DELETIONS AND POINT MUTATIONS OF p16,p15 GENE IN PRIMARY TUMORS AND TUMOR CELL LINES

Aberrations of chromosome 9 p21 22 are involved in the genesis of many forms of cancer.The gene p16 and p15 have been assigned to this region.Both p16 and p15 are an inhibitor of cyclin D cdk4,cyclin D cdk6 complex and have been implicated in a wide variety of cancer types,including the germline of patients with familial melanoma.In order to investigate and compare the status of p16,p15 gene in primary tumors and cell lines,we examined 357 primary tumors and 29 cell lines derived from diverse tumor types.In addition to analysis of these primary tumors and cell lines,blood specimens from 91 patients either with sporadic multiple cancers or from cancer prone families were also analyzed.The data showed the following:1)Homozygous deletions of p16,p15 were comparatively rare and far less common than previously reported,although hemizygous deletions were observed in a significant fraction of many tumor types;2)the incidence of p16,p15 deletions(either homozygous deletions or heterozygous deletions)varied significantly among different tumor types;3)most deletions involved in both p16 and p15 genes;4)sequence variations in the coding sequence of p16,p15 were comparatively rare among these tumor types,though mutations and polymorphisms were identified;5)some tumors which showed LOH at 9p,containing p16 and p15 gene,did not show deletions or point mutations in the p16,p15 gene.6)In a subset of retinoblastoma and osteosarcoma where no Rb gene mutations were present a significant fraction was found to contain p16,p15 gene deletions.

Effect of 5-Aza-2'-deoxycytidine on the P16 tumor suppressor gene in hepatocellular carcinoma cell line HepG2

INTRODUCTIONHepatocellular carcinoma (HCC) is one of the mostcommon human malignancies worldwide[1,2], and isclosely associated with infection of HBV and HCVand contamination of aflatoxin B1[3-6]. Althoughthe molecular mechanisms of hepatocarcinogenesisremain poorly understood, an increasing number ofgenetic abnormalities have been recognized[7-10],for example, the p16 gene[11,12] the p53gene[13-18], the E-cadherin gene[19], and the c-mycgene[20].

FREQUENT DELETION OF MTS1/p16 GENE AND CORRELATION WITH CLINICOPATHOLOGICAL PARAMETERS IN ENDOMETRIAL CARCINOMA

Objective: To investigate the possible relationship between deletion of MTS/p16 gene and progression of endometrial carcinoma Methods: Forty six primary endometrial carcinoma, 7 tumor adjacent endometrial tissue, 10 normal endometrial tissue specimen and 5 xenografts from patients with endometrial carcinoma were examined for homozygous deletion of MTS/p16 gene by polymerase chain reaction based analysis Results: Of 46 endometrial cancer specimens, 9 showed homozygous deletion, no deletion was detected in the tumor adjacent and normal endometial tissues Nor was it detected in well differentiated endometrial carcinoma and all xenografts Conclusions: Deletion of MTS1/p16 gene might contribute to the progression of endometrial carcinoma and could be served as indicator for predicting prognosis

Expression and significance of tumor suppressor gene p16 in human ovarian neoplasm

To observe the relationship between tumor suppressor gene p16 expression and ovarian cancer occurrence and development. Metbods: Using ABC immunohistochemistry method, we investigated the expression of p16 in 72 cases of ovarian neoplasm. Results: The positive rates of p16 in malignant, benign, borderline tumors and normal ovarian tissue were 7. 89%, 60.00%, 66. 67% and 83. 33%, respectively (P<0.01). In the cases whose tumors were more malignant and poorly differentiated, and who relapsed and died, the positive stainings were not discovered. Conclusiou: p16 is well related with the occurrence and development of malignant ovarian tumor.

Alterations of FHIT Gene and P16 Gene in Nickel Transformed Human Bronchial Epithelial Cells

