p53的两种结合蛋白:53BP1和53BP2

p53基因是一种广谱的肿瘤抑制基因,其产物p53为一多功能的转录调节因子,可以发挥调节细胞生长、细胞凋亡和DNA修复的作用。在人类肿瘤已发现多种p53基因的点突变,但突变并不是p53蛋白质丧失功能的唯一途径。胞内蛋白可能影响其活性和功能。53BP1和53BP2是p53在胞浆中的两种结合蛋白。本文阐述了有关这两种蛋白质的研究进展。     

非整倍体,MSI,TLS与肿瘤

肿瘤发生的机制多种多样,其中非整倍体细胞导致的肿瘤占了相当大的比例。然而,在某些情况下,非整倍体的细胞可以抑制肿瘤的发生。某些致癌因素可以引起细胞微卫星不稳定性(microsatellite instability,MSI)的发生,而MSI可以通过框移突变促进肿瘤的发生。当DNA损伤无法修复时,细胞可以利用跨损伤DNA合成(translesion DNA synthesis,TLS)提高其对DNA损伤的耐受性。非整倍体、MSI和TLS与肿瘤发生的机制有关,对肿瘤的治疗也有一定的影响。     

核糖体蛋白基因与人类疾病的相关研究

核糖体是细胞内合成蛋白质的重要细胞器,由核糖体RNA和核糖体蛋白质组成.核糖体蛋白除了参与蛋白质的生物合成还表现出一些其他生理功能,如参与DNA复制、转录和损伤修复,调控细胞生长、增殖、凋亡和发育以及细胞转化等.最近研究发现,某些核糖体蛋白的表达不足或缺失以及核糖体蛋白基因发生突变与某些先天性遗传性疾病的发生有关.在许多恶性肿瘤细胞中也存在某些核糖体蛋白基因异常表达.此文对核糖体蛋白基因与人类疾病发生(某些先天性遗传性疾病和肿瘤)之间的关系进行了详细阐述.     

DNA修复基因甲基化与肿瘤发生关系的研究进展

肿瘤抑制基因的异常甲基化可能导致基因的表达沉默,当DNA修复基因包括MGMT、hMLH及BRCA等发生甲基化会导致修复蛋白合成减少,细胞修复损伤能力降低,基因组稳定性降低,基因突变的概率增高最终导致肿瘤发生.     

用微小细胞介导人的DNA修复基因转移到着色性干皮病的细胞中

着色性干皮病(XP)是一种人类遗传疾病,患者细胞中缺乏DNA修复基因,当这种细胞中的DNA被紫外线(UV)损伤后,DNA就不能再合成。本文作者用微小细胞(microcell)技术,将正常人的DNA修复基因转移到XP细胞中,得到了抗紫外线克隆,并证明了这种克隆内有正常人的一部分基因组。微小细胞是从一个33岁男人皮肤正常二     

人恶性畸胎瘤PA-1细胞染色体特性及其影响因素的探讨

目的:探讨人类恶性畸胎瘤PA-1细胞株染色体特性及其影响因素。方法:采用G带核型分析、DNA碱基序列分析及Western blot(蛋白印迹分析)等方法对经20余年407-445继代培养的PA-1细胞株染色体核型及p 53 基因状态进行了研究。结果:PA-1细胞株80%以上仍然保持近乎二倍体的核型,30代以后的细胞由于第15号染色体与20号染色体间的相互易位形成了M1及M2标识染色体。RT-PCR产物DNA定向序列分析显示具有野生及突变两个带(p53密码子239突变),Western blot 未检测出突变的p53基因蛋白,而p21蛋白的表达水平比正常成纤维细胞低。结论:人卵巢恶性畸胎瘤PA-1细胞株经20年的继代培养后,一个p53 等位基因发生错义突变,另一个仍然是野生型的。仅一个p53 等位基因发生突变,不足以引起细胞的染色体不稳定性。     

