Preliminary study on the loss of heterozygosity at 17p13 in gastric and colorectal cancers

AIM: To evaluate the role of p53 in the development and progression of colorectal cancer and gastric carcinoma by analyzing the loss of heterozygosity (LOH) at 17p13.1 and 17p13.3.METHODS: LOH at the p53 gene locus and 17p13.3 were examined in 22 cases of gastric carcinoma and 14 cases of colorectal cancer by Southern blot analysis.RESULTS: Of the 22 gastrocarcinoma cases, 12 (54%) were heterozygous and LOH was detected in 6 (50%) of the 12 informative cases. In the 14 colorectal cancer cases, 10 (71%) were heterozygous, and LOH was detected in 6 (60%) of the 10 informative cases.CONCLUSION: LOH at the p53 gene locus is a frequent event in multiple step carcinogenesis progression. The high frequency of LOH at 17p13.3 suggests that there may be another tumor suppresser gene in that chromosome region.     

肿瘤抑制基因p53及p16与胃癌生物学行为的关系

目的探讨肿瘤抑制基因ｐ５３和ｐ１６异常与胃癌生物学行为的关系．方法采用免疫组化ＡＢＣ法检测５８例原发性胃癌（男３８例，女２０例，年龄３７岁～７６岁），Ｐ５３和Ｐ１６蛋白的表达变化．所有组织均新鲜取材，并迅速用８５０ｍｌ／Ｌ酒精固定，石蜡包埋，连续切片．结果受检组织中ｐ５３和ｐ１６阳性表达率分别为５１７％（３０／５８）和４８３％（２８／５８）．Ｐ５３蛋白在低分化胃癌（７００％）、进展期胃癌（５６９％）、淋巴结阳性胃癌（７４１％）中的表达率高于相应的高分化、早期、淋巴结阴性胃癌的表达率（２７３％，１４３％，３２３％）（Ｐ＜００５），且ｐ５３高表达多见于弥散型胃癌（同肠型胃癌比）、累及浆膜的胃癌也较局限于粘膜层的胃癌有更高的Ｐ５３蛋白表达（Ｐ＜００５）；Ｐ１６蛋白表达与胃癌大多数生物学行为无明显关系，但其在淋巴结阳性胃癌中的表达率（３３３％），低于淋巴结阴性胃癌中的表达率（６１３％）；相关性分析显示；ｐ５３阳性组织大多伴有Ｐ１６蛋白阳性表达（Ｐ＜００５）．结论Ｐ５３蛋白异常表达对胃癌生物学行为有广泛影响，Ｐ１６蛋白表达缺失可能是胃癌淋巴结转移的重要促发因素．     

Genome-wide search for loss of heterozygosity in Chinese patients with sporadic colorectal cancer

In an attempt to integrally investigate the loss of tumor suppressor genes and search for putative suppressor loci associated with tumor occurrence and progression, we conducted a genome-wide loss of heterozygosity (LOH) study of 83 tumor samples obtained from Chinese patients with sporadic colorectal cancer. We employed 400 fluorescence-labeled microsatellite marker primers to amplify the corresponding loci of the genomic DNA and then electrophoresed the polymerase chain reaction products and analyzed the fluorescent signals. The LOH frequencies were high (>35%) but were not associated with the tumor stage and progression in 20 loci, including the regions where TP53, E-cadherin, deleted in colorectal carcinoma (DCC), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), mothers against decapentaplegic, Drosophila, homolog of 2 (MADH2) and mothers against decapentaplegic, Drosophila, homolog of 4 (MADH4) reside. Loss of other loci, including two narrow regions on chromosome 2, was found to relate to the tumor stage, suggesting that this genomic instability may contribute to tumor progression.     

