OBJECTIVEThe recombinant adenovirus vector carrying p14ARF gene was constructed for using in the interference therapy in signal transduction of laryngeal squamous cell carcinoma.

METHODSThe total cDNA fragment of p14ARF was cloned into the shuttle plasmid pAdTrack-CMV, with the resultant plasmid and the backbone plasmid pAdEasy-1, the homologous recombination took place in the E.Coli BJ5183 and the recombinant adenoviral plasmid was generated. The adenoviruses were packaged and amplified in the 293 cells. Then the viral titer was checked by GFP.

RESULTSThe recombinant adenovirus vector carrying p14ARF was constructed successfully. The viral titer was 2.3 x 10(9).

CONCLUSIONThe recombinant adenovirus vector could introduce p14ARF gene into the laryngeal squamous cell carcinoma line or tumor tissue effectively, which would provide experimental basis for the mechanisms and further study of the interference therapy in signal transduction of laryngeal squamous cell carcinoma.

Adenoviridae ; Cell Cycle ; Genetic Vectors ; Humans ; Plasmids ; Recombination, Genetic ; Tumor Suppressor Protein p14ARF

OBJECTIVETo investigate the synergistic antitumor effects of combined use of p14ARF gene and 5-fluorouracil (5-Fu) in pancreatic cancer.

METHODSA human pancreatic cancer cell line PC-3 was transfected with lipofectin-mediated recombinant p14ARF gene, and was then administered with 5-Fu. Cell growth, morphological changes, cell cycle, apoptosis, and molecular changes were measured using the MTT assay, flow cytometry, RT-PCR, Western blotting, and immunocytochemical assays.

RESULTSAfter transfection of p14ARF, cell growth was obviously inhibited, resulting in an accumulation of cells in the G(1) phase. The proportion of cells in the G(1) phase was significantly increased from 58.51% to 75.92%, and in the S and G(2)/M phases decreased significantly from 20.05% to 12.60%, and from 21.44% to 11.48%, respectively, as compared with those of the control groups. PC-3/p14ARF cells that underwent 5-Fu treatment had significantly greater G(2)/M phase accumulation, from 11.48% to 53.47%. The apoptopic index was increased in PC-3/p14ARF cells from 3.64% to 19.62%. The MTT assay showed p14ARF-expressing cells were significantly more sensitive to 5-Fu (0.01 - 10 mg/L) than those devoid of p14ARF expression (P < 0.01). Western blotting showed p14ARF upregulates p53 expression.

CONCLUSIONCombined use of p14ARF gene and 5-Fu acts synergistically to inhibit pancreatic cancer cell proliferation, suggesting a new anticancer strategy.

Fluorouracil ; pharmacology ; Humans ; Pancreatic Neoplasms ; genetics ; therapy ; Transfection ; Tumor Cells, Cultured ; Tumor Suppressor Protein p14ARF ; genetics ; Tumor Suppressor Protein p53 ; genetics ; Up-Regulation ; physiology

OBJECTIVETo investigate the correlations between p14(ARF) and E2F-1, and the role of their alterations in the tumorigenesis of the lung cancer in Gejiu and Xuanwei regions in Yunnan Province for providing the important experiment basis in revealing the molecular mechanism and looking for new markers for early diagnosis of lung cancer.

METHODSThe expression of p14(ARF) and E2F-1 was detected at theirs protein level by Immunohistochemistry S-P method in 30 specimens of lung cancer of Gejiu tin miners, 30 specimens of lung cancer of Xuanwei peasants and 20 specimens of normal lung tissue. E2F-1 mRNA was detected by ISH in 25 specimens of lung cancer of Gejiu tin miners, 25 specimens of lung cancer of Xuanwei peasants and 10 specimens of normal lung tissue. The positive signals were quantitatively analysed by HPIAS-100.

RESULTSThe positive unit (PU) of p14(ARF) and E2F-1 was 16.44 +/- 4.85 and 47.39 +/- 5.43 in Gejiu group, and 16.79 +/- 3.55 and 48.15 +/- 9.11 in Xuanwei group. Expression of p14(ARF) and E2F-1 protein in lung cancer of Gejiu and Xuanwei were statistically different compared with that in the normal lung (P < 0.01) respectively; The PU of E2F-1 mRNA was 48.58 +/- 7.75 in Gejiu group, and 49.41 +/- 8.53 in Xuanwei group, which was higher than that in normal tissue group. The differences were significant (P < 0.01). There was positive correlation between the expression of E2F-1 protein and E2F-1 mRNA in Gejiu group, Xuanwei group and normal group (P < 0.01, r = 0.833). The expression of p14(ARF) protein was significantly negatively correlated with the expression of E2F-1 protein (P < 0.01, r = -0.830).