Rad9、Rad1、Hus1与肿瘤发生的研究进展

基因的稳定性对维持机体的正常生长具有重要作用。DNA随时面临损伤因子的威胁,一旦DNA发生不可逆损伤将会导致基因的不稳定,甚至诱发肿瘤形成。但机体具有DNA损伤修复、细胞凋亡调控系统可以及时修复损伤的DNA以维持基因的稳定性。有研究表明Rad9、Rad1、Hus1基因参与DNA的损伤修复和细胞凋亡并与肿瘤的发生、发展有明确关系。本文将对目前Rad9、Rad1、Hus1基因的研究以及与肿瘤发生的关系进行综述。     

胃肠道肿瘤与胰岛素样生长因子Ⅱ受体基因中微卫星DNA的不稳定性的关系

微卫星DNA是出现于整个人类基因组中的寡核着酸重复序列。这些区域的突变，包括重复单位的增加或缺失，被称为复制／修复错误阳性表现型（RER＋），或微卫星DNA不稳定（MI）。MI已在人类多数原发肿瘤中被证实。但是，在不编码的DNA，即内含子或基因组的基因间区域中的许多MI还未被了解。最近，在编码区域--结肠癌细胞的变异的生长因子BI型受体（TGF01RI）基因中定位了第一个MI。在其。DNA中，上游的一个多脱氧腺膘吟序列中的一、两个碱基的缺失或插入已有报道。这些突变将在下游导致无义突变，并生成截断的无功能的蛋白质。除散…     

微卫星不稳定型大肠癌中基因突变及其产物的检测

目的 通过检测基因突变及其产物在微卫星不稳定型大肠癌中的表达,探讨微卫星不稳定型大肠癌发生发展的分子生物学机制.方法 利用PCR、RT-PCR及蛋白印迹杂交方法,在11个大肠癌细胞株中检测微卫星稳定程度;9个蛋白质编码区域存在重复碱基序列的,与细胞生长、DNA 修复及细胞凋亡相关基因中检测基因突变及其mRNA和蛋白质表达.结果 检测结果发现了7个大肠癌细胞株表现高度微卫星不稳定,1个大肠癌细胞株表现低度微卫星不稳定,3个大肠癌细胞株表现微卫星稳定.与微卫星稳定的细胞株相比,微卫星不稳定型大肠癌中基因突变率显著增高.ACVRⅡ、Bax、hMSH6、hRad50、RIZ和TGFβRⅡ等6个基因突变率超过50%;ACVRⅡ、ATR、Bax、MBD4、hMSH3、RIZ和TGFβRⅡ 7个基因表达突变型mRNA;而hMSH6和hRad50不表达突变型mRNA;同时hRad50的蛋白质表达与无突变的细胞株相比显著下降.结论 与细胞生长,DNA修复,细胞凋亡相关基因的高度突变及其突变基因产物与微卫星不稳定型大肠癌的发生有密切相关,hRad50蛋白表达降低可能与微卫星不稳定型大肠癌的染色体不稳定有密切关系.     

人类恶性畸胎瘤PA-1细胞的p53基因结构、功能状态及其意义

目的:探讨人类恶性畸胎瘤PA-1细胞株p53基因的结构、功能状态及其意义。方法:采用DNA碱基序列分析、FASAY（酵母细胞中单个等位基因的功能分析）及Western blot（蛋白印迹分析）等方法对经20余年407-445继代培养的PA-1细胞株p53的基因结构、功能状态进行了研究。结果:RT-PCR产物DNA定向序列分析显示具有野生及突变2个带（p53密码子239突变）,FASAY结果也显示p53一个等位基因是有活性的（野生的）,而另一个是非活性的（突变的）。Western blot未检测出突变的p53基因蛋白,而p21蛋白的表达水平比正常成纤维细胞低。结论:人卵巢恶性畸胎瘤PA-1细胞株经20年的继代培养后,一个p53等位基因发生错义突变,另一个仍然是野生型的。仅一个p53等位基因发生突变,不足以引起细胞的染色体不稳定性。因此,研究PA-1细胞稳定核型的维持结构,在确定有关染色体的稳定性与不稳定性的基因的方面是非常重要的。     