Objective To study the alterations of FHIT gene and P16 gene in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide using an immortal human bronchial epithelial cell line, and to explore the molecular mechanism of nickel carcinogenesis. Methods 16HBE cells were treated 6 times with different concentrations of NiS in vitro, and the degree of malignant transformation was determined by assaying the anchorage-independent growth and tumorigenicity. Malignant transformed cells and tumorigenic cells were examined for alterations of FHIT gene and P16 gene using RT-PCR, DNA sequencing, silver staining PCR-SSCP and Western blotting. Results NiS-treated cells exhibited overlapping growth. Compared wkh that of negative control cells, soft agar colony formation efficiency of NiS-treated cells showed significant increases （P〈0.01） and dose-dependent effects. NiS-treated cells could form tumors in nude mice, and a squamous cell carcinoma was confirmed by histopathological examination. No mutation of exon 2 and exons 2-3, no abnormal expression in pl6 gene and mutation of FHIT exons 5-8 and exons 1-4 or exons 5-9 were observed in transformed cells and tumorigenic cells. However, aberrant transcripts or loss of expression of the FHIT gene and Fhit protein was observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in the FHIT gene was confirmed to have a deletion of exon 6, exon 7, exon 8, and an insertion of a 36 bp sequence replacing exon 6-8. Conclusions The FHIT gene rather than the P16 gene, plays a definite role in nickel carcinogenesis. Alterations of the FHIT gene induced by crystalline NiS may be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation. FHIT may be an important target gene activated by nickel and other exotic carcinogens.

Loss of chromosome 9p21 and decreased p16 expression correlate with malignant gastrointestinal stromal tumor

AIM: To investigate loss of heterozygosity (LOH) of chromosome 9p21 and the prognostic relevance of p16 expression in gastrointestinal stromal tumor (GIST). METHODS: Fifty-one GIST patients (30 men and 21 women; median age 59 years; range 29-80 years) treated surgically within a 10-year period were grouped by aggressive behavior risk (17 with very low and low, 14 intermediate, and 20 high risk). GISTs were characterized immunohistochemically and evaluated for LOH of 9p21 by microsatellite analysis at D9S1751, D9S1846, D9S942, and D9S1748. LOH of 9p21 and immunohistochemicalexpression of p16 protein encoded at 9p21 were correlated with clinicopathological parameters, and the prognostic significance of p16 alterations was evaluated. RESULTS: Thirty-one (63.3%) cases showed LOH with at least one microsatellite marker. LOH frequency was 37.0% at D9S1751, 37.5% at D9S1846, 42.1% at D9S942, and 24.2% at D9S1748. There was a higher LOH frequency of D9S942 in high-risk than in non-highrisk tumors (P < 0.05, χ 2 = 4.47). Gender, age, tumor size and site were not correlated with allelic loss. Ninety percent (18/20) of the GIST patients in the high risk group showed LOH with at least one of the 9p21 markers, while 57.1% (8/14) in the intermediate risk group and 33.3% (5/15) in the very low and low risk groups, respectively (P < 0.05, χ 2 = 12.16). Eight (28.5%) of 31 patients with LOH and 1 (5.6%) of 18 patients without LOH died of the disease during the follow-up period. Loss of p16 protein expression occurred in 41.2%, but in 60% of the high risk group and 23.5% of the very low and low risk groups (P < 0.05, χ 2 = 4.98). p16 loss was associated with poor prognosis (P < 0.05, χ 2 = 4.18): the 3and 5-year overall survival rates were 84.8% and 70.8% for p16-negative and 100% and 92.0% for p16-positive patients, respectively. CONCLUSION: LOH at 9p21 appears to play an important role in GIST progression; decreased p16 expression in GIST is highly predictive of poor outcome.

Exogenous p16 gene therapy combined with X-ray irradiation suppresses the growth of human glioma cells

In this study, we infected human glioma U251 cells with a replication-defective recombinant adenovirus carrying the p16 gene. This adenovirus constructed was able to transfect exogenous p16 into the human glioma cells efficiently, and direct a high level of p16 protein expression. Tumor-inhibition experiments demonstrated that treatment with the adenovirus-p16 significantly inhibited the growth of glioma cells in vitro as well as the in vivo development of tumors in nude mice bearing a brain glioma. The combination of adenovirus-p16 gene treatment and X-ray irradiation resulted in a greater inhibition of tumor growth. Adenovirus-mediated p16 gene therapy conferred a significant antitumor effect against human glioma cells both in vitro and in vivo, and that there was a synergistic effect when X-ray irradiation was also used.