散发性结直肠癌染色体3q24-25区杂合性缺失的精细定位

目的探讨散发性结直肠癌3号染色体杂合性缺失,对3q24-25区进行精细定位,寻找新的结直肠癌抑癌基因。方法将3号染色体采用13个微卫星DNA标记,在3q24-25区另取6个微卫星标记对83例结直肠癌患者的肿瘤和正常组织进行PCR反应。PCR产物在ABI Prism 377自动荧光测序仪进行电泳3 h,以Genescan3.1和Genotyper 2.1软件进行基因分型。结果 3号染色体上发现了2个高频杂合缺失区即3p14区和3q24-25区,在3q24-25区界定了1个大约2 cm的跨越D3S1279位点的杂合缺失区。结论 3q24-25区存在1个精细的杂合缺失区域,该区很可能存在1个或多个与结直肠癌相关的新抑癌基因。     

胃癌p53基因表达与转移及预后的研究

目的观察胃癌组织ｐ５３基因的超表达及其与预后的关系。方法用抗人Ｐ５３基因蛋白单克隆抗体Ｓ＿Ｐ免疫组织化学方法，观察１２８例胃癌组织ｐ５３表达状况，并对ｐ５３表达与胃癌淋巴结转移状态和术后５年生存率进行比较分析。结果胃癌组织１２８例的ｐ５３表达阳性率为４３８％（５６／１２８）；ｐ５３表达阳性和阴性组的胃癌局部和远处淋巴结转移率分别为６７９％（３８／５６）和５１４％（３７／７２），两者经统计学处理无显著性差异（Ｐ＞００５）。获得随访９８例，胃癌术后５年生存率的随访结果显示，ｐ５３阳性和阴性组分别为３８１％（１６／４２）和３０１％（１７／５６），两组间无统计学意义（Ｐ＞００５）。结论胃癌的发生与ｐ５３基因突变关系密切，并可用免疫组化检测，但Ｐ５３基因蛋白在胃癌组织中的超表达，似不能作为判断胃癌预后的参考指标，应进一步探讨     

胃癌微卫星不稳定性和抑癌基因杂合缺失

目的研究微卫星不稳和抑癌基因缺失在胃癌发生中的作用．方法采用ＰＣＲ为基础的方法,检测了５３例胃癌中６个微卫星标记突变及ＡＰＣ／ＭＣＣ和ＤＣＣ基因杂合缺失（ＬＯＨ）．结果胃癌微卫星不稳的检出率为３２１％（１７／５３）．７例（１３２％）为微卫星高频率不稳（３个以上微卫星标志）,１０例（１８９％）为微卫星低频率不稳（１或２个微卫星标记）．肠型胃癌微卫星高频率不稳的发生率（２５０％）显著高于弥漫型胃癌（３４％）（Ｐ＜００５）．高频率不稳组未发现有ＡＰＣ,ＭＣＣ和ＤＣＣ基因ＬＯＨ,微卫星高频率不稳与ＡＰＣ／ＭＣＣ和ＤＣＣ基因ＬＯＨ呈负相关．结论微卫星不稳在部分胃癌,特别是肠型胃癌早期发生中起重要作用,高频率不稳胃癌与遗传性非息肉大肠癌有共同的特点．与此相反,低频率不稳和无不稳胃癌可能通过ＬＯＨ病理途径发生     

Refined mapping of loss of heterozygosity on 1q31.1-32.1 in sporadic colorectal carcinoma

AIM： To explore precise deleted regions and screen the candidate tumor suppressor genes related to sporadic colorectal carcinoma. METHODS： Six markers on 1q31.1-32.1 were chosen. These polymorphic microsatellite markers in 83 colorectal cancer patients tumor and normal DNA were analyzed via PCR. PCR products were electrophoresed on an ABI 377 DNA sequencer. Genescan 3.1 and Genotype 2.1 software were used for Loss of heterozygosity （LOH） scanning and analysis. Comparison between LOH frequency and clinicopathological factors was performed by χ2 test. RESULTS： 1q31.1-32.1 exhibited higher LOH frequency in colorectal carcinoma. The average LOH frequency of 1q31.1-32.1 was 23.0%, with the highest frequency of 36.7% （18/49） at D1S2622, and the lowest of 16.4% （11/67） at D1S412, respectively. A minimal region of frequent deletion was located within a 2 cM genomic segment at D1S413-D1S2622 （1q31.3-32.1）. There was no significant association between LOH of each marker on 1q31.1-32.1 and the clinicopathological data （patient sex, age, tumor size, growth pattern or Dukes stage）, which indicated that on 1q31.1-32.1, LOH was a common phenomenon in all kinds of sporadic colorectal carcinoma. CONCLUSION： Through our refined deletion mapping,the critical and precise deleted region was located within 2 cM chromosomal segment encompassing 2 loci （D1S413, D1S2622）. No significant association was found between LOH and clinicopathologic features in 1q31.1-32.1.     

