胃癌组织大肠癌丢失基因杂合性丢失及表达缺失的研究

为探讨大肠癌丢失基因（ＤＣＣ）改变在胃癌发生发展中的作用，应用ＰＣＲ－ＲＦＬＰ及ＲＴ－ＰＣＲ技术对手术切除胃癌组织ＤＣＣ基因的杂合性丢失及ｍＲＮＡ表达进行分析。５１例胃癌中发现杂合丢失１８例，占３５．３％。临床Ⅲ～Ⅳ期组杂合丢失率（５０．０％）显著高于Ⅰ～Ⅱ期组（１４．３％，Ｐ值＜０．０５）。杂合丢失与组织学类型、肿瘤大小、侵袭深度、淋巴转移无显著相关（Ｐ＞０．０５）。５１例胃癌中有２６例检测了ＤＣＣ基因ｍＲＮＡ表达，发现表达缺失８例，占３０．７％。表达缺失与杂合丢失及临床病理参数无关（Ｐ＞０．０５）。胃癌组织中存在着ＤＣＣ基因杂合丢失和ｍＲＮＡ表达缺失。杂合丢失是胃癌的晚期改变     

nm23－H1等位基因缺失与大肠癌转移相关性研究

ｎｍ２３基因是近年来发现的与肿瘤转移表型抑制相关的基因，称之为转移抑制基因。ｎｍ２３基因的等位基因缺失、突变和低表达与多种人类肿瘤转移相关。ｎｍ２３基因包括ｎｍ２３－Ｈ１及ｎｍ２３－Ｈ２两种亚型。我们利用Ｓｏｕｔｈｅｒｎ杂交技术检测了２３例大肠癌及其相应正常粘膜基因组ＤＮＡｎｍ２３－Ｈ１等位基因缺失情况，结果发现５例存在ｎｍ２３－Ｈ１等位基因缺失。有淋巴结、肝或其它脏器转移者，ｎｍ２３－Ｈ１等位基因缺失率为５７．１％（４／７），而无转移者为６．２％（１／１６）。两组比较差异有显著性意义（Ｐ＜０．００５）。ｎｍ２３－Ｈ１等位基因缺失与肿瘤大小、部位及分化程度无显著相关（Ｐ＞０．０５）。结果表明，ｎｍ２３－Ｈ１基因在抑制大肠癌转移方面起重要作用。     

大肠癌CD44V6,E—cadherin表达及临床意义

目的：探讨大肠癌ＣＤ４４Ｖ６,Ｅ－ｃａｄｈｅｒｉｎ的表达对大肠癌的进展和转移的意义。方法：应用ＳＰ免疫组化技术检测５６例大肠癌标本的ＣＤ４４Ｖ６,Ｅ０ｅａｄｈｅｒｉｎ的表达。结果：ＣＤ４４Ｖ６表达有转移者８４．９％（２８／３３）,无转移者３９．１％（９／２３）。Ｅ０ｃａｄｂｈｅｒｉｎ的表达有转移者２４．２％（８／２３）,无转移者５２．２％（１２／２３）,两者与肿瘤转移明显相关（Ｐ〈０．０５）。结论     

TSP、VEGF与大肠癌血管生成、转移关系的研究

目的 分析小板反应素（ｔｈｒｏｍｂｏｓｐｏｎｄｉｎ,ＴＳＰ）、血管内皮生长因子（ｖａｓｃｕｌａｒ ｅｎｄｏｔｈｅｌｉａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ,ＶＥＧＦ）表达和大肠癌血管生成、远处转移之间的关系。方法 对４７例大肠癌手术标本采用逆转录多聚酶链反应（ＲＴ－ＰＣＲ）检测ＴＳＰ１和ＴＳＰ２ｍＲＮＡ表达,并采用免疫组化法检测微血管计数（ｍｉｃｒｏｖｅｓｓｅｌ ｃｏｕｎｔ,ＭＶＣ）和ＶＥＧＦ蛋白表达。结果 大肠癌ＭＶＣ和ＶＥＧＦ表达的程度呈正相关（ｒ＝０．４３８,Ｐ＝０．００２）,在淋巴结转移和远处转移者高于无转移者（Ｐ〈０．０５）。ＴＳＰ２ｍＲＮＡ表害和ＭＶＣ（ｒ＝－０．３６２,Ｐ＝０．０１）、ＶＥＧＦ表达（ｒ＝－０．３２２,Ｐ〈０．０５）有明显的负相关。ＴＳＰ２ｍＲＮＡ表达率在远处转移病人中低于没有远处转移者（     

