Telomere shortening and mitotic dysfunction generate cytogenetic heterogeneity in a subgroup of renal cell carcinomas

Most renal cell carcinomas (RCC) show only simple chromosomal changes. However, a more complex cytogenetic pattern has been found in a subgroup of aggressive RCC, indicating that further accumulation of chromosome changes could play a role in tumour progression. To explore the possible mechanisms behind cytogenetic evolution in RCC, a parallel assessment of chromosome mutations and mitotic segregation pattern in eight tumours was performed. In the majority of cases, no abnormalities in the cell division machinery were found and the rate of alterations in chromosome copy number, as measured by interphase FISH, was similar to that in non-neoplastic cells. This was reflected by relatively simple karyotypes, with little cytogenetic intratumour heterogeneity. In contrast, another group of tumours exhibited several cytogenetically related clones with additional structural chromosomal changes at two or more ploidy levels and a frequency of copy number alterations that was higher than in normal cells. In these cases, the telomere repeat sequences were abnormally short and chromosomal breakage-fusion-bridge events were observed at cell division, as well as multipolar configurations and supernumerary centrosomes. Abnormalities of the cell division machinery may thus contribute to the evolution of complex karyotypes and genetic intratumour heterogeneity in a subgroup of RCC.     

Telomere shortening is correlated with the DNA damage response and telomeric protein down-regulation in colorectal preneoplastic lesions

BackgroundA relation between telomere attrition in early carcinogenesis and activation of DNA damage response (DDR) has been proposed. We explored telomere length and its link with DDR in colorectal multistep carcinogenesis.Patients and methodsWe studied normal mucosa, low-grade dysplasia (LGD) and high-grade dysplasia (HGD) and invasive carcinoma (IC) in matched human colon specimens by evaluating p-ataxia telangiectasia mutated (ATM), p-checkpoint kinase 2 (Chk2), c-H2AX, TRF1 and TRF2 expressions by immunohistochemistry. FISH was used to assess telomere length.ResultsTelomeres shortened significantly from normal (N) to LGD and HGD (P < 0.0001; P = 0.012), then increased in length in IC (P = 0.006). TRF1 and TRF2 expressions were diminished from N to LGD and HGD (P = 0.004, P < 0.0001, ns) and were reexpressed at the invasive stage (P = 0.053 and P = 0.046). Phosphorylated ATM, Chk2 and H2AX appeared already in LGD (respectively, P = 0.001, P = 0.002 and P = 0.02). Their expression decreased from HGD to IC (respectively, P = 0.03, P = 0.02 and P = 0.37). These activating phosphorylations were inversely correlated with telomere length and TRF1/2 expression.ConclusionIn a model of colon multistep carcinogenesis, our data indicate that telomeric length and protein expression levels are inversely correlated with the activation of the DDR pathway.     

Telomere shortening and chromosomal abnormalities in intestinal metaplasia of the urinary bladder.

