老年人胃癌肿瘤抑制基因启动子区甲基化状态

目的分析老年人胃癌组织及其癌旁小凹上皮和慢性胃炎小凹上皮中肿瘤抑制基因启动子区甲基化状态。方法采用甲基化特异性聚合酶链反应(MSP)方法,检测51例老年人胃癌及其癌旁小凹上皮和15例慢性胃炎小凹上皮石蜡标本中E钙黏素(E-CD)、错配修复酶hMLH1、APC和6-氧-甲基鸟嘌呤-DNA甲基转移酶(MGMT)基因启动子区甲基化状态。结果在胃癌组织及其癌旁小凹上皮和慢性胃炎小凹上皮中,基因总的甲基化率分别为70.6%(36/51例)、47.0%(24/51例)和6.6%(1/15例),三者之间差别有统计学意义(P<0.01)。Lauren弥漫型、Ming浸润型、低分化和未分化、Ⅲ期和Ⅳ期、T3和T4以及有淋巴结转移胃癌的甲基化率分别高于Lauren肠型、Ming膨胀型、高和中分化、Ⅰ期和Ⅱ期、T1和T2以及无淋巴结转移胃癌的甲基化率(均P<0.05)。结论E-CD、hMLH1、APC和MGMT基因启动子区甲基化在慢性胃炎小凹上皮中少见,在癌旁小凹上皮中频繁出现,在胃癌组织中普遍存在,提示甲基化可能是胃癌发生的早期事件。

NF-κBp65和PTEN在子宫内膜样腺癌中的表达及临床意义

目的探讨核转录因子-κB(NF-κBp65)和肿瘤抑制基因(gene of phosphate and tension homology deleted on chromsome ten,PTEN)在子宫内膜样腺癌中的表达及其临床意义。方法用免疫组化方法对42例子宫内膜癌,11例子宫内膜不典型增生和10例正常子宫内膜的表达进行检验。结果 NF-κBp65及PTEN蛋白在正常子宫内膜、不典型增生子宫内膜及子宫内膜样腺癌组织中的阳性表达率差异均有统计学意义(P均<0.05);NF-κBp65蛋白的阳性表达率在子宫内膜样腺癌不同手术病理分期、组织分化程度的差异均有统计学意义(P均<0.05),与年龄无关(P>0.05),而PTEN的阳性表达率在子宫内膜样腺癌不同手术病理分期中差异无统计学意义(P>0.05),而在不同组织分化程度上的差异有统计学意义(P<0.05)。结论 NF-κBp65是与子宫内膜癌相关的癌蛋白,可能在子宫内膜癌的发生、发展中发挥重要作用。PTEN与子宫内膜癌发生、发展密切相关,可能是子宫内膜癌发生的相对早期的分子事件。

The Relationship between Methylation and Expression Defect of Tumor Suppressor Gene p16INK4A in Epithelial Ovarian Cancer

Objective： To evaluate the expression of p16INK4A gene in ovarian cancer and analyze the relation between this alteration and the promoter methylation of p16INK4A DNA. Methods： Seven ovarian cancer cell lines and eighteen ovarian cancer specimens were selected for the study. Genomic DNA and RNA were extracted from fresh tissues and cell lines, DNA was treated with sodium bisulfite and then analyzed with methylation-specific PCR （MSP） to detect p16INK4A methylation. The expression of p16INK4A mRNA was detected by reverse transcription-polymerase chain reaction （RT-PCR）. In addition, the proliferation of methylated cell lines before and after treatment of demethylating agent 5-Aza-2＇-deoxycytidine （5-ADC） was examined with 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay in vivo. Results： Compared with the control, the expression of p16INK4A mRNA decreased significantly or absolutely defaulted in 10 of 18 （55.56%） ovarian cancer specimens and 71.4% （5/7） ovarian cancer cell lines （P〈0.05）, and the expression of p16INK4A protein also decreased （P〈0.05）. The decrease of p16INK4A was due, in part, to p16INK4A methylation, which was found in the first exon of three cell lines and six ovarian cancer specimens and the rate was 42.86% and 33.33% in ovarian cancer cell lines and specimens respectively. All the methylated cells and tissues showed expression defect of p16INK4A, but the treatment of 5-ADC reactivated the expression of p16INK4A in methylated cells and decreased the proliferation of tumor cells in vitro and in vivo. Conclusion： The expression defect of p16INK4A gene possibly has an important role in the development of ovarian cancer, and this alteration is due, in part, to the methylation of the first exon in p16INK4A.

