原发性与复发性成胶质细胞瘤中DNA错配修复蛋白、p53蛋白表达和DNA倍性的差异

目的探讨原发与复发性成胶质细胞瘤的临床病理特点和分子遗传学差异。方法应用免疫组化和流式细胞学方法对32例广东籍成胶质细胞瘤hMSH2，hMLH1和p53蛋白表达及DNA倍性进行检测，结合临床病理学资料，分析它们之间的相关性。结果32例成胶质细胞瘤患者的DNA倍性与p53的表达存在显著的相关性(P<0.05)，23例非整倍体DNA含量的肿瘤中，有20例(87.0％)出现p53蛋白的过度表达。在原发与复发性成胶质细胞瘤中，肿瘤的DNA含量和p53蛋白表达有显著的差异性(P<0.05)，复发性成胶质细胞瘤中，有87.5％(14/16)出现了p53蛋白的过度表达，93.8％(15/16)为非整倍体的DNA含量；而原发性成胶质细胞瘤，则有56.3％(9/16)呈p53表达正常表达，50.0％(8/16)是二倍体或近二倍体DNA含量。另外，丢失了hMSH2蛋白的2例成胶质细胞瘤，全部是复发性的肿瘤，均呈p53蛋白的过度表达和非整倍体的DNA含量。结论广东籍成胶质细胞瘤患者的发病年龄明显低于欧美人。绝大多数的复发性成胶质细胞瘤是沿染色体不稳定性(Chromosomal     

胶质瘤染色体1p和19q杂合性缺失与O6-甲基鸟嘌呤DNA甲基转移酶p53和Ki-67蛋白表达的关系

目的 探讨胶质瘤染色体1p和19q杂合性缺失(LOH)与O6-甲基鸟嘌呤DNA甲基转移酶(MGMT)、p53和Ki-67蛋白表达的关系.方法 采集146例胶质瘤(45例少突胶质细胞瘤、42例少突星形细胞瘤和59例星形细胞瘤)的肿瘤组织和血液标本,采用聚合酶链反应结合变性高效液相色谱技术检测染色体1p和19q LOH,免疫组化法检测肿瘤组织中MGMT、p53和Ki-67蛋白的表达,并进一步分析其与胶质瘤临床病理特征的关系.结果 少突胶质细胞肿瘤和星形细胞瘤中,1p LOH的发生率分别为59.8％和33.9％,差异有统计学意义(P=0.002);1p和19q LOH的发生率分别为42.5％和16.9％,差异有统计学意义(P=0.001).MGMT低表达和Ki-67高表达多发生于少突胶质细胞肿瘤中,发生率分别为65.5％和54.0％,而p53高表达多发生于星形细胞瘤和少突星形细胞瘤中,发生率为75.2％.在87例少突胶质细胞肿瘤中,1p LOH和MGMT蛋白低表达多发生于Ⅱ级少突胶质细胞肿瘤中,发生率分别为72.5％和87.5％,而p53和Ki-67蛋白高表达多发生于Ⅲ级少突胶质细胞肿瘤中,发生率分别为83.0％和76.6％.1p和19q LOH在非颞叶和颞叶肿瘤的发生率分别为55.6％和21.2％(P=0.002).1p LOH与19q LOH、MGMT蛋白表达与p53蛋白表达、MGMT蛋白表达与Ki-67蛋白表达、1p和19q LOH与p53蛋白表达、1p LOH与Ki-67蛋白表达均有关(均P＜0.05).结论 1p和19q LOH及MGMT、p53和Ki-67的蛋白表达与胶质瘤的临床病理学特征有关,检测其LOH状态和表达水平对胶质瘤的诊断和治疗具有指导作用.     