PURPOSE: Although intestinal metaplasia is often found in association with adenocarcinoma of the urinary bladder, it is unclear whether intestinal metaplasia of the bladder is a premalignant lesion. Telomere shortening has recently been implicated in epithelial carcinogenesis. We used quantitative fluorescent in situ hybridization (FISH) to measure telomere length and UroVysion FISH to detect cytogenetic abnormalities in urinary bladder specimens with intestinal metaplasia. EXPERIMENTAL DESIGN: Paraffin-embedded tissue blocks from 34 patients with intestinal metaplasia of the urinary bladder were evaluated. Twelve of the 34 patients had coexistent cystitis glandularis, and telomere length was measured in these lesions for comparison. Tissue sections were prepared and hybridized with a telomere-specific peptide nucleic acid probe. Quantitative FISH on interphase nuclei was used to assess telomere signal intensity. Additional sections were hybridized with centromeric probes for chromosomes 3, 7, and 17 and a locus-specific probe 9p21. Multicolor FISH was used to analyze for cytogenic abnormalities in the interphase nuclei of intestinal metaplasia. RESULTS: In all 34 cases, reduced average telomere signal intensity was observed in the nuclei of intestinal metaplasia cells compared with adjacent control nuclei to produce a mean relative intensity of 48.5% (P < 0.0001). When cystitis glandularis was present, significant differences in the telomere-specific signal intensity existed between cystitis glandularis and normal cells (P = 0.0005) and between cystitis glandularis and intestinal metaplasia cells (P = 0.0015). Three of the 34 cases showed chromosomal gains in the UroVysion FISH assay. CONCLUSIONS: Our findings indicate that intestinal metaplasia in the urinary bladder is associated with significant telomere shortening relative to telomere length in adjacent normal urothelial cells. These lesions also occasionally showed cytogenetic abnormalities associated with telomere shortening. Our findings support the hypothesis that intestinal metaplasia of the urinary bladder is a precursor lesion to and could be a marker in the development of adenocarcinoma of the urinary bladder.     

Telomere shortening is an early somatic DNA alteration in human prostate tumorigenesis

Chromosomal instability appears to be key to the pathogenesis of malignant transformation in human cancers, yet the precise molecular mechanisms underlying chromosomal rearrangements remain largely unknown. Telomeres stabilize and protect the ends of chromosomes, but shorten because of cell division and/or oxidative damage. Critically short telomeres, in the setting of abrogated DNA damage checkpoints, have been shown to cause chromosomal instability in vitro and in animal models, leading to an increased cancer incidence as a result of chromosome fusions, subsequent breakage, and rearrangement. We present results from a quantitative, high-resolution, in situ method for telomere length assessment used to test the hypothesis that telomere shortening is an early contributor to human tumorigenesis. High-grade prostatic intraepithelial neoplasia (HGPIN) is a putative preinvasive precursor of prostatic adenocarcinoma, the most common noncutaneous malignancy in Western men. The telomere lengths of epithelial cells within HGPIN lesions were strikingly shorter than those of adjacent normal appearing epithelial cells in 93% (28 of 30) of lesions examined. This shortening is similar to what has been shown in fully invasive prostate adenocarcinomas. Interestingly, telomere shortening was restricted to the luminal epithelial cells of HGPIN and was not present in the underlying basal epithelial cells; this provides strong evidence that basal cells are most likely not the direct targets of neoplastic transformation. These findings reveal that telomere shortening is a defining somatic DNA alteration characterizing HGPIN. The implications of this are that the earliest phase of human prostate carcinogenesis may proceed as a consequence of chromosomal instability mediated by shortened, dysfunctional telomeres.     

Telomere shortening alters the kinetics of the DNA damage response after ionizing radiation in human cells

Studies of telomerase-deficient mice and human cell lines have showed that telomere shortening enhances sensitivity to ionizing radiation (IR). The molecular basis for this observation remains unclear. To better understand the connection between telomere shortening and radiation sensitivity, we evaluated components of the DNA damage response pathway in normal human fibroblasts with short and long telomeres. Late-passage cells with short telomeres showed enhanced sensitivity to IR compared with early-passage cells with longer telomeres. Compared with early-passage cells, late-passage cells had a higher baseline level of phosphorylated H2AX protein (γH2AX) before IR but diminished peak levels of H2AX phosphorylation after treatment with IR. Both the appearance and disappearance of γH2AX foci were delayed in late-passage cells, indicative of delayed DNA repair. In contrast to the situation with H2AX, ATM and p53 phosphorylation kinetics were similar in early- and late-passage cells, but phosphorylation of the chromatin-bound ATM targets SMC1 and NBS1 was delayed in late-passage cells. Because impaired phosphorylation associated with short telomeres was restricted to chromatin-bound ATM targets, chromatin structure was assessed. DNA from cells with short telomeres was more resistant to digestion with micrococcal nuclease, indicative of compacted chromatin. Moreover, cells with short telomeres showed histone acetylation and methylation profiles consistent with heterochromatin. Together our data suggest a model in which short telomeres induce chromatin structure changes that limit access of activated ATM to its downstream targets on the chromatin, thereby providing a potential explanation for the increased radiation sensitivity seen with telomere shortening.     