CLINICOPATHOLOGICAL SIGNIFICANCE OF PTEN AND CASPASE-3 EXPRESSIONS IN BREAST CANCER

Objective To investigate the expressions of PTEN and Caspase-3 proteins in human breast carcinoma,and to evaluate their clinicopathological implications during the tumorigenesis and progression of breast cancer.Methods The expressions of PTEN and Caspase-3 proteins in 95 cases of breast cancer and 15 cases of benign breast diseases were investigated immunohistochemically.Correlations between the expression of PTEN protein,Caspase-3 protein,and clinicopathological features of breast cancers were analyzed.Results The loss expression rate of PTEN protein in tumor tissues was significantly higher than that in benign breast diseases(33.7% vs.0,P<0.01).Analysis of the clinicopathological features showed that PTEN expression level was negatively correlated with TNM stage,histological grade,axillary lymph node status,recurrence,and metastasis(P<0.05).The positive expression level of Caspase-3 was negatively correlated with TNM stage(P<0.01),but not related with histological grade,axillary lymph node status,recurrence,or metastasis(P>0.05).In addition,the expression of PTEN protein had significantly positive correlation with the expression of Caspase-3 protein in breast cancer(P<0.01).Conclusion The combination detection of PTEN and Caspase-3 may serve as an important index to estimate the pathobiological behavior and prognosis of breast cancer.

Loss of heterozygosity on 10q23.3 and mutation of tumor suppressor gene PTEN in gastric cancer and precancerous lesions

AIM: To investigate the loss of heterozygosity (LOH) and mutation of tumor suppressor gene PTEN in gastric cancer and precancerous lesions. METHODS: Thirty cases of normal gastric mucosa, advanced and early stage gastric cancer, intestinal metaplasia, atrophic gastritis, and atypical hyperplasia were analyzed for PTEN LOH and mutations within the entire coding region of PTEN gene by PCR-SSCP denaturing PAGE gel electrophoresis, and PTEN mutation was detected by PCR-SSCP sequencing followed by silver staining. RESULTS: LOH rate found in respectively atrophic gastritis was 10% (3/30), intestinal metaplasia 10% (3/30), atypical hyperplasia 13.3% (4/30), early stage gastric cancer 20% (6/30), and advanced stage gastric cancer 33.3% (9/30), None of the precancerous lesions and early stage gastric cancer showed PTEN mutations, but 10% (3/30) of the advanced stage gastric cancers, which were all positive for LOH, showed PTEN mutation. CONCLUSION: LOH of PTEN gene appears in precancerous lesions, and PTEN mutations are restricted to advanced gastric cancer, LOH and mutation of PTEN gene are closely related to the infiltration and metastasis of gastric cancer.

Review:Mechanisms of inherited cancer susceptibility

A small proportion of many cancers are due to inherited mutations in genes,which result in a high risk to the indi-vidual of developing specific cancers. There are several classes of genes that may be involved: tumour suppressor genes,onco-genes,genes encoding proteins involved in DNA repair and cell cycle control,and genes involved in stimulating the angiogenic pathway. Alterations in susceptibility to cancer may also be due to variations in genes involved in carcinogen metabolism. This review discusses examples of some of these genes and the associated clinical conditions caused by the inheritance of mutations in such genes.