MDM2和p53在反应性及肿瘤性星形胶质细胞中的表达

目的：检测MDM2和p53蛋白在星形胶质细胞反应性增生与星形胶质细胞瘤中的表达，探讨二者在胶质瘤形成和发展中的作用及其相关性。方法：应用组织芯片和免疫组化染色技术检测正常脑组织、星形胶质细胞反应性增生、低级别（Ⅰ-Ⅱ级）和高级别（Ⅲ-Ⅳ级）星形胶质细胞瘤中MDM2和p53蛋白的表达情况。结果：正常脑组织中MDM2和p53蛋白均呈阴性表达；反应性增生组、低级别肿瘤组及高级别肿瘤组中MDM2蛋白的阳性率分别为32.7％（16／49）、59．2％（29／49）、80．0％（40／50）；p53蛋白的阳性率分别为27．3％（12／49）、57．1％（28／49）、82．0％（41／50）。二者阳性表达率均随着病变恶性程度的增加而升高，MDM2和p53在各实验组间的比较差异均有统计学意义（P〈0．05）；且MDM2和p53表达密切相关（P〈0．05）。结论：MDM2和p53在星形胶质细胞反应性增生及星形胶质细胞瘤中均呈不同程度的过度表达，且随着病变恶性程度的增加表达水平增高，MDM2扩增和p53突变是胶质瘤发生的早期事件，二者的联合检测可能会对星形胶质细胞反应性增生与低级别胶质细胞瘤的鉴别诊断以及星形胶质细胞瘤的早期诊断提供一定的依据。     

散发性微卫星不稳定性结直肠癌患者Mt-p53、bcl-2、hMLH1和hMSH2蛋白表达及意义

目的探讨散发性微卫星不稳定性结直肠癌患者突变型P53基因(Mt-p53)、B淋巴细胞瘤-2基因(bcl-2)、人类错配修复基因1(hMLH1)和人类错配修复基因2(hMSH2)蛋白表达及临床意义。方法选取2017年10月至2018年11月间山东大学齐鲁医院收治的149例经手术切除的散发性结直肠癌(SRC)标本作为肠癌组,另收集距癌组织边缘10cm处的健康肠组织标本149例为健康组。采用PCR仪及遗传分析仪对肠癌组标本进行微卫星不稳定(MSI)检测,采用免疫组化检测试剂盒检测两组标本Mt-p53、bcl-2、hMLH1和hMSH2蛋白表达情况。结果 149例SRC标本中,位点D2S123 MSI阳性率为12. 1%,位点BAT26MSI阳性率为18. 1%,位点D17S261 MSI阳性率为10. 1%,位点D17S799 MSI阳性率为8. 1%。其中MSI-L为18例,MSI-H为22例,MSI总阳性率为26. 8%。149例肠癌组中Mt-p53蛋白表达阳性率为67. 1%(100例),bcl-2蛋白表达阳性率为77. 9%(116例),hMLH1蛋白表达阳性率为73. 2%(109例),hMSH2蛋白表达阳性率为71. 1%(106例)。149例健康组中Mt-p53、bcl-2、hMLH1和hMSH2蛋白表达阳性率均为100. 0%(149例)。两组标本各蛋白表达情况比较,差异均有统计学意义(均P <0. 01)。MSI阳性标本及阴性标本中Mt-p53、bcl-2和hMSH2蛋白表达阳性率比较,差异均无统计学意义(均P> 0. 05)。MSI阳性标本及阴性标本中hMLH1蛋白表达阳性率比较,差异有统计学意义(P <0. 05)。结论 SRC的发生不仅与Mt-p53的失活和bcl-2的激活有关,还与错配修复(MMR)基因突变引发的MSI相关,是SRC新的重要发生机制。MSI与Mt-p53、bcl-2和hMSH2蛋白表达情况相关性不大,但部分MSI与hMLH1蛋白表达显著相关,其余MSI可能涉及其他MMR基因。     

胆囊癌中ER、PR、p53 表达及DNA 含量的研究

 目的 探讨ER、PR、p53表达及DNA倍性在胆囊癌发生发展中的意义。方法 用免疫组化二步法及HPIAS-1000DAN图像分析系统检测27例胆囊癌组织中ER、PR、p53的表达及DNA倍性。结果 ①胆囊癌中ER、PR及p53的阳性表达率分别为77．78％(21／27)、74．07％(20／27)、66．67％(18／27)，②胆囊高分化腺癌中DNA倍体为高二倍体；中分化腺癌为高二倍体至亚四倍体；低分化腺癌为四倍体。③胆囊癌分化越差，浸润越深，其ER、PR、p53的阳性表达率越高(P<0．05)，DNA含量越高，多倍体越明显(P<0．05)，预后越差。结论 ER、PR、p53阳性表达率及DNA倍性改变与胆囊癌的分化程度和浸润深度有关。ER、PR过度表达可能会诱发p53基因突变，两者在胆囊癌的发生发展中有密切的内在联系。     

