Identification of tumor suppressive activity by irradiation microcell-mediated chromosome transfer and involvement of alpha B-crystallin in nasopharyngeal carcinoma

In previous studies, we successfully refined nasopharyngeal carcinoma (NPC) critical regions (CRs) mapping to chromosome 11q13 and 11q22-23. The chromosome 11 fragment containing the 1.8 Mb NPC CR at 11q13 (CR1), the CR at 11q22.3 mapped near D11S2000 (CR2), part of the CR at 11q23.1-11q23.2 overlapping with D11S1300 and D11S1391 (CR3), and the CR at cell adhesion molecule 1 (CADM1) locus (CR4), was chosen as the chromosome 11 donor cell line for the present study. Gamma irradiation was applied to cleave this truncated chromosome into smaller fragments and a new panel of donor cells containing further deleted fragments was produced. Subclones XMCH3.2 and XMCH3.4 were chosen for subsequent transfer to HONE1 cells; each contains a single copy of deleted chromosome 11 fragment with or without CR2 and the THY1 locus, previously shown to be involved in NPC. Both resultant chromosome 11 fragments in XMCH3.2 and XMCH3.4 caused tumor suppression. The association of alpha B-crystallin (CRYAB), a gene identified as being differentially expressed by gene profiling of NPC and an immortalized nasopharyngeal epithelial cell line, and which is located near CR3, was found to be associated with tumor suppression in all the tumor-suppressive hybrids. In addition, the expression level of this gene was down-regulated in the 7 NPC cell lines and in 5 out of 14 normal/tumor tissue pairs in the present study. Both promoter hypermethylation and allelic loss may be involved in the inactivation of this gene, suggesting its possible role in NPC development.     

Chromosome 13q12 region critical for the viability and growth of nasopharyngeal carcinoma hybrids

Allelic losses of chromosome 13 are often detected in nasopharyngeal carcinoma (NPC) and other cancers, implicating the presence of possible tumor suppressor genes (TSGs) on this chromosome. To identify candidate regions from larger and multiple lost areas observed from direct tumor studies, the technique of monochromosome transfer was utilized to provide functional evidence to verify and define these deletion findings. An intact chromosome 13 was transferred into the NPC HONE1 cell line. Resultant hybrids were used to map putative TSG activity. A critical region at 13q12 was non-randomly eliminated in all surviving microcell hybrids around the marker D13S893; these hybrids were uniformly tumorigenic. Although a known TSG, BRCA2, is mapped close to this critical region, no aberrant expression of this gene was detected in microcell hybrids and other NPC cell lines. These results suggest that at least one novel growth control gene on chromosome 13q12, which is not the BRCA2 gene, is essential for hybrid selection and may play a critical role in tumorigenicity.     

Expression of TSLC1, a candidate tumor suppressor gene mapped to chromosome 11q23, is downregulated in unfavorable neuroblastoma without promoter hypermethylation

Although it has been well documented that loss of human chromosome 11q is frequently observed in primary neuroblastomas, the smallest region of overlap (SRO) has not yet been precisely identified. Previously, we performed array-comparative genomic hybridization (array-CGH) analysis for 236 primary neuroblastomas to search for genomic aberrations with high-resolution. In our study, we have identified the SRO of deletion (10-Mb or less) at 11q23. Within this region, there exists a TSLC1/IGSF4/CADM1 gene (Tumor suppressor in lung cancer 1/Immunoglobulin superfamily 4/Cell adhesion molecule 1), which has been identified as a putative tumor suppressor gene for lung and some other cancers. Consistent with previous observations, we have found that 35% of primary neuroblastomas harbor loss of heterozygosity (LOH) on TSLC1 locus. In contrast to other cancers, we could not detect the hypermethylation in its promoter region in primary neuroblastomas as well as neuroblastoma-derived cell lines. The clinicopathological analysis demonstrated that TSLC1 expression levels significantly correlate with stage, Shimada's pathological classification, MYCN amplification status, TrkA expression levels and DNA index in primary neuroblastomas. The immunohistochemical analysis showed that TSLC1 is remarkably reduced in unfavorable neuroblastomas. Furthermore, decreased expression levels of TSLC1 were significantly associated with a poor prognosis in 108 patients with neuroblastoma. Additionally, TSLC1 reduced cell proliferation in human neuroblastoma SH-SY5Y cells. Collectively, our present findings suggest that TSLC1 acts as a candidate tumor suppressor gene for neuroblastoma.     