Decreased expression of Neurensin-2 correlates with poor prognosis in hepatocellular carcinoma

AIM： To investigate the expression of Neurensin-2 （NRSN2） in hepatocellular carcinoma （HCC） and its prognostic values in predicting survival. METHODS： The expression and prognostic significance of NRSN2 in HCC was examined by performing immunohistochemical analysis using a total of 110 HCC clinical tissue samples, and Western blotting analysis to further confirm the result. RESULTS： Decreased NRSN2 expression was shown in 70.9% cases. Loss of NRSN2 expression in HCC was significantly related to tumor size （P = 0.006）. Larger tumor size was related to negative expression of NRSN2. Patients showing negative NRSN2 expression had a significantly shorter overall survival than those with positive expression （P = 0.008）. Multivariate Cox regression analysis indicated that NRSN2 expression level was an independent factor of survival （P = 0.013）. Western blotting analysis further confirmed decreased expression of NRSN2 in tumor tissues compared with non-tumorous tissues. CONCLUSION： Our study indicated that NRSIV2 could be a tumor suppressor gene for HCC and a candidate biomarker for long-term survival in HCC.

Science Letters:IGFBP7 plays a potential tumor suppressor role against colorectal carcinogenesis with its expression associated with DNA hypomethylation of exon 1

Insulin-like growth factor binding-protein-7 （IGFBP7） was obtained from our previous colonic adenocarcinoma （CRC） and normal mucosa suppression subtraction hybridization （SSH） cDNA libraries. By RT-PCR and immunohistochemistry, we found that IGFBP7 was overexpressed in CRC tissue compared to normal tissue. However, our in vitro experiments performed in 10 CRC cell lines showed that IGFBP7 expressed only in SW480 and Caco2 cell lines, which implied an underlying reversible regulatory mechanism. Using methylation-specific PCR （MSP） and bisulfite sodium PCR （BSP）, we found that its expression was associated with DNA hypomethylation of exonl. This was further supported by the in vitro study which showed restored IGFBP7 expression after demethylation agent 5-aza-2＇-deoxycytidine treatment. Correlation analysis between IGFBP7 expression and prognosis indicated that overexpression of IGFBP7 in CRC tissue correlated with favourable survival. Investigation of the functional role of IGFBP7 through transfection studies showed that IGFBP7 protein could inhibit growth rate, decrease colony formation activity, and induce apoptosis in RKO and SW620 cells, suggesting it a potential tumor suppressor protein in colorectal carcinogenesis. In conclusion, our study clearly demonstrated that IGFBP7 plays a potential tumor suppressor role against colorectal carcinogenesis and its expression is associated with DNA hypomethylation of exon 1.

Is NEDD4-1 a negative regulator of phosphatase and tensin homolog in gastric carcinogenesis?

The expression of phosphatase and tensin homolog (PTEN ), a tumor suppressor gene, is frequently downregulated in gastric carcinomas due to mutation, loss of heterozygosity, and promoter hypermethylation. However, it is unknown if additional mechanisms may account for the down-regulation of PTEN expression. While neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1) is believed to be a potential dual regulator of PTEN, there are conflicting reports regarding their interaction. To gain further insight into the role of NEDD4-1 and its association with PTEN in gastric carcinoma development, we measured the protein expression of NEDD4-1 and PTEN in gastric mucosae with various pathological lesions and found that NEDD4-1 increased from normal gastric mucosa to intestinal metaplasia and decreased from dysplasia to gastric carcinoma. These changes did not correlate with PTEN expression changes during gastric carcinogenesis. Moreover, we found similar results in protein levels in the primary tumors and adjacent non-tumorous tissues. These results differ from a previous report showing that expression of NEDD4-1 is up-regulated in gastric carcinomas, and show a more complex pattern of NEDD4-1 gene expression during gastric carcinogenesis.