转移相关基因CD44v在大肠癌中的表达及其临床意义

用ＲＴ－ＰＣＲ及ｃＤＮＡ印迹杂交技术，分别检测大肠正常粘膜、癌前病变及癌的ＣＤ４４ｖｍＲＮＡ表达，探讨ＣＤ４４ｖｍＲＮＡ异常表达和拼接与大肠癌临床病理之间的相关意义。通过对６７份大肠原发癌、２０份转移癌、７份癌前病变及１５份正常粘膜的研究，结果显示：ＣＤ４４ｖｍＲＮＡ异常表达者原发癌６５／６７、转移癌２０／２０、癌前病变５／７、正常粘膜０／１５；经ｃＤＮＡ杂交发现ＣＤ４４ｖｍＲＮＡ分子异常拼接和高分子量表达（简称高表达）者在原发癌为１０／２０（５０％），转移癌为１８／２０（９０％）；１０例原发癌ＣＤ４４ｖｍＲＮＡ高表达者中，８例有转移，另１０例原发癌弱表达者中，２例有转移。提示ＣＤ４４ｖｍＲＮＡ在大肠癌中的异常表达具有普遍性，既可能是早期癌变伴随出现的生物学标志，其高表达也可能是判断大肠癌转移的有用指标     

大肠恶性肿瘤VEGF和CD44基因mRNA表达及其临床意义

目的：研究大肠恶性肿瘤的转移相关基因血管内皮细胞生长因子（ＶＥＧＦ）和ＣＤ４４粘附分子的表达及其临床意义。方法：应用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）方法检测１９９７年１２月－１９９８年５月间收治的２１例大肠恶性肿瘤。其中１９例为大肠癌，１例为平滑肌肉瘤，１例为转移性腺样腺样囊腺癌。大肠癌Ｄｕｋｅｓ分期为Ｂ期６例，Ｃ期９例及Ｄ期４例。上述标本采用ＲＴ－ＰＣＲ方法检测各类基因的ｍＲＮＡ表达。结果：     

血管内皮细胞生长因子的表达与肝细胞癌侵袭和转移的关系

目的了解血管内皮细胞生长因子（ＶＥＧＦ）ｍＲＮＡ在肝细胞癌（ＨＣＣ）和癌周组织中的表达,探讨ＶＥＧＦ与ＨＣＣ侵袭和转移的关系,为在分子水平干预肿瘤血管形成,预防ＨＣＣ复发和转移打下基础。方法采用逆转录—聚合酶链反应（ＲＴ－ＰＣＲ）检测方法,对４３例ＨＣＣ手术标本的癌和癌周组织中ＶＥＧＦｍＲＮＡ的表达水平进行了相对定量研究。结果肿瘤组织中ＶＥＧＦｍＲＮＡ的表达率为７９．１％（３４／４３）,癌周组织表达率仅为２０．９％（９／４３）;肿瘤组织中ＶＥＧＦｍＲＮＡ表达水平癌栓组（０．６９７９±０．２３６３）和包膜不完整组（０．４７０２±０．２８８３）分别显著高于无癌栓组（０．３４３６±０．２３９１）和包膜完整组（０．２８７０±０．２５１０,Ｐ＜０．０５）;肿瘤组织中ＶＥＧＦｍＲＮＡ表达水平大肝癌组（０．４０７２±０．２８８４）与小肝癌组（０．３７３４±０．２９００）相比差异无显著性（Ｐ＞０．０５）。结论ＶＥＧＦ在ＨＣＣ浸润和转移过程中发挥重要作用,肿瘤血管形成与ＨＣＣ浸润和转移关系密切。     

大肠癌的p53基因点突变

作者以改良的ＰＣＲ－ＳＳＣＰ的ＰＣＲ直接测序方法，分析了２２例大肠癌原发灶和１例转移淋巴结组织中ｐ５３基因第七外显子的基因结构变化。发现２７％（６／２２）大肠癌原发灶和检测的１例转移淋巴结中存在ｐ５３基因第七外显子点突变，突变位点分别为２４５、２５１、２５９和２６０密码子。５０％点突变是２４５密码子中碱基Ｇ∶Ｃ向Ａ∶Ｔ转换，其它突变形式是碱基插入和碱基缺失。本研究ｐ５３点突变阳性标本均为结肠癌，其中低分化腺癌突变率高于高、中分化腺癌（Ｐ＝０．０１７８），ＤｕｋｅｓＣ１期突变率高于ＤｕｋｅｓＡ２、Ｂ期（Ｐ＝０．０３６１）。此结果提示，临床检测大肠癌原发灶和转移淋巴结中是否存在ｐ５３基因第七外显子突变，可有助于识别高度恶性的大肠癌和判断大肠癌病员的预后。     

