乳腺癌组织染色体17p13的杂合性缺失分析

目的检测乳腺癌组织在染色体１７ｐ１３区各位点的杂合性缺失。用方法通过Ｓｏｕｔｈｅｒｎｂｌｏｔ分析ＶＮＴＲ探针ＹＮＺ２２的杂合性缺失。用ＰＣＲ扩增微卫星重复序列后，同位素标记、变性胶分离分析微卫星ｍａｒｋｅｒ的杂合性缺失。结果１１例乳腺癌组织中有３例在染色体１７ｐ１３．３区有杂合性缺失，占２７％。缺失的上限为紧邻端粒的Ｄ１７Ｓ１８６６位点，缺失至Ｄ１７Ｓ１８４０位点，缺失的下限尚待确定。位于染色体１７ｐ１３．１区的ＴＰ５３位点有１例变化产生新长度的微卫星重复序列，４例存在杂合性缺失，其中２例在１７ｐ１３．３区也有杂合性缺失。结论乳腺癌组织在染色体１７ｐ１３．３区有较高的杂合性缺失。预示该区可能存在有关的抑癌基因。１７ｐ１３．１区的ＴＰ５３位点也有变化，说明ｐ５３基因及产物在乳腺癌的癌变过程中也起着一定的作用     

11株肝癌细胞系染色体17P13.3和p53基因状况的研究

目的了解１１株肝癌细胞系染色体１７ｐ１３．３区基因组结构和ｐ５３基因状况，为今后更好使用这些细胞株提供信息。方法应用染色体微卫星标志ＰＣＲ扩增，同位素标记，变性聚丙烯酰胺凝胶电泳分析以及染色体可变数目重复排列序列ＶＮＴＲ标志ＹＮＺ２２的Ｓｏｕｔｈｅｒｎ杂交检测各细胞株染色体１７ｐ１３．３区基因组结构和ｐ５３基因状况。结果通过上述检测，获得了１１株肝癌细胞在染色体１７ｐ１３．３区８个位点的结构状况。发现来源于我国上海地区构建的６株细胞系在染色体１７ｐ１３．３区各个位点的结构状况基本一致。ＣＣＬ细胞株在该区的两个位点呈纯合子缺失。上述６株细胞系在染色体１７ｐ１３．１区的ＴＰ５３位点呈杂合子，且微卫星重复序列的大小一致，ＣＣＬ及Ｈｅｐ３Ｂ细胞系在ｐ５３基因外显子５，６，７，８基因组片段有缺失，其它细胞系在该区有正常大小的基因组片段。结论本研究提供了１１株肝癌细胞系在染色体１７ｐ１３．３区和ｐ５３基因的结构状况信息     

卵巢癌及宫颈癌中17p13.3的杂合性丢失

目的探讨染色体１７ｐ１３．３的杂合性丢失（ＬＯＨ）与卵巢癌、宫颈癌发生及发展之间的相关性。方法采用ＰＹＮＺ．２２探针做Ｓｏｕｔｈｅｒｎ印迹技术,检测２４例卵巢癌、９例宫颈癌及１３例妇科非癌患者手术切除组织染色体１７ｐ１３．３的ＬＯＨ。结果１２例卵巢癌（包括１例交界性粘液性囊腺癌）和４例宫颈癌发生１７ｐ１３．３的ＬＯＨ,丢失频率分别为５０．０％和４４．４％。１３例非癌组织中,仅１例（７．７％）发生丢失,该例经病理证实为宫颈上皮内瘤变Ⅲ级,属癌前期病变（Ｐ＜０．０１）。结论染色体１７ｐ１３．３的ＬＯＨ可能与宫颈癌和卵巢癌的发生相关,检测１７ｐ１３．３的杂合性丢失将有助于深入了解卵巢癌和宫颈癌发生及发展的分子基础。     