Epigenetic inactivation of TSLC1 gene in nasopharyngeal carcinoma

Deletion of 11q23 is a common genetic aberration in nasopharyngeal carcinoma (NPC). Multiple candidate tumor suppressor genes (TSG) were mapped to this region but few of them were investigated in NPC. TSLC1 (tumor suppressor in lung cancer) is recently reported to be a putative TSG on 11q23. This gene was found to be inactivated by promoter hypermethylation in non-small cell lung carcinoma (NSCLC), liver cancer, and breast cancer. To study the role of TSLC1 gene in NPC tumorigenesis, we screened for mutations and aberrant methylation of TSLC1 gene in 5 NPC cell lines, 3 NPC xenografts, and 38 primary NPC cases. No somatic mutations of TSLC1 were detected in the NPC samples, but a 9-bp (CCACCACCA) deletion in exon 8 was found in a primary NPC and its corresponding blood sample. Bisulfite sequencing revealed aberrant methylation of TSLC1 promoter in four NPC cell lines. Loss of TSLC1 gene expression was found in two cell lines (HK-1 and CNE-2) with dense methylation. Expression of this gene was restored in these cell lines after treatment with demethylating agent 5-aza-2'-deoxycytidine. Our results showed that silencing of TSLC1 gene expression in NPC was associated with promoter hypermethylation. Promoter hypermethylation of TSLC1 gene was further illustrated in 34.2% (13/38) of primary NPCs. No aberrant promoter methylation was found in any of the four investigated normal nasopharyngeal epithelia. Frequent epigenetic inactivation of TSLC1 gene in NPC suggested that this gene is one of the target tumor suppressor genes of this endemic cancer.     

Detailed deletion mapping at chromosome 11q23 in colorectal carcinoma

Loss of heterozygosity (LOH) is frequent at the chromosomal region 11q22-q23 in several types of tumours of diverse cell origin. Previous investigations of LOH at this chromosomal region in colorectal carcinoma have been contradictory in their findings, and have only included between 1-4 loci. In order to define any regions of LOH on 11q23, we investigated 16 loci between D11S940 and D11S934 on the long arm of chromosome 11 using microsatellite analysis. Of 57 colorectal carcinomas specimens, 36 (63.2%) demonstrated LOH at one or more marker, with the highest frequencies of LOH at D11S1340 (41.0%), located between 105.13-111.97 Mb from the centromere, and D11S924 (37.1%) and D11S4107 (40.5%), both located approximately 113 Mb from the centromere. No statistically significant associations between LOH and age-of-presentation or Dukes' stage were found. LOH was observed in colorectal tumours of all Dukes' stages, including Dukes' stages A and B, suggesting that the inactivation of a tumour suppressor gene(s) on 11q23 occurs in the early stages of colorectal carcinoma. These results confirm the presence of putative tumour suppressor gene(s) at chromosome 11q23, involved in the carcinogenesis of colorectal carcinoma, and will facilitate future identification of candidate genes.     

Fine mapping of chromosome 22q tumor suppressor gene candidate regions in astrocytoma

Astrocytomas and glioblastomas are the most frequent primary brain tumors in adults. Mutations and altered expression of multiple genes have been found to contribute to the genesis of these tumors. However, many factors in the genesis of astrocytic gliomas are not resolved yet. The frequent losses on several chromosomes indicate the role of still unidentified tumor suppressor genes. Loss of heterozygosity (LOH) on 22q has been described in up to 30% of astrocytic tumors and may be associated with progression to anaplasia. In a first step, information from the nearly finished physical sequence of chromosome 22 were used to map LOH data from 22q deletion studies on different tumor entities to identify potential tumor suppressor gene candidate regions. Next, a series of 153 astrocytic gliomas was examined with 11 polymorphic markers spanning these regions. Forty-nine (32%) astrocytic gliomas exhibited LOH on 22q, 17 (35%) of which lost heterozygosity for all markers and 32 (65%) of which carried interstitial or partial deletions. Two regions were identified on the physical DNA sequence. The centromeric region spans 3 Mb and the telomeric region 2.7 Mb. The reduced size of these regions now allows direct analysis of all genes included. We already performed mutation analysis on 4 candidate genes from these regions (MYO18B, DJ1042K10.2, MKL1 and EP300), but did not find any mutations in astrocytic tumors.     

