原发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 instability,CI)途径而发展,多数出现 p53基因的异常表达,少部分肿瘤可同时涉及微卫星不稳定(Microsatellite.instability,MSI)途径;约一半的原发性成胶质细胞瘤既不沿 CI途径发展,也未出现 p53基因的异常,同时又无证据显示与 MSI途径有关     

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.     

大肠癌 p27蛋白表达与 DNA倍体分析的研究

背景与目的细胞周期调控的改变存在于绝大多数肿瘤中,因此可以认为肿瘤是一类细胞周期疾病.p27蛋白是细胞周期负性调控因子的主要成员,同时也是肿瘤的重要预后因子.p27蛋白表达下降及亚细胞定位改变在大肠癌发展中的意义尚不清楚.大肠癌中 p27蛋白表达与 DNA倍体关系尚未见报道.本实验研究大肠癌 p27蛋白表达与临床病理特征及 DNA倍体分析的关系.方法应用免疫组化方法检测 p27蛋白在 40例大肠癌中的表达水平,同时应用病理图像 DNA测量系统对癌组织进行倍体分析.结果p27蛋白在大肠癌中的高表达率为 62.5%(25/40),低表达率为 37.5%(15/40),p27蛋白低表达与大肠癌分化程度低显著相关(P< 0.05).除胞核着色外,32.5%(13/40)的大肠癌出现胞浆免疫阳性反应.p27蛋白胞浆表达与 Dukes分期晚(P< 0.01)、易发生淋巴结转移显著相关(P< 0.05).p27蛋白低表达组 DNA多倍体细胞检出率(22.2± 11.3)% 明显高于 p27蛋白高表达组 [(8.0± 7.7)%,P< 0.001]. 结论p27蛋白表达的下降及亚细胞定位改变促进大肠癌的发展 ; 抑制 DNA多倍体形成可能是 p27蛋白的抑癌机理之一.     

NSCLC中DNA—PKcs的表达及其与凋亡相关蛋白关系的研究

目的检测非小细胞肺癌(NSCLC)中DNA依赖蛋白激酶催化亚基(DNA-PKcs)的表达,探讨其与凋亡相关蛋白表达之间的关系.方法采用免疫组化SP法检测113例NSCLC患者肿瘤组织中DNA-PKcs、p53和bcl-2的表达.结果 NSCLC中DNA-PKcs、p53和bcl-2的检出率分别为89.38%、61.95%及59.29%;DNA-PKcs的表达顺序依次为细支气管肺泡癌>腺癌>腺鳞癌>鳞癌,而且DNA-PKcs表达水平随肿瘤分化程度的增高亦逐渐增高,差异均具有高度显著性(P<0.05),但其表达与淋巴结转移无明显关系;p53在不同临床病理类型NSCLC中的表达无明显差别;bcl-2在不同组织学类型NSCLC中的表达顺序依次为鳞癌>腺鳞癌>腺癌>细支气管肺泡癌,差异具有高度显著性(P<0.01),但其表达与肿瘤分化程度及淋巴结转移无关;DNA-PKcs与p53(P<0.01)、p53和bcl-2(P<0.05)的表达之间有显著相关性.结论 DNA-PKcs高表达导致的DNA损伤修复系统活性增高以及突变型p53和bcl-2过表达导致的凋亡抑制相互影响、共同作用,可能是形成NSCLC放疗抗拒性的重要因素.     

胃癌表皮生长因子受体和p53蛋白的表达及意义

[目的]研究表皮生长因子受体(EGFR)、p53蛋白在胃癌中的表达及其临床意义。[方法]应用ABC免疫组化方法检测75例胃癌中EGFR、p53蛋白的表达。[结果]正常胃粘膜EGFR、p53均呈阴性表达。而胃癌中EGFR、p53的阳性表达率分别为50.1％和41.3％,EGFR的表达与胃癌的分化程度<0.05)相关,与淋巴结转移、浸润程度显著相关(P<0.01);同时p53的表达亦与胃癌的分化程度、淋巴结转移相关(P<0.05),但与浸润深度无显著相关(P<0.05)。[结论]本实验提示检测EGFR和p53的表达可作为判断胃癌预后的一项有用指标。     