CONCLUSIONThere is the over-expression of E2F-1 gene and the deletion of p14(ARF) gene in the tumorigenesis of the lung cancer in Gejiu and Xuanwei regions in Yunnan Province. Over-expression of E2F-1 protein in lung cancer may be caused by enhanced transcription.

China ; E2F1 Transcription Factor ; genetics ; metabolism ; Female ; Humans ; Lung Neoplasms ; metabolism ; Male ; RNA, Messenger ; genetics ; Tumor Suppressor Protein p14ARF ; metabolism

BACKGROUND: Glioblastomas are one of the most common and aggressive malignant glial tumors occuring in the central nervous system. This study analyzed the status of p15INK4b, p14ARF, p16INK4a, MTAP, IFNA, and IFNB genes in 36 primary glioblastomas to investigate whether the inactivation of these genes participate in primary glioblastoma tumorigenesis. METHODS: We used polymerase chain reaction, polymerase chain reaction/single strand conformational polymorphism (PCR/SSCP) analysis, and methylation-specific PCR. RESULTS: Homozygous deletions at the p16INK4a gene were detected in 11 cases (30.5%) of 36 primary glioblastomas, and the promoter hypermethylation was found in 3 cases (8.3%) of 36 primary glioblastomas. In mutational analysis for the p16INK4a gene by PCR/SSCP, there was no abnormal mobility-shifted band in 36 cases of primary glioblastomas. The overall frequency of p16INK4a alterations including homozygous deletion and promoter hypermethylation in 36 primary glioblastomas was 38.8% (14 of 36). Deletions of p15INK4b were noted in 4 cases (11.1%), whereas deletions of the p14ARF and MTAP genes were detected in 1 case of 36 cases of primary glioblastomas. But deletions of the INFA and B genes were not found. CONCLUSIONS: These results suggest that alterations of the p16INK4a gene can be important mechanisms of the tumorigenesis of primary glioblastomas, and the p16INK4a gene is inactivated by mechanisms including homozygous deletion and promoter hypermethylation.
Brain Neoplasms ; Carcinogenesis ; Central Nervous System ; Genes, p16 ; Glioblastoma* ; Humans ; Polymerase Chain Reaction ; Tumor Suppressor Protein p14ARF

PURPOSE: Promoter methylation provides an alternative pathway for the loss of tumor suppressor gene functions. This epigenetic change is a new marker for human cancers. We herein investigated the aberrant methylation profile of bladder cancer to identify the epigenetic markers that are useful for the diagnosis of bladder cancer. MATERIALS AND METHODS: Gene promoter methylation was analyzed in 50 bladder transitional cell cancer (TCC) tissues and 30 nonmalignant bladder mucosal tissues including 21 tissue samples with normal histology and 9 tissue samples with inflammation. The methylation status of 13 tumor suppressor genes was analyzed by methylation specific polymerase chain reaction and bisulfite genomic sequencing. The methylation frequency and methylation index were comparatively analyzed in each group of tissues. RESULTS: Bladder TCC showed a high frequency of promoter hypermethylation for RASSF1A(62.0%), RARbeta2(54.0%), E-cadherin(48.0%), p16INK4A(46.0%), p14ARF(34.0%) and H-cadherin(32.0%), whereas, methylation was less common for MGMT(18.0%), DAPK(14.0%) and p15INK4B(10.0%), and methylation was rare for GSTP1(4.0%), FHIT(2.0%), APC(2.0%) and MLH1(2.0%). Benign bladder mucosa rarely showed aberrant methylation except for E-cadherin (10.0%) and RARbeta2(10.0%). The methylation index of bladder TCC(0.25) was significantly higher than that of the benign bladder mucosal tissues(0.03, p<0.01). Remarkably, all of the bladder cancer tissues showed aberrant methylation of at least one of 6 genes including RASSF1A, RARbeta2, p16INK4A, p14ARF, E-cadherin and H-cadherin, whereas only 5 of 30 (16.7%) benign bladder mucosa tissues showed the same findings. CONCLUSIONS: Aberrant promoter methylation of tumor suppressor genes may be a critical step in the development of bladder TCC. Aberrant promoter methylations of RASSF1A, RARbeta2, p16INK4A, p14ARF, E-cadherin and H-cadherin may be promising epigenetic markers for the diagnosis and follow up of bladder cancer.
Cadherins ; Diagnosis ; Epigenomics* ; Follow-Up Studies ; Genes, Tumor Suppressor ; Humans ; Inflammation ; Methylation ; Mucous Membrane ; Polymerase Chain Reaction ; Tumor Suppressor Protein p14ARF ; Urinary Bladder Neoplasms* ; Urinary Bladder*

OBJECTIVETo explore the effects of hepatitis B virus X protein (HBx) on hepatoma cell growth through p14(ARF)-dependent and p14(ARF)-independent pathways.