CD44v6、p27表达与大肠癌临床病理学因素的关系

Objective To observe the expression and reveal clinical significance of CD44v6 and p27 in colorectal carcinoma.Methods Collecting 50 cases of fresh specimen of colorectal carcinoma and normal mucous from excision operation.CD44v6 expression was detected by means of FCM in 50 cages of colorectal carcinoma and corresponding normal mucous.p27 protein was studied by SP immunohistochemical method.Analysis of statistics was performed by combining clinical and patholoical materials.Results CD44v6 and p27 expression in colorectal carcinoma was significantly higher than that in normal mucous (P＜0.01).As well as the expression of CD44v6 and p27 was obviously associated with differentiated grade,lymph node metastasis,distant metastasis and Dukes stage(P＜0.05).The expression of CD44v6 was not evidently correlated with the depth of tumor invasion(P＞0.05).but the expression of p27 Was correlated with the depth of tumor invasion(P＜0.05).Conclusion The expression of CD44v6 and p27 is markedly related with clinicopathological characters of colorectal carcinoma.CD44v6 expression and p27 expression axe obvious negative relationship in colorectal carcinoma.There is a certain value of reference for assessing the malignant grade and the potential of recurrence and metastasis,evaluating patient prognosis and selecting rational treatment after operation.     

CD44v6、p27表达与大肠癌临床病理学因素的关系

目的探讨CD44v6和p27在大肠癌中的表达及其临床意义。方法对50例手术切除的大肠癌新鲜标本及相应的正常大肠黏膜,应用流式细胞术（FCM）检测CD44v6的表达;同步制备上述病例大肠癌组织石蜡标本,采用免疫组织化学方法检测大肠癌组织中p27的表达,结合病理及临床资料进行统计学分析。结果CD44v6和p27在大肠癌组织及正常黏膜组织中表达存在明显差异（P〈0．01）,而且CD44v6和p27的表达与大肠癌的分化程度、有无淋巴结转移、有无远端器官转移、Duke分期及预后相关（P值均〈0．05）,CD44v6。的表达与大肠癌的浸润深度无明显相关（P〉0．05）,但p27的表达与大肠癌的浸润深度明显相关（P〈0．05）。结论CD44v6和p27的表达与大肠癌的临床病理学特征密切相关,两者在大肠癌组织中的表达呈一定的负相关,同时检测二者对判断大肠癌的恶性程度、复发转移潜能,评价患者的预后及术后选择合理的治疗方案具有一定的参考价值。     

Influence of biogenic amines on the growth of xenografted human colorectal carcinomas.

The influence of some biogenic amines and amine-receptor-blocking drugs in the growth rate of human colorectal carcinomas propagated as s.c. xenografts in immune-deprived mice was studied. In mice treated with adrenaline, a beta-adrenergic agonist, the growth of xenografts was suppressed for 2 days, after which growth was resumed at a rate similar to that in control animals. Treatment with the phosphodiesterase inhibitor theophylline prolonged the adrenaline-induced inhibition of growth. Treatment with the beta-adrenergic antagonist sotalol or practolol increased the rate of tumour growth. Treatment with either serotonin or the histamine H2-receptor agonist Dimiprit had no effect on tumour growth rate. However, the antiserotoninergic drug BW 501C and the histamine H2-receptor antagonist cimetidine each caused short-term suppression of tumour growth.     

Transglutaminase activity in human colorectal carcinomas of differing metastatic potential.

Transglutaminase (TGA) activity in four human colorectal carcinoma cell lines of differing metastatic potential, and the effects of mild proteolysis on this activity, was investigated. Rank order of metastatic activity measured in nude mice (intrasplenic injection) was found to be LS174T greater than SW620 greater than WiDr greater than SW480. Rank orders of TGA activity were SW480 greater than WiDr greater than SW620 greater than LS174T. Proteolysis of cell lysates increased LS174T TGA activity 42-fold, SW620 2-fold without affecting WiDr or SW480 activity. Hence a negative association exists between metastatic potential and TGA activity in human colorectal carcinoma cells. Furthermore, a positive association exists between proteolytic activation of TGA and metastatic potential.     

Cryopreservation of human colorectal carcinomas prior to xenografting

Background  

Molecular heterogeneity of colorectal carcinoma (CRC) is well recognized, forming the rationale for molecular tests required before administration of some of the novel targeted therapies that now are rapidly entering the clinics. For clinical research at least, but possibly even for future individualized tumor treatment on a routine basis, propagation of patients' CRC tissue may be highly desirable for detailed molecular, biochemical or functional analyses. However, complex logistics requiring close liaison between surgery, pathology, laboratory researchers and animal care facilities are a major drawback in this. We here describe and evaluate a very simple cryopreservation procedure for colorectal carcinoma tissue prior to xenografting that will considerably reduce this logistic complexity.     

A study of chromosome 6 allele loss in human colorectal carcinomas.

Matched normal/tumor DNA pairs from patients with sporadic and hereditary (FAP = familial adenomatous polyposis) colorectal carcinoma were examined for tumor-specific allele loss on chromosome 6 using cDNA probes for the avian myeloblastosis viral oncogene homologue (MYB on 6q22-q23), the estrogen receptor (ESR on 6q24-q27), and for the alpha polypeptide of human chorionic gonadotropin (CGA on 6q14-q21). No chromosome 6 allele loss was observed at these gene loci among 22 colorectal carcinomas examined, although such losses were relatively frequent (37.5% of informative individuals) at the D17S28 locus on chromosome 17. These results are consistent with karyological studies and indicate that chromosome 6 allele loss from colorectal carcinomas may occur less frequently than previously reported.     

Ki-ras CODON 12 MUTATION IN HUMAN COLORECTAL CARCINOMAS

We examined the incidence of point mutation in codon 12 of Ki-ras oncogene in human colorectal carcinomas by polymerase chain reaction in combination with alot-blot hybridization using mutation-specific ollgodeoxynucleotide as probes. Among 72 colorectal carcinomas, point mutations were found in 36 samples, GGT to TGT in 16 cases and to AGT In 21 cases, one sample contain two different mutations. One of five normal mucosa contains the same mutation as in the adjacent carcinoma, suggesting that genetic alterations may also exist in the regions from which such carcinomas arise.     

Investigation of human papillomavirus DNA in colorectal carcinomas and adenomas

Human papillomavirus (HPV) has been considered to be an etiological agent for anogenital cancers, such as cervical cancer and possibly a subset of cancers of the aerodigestive tract. The aim of the study was to evaluate the presence of human papillomavirus DNA in colorectal carcinomas and adenomas. Formalin-fixed and paraffin-embedded archival tissue samples were used for DNA extraction. One hundred and six colorectal carcinomas and 62 adenomas were screened by nested polymerase chain reaction (PCR) for HPV DNA with a control group of 49 cervical tissues with invasive cervical carcinoma and cervical intraepithelial neoplasia (CIN). In the study group, we did not find HPV DNA positivity in any of all the colorectal carcinomas and adenomas. In the control group with cervical lesions, 34 out of 49 (69.4%) samples were positive for the HPV DNA. These results indicated that there was no correlation between HPV infection and colorectal carcinomas and adenomas.     

Expression of the MDR1 gene in human gastric and colorectal carcinomas

We measured expression of the MDR1 gene (also known as the PGY1 gene) in the human gastrointestinal tract. MDR1 messenger RNA (mRNA) levels were elevated in 13 of 15 colorectal carcinoma specimens and in six of 13 gastric carcinoma specimens. Well-differentiated colorectal carcinomas contained significantly higher concentrations of MDR1 mRNA than moderately differentiated colorectal carcinomas. Similarly, moderately differentiated gastric carcinomas contained higher concentrations of MDR1 mRNA than poorly differentiated gastric carcinomas. MDR1 gene expression in normal colorectal and gastric tissues adjacent to carcinomas was similar to that in the carcinomas. MDR1 gene expression in xenografts of colorectal and gastric carcinomas in nude mice was also investigated. Elevated expression of the MDR1 gene was seen in only four of 18 xenografts of colorectal carcinoma and was not seen in any xenografts of gastric carcinoma. P-glycoprotein was distributed over the luminal surface of the colorectal carcinoma. These results imply that the higher levels of MDR1 mRNA found in well-differentiated carcinomas derived from colorectal tissues are the results of increased expression of the MDR1 gene in the luminal surface cells. The level of expression of the MDR1 gene in colorectal and gastric carcinomas appears to correlate with the degree of differentiation and also appears to be affected by transplantation into nude mice.     

结直肠癌化疗敏感性相关蛋白的蛋白质组学初步研究

目的:本研究应用蛋白质组学技术建立化疗敏感性不同的结直肠癌组织总蛋白双向凝胶电泳(two-dimensional gel electrophoresis,2-DE)图谱,并鉴定部分差异表达蛋白,以发现与结直肠癌化疗敏感性有关的蛋白.方法:收集临床诊断为晚期结直肠癌的病例,肠镜活检获取新鲜结直肠癌标本后液氮保存备用,根据肿瘤药物敏感实验分为化疗高敏感组和化疗低敏感组.提取组织总蛋白,采用双向凝胶电泳技术得到各组凝胶图谱;采用PD-quest 7.3软件进行图像的合成、对比和差异分析,识别化疗高敏感组和化疗低敏感组之间差异表达的蛋白斑点;选取差异蛋白质点,胶内酶解后行肽指纹图分析及网上数据库检索,鉴定差异蛋白质;应用Western印迹法检测部分差异蛋白的表达情况.结果: 建立了化疗高敏感组和化疗低敏感组的双向凝胶电泳图谱,多数蛋白质点集中于pH 4～8、相对分子质量为(20～100)×10~3.高敏感组和低敏感组的电泳图谱中平均蛋白质点数分别为(842±23)个和(793±19)个,2组平均匹配率为90.7%,2组间差异表达蛋白质点数为(79.00±13.56)个;选择30个差异蛋白质点进行质谱分析,经数据库查询鉴定出9个差异表达蛋白.结论:在化疗敏感性不同的结直肠癌中存在蛋白质表达的差异,这些差异表达蛋白可能与化疗敏感性有关,并可能用于化疗敏感性的预测.     

K-ras point mutations in human colorectal carcinomas: relation to aneuploidy and metastasis.

Material from paraffin sections of 109 human colorectal carcinomas, mostly obtained at autopsy, was analyzed for the presence of K-ras point mutations at codon 12, position 2. Mutations at this position were found in 23 cases (21.1%). Aneuploid colorectal carcinomas showed a significantly higher prevalence of K-ras point mutations than diploid tumors, suggesting an involvement of ras mutations in the development of aneuploidy. No differences in the prevalence of K-ras mutations were observed with respect to the patients' age, sex and tumor type. In metastases, the type of ras gene mutation was always identical to that of the respective primary tumor. Mutations were not found in metastases from primary tumors devoid of ras mutations. This renders a clonal selection of K-ras mutated cells from a wild-type primary tumor during the metastatic process unlikely. However, nearly twice as many ras gene mutations were seen in metastatic than in non-metastatic primary tumors.