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.

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.

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.

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 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.

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.

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

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.

A Quantitative DNA Methylation Assay Using Mismatch Hybridization and Chemiluminescence

To develop a quantitative method for methylation analysis of the p16 gene based on mismatch hybridization and chemiluminescence. Methods Genomic DNA was modified by sodium bisulfite to convert all unmethylated but not methylated cytosines to uracil, and subsequently a pair of primer having no CpG sites was designed for amplification target DNA containing methylated or unmethylated CpG sites. The PCR product spanning CpG sites were hybridized with two oligonucleotide probes which perfectly matched the methylated and unmethylated CpG sequences respectively, and the hybrids were detected by chemiluminescent method. The percentage of methylated target sequences could be estimated by calculating the ratio of signals obtained with two probes. Results The percentage of methylation of artificial mixtures DNA showed a linear relation. There was a negative correlation between the methyaltion index with p16 transcriptional mRNA of p16 gene in tumor cell lines. Conclusion Compared with existing methods, this assay is nonisotopic, rapid, simple, and can be widely applied to the study of DNA methylation.

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

Aberrant p16 promoter hypermethylation in bronchial mucosae as a biomarker for the early detection of lung cancer

Lung cancer is the leading cause of cancer related death in the world and its mortality could be greatly reduced by diagnosis and treatment in its early stages. Effective tools for the early detection of lung cancer and its high risk factors remain a major challenge. Biomarkers that detect lung cancer in its early stages or identify its pretumour lesions, enabling early therapeutic intervention, would be invaluable to improve its dismal prognosis.