Association of genetic polymorphisms at 1q22 but not 10q23 with gastric cancer in a southern Chinese population

Objective: Data from a recent genome-wide association studiesy of gastric cancer (GC) and oesophagealsquamous cell carcinoma in Chinese living in the Taihang Mountains of north-central China suggest that 1q22and 10q23 are susceptibility-associated regions for GC. However, this has not been confirmed in southernChinese populations. The aim of this study was to investigate whether these polymorphisms at 1q22 and 10q23are associated with the risk of GC in a southern Chinese population. Methods: We selected seven top significantassociated single nucleotide polymorphisms (SNPs) at 1q22 and 10q23 and conducted a population-based casecontrolstudy in a southern Chinese population. Genotypes were determined using MassARRAYTM system(Sequenome, San Diego, CA). Results: Two SNPs at 1q22, rs4072037 and rs4460629, were significantly associatedwith a reduced risk of GC, best fitting the dominant genetic model. Logistic regression models adjusted for ageand sex showed that rs4072037 AG and GG (OR=0.64, P=0.017, compared with AA) and rs4460629 CT and TT(OR=0.54, P=0.0016, compared with TT) significantly reduced the risk of GC. However, no significant results forthe five SNPs at 10q23 were obtained in this study. Conclusion: These outcomes indicate that 1q22 is associatedwith GC susceptibility in this southern Chinese population, while an association for the locus at 10q23 was notconfirmed.     

鼻咽癌原发灶与颈部淋巴结转移癌组织中nm23-H1、MMP-9蛋白表达及其意义

目的：研究鼻咽癌原发灶和颈部淋巴结转移癌组织中nm23-H1（non—metastatic cloneNo．23）、MMP-9（matrix metalloproteinase9）基因蛋白的表达及其意义。方法：采用免疫组化二步法检测22例鼻咽癌患者自身原发灶和颈部淋巴结转移癌组织中nm23-H1、MMP-9蛋白的表达。结果：淋巴结转移癌组织的nm23-H1阳性表达率（17／22，77．3％）低于原发灶组织中的表达水平（18／22，81．8％），但未见显著性（P〉0．05）；MMP-9在淋巴结转移癌组织中，癌细胞阳性表达率（19／22，86．4％）高于原发灶组织中的表达水平（13／22，59．1％），差异有显著性（P〈0．05）；原发灶与转移灶中nm23-H1呈正相关，转移癌组织中nm23-H1和MMP-9呈负相关，均有统计学意义（P〈0．05）。结论：在鼻咽癌组织中，nm23-H1蛋白的表达下调可能是导致癌细胞侵袭转移的-个重要因素。MMP-9在鼻咽癌组织其表达强度可能与鼻咽癌病期进展和颈部淋巴结转移有关。     

MTA1和TSLC1表达与结肠腺瘤癌变相关性研究

目的 结肠腺瘤是结肠癌的癌前病变,积极诊断和治疗结肠腺瘤是控制、减少结肠癌发病的重要途径.本研究探讨肿瘤转移相关基因1(metastsis associated gene 1,MTA1)和肺癌肿瘤抑制因子1(tumor suppressor in lung cancer 1,TSLC1)在结肠腺瘤和结肠癌组织中的表达,以及MTA1和TSLC1在结肠腺瘤癌变过程中的作用.方法 选择2010-04-01-2015-12-31河北医科大学附属邢台市人民医院手术切除的结肠癌标本104例、肠镜活检结肠腺瘤标本114例及癌旁正常结肠组织(距离癌组织>3 cm)30例为研究对象,应用免疫组织化学法检测3组标本中MTA1和TSLC1蛋白的表达.结果 正常结肠组织中MTA1阳性表达率为6.67%,管状腺瘤为42.86%,绒毛状腺瘤为63.64%,结肠癌为84.62%,呈逐渐上升趋势,χ2=68.668,P<0.001;中重度异型增生结肠腺瘤中MTA1的阳性表达率(81.25%)高于轻度异型增生结肠腺瘤(39.02%),χ2=16.421,P<0.001;正常结肠组织中TSLC1阳性表达率为96.67%,管状腺瘤为88.57%,绒毛状腺瘤为63.64%,结肠癌组为42.31%,呈逐渐下降趋势,χ2=52.679,P<0.001;中重度异型增生结肠腺瘤中TSLC1阳性表达率(37.50%)明显低于轻度异型增生结肠腺瘤(95.12%),差异有统计学意义,χ2=45.982,P<0.001.结论 结肠癌组织中MTA1呈高表达,TSLC1表达缺失.MTA1和TSLC1参与正常组织、癌前病变和癌组织的发生发展,在结肠腺瘤的癌变过程中起着重要的作用.     