METHODSHBx and p14(ARF) were transfected either separately or in combination into HepG2 cells containing wt-p53 but not expressing p14(ARF). The cells were divided into 4 groups, namely pcDNA3 (control), pcDNA3HBx, pcDNA3p14(ARF), and pcDNA3HBx + pcDNA3p14(ARF) groups. Flow cytometry was used to examine the apoptosis rates and cell cycle progression of HepG2 cells in different groups. The expression of p14(ARF), MDM2, p53, and p21(WAF1) proteins were investigated by detecting the activity of p21(WAF1) promoter-luciferase and using Western blotting.

RESULTSThe apoptosis rates of HepG2 cells in pcDNA3HBx and pcDNA3p14(ARF) groups were significantly higher than that in the control group (14.11%, 13.72% vs 10.66%). Compared with the control group, pcDNA3HBx and pcDNA3p14(ARF) groups also showed significantly higher cell percentages arrested at G(0)/G(1) phase (63.62%, 61.75% vs 57.42%), luciferase activity of p21 promoter (1.25-/+0.05, 1.09-/+0.06 vs 0.77-/+0.03) and expressions of p53 and p21(WAF1). The cell apoptosis rate, percentage of cells in G(0)/G(1) phase and expression level of p14(ARF) were even higher in pcDNA3HBx+pcDNA3p14(ARF) group (18.61%, 66.74%, and 3.53-/+0.43, respectively) than in either p14(ARF) or HBx group.

CONCLUSIONHBx induces p53 expression through p14(ARF)-dependent and independent pathways to activate p21(WAF1) promoter, leading to G(0)/G(1) arrest and apoptosis of HepG2 cells.

Carcinoma, Hepatocellular ; genetics ; pathology ; virology ; Cell Line, Tumor ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Humans ; Liver Neoplasms ; genetics ; pathology ; virology ; Promoter Regions, Genetic ; Trans-Activators ; genetics ; Transfection ; Tumor Suppressor Protein p14ARF ; genetics ; Tumor Suppressor Protein p53 ; genetics

OBJECTIVETo investigate the effect of up-regulated expression of tumor suppressor gene p14(ARF) on apoptosis of chronic myeloid leukemia (CML) cells and its interaction with imatinib.

METHODSTumor suppressor gene p14(ARF) was transduced into K562 (K562-p14(ARF)) and 4 blast crisis primary CML cells (CML-BC 1-4) using vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vector with cells transduced by empty vector as control. Fluorescence microscopy and flow cytometry were applied to measure transduction efficiency, and Western blotting assay was used to detect p14(ARF) protein of K562 cells. WST-8 method was used to determine cell growth inhibition rate of K562 cells transduced by the target gene under different concentrations of imatinib (0, 0.015, 0.062, 0.125, 0.25, 0.5, 1.0, 2.0 μmol/L). Cell apoptosis and leukemic cellular colony-forming ability were detected by Annexin V-FITC/PI dyeing using flow cytometry (FCM) and semi-solid culture method respectively.

RESULTSFluorescence microscopy and FCM showed that transduction efficiency (GFP positive cells) of K562-p14(ARF), K562-VSV and CML-BC1 cells were close to 100%, and CML-BC 2-4 cells were 80% to 90% on average. Results of Western blotting showed that the levels of ARF protein expression of K562 cells transduced by p14(ARF) were significantly higher than of untransduced cells; the apoptosis rate of K562-p14(ARF) was 20%; the mean apoptosis rate of 4 primary leukemic cells transduced by the p14(ARF) [(71.1±22.4)%] was significantly higher than of control group [(12.4±6.2)%] (P<0.05). Imatinib significantly inhibited the proliferation of K562-p14(ARF) cells in a dose-dependent manner. The mean leukemic cellular colony-forming unit of 4 primary leukemic cells transduced by the p14(ARF) (41.5±13.2) was significantly lower than of the control group (88.5±7.9) (P<0.05).