INACTIVATION OF P16 GENE IN LEUKEMIA

To determine the frequency of p16 gene inactivation in leukemia cells, and to evaluate their value in the prediction of their clinical outcome. Bone marrow or peripheral blood samples from 48 patients with leukemia were examined by multiplex polymerase chain reaction(MPCR) to detect p16 gene homozygous deletion, and restriction enzyme PCR to detect p16 gene methylation. p16 gene inactivation were detected in 10 of the 48 patients(20.4%). They were five patients with p16 homozygous deletion, and five patients with p16 methylation, respectively. p16 gene inactivation correlates with adverse prognosis features. The patients with p16 inactivation had poor response to chemotherapy, and had significantly shorter survival times than the patients in whom p16 gene was preserved(P<0.001). The inactivation of p16 gene play a key role in the pathogenesis and the progression of some leukemia. The detection of p16 gene is reliable prognostic factor that predict shortened survival times.

Expression,deleton and mnutation of ρ16 gene in human gastric cancer

AIM To investigate the relationship between the expression of p16 gene and the gastric carcinogenesis,depth of invasion and lymph node metastases, and to evaluate the deletion and mutation of exon 2 in p16 gene in gastric carcinoma.METHODS The expression of P16 protein was examined by streptavidin-peroxidase conjugated method (S-P); the deletion and mutation of p16 gene were respectively examined by polymerase chain reaction (PCR) and polymerase chain reaction single-strand conformation polymorphism analysis (PCR-SSCP) in gastric carcinoma.RESULTS Expression of P16 protein was detected in 96.25% (77/80) of the normal gastric mucosa, in 92.00% (45/50) of the dysplastic gastric mucosa and in 47.54% (58/122) of the gastric carcinoma. The positive rate of P16 protein expression in gastric carcinoma was significantly lower than that in normal gastric mucosa and dysplastic gastric mucosa (P＜0.05). The positive rate of P16 protein expression in mucoid carcinoma 10.00% (1/ 10) was significantly lower than that in poorly differentiated carcinoma 51.22% ( 21/ 41 ),undifferentiated carcinoma 57.69% (15/26) and signet ring cell carcinoma 62.50% (10/ 16) (P＜0.05). The positive rate of p16 protein in 30 cases paired primary and lymph node metastatic gastric carcinoma: There was 46.67% (14/30) in primary gastric carcinoma, 16.67% (5/30) in lymph node metastatic gastric carcinoma. The positive rate of lymph node metastatic carcinoma was significantly lower than that of primary carcinoma (P＜0.05). There was of p16 gene mutation in exon 2, but 5 cases displayed deletion of p16 gene in exon 2 in the 25 primary gastric carcinomas.CONCLUSIONS The expression loss of P16 protein related to the gastric carcinogenesis, gastric carcinoma histopathological subtypes and lymph metastasis. The mutation of p16 gene in exon 2 may not be involved in gastric carcinogenesis. But the deletion of p16 gene in exon 2 may be involved in gastric carcinogenesis.

Detection of Serum Aberrant CDKN2/P16 DNA in Colorectal Cancer

Objective： To search for a biomarker for colorectal cancer. Methods： The MSP, SSCP and deletion tests with serum have been taken simultaneously in 100 cases of colorectal cancer and 2 groups of controls, as well as the specimens of 26 cancer tissues and 22 paracancerous tissues and 29 cases of benign disease tissues for a contrast. Results： The aberrant methylation rate of P16 in the serum was 69.00%, deletion rate 4.00% and suspicious point mutation rate 15.00% in colorectal cancer patients. The data of cancer tissues were the same as those of the serum, but in paracancerous tissue those were significantly lower. In 10 cases, sequencing analysis revealed that there were 3 cases of missense, one case of frameshift and one case of nonsense. Among them, four cases had P16 protein deletion. As a tumor marker, the sensitivity of combined use of three methods was 88.00%, specificity 96.87% and accuracy 90.15%. The combined use of MSP and SSCP could obtain the same results. Conclusion： The content of DNA in serum is minimal, but it reflects the tumor burden of patients. The 10^-3 fragments of DNA could be detected in the serum by MSP. It can be used in the clinical diagnosis or popular investigation, and long-term postoperative follow-up.

原发恶性肿瘤MTS1/p16基因缺失的研究

目的:研究MTS1/p16基因在人类恶性肿瘤组织中的变化.方法:采用Southern杂交和多重PCR技术,分析了68例原发肿瘤组织标本,包括非小细胞肺癌31例、食管癌15例、胃癌13例、乳腺癌9例.结果:PCR检测癌旁组织未见p16基因缺失,而不同肿瘤组织标本的p16基因缺失差别很大,总缺失率为13.2%(9/68).Southern杂交检测p16基因的缺失率为17.7%(12/68).结论:MTS1/P16基因缺失在人类恶性肿瘤发生发展中起重要作用,是重要的多肿瘤抑制基因.

