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     

大肠癌组织DCC基因启动子甲基化及表达水平相关性研究

目的:探讨结直肠癌缺失基因 (DCC)在大肠癌组织中的表达特征及其与启动子甲基化的相关性.方法:分别采用免疫组织化学和甲基化特异性PCR (MSP)方法检测71例大肠癌及其相应癌旁组织中DCC蛋白的表达情况及DCC基因启动子甲基化状态.结果:大肠癌组织中DCC蛋白表达水平 (35/71, 49.3%) 显著低于癌旁正常组织 (34/39, 87.2%, P<0.001),且与临床Duke分期相关,P=0.023;大肠癌组织中DCC基因启动子甲基化发生率 (54/71, 76.1%)高于癌旁正常组织 (14/39, 35.9%, P<0.001)和癌旁炎性组织 (10/22, 45.5%, P=0.007),且直肠癌患者DCC基因启动子甲基化发生率 (28/31, 90.3%)高于结肠癌患者 (27/40, 67.5%, P=0.022);大肠癌组织中DCC蛋白失表达组甲基化发生率 (34/36, 94.4%)显著高于DCC蛋白表达组 (20/35, 57.1%, P<0.001).结论:DCC基因启动子甲基化可能是大肠癌中DCC失表达的主要机制之一.     

Codon 201 polymorphism of DCC gene is a prognostic factor in patients with colorectal cancer

The polymorphism at codon 201 of the "deleted in colorectal carcinoma" (DCC) gene has been liked to susceptibility to colorectal cancer. However, its clinicopathological significance has not been reported. We examined the codon 201 polymorphism and loss of heterozygosity (LOH) by PCR-restriction fragment length polymorphism (PCR-RFLP) in 59 colorectal cancers, 48 samples from transitional mucosa and 67 samples from normal mucosa. The frequencies of the polymorphism did not significantly differ from normal to transitional mucosa and to tumor, but LOH was increased from transitional mucosa to tumor. Almost all of the LOH cases showed the polymorphism. The polymorphism was increased from well/moderately to poorly differentiated and to mucinous carcinoma (P=0.03). The polymorphism was more frequently seen in advanced stages than in earlier stages (P=0.02), and further predicted worse survival (P=0.04). The data suggest that the codon 201 polymorphism of the DCC gene was a target of LOH, and predicted prognosis in colorectal cancer patients.     

Detection of k-ras mutation in normal and malignant colonic tissues by an enriched PCR method

ras oncogene mutations appear in over 50% of colon tumors in humans. Studies in animal systems have revealed that ras mutations are also present in preneoplastic lesions, suggesting the possibility of early detection of ras mutation in morphologically normal colon tissues for diagnostic purposes. An Enriched PCR, developed by us, eliminates most of the normal ras alleles prior to amplification; subsequent analysis via RFLP enables the detection of one mutant allele within 10(4) normal alleles. Using the Enriched PCR, we have determined the frequency of mutant ras alleles in normal mucosae and in adenomatous polyps of patients with or without adenocarcinoma. Of the 42 patients who had colon tumors, 15 were found to harbor K-ras oncogene mutation (35%). In two of the 14 cases with mutant K-ras in the tumor tissue we were able to identify mutations in tissues that had been obtained from a site at considerable distance from the tumor (13%); Analysis of 7 adenomas identified one as a carrier of the mutant ras allele (14%). Of 11 normal colonic mucosa obtained from patients without neoplasia, one specimen contained K-ras mutation. Thus, mutated alleles of K-ras may be present, at low frequency, throughout the 'normal appearing' tissue. Cells of normal appearance that harbor such mutation, have the potential to undergo further changes and to develop into the transformed phenotype. Overall, our findings suggest that mutant ras alleles can be detected in preneoplastic mucosa that is morphologically normal, and in adenomas, suggesting the occurrence of an initiation event, and possibly enabling the identification of patients who may be at high risk for developing malignant tumors.     