CONCLUSIONIncreased p14(ARF) gene expression could induce apoptosis of CML cells; Moreover, it could enhance inhibitory effect on cell proliferation when combined with imatinib.

Apoptosis ; Gene Expression Regulation, Leukemic ; Genetic Vectors ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; pathology ; Tumor Suppressor Protein p14ARF ; metabolism ; Up-Regulation

PURPOSE: In this study, the level of expression of p14(ARF), p16(INK4a), p53 and pRb was immunohistochemically examined according to the stage of gastric cancer, lymph node metastasis, cell differentiation and Lauren's classification. The effect on survival rate and the associations between the components were examined as well. METHODS: One hundred and fourteen patients who underwent surgery for gastric cancer were studied retrospectively using their paraffin embedded tissue and medical records. Using antibodies of p14(ARF), p16(INK4a), p53 and pRb, immunohistochemical stain was applied and their level of expression examined. RESULTS: The level of p53 expression was high when the stage of gastric cancer was more progressed, the invasiveness higher, lymph node metastasis present and cell differentiation poorer. In contrast, the level of p14ARF expression tended to be lower as the stage was more progressed, but this was not statistically significant. Expression of p16 and pRb did not show any association with stage or other pathologic findings. Expression of p53 and p14(ARF) also had a significant association with survival rate. Survival rate was lower in patients who expressed p53 than in those who did not, but it was higher in who expressed p14ARF than in those who did not. When these two were combined, patients with p14(ARF)(+)/p53(-) had the highest survival rate, whereas those with p14(ARF)(-)/p53(+)had the lowest. This demonstrated that the expressions of p14ART and p53 have value as prognostic indicators. CONCLUSION: From these results, p53 seems closely related to stage and other pathologic findings. Furthermore, p14(ARF) and p53 showed a statistically significant relationship with survival rate, making them valuable as prognostic indicators after surgery. In combination, it would be possible to predict a more accurate prognosis.
Antibodies ; Cell Differentiation ; Classification ; Cyclin-Dependent Kinase Inhibitor p16* ; Humans ; Lymph Nodes ; Medical Records ; Neoplasm Metastasis ; Paraffin ; Prognosis ; Retrospective Studies ; Stomach Neoplasms* ; Survival Rate ; Tumor Suppressor Protein p14ARF*

PURPOSE: The methylation status of the CpG promoter regions of the p16INK4A and p14ARF genes, mutations of 4 exons of the CDKN2A gene, and the expression of the corresponding proteins were examined. Prognostic implications were assessed in osteosarcoma. MATERIALS AND METHODS: Methylation-specific PCR, sequence analysis, and immunohistochemical staining were performed upon 32 frozen osteosarcoma tissues. RESULTS: Methylation of p16INK4A was found in 16%, and methylation of p14ARF in 47%. Metastasis and poor survival was statistically related to the methylation of p14ARF. The methylation of p14ARF correlated with the repression of the corresponding protein, and repression of p14ARF with the repression of p21 and of wild type of p53. No sequence alterations were found in the four exons of the CDKN2A gene. Methylation of p14 showed highest hazard ratio by multivariate survival analysis. CONCLUSION: Our data suggest that methylation of the CDKN2A gene seems to be the main mechanism of protein repression. For p14ARF, the methylation of its promoter region was related to the repression of p21 and wild type p53, distant metastasis and a poor prognosis. Further study regarding cell cycle regulatory factors should shed light on oncogenesis and the possibility of a new treatment strategy for osteosarcoma.
Carcinogenesis ; Cell Cycle ; Exons ; Genes, p16 ; Methylation* ; Neoplasm Metastasis ; Osteosarcoma* ; Phosphotransferases* ; Polymerase Chain Reaction ; Prognosis* ; Promoter Regions, Genetic ; Repression, Psychology ; Sequence Analysis ; Tumor Suppressor Protein p14ARF

OBJECTIVETo investigate the relationship between alterations of p16(INK4a) and p14(ARF) genes and gastric carcinogenesis.

METHODSThe 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 1alpha 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 1beta 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).

CONCLUSIONp16(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.

Adolescent ; Adult ; Aged ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; DNA Methylation ; Gene Deletion ; Genes, p16 ; Humans ; Middle Aged ; RNA, Messenger ; analysis ; Stomach Neoplasms ; genetics ; Tumor Suppressor Protein p14ARF ; genetics