甲状腺癌组织端粒酶激活与p16基因失活的关系

目的探讨甲状腺癌细胞增殖、分化与端粒酶激活及抑癌基因p16失活(缺失突变)之间可能存在的关系。方法应用TRAP、多重PCR、免疫组化法检测42例甲状腺癌与16例癌旁组织端粒酶活性、P16基因外显子2缺失、P16蛋白表达。结果甲状腺癌组端粒酶活性90.48%,高于癌旁组织(P<0.01);甲状腺癌p16基因外显子2纯合缺失率28.57%,相应癌旁组未检出(P<0.01);甲状腺癌P16蛋白表达缺失率40.48%,高于癌旁组(P<0.05);甲状腺癌P16蛋白表达缺失率高于p16基因外显子2缺失率。结论端粒酶激活与p16基因失活以及P16蛋白表达下调可能是甲状腺癌变过程中的重要分子事件,甲状腺癌中p16基因失活可能是端粒酶激活的一种途径。

MUTATION AND ABNORMAL EXPRESSION OF P16^(INK4a) INHEPATOCELLULAR CARCINOMA

Objective:To investigate the relationship betweenp16INK4a and primary hepatocellular carcinoma (HCC),especially hepatitis B-related HCC. Methods: p16INK4a andits protein in HCC were analyzed with PCR-SSCP and theimmunohistochemistry methods respectively. Results: Thepositive incidence of p16INK4 protein expressing in HCC waslower than that of normal liver tissue (P<0.05), and theabsence of p16INK4 protein was associated with HCCmetastasis (P<0.05). The low frequency of mutation ofp16INK4 exonl and exon2 upstream fragment was found inHCC. Conclusion: Absence of p16INK4 protein in HCC wasnot associated with HBV-infection.

Replacement of the p16 gene in human ovarian cancer cells

Objective To study the inhibitory effects of retrovirus-mediated p16 gene on the human ovarian cancer cell line CAOV3.Methods The recombinant eukaryotic expression vector pDOR-p16 containing exogenous human wt-p16 cDNA and vector with neomycin resistance gene only were introduced into a CAOV3 cell line which does not express p16 endogenously by lipofectamine-mediated gene transfection. By using polymerase chain reaction amplification, mRNA in situ hybridization and immunocytochemistry, the clones obtained were tested for their efficiency of transfection and effects of vector expression. Their biologic behavior was observed further.Results Exogenous wt-p16 was transferred into CAOV3 cells successfully and permanent expression was obtained. The growth rate of the transfected CAOV3 cells in regular medium and soft agar was inhibited, and the tumorigenicity in nude mice showed that two of four mice failed to form tumors, and the others developed tumors 7 to 14 days later than mice of the contrast group. The percentage of phase G1 cells increased and that of phase S cells decreased. Under electron microscope, the ultrastructural changes of the cells revealed necrosis and growth retardation.Conclusions The p16 gene plays an important role in the generation and development of ovarian carcinoma. This study might provide experimental evidence for gene therapy in human ovarian cancer.

Molecular and immunohistochemical study of the inactivation of the p16 gene in primary hepatocellular carcinoma

Obejctive To determine whether p16 gene is involved in the genesis of primary hepatocellular carcinoma (HCC) Methods Twenty five HCC tumor samples with corresponding non tumor liver tissue specimens were examined for p16 gene alterations The identification of deletion of exon 1 and exon 2 in p16 gene was performed using comparative multiplex polymerase chain reaction (PCR) analysis The point mutation of exon 2 in p16 gene was investigated by single strand conformational polymorphism (SSCP) analysis, and the status of p16 gene methylation was screened using a PCR based methylation analysis 35 parafin embedded specimens of HCC with corresponding non tumor liver tissues, including the 25 cases described above for screening p16 gene alterations, were investigated for p16 protein expression using immunohistochemical analysis Results Among 25 cases, 2 homozygous deletions and 1 hemizygous deletion were found in HCC samples No point mutation was identified in the remaining 22 tumor samples without p16 gene deletions Hypermethylation was detected in 24% (6/25) of tumor samples However, the corresponding non tumor liver tissue specimens were always unmethylated at the p16 locus Loss of p16 protein expression occurred in 16 of 35 (45 7%) tumor samples, and all the non tumor liver tissue specimens showed positive p16 staining For the 25 cases examined for p16 gene alterations, the loss of p16 protein expression was observed in all tumors with p16 gene alterations and also in 3 tumors without p16 gene alterations Conclusion Inactivation of the p16 gene may play an important role in the genesis of primary hepatocellular carcinoma