Loss of heterozygosity and analysis of mutation of p53 in hepatocellular carcinoma

Abstract Thirty-six hepatocellular carcinoma (HCC) tissues obtained from 34 patients were classified according to histological diagnosis into six well-differentiated HCC, 20 moderately differentiated HCC and 10 poorly differentiated HCC. High molecular weight DNA was prepared from each tumour and the corresponding non-tumour tissue. Loss of heterozygosity (LOH) on chromosomes 4q, 5q, 10q, 11p, 16q, 17p, mutation of the p53 gene and polymorphism of intron 25 of the retinoblastoma (RB) gene were simultaneously analysed. The patients were composed of three cases of small HCC (the diameter of which was < 3 cm) and 31 cases of advanced HCC. Twenty-nine of 34 (85.3%) patients analysed had been exposed to hepatitis B virus and/or hepatitis C virus. The frequencies of LOH on seven chromosomes were 57.9% in 17p13.3, 45.1% in 17p, 45.1% in 11p, 41.9% in 5q, 41.9% in 16q24, 29.0% in 4q, 25.8% in 10q in advanced HCC (four of well differentiated, 18 of moderately differentiated and nine of poorly differentiated carcinoma). In contrast, LOH was observed on 4q, 5q, 16q and 17p in 33% (1/3) of the small HCC (two of well differentiated and one of moderately differentiated carcinoma). The mutation of the p53 genes and polymorphism of the RB gene were present in 25.8% (8/31) and 12.9% (4/31) of the advanced tumours, respectively, but the mutation was not found in small HCC. LOH on every chromosome and the p53 mutation were observed more frequently in more advanced tumours, and the genetic changes accumulated with the increase of the histopathological grade. These findings suggest that the accumulation of genetic changes in multiple tumour suppressor genes is involved in the progression of HCC.     

Loss of heterozygosity and mRNA expression at DCC locus in gastric cancer

ＬｏｓｓｏｆｈｅｔｅｒｏｚｙｇｏｓｉｔｙａｎｄｍＲＮＡｅｘｐｒｅｓｓｉｏｎａｔＤＣＣｌｏｃｕｓｉｎｇａｓｔｒｉｃｃａｎｃｅｒＷＡＮＧＤｏｎｇＸｕ，ＦＡＮＧＤｉａｎＣｈｕｎ，ＬＵＯＹｕａｎＨｕｉａｎｄＬＩＵＷｅｉＷｅｎＳｕｂｊｅｃｔｈｅａｄｉ...     

Frequent loss of heterozygosity at 8p22 chromosomal region in diffuse type of gastric cancer

AIM： TO study the loss of heterozygosity （LOH） at 8p21-23 locus in diffuse gastric cancer.
METHODS： To evaluate the involvement of this region in gastric cancer, we used eight microsatellite markers covering two Mb of mentioned region, to perform a high-resolution analysis of allele loss in 42 cases of late diffuse gastric adenocarcinoma.
RESULTS： Six of these STS makers： D8S1149, D8S1645, D8S1643, D8S1508, D8S1591, and D8S1145 showed 36%, 28%, 37%, 41%, 44% and 53% LOH, respectively.
CONCLUSION： A critical region of loss, close to the NAT2 locus and relatively far from FEZ1 gene currently postulated as tumor suppressor gene in this region.     

hTERT、P53在胃癌及癌前病变中的表达及相关性研究

目的探讨端粒酶逆转录酶（hTERT）基因、p53蛋白在胃癌（GC）及癌前病变中的表达及相关性。方法采用免疫组化和原位杂交方法分别检测130例GC及癌前病变组织标本中p53和hTERT mRNA的表达。结果p53蛋白在慢性表浅性胃炎（CSG）、慢性萎缩性胃炎（CAG）、非典型增生（DYS）、GC中的表达率分别为5％、25％、50％、62．5％,其中GC与CSG、CAG比较有统计学差异：hTERT mRNA在CSG、CAG、DYS及GC中的表达率分别是0、10％、30％、78．75％,GC与CSG、CAG、DYS比较有统计学差异。p53阳性的GC组织中hTERT表达均为阳性,p53阴性的GC组织中hTERT阳性表达率为66．7％。结论hTERT是一个比p53更好的恶性肿瘤标记物;p53基因突变可导致端粒酶活化,但端粒酶激活可能不完全依赖于p53基因的调控。     