大肠癌中C－erbB－2癌基因产物的表达及意义

应用ＡＢＣ免疫组化方法和Ｃ－ｅｒｂＢ－２癌基因的单克隆抗体对９３例大肠组织标本进行了免疫组化染色。结果表明：在６８例大肠癌中，Ｃ－ｅｒｂＢ－２阳性表达为６１．８％。其中高分化腺癌组Ｃ－ｅｒｂＢ－２阳性表达为４０％；中分化腺癌组阳性表达为５０％。低分化腺癌组阳性表达为８１．２％；粘液细胞癌组阳性表达为８５．７％。粘液细胞癌组和低分化癌组Ｃ－ｅｒＢ－２阳性表达明显高于其它２组（Ｐ＜０．０１）。在已有淋巴结转移的大肠癌中，Ｃ－ｅｒｂＢ－２癌基因阳性表达为７６．２％（３２／４２）。在无淋巴结转移的大肠癌中，Ｃ－ｅｒｂＢ－２阳性表达为３８．５％（１０／２６）。二者比较，Ｃ－ｅｒｂＢ－２癌基因表达有显著性差异（Ｐ＜０．０１）。在１５例大肠腺瘤标本中，Ｃ－ｅｒｂＢ－２癌基因阳性表达为６．７％。１０例慢性炎症和１０例正常肠粘膜Ｃ－ｅｒｂＢ－２则均为阴性。以上这些结果提示：Ｃ－ｅｒｂＢ－２癌基因产物不仅可视为肿瘤的一种标志，而且与大肠癌的分化程度、淋巴结的转移密切相关，这证明Ｃ－ｅｒｂＢ－２癌基因的检测对大肠癌的病理诊断和预后判断有很高的实用价值。     

大肠癌中DCC基因201密码子点突变的研究

目的：探寻结直肠癌缺陷基因（ＤＣＣ基因）２０１密码子在大肠癌中的突变规律。方法：采用等位基因特异性ＰＣＲＡＳ－ＰＧＲ结合ＳａｌⅠ酶切方法检测３５例大肠癌组织及配对的癌旁粘膜ＤＣＣ基因２０１密友子突变情况。结果：ＤＣＣ基因２０１密码子纯合突变率大肠癌（４０％）显高于癌旁粘膜（２．８％）,（Ｐ〈０．０５）。且与肿瘤侵袭深度、Ｄｕｋｅｓ分期相关,至少有１７例（４９％）大肠癌与相应癌旁粘膜相比增获一个密     

Mapping of a Breast Cancer Tumor Suppressor Gene Locus to a 4-cM Interval on Chromosome 18q21

DPC4 and DCC , putative tumor suppressor genes implicated in the genesis of several types of human cancer, lie on the long arm of human chromosome IS. We examined 200 primary breast cancers for allelic losses on chromosome IS, using 15 microsatellite markers distributed along the long arm. Allelic loss was detected most frequently (29–30%) at loci mapped to 18q21. Deletion mapping of the 34 tumors showing partial or interstitial deletions identified a commonly deleted region within the 4-cM interval flanked by D18S474 and D18S487 at 18q21.1-q21.3. Although this interval included the DPC4 and DCC genes, we excluded DPC4 from candidacy when polymerase chain reaction-single-strand conformation polymorphism analysis of each exon failed to detect abnormalities in any of the 54 breast cancers that exhibited loss of heterozygosity involving 18q. Allelic loss on 18q was found more frequently in tumors of the solid tubular histological type (24 of 55, 44%) than in other types (24 of 113, 21%) ( P= 0.0049). The results suggest that a tumor suppressor gene located within the 4-cM region at 18q21, either DCC or another gene not yet identified, may play a role in the development of some sporadic breast cancers, particularly those of the solid tubular type.     

Mutation and expression of the DCC gene in human lung cancer

Chromosome 18q is frequently deleted in lung cancers, and a common region of 18q deletions was mapped to chromosome 18q21. Since the DCC candidate tumor suppressor gene has been mapped in this region, mutation and expression of the DCC gene were examined in 46 lung cancer cell lines, consisting of 14 small cell lung carcinomas (SCLCs) and 32 non-small cell lung carcinomas (NSCLCs), to elucidate the pathogenetic significance of DCC alterations in human lung carcinogenesis. A heterozygous missense mutation was detected in a NSCLC cell line, Ma26, while homozygous deletion was not detected in any of the cell lines. The DCC gene was expressed in 11 (24%) of the 46 cell lines, and the incidence of DCC expression was significantly higher in SCLCs (7/14, 50%) than in NSCLCs (4/32, 13%) (P = .01, Fisher's exact test). Therefore, genetic alterations of DCC are infrequent; however, the levels of DCC expression vary among lung cancer cells, in particular, between SCLCs and NSCLCs. The present result does not implicate DCC as a specific mutational target of 18q deletions in human lung cancer; however, it suggests that DCC is a potential target of inactivation by genetic defects including intron or promoter mutations and/or epigenetic alterations. The present result also suggests that DCC expression is associated with some properties of SCLCs, such as a neuroendocrine (NE) feature.     