Tumor suppress genes screening analysis on 4q in sporadic colorectal carcinoma

AIM: To search candidate tumor suppressor genes (TSGs) on chromosome 4q through detecting high loss of heterozygosity (LOH) regions in sporadic colorectal carcinoma in Chinese patients. METHODS: Thirteen fluorescent labeled polymorphic microsatellite markers were analyzed in 83 cases of colorectal carcinoma and matched normal tissue DNA by polymerase chain reaction (PCR). PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan 3.7 and Genotype 3.7 software were used for LOH scanning and analysis. Comparison between LOH frequency and clinicopathological factors were performed by χ2 test. RESULTS: Data were collected on all informative loci. The average LOH frequency on 4q was 28.56%. The D4S2915 locus showed highest LOH frequency (36.17%). Two obvious deletion regions were detected: one between D4S3000 and D4S2915 locus (4q12-21.1), another flanked by D4S407 and D4S2939 locus (4q25-31.1). None case showed complete deletion of 4q, most cases displayed interstitial deletion pattern solely. Furthermore, compared with clinicopathological features, a significant relationship was observed between LOH frequencies on D4S3018locus. In tumors larger than 5 cm in diameter, LOH frequency was significantly higher than tumors that were less than 5 cm (56% vs 13.79%, P = 0.01). On D4S1534 locus, LOH was significantly associated with liver metastasis (80% vs 17.25%, P = 0.012). No relationship was detected on other locus compared with clinicopathologial features. CONCLUSION: By high resolution deletion mapping, two high frequency regions of LOH (4q12-21.1 and 4q25-31.1) were detected, which may contribute to locate TSGs on chromosome 4q involved in carcinogenesis and progression of sporadic colorectal carcinoma.

Effect of breast-cancer metastasis suppressor 1 (BRMS1) on growth and metastasis of human gastric cancer cells in vivo

Objective： The aim of the study was to investigate inhibitory effects of breast-cancer metastasis suppressor 1 protein （BRMS1） on primary tumor growth and metastasis of human gastric cancer （GC） cells in nude mice. Methods： We compared the expression of BRMS1 in the primary gastric tumor and metastatic gastric tumor by immunohistochemistry. Expression of BRMS1 also was detected in the GC cells by RT-PCR and Western blot. Three groups of cultured human GC cell line SGC-7901, were maintained： transfected cells with pcDNA3.1（-）B/myc-BRMS1; negative control cells with pcD- NA3.1/myc-his（-）B; and blank control ceils without any transfection. Histologically intact samples of the cells, maintained by passage in the subcutis of nude mice, were transplanted orthotopically into stomach walls of nude mice to establish a nude mouse model of human gastric carcinoma. Their primary tumor growth and metastasis were then observed. Results： The expression of BRMS1 was markedly stronger in the primary gastric tumor compared with metastatic gastric tumor. We also detected BRMS1 gene and protein in the gastric cancer cell tines. Numbers of metastasis tumors significantly differed among mice infected with transfected cells, with negative controls and with blank controls （4.38 ± 0.60, 7.75 ± 0.59, and 7.63± 0.65, respectively; P 〈 0.05）. However, there were no significant differences in the size of orthotopic tumors among mice infected with transfected, negative control and blank control cells [（12.02 ± 0.70）, （12.71 ± 0.63） and （12.89 ± 0.71） mm, respectively; P 〉 0.05]. Conclusion： BRMS1 suppresses metastasis of GC cells, but does not inhibit growth of gastric tumors.

Refined mapping of loss of heterozygosity on 1q31.1-32.1 in sporadic colorectal carcinoma

AIM: To explore precise deleted regions and screen the candidate tumor suppressor genes related to sporadic colorectal carcinoma. METHODS: Six markers on 1q31.1-32.1 were chosen. These polymorphic microsatellite markers in 83 colorectal cancer patients tumor and normal DNA were analyzed via PCR. PCR products were electrophoresed on an ABI 377 DNA sequencer. Genescan 3.1 and Genotype 2.1 software were used for Loss of heterozygosity (LOH) scanning and analysis. Comparison between LOH frequency and clinicopathological factors was performed by χ2 test. RESULTS: 1q31.1-32.1 exhibited higher LOH frequency in colorectal carcinoma. The average LOH frequency of 1q31.1-32.1 was 23.0%, with the highest frequency of 36.7% (18/49) at D1S2622, and the lowest of 16.4% (11/67) at D1S412, respectively. A minimal region of frequent deletion was located within a 2 cM genomic segment at D1S413-D1S2622 (1q31.3-32.1). There was no significant association between LOH of each marker on 1q31.1-32.1 and the clinicopathological data (patient sex, age, tumor size, growth pattern or Dukes stage), which indicated that on 1q31.1-32.1, LOH was a common phenomenon in all kinds of sporadic colorectal carcinoma. CONCLUSION: Through our refined deletion mapping,the critical and precise deleted region was located within 2 cM chromosomal segment encompassing 2 loci (D1S413, D1S2622). No significant association was found between LOH and clinicopathologic features in 1q31.1-32.1.

