散发性结直肠癌中SMAD4基因的突变

目的观察SMAD4基因在散发性结直肠癌中的突变，并探讨其对散发性结直肠癌发生发展的意义。方法对本院83例散发性结直肠癌患者，应用聚合酶链反应-单链构象多态性（PCR—SSCP）分析癌组织中SMAD4基因各外显子的突变情况，基因突变率与临床病理参数间采用，检验；应用多态性微卫星标记研究SMAD4基因所在18q21区的杂合性缺失。结果SMAD4基因在83例散发性结肠癌的患者中共有9例发生突变，平均突变率为10．8％。经Χ^2检验SMAD4基因突变率在肿瘤的Dukes分期及有无远处转移间差异有统计学意义（P〈0．05），而与肿瘤的分化程度、发生部位及患者的性别差异无统计学意义（P〉0．05）。18q21区的杂合性缺失率为62．71％，其中发生突变的9例均存在18q21区的杂合性缺失。结论SMAD4基因的突变可能介导了散发性结直肠癌后期的发生发展。     

食管癌3p24等位基因LOH及其扩增产物克隆的研究

目的；研究3p24EAβMD位点与食管癌的关系。为寻找该位点附近可能存在的抑癌基因奠定基础。方法：采用PCR—RFLP法检测45例食管癌患者3p24位点杂合缺失情况。并对该片段进行克隆。结果：在22例食管癌信息个体中共检出9例杂合缺失。并通过序列分析可知变化为点突变。结论：3p24EAβMD较高的杂合缺失率显示该位点附近可能存在潜在的抑癌基因。     

胶质瘤中LGI 1的突变及微卫星不稳定性研究

目的 为了检测人脑胶质瘤组织及胶质瘤细胞系LGI 1基因编码区中有无碱基突变及微卫星不稳定性(MSI)和杂合性缺失(LOH).方法 收集30例胶质瘤标本,2例脑膜瘤、2例瘤旁及2例颅脑损伤内减压脑组织标本及体外培养5个脑胶质瘤细胞系.提取组织标本及培养细胞基因组DNA.设计特异性引物分别扩增LGI 1各外显子序列,采用SSCP银染分析;扩增LGI 1微卫星序列,凝胶电泳银染分析;发现异常泳动条带进行DNA序列分析.结果 ①PCR-SSCP分析琼脂糖凝胶电泳检测有1例Ⅱ级胶质瘤标本未检测到第1 a外显子处扩增产物以及Ⅱ、Ⅲ级各1例胶质瘤标本均未检测到第8 c外显子处扩增产物.未在30例胶质瘤标本和4个脑胶质瘤细胞系中检测到电泳条带异常,在细胞系TJ905第5外显子处检测到电泳条带异常,经DNA测序分析证实确有突变.②在30例胶质瘤标本及5个细胞系中均未检测到MSI和LOH.结论 基因突变及MSI和LOH可能不是LGI 1在胶质瘤恶性进展过程中失活的主要原因.     

Microsatellite analysis of breast carcinoma and corresponding local recurrences

Local recurrence is a serious complication of breast carcinoma that reduces quality of life and influences prognosis. The aim of this study was to determine whether local recurrences of breast carcinoma are genetically related to the primary tumours. Forty cases of locally recurrent breast carcinomas (median onset: 3.6 years after primary surgery) were analysed: 22 patients had undergone breast-conserving therapy and 18 mastectomy. Eighteen microsatellites on chromosomes 2p, 3p, 5q, 10q, 11p, 11q, 13q, 17q, 17p, 18p were amplified by PCR using fluorescent-labelled primers, automatically detected after polyacrylamide gel electrophoresis and analysed for loss of heterozygosity (LOH) or microsatellite instability (MSI). Follow-up data were available for 39 cases with a median value of 89 months. All LOH and MSI found in the primary tumours were also present in the corresponding recurrences, indicating that they are genetically related to the primary tumours and not secondary malignancies in the same breast. MSI was found in three cases, of which one harboured MSI at more than two loci. The median value of LOH per case was significantly higher in the recurrent (four per case) compared to the primary tumours (two per case; p < 0.001, Mann-Whitney test), reflecting the genotype of tumour progression. Early local recurrence was associated with specific LOH for TP53.15 (p = 0.018, log-rank test) in the primary tumours. LOH on D13S1699 or D17S855 was associated with lymph node metastases (p = 0.024 and p = 0.019, respectively; chi-square test). In addition, tumour grade, lack of oestrogen or progesterone receptor expression, young patient age and early appearance of local recurrence significantly correlated with poor survival. The development of local recurrence despite clear resection margins may result from residual DCIS distant from the invasive carcinoma, homing of circulating tumour cells, or genetically altered, histologically normal breast tissue not immediately adjacent to the invasive carcinoma.     

