OBJECTIVE: To investigate the effect of gene silencing of Bmi-1 on proliferation regulation of CD44+ nasopharyngeal carcinoma cancer stem-like cells (CSC-LCs). METHOD: The sequence-specific short hairpin RNA lentivirus targeting at human Bmi-1 gene (LV-Bmi-1shRNA) was constructed and was used to infect CD44+ nasopharyngeal carcinoma cells which were sorted by flow cytometry. A lentiviral which included a random sequence was also designed to serve as a negative control. We employed fluorescence microscope and flow cytometry to detect infection efficiency; real-time PCR was used to detect Bmi-1 and its downstream gene while each protein expression level was confirmed by western blotting protocol; CCK-8 proliferation assay was applied to measure proliferation capacity; tumor spheroid assay was used to evaluate the self-renewal capacity. Colony formation assay was used to measure cell colony formation capability; flow cytometry analyzed cell cycle distribution. RESULT: The constructed LV-Bmi-1shRNA successfully infected into the CD44+ nasopharyngeal carcinoma cells. The infection efficiency could reach above 95%; LV-Bmi-lshRNA effectively inhibited Bmi-1 mRNA and protein expression, while the downstream gene p16INK4a and p14ARF mRNA as well as protein expression level were upregulated (P < 0.05). Notablely, the proliferation, colony formation, self-renewal capabilities of the experimental group decreased significantly (P < 0.05). In addition, the cell cycle arrested at the G0-G1 phase. CONCLUSION Gene silencing of Bmi-1 inhibited the proliferation, colony formation and self-renewal capabilities of the CD44+ nasopharyngeal carcinoma CSC-LCs, inhibited the cell cycle processes, which may mediate through Bmi1-p16INK4a/p14ARF-p53 pathway. Our experimental results indicated that Bmi-1 gene may play an important role in the maintenance of the stem cell-like characteristics of CD44+ nasopharyngeal carcinoma cells. Bmi-1 gene may be a potential new target for the treatment of nasopharyng al carcinoma in the future.
Carcinoma ; Cell Cycle ; Cell Division ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Gene Silencing ; Humans ; Hyaluronan Receptors ; metabolism ; Lentivirus ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; genetics ; pathology ; Neoplastic Stem Cells ; cytology ; Polycomb Repressive Complex 1 ; genetics ; RNA, Messenger ; RNA, Small Interfering ; Tumor Suppressor Protein p14ARF ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

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

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 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

OBJECTIVETo investigate the CpG methylation locus and frequency pattern on p16 INK4a gene promoter in epidermis of p16 INK4a methylated patients with psoriasis vulgaris.

METHODSThe DNA specimens were obtained from epidermal lesion of 50 plaque psoriatic patients. Methylation specific PCR and DNA sequencing were used to detect the frequency and locus of methylation in p16 INK4a gene promoter region.

RESULTSApproximately 50% CpG was methylated in p16 INK4a methylated patients, methylation was found in specifical locus of p16 INK4a gene promoter.

CONCLUSIONThe distinct methylation pattern is showed on the p16 INK4a gene promoter region in patients with psoriasis.

Adolescent ; Adult ; Aged ; Base Sequence ; CpG Islands ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; DNA Methylation ; genetics ; Epidermis ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; Psoriasis ; genetics ; Sequence Alignment ; Tumor Suppressor Protein p14ARF ; genetics ; Young Adult

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 explore the feasibility and mechanism of recombinant adenovirus Ad-pl4ARF in cancer gene therapy.

METHODSThe proliferation of different liver cancer cells was assessed by morphology and trypan blue assay. Cell apoptosis was confirmed by detecting phosphatidylserine (PS) externalization with Annexin V/PI double staining. The expression of related proteins was analyzed by Western bloting. Nude mouse model bearing subcutaneous transplanted BEL7402 tumor was established to study the therapeutic ability of Ad-pl4ARF.