原发性胃癌p53基因突变

目的 p53基因是当前抑癌基因研究中的热点之一。迄今,有关 p53基因异常与胃癌临床病理学参数如大体类型、临床分期、组织分化程度,浸润深度及淋巴结转移之间的关系尚无定论。Tumura 报告p53基因改变主要发生于异倍体瘤,国内尚无报道。本实验目的主要是分析中国人原发性胃癌 p53基因突变与这些病理参数,包括 DNA 倍体之间的关系。方法用聚合酶链式反应—单构象多态分析(PCR—SSCP)技术对20例原发性胃癌 p53基因外显子5—8突变进行检测。结果 8例(40%)发生了突变,其中2例发生在外显子7,4例发生在外显子8。0至Ⅲ期均有突变存在。66.7%(6/9)的异倍体瘤检测到了p53突变,而二倍体瘤中只有18.2%(2/11)发生了 p53突变。结论 p53基因突变与胃癌临床病理参数如大体类型、分期、组织分化程度、浸润深度及淋巴结转移之间无明显关系,而与胃癌 DNA 倍体改变有关。     

大肠癌组织APC/MCC基因杂合的缺失

目的研究大肠癌ＡＰＣ／ＭＣＣ基因杂合缺失的作用。方法采用ＰＣＲ技术，并配合限制性片段长度多态现象（ＲＦＬＰ）分析，对４１例外科手术切除大肠癌组织ＡＰＣ／ＭＣＣ基因杂合缺失（ＬＯＨ）进行检测。结果在大肠癌４１例中ＡＰＣ基因属信息个体者２５例，检出ＬＯＨ７例，占２８．０％；ＭＣＣ基因属信息个体者２２例，检出ＬＯＨ８例，占３６．４％。若将ＡＰＣ和ＭＣＣ基因进行综合分析，则信息个体者３６例，检出ＬＯＨ１４例，占３８．９％。ＡＰＣ和ＭＣＣ基因的ＬＯＨ与肿瘤大小、组织学类型、浆膜浸润、淋巴结转移及Ｄｕｋｅｓ分期无关（Ｐ＞０．０５）。结论ＡＰＣ和ＭＣＣ基因ＬＯＨ是大肠癌的常见改变     

5'-CpG island methylation of the LKB1/STK11 promoter and allelic loss at chromosome 19p13.3 in sporadic colorectal cancer

BACKGROUND: In patients with Peutz-Jeghers syndrome (PJS), causative germline mutations in the LKB1/STK11 gene on chromosome 19p13.3 have been identified. Because of the loss of heterozygosity (LOH) at 19p13.3 in hamartomas and the cancer susceptibility of patients with PJS, LKB1/STK11 is suggested to act as a tumour suppressor. However, the frequency of genetic and epigenetic inactivation of LKB1/STK11 in sporadic tumours is unclear. AIMS: To investigate the LKB1/STK11 gene for promoter hypermethylation and allelic loss in tumour specimens of patients with sporadic colorectal cancer. METHODS: DNA from 50 consecutive paraffin embedded sporadic colorectal adenocarcinomas and corresponding normal epithelium was extracted. After bisulphite treatment, specimens were analysed for methylation of the LKB1/STK11 promoter 5'-CpG island by methylation specific polymerase chain reaction (MSP). In addition, tumours were analysed for LOH of chromosome 19p13.3. In tumours exhibiting LOH, LKB1/STK11 was sequenced. RESULTS: MSP was successful in 48 of 50 tumour specimens. Of those, four (8%) demonstrated hypermethylation of the LKB1/STK11 promoter 5'-CpG island. Moreover, LOH at either D19S886 or D19S878 was observed in five of 38 (13%) informative tumours. All five tumours showing LOH at 19p13.3 were advanced and four of five were located in the left sided colon. There was no correlation between LOH and LKB1/STK11 promoter hypermethylation or somatic mutation. CONCLUSIONS: In sporadic colorectal cancer, hypermethylation of the LKB1/STK11 promoter and allelic loss at the STK 11 gene locus are rare events. LOH at 19p13.3 was associated with advanced tumour stage and left sided location but not with LKB1/STK11 promoter hypermethylation or somatic mutation.     

Changes of p53 and Waf1p21 and cell proliferation in esophageal carcinogenesis

Ｃｈａｎｇｅｓｏｆｐ５３ａｎｄＷａｆ１ｐ２１ａｎｄｃｅｌｐｒｏｌｉｆｅｒａｔｉｏｎｉｎｅｓｏｐｈａｇｅａｌｃａｒｃｉｎｏｇｅｎｅｓｉｓＷＡＮＧＬｉＤｏｎｇ１，ＹＡＮＧＷａｎＣａｉ１，ＺＨＯＵＱｉ１，ＸＩＮＧＹｉｎｇ１，ＪＩＡＹｕｎＹｉｎｇ２ａ...     

肝细胞癌肿瘤抑制基因p53过度表达及点突变的研究

目的检测重庆地区肝细胞癌 p53突变发生率,并进一步探讨 p53突变与肝细胞癌临床病理及相关危险因素的关系.方法应用一种敏感的 ARF 免疫组化和 PCR、银染 PCR-SSCP 方法检测本地区38例肝细胞癌(HCC)组织中肿瘤抑制基因p53的过度表达及点突变.结果 16例有P53蛋白过度表达(41.2%),7例有 p53基因249位密码子点突变(18.4%),2例249位密码子外第7外显子点突变.9例 p53基因有突变的肝癌中8例 P53蛋白阳性,两者符合率为88.9%.p53基因蛋白过度表达和点突变与 HCC 分化和转移有关.本组 HCC p53基因突变率与该地区黄曲霉素(AFB1)含量及乙型肝炎病毒(HBV)感染分布一致.结论该结果提示 p53基因突变与 AFB1和 HBV 等环境因素的协同作用有关,其中 AFB1主要与 p53基因249位密码子特异型突变有关,而 HBV 可能在散发型突变中发挥重要作用.     

大肠癌MCC、DCC基因和YNZ22位点串联重复序列的杂合性丢失现象

目的：研究MCC、DCC基因和YNZ22位点杂合性丢失（LOH）与大肠癌发生发展的关系。方法：应用多聚酶链反应技术对41例大肠癌MCC、DCC基因和YNZ22位点的数量可变的重复序列（VNTR）区进行了分析。结果：大肠癌MCC基因LOH率为34．8％；DCC基因为36．0％，YNZ22位点为43．3％；若将以上基因综合分析，大肠癌的LOH率则为56．1％。分析LOH与临床病理参数间的关系发现，大肠癌DukesC、D期DCC基因的LOH率（53．3％）显著高于A、B期组（10．0％）（P＜0．05），其它各组间的LOH率则无显著差别。结论：MCC、DCC基因和YNZ22位点的LOH与大肠癌发生、发展有关，DCC基因LOH多为大肠癌的晚期改变。     

Hp感染与胃癌和癌前病变中p53、ras、c-myc基因表达的关系

目的研究幽门螺杆菌（Hp）感染与胃癌（GC）和癌前病变中p53、ras、c-myc基因表达的关系,以探讨其致病机制。方法用美兰和W-S特殊染色方法确定Hp感染,免疫组化SP法检测p53、ras、c-myc基因的表达。结果慢性萎缩性胃炎（CAG）、肠化生（IM）、异型增生（DYS）、GC的Hp感染率均高于慢性浅表性胃炎（CSG）（P均〈0.05）;p53、ras、c-myc基因在GC、DYS中的表达均高于CAG（P均〈0.05）,p53、ras基因在IM中的表达均高于CAG（P〈0.05）;IM中Hp阳性者的p53阳性表达率高于Hp阴性者（P〈0.05）,DYS、GC中Hp阳性者p53、ras、c-myc的表达率高于Hp阴性者（P均〈0.05）。结论Hp感染可能通过调节p53、ras、c-myc基因的表达而促进GC的发生。