Infrequent genetic alterations of the PTEN gene in Japanese patients with sporadic prostate cancer

Prostate cancer is a major cause of cancer death among elderly men in America, Europe, and Japan. However, the molecular mechanism of carcinogenesis is not yet well characterized. Frequent loss of heterozygosity (LOH) on chromosome 10q was reported in prostate cancer, and a candidate tumor suppressor gene, PTEN, was isolated on chromosome band 10q23.3. To investigate the genetic alterations of PTEN, we examined 45 primary prostate cancer specimens. LOH at the PTEN locus was observed in two (11.1%) of 18 tumors. However, no mutations were observed in any of the primary prostate cancers. These data suggest that mutation of the PTEN gene does not play a major role in prostate carcinogenesis of Japanese patients. Received: February 6, 1998 / Accepted: July, 3, 1998     

Mutation analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis

Pulmonary lymphangioleiomyomatosis (LAM) is a destructive lung disease characterized by a diffuse hamartomatous proliferation of smooth muscle cells (LAM cells) in the lungs. Pulmonary LAM can occur as an isolated form (sporadic LAM) or in association with tuberous sclerosis complex (TSC) (TSC-LAM), a genetic disorder with autosomal dominant inheritance with various expressivity resulting from mutations of either the TSC1 or TSC2 gene. We examined mutations of both TSC genes in 6 Japanese patients with TSC-LAM and 22 patients with sporadic LAM and identified six unique and novel mutations. TSC2 germline mutations were detected in 2 (33.3%) of 6 patients with TSC-LAM and TSC1 germline mutation in 1 (4.5%) of 22 sporadic LAM patients. In accordance with the tumor-suppressor model, loss of heterozygosity (LOH) was detected in LAM cells from 3 of 4 patients with TSC-LAM and from 4 of 8 patients with sporadic LAM. Furthermore, an identical LOH or two identical somatic mutations were demonstrated in LAM cells microdissected from several tissues, suggesting LAM cells can spread from one lesion to another. Our results from Japanese patients with LAM confirmed the current concept of pathogenesis of LAM: TSC-LAM has a germline mutation but sporadic LAM does not; sporadic LAM is a TSC2 disease with two somatic mutations; and a variety of TSC mutations causes LAM. However, our study indicates that a fraction of sporadic LAM can be a TSC1 disease; therefore, both TSC genes should be examined, even for patients with sporadic LAM. Received: August 30, 2001 / Accepted: November 2, 2001     

Expression of BRCA1 protein in benign, borderline, and malignant epithelial ovarian neoplasms and its relationship to methylation and allelic loss of the BRCA1 gene