Are there tumor suppressor genes on chromosome 4p in sporadic colorectal carcinoma?

AIM: To study the candidate tumor suppressor genes (TSG) on chromosome 4p by detecting the high frequency of loss of heterozygosity (LOH) in sporadic colorectal carcinoma in Chinese patients.METHODS: Seven fluorescent labeled polymorphic microsatellite markers were analyzed in 83 cases of colorectal carcinoma and matched normal tissue DNA by PCR. PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan 3.7 and Genotype 3.7 software were used for LOH scanning and analysis. The same procedure was performed by the other six microsatellite markers spanning D4S3013 locus to make further detailed deletion mapping. Comparison between LOH frequency and clinicopathological factors was performed by χ2 test.RESULTS: Data were collected from all informative loci. The average LOH frequency on 4p was 24.25%, and 42.3% and 35.62% on D4S405 and D4S3013 locus, respectively. Adjacent markers of D4S3013 displayed a low LOH frequency (< 30%) by detailed deletion mapping. Significant opposite difference was observed between LOH frequency and tumor diameter on D4S412 and D4S1546 locus (0% vs 16.67%, P = 0.041; 54.55% vs 11.11%, P = 0.034, respectively). On D4S403 locus, LOH was significantly associated with tumor gross pattern (11.11%, 0, 33.33%, P = 0.030). No relationship was detected on other loci compared with clinicopathologial features.CONCLUSION: By deletion mapping, two obvious high frequency LOH regions spanning D4S3013 (4p15.2) and D4S405 (4p14) locus are detected. Candidate TSG, which is involved in carcinogenesis and progression of sporadic colorectal carcinoma on chromosome 4p, may be located between D4S3017 and D4S2933 (about 1.7 cm).

Human cancer genetics

The short report will be focused on the genetic basis and possible mechanisms of tumorigenesis, common types of cancer, the importance of genetic diagnosis of cancer, and the methodology of cancer genetic diagnosis. They will also review presymptomatic testing of hereditary cancers, and the application of expression profiling to identify patients likely to benefit from particular therapeutic approaches.

Effect of tumor suppressor in lung cancer-1 on growth inhibition of MG63 cell line

Objective： The aim of this study was to establish the osteosarcoma cell sublines which stably expressing tumor suppressor in lung cancer-1 （TSLC1） gene and evaluate its effect on growth inhibition of human osteosarcoma cell line MG63. Methods： The recombinant plasmid pCI-TSLC1 was stably transfected into MG63 cells with Lipofectamine 2000. The posi- tive clones were developed by selection by G418. Biological characteristics of one of the 6 cell lines which highly expressing TSLC1, namely, the M8T were studied. Cell growth was analyzed with MTT assay. 2 x 10^7cells suspended in 0.2 mL phosphate buffered saline （PBS） were injected into the two flanks of 5-6-week-old female BALB/C nu/nu athymic nude mice. The volumes of subcutaneous of tumor growth were evaluated and calculated by the formula V= Length x Width x Height x 0.5 once a week. Results： The MST cell subline which stably expressing TSLC1 was characterized by Western blot. The genetic stability and purity of M8T cells were stable. TSLC1 significantly suppressed the growth of M8T cells in vitro. Moreover, the tumorigenicity of MST cells was suppressed in vivo. Conclusion： The osteosarcoma cell sublines MST which stably expressing TSLC1 had been successfully established. The ability of growth and metastasis of MST was significantly suppressed both in vitro and in vivo.