Establishment of a chemical synthetic lethality screen in cultured human cells

The synthetic lethality screen is a powerful genetic method for unraveling functional interactions between proteins in yeast. Here we demonstrate the feasibility of a chemical synthetic lethality screen in cultured human cells, based in part on the concept of the yeast method. The technology employs both an immortalized human cell line, deficient in a gene of interest, which is complemented by an episomal survival plasmid expressing the gene of interest, and the use of a novel double-label fluorescence system. Selective pressure imposed by any one of several synthetic lethal metabolic inhibitors prevented the spontaneous loss of the episomal survival plasmid. Retention or loss over time of this plasmid could be sensitively detected in a blind test, while cells were grown in microtiter plates. Application of this method should thus permit high throughput screening of drugs, which are synthetically lethal with any mutant human gene of interest, whose normal counterpart can be expressed. This usage is particularly attractive for the search of drugs, which kill malignant cells in a gene-specific manner, based on their predetermined cellular genotype. Moreover, by replacing the chemicals used in this example with a library of either DNA oligonucleotides or expressible dominant negative genetic elements, one should be able to identify synthetic lethal human genes.     

PARP inhibition induces BAX/BAK-independent synthetic lethality of BRCA1-deficient non-small cell lung cancer

Evasion of apoptosis contributes to both tumourigenesis and drug resistance in non-small cell lung carcinoma (NSCLC). The pro-apoptotic BCL-2 family proteins BAX and BAK are critical regulators of mitochondrial apoptosis. New strategies for targeting NSCLC in a mitochondria-independent manner should bypass this common mechanism of apoptosis block. BRCA1 mutation frequency in lung cancer is low; however, decreased BRCA1 mRNA and protein expression levels have been reported in a significant proportion of lung adenocarcinomas. BRCA1 mutation/deficiency confers a defect in homologous recombination DNA repair that has been exploited by synthetic lethality through inhibition of PARP (PARPi) in breast and ovarian cells; however, it is not known whether this same synthetic lethal mechanism exists in NSCLC cells. Additionally, it is unknown whether the mitochondrial apoptotic pathway is required for BRCA1/PARPi-mediated synthetic lethality. Here we demonstrate that silencing of BRCA1 expression by RNA interference sensitizes NSCLC cells to PARP inhibition. Importantly, this sensitivity was not attenuated in cells harbouring mitochondrial apoptosis block induced by co-depletion of BAX and BAK. Furthermore, we demonstrate that BRCA1 inhibition cannot override platinum resistance, which is often mediated by loss of mitochondrial apoptosis signalling, but can still sensitize to PARP inhibition. Finally we demonstrate the existence of a BRCA1-deficient subgroup (11-19%) of NSCLC patients by analysing BRCA1 protein levels using immunohistochemistry in two independent primary NSCLC cohorts. Taken together, the existence of BRCA1-immunodeficient NSCLC suggests that this molecular subgroup could be effectively targeted by PARP inhibitors in the clinic and that PARP inhibitors could be used for the treatment of BRCA1-immunodeficient, platinum-resistant tumours.     

The enigmatic oncogene and tumor suppressor‐like properties of RAD54B: Insights into genome instability and cancer

One of the major challenges to the cell is to ensure genome stability, which can be compromised through endogenous errors or exogenous DNA damaging agents, such as ionizing radiation or common chemotherapeutic agents. To maintain genome stability the cell has a multifaceted line of defense, including cell cycle checkpoints and DNA damage repair pathways. RAD54B is involved in many of these pathways and thus exhibits a role in maintaining and repairing genome stability following DNA damage. RAD54B is involved in cell cycle regulation after DNA damage and participates in homologous recombinational repair, which ensures the precise repair of the most deleterious DNA lesions, double‐stranded breaks. This review focuses on structural aspects of RAD54B, molecular functions associated with its cellular roles in preventing genome instability, and how aberrant function contributes to oncogenesis. By understanding how aberrant RAD54B expression and/or function can contribute to oncogenesis, novel therapeutic approaches that specifically exploit these aberrant genetics are now being explored for precision medicine targeting. RAD54B represents an ideal candidate for synthetic genetic therapeutic approaches (synthetic dosage lethality or synthetic lethality), which are designed to target the specific genetics associated with cancer formation. These therapeutic approaches represent a precision‐based approach, which is ideal as we are now entering the era of precision medicine.     

Tissue-specific genotoxic effects of acrylamide and acrylonitrile.