BRCA1 is a putative tumour suppressor gene responsible for a hereditary ovarian cancer syndrome. To clarify the possible involvement of BRCA1 in the development of sporadic ovarian neoplasms, this study analysed the immunohistochemical expression of BRCA1 protein in normal ovarian surface epithelium and 119 epithelial ovarian tumours (19 benign, 24 borderline, and 76 malignant tumours). Loss of heterozygosity (LOH) of BRCA1 was examined using three microsatellite markers to analyse the relationship between BRCA1 expression and alterations of the BRCA1 gene. Methylation of the BRCA1 promoter was also analysed by methylation-specific PCR. In ovarian carcinomas showing heterogeneous expression of BRCA1 protein in the same tumour, LOH and methylation status were analysed using microdissection techniques. Finally, the relationship of BRCA1 expression or its genetic alteration to clinicopathological parameters and patient survival was analysed. Ovarian surface epithelial cells expressed BRCA1 protein. Decreased expression of BRCA1 was found in 16% of benign tumours, 38% of borderline tumours, and 72% of carcinomas. LOH of BRCA1 was demonstrated in no benign tumours, 15% of borderline tumours, and 66% of carcinomas. Methylation of BRCA1 was not detected in benign or borderline tumours, but was present in 31% of carcinomas. Reduced expression of BRCA1 correlated with the presence of gene methylation. The frequency of BRCA1 methylation and LOH was higher in serous carcinomas than in other types. In one of the three serous carcinomas that showed heterogeneous expression of BRCA1, BRCA1-positive borderline-like tumour cells were LOH-positive and methylation-negative, whereas adjacent BRCA1-negative carcinoma cells were LOH-positive and methylation-positive. The prognosis of carcinoma patients did not correlate with BRCA1 expression or genetic status. These findings suggest that reduced expression of BRCA1 protein along with genetic and epigenetic changes of the BRCA1 gene play an important role in the development of sporadic ovarian carcinomas, particularly those of serous histology.     

Cordyceps militaris Fruit Body Extract Decreases Testosterone Catabolism and Testosterone-Stimulated Prostate Hypertrophy

The androgens testosterone and dihydrotestosterone (DHT) are essential for a variety of systemic functions in mature males. Alteration of these hormones results in late-onset hypogonadism (LOH) and benign prostate hyperplasia (BPH). The fruit bodies of fungi of the genus Cordyceps have been regarded as folk medicine or health food with tonic and antifatigue effects. The extract from the fruit body of Cordyceps militaris parasitizing Samia cynthia ricini (CM) was evaluated as a novel-candidate natural product for ameliorating male andropause symptoms. To explore the effects of CM on LOH and BPH, CM was applied to rat models and cultured testicular cells and prostate cells. The concentrations of androgens in the serum and culture media were determined by ELISA. Expression of steroidogenic enzymes and androgen-related genes was evaluated by qPCR, and prostatic cell proliferation was assessed with the cell-viability assay. CM maintained the serum levels of testosterone and DHT, but inhibited testosterone-induced prostate hypertrophy. CM also increased the secretion of testosterone and DHT by primary testicular cells, with no changes in the mRNA expression of steroidogenic enzymes, but decreased the growth of prostatic cell lines. Our data suggest that CM could improve both LOH and BPH in males.     

Molecular genetic studies of early breast cancer evolution

Summary In the past few years there has been an explosion in the number of patients diagnosed with hyperplastic breast disease andin situ breast cancer. Based on epidemiological data, these morphologically defined lesions may be categorized as those with little malignant potential (e.g. typical hyperplasia or proliferative disease without atypia [PDWA]), those with significant malignant potential which may already be initiated (e.g. atypical ductal hyperplasia [ADH]), and early transformed lesions which are malignant but not yet invasive (e.g. ductal carcinomain situ [DCIS]). They may represent sequential evolutionary stages in the ontogeny of invasive breast cancer, with each morphologically defined stage resulting from accumulating genetic changes culminating in a transformed clonal lineage capable of invasion and metastasis. Using loss-of-heterozygosity (LOH) analysis, we are studying the genetic changes associated with these lesions in archival tissue samples. 50% (6/12) of the proliferative lesions (PDWA and ADH) and 80% of the DCIS shared their LOH patterns with more advanced lesions from the same breast, strongly supporting a precursor/product relationship between these lesions and the cancers they accompany.     

散发内淋巴囊瘤VHL基因位点微卫星标志杂合性丢失的研究

目的 探讨散发内淋巴囊瘤发病与VHL基因异常之间的关系。方法 采用组织微切割技术和多聚酶链式反应等方法对3例散发内淋巴囊瘤肿瘤细胞VHL基因位点染色体微卫星标志的杂合性丢失进行分析。结果 3例散发内淋巴囊瘤中有2例发生VHL基因位点微卫星标志的杂合性丢失,进一步的研究证实,该两例肿瘤细胞中分别存在着VHL基因第二外显子的异常。结论 VHL基因的异常导致其功能改变不但是VHL的致病原因,而且是散发性内淋巴囊瘤发病的重要的基因遗传学基础。