肝癌染色体17p13.3区的杂合性缺失分析及缺失区连续克隆群？…

目的 检测原发性肝癌在染色体１７ｐ１３．３区的杂合性缺失状况,确定其共同缺失范围和最小热点缺失范围,并获得缺失范围内基因组克隆和构建连续克隆群。方法 应用Ｓｏｕｔｈｅｍ杂交分析ＶＮＴＲ（ｖａｒｉａｂｌｅ ｎｕｍｂｅｒ ｏｆ ｔａｎｄｅｍ ｒｅｐｅａｔ,ＶＮＴＲ）和ＲＦＬＰ标志在肝癌中杂合性缺失（ＬＯＨ）状况。应用ＰＣＲ扩增微卫星标志,变性聚丙烯酰胺凝胶电泳分析各个微卫星标志的ＬＯＨ状况。以微卫星标     

染色体3p14.2-14.3区域一个与鼻咽癌相关新基因的克隆

目的：在染色体３ｐ１４．２－１４．３区域寻找与鼻咽癌发生发展密切相关的新基因。方法：运用ＲＡＣＥ方法获取基因全长ｃＤＮＡ序列,ｐＥＧＦＰ质粒和脂质体共转染ＣＯＳ７细胞进行新基因的蛋白质定位。结果：在３ｐ１４．２－１４．３区域克隆了一个在鼻咽癌中表达不调新基因的全长ｃＤＮＡ序列,被命名为ＣＰＣＤＲ１,编码１０９个氨基酸,其蛋白质聚集在细胞核内,其基因组由２个外显子和１个内含子组成。Ｎｏｒｔｈｅｒｎ印     

鼻咽癌染色体3p21-26的等位基因杂合性丢失研究

目的细胞遗传学研究表明,３号染色体缺失是鼻咽癌常见的染色体异常之一。分子生物学研究表明,染色体３ｐ在鼻咽癌中出现高频率的等位基因杂合性丢失（ＬＯＨ）。本研究将进一步确定鼻咽癌３ｐ等位基因杂合性丢失的频率及范围。方法应用位于３ｐ２１２６区域１６个微卫星多态性位点,对２４例低分化鼻咽癌患者进行了ＬＯＨ分析。结果２４例患者中有１６例存在杂合性丢失（６６．７％）。丢失频率最高的两个位点是Ｄ３Ｓ１５６０（５０％,１１／２１）和Ｄ３Ｓ１６２０（５０％,９／１８）。在具有丢失的１６例患者中,８例显示为１个连续的多个相邻位点的杂合性丢失区域,５例患者存在２个或２个以上的杂合性丢失区。病例１,３,４,７,８,１０,１６,１７,１８,１９和２２在Ｄ３Ｓ１５９７和Ｄ３Ｓ１２９７之间,表现为一个不同大小的杂合性丢失区。结论最小共同丢失区位于Ｄ３Ｓ１５６０Ｄ３Ｓ１６２０（３ｐ２５．３２６．２）之间,提示该区域有一个尚未克隆的、与晚期鼻咽癌明显相关的抑癌基因。     

肝癌中具有高突变率位于染色体17p13.3区的新报癌基因

目的：以往研究表明肝癌中染色体17p13.3区有高频率的杂合性缺失，其最小杂合性缺失范围已被确定在D17S643至D17S1574位点间，而且其中的D17S926位点有最高的杂合性缺失率，含有该位点的基因组克隆P579已被测序分析，在P579范围共有13个新基因，这里报告其中的一个新基因（命名为肝癌抑癌基因1，HCCS1）的克隆和特性研究结果。方法：利用直接杂交筛选方法获得基因组克隆P579中的基因克隆，根据HCCS1基因克隆的cDNA序列与基因组序列进行比较确定基因的外显子与内含子，应用RT－PCR扩增组织中的HCCS1基因，序列测定检查突变，应用免疫组化检测HCCS1在组织中的表达，应用克隆形成试验和裸鼠成瘤试验检测HCCS1的生物学功能。结果：HCCS1有18个外显子，cDNA全长约2．0kb，蛋白产物定位于线粒体，HCCS1在肝癌细胞中有高频率的突变，免疫组化检测表明HCCS1在癌旁组织的表达明显高于癌组织，HCCS1转染肝癌细胞明显抑制其克隆的形成及在裸鼠体内的成瘤，结论：上述发现表明HCCS1具有肝癌抑癌基因的作用。     