胃癌组织中p53蛋白表达和微卫星不稳定及其与预后的相关性研究

摘 要：［目的］ 探讨胃癌组织中 p53 蛋白表达与微卫星不稳定(microsatellite instability,MSI)与胃癌临床病理特征及预后的关系。［方法］ 选取行胃癌手术患者104例,采用免疫组织化学法检测肿瘤组织中p53蛋白和错配修复蛋白(mismatch repair protein,MMRP)（MLH1、MSH2、MSH6和PMS2）表达,分析p53表达和MSI与患者临床病理参数的关系。术后患者随访2年,采用Logistics回归分析影响患者预后的危险因素。［结果］ 胃癌组织中p53、MLH1、MSH2、MSH6和PMS2均定位于肿瘤细胞核。104例胃癌中p53阳性46例(44.23%);MMRP阳性65例(62.50%),39例MMRP表达缺失(37.50%),其中MLH1、MSH2、MSH62及PMS表达缺失分别为25例(24.04%)、19例(18.27%)、18例(17.31%)和 22例(21.15%)。胃癌低分化组及Ⅲ期组中p53阳性表达率显著降低(P<0.05)。p53在癌组织中的表达与MSI呈负相关(r=-0.486,P<0.05)。Logistic多因素回归分析显示,TNM分期、MSI和p53阴性是影响患者预后的独立危险因素。［结论］ 胃癌组织中p53与MSI呈负相关。对胃癌组织监测p53与MSI对制定给药方案及评估预后具有重要意义。     

食管鳞癌组织中肺耐药蛋白表达和DNA含量的定量分析

目的：探讨食管鳞癌组织中肺耐药相关蛋白（lung resistance-related protein,LRP）的表达和DNA含量检测的临床意义。方法：应用流式细胞术（flow cytometry,FCM）定量分析52例原发食管鳞癌组织和相应癌旁组织中LRP蛋白表达及DNA含量的变化状况。结果：食管鳞癌组织LRP表达的相对荧光强度（RFI）的中位数（M）为1.39,而相应癌旁组织为0.75;差异有统计学意义,P〈0.05。癌组织DNA指数（DI）、SPF和PI显著高于相应癌旁组织,P〈0.05。LRP蛋白表达在不同的性别、病理分级、临床分期和有无淋巴结转移间的表达均差异无统计学意义,P〉0.05。DNA倍体和SPF、PI均与性别、病理分级和临床分期无明显关系,P〉0.05;但与有无淋巴结转移有关,P〈0.05。非整倍体肿瘤患者淋巴结转移率（63.9%,23/36）高于二倍体肿瘤患者淋巴结转移率（18.8%,3/16）,P〈0.05。有淋巴结转移患者SPF和PI显著高于无淋巴结转移患者,P〈0.05。非整倍体肿瘤LRP蛋白表达水平（Median=1.42）略高于二倍体肿瘤（Median=1.35）,但两者差异无统计学意义,P〉0.05。结论：LRP蛋白表达和DNA含量在食管鳞癌发生中起重要作用。DNA含量与淋巴结转移有关,可作为预测食管鳞癌预后的指标。     

FCM对星形细胞瘤石蜡包埋标本的DNA含量分析

本实验利用FCM对36例不同病理分级的星形细胞瘤石蜡包埋标本进行DNA含量分析,并对其与肿瘤病理组织学诊断及预后之间的关系进行了初步探讨。结果表明:随着星形细胞瘤恶性度的增高,异倍体细胞的出现率亦有上升趋向。异倍体细胞的出现确是恶性星形细胞瘤的特征性表现之一,但恶性星形细胞瘤其DNA含量也可以是二倍体或近二倍体,此种情况在多形性胶质母细胞瘤中尤为明显。而标志着肿瘤增殖活性的S期细胞百分此含量(S期％)在星形细胞瘤Ⅱ级中虽有升高趋势,但各级之间并无显著差异。DNA异倍体性与星形细胞瘤的预后之间也无明确关系。     