长链非编码RNA SNHG16与消化系统恶性肿瘤关系的研究进展

消化系统恶性肿瘤的发病率和病死率在我国恶性肿瘤中位居前列,造成了巨大医疗及社会负担。长链非编码RNA(lncRNA)可通过转录干扰、转录后调控和表观遗传修饰等调控基因表达,有作为肿瘤标志物的巨大潜力。lncRNA小核仁RNA宿主基因16(SNHG16)在胃癌、结直肠癌、肝癌、胰腺癌、食管癌等常见消化系统恶性肿瘤中高表达,且具有致癌特性,可影响肿瘤细胞的增殖、凋亡、迁移和侵袭等生物学过程,在肿瘤发生发展中发挥重要作用。lncRNA SNHG16可能在消化系统恶性肿瘤临床诊断和预后预测方面具有重要价值。

多药耐药基因在睾丸恶性肿瘤血睾屏障中的作用研究

目的 探讨睾丸恶性肿瘤血睾屏障产生机制 ,为采用分子生物学方法克服血睾屏障从而提高化疗疗效提供依据。方法 采用P 糖蛋白 (P gp)单克隆抗体、免疫组化法检测睾丸恶性肿瘤组织P gp表达部位及强度。进行统计分析 ,对比睾丸组织各类细胞间的P gp表达情况。结果 睾丸恶性肿瘤细胞P gp表达率及程度远低于睾丸肿瘤毛细血管内皮细胞P gp表达 (P<0 .0 1 )。结论 睾丸恶性肿瘤病人存在抗癌药物血睾屏障 ,其机制为肿瘤间质毛细血管内皮细胞过量表达P gp所引起 ,而非肿瘤细胞自身表达P

鼻咽癌组织中脑恶性肿瘤缺失基因1的表达及意义

目的:探讨鼻咽癌组织中脑恶性肿瘤缺失基因1(DMBT1)的表达与临床病理的关系。方法:选取48例鼻咽癌患者的鼻咽癌组织、48例正常鼻咽部组织,采用免疫组织化学方法及实时定量PCR方法检测组织中DMBT1蛋白及mRNA的表达水平,分析DMBT1蛋白表达与鼻咽癌患者性别、年龄,鼻咽癌的临床分期、分化程度及颈部淋巴转移的关系。结果:DMBT1蛋白在鼻咽癌组织中的表达阳性率(31.25%)低于正常鼻咽部组织(79.17%),差异有统计学意义(P<0.01);鼻咽癌组织中DMBT1 mRNA表达明显低于正常鼻咽部组织,差异有统计学意义(P<0.05);DMBT1蛋白表达与鼻咽癌的临床分期、细胞分化程度及有无颈淋巴转移有关(P<0.05),与患者的年龄和性别无关(P>0.05)。结论:DMBT1在鼻咽癌组织中表达水平降低,与鼻咽癌的浸润转移及发生发展有关。

宫颈癌组织中TESTIN和DMBT1蛋白表达水平与其病理特征的关系

目的:探讨宫颈癌组织中TESTIN和脑恶性肿瘤缺失基因1(DMBT1)蛋白表达水平与其病理特征的关系。方法:采用免疫组化SP法检测宫颈癌(宫颈癌组,47例)、同期的宫颈内瘤样病变(CIN组,40例)及正常宫颈(对照组,40例)组织中TESTIN及DMBT1蛋白表达阳性率,比较不同病理特征的宫颈癌组织中TESTIN及DMBT1表达水平;采用Spearman方法分析宫颈癌组织中TESTIN及DMBT1表达与其临床病理特征及预后的相关性。结果:宫颈癌组TESTIN和DMBT1蛋白阳性表达率显著低于CIN组和对照组(P<0.05);低分化宫颈癌组织中TESTIN级DMBT1表达水平显著低于中、高分化宫颈癌组织(P<0.05),有淋巴转移宫颈癌组织的TESTIN和DMBT1表达显著低于无转移宫颈癌组织(P<0.05);Spearman分析显示,宫颈癌组织TESTIN及DMBT1阴性表达淋巴结转移和低分化显著相关。结论:宫颈癌组织中TESTIN和DMBT1蛋白明显低表达,且与宫颈癌分期、转移及侵袭密切有关。