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.

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].

PROMOTER HYPERMETHYLATION OF p16 GENE AND DAPK GENE IN SERA FROM HEPATOCELLULAR CARCINOMA (HCC) PATIENTS

Objective： To analyze the aberrant methylation of p16 gene and DAPK gene in sera from primary liver cancer patients ad to evaluate the clinical significance. Methods： A methylation-specific PCR was performed for the detection of promoter hypermethylation of p16 gene and DAPK gene in blood DNA from 64 cases of HCC patients, and to analyze the relation of the aberrant methylation of p16 gene and KAPK gene and the clinical pathological data. Results： 76.6%（49/64） of the sera from 64 cases of HCC patients showed hypermethylation for p16 promoter and 40.6% （26/64） for KAPK promoter, whereas no methylated p16 gene promoter and DAPK gene promoter were found in sera from benign liver diseases patients and normal control. Methylated p16 gene and KAPK gene promoters in sera did not strongly correlated with HBsAg, stage, metastasis and differentiation in HCC; but strongly correlated with AFP. Conclusion： Detection of the aberrant methylation of p16 gene and KAPK gene in blood DNA from HCC patients might offer an effective means for the earlier auxiliary diagnosis of the malignancy.

Gene Product Expression of Cyclin D_2 and p16 During the Transition from Cardiac Myocyte Hyperplasia to Hypertrophy

The current study was to investigate mRNA expression of cyclin D 2 and p16 during the transition from cardiac myocyte hyperplasia to hypertrophy. Cultured cardiac myocytes (CM) and fibroblasts (FC) obtained from 1 day old Sparague Dawley rats were used in this study. We have determined (1) hyperplasia by cell growth curve and fluorescence activated cell sorting (FACS); and (2) ultrastructure by electron microscope observation; and (3) expressions of cyclin D 2 mRNA and p16 mRNA by using in situ hybridization and image analysis. The results were shown (1) Results of cell growth curve and FACS analysis showed CM could proliferate in the first 3 cultured days (4 days in postnatal development). But the ability decreased quickly, concomitant with the differentiation. (2) The ultrastructure of CM showed the large amount of myofilaments and mitochondrion and FC showed moderate amount of rough endoplasmic reticulum. (3) The expression of cyclin D 2 mRNA in 3 , 4 , 5 day CM group was 0.89 times(p<0.05), 0.80 times (p<0.05)and 0.56 times (p<0.01)of that in 1 day group respectively. P16 mRNA in 2 , 3 , 4 , 5 day CM group were 1.63 times(p<0.01),1.72 times(p<0.01),1.99 times (p<0.01)and 2.84 times (p<0.01) of that in 1 day group respectively. It can be concluded that cultured neonatal rat cardiac myocytes could proliferate during the first 3 cultured days, but the ability of proliferation decreased, from the fourth day, concomitant with differentiation. Cyclin D 2 and p16 have the key roles during the transition from myocyte hyperplasia to hypertrophy.

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.

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.

Effects of Exogenous p16^(ink4a) Gene on Biological Behaviors of Human Lung Cancer Cells

The effects of exogenous p16^ink4a gene on biological behaviors of human lung cancer cell line with homozygous deletion of p16^ink4a gene were investigated. Exogenous p16^ink4a gene was transfected by lipofectin into human lung cell line A549, in which p16^ink4a gene was homozygously deleted. The expression of p16^ink4a mRNA and protein was detected by RT-PCR and immunocytochemistry, respectively. The changes in the behaviors of the transfected cell lines in vitro and in vivo were observed. In the transfected cell line A549, the exogenous p16^ink4a gene could be stably expressed. The growth of A549 cells transfected with p16^ink4a gene was obviously slowed down. Flow cytometry revealed that transfection of the exogenous p16^ink4a gene resulted in A549 cell lines arrest in G1 phase of cell cycle. The tumorigenicity of these transfected cells in nude mice could be inhibited, and the tumor growth of nude mice was significantly suppressed. It was concluded that exogenous p16^ink4a gene may be stably expressed in human lung cancer cell line A549. The expression of the introduced p16^ink4a could block lung cancer cells to entry into S phase of cell cycle and inhibit tumor malignant growth both in vitro and in vivo.