p53和hMSH2蛋白在大肠癌组织表达的意义

目的:探讨抑癌基因p53和错配修复基因hMSH2蛋白表达在大肠癌发生中的意义. 方法:用免疫组化S-P法检测89例大肠癌、 37例结肠炎组织中p53和hMSH2蛋白的表达情况.结果:p53蛋白在大肠癌组表达64.04%(57/89)明显高于结肠炎组8.11%(3/37)(P<0.01);hMSH2蛋白在大肠癌组表达84.27%(75/89) 明显高于结肠炎组21.62%(8/37)(P<0.05).p53 蛋白阳性与阴性大肠癌组hMSH2蛋白阳性表达率分别为50.56%和35.95%,两种蛋白表达呈正相关(P<0.05).p53 蛋白阳性与阴性结肠炎组hMSH2蛋白阳性表达率分别为100.0%和14.71%,两种蛋白表达呈正相关(P<0.01).结论:碱基错配的增多导致突变的积累可能是大肠癌的发病原因之一.     

肺癌微卫星不稳定性与错配修复基因缺陷的相关性研究

目的 通过对肺癌微卫星不稳定性(MSI)的分析与错配修复基因蛋白表达的检测,探讨肺癌发病的分子机制.方法 从50例肺癌患者的正常肺组织、癌组织中提取DNA;SSCP法检测标本中MSI发生情况;免疫组织化学法检测错配修复基因hMLH1及hMSH2在肺癌中的表达情况.结果 50例肺癌中微卫星高度不稳定(MSI-H)14例,低度不稳定(MSI-I)21例,稳定(MSS)15例,正常组织中未出现MSI,两者之间差异有统计学意义(P=0.000);免疫组化结果显示hMLH1在MSI肺癌组织中常为缺失表达,表达率为74%(37/50);hMSH2在MSI肺癌组织中也呈缺失表达,表达率为32%(16/50);而在MSS肺癌组织中均显示hMLH1、hMSH2基因蛋白表达阳性.结论 肺癌的发生可能存在MSI途径,而hMLH1、hMSH2的表达失活则可能导致MSI的发生,因此,MSI可作为肺癌诊断的指标之一.     

Effect of deferoxamine on DNA synthesis, DNA repair, cell proliferation, and differentiation of HL-60 cells

The ribonucleotide reductase inhibitors deferoxamine and hydroxyurea induce monocyte-macrophage cell differentiation in the leukemic cell line HL-60 as judged by the expression of cell surface antigens, nonspecific esterase activity, and morphological changes. Treatment of HL-60 cells with deferoxamine results in inhibition of DNA synthesis and irreversible loss of colony-forming ability. In addition, both deferoxamine and hydroxyurea caused an increase in the number of DNA strand breaks in HL-60 cells. A DNA methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine, also caused cellular differentiation in HL-60 cells associated with DNA strand breaks. These observations are consistent with a role for DNA damage or for inhibition of DNA synthesis and repair in the differentiation process of HL-60 cells.     

The Origins of DNA Breaks: A Consequence of DNA Damage, DNA Repair, or Apoptosis?

DNA breaks can arise from many sources after incubation of cells with toxic agents. Very few agents break DNA directly, rather most breaks occur as a result of metabolic participation by the cell, such as during attempts to repair the damage. It is now realized that many DNA breaks arise as a consequence of steps in the pathway of cell death. Upon reanalyzing the methodology commonly used to detect DNA breaks, it is evident that many studies would not have observed DNA breaks associated with cell death. Frequently experimental conditions have been used that are extremely toxic to cells with the justification that the cells were still viable as measured by their ability to exclude dyes such as trypan blue. However, the DNA digestion associated with cell death by apoptosis occurs prior to changes in membrane integrity. Because the possibility of endogenous endonuclease activity was not realized, many studies may have inaccurately assumed that DNA breaks arose during, for example, inhibition of DNA repair or as intermediates in recombination. In light of the new understanding of apoptosis and the formation of DNA breaks as an early event in cell death, it is important to both reevaluate past conclusions and to ensure that future studies fully consider the breaks derived from the cytotoxicity of every agent under investigation.     