结直肠癌NRAS基因突变检测及其临床意义结直肠癌NRAS基因突变检测及其临床意义

目的:探索结直肠癌NRAS基因突变的特征及其临床意义。方法:收集214例江南大学附属医院2014年3月至2015年1月收治的结直肠癌患者术后石蜡包埋组织标本,采用扩增受阻突变体系检测上述标本的NRAS基因第二、三、四外显子的突变情况,结合临床病理参数分析该项检测的意义。结果:214例结直肠癌患者中有8例NRAS基因发生突变,NRAS基因突变率3.74%。第二、三外显子均突变四例,尚未发现第四号外显子的突变。NRAS突变状况在不同的性别、分化程度、组织学类型和淋巴结转移情况中尚未观察到明显差异。结直肠癌患者中NRAS基因的突变状况与其免疫表型ALK、Ki67、CAM5.2、Her-2无明显相关性。结论:结直肠癌NRAS基因的突变较低,主要为第二、三外显子的突变。NRAS基因的突变状况与性别、年龄、分化程度、免疫表型的相关性有待进一步证实。     

胃癌组织中Caveolin-1基因突变的检测及意义

目的探讨胃癌组织中Caveolin-1基因异常的可能原因及该候选抑癌基因Caveolin-1在胃癌发生发展过程中的作用。方法通过PCR-RFLP技术分析和PCR扩增产物进行基因测序,检测胃癌中Caveolin-1基因的突变情况。结果PCR扩增产物经纯化回收后测序结果证明了132位密码cct突变为cat,即由脯氨酸（Pro）的密码子突变为亮氨酸（Leu）的密码子。结论DNA测序结果显示,第132位脯氨酸密码子突变为亮氨酸密码子,这种突变可能是导致胃癌组织中Caveolin-1基因表达降低的可能原因。     

Overexpression of p53 mRNA in colorectal cancer and its relationship to p53 gene mutation.

We analysed the frequency of p53 mRNA overexpression in a series of 109 primary colorectal carcinomas and its association with p53 gene mutation, which has been correlated with short survival. Sixty-nine of the 109 cases (63%) demonstrated p53 mRNA overexpression, without any correlation with stage or site of disease. Comparison with p53 gene mutation indicated that, besides cases in which p53 gene mutation and p53 mRNA overexpression were either both present (40 cases) or both absent (36 cases), there were also cases in which p53 mRNA was overexpressed in the absence of any mutation (29 cases) and those with a mutant gene in which the mRNA was not overexpressed (four cases). Moreover, the mutant p53 tumours exhibited an increase of p53 mRNA expression, which was significantly higher in tumours expressing the mutated allele alone than in tumours expressing both wild- and mutated-type alleles. These data (1) show that p53 mRNA overexpression is a frequent event in colorectal tumours and is not predictive of the status of the gene, i.e. whether or not a mutation is present; (2) provide further evidence that p53 protein overexpression does not only result from an increase in the half-life of mutated p53 and suggest that inactivation of the p53 function in colorectal cancers involves at least two distinct mechanisms, including p53 overexpression and/or mutation; and (3) suggest that p53 mRNA overexpression is an early event, since it is not correlated with Dukes stage.     

Immunolocalization of hCDC47 protein in normal and neoplastic human tissues and its relation to growth

hCDC47 is a human member of the MCM family, which has been implicated in the regulatory machinery causing DNA to replicate once per cell cycle. We examined its protein expression and localization in normal human tissues, using immunostaining with polyclonal antibodies. Positive nuclei were found in the proliferative components of lymph nodes, bone marrow, epidermis and mucosa. Immunohistochemical analysis was also performed for 3 types of cutaneous keratinocytic tumor originating from same cell type but showing different grades of malignancy. In seborrheic keratosis, a benign condition, cells with hCDC47-positive nuclei were located in the outermost layers of the tumor lobules, while in Bowen's disease, carcinomas in situ and squamous-cell carcinomas, they were present throughout the lesions. The percentages of hCDC47-positive cells were 65.4% in squamous-cell carcinomas, 60.9% in Bowen's disease, 12.6% in seborrheic keratosis and 3.9% in normal epidermis (n = 5 in all cases). Further expansion of the analysis to include malignant tumors from several other organs revealed that all malignant lesions tested contained more nuclear hCDC47-positive cells than their normal counterparts. Our findings indicate that hCDC47 plays a role in normal and neoplastic cell growth in vivo and that hCDC47 immunolocalization could be used as an index of cell proliferation in tissue sections.Int. J. Cancer 74:180–184, 1997. © 1997 Wiley-Liss, Inc.     

大肠癌生长抑制基因ST13在恶性肿瘤及其相邻正常组织中的表达

目的 研究恶性肿瘤及其相邻正常组织中ＳＴ１３基因的表达差异。方法 本研究采用逆转录聚合链反应酶标免疫法（ＲＴ－ＰＣＲ－ＥＬＩＳＡ）对５７例恶性肿瘤（其中大肠癌２０例，胃癌１１例，乳腺癌１０例，肝癌９例，肺癌７例）及其相邻正常组织中ＳＴ１３基因的表达进行检测。结果 ＳＴ１３基因在大肠癌，胃癌中呈低表达趋势，平均值，大肠癌１．１９６，相邻正常组织０．９０２，Ｐ＝０．０９。胃癌０．９０９，相邻正常组织０     

Characterization of pyrimidine nucleoside monophosphokinase in normal and malignant tissues.