TSLC1在食管鳞癌中的表达及临床意义

目的：研究食管鳞状细胞癌组织中肺癌肿瘤抑制因子1（TSLC1）基因的表达及临床病理意义,探讨其在食管鳞状细胞癌发生、发展过程中的作用。方法：应用免疫组化（SP法）检测50例食管鳞状细胞癌标本及癌旁正常食管组织中TSLC1基因的表达情况,分析其与食管鳞状细胞癌患者临床病理学参数之间的关系。结果：50例食管鳞状细胞癌组织中TSLC1基因表达阳性率为48.0%（24/50）,其表达与癌组织浸润深度、区域淋巴结转移及TNM分期有关（P〈0.05）。与性别、年龄、肿瘤大体分型、肿瘤大小、位置、分化程度无关（P〉0.05）。50例癌旁正常食管组织中TSLC1基因高表达,阳性率为94.0%（47/50）。食管鳞状细胞癌组织中的阳性表达率显著低于癌旁正常食管组织,两者相比差异有统计学意义（P〈0.01）。结论：食管鳞状细胞癌组织中有明显的TSLC1基因的表达缺失,TSLC1蛋白表达缺失与食管鳞状细胞癌的发生、发展有关。     

nm23-H1基因表达与鼻咽癌的早发转移

目的探讨nm23-H1基因产物的表达与鼻咽癌早发转移的关系.方法应用免疫组化S-P法对95例鼻咽癌组织蜡块进行nm23-H1检测.结果nm23-H1的阳性表达率为47.4%(45/95),早发转移组nm23-H1阳性率(26.7%)低于无转移组(60.0%),差异有统计学意义(P＜0.05).nm23-H1表达与临床分期无关.结论nm23-H1阴性表达与鼻咽癌早发转移有关,nm23-H1的表达水平可能是预测中晚期鼻咽癌远处转移的一个重要的生物学指标.     

RTQ—PCR检测鼻咽癌组织中nm23-H1基因表达研究

目的：建立实时荧光定量RTQ—PCR方法检测鼻咽癌患者组织中nm23-H1mRNA的表达，探讨nm23-H1mRNA的表达与鼻咽癌发生发展关系。方法：用TRIZOL方法提取鼻咽癌组织总RNA后，将其反转录为cDNA，以实时荧光定量RTQ—PCR方法检测人鼻咽癌nm23-H1mRNA。结果：nm23-H1mRNA在鼻咽癌组织中表达低于慢性鼻咽炎患者，在转移性鼻咽癌组织中nm23～H1mRNA表达也低于非转移的鼻咽癌患者，二组结果比较均具有显著性差异。结论：实时荧光定量RTQ—PCR法用于检测人鼻咽癌nm23-H1mRNA的表达是-种快速有效、灵敏度高、特异性好的定量检测方法，nm23-H1mRNA基因表达水平可能与鼻咽癌发生发展和肿瘤转移有关。     

Cyclin D1 amplification in chromosomal band 11q13 is associated with overrepresentation of 3q21-q29 in head and neck carcinomas

Eight cytogenetically characterized head and neck squamous cell carcinomas (HNSCCs) with CCND1 amplification in the form of a homogeneously staining region (hsr) in 11q13 were studied by COBRA FISH and FISH with specific probes to identify and characterize chromosomal segments added to the derivative chromosomes 11. In 4 of the tumors, it could be recognized that the material added was derived from the long arm of chromosome 3. The rearrangements were interpreted as der(11)hsr(11)(q13)t(3;11)(q21;q13) in 3 cases and as der(11)hsr(11)(q13)t(3;11)(q14;q13) in 1 case. In the other 4 cases, material from chromosomes 1, 16, or 19 was added to the derivative chromosomes 11. By further FISH analysis with 14 YAC clones spanning 3q13-q21 in the 4 tumors with der(11)hsr(11)t(3;11), it could be shown that they had different breakpoints at the molecular level, excluding the possibility that a particular gene was rearranged by the translocations. More surprisingly, gain of the 3q21-q29 segment was found in all 8 tumors with hsr in 11q13 and loss of 3p was seen in 7 of the tumors. These findings strongly indicate a synergistic effect of CCND1 amplification, loss of distal 11q, 3q gain and 3p deletion in HNSCC development and also suggests a mechanistic link between intrachromosomal amplification at 11q13 and recombination with distal 3q.     