RESULTSAd-pl4ARF suppressed cell growth and proliferation, and promoted cell apoptosis of cancer cell lines with different genetic background. Ad-pl4ARF inhibited growth of liver cancer cells ( HepG2, BEL7402) in a dose-dependent manner. Ad-pl4ARF lead to overexpression of Bax and p21, the downstream regulating genes of p53. In the experimental therapy on nude mice bearing subcutaneous transplanted BEL7402 tumor, Ad-pl4ARF suppressed tumor growth significantly.

CONCLUSIONpl4ARF is a short gene and with powerful function, which are consistent with the requirements for tumor suppressor genes used in gene therapy. It may play an important role in gene therapy against malignancies in the future.

Adenoviridae ; genetics ; Animals ; Apoptosis ; Blotting, Western ; Carcinoma, Hepatocellular ; metabolism ; pathology ; therapy ; Cell Line, Tumor ; Cell Proliferation ; Genetic Therapy ; methods ; Humans ; Liver Neoplasms, Experimental ; metabolism ; pathology ; therapy ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Proto-Oncogene Proteins p21(ras) ; metabolism ; Recombinant Fusion Proteins ; genetics ; Transfection ; Tumor Suppressor Protein p14ARF ; genetics ; Tumor Suppressor Protein p53 ; metabolism ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; metabolism

Carcinoma of the uterine cervix is one of the most common malignancies among women worldwide. Human papillomaviruses (HPV) have been identified as the major etiological factor in cervical carcinogenesis. However, the time lag between HPV infection and the diagnosis of cancer indicates that multiple steps, as well as multiple factors, may be necessary for the development of cervical cancer. The development and progression of cervical carcinoma have been shown to be dependent on various genetic and epigenetic events, especially alterations in the cell cycle checkpoint machinery. In mammalian cells, control of the cell cycle is regulated by the activity of cyclin-dependent kinases (CDKs) and their essential activating coenzymes, the cyclins. Generally, CDKs, cyclins, and CDK inhibitors function within several pathways, including the p16INK4A-cyclin D1-CDK4/6-pRb-E2F, p21WAF1-p27KIP1-cyclinE-CDK2, and p14ARF-MDM2-p53 pathways. The results from several studies showed aberrant regulation of several cell cycle proteins, such as cyclin D, cyclin E, p16 INK4A, p21WAF1, and p27KIP1, as characteristic features of HPV- infected and HPV E6/E7 oncogene-expressing cervical carcinomas and their precursors. These data suggested further that interactions of viral proteins with host cellular proteins, particularly cell cycle proteins, are involved in the activation or repression of cell cycle progression in cervical carcinogenesis.
Uterine Cervical Neoplasms/*pathology ; Tumor Suppressor Protein p53/physiology ; Tumor Suppressor Protein p14ARF/physiology ; Retinoblastoma Protein/physiology ; Proto-Oncogene Proteins c-mdm2/physiology ; Humans ; Female ; E2F Transcription Factors/physiology ; Cyclin-Dependent Kinase Inhibitor p27/physiology ; Cyclin-Dependent Kinase Inhibitor p21/physiology ; Cyclin-Dependent Kinase Inhibitor p16/physiology ; Cyclin-Dependent Kinase 4/physiology ; Cyclin-Dependent Kinase 2/physiology ; Cyclin E/physiology ; Cyclin D1/physiology ; Cell Cycle/*physiology

BACKGROUND/AIMS: Mucin-hypersecreting bile duct tumor is rare, and has an unusual histologic characteristic of having various degrees of cellular atypia ranging from dysplasia to invasive carcinoma in the same specimen. To gain insight into the role of p16, p14 and p53 in the carcinogenic process of bile duct tumor, we analyzed the expression status of these proteins in mucin-hypersecreting bile duct tumor. METHODS: Immunohistochemical staining of p16, p14 and p53 were performed in 34 paraffin embedded tissues obtained from 22 patients of mucin-hypersecreting bile duct tumor. RESULTS: Thirty-four specimens were categorized into low-grade dysