Analysis of the p21 gene in gliomas

The p21 gene encodes a cyclin dependent kinase inhibitor protein (p21) which has a tumor suppressive activity in a variety of tumor cell lines. Since, the p21 gene is up-regulated by the p53 tumor suppressor gene, which is frequently mutated in gliomas, acting therefore in the same control pathway, it constitutes a good candidate gene to be also inactivated in these tumors. To test this hypothesis, DNAs from 81 gliomas (48 glioblastomas, 11 anaplastic astrocytomas, 10 low-grade astrocytomas, 12 oligodendrogliomas and mixed gliomas), were investigated for mutations in the p21 coding sequence by denaturant gradient gel electrophoresis followed by sequencing. All these tumors have been previously screened for p53 mutations. Three different DNA variants were identified on codon 31 (17 cases), 27 (1 case) and 117 (1 case) and shown to be also present in matching constitutional DNA, suggesting they were polymorphisms. None of the tumors demonstrated a somatic mutation. No significant correlation between the presence of a p21 variant and the p53 mutation tumor status was observed. In conclusion, mutation in the p21 gene unlikely contributes to the development of gliomas.     

Detection of p21WAF1/Cip1 in brain metastases

The p21^WAF1/Cip1 (p21) protein, a negative regulator of G₁ checkpoint control, was overexpressed in the majority of human gliomas. To investigate whether p21 expression in brain metastases from various systemic origins is similar to that in gliomas and whether p21 expression is regulated differently in brain metastases and in corresponding primary tumors, we used immunohistochemical staining to examine the expression of p21 in paraffin-embedded sections prepared from primary colon and breast carcinomas and from metastatic brain tumors that originated from colon, breast, lung, and kidney cancers and from melanoma. Our results showed that 56% (28 of 50) of the brain metastases samples have more than 1% p21-positive staining cells compared with 87% of primary gliomas reported previously. Among the samples analyzed, p21 expression in brain metastases from breast carcinomas was much higher than in primary breast carcinomas. In contrast, p21 expression in brain metastases from colon carcinomas was less than primary colon carcinomas. The results from this pilot study suggest that p21 expression is regulated differently in metastatic and primary tumors.     

Fluorescence in situ Hybridization Analysis of 12;21 Translocation in Japanese Childhood Acute Lymphoblastic Leukemia

Fluorescence in situ hybridization (FISH) analysis was applied to detect t(12;21) using two yeast artificial chromosome probes and cosmid probes covering the TEL(ETV6) and the AML1 gene to clarify the incidence of abnormality of t(12;21) in Japanese childhood acute lymphoblastic leukemia (ALL). We detected seven TEL/AML1 fusion positive patients (9.5%), all of whom were diagnosed as B-lineage ALL, among 74 childhood ALL. On the other hand, no TEL/AML1 fusion positive patients were found among 37 adult ALL. The incidence among Japanese seemed to be lower than that among other nations. Of the seven patients with the TEL/AML1 fusion, five exhibited normal karyotype, one was t(8;12)(q11;p13), i(21q) and the remaining one exhibited a near-triploid karyotype in conventional G-banding. The FISH method clearly demonstrated that all patients with the TEL/AML1 fusion had subpopulations of leukemic cells with deletion of the normal TEL allele, which is significant for understanding the progression of leukemia with t(12;21).     

原发性肺癌患者外周血肿瘤标记物联合检测的临床意义

目的探讨外周血CEA、CYFRA21-1、NSE联合检测对原发性肺癌的诊断、病理分型、分期和疗效判断的临床应用价值。方法采用放射免疫技术,检测72例原发性肺癌患者化学治疗前后、38例良性肺部疾病患者及30例健康人血清CEA、CYFRA21-1、NSE水平。结果肺癌组治疗前CEA、CYFRA21-1、NSE水平显著高于良性肺部疾病组及正常对照组(P均<0.01),且不同病理类型肺癌3种肿瘤标记物的水平有显著性差异。3种肿瘤标记物联合检测与各单项指标检测比较,对肺癌诊断的敏感性显著增高(P<0.01),肺癌NSE检出阳性率最高(63.9%),但特异性有所下降。肺癌组化疗有效者3种肿瘤标记物水平显著下降,病情稳定和进展者三者水平无变化或升高。结论血清CEA、CYFRA21-1、NSE联合检测用于肺癌的诊断有较好的临床参考价值,并有助于肺癌病理类型的评估,亦可作为肺癌病情监测和疗效判断方面有价值的指标。     

Cyclin E、CDK2和p21WAF1在食管上皮癌变过程中的表达及意义

目的探讨食管上皮癌变过程中细胞周期调控因子cyclin E、CDK2和p21WAF1的表达状况及其意义.方法应用免疫组化SP法和原位杂交方法分别检测48例食管癌组织、31例非典型增生组织和17例正常食管粘膜中cyclin E、CDK2和p21WAF1蛋白及mRNA表达.应用半定量RT-PCR和Western blot检测22例新鲜食管癌及相应癌旁组织的mRNA和蛋白表达.结果从食管正常粘膜、非典型增生组织到癌组织,cyclin E和CDK2蛋白和mRNA阳性表达率逐渐上升,差异具有统计学意义(P＜0.01或P＜0.05).食管癌组织中cyclin E、CDK2和p21WAF1蛋白及mRNA高表达,与癌旁组织或切缘正常食管粘膜有显著性差异(P＜0.01).cyclin E、CDK2和p21WAF1基因表达显著正相关(P＜0.01或P＜0.05).结论食管上皮癌变过程中,细胞周期相关基因cyclin E和CDK2表达逐渐增强.cyclin E基因表达异常是食管癌变过程中的早期事件.p21WAF1基因在食管癌中高表达,可能与细胞周期调控的反馈机制有关.     

Xa21转基因大米对大鼠致畸作用的实验研究

目的　观察Xa2 1转基因大米对大鼠胚胎生长、发育的影响。方法　将初断乳Wistar大鼠按雌雄分别随机分为 4组 :转基因大米组、非转基因大米组、AIN93G对照组和敌枯双阳性对照组。单笼喂养 ,饲相应鼠料 ,喂满 90天 ,雌雄合笼。观察母鼠和胎鼠的生长发育情况。结果　转基因大米组孕鼠增重、活胎体重、身长、尾长均显著高于阳性对照组 ,而死胎数、吸收胎数、畸形率 (外观、内脏、骨骼 )均显著低于阳性对照组。转基因大米组与非转基因大米组、AIN93G对照组相比 ,所有观察指标均无统计学差异。结论　转Xa2 1基因大米与非转基因大米相比 ,对大鼠受孕率、胚胎生长发育无显著性差异。     

胃肠道类癌中MMP-2和p21WAF1/CIP1蛋白表达的意义

目的 探讨MMP-2和p21WAF1/CIP1蛋白阳性表达与胃肠道类癌组织分化、浸润和转移的关系.方法 采用免疫组化S-P法对36例胃肠道类癌组织MMP-2和p21WAF1/CIP1蛋白的表达进行检测.结果 36例类癌组织中,MMP-2蛋白在低分化类癌组高表达,MMP-2和p21WAF1/CIP1蛋白在高分化类癌组高表达(P＜0.05),随着肿瘤的浸润和淋巴结转移MMP-2阳性表达率显著增加(P＜0.01),MMP-2和p21WAF1/CIP1阳性表达降低(P＜0.05).结论 MMP-2和MMP-2和p21WAF1/CIP1在类癌组织中的表达与组织分化、浸润和淋巴结转移关系密切,可用于临床对病人进行预后判断.     

胡椒碱对SF-21细胞株的杀伤作用及对其形态影响的研究

目的检测胡椒碱对草地贪夜蛾SF-21细胞的毒性作用和生长抑制作用,以便进一步研究其对昆虫细胞离子通道的影响。方法胡椒碱处理SF-21细胞后,用MTT微量酶反应比色法、改良微波Giemsa染色法检测细胞死亡率及形态学改变,用冲击式测试法检测细胞生长率及受损细胞的恢复。结果胡椒碱处理24h后,对SF-21细胞的半数毒性浓度(IC50)为95.37μg/ml,且毒性作用强度随着药物浓度增加而增强。胡椒碱浓度在10μg/ml以上时可诱发SF-21细胞形态学改变,表现为细胞呈多形性、细胞间有间隙、胞质内充满颗粒,以后随药物浓度的升高,胞质出现空泡、染色质凝成粗大颗粒或无结构大块、大片细胞脱落、崩解、死亡。低浓度胡椒碱(5μg/ml)对SF-21细胞增殖没有影响,较高浓度胡椒碱(10、20、40μg/ml)则对SF-21细胞增殖有明显的抑制作用,抑制作用强度随药物浓度的增加而增加,去除药物作用后,受抑制的细胞又重新恢复生长、分裂。结论胡椒碱对昆虫细胞有较强的毒性作用和生长抑制作用,可作为新型杀虫剂的功能物质。     

肉桂氨茴酸对血管平滑肌细胞生长及周期蛋白P21和P53表达的影响

目的探讨肉桂氨茴酸对血管平滑肌细胞(VSMC)生长及周期蛋白P21和P53表达的影响。方法以培养的兔主动脉VSMC株为模型,应用活细胞计数进行细胞增殖和迁移检测,计算半数抑制浓度(IC50)。采用流式细胞仪分析肉桂氨菌酸作用24h后VSMC周期的变化和细胞凋亡率,以western印迹法检测细胞周期蛋白P21和P53的表达。结果20%的胎牛血清条件下,肉桂氨茴酸显著抑制VSMC的增殖和迁移,在9．6～305．8/μmol/L范围内,随剂量增加其抑制率增加。IC-(50)为75．6/μmol/L。IC-(50)的肉桂氨茴酸作用VSMC 24h后,G1期细胞数占24%,显著低于作用前的51%(P＜0．01),形成明显的亚二倍体峰,细胞凋亡率为36．7%。经IC_(50)的肉桂氨茴酸作用VSMC 24h后出现明显的P21和P53条带,而空白对照组无表达。结论肉桂氨茴酸浓度依赖性抑制VSMC的生长,其抑制作用主要通过细胞凋亡的方式。肉桂氨茴酸抑制VSMC增殖与细胞周期蛋白P21和P53的表达有关。     

A mechanistic basis for the role of cycle arrest in the genetic toxicology of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed during the cooking of meat, induces tumors of the prostate, colon, and mammary gland when fed to rats. PhIP is readily absorbed and efficiently metabolized to a genotoxic derivative by CYP1 enzymes. Although metabolism and mutational potential of PhIP have previously been well characterized, the intervening cellular and genomic responses to the chemical are not fully understood. We have examined the cellular response to PhIP exposure in human mammary epithelial MCF10A cells, which retain characteristics of normal breast epithelial cells. Because these cells fail to activate PhIP, they were cocultured with a human lymphoblastoid cell line MCL-5, which constitutively expresses CYP1A1, and have been transfected to express human CYPs1A2, 2A6, 3A4, and 2E1. The MCL-5 cells were irradiated (2,000 rads) prior to coculture, rendering them unable to replicate yet still retaining metabolic competency. MCF10A cells were treated (in the presence of MCL-5 cells) with PhIP (1-100 microM) and harvested at various time-points. Compared to DMSO control, treatment (24 or 48 h) with PhIP resulted in a significant dose-dependent fall in cell number. Cells treated for 48 h then cultured in the absence of PhIP (and MCL-5 cells) for a further 6 days showed a much greater dose-dependent reduction in cell number. Flow cytometric analysis indicated that PhIP treatment (48 h) resulted in a dose-dependent accumulation of cells in the G1 population. Western blotting revealed elevated expression of p53 and the cyclin dependent kinase inhibitor p21WAF1/CIP1 after PhIP treatment. Levels of MDM2, a negative regulator of p53, and the hypophosphorylated form of RB were also elevated, consistent with the triggering of G1 cell cycle checkpoint. These cell cycle effects are critical, as they enable cells to effect genome repair, accept mutation, or eliminate excessively damaged cells.