Expression of nm23-H1 gene product in nasopharyngeal carcinoma and its clinical significance

Objective: The nm23 gene is one of the tumor metastatic suppressor genes. The expression of nm23-H1 has been reported to be inversely associated with metastatic potentiality in a number of human carcinomas, including breast, colorectal, gastric, hepatocellular and gallbladder carcinomas. In this study, the immunohisto-chemical staining of nm23-H1 protein in human naso-pharyngeal carcinoma (NPC) was examined, and the relationship between nm23-H1 and both metastasis and prognosis of patients with NPC was also investigated. Methods: Routine LSAB immunohistochemistry with the nm23-H1 monoclonal murine antibody was employed to study the expression of nm23-H1 protein in 95 paraffin-embedded specimens of NPC treated at our hospital. The clinical pathologic data and results of follow-up were also retrieved. Comparisons between patients with and without expression of nm23-H1 protein with respect to metastasis, loco-regional recurrence and survival were performed using Log rank test. Multivariate prognostic analyses were performed by using Cox’s regression model. Results: Nm23-H1 negative expressive tumors were associated with a higher incidence of lymph-node metastasis (86.7%) than those of nm23-H1 positive (48.6%,P<0.01). Nm23-Hl negative expressive tumors were associated with a high incidence of recurrence and distant metastasis after radiotherapy (P<0.05). A significant association was found between expression of nm23-H1 and prognosis (P<0.01). The expression of nm23-H1 indicated favorable prognosis. Conclusion: It was suggested that nm23-H1 negative expression was significantly associated with lymph-node metastasis, recurrence and distant metastasis. Nm23-H1 may have value for predicting the prognosis of NPC.     

鼻咽癌原发灶与颈部淋巴结转移癌组织中nm23-H1、MMP-9蛋白表达及其意义

目的:研究鼻咽癌原发灶和颈部淋巴结转移癌组织中nm23-H1(non-metastatic clone No.23)、MMP-9(matrix metalloproteinase9)基因蛋白的表达及其意义。方法:采用免疫组化二步法检测22例鼻咽癌患者自身原发灶和颈部淋巴结转移癌组织中nm23-H1、MMP-9蛋白的表达。结果:淋巴结转移癌组织的nm23-H1阳性表达率(17/22,77.3%)低于原发灶组织中的表达水平(18/22,81.8%),但未见显著性(P>0.05);MMP-9在淋巴结转移癌组织中,癌细胞阳性表达率(19/22,86.4%)高于原发灶组织中的表达水平(13/22,59.1%),差异有显著性(P<0.05);原发灶与转移灶中nm23-H1呈正相关,转移癌组织中nm23-H1和MMP-9呈负相关,均有统计学意义(P<0.05)。结论:在鼻咽癌组织中,nm23-H1蛋白的表达下调可能是导致癌细胞侵袭转移的一个重要因素。MMP-9在鼻咽癌组织其表达强度可能与鼻咽癌病期进展和颈部淋巴结转移有关。     

Frequent alterations of LOH11CR2A, PIG8 and CHEK1 genes at chromosomal 11q24.1-24.2 region in breast carcinoma: clinical and prognostic implications

To understand the importance of frequent deletions at chromosome 11q24.1-24.2 region in breast carcinoma, alterations (deletion/methylation) of the candidate genes LOH11CR2A, ROBO3, ROBO4, HEPACAM, PIG8 and CHEK1 located in this region were analyzed in 106 breast carcinoma samples. Among these genes, LOH11CR2A showed highest frequency of deletion (56%), followed by PIG8 (35%), CHEK1 (31%) and ROBO3/ROBO4/HEPACAM loci (28%). Comparable frequency of promoter methylation (26–35%) was observed for LOH11CR2A, CHEK1 and PIG8. Overall alterations (deletion/methylation) of these genes were in the following order: LOH11CR2A (60%) > PIG8 (46%) > CHEK1 (41%) and showed significant association with each other. Breast carcinoma samples that were estrogen/progesterone receptor negative showed significantly high deletion and overall alterations than estrogen/progesterone receptor positive samples for LOH11CR2A, CHEK1 and PIG8. The methylation and overall alteration of LOH11CR2A were significantly associated with tumor stages in breast carcinoma. However, in early/late onset and estrogen/progesterone receptor positive/negative breast carcinoma, the overall alterations of LOH11CR2A, PIG8 and CHEK1 were differentially associated with advanced stages, tumor grade and lymph node metastasis. Alterations of PIG8 and CHEK1 were significantly associated with poor prognosis in patients with early age of onset of the disease indicating significant prognostic importance. Quantitative mRNA expression analysis detected reduced expression of the genes in the order LOH11CR2A > CHEK1 > PIG8. Immunohistochemical analysis showed reduced protein expression of PIG8 and CHEK1 that was concordant with their molecular alterations. Thus, our study suggests that LOH11CR2A, PIG8 and CHEK1 are candidate tumor suppressor genes associated with breast carcinoma and have significant clinical as well as prognostic importance.