STUDY OF DELETION OF P16 GENE IN THE PROGRESSION OF BRAIN ASTROCYTOMAS

Objective: To study the relationship between deletion of P16 gene and occurrence and progression of astrocytomas Methods: The techniques of polymerase chain reaction (PCR) and immunohistochemistry were used to detect the deletion of exon2 of P16 gene and expression of P16 gene in 52 cases of Brain astrocytoma Results: The deletion rate of exon2 of P16 gene in the tumors analyzed was 34 6% Most of them with deletion of exon2 of p16 gene were high grade astrocytomas (grade III 42%, grade IV 50%) 61 5% of the tumors were absent from expression of p16 and the deletion rate of p16 protein increased with the grade of astrocytoma (X 2=10 83, P <0 005) Conclusion: Deletion of p16 gene and protein may correlate with the malignant progression of astrocytoma

EFFECTS OF p16^(INK4) GENE ON CHEMOSENSITIVITY OF HUMAN GLIOMA U251 CELL LINE TO TENIPOSIDE

Objective: To determine the effects on the cell growth, tumorigenicity and chemosensitivity of p16/CDK4I in human glioma. Methods: p16 gene was transfected into U251 cells by lipofectin. Expression of exogenous p16 gene was confirmed by immunohistochemistry and Northern blot. The effects of exogenous p16 gene on the growth and chemosensitivity to teniposide were examined. Results: Expression of exogenous p16 gene inhibited the growth dramatically in vitro. G1 arrest of tumor cells was observed. However, wt p16-positive U251 was less sensitive than control cell lines and the number of apoptotic cells after chemotherapy was reduced. Conclusion: The expression of exogenous p16 gene could inhibit the growth of glioma. On the other hand, the chemosensitivity to teniposide of p16-positive U251 was decreased.

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.

IN SITU PCR AND IMMUNOHISTOCHEMICAL STUDIES ON p16 GENE IN PITUITARY ADENOMAS

Objective: To examine the occurrence of p16 gene deletion and to analyze p16 expression on paraffin-embedded human pituitary adenoma specimens. Efforts were made to optimize the technical conditions forin situ PCR. Methods:In situ PCR techniques and inimuno-histochemistry were used. Results: Immunohistochemically, p16-positive tumor cells were observed in all cases with various proportions. The majority of the stromal cells and part of tumor cells was devoid of p16 immunostaining, but signal ofin situ PCR for p16 gene, exon 2, was displayed in these cells. Conclusion: The results implied that p16 gene might not be deleted in these pituitary adenomas. It also indicated thatin situ PCR, both direct and indirect methods, proved feasible and informative to the aim of DNA detection. It is critical to overcome non-specific amplification in directin situ PCR by means of higher annealing temperature, fewer cycle, lower magnesium concentration and stringent washing. A target DNA-deleted sample as the negative control is extremely necessary. For the indirect method, the way to improve the sensitivity is to loosen the conditions for amplification and washing, so that more amplification products are subject to hybridization, and signal detection is facilitated.

Study on P16 Gene in Acute Leukemia

To stddy the chang of suppressing cancer gene P16 in acute leukemia, the P16 antigen expression of leukemia cell surfaces in 61 cases were investigated with ABC assay and gene structural defects in 51 cases of acute leukemia were examined with multiple comparative PCR method. It was found that antigen expression of P16 in leukemia was obviously lower than that innormal subjects ( P <0 001). At the same time, antigen expression in All was lower than that AML ( P <0 05). No significant difference was found betwee the complete reission (CR) and non remission (NR) subjects from AML and ALL groups ( P >0 05). THe exon 2 of P16 gene showed homozygous deletion only inn4 cases out of 30 cases in ALL. No stuctural defect was revealed in 21 cases of AML. It was suggested that expression defect of P16 gene was a main cause in development and progression of acute leukemia, and structural defect of exon 2 was not a primary molecular event.

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.

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.

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,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.

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

FISH检测9号染色体GLP9 p16基因扩增在膀胱癌临床诊断中的意义

目的探讨GLP9 p16基因扩增在诊断膀胱癌中的临床意义。方法应用荧光标记的p16基因探针检测35例膀胱癌和10例腺性膀胱炎患者尿液脱落细胞的染色体及基因畸变情况。结果荧光显微镜下细胞核大且不规则者易观察到染色体基因异常,GLP9 p16多倍体细胞数在膀胱癌中的阳性率为85.7%(30/35),显著高于腺性膀胱炎中的阳性率10.0%(1/10)(P<0.05)。在各分期分级中GLP9 p16多倍体细胞数分别为Ta期17.3±14.9,T1期36.5±27.3,T2期61.8±21.9,T2vs.Ta(P<0.05);G1级14.5±12.0,G2级38.1±24.8,G3级73.5±12.7,随着病理分级的增高GLP9 p16多倍体细胞数显著增加(P<0.05)。结论 FISH检测GLP9 p16基因扩增对膀胱癌的临床诊断有辅助价值,同时也可以作为临床监测膀胱癌进展的一个生物学指标。

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