Acrylamide (AA) has been reported to induce dominant lethal mutations in male rat germ cells and tumors in a variety of organs, including the scrotum, thyroid and mammary glands, but not the liver of rats. The structurally similar vinyl monomer acrylonitrile (ACN) does not induce dominant lethal mutations but does induce tumors of the brain, Zymbal gland, forestomach and mammary gland, but not the liver of rats. Several in vitro and/or in vivo unscheduled DNA synthesis (UDS) assays were employed to examine the potential tissue-specific genotoxic activity of these compounds. Neither AA nor ACN induced DNA repair in either the in vitro or in vivo hepatocyte DNA repair assays. Glycidamide (GA), a mutagenic metabolite of AA, induced DNA repair in the in vitro hepatocyte DNA repair assay. Cyanoethylene oxide (CEO), a mutagenic metabolite of ACN, did not yield a DNA repair response in the in vitro hepatocyte DNA repair assay, but was highly toxic and could not be tested at doses equivalent to GA. AA, but not ACN, produced a DNA repair response in the in vivo spermatocyte DNA repair assay. AA produced a slight response in the in vitro human mammary epithelial cell (HMEC) DNA repair assay in normal cells derived from discarded surgical samples from five different women. GA produced a strong UDS response in all cases in the same assay. CEO, but not its parent compound ACN, produced a response in the HMEC DNA repair assay. These results show a highly tissue-specific pattern of genotoxic activity for AA and ACN that correlates, to the extent that it has been examined, with the tissue-specific pattern of carcinogenic and dominant lethal activity. The induction of DNA repair by GA and CEO confirms the genotoxic potential of these metabolites. While the observation of genotoxic activity of AA in the HMEC DNA repair assay suggests that mammary cells might be a target for carcinogenic activity of this compound in humans, other factors such as pharmacokinetics and epidemiology must be evaluated to establish that effect.     

Kinase-impaired BRAF mutations in lung cancer confer sensitivity to dasatinib

During a clinical trial of the tyrosine kinase inhibitor dasatinib for advanced non-small cell lung cancer (NSCLC), one patient responded dramatically and remains cancer-free 4 years later. A comprehensive analysis of his tumor revealed a previously undescribed, kinase-inactivating BRAF mutation ((Y472C)BRAF); no inactivating BRAF mutations were found in the nonresponding tumors taken from other patients. Cells transfected with (Y472C)BRAF exhibited CRAF, MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase), and ERK (extracellular signal-regulated kinase) activation-characteristics identical to signaling changes that occur with previously known kinase-inactivating BRAF mutants. Dasatinib selectively induced senescence in NSCLC cells with inactivating BRAF mutations. Transfection of other NSCLC cells with these BRAF mutations also increased these cells' dasatinib sensitivity, whereas transfection with an activating BRAF mutation led to their increased dasatinib resistance. The sensitivity induced by (Y472C)BRAF was reversed by the introduction of a BRAF mutation that impairs RAF dimerization. Dasatinib inhibited CRAF modestly, but concurrently induced RAF dimerization, resulting in ERK activation in NSCLC cells with kinase-inactivating BRAF mutations. The sensitivity of NSCLC with kinase-impaired BRAF to dasatinib suggested synthetic lethality of BRAF and an unknown dasatinib target. Inhibiting BRAF in NSCLC cells expressing wild-type BRAF likewise enhanced these cells' dasatinib sensitivity. Thus, the patient's BRAF mutation was likely responsible for his tumor's marked response to dasatinib, suggesting that tumors bearing kinase-impaired BRAF mutations may be exquisitely sensitive to dasatinib. Moreover, the potential synthetic lethality of combination therapy including dasatinib and BRAF inhibitors may lead to additional therapeutic options against cancers with wild-type BRAF.     