It was found that there are two kinds of pyrimidine nucleoside, monophosphokinase deoxythymidine 5'-monophosphate-deoxyuridine 5'-monophosphate (dTMP-dUMP) kinase and cytidine 5'-monophosphate-deoxycytidine 5'-monophosphate-uridine 5'-monophosphate-doexyuridine 5'-monophosphate (CMP-dCMP-UMP-dUMP) kinase, and their molecular weights were calculated to be 46,000 and 26,000, respectively, by gel filtration. dTMP-dUMP kinase phosphorylated dTMP with a Km of 3.1 X 10(-5)M and dUMP with a Km of 7.7 X 10(-4) M. dTMP phosphorylation catalyzed by dTMP-dUMP kinase was inhibited competively by dUMP with a Ki of 2.0 X 10(-3) M. Similarly, phosphorylation of dUMP by this enzyme was inhibited competively by dTMP with a Ki of 2.5 X 10 (-5) M. CMP-dCMP-UMP-dUMP kinase of Yoshida sarcoma phosphorylated dUMP with a Km of 3.1 X 10(-3) M and dCMP with a Km of 7.1 X 10 (-4) M, but it did not phosphorylate dTMP. Phosphorylation of dUMP BY CMP-dCMP-UMP-dUMP kinase was inhibited competitively by DCMP and dTMP with Ki's of 6.9 X 10(-4) and 3.0 X 10(-3) M, respectively, and phosphorylation of dCMP was inhibited completely by dUMP a Ki of 2.2 X 10(-3) M. Relative Vmax activity of this enzyme was 345 nmoles/mg protein with dCMP and 127 nmoles/mg protein with dUMP.     

Detection of codon 61 point mutations of the K-ras gene in lung and colorectal cancers by enriched PCR

Mutation of the Kirsten ras (K-ras) gene is one of most common alterations in solid tumors including lung and colorectal cancers. We developed new enriched PCR-RFLP assay to detect mutations of K-ras codon 61 at the 1st and 2nd letters and non-enriched PCR-RFLP assay to detect the 3rd letter mutation. One mutant allele among 10(3) wild-type alleles was detected by enriched PCR-RFLP assay, while one mutant in 10 wild-type alleles was detected by non-enriched PCR-RFLP assay for codon 61 3rd letter. We then examined K-ras codon 12, 13 and 61 mutations in lung and colorectal cancers using these assays. K-ras codon 12 mutation was detected in 10 of 109 (9%) lung cancer and 19 of 83 (23%) colorectal cancer cases. K-ras codon 13 mutation was detected in 2 of 83 (2%) colorectal and 0 of 109 NSCLC cases, respectively. There was no K-ras codon 61 mutation in either type of cancer. Our results demonstrate that enriched PCR-RFLP is a sensitive assay to detect K-ras codon 61 mutation, however, it was extremely rare in lung and colorectal cancers, suggesting organ-specific pathways in mutagenesis of the ras gene family.     

The distribution of the c-myc oncogene product in malignant lymphomas and various normal tissues as demonstrated by immunocytochemistry

The expression of c-myc was studied in 51 malignant lymphomas and in a variety of normal tissues by immunocytochemistry using monoclonal antibodies raised to different synthetic peptides and reacting monospecifically with the c-myc product (p62c-myc). The c-myc product was detected in only a minority of malignant lymphomas principally those containing cells with immunoblastic characteristics, and was predominantly localised to the cytoplasm. In normal lymphoid tissues only plasma cells and histiocytes were found to have immunoreactivity. In non-lymphoid normal tissues, however, the c-myc product was distributed widely. Marked differences in its intracellular distribution were apparent in different tissues. These findings suggest that the relationship of p62c-myc expression to cell division may be more complex than previously suggested by in vitro studies, and raise the possibility that it may have other functions within the cell.     