CD44V6、nm23蛋白、p53的表达与胃癌淋巴结转移关系的研究

目的探讨CD44V6、nm23蛋白、p53的过度表达与胃癌生物学行为及淋巴结转移的关系。方法应用免疫组化SP法对82例胃癌标本进行CD44V6、nm23蛋白、p53的测定。结果胃癌中CD44V6、nm23蛋白、p53阳性率分别为61.0%、56.1%、68.3%;CD44V6、p53过度表达及nm23低表达与淋巴结转移有关(P<0.05)。胃癌中CD44V6和nm23表达呈负相关(P<0.05)。CD44V6的表达与p53的表达之间有显著相关(P<0.05)。结论CD44V6、nm23蛋白、p53在胃癌中的表达与胃癌的淋巴结转移密切相关,CD44V6与nm23蛋白和p53表达在胃癌淋巴结转移中起协同作用。可为临床预测转移和估计预后提供重要参考指标。     

p27蛋白在喉癌组织中的表达及其临床意义

目的：探讨细胞周期调控因子p27蛋白在喉癌组织中的表达及临床意义。方法：采用免疫组织化学SP法检测41例喉癌组织中p27蛋白的表达，并与12例癌旁正常组织作对照，进行比较分析。结果:41例喉癌组织中p27蛋白的阳性表达率为26．8％(11／41)，12例癌旁正常组织阳性率为58．3％(7／12)，二者差异有统计学意义，P<0．05。p27蛋白阳性表达率与喉癌患者的不同性别、年龄、病程、肿瘤部位、TNM分期以及淋巴结有无转移无关，P>0．05；患者吸烟(14．3％)和饮酒组(10．0％)与各自对照组(53．8％和45．0％)差异有统计学意义，P<0．05；与喉癌组织的病理分级差异有统计学意义，P<0.01。结论:p27蛋白在喉癌的发生、发展过程中起着一定的作用，可作为判断喉癌恶性程度的一个重要参考指标。     

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。     

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。     

鼻咽癌及癌前病变组织中p16蛋白表达的研究

目的观察p16蛋白在鼻咽癌组织中的表达状态,探讨其与鼻咽癌的发生、发展关系。方法应用S-P免疫组化法检测p16蛋白在50例鼻咽癌、20例鼻咽粘膜慢性炎组织中的表达。结果鼻咽癌p16蛋白表达率为24.0%,显著低于鼻咽粘膜慢性炎组(P<0.01)。角化性鳞癌p16蛋白的表达率明显高于分化型非角化性癌和未分化癌(P<0.05)。20例淋巴结转移阳性组中p16蛋白表达率显著低于14例阴性组(P<0.05)。结论p16蛋白的缺失可能涉及鼻咽癌的发生、发展过程,且与鼻咽癌的转移、组织学分型有关。     

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.     

Evaluation of cell survival, DNA double strand breaks, and DNA synthesis during concurrent camptothecin and X-radiation treatments

We evaluated cell survival, DNA double strand breaks (dsbs), and DNA synthesis following camptothecin (CPT) alone or concurrent CPT and X-radiation treatments in exponential-phase cultures of a radioresistant human melanoma cell line. Cell survival was measured by a clonogenic assay. DNA dsbs were measured by pulsed-field gel electrophoresis. DNA synthesis was measured by incorporation of (3)H-thymidine. We found that (i) concurrent CPT and X-radiation interacted additively, unlike previous results with plateau-phase cultures of these cells, which showed synergistic interaction; (ii) there were strong negative correlations (correlation coefficients of at least 0.82) between clonogenic surviving fractions and DNA dsbs following CPT alone or concurrent CPT and radiation treatments; and (iii) concurrent CPT and radiation (10 Gy) treatment did not completely inhibit DNA synthesis, even though addition of radiation to CPT did further decrease DNA synthesis (relative to CPT alone) at CPT concentrations below 20 microM. Our results suggest that during concurrent CPT and radiation treatment residual DNA dsb levels were good indicators of cell killing and that the absence of complete inhibition of DNA synthesis could at least in part explain the additive interaction between CPT and radiation.