Tumor suppressor gene p16 and Rb expression in gastric cardia precancerouslesions from subjects at a high incidence area in northern China

AIM:To further understand the molecular basis for gastric cardia carcinogenesis and to provide etiological clues. METHODS: Endoscopic mucosa biopsy and histopathological examinations were made on 37 subjects from a high incidence area for both esophageal and gastric cardia carcinomas in northern China. All the biopsy samples were fixed in 850 ml. (-1)L alcohol and embedded in paraffin. Each block contained one piece of tissue and was serially section at 5 microm. Immunohistochemistry (ABC) was carried out on these gastric cardia samples to determine the alterations of p16 and Rb. RESULTS: Based on the histopathlogical examination there were 11 cases of chronic superficial gastritis, 12 cases of chronic atrophic gastritis and 14 cases of dysplasia. The immunostaining demonstrated different levels of unclear immunostaining of p16 and Rb in normal gastric cardia tissue and the tissues with different severity of lesions. With the lesions progressing, the positive immunostaining rates for p16 protein had a decreasing tendency. In contrast, the positive immunostaining rate for Rb protein had an increasing tendency. There was a significant negative relationship between the two parameters. Changes of p16 was CSG 11(100%), CAG 7(58%), DYS 4(29%) and changes of Rb was CSG 2(18%), CAG 8(67%) and DYS 12(86%), (P【0.05). CONCLUSION: The alterations of p16 and Rb protein may play a role in the early stages of gastric cardia carcinogenesis.

Quantitative Study on Expression of P16 Multiple Tumor Suppressor Gene in Salivary Gland Neoplasm

The expression of P16 gene were found in all 3 groups. The positive unit (PU) was higher in tumor group and cancer group than that in normal group ( P <0.01). Furthermore, the PU of P16 was stronger in cytoplasm than in nucleus. Malignant tumors and acini surrounding the tumor revealed strong positives and week positives respectively. The PU of P16 gene was higher in deep lobe of recurrent parotid neoplasm with incomplete capsule than that in shallow lobe of primary parotid neoplasm with complete capsule. Our findings suggests that P16 gene plays equally important role in the salivary gland tumors and tumors in other part of the body.

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.

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].

Alteration of tumor suppressor gene p16 and Rb in gastric cancinogesis

IM To study the alterations of tumor suppressor gene p16 and Rb in the carcinogenesis of the stomach. METHODS Different mucosal biopsies were endoscopically obtained, all samples were immediately fixed with 10% buffered formalin, embedded with paraffin and sectioned serielly. Alterations of p16 and Rb protein in 12 cases of superficial gastritis, 15 atrophic gastritis, 20 atypical hyperplasia and 40 cancerous tissues were detected by the immunohistochemical method (ABC). RESULTS Different degrees of nuclear immunostaining of p16 and Rb occurred on gastric epithelium in different stages of lesions. With the lesions progressing, the positive immunostaining rate of p16 protein had a decreasing tendency (833%→733%→300%→275%), and on the other hand, that of Rb protein had an increasing tendency (250%→467%→600%→675%). A negative correlationship was found between these two parameters in the gastric cancer. Of 40 cases of gastric cancer, a negative relationship was observed in 20 cases. In comparison with both positive (9 cases) and both negative tissues (11 cases), there was a significant difference (500%,225%,275%) (P<005).CONCLUSION Abnormal expression of p16 and Rb plays an important role in gastric carcinogenesis.

The effects of CYP1A1 gene polymorphism and p16 gene methylation on the risk of lung cancer in a Chinese population

Objective： To investigate the relationship between the genetic polymorphism of CYP1A1 and the genetic susceptibility to lung cancer as well as to study the effects of the methyiation in p16 gene on the risk of lung cancer in a Chinese population. Methods： A case control study was conducted among 47 cases of lung cancer and 94 controls. The genetic polymorphism of CYP1A1 was tested with method of PCR-RFLP, and a methylation-specific PCR （MSP） was performed to detect p16 methylation. Results： It showed that there was no significant difference in frequencies of the genotypes of CYP1A1 between the two groups （P 〉 0.05）. Synergistic effects were not found between smoking and CYP1AI. Methylated p16 gene was found in 44.7% （21/47） of lung cancer tissues and in 17.0% （8/47） of normal lung tissues with significant difference （P 〈 0.05）. Conclusion： The genetic polymorphism of CYP1A1 does not increase the risk of lung cancer in a Chinese population. The methylation in p16 gene may be the most common mechanism to inactivate p16 gene in lung cancer, and is not significantly associated with genotype of CYP1A1,

PROMOTER HYPERMETHYLATION OF p16 GENE IN PRE- AND POST-OPERATIVE PLASMA OF PATIENTS WITH GASTRIC ADENOCARCINOMA

Objective: To detect promoter hypermethylation of p16 gene in matched pre- and post-operative plasma of patients with gastric adenocarcinoma for evaluating the effectiveness of therapeutic intervention. Methods: Tissue samples, pre- and post-operative plasma of 84 patients were collected. Plasma of 15 healthy people was collected as control. After sodium-bisulfite treatment, extracted DNA was amplified for p16 promoter by methylation-specific polymerase chain reaction (MSP). The PCR products were detected by both gel-ethidium bromide electrophoresis and high performance liquid chromatogram (HPLC). Results: Among 84 patients, p16 hypermethylation was detected in 26 (31.0%) cancer tissues and 2 (0.02%) tumor-adjacent tissues and 12 (14.3%) pre-operative plasma, while negative in plasma of healthy people. For positive plasma cases, the paired tumor tissues were confirmed to be methylated. Within available 30 pairs of matched pre- and post-operative plasma, 6 pre-operative plasma was positive, and only 1 of 6 plasma remained hypermethylated after surgery. The results detected by HPLC exactly matched those by gel-electrophoresis. Conclusion: The alteration of status of p16 hypermethylation in post-operative plasma is considered the consequences of surgical intervention. Although p16 hypermethylation has no role in pre-operative staging of gastric cancer, detecting hypermethylated p16 in plasma could be utilized in monitoring patients after surgery.

Effects of endotoxin on expression of ras, p53 and bcl-2 oncoprotein in hepatocarcinogenesis induced by thioacetamide in rats

AIM To evaluate the relationship between expression of ras, p53, bcl 2 gene products, and hepatocarcinogenesis since endotoxemia produced from lipopolysaccharide admi nistration and/or the hypophagocytic state of splenectomy significantly accelerated hepatocarcinogenesis induced by thioacetamide. 〖WTH4〗METHODS〓〖WTXFZ〗The hepatocarcinoma model was induced by oral intake of 0 03% thioacetamide for six months. During the induction of hepatocarcinoma model, rats were additionally treated with splenectomy and/or lipopolysaccharide administration. The techniques of flow cytometry, immunohistochemistry and immunoelectronmicroscopy were applied to quantitative analysis of the expression of oncogene proteins. RESULTS In this model system, overexpression of ras p21 protein mainly occurred on precancerous cell population or in early stage of hepatocyte transformation. And the levels of ras p21 declined when nuclear DNA aneuploid increased. Expression of bcl 2 protein slowly and steadily rose with more hepatocytes staying in S+G2M phases as the hepatocarcinoma became more malignant. P53 was moderately expressed during the hepatocarcinogenesis. There was no statistical correlation between endotoxemia levels and the changes of ras, p53 and bcl 2 gene products. CONCLUSION Over expression of oncogene ras p21 was likely to be a precursor of the premalignant hepatocytes and it might be responsible for the initiation of hepatocarcinogenesis. Bcl 2 protein expression is proportional to the severity of the malignancies. P53 may be a key pathway on the transformation and development of hepatocarcinoma. This study confirmed the hypothesis that there are multiple genes and multiple steps involved in hepatocarcinogenesis. Expressions of oncogene proteins reflected the properties of the premalignant and malignant cells, but not directly related to endotoxemia statistically.[JP]

Relationship between alterations of p16^( INK4a) and p14^(ARF) genes of CDKN2A locus and gastric carcinogenesis

Objective To investigate the relationship between alterations of p16 INK4a and p14 ARF genes and gastric carcinogenesis Methods The tumors and neighboring gastric tissues from 48 patients with gastric cancer were studied The homozygous deletion, mutation, methylation of the CpG islands, and mRNA expression of p16 INK4a and p14 ARF genes were assessed by PCR, PCR SSCP, PCR based methylation assay, and RT PCR Results ① The homozygous deletion rate of p16 INK4a and p14 ARF was 35 4% (17/48), and no homozygous deletion was examined in any gastric tissue neighboring the tumor ② There was no point mutation of p16 INK4a and p14 ARF in 31 gastric cancers without homozygous deletion or in the matched gastric tissues adjacent to the tumor ③ Methylation of the CpG islands of p16 INK4a and p14 ARF was detected in 47 9% (23/48) of gastric cancers, while methylation was observed only in 2 of 48 gastric tissues neighboring the cancer with a significant difference ( P <0 01) ④ The loss rate of p16 INK4a mRNA was 47 9% (23/48) in gastric cancer, and the patients of the combined methylation of exons 1α and 2 had a higher loss rate (100%, 6/6) of p16 INK4a mRNA than those of the methylation of the other exons (11 8%, 2/17, P <0 01); the loss rate of p14 ARF mRNA was 45 8%(22/48) in gastric cancer, and patients with the combined methylation of exons 1β and 2 had a higher loss rate (100%, 3/3) of p14 ARF mRNA than those of the methylation of the other exons (15%, 3/20, P <0 05) ⑤ The combined loss of p16 INK4a and p14 ARF mRNAs was examined in 1 (5 6%) of 18 patients of well and moderately differentiated carcinomas, and 11 (36 7%) of 30 patients of poorly and not differentiated carcinomas with a significant difference ( P <0 05) Conclusion p16 INK4a and p14 ARF genes are frequently inactivated by homozygous deletion and methylation of the 5'CpG islands in gastric cancer, which may play an important role in the carcinogenesis of gastric cancer

Expression of vascular endothelial growth factor and its role in oncogenesis of human gastric carcinoma

AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.

DNA methylation and carcinogenesis in digestive neoplasms

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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.

p16 gene methylation in colorectal cancers associated with Duke's staging

AIM: To explore the association of methylation of the CpG island in the promotor of the p16 tumor suppressor gene with the clinicopathological characteristics of the colorectal cancers. METHODS: Methylation-specific PCR (MSP) was used to detect p16 methylation of 62 sporadic colorectal cancer specimens. RESULTS: p16 methylation was detected in 42% of the tumors.Dukes'staging was associated with p16 methylation status.p16 methylation occurred more frequently in Dukes'C and D patients (75.9%) than in Dukes'A and B patients (12.1%). CONCLUSION: p16 methylation plays a role in the carcinogenesis of a subset of colorectal cancer, and it might be linked to poor prognosis.

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.

Expression and significance of proapoptotic gene Bax in gastric carcinoma

AIM To study the expression of proapoptotic gene Bax in human gastric carcinoma and its significance. METHODS Using immunohistochemistry methods, the Bax protein expression in 57 specimens of gastric carcinoma and its relationship with clinical status and pathomorphological parameters were observed. RESULTS Thirty three (57 89%) cases were positive for Bax protein staining which was mainly located in the cytoplasm of tumor cells. The rate of Bax protein expression was not correlated with the tumor size, lymph node metastasis, depth of invasion, clinical stages of tumors and age and sex of patients ( P >0 05), but strongly associated with the morphological type and differentiation degree of tumors. It was significantly higher in intestinal type and well/moderately differentiated gastric carcinoma than in diffuse type and poorly differentiated gastric carcinoma ( P <0 05 and P <0 01). CONCLUSION The proapoptotic gene Bax is differentially expressed in most of gastric carcinoma and may take part in the modulation of apoptosis in gastric carcinoma. The expression of Bax might be associated with the occurrence of intestinal type gastric carcinoma and the differentiation of gastric carcinoma.

Apoptosis,proliferation and p53 gene expression of H.pylori associated gastric epithelial lesions

AIM: To study the relationship between Helicobacter pylori (H. pylori) and gastric carcinoma and its possible pathogenesis by H. pylori. METHODS: DNEL technique and immunohistochemical technique were used to study the state of apoptosis, proliferation and p53 gene expression. A total of 100 gastric mucosal biopsy specimens, including 20 normal mucosa, 30 H. pylori-negative and 30 H. pylori-positive gastric precancerous lesions along with 20 gastric carcinomas were studied. RESULTS: There were several apoptotic cells in the superficial epithelium and a few proliferative cells within the neck of gastric glands, and no p53 protein expression in normal mucosa. In gastric carcinoma, there were few apoptotic cells, while there were a large number of proliferative cells, and expression of p53 protein significantly was increased. In the phase of metaplasia, the apoptotic index (AI, 4.36%+/-1.95%), proliferative index (PI, 19.11%+/-6.79%) and positivity of p53 expression (46.7%) in H. pylori-positive group were higher than those in normal mucosa (P【0.01). AI in H. pylori-positive group was higher than that in H. pylori-negative group (3.81%+/-1.76%), PI in H. pylori-positive group was higher than that in H. pylori-negative group (12.25%+/-5.63%, P【0.01). In the phase of dysplasia, AI (2.31%+/-1.10%) in H. pylori-positive group was lower (3.05%+/-1.29%) than that in H. pylori-negative group, but PI (33.89%+/-11.65%) was significantly higher (22.09+/-8018%, P【0.01). In phases of metaplasia, dysplasia and gastric cancer in the H. pylori-positive group, AIs had an evidently graduall decreasing trend (P【0.01), while PIs had an evidently gradual increasing trend (P【0.05 or P【0.01), and there was also a trend of gradual increase in the expression of p53 gene. CONCLUSION: In the course of the formation of gastric carcinoma, proliferation of gastric mucosa can be greatly increased by H. pylori, and H. pylori can induce apoptosis in the phase of metaplasia, but in the phase of dysplasia H. pylori can inhibit cellular apoptosis. And H. pylori infection can strengthen the expression of mutated p53 gene.

Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.

Analysis of N-ras gene mutation and p53 gene expression in human hepatocellular carcinomas

IM To study the relationship between Nras gene mutation and p53 gene expression in the carcinogenesis and the development of human hepatocellular carcinomas (HCC).METHODS The Nras gene mutation and the p53 gene expression were analyzed in 29 cases of HCC by polymerase chain reactionsingle strand conformation polymorphism (PCRSSCP) and immunohistochemistry.RESULTS Thirteen cases of HCCs were p53 positive (448%), which showed a rather high percentage of p53 gene mutation in Guangxi. The aberrations at Nras codon 2-37 were found in 7931% of HCCs and 8077% of adjacent nontumorous liver tissues. More than 2 point mutations of Nras gene were observed in 22 cases (7586%). Twelve cases (4137%) of HCCs showed both Nras gene mutation and p53 gene expression.CONCLUSIONS Nras gene and p53 gene may be involved in the carcinogenesis and the development of HCC. That 38% of HCCs with Nras gene mutation did not express p53 protein indicates that some other genes or factors may participate in the carcinogenesis and the development of HCC.

HER2 gene status and the relationship with p21 protein expression in gastric cancer

Objective： We aimed to analysis the HER2 gene status and its relationship with p21 protein expression in gastric carcinoma. Methods： Fluorescence in situ hybridisation （FISH） and immunohistochemistry （IHC） techniques were used to detect HER2 gene status and p53 protein in 59 cases of gastric cancer. Results： FISH detection of HER2 gene amplification rate was 16.9% （10/59）, HER2 gene amplification in 49 cases without copy number gain and gene amplification were a total of 49.2% （29/59）. HER2 protein expression was 42.4% （25/59）, HER2 gene amplification rates in patients with ＋＋＋, ＋＋ HER2 protein expression were 3/3 and 5/8, while in patients with ＋ HER2 protein expression, it was 2/14, there was significant difference （P 0.05）. p21 protein expression rate was 49.2% （29/59）, HER2 gene amplification rates and p21 protein expression had significant difference in tumor invasion depth, lymph node metastasis （P 0.05）; had no statistical significance in histological type, age, gender differences （P 0.05）. Conclusion： HER2 gene amplification rate and gene copy number had positively correlation with p21 protein expression, HER2 gene status and expression of p21 protein combined detection can provide a reference value in gastric cancer metastasis, patient’s condition development and prognosis, it also can guide clinical development of individual treatment.