卵巢上皮性癌p53基因改变,蛋白过度表达及临床意义

目的：检测卵巢上皮性癌ｐ５３基因改变、蛋白过度表达，探讨其与临床病理和预后的关系。方法：应用ＡＢＣ免疫组化法检测５６例卵巢上皮性肿瘤ｐ５３蛋白表达，对其中４０例应用聚合酶链反应单链构象多态性（ＰＣＲＳＳＣＰ）溴乙锭染色法检测ｐ５３基因５～８外显子突变及杂合性缺失（ＬＯＨ）。结果：（１）ｐ５３蛋白表达阳性率：良性肿瘤０／１０；交界性肿瘤１／４；卵巢上皮性癌２２／４２（５２．３８％），浆液性与粘液性癌表达相似，且３例原发灶与转移灶表达一致。（２）ｐ５３基因突变率：良性肿瘤０／１０；交界性肿瘤１／３；卵巢上皮性癌１３／２７（４８．１５％），其中８例杂合型基因的病例中４例基因突变伴等位基因杂合性缺失。本研究１３例ｐ５３基因突变者中１２例ｐ５３蛋白过度表达。ｐ５３蛋白过度表达与卵巢上皮性癌组织学类型、临床分期及预后无关（Ｐ＞０．０５）。而与卵巢上皮性癌病理有关（Ｐ＜０００５）。结论：ｐ５３基因改变及蛋白过度表达在卵巢癌中较常发生，且与卵巢上皮性癌病理分级有关，这些改变可能是卵巢上皮性癌的早发事件，亦在卵巢上皮性癌的发生发展过程中起重要作用。     

非小细胞肺癌pl6/CDKN2基因失活的研究

目的：分析中国人非小细胞肺癌中ｐ１６／ＣＤＫＮ２基因失活的情况,探讨该基因在肺癌发生中的作用。方法：选取与ｐ１６基因紧密连锁的Ｄ９Ｓ１７４８位点,对１７例临床切除的原发性肺癌标本进行微卫星不稳定性分析,用甲基化特异性ＰＣＲ（ｍｅｔｈｙｌａｔｉｏｎ－ｓｐｅｃｉｆｉｃ ＰＣＲ, ＭＳＰ）检测 ｐｌ６基因启动子区 ＣｐＧ岛甲基化状况,用免疫组织化学法检测Ｐ１６蛋白表达情况。结果：微卫星不稳定性分析结果表明,在１３例Ｄ９Ｓ１７４８位点存在多态性的肿瘤ＤＮＡ中有 ９例（６９ ．２％）发生了杂合性缺失（ｌｏｓｓ ｏｆ ｈｅｔｅｒｏｚｙｇｏｓｉｔｙ, ＬＯＨ）。 ＭＳＰ的结果显示,有 ７０． ６％（１２／１７）的肿瘤组织存在ｐ１６启动子区的异常高甲基化。在本研究中,８２．４％（１４／１７）的肿瘤组织可以检测出一种或两种ｐ１６基因的异常改变。对此１７例肿瘤标本进行的免疫组织化学分析显示,有 １３例 Ｐ１６蛋白表达阴性,其中 ９２ ３％（１２／１３）存在一种或两种ｐ１６基因的异常改变。免疫组化结果与ｐ１６基因分子遗传学改变情况基本吻合。结论：作为一种抑癌基因,ｐ１６在多种肿瘤组织中都有异常改变。我们的研究表明,ｐ１６基因失表达是非小细胞肺癌     