Variation in the extent of microsatellite instability in human cell lines with defects in different mismatch repair genes

Mismatch repair deficiency results in the elevation of mutation rates in tumors, which is especially pronounced in simple repeat sequences (microsatellites). We have investigated the relationship between microsatellite mutagenesis and certain combinations of mutations in mismatch repair genes, using a frameshift reversion assay to determine the spontaneous mutation rates of a dinucleotide microsatellite in two cancer cell lines, HCT116, which has defects in hMLH1 and hMSH3, and HEC-1-A, which has defects in hPMS2 and hMSH6. We found a 10-fold difference in mutation rates between these two cell lines. In addition, a mutant hPMS2 allele, PMS134, which has been reported to have a dominant negative effect, was expressed in mismatch repair-proficient telomerase-immortalized hTERT-1604 fibroblasts and mutation rates were determined. Expression of PMS134 did not elevate mutation rates in hTERT-1604. Combined, these results suggest that mutations in different mismatch repair genes can lead to varying degrees of microsatellite instability. It is also likely that there is heterogeneity in the mutations that are acquired in the absence of mismatch repair, such that some mismatch repair-defective cancer cells also contain mutations in other genes coding for proteins involved in the maintenance of genetic stability.     

Pitfalls of the synthetic lethality screen in Saccharomyces cerevisiae: an improved design

The colony color assay in yeast enables the visual identification of plasmid-loss events. In combination with a plasmid-dependence assay, it is commonly used to identify synthetic interactions between functionally related genes. Frequently, the plasmid carries the ADE3 gene and mutants are recognized as red colonies that fail to produce sectors. In these assays, a high percentage of false-positives is obtained, most of which result from synthetic lethality with the ade3 mutation. Here, we study the nature of these mutants. We report that mutations in the HIP1 and SHM1 genes exhibit synthetic lethality with ade3 deletions. A similar interaction is found between the fur1 and ura3 mutations. Lethality in the absence of the mitochondrial Shm1 and the cytoplasmic Ade3 enzymes indicates that, under certain circumstances, these cellular compartments cooperate in carrying out essential metabolic processes. In addition, we report the identification of a truncated ADE3 allele with a unique coloration phenotype and show that it can be used to improve synthetic lethal screens.     

Alterations of DNA damage-response genes ATM and ATR in pyothorax-associated lymphoma

Pyothorax-associated lymphoma (PAL) is non-Hodgkin's lymphoma that develops from chronic inflammation. Free radicals and oxidative stress generated in the inflammatory lesions could cause DNA damage and thus provide a basis for lymphomagenesis. Ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) genes are responsive genes for DNA damage, therefore potential involvement of these genes in PAL lymphomagenesis was examined in eight PAL cell lines and clinical samples from five cases. ATM mutations were detected in five of eight PAL lines. All but one of these mutations affected the phosphatidylinositol 3-kinase domain, indicating the loss-of-function mutation of ATM gene. Heterozygous mutations of ATR were found in two of eight lines; one a missense and the other a truncation mutation. ATR mutations were also detected in two of five cases in clinical samples from PAL. PAL cells with ATR mutation showed a delay or abrogation in repair for ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) or ultraviolet (UV)-induced DNA single-strand breaks (SSBs), and exhibited a defect in p53 accumulation and failure in cell cycle checkpoint at G1-S phase. These findings showed that mutations of ATR gene result in failure for DNA DSB and SSB repair, suggesting the role of ATM and ATR gene mutations in PAL lymphomagenesis.     

Effect of mutagen-induced cell lethality on the dose response of germline mutations.

Molecular tests for mutations require a sample of tissue from which DNA is extracted, to determine the presence or absence of one or more mutations per sample. To ensure mutation fixation each sample must consist of an equal number of cells that have had one or more DNA replications. In an in vivo test, surviving stem cells compensate to give the same number of cells per sample, leaving as the only evidence for stem cell lethality the increase in mutants of clonal origin because the mutant clone developed from a population of fewer stem cells. A problem is that an increase in mutagen dose increases stem cell death, resulting in a decreased number of surviving target cells, thus giving a downward bias of samples with one or more mutations per sample. To compare in vivo tests with molecular tests we will use as a model system the sex-linked recessive lethal (SLRL) test for germ cell mutations in Drosophila melanogaster. Spermatogonia cells in male larvae were exposed to ENU and mutations detected in sperm cells from adults. The same SLRL data were analyzed by two methods: (1) The conventional analysis of SLRL data, in which each mutation of a cluster of mutations of common origin was counted. (2) An analysis was used to simulate a sample for molecular analysis by determining mutations per male with an equal size sample of progeny per male. With this second analysis a correction factor is required based on the change in cluster size of mutants of common origin.