结直肠癌组织缺陷基因的杂合性缺失及其意义

目的:探讨结直肠癌组织缺陷基因DCC的缺失与结直肠癌临床病理特征之间的关系。方法:采用聚合酶链反应方法(polymerasechainreaction,PCR)对23例结直肠癌组织新鲜标本DCC基因杂合性缺失(lossofheterozygosity,LOH)进行研究。结果:1)结直肠癌患者标本中18例为杂合子,其中7例存在DCC基因杂合缺失情况;2)其DCC杂合缺失与肿瘤淋巴结转移的情况关系密切,有淋巴结转移的患者DCC的杂合缺失为6例,占所有存在杂合病例数的85.7%。结论:DCC基因的杂合缺失可能对肿瘤的转移起促进作用。     

结直肠癌患者血清外泌体代替肿瘤组织检测K-Ras基因突变的研究*

目的 探讨结直肠癌患者血清外泌体代替肿瘤组织进行DNA中K-Ras基因12密码子突变检测的价值。方法 收集2014年5月至2015年9月于我院病理组织学诊断为局部晚期或转移性结直肠癌患者的外周血标本90例,使用ExoQuick试剂提取血清外泌体,盐析法提取外泌体及血细胞DNA,采用PCR方法扩增K-Ras基因12密码子并通过高通量测序法检测突变,比较血清外泌体DNA与血细胞DNA突变的特异性,比较外泌体与组织学检测的K-Ras基因12密码子突变情况。结果 90例结直肠癌患者中检测出外泌体K-Ras基因12密码子突变43例,突变率为47.8％。全组共检出4种突变类型,以G12D突变率为最高。与组织学检测比较,灵敏度为90.6％,一致性为81.1％（Kappa值=0.62,P＜0.05）。结论 血清外泌体DNA用于检测肿瘤相关突变,与肿瘤组织相比一致性较高,可作为液体活检的来源指导肿瘤的个体化治疗。     

Codon 201(Gly) polymorphic type of the DCC gene is related to disseminated neuroblastoma

The deleted in colorectal carcinoma (DCC) gene is a potential tumor-suppressor gene on chromosome 18q21.3. The relatively high frequency of loss of heterozygosity (LOH) and loss of expression of this gene in neuroblastoma, especially in the advanced stages, imply the possibility of involvement of the DCC gene in progression of neuroblastoma. However, only few typical mutations have been identified in this gene, indicating that other possible mechanisms for the inactivation of this gene may exist. A polymorphic change (Arg to Gly) at DCC codon 201 is related to advanced colorectal carcinoma and increases in the tumors with absent DCC protein expression. In order to understand whether this change is associated with the development or progression of neuroblastoma, we investigated codon 201 polymorphism of the DCC gene in 102 primary neuroblastomas by polymerase chain reaction single-strand conformation polymorphism. We found no missense or nonsense mutations, but a polymorphic change from CGA (Arg) to GGA (Gly) at codon 201 resulting in three types of polymorphism: codon 201(Gly) type, codon 201(Arg/Gly) type, and codon 201(Arg) type. The codon 201(Gly) type occurred more frequently in disseminated (stages IV and IVs) neuroblastomas (72%) than in localized (stages I, II, and III) tumors (48%) (P=.035), and normal controls (38%) (P=.024). In addition, the codon 201(Gly) type was significantly more common in tumors found clinically (65%) than in those found by mass screening (35%) (P=.002). The results suggested that the codon 201(Gly) type of the DCC gene might be associated with a higher risk of disseminating neuroblastoma.     

Expression of human regenerating gene mRNA and its product in normal and neoplastic human pancreas.

BACKGROUND. Localization of human regenerating gene (reg) mRNA and its product was investigated in normal and neoplastic human pancreas with the in situ hybridization method and immunohistochemical studies. METHODS. Both reg mRNA and reg protein were observed in acinar cells of the pancreas, but neither was found in ductal or islet cells. Immunoreactive reg was observed in an acinar cell carcinoma, a pancreatoblastoma, a solid and cystic tumor, and 5 of 20 duct cell carcinomas, but it was not found in 15 endocrine tumors and 2 microcystic adenomas. RESULTS. For comparison with reg, alpha-1-antitrypsin (AAT), lysozyme, chromogranin A (CMG), CA 19-9, carcinoembryonic antigen (CEA), cytokeratin, vimentin, and alpha-fetoprotein (AFP) were assessed in those tumors. An acinar cell carcinoma and a pancreatoblastoma had positive results for AAT, lysozyme, cytokeratin, and AFP but negative results for vimentin. An acinar cell carcinoma showed cells focally immunoreactive for CMG and CEA. A solid and cystic tumor had strongly positive results for AAT and vimentin and focally positive results for CMG and pancreatic hormones. Microcystic adenomas had abundant glycogen and strong immunoreactivity for cytokeratin. Ductal cell carcinomas showed cells focally positive for AAT, lysozyme, CMG, CA 19-9, and CEA. CONCLUSIONS. The localization of reg protein was not consist with that of any other proteins examined in the current study. Thus, reg protein was considered a useful marker for acinar cell differentiation; however, ectopic expression of reg also was observed in ductal cell carcinomas. In ductal cell carcinomas, expression of reg immunoreactivity was considered as one of phenotypic heterogeneity, as seen in AAT, lysozyme, and CMG immunoreactivity.     

