High frequency of deletion on chromosome 9p21 may harbor several tumor-suppressor genes in human prostate cancer.

Chromosome 9p has been reported to be a critical region of loss in various cancers. Our present study was designed to determine the frequency of deletions at different loci of chromosome 9p in microdissected samples of normal prostatic epithelium and carcinoma from the same patients. For this purpose, DNA was extracted from the microdissected sections of normal and tumor cells of 40 prostate specimens, amplified by PCR and analyzed for loss of heterozygosity (LOH) on chromosome 9p using 15 microsatellite markers. Only 6 of 15 microsatellite markers exhibited LOH in prostate cancer specimens (D9S162, D9S1748, D9S171, D9S270, D9S273 and D9S153). LOH on chromosome 9p was identified in 29 of 40 cases (72.5%) with at least 1 marker. The main deletion was found on 9p21, at loci D9S1748 (50%), D9S171 (51.4%) and D9S270 (21.8%). There was also a deletion on 9p22 at locus D9S162 (8.3%), on 9p13 at locus D9S273 (13.8%) and on 9p11 at locus D9S153 (7.7%). LOH data were correlated with stage of prostate cancer and revealed a high frequency of LOH at 3 or more loci in samples with stage T(3)N(0)M(0) (46%) compared with stage T(2)N(0)M(0) (15%), which suggests a higher incidence of LOH in the advanced stage of prostate cancer. One of the candidate target tumor-suppressor genes, p16 (MTS-1/CDKN2), has been identified within the 9p21 deleted region in tumor cell lines. Expression of P16 protein was either absent or very low in prostate cancer samples, suggesting that loss of the p16 gene may be involved in prostatic carcinogenesis.     

Evidence for three tumor suppressor loci on chromosome 9p involved in melanoma development

Cytogenetic and loss of heterozygosity (LOH) studies have long indicated the presence of a tumor suppressor gene (TSG) on 9p involved in the development of melanoma. Although LOH at 9p has been reported in approximately 60% of melanoma tumors, only 5-10% of these tumors have been shown to carry CDKN2A mutations, raising the possibility that another TSG involved in melanoma maps to chromosome 9p. To investigate this possibility, a panel of 37 melanomas derived from 35 individuals was analyzed for CDKN2A mutations by single-strand conformation polymorphism analysis and sequencing. The melanoma samples were then typed for 15 markers that map to 9p13-24 to investigate LOH trends in this region. In those tumors demonstrating retention of heterozygosity at markers flanking CDKN2A and LOH on one or both sides of the gene, multiplex microsatellite PCR was performed to rule out homozygous deletion of the region encompassing CDKN2A. CDKN2A mutations were found in tumors from 5 patients [5 (14%) of 35], 4 of which demonstrated LOH across the entire region examined. The remaining tumor with no observed LOH carried two point mutations, one on each allele. Although LOH was identified at one or more markers in 22 (59%) of 37 melanoma tumors corresponding to 20 (57%) of 35 individuals, only 11 tumors from 9 individuals [9 (26%) of 35] demonstrated LOH at D9S942 and D9S1748 the markers closest to CDKN2A. Of the remaining 11 tumors with LOH 9 demonstrated LOH at two or more contiguous markers either centromeric and/or telomeric to CDKN2A while retaining heterozygosity at several markers adjacent to CDKN2A. Multiplex PCR revealed one tumor carried a homozygous deletion extending from D9S1748 to the IFN-alpha locus. In the remaining eight tumors, multiplex PCR demonstrated that the observed heterozygosity was not attributable to homozygous deletion and stromal contamination at D9S1748, D9S942, or D9S974, as measured by comparative amplification strengths, which indicates that retention of heterozygosity with flanking LOH does not always indicate a homozygous deletion. This report supports the conclusions of previous studies that a least two TSGs involved in melanoma development in addition to CDKN2A may reside on chromosome 9p.     