肝癌中具有高突变率位于染色体17p13.3区的新抑癌基因

目的　以往研究表明肝癌中染色体 1 7p1 3 .3区有高频率的杂合性缺失。其最小杂合性缺失范围已被确定在D1 7S643至D1 7S1 574位点间 ,而且其中的D1 7S92 6位点有最高的杂合性缺失率。含有该位点的基因组克隆P579已被测序分析 ,在P579范围共有 1 3个新基因。这里报告其中的一个新基因 (命名为肝癌抑癌基因 1 ,HCCS1 )的克隆和特性研究结果。方法　利用直接杂交筛选方法获得基因组克隆P579中的基因克隆。根据HCCS1基因克隆的cDNA序列与基因组序列进行比较确定基因的外显子与内含子。应用RT PCR扩增组织中的HCCS1基因 ,序列测定检查突变。应用免疫组化检测HCCS1在组织中的表达。应用克隆形成试验和裸鼠成瘤试验检测HCCS1的生物学功能。结果　HCCS1有 1 8个外显子 ,cDNA全长约2 .0kb ,蛋白产物定位于线粒体。HCCS1在肝癌组织中有高频率的突变 ,免疫组化检测表明HCCS1在癌旁组织的表达明显高于癌组织。HCCS1转染肝癌细胞明显抑制其克隆的形成及在裸鼠体内的成瘤。结论　上述发现表明HCCS1具有肝癌抑癌基因的作用     

Loss of heterozygosity at chromosome 11 in breast cancer: association of prognostic factors with genetic alterations.

We examined DNA from 116 female and four male breast cancer patients for loss of heterozygosity (LOH). DNA was analysed by polymerase chain reaction using ten microsatellite markers on chromosome 11. Three distinct regions of LOH were identified: 11p15.5, 11q13 and 11q22-qter with a LOH frequency of 19, 23 and 37-43% respectively. The marker D11S969 showing the highest frequency of LOH (43%) is located at the 11q24.1-q25 region. No previous molecular genetic studies have shown frequent LOH at the region telomeric to q23 on chromosome 11. Southern analysis revealed that LOH at 11q13 was due to amplification, whereas LOH at 11q22qter was due to deletion. LOH at 11p15.5 was associated with paucity of hormone receptor proteins, high S-phase and positive node status. An association was found between LOH at 11q13 and positive node status. LOH at the 11q22-qter region correlated with a high S-phase fraction. A significant association was found between LOH at 11p15 and chromosome regions 17q21 (the BRCA1 region) and 3p.     

LOSS OF HETEROZYGOSITY ON CHROMOSOME 17p13.3 IN OVARIAN CANCER AND CERVICAL CANCER

ＣａｒｃｉｎｏｇｅｎｅｓｉｓｃｏｎｓｉｓｔｓｏｆｍｕｌｔｉｐｌｅｑｕａｌｉｔａｔｉｖｅｌｙｄｉｆｆｅｒｅｎｔｓｔｅｐｓｉｎｗｈｉｃｈａｃｃｕｍｍｕｌａｔｉｏｎｏｆＤＮＡａｌｔｅｒａｔｉｏｎｏｃｕｒｓａｎｄｃｒｉｔｉｃａｌｅｖｅｎｔｓ，ｉｎｖｏｌｖｉ...     

Loss of heterozygosity at chromosome 1p in human breast cancer

232 human primary invasive breast tumors were analyzed with 13 polymorphic microsatellite markers specific to chromosome 1p. Loss of heterozygosity (LOH) was observed in 126 cases or 54% of the tumors. One marker, D1S496, at the 1p35 region showed the highest LOH, 28%. High frequencies of LOH were also detected by the markers, D1S488, D1S167 and D1S435, at the 1p31 region, 25%, 24% and 26% LOH, respectively. This suggests the presence of tumor suppressor genes at these two regions. Tumors with and without LOH at 1p were tested for association with clinico-pathological features of the tumors such as estrogen- and progesterone-receptor content (ER and PgR), age at diagnosis, tumor size, node status, histological type, S-phase fraction, ploidy, survival and LOH at chromosomes 3p, 6q, 9p, 11p, 11q, 13q, 16q, 17p and 17q. A significant association was found between LOH at chromosome 1p and high S-phase fraction and lower survival rate. Association was also found between LOH at 1p and chromosome regions 3p, 6q, 9p and 17q. A multivariate model including prognostic variables, showed that LOH at 1p is an independent prognostic variable and patients who have breast tumors with LOH at 1p have approximately a two-fold increase in relative risk of death. We conclude that screening for 1p deletions gives additional prognostic information that might be useful in breast cancer treatment.     

