin-positive cells, which was assessed by flow cytometry. Second, a dominant-negative form of JNK1 (dnJNK1) also reduced these apoptotic events, while a constitutively active form of JNK1, MKK7-JNK1beta, enhanced them. Finally, treatment with IFN-alpha enhanced the promoter activity of the TRAIL gene, which was partially abrogated by either JNK inhibitor or dnJNK1, while it was moderately enhanced by MKK7-JNK1beta. These findings are useful for understanding molecular mechanisms of IFN-alpha-induced apoptosis and also for development of treatment modalities of some tumors with IFN-alpha.     

RNA-cleaving properties of human apurinic/apyrimidinic endonuclease 1 (APE1)

We have recently identified apurinic/apyrimidinic endonuclease 1 (APE1) as an endoribonuclease that cleaves c-myc mRNA in vitro and regulates c-myc mRNA levels and half-life in cells. This study was undertaken to further unravel the RNA-cleaving properties of APE1. Here, we show that APE1 cleaves RNA in the absence of divalent metal ions and, at 2 mM, Zn²⁺, Ni²⁺, Cu²⁺, or Co²⁺ inhibited the endoribonuclease activity of APE1. APE1 is able to cleave CD44 mRNA, microRNAs (miR-21, miR-10b), and three RNA components of SARS-corona virus (orf1b, orf3, spike) suggesting that, when challenged, it can cleave any RNAs in vitro. APE1 does not cleave strong doublestranded regions of RNA and it has a strong preference for 3’ of pyrimidine, especially towards UA, CA, and UG sites at single-stranded or weakly paired regions. It also cleaves RNA weakly at UC, CU, AC, and AU sites in single-stranded or weakly paired regions. Finally, we found that APE1 can reduce the ability of the Dicer enzyme to process premiRNAs in vitro. Overall, this study has revealed some previously unknown biochemical properties of APE1 which has implications for its role in vivo.     

Role of p53 and mismatch repair in PhIP-induced perturbations of the cell cycle

Heterocyclic amines, found ubiquitously in our diet, are carcinogenic and mutagenic. Among this class of compounds, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) is the most abundant. To further understand the carcinogenesis of this compound, we studied the effects of PhIP on the progression of human lymphoblastoid cells through the cell-cycle. Cells differing in p53 or mismatch repair status were used to evaluate the role of those proteins. Following PhIP-treatment, a dose and time-dependent accumulation of p53 was found in cells containing functional p53. The augmentation of the p53 protein, accompanied by increases in p21-WAF1, confirms that the p53 is activated. The increase in p53 was independent of the mismatch repair status of the cells. Perturbations in the cell-cycle were also observed. Twenty-four hours after PhIP treatment, the activation of the G2-M checkpoint was evident. Functional p53 and mismatch repair were not required for the PhIP-induced G2-M arrest. The G2-M arrests were reversible and are interpreted as necessary for the repair of the PhIP-DNA lesions. Under treatment conditions where less than 5% of the cells survived, the G2-M arrests were absent.     

Erythropoietin-dependent autocrine secretion of tumor necrosis factor-alpha in hematopoietic cells modulates proliferation via MAP kinase--ERK-1/2 and does not require tyrosine docking sites in the EPO receptor

Primary erythroid cells and erythroid cell lines may synthesize and secrete tumor necrosis factor-alpha (TNF-alpha) following stimulation with erythropoietin (EPO). The effect of triggering TNF-alpha synthesis and secretion was investigated in erythroleukemia and myeloid cell lines: HCD57, DA3-EPOR, and BAF3-EPOR. The EPO-induced, membrane-bound form of autocrine TNF-alpha seemed to enhance proliferation of HCD57 and DA3-EPOR cells; however, the concentration of secreted autocrine/paracrine TNF-alpha was never sufficient to have an effect. Autocrine TNF-alpha acts through TNFRII receptors to stimulate proliferation. Modulation of mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK-1/2) activity by the membrane-bound form of autocrine TNF-alpha apparently played a central role in the control of EPO-dependent proliferation of HCD57 and DA3-EPOR cells. Primary erythroid cells and DA3-EPOR cells were found to express similar, high levels of both TNFRI and TNFRII, showing that differential expression of TNF-alpha receptors does not explain why primary cells are inhibited and DA3-EPOR cells are stimulated by autocrine TNF-alpha. BAF3 cells expressing a mutant EPOR with no cytoplasmic tyrosine residues were capable of triggering EPO-dependent TNF-alpha synthesis and secretion, indicating that tyrosine-docking sites in the EPOR were not required for EPO-dependent TNF-alpha secretion.