鼻咽癌染色体9p21～22区域精细缺失图谱的构建

目的 进一步精细限定鼻咽癌９ｐ２１－２２区域等位基因杂合性丢失的频率和范围,为发现和分离克隆该区域内的鼻咽癌抑瘤提供新线索和依据。方法 应用１１个定位于９ｐ－２１－２２区域的高密度微卫星位点,检测２５例低分化鼻咽癌患者的杂合性丢失。结果 ２５例患者中,有１７例存在一个或多个位点的杂合性丢失,占６８．０％。     

Detailed deletion mapping at chromosome 9p21 in non-small cell lung cancer by microsatellite analysis and fluorescence in situ hybridization

We examined 82 cases of primary non-small cell lung cancer (NSCLC) for loss of heterozygosity (LOH) at the chromosome 9p21-24 region using 16 microsatellite markers. A total of 52 tumors (63%) displayed LOH, and 25 of these cases displayed LOH for all markers. Two cases had small hemizygous losses confined to the p16 gene and more distal markers, whereas 3 cases had loss proximal to p16 and extended through marker D9S126. This latter region has recently been described as another minimal region of loss at 9p21 in lung cancer. However, homozygous deletion of the p16 gene was observed in 18 of 85 cases, with only 5 cases having large deletions extended into the D9S126 region. Furthermore, we did not observe homozygous deletion at the 9p21 region that excluded the p16 gene. Fluorescence in situ hybridization (FISH) analysis using genomic probes spanning either the p16 or Hel-N1 (located at D9S126) gene was performed in 14 tumors. The results from FISH correlated with the chromosomal mapping data, suggesting that the p16 region is the major target of deletion at chromosome 9p21 in primary NSCLC. Int. J. Cancer 74:588–592, 1997.© 1997 Wiley-Liss, Inc.     

Detailed Deletion Mapping of Chromosome 9p21-22 in Nasopharyngeal Carcinoma

Previous studies have showed that Epstein-Barr virus (EBV) infection, certain environmental factors and genetic factors were found to be closely associated with nasopharyngeal carcinoma. The rates of NPC in southern China and southeast Asia are 25 times higher than that of in western countries. The statistic analysis revealed 5%-10% NPC patients have family history, there, genetic susceptibility might be an important factor in the pathogenesis of NPC. Unfortunately the alterations of com…     

Allelic loss on chromosome 17 in human ovarian cancer

In order to identify a common region of deletion on chromosome 17 potentially containing a tumor-suppressor gene, 27 ovarian carcinomas and 3 ovarian tumors of low malignant potential (LMP) were examined for loss of heterozygosity (LOH) at 6 p arm and 10 q arm loci. Ninety percent of all tumors had deletions at one or more loci. On the p arm, there was a single near-common region of deletion on 17p 13.3 (D/7S30/ pYNZ22.1; 86% LOH), an intervening locus with a low LOH rate, and a more proximal locus on 17p11.2 (D/7S58/pEW301; 82% LOH) with a high LOH rate. In less aggressive tumors, LOH at Df 7S30 was not accompanied by LOH at p53. The q arm had a common region of deletion for high-stage carcinoma at D/7S579 (Mfd 188; 74% LOH) on q21, a locus tightly linked to the familial breast-ovarian-cancer syndrome (BRCAI) locus. D/7S579 was lost in all informative high-stage carcinomas and retained in all low-stage carcinomas and tumors of LMP. There may be at least 2 tumor-suppressor genes, an early-acting gene on the p arm and a gene on the q arm involved in tumor progression and metastasis.     

Frequent loss of heterozygosity in early non-small cell lung cancers at chromosome 9p21 proximal to the CDKN2a gene

Deletions involving the chromosome 9p21 region have been reported as frequent events in non-small cell lung cancer (NSCLC). To investigate potential tumor-suppressor gene (TSG) loci within the 9p21 region, which also harbors the candidate TSG locus CDKN2a, we are studied 32 cases of primary NSCLC for loss of heterozygosity (LOH). Tumor and paired normal lung cells were microdissected from lung tissue imprints and all samples screened using PCR-LOH analysis with 15 9p markers. In addition, 3 NSCLC cell lines and their matched normal lung and tumor DNA were similarly analyzed. LOH at the marker D9S259, which is proximal to the CDKN2a locus, was found most frequently (52%) while LOH at D9S942, the marker closest (5 kb) to the CDKN2a gene, was seen in only 17%. Homozygous loss of markers close to CDKN2a gene was, however, detected in 2 of the cell lines and one accompanying tumor sample. We propose that a TSG in the region of deletion proximal to the CDKN2a gene within 9p21 may play a significant role in the pathogenesis and progression of NSCLC. Int. J. Cancer 71:213–217, 1997. © 1997 Wiley-Liss, Inc.     

精细定位和克隆9p21-22区域内鼻咽癌候选抑瘤基因

目的：进一步精细限定鼻咽癌９p２１－２２区域等位基因杂合性丢失的频率和范围,筛选 和克隆其共同缺失区内鼻咽癌相关的候选抑瘤基因。方法：应用１１个定位于９p２１－２ ２区域的高密度微卫星位点,检测２５例低分化鼻咽癌患者的杂合笥技失,确定其共同缺失区;用ＲＴ－ＰＣＲ和Ｎｏｒｔｈｅｒｎ筛出在鼻咽癌细胞株ＨＮＥ１和鼻咽癌活检组织中表达下调的、定位于共同缺失区内的３’末端ＥＳＴs（Ｅｘｐｒｉｓｓ Ｓｅｑｕｅｎｃ     

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     

Intragenic Homozygous Deletions of MTS1 Gene in Gastric Cancer in Taiwan

The multiple tumor suppressor 1 ( MTS1 ) and 2 ( MTS2 ) genes, located on chromosome 9p21, have been reported to be deleted or mutated in many malignant cell lines and in a high percentage of some primary carcinomas. To determine whether these genes are altered, and if so, what is the nature of the alterations, in human gastric adenocarcinoma, we investigated their frequency of mutation by Southern blotting, polymerase chain reaction (PCR) and direct sequencing in 55 patients. Furthermore, loss of heterozygosity (LOH) of chromosome 9p21 at the IFNA locus and D9S171 was assessed. Homozygous deletions of exon 1 of the MTS1 gene were identified in 5 of 55 (9.1%) primary tumors. No deletion of MTS2 gene was noted. LOH was observed in 7 (14.3%) of 49 informative cases (5 cases at IFNA locus, 2 cases at D9S171 and one case with combined LOH at D9S171 and homozygous deletion at exon 1 of MTS1 ). Direct sequencing of PCR products of the MTS1 and MTS2 gene did not reveal any point mutation in these 55 patients. These data indicate that alterations of the MTS1 and MTS2 genes are infrequently encountered. Additional studies of LOH with more micro-satellite markers near 9p21 are mandatory to elucidate whether another tumor suppressor gene exists in the vicinity of MTS1 in primary gastric adenocarcinoma.     

Detailed deletion mapping of chromosome 9p21–22 in nasopharyngeal carcinoma

Objective: To further refine the extent of deletion on chromosome 9p21–22 in nasopharyngeal carcinoma (NPC) and provide evidence for discovering new tumor suppressor gene. Methods: Loss of heterozygosity (LOH) on chromosome 9p21–22 was analyzed in 25 paired blood and tumor samples by using 11 high-density microsatellite polymorphic markers. Results: 17 of 25 cases (68.0%) showed LOH at one or more loci. Higher frequencies of LOH were found at four loci: D9S161 (35.0%), D9S1678 (31.5%), D9S263 (33.3%) and D9S1853 (33.3%), where 6 cases had a contiguous stretch of allelic loss. Conclusion: The minimal common region of deletion might be defined between D9S161 and D9S1853 (estimated about 2.7 cM in extent) at 9p21.1, suggesting that inactivation of one or more tumor suppressor genes located in this region may be an important step in NPC. This work was supported by a grant from the National “863” Project of China (No. 102-10-01-05).     

Frequent Loss of 1p32 Region but no Mutation if the p18 Tumor Suppressor Gene in Meningiomas

After chromosome 22 and NF2 inactivation, the loss of chromosome 1p is one of the most frequent abnormalities encountered in meningiomas. However the putative tumor suppressor gene located on 1p inactivated in meningiomas has still to be identified. We screened 68 meningiomas for LOH on chromosome 22 and 1. We found 34 LOH on the NF2 region on chromosome 22 (50%) and 19 LOH on 1p (28%), 16 being associated with loss of chromosome 22. Partial deletions delimited a candidate region located between D1S234 and D1S2797. The p18 ^INK4C tumor suppressor gene, a member of the genes family coding for inhibitors of cyclin-dependent kinases, is located in this region. To determine whether p18 is involved in development of meningiomas, we performed a mutation analysis of the p18 gene and a search for homozygous deletion in the 19 meningiomas with 1p loss. Sequencing analysis of the p18 gene revealed one polymorphism, but no somatic mutations and no homozygous deletions were found. These results confirm that the loss of chromosome 1p32 is a frequent feature in meningiomas, however the p18 tumor suppressor gene which is located in this region, does not seem to be involved.     

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。     

Detailed characterization of a homozygously deleted region corresponding to a candidate tumor suppressor locus at distal 17p13.3 in human lung cancer

17p13.3 is one of the chromosomal regions most frequently affected by allelic loss in a variety of human neoplasms including lung cancer. A number of loss of heterozygosity (LOH) analyses have suggested the existence of a tumor suppressor gene at 17p13.3, distal to the p53 locus at 17p13.1. In the present study, we characterized a homozygous deletion at 17p13.3 in a small cell lung cancer cell line by constructing a bacterial artificial chromosome (BAC) contig and a restriction map surrounding the region, as well as by utilizing publicly available draft sequences. We defined the breakpoint, assigned and analysed two known genes, 14-3-3 epsilon and CRK, and a novel gene LOST1 within or at the end of the homozygous deletion of about 170 kb in size. Marked reduction of LOST1 expression was detected in 69% (11/16) of lung cancer specimens by quantitative real-time RT-PCR, while significant DNA hypermethylation was observed at the 5' end of the LOST1 gene in four of six lung cancer cell lines with negligible LOST1 expression. We also show here that a polymorphic marker D17S1174, which resides within the homozygous deletion, was apparently located in the middle of the minimum LOH region, providing further supportive evidence for the presence of a tumor suppressor gene(s) in this region.     

Homozygous deletions but no sequence mutations in coding regions of p15 or p16 in human primary bladder tumors

Chromosome 9p21 appears to harbor a tumor suppressor gene, as evidenced by deletions in this region in a variety of human primary tumors and cell lines. To map the deletion at 9p21 in bladder tumors, we analyzed DNA from 28 tumor and normal pairs at five microsatellite markers that flank the region occupied by the putative tumor suppressor genes p16 and p15. Loss of heterozygosity (a) at the markers human interferon (a) α and D9S171, which are adjacent to the p15 and p16 loci, was detected in 41% and 33%, respectively, of informative cases of bladder tumors. No sequence mutations were detected in exons 1 or 2 of either p15 or p16 in any of the bladder tumors. Three sequence-tagged site markers in the region bordered by HIFNα and D9S171 were used to further map the deleted region by multiplex polymerase chain reaction with the HIFNγ marker (on chromosome 12) as a control for amplification. Six of 11 tumors with LOH at surrounding markers had homozygous deletions of the marker c5.1, which is located within the p16 gene; and two tumors appeared to have homozygous deletions within p15(RN1.1) but not p16 (c5.1). A recently identified microsatellite marker, p16-CA-1, located 16 kb distal to p16, proved valuable in defining the minimal deletion involved in these bladder tumors. Five tumors exhibited homozygous deletions of this marker but not HIFNα and two tumors showed LOH at this marker and homozygous deletion of p16. Although these data could not be used to identify p16 or p15 as the definitive tumor suppressor gene in this region that is involved in bladder carcinogenesis, they suggest that homozygous deletion is a common mechanism of loss of tumor suppressor gene function in this region. © 1995 Wiley- Liss, Inc.     

A novel growth suppressor gene on chromosome 17p13.3 with a high frequency of mutation in human hepatocellular carcinoma

Previous studies have shown that there is a high frequency of loss of heterozygosity (LOH) on chromosome 17p13.3 in hepatocellular carcinoma [HCC (M. Fujimori et al., Cancer Res., 51: 89-93, 1991; H. Nagai et al., Oncogene, 14: 2927-2933, 1997; V. Boige et al., Cancer Res., 57: 1986-1990, 1997; Z. Piao et al., Int. J. Cancer, 75: 29-33, 1998; and B. Charroux et al., J. Cell Biol., 148: 1177-1186, 2000)]. The minimum region of LOH on chromosome 17p13.3 in HCC has been defined within the region between D17S643 and D17S1574. Moreover, D17S926 in the minimum region of LOH has the highest frequency of LOH, and its sequencing analysis has been accomplished. In this region, 6 of 13 novel genes have been characterized (X. Zhao, D. Wan, M. He, Yu. Ye, Yi. He, L. Han, M. Guo, Y. Huang, W. Qin, M-W. Wang, W. Chong, J. Chen, L. Zhang, N. Yang, B. Xu, M. Wu, L. Zuo, and J. Gu. A high frequency LOH region on chromosome 17p13.3 in human HCC with densely clustered genes identified, submitted for publication). Here we describe the cloning and characterization of one of these novel genes, designated HCC suppressor 1 (HCCS1), located at this region. HCCS1 had 18 exons, and its full-length cDNA was 2.0 kb. The protein expression product of HCCS1 was located in mitochondria. HCCS1 had a high frequency of mutations in HCC samples, whereas no alteration has been found in matched noncancerous liver tissues. Immunohistochemistry revealed a significantly higher expression of HCCS1 in the noncancerous liver tissues (33 of 35 samples) than in the HCC samples (2 of 35 samples). Transfection of HCCS1 cDNA into the HCC cell line remarkably reduced the efficiency of its colony formation and inhibited tumor growth in nude mice. Taken together, these findings strongly suggest a potential role of HCCS1 as a HCC putative suppressor gene.     

Cytogenetic and molecular analyses of multiple endocrine neoplasias of the MEN1 syndrome

A pancreas carcinoid, an adrenocortical adenoma, three parathyroid adenomas and a parathyroid hyperplasia of 5 MEN1 patients were used for loss of heterozygosity (LOH) and comparative genomic hybridization (CGH) studies. The MEN1 gene is located in the region 11q13, approximately 30 kb distal to PGYM. Four tumors showed LOH on chromosome 11q13 (D11S11335, PGYM, D11S1883, FGF3, D11S937) however LOH was also found beyond 11q13. The pancreas carcinoid and adrenocortical adenoma, both from the same patient, showed LOH at marker 11q23.3 and 11q25. In the three parathyroid adenomas LOH was detected in five different markers: 11q21, 11q22.3, 11q23.2, 11q23.3 and 11q25. No LOH was found in parathyroid hyperplasia. CGH analysis showed in case of the pancreas carcinoid losses on chromosomes 1p, 2q, 3, 6, 9p, 11 and 12p. Gains were found at 4, 5, 7, 8, 13, 14, 15q, 18, 19. The parathyroid adenoma of the third patient showed losses only on chromosome 11 in the region 11p12-p15 and 11q12-q23. Our data indicate that other genes are involved in the tumorigenesis of the MEN1 syndrome. Especially the numerous allelic losses between markers 11q23 and 11q25 (D11S938 and D11S910) are a hint for further tumor suppressor genes on chromosome 11.     

Two regions of deletion in 9p23-24 in sporadic breast cancer.

Allelic deletions of 9p including band 21-22 are common in various types of human carcinomas including breast cancer. Our previous cytogenetic studies had identified constitutional chromosomal changes in 9p23-24 in patients of a male-breast-cancer family and 9p23-24 alterations in a cell line established from a sporadic female breast cancer. To find out whether this genomic region is involved more frequently in alterations in sporadic breast cancers, we have surveyed 80 microdissected tumor samples for both loss of heterozygosity (LOH) and homozygous deletion at 22 microsatellite loci spanning 9p22 to 9p24 using fluorescent multiplex PCR. LOH at one or more loci was observed in 32 (40%) of these tumors. Homozygous deletion was detected in four cases. Eleven tumors had LOH at all of the informative loci analyzed, whereas 21 tumors showed partial-terminal or interstitial allelic loss of 9p. Deletion mapping identified two common regions of deletion: (a) 4 cM including D9S281 to D9S286; and (b) 1 cM including D9S1808 to D9S268.     

Homozygous deletion,rearrangement and hypermethylation implicate chromosome region 3p14.3-3p21.3 in sporadic breast-cancer development

DNAs from 19 malignant human breast tumors and 2 benign fibroadenomas were analyzed for heterozygosity at 5 polymorphic loci on the short arm of chromosome 3. One homozygous deletion and one rearrangement were identified using probe D3S2 which maps to 3p14.3-3p21.1. This probe also detected novel hybridizing fragments of 2.0 kb and/or 3.4 kb in 6/18 (33%) of the malignant tumor samples that hybridized with the D3S2 probe following digestion with the 5′-methylcytosine-insensitive enzyme Mspl. Comparisons of Hpall and Mspl digestion showed that all but one of the tumor DNAs analyzed were hypermethylated. The two fibroadenoma DNAs were not as highly methylated and had hybridizing fragments of 3.4 kb after Hpall digestion. These malignant breast-tumor DNAs exhibit 3 mechanisms by which a tumor-suppressor gene hypothesized to reside at 3p 14-3p21 could be inactivated: homozygous deletion, rearrangement and hypermethylation, and strongly implicate this 3p chromosome region in breast-tumor development. © 1994 Wiley-Liss, Inc.