DCC promoter hypermethylation in esophageal squamous cell carcinoma

Deleted in Colorectal Cancer (DCC) is a putative tumor suppressor gene, whose loss has been implicated in colorectal tumorigenesis. Decreased or loss of DCC expression has been demonstrated in a number of human cancers, including esophageal cancer. In this study, we analyzed esophageal squamous cell carcinoma (ESCC) cell lines and primary ESCCs as well as normal esophageal tissues for DCC methylation by bisulfite sequencing, methylation-specific PCR (MSP) and/or quantitative methylation-specific PCR (qMSP). When a qMSP cut-off value for positivity was set to 1.0, DCC methylation was detected in 10 of 12 ESCC cell lines tested, 74% of primary ESCCs (n = 70), 0% of corresponding normal esophageal tissues (n = 20) and 0% of normal esophagus from healthy individuals (n = 19). DCC expression was undetectable in the majority of ESCC cell lines, and treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reactivated gene expression. DCC overexpression suppressed colony formation in ESCC cell lines, suggesting that DCC may function as a tumor suppressor gene in the esophagus. However, DCC methylation was not associated with any clinical or pathologic parameters measured. We have demonstrated that DCC methylation is a frequent and cancer-specific event in primary ESCCs, suggesting that DCC and associated pathways may represent a new diagnostical therapeutic target.     

Expression of the deleted in colorectal cancer gene is related to prognosis in DNA diploid and low proliferative colorectal adenocarcinoma.

PURPOSE: Whether or not the deleted in colorectal cancer (DCC) gene is implicated in metastases or in predicting prognosis in patients with colorectal cancer has not previously been substantiated. Our aims were to investigate DCC expression in primary colorectal cancers and in metastases to identify any prognostic significance. PATIENTS AND METHODS: DCC expression was examined immunohistochemically in 195 primary colorectal adenocarcinomas and in 23 paired primary tumors and lymph node metastases. DNA content and S-phase fraction were measured by flow cytometry. RESULTS: The absence of DCC expression was observed in 55 primary tumors (28%). DCC negativity was significantly related to poor prognosis in patients with DNA diploid tumors (P =.03) and those with a low S-phase fraction (< 5%, P =.02) but not in patients with nondiploid tumors or those with a higher S-phase fraction. Furthermore, DCC expression retained its prognostic significance in the diploid subgroup after adjusting for sex, age, site, stage, growth pattern, and differentiation (P =.01). DCC expression was similar in primary tumors and their metastases. CONCLUSION: The absence of DCC predicted a poor outcome in the patients with diploid tumors and those tumors with a low S-phase fraction. Immunohistochemistry may be considered as a practical test to assess prognosis in this subgroup of patients.     

Role of the dependence receptor DCC in colorectal cancer pathogenesis.

More than a decade ago, the DCC (deleted in colorectal cancer) gene was proposed as a putative tumor suppressor gene. Data supporting this proposal included observations that one DCC allele was deleted in roughly 70% of colorectal cancers, some cancers had somatic mutations of the DCC gene, and DCC expression was often reduced or absent in colorectal cancer tissues and cell lines. Despite subsequent studies which have supported DCC's potential role as a tumor suppressor gene, the rarity of point mutations identified in DCC coding sequences, the lack of a tumor predisposition phenotype in mice heterozygous for DCC inactivating mutations, and the presence of other known and candidate tumor suppressor genes on chromosome 18q have raised questions about DCC's candidacy. Following its initial characterization, the DCC protein was identified as a transmembrane receptor for netrins, key factors in axon guidance in the developing nervous system. At first glance, the established role of DCC and netrin-1 during organization of the spinal cord could be viewed as a further challenge to the position that DCC inactivation might play a significant role in tumorigenesis. However, recent observations on DCC's functions in intracellular signaling have renewed interest in the potential contribution of DCC inactivation to cancer. In particular, data indicate that, when engaged by netrin ligands, DCC may activate downstream signaling pathways. Moreover, in settings where netrin is absent or at low levels, DCC can promote apoptosis. Here, we review DCC's candidacy as a tumor suppressor gene, with an emphasis on how recent molecular analyses of DCC have offered support for the notion that DCC may function as a tumor suppressor gene.