DNA adducts, mutations and cancer

Anyone having lectured on DNA adducts is likely to be familiarwith the first question after the presentation: ‘Whatis the relationship of DNA adducts and cancer?’. Althoughthere are a number of reviews on the topic (1–5), my personalresponse, instead of going into lengthy circumstantial argumentationand hand-waving, has been a quotation from Mortimer Mendelson:‘I wouldn't like to have my DNA messed up’. In thiscommentary I will indulge in this still circumstantial argumentation.The reason being that never before has it appeared to be soeasy, or less difficult, to entertain the question. Hopefullythis effort will spare colleagues confronting the same questionsome time. At the same time I will take the opportunity to describedevelopments in the identification of DNA adducts. A vivid historyof the development of ideas on chemical causes of cancer hasbeen published by Lawley (2).     

Overexpression of the DNA mismatch repair factor, PMS2, confers hypermutability and DNA damage tolerance

Inherited defects in genes associated with DNA mismatch repair (MMR) have been linked to familial colorectal cancer. Cells deficient in MMR are genetically unstable and demonstrate a tolerance phenotype in response to certain classes of DNA damage. Some sporadic human cancers also show abnormalities in MMR gene function, typically due to diminished expression of one of the MutL homologs, MLH1. Here, we report that overexpression of the MutL homolog, human PMS2, can also cause a disruption of the MMR pathway in mammalian cells, resulting in hypermutability and DNA damage tolerance. A mouse fibroblast cell line carrying a recoverable lambda phage shuttle vector for mutation detection was transfected with either a vector designed to express hPMS2 or with an empty vector control. Cells overexpressing hPMS2 were found to have elevated spontaneous mutation frequencies at the cII reporter gene locus. They also showed an increase in the level of mutations induced by the alkylating agent, methynitrosourea (MNU). Clonogenic survival assays demonstrated increased survival of the PMS2-overexpressing cells following exposure to MNU, consistent with the induction of a damage tolerance phenotype. Similar results were seen in cells expressing a mutant PMS2 gene, containing a premature stop codon at position 134 and representing a variant found in an individual with familial colon cancer. These results show that dysregulation of PMS2 gene expression can disrupt MMR function in mammalian cells and establish an additional carcinogenic mechanism by which cells can develop genetic instability and acquire resistance to cytotoxic cancer therapies.     

Sequence-selective depurination, DNA interstrand cross-linking and DNA strand break formation associated with alkylated DNA.

Alkylation of DNA by chloroethylnitrosourea (CNU) at the guanine N7 position has been shown to occur in a sequence-selective fashion. In this report we find that the depurination of these alkylated sites occurs with two distinct kinetic components--GG sequences depurinate within 30 min of exposure to CNU, while depurination at GT sequences is first observed after 1 h and continues to increase 16 h after drug exposure. These apurinic sites are converted to DNA strand breaks and constitute less than 10% of the total sites of guanine N7 alkylation. Spermidine was found to decrease alkylation in 5'-GG-3' sequences but increases alkylation at 5'-GTC-3' sequences. These findings suggest that the majority of the guanine N7 alkylations formed by CNU are stable, with a minor adduct being responsible for the slow depurination event. We propose that the rapid depurination induced by CNU occurs from an initial guanine O6 alkylation, which then depurinates via a guanine O6-N7 cyclized intermediate. We also propose that the resulting apurinic sites may lead to DNA interstrand cross-linking (ISC). In support of these hypotheses we show that (i) DNA modified with the monoalkylating agent dimethylsulfate forms DNA ISC upon depurination; (ii) ellagic acid enhances the level of guanine N7 alkylation and alters the pattern of sequence selectivity shown by three bifunctional chloroethylating agents CNU, mitozolomide and methyl 3-(2-chloroethyl)-4-oxoimidazo[5,1-d]-1,2,3,5-tetrazine-8-ca rboxylate but not with nitrogen mustard; (iii) ellagic acid has no effect upon the frequency of alkylation observed with the monofunctional alkylators N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and methylmethanesulfonate; (iv) ellagic acid increases the frequency of depurination and strand break formation induced by CNU without affecting the sequence-selective pattern of depurination.