Loss of heterozygosity for defined regions on chromosomes 3, 11 and 17 in carcinomas of the uterine cervix.

Loss of heterozygosity (LOH) frequently occurs in squamous cell carcinomas of the uterine cervix and indicates the probable sites of tumour-suppressor genes that play a role in the development of this tumour. To define the localization of these tumour-suppressor genes, we studied loss of heterozygosity in 64 invasive cervical carcinomas (stage IB and IIA) using the polymerase chain reaction with 24 primers for polymorphic repeats of known chromosomal localization. Chromosomes 3, 11, 13, 16 and 17, in particular, were studied. LOH was frequently found on chromosome 11, in particular at 11q22 (46%) and 11q23.3 (43%). LOH on chromosome 11p was not frequent. On chromosome 17p13.3, a marker (D17S513) distal to p53 showed 38% LOH, whereas p53 itself showed only 20% LOH. On the short arm of chromosome 3, LOH was frequently found (41%) at 3p21.1. The beta-catenin gene is located in this chromosomal region. Therefore, expression of beta-catenin protein was studied in 39 cases using immunohistochemistry. Staining of beta-catenin at the plasma membrane of tumour cells was present in 38 cases and completely absent in only one case. The tumour-suppressor gene on chromosome 3p21.1 may be beta-catenin in this one case, but (an)other tumour-suppressor gene(s) must also be present in this region. For the other chromosomes studied, 13q (BRCA-2) and 16q (E-cadherin), only sporadic losses (< 15% of cases) were found. Expression of E-cadherin was found in all of 37 cases but in six cases the staining was very weak. No correlation was found between clinical and histological parameters and losses on chromosome 3p, 11q and 17p. In addition to LOH, microsatellite instability was found in one tumour for almost all loci and in eight tumours for one to three loci. In conclusion, we have identified three loci with frequent LOH, which may harbour new tumour-suppressor genes, and found microsatellite instability in 14% of cervical carcinomas.     

Molecular genetic investigation of sporadic renal cell carcinoma: analysis of allele loss on chromosomes 3p, 5q, 11p, 17 and 22.

To investigate the role of tumour-suppressor genes on the short arm of chromosome 3 in the mechanism of tumorigenesis in non-familial renal cell carcinoma, we analysed 55 paired blood-tumour DNA samples for allele loss on chromosome 3p and in the region of known or putative tumour-suppressor genes on chromosomes 5, 11, 17 and 22. Sixty-four per cent (35/55) of informative tumours showed loss of heterozygosity (LOH) of at least one locus on the short arm of chromosome 3, compared with only 13% at the p53 tumour-suppressor gene and 6% at 17q21. LOH at chromosome 5q21 and 22q was uncommon (2-3%). Detailed analysis of the regions of LOH on chromosome 3p suggested that, in addition to the VHL gene in chromosome 3p25-p26, mutations in one or more tumour-suppressor genes in chromosome 3p13-p24 may be involved in the pathogenesis of sporadic renal cell carcinoma (RCC). We also confirmed previous suggestions that chromosome 3p allele loss is not a feature of papillary RCC (P < 0.05).     

Frequent loss of heterozygosity at the DCC locus in gastric cancer.