Suppression subtractive hybridization for identifying differentially expressed genes in renal cell carcinoma

Objective To construct a renal cell carcinoma (RCC) cDNA subtractive library using suppression subtractive hybridization.Methods Polyadenylated RNA ［Poly (A)+ RNA］ was isolated from tissues of RCC and normal kidney, and single-strand cDNAs and double-strand cDNAs were synthesized in turn. RCC cDNAs were divided into two groups and ligated to the specific adaptors l and 2, and then hybridized with normal kidney cDNA twice with two rounds of suppression PCR. Second round PCR products were cloned to T/A plasmid vectors to set up the subtractive library. One hundred clones were randomly picked to perform enzyme digest analysis, and some underwent sequence analysis and Northern blot to identify RCC specifically expressed genes. SMART RACE procedure was operated to clone full length novel RCC specifically expressed genes.Results A human RCC subtractive library with high subtractive efficiency was successfully set up. The amplified library contains 350 positive clones. Random analysis of 100 clones with enzyme restriction showed that 85 plasmids in the clones contained 50-400?bp inserts. Sequence analysis was performed for 10 clones. All the 10 sequences were unknown before and derived from 6 unique, novel genes among which the cDNA insert RCC18 had five copies. Northern blot analysis showed that RCC18 cDNA was highly expressed in RCC, but no signal could be detected in normal kidney. Using SMART RACE technique, we obtained the full length of the novel gene RCC18.Conclusions The constructed cDNA subtractive library of human RCC is a highly efficient one and lays a solid foundation for large scale screening and cloning new and specific oncogenes or tumor suppressor genes of RCC. The novel specifically expressed genes provided an important clue for studying the mechanisms of occurrence and development of RCC.

The p53/miR-34 axis in development and disease

The tumor suppressor p53 is one of the most frequently mutated genes in human cancers. MicroRNAs （miRNAs） are small non-protein coding RNAs that regulate gene expression on the post-transcriptional level. Recently, it was shown that p53 regulates the expression of several miRNAs, thereby representing an important mechanism of p53 signaling. Several independent studies identified the members of the miR-34 family as the most prevalent p53-induced miRNAs, miR-34s are frequently silenced in variety of tumor entities, suggesting that they are important tumor suppressors. Indeed, ectopic expression of miR-34s inhibits proliferation, epithelial to mes- enchymat transition, migration, invasion, and metastasis of various cancer celt entities. Moreover, delivery or re-expression of miR-34 leads to notable repression of tumor growth and metastasis in cancer mouse models, and may therefore represent an efficient strategy for future cancer therapeutics. Besides their crucial functions in cancer, members of the miR-34 family also play important roles in spermatogenesis, stem cell differentiation, neuronal development, aging, and cardiovascular functions. Consequently, miR-34 has also been implicated in various non-cancerous diseases, such as brain disorders, osteoporosis, and cardiovascular complications.

miR-29b upregulates miR-195 by targeting DNMT3B in tongue squamous cell carcinoma

MicroRNAs play important roles in the devel- opment and progression of various cancers, including tongue squamous cell carcinoma （TSCC）. miR-29b and miR-195 have been reported to be tumor suppressors in TSCC. Here, we investigated the expression of miR-29b and miR- 195 and their relationship in TSCC. Our data showed that miR-29b and miR-195 were significantly downregulated in TSCC com- pared with their matched nonmalignant tissues in 60 paired samples. The level of miR-29b was positively correlated with that of miR-195 in TSCC and the matched nonmalignant tissues. Moreover, miR-29b overexpression induced the demethylation of CpG islands upstream of miR-195 via targeting DNMT3B, leading to the upregulation of miR-195 in TSCC cell lines. Following DNMT3B silencing, the expression of miR-195 was increased and the methylation of CpG islands upstream of miR-195 was reduced. Although overexpression of miR-29b alone significantly increased miR- 195 expression, co-transfection of miR-29b with DNMT3B resulted in no change in miR-195 expression. Taken together, our results demonstrated that miR-29b could upregulate miR- 195 by directly targeting DNMT3B in TSCC. The interaction between miR-29b and miR-195 might provide new insights in developing novel therapeutic approaches of TSCC.