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.     

The retinoblastoma susceptibility gene product: a characteristic pattern in normal cells and abnormal expression in malignant cells

Immunoprecipitation and Western immunoblotting studies were undertaken using purified high-affinity antibodies against a synthetic peptide corresponding to a portion of the deduced retinoblastoma (RB) protein. On SDS-PAGE, normal human cells showed an RB protein pattern consisting of a lower sharp band with a Mr of 110 kD and a more variable region above this band with a Mr ranging from 110 kD to 116 kD. The 110 kD band represents the unphosphorylated Rb protein whereas the broader, less well defined region is the phosphorylated RB protein in which molecular mass heterogenicity results from varying amount of phosphorylation. This pattern repeats once at a lower Mr in which a 98 kD band and 98-104 kD variable region can be visualized. This latter conformation seems to represent the unphosphorylated and phosphorylated RB protein translated from the second AUG codon of the RB mRNA. Cellular RB mRNA extracted from normal fibroblasts was translated in vitro reinforcing the usage of this second start codon. A higher ratio of phosphorylated to unphosphorylated Rb protein was seen in cells growing in log phase compared to those arrested in G1 phase. Our present studies also detected two candidates for RB-associated cellular proteins with a Mr of 124 kD and 55 kD respectively. In addition, shortened versions of RB-isoantigenic proteins were found in retinoblastoma and osteosarcoma cell lines.     

Enhancement of the cytotoxicity of SR 4233 to normal and malignant tissues by hypoxic breathing.

The bioreductive cytotoxic agent SR 4233 (1,2,4-benzotriazine 3-amine 1,4-dioxide) has been shown to markedly potentiate the cell killing of mouse tumours when combined with fractionated radiation therapy. Differential metabolism under oxic compared to hypoxic conditions results in SR 4233 exhibiting selective cytotoxicity to hypoxic cells. This is thought to result from the production of a cytotoxic free radical which is generated predominantly in the absence of oxygen. We have examined a way of enhancing the effectiveness of this antitumour agent in vivo by artificially increasing the hypoxic fraction of tumours by hypoxic breathing. Mice are placed in a chamber containing 10% Oxygen 90% Nitrogen for 1 h after each administration of SR 4233. Our results in the SCCVII tumour model indicate that this manoeuvre results in a 10-fold increase in antitumour effectiveness of SR 4233 when administered in a fractionated regime with radiotherapy (8 x 2.5 Gy and 0.08 mmol kg-1), but not when a single treatment regime (1 x 20 Gy and 0.3 mmol kg-1) is used. Mathematical modelling of this effect is used to illustrate this phenomenon and can be used to predict the dependence of this type of therapy on the modification of tumour oxygenation.     

Immunohistochemical expression of the c-kit proto-oncogene product in human malignant and non-malignant breast tissues.

The immunohistochemical expression of c-kit proto-oncogene product in 57 breast cancer tissues was studied using anti-c-kit proto-oncogene product antibody in comparison with 20 normal breast tissues and 58 benign breast tumours. In normal breast tissues, the c-kit proto-oncogene product was strongly expressed on cell membrane and/or cytoplasm of alveolar and ductal cells. The immunoreactive score (IRS) of c-kit proto-oncogene product in normal mammary epithelia was 6.22 +/- 2.11 (mean +/- s.d.). In benign breast diseases, the c-kit proto-oncogene product was detected heterogeneously with a reduced IRS (3.33 +/- 2.44). In breast cancer tissues, the expression of the immunoreactive c-kit proto-oncogene product was often deleted and the average IRS was significantly reduced compared to those of normal breast tissues or benign breast diseases tissues. Among benign diseases, the average IRS of intraductal papilloma was significantly reduced (1.34 +/- 1.70) and the staining intensity and pattern were found to be similar to those seen in breast cancer. The results in this study suggested that the c-kit proto-oncogene product is correlated with the growth control or the differentiation of normal breast epithelium. Also, the loss of the expression of this protein may indicate the change of the signal transduction in relation to malignant transformation in human mammary epithelium.