We examined 28 cases of surgically resected gastric cancer, excluding the diffuse type, for loss of heterozygosity (LOH) on 12 chromosomal arms using polymorphic DNA markers. LOH on chromosome 18q was detected in 61% (14 of 23) of the cases by the probes OLVIIA8, OLVIIE10, p15-65, SAM 1.1, and OS-4, and a putative common region showing LOH included the locus of the DCC tumor suppressor gene. LOH on chromosome 17p was also frequently found (8 of 19 or 42% of the cases) by the probes p10-3 and pHF12-1, and in 5 of these 6 cases the LOH on chromosome 17p was accompanied by LOH on chromosome 18q. On the other hand, the incidence of LOH was 30% or less using probes pHRnES, pHF12-65, p-c-mybE2.6, NJ3 3.2, pHF12-8, pHINS6.0, p9D11, hp2-alpha, pCMM6, and P1A5 on chromosomes 1q, 5, 6q, 7q, 9, 11p, 13q, 16q, 20, and 22q, respectively. LOH on chromosome 18q was frequent irrespective of the depth of tumor invasion, whereas the incidence of LOH on chromosome 17p was higher in the cases in which the tumor invaded beyond the muscularis propria than in those in which tumor invasion was limited to the submucosa and muscularis propria. These results suggest that LOH on chromosome 18q occurs at an earlier stage than LOH on chromosome 17p and that the inactivation of tumor suppressor genes located on chromosome 17p and 18q (e.g., the p53 and DCC genes) is critically involved in the development of the majority of gastric cancers. While alteration of the p53 gene is observed in various human cancers, that of the DCC gene is considered to occur more selectively in gastrointestinal cancers.     

肝癌染色体17p13.3区的杂合性缺失分析及缺失区连续克隆群的构建

目的　检测原发性肝癌在染色体 17p13.3区的杂合性缺失状况 ,确定其共同缺失范围和最小热点缺失范围 ,并获得缺失范围内基因组克隆和构建连续克隆群。方法　应用Southern杂交分析VNTR(variablenumberoftandemrepeat,VNTR)和RFLP标志在肝癌中杂合性缺失 (LOH)状况。应用PCR扩增微卫星标志 ,变性聚丙烯酰胺凝胶电泳分析各个微卫星标志的LOH状况。以微卫星标志为引物 ,经过 3轮PCR筛选阳性基因组克隆。通过检测各位点标志对基因组克隆的反应 ,构建连续克隆群。结果　检测了 5 4份原发性肝癌样品在染色体 17p13.3区 16个位点标志和染色体 17p13.1区的p5 3基因的TP5 3位点标志的LOH情况 ,发现从D17S5位点至D17S34位点间的各个标志都有较高LOH ,频率 >6 3%。而从D17S5位点起、近着丝粒方向的 3个标志LOH率都较低或无LOH。染色体17p13.1的TP5 3标志只有 31%的LOH ,低于染色体 17p13.3区的D17S5至D17S34位点间各标志的LOH率。有 2例肝癌样品在近端粒的D17S34、D17S186 6位点和近着丝粒的D17S5、D17S15 74位点均无LOH ,但在D17S849至D17S15 74间的各位点上均呈LOH或为纯合子。在缺失范围内 ,共筛选了 18个位点的基因组克隆 ,获得了相对应的阳性基因组克隆 ,经过检测各位点对基因组克隆的反应性 ,构建了覆盖 9个标     

Multiple target sites of allelic imbalance on chromosome 17 in Barrett's oesophageal cancer

Twelve Barrett's adenocarcinomas have been analysed for the occurrence of allelic imbalance (LOH) on chromosome 17 using 41 microsatellite markers. This study provides evidence for 13 minimal regions of LOH, six on 17p and seven on 17q. Four of these centre in the vicinity of the known tumour suppressor genes (TSGs) TP53 (17p13.1), NFI (17q11.2), BRCA1 (17q21.1), and a putative TSG (17p13.3). The tumours all displayed relatively small regions of LOH (1-10 cM), and in several tumours extensive regions of LOH were detected. One tumour displayed only two very small regions of LOH; 17p11.2 and 17p13.1. The frequency of allelic imbalance has been calculated based on the LOH encompassing only one minimal region, and based on all the LOH observations. By both evaluations the highest LOH frequencies were found for regions II (p53), III (17p13.1 centromeric to p53), IV (17p12), V (17p11.2) and VII (NF1, 17q11.2). Our data supports the existence of multiple TSGs on chromosome 17 and challenges the view that p53 is the sole target of LOH on 17p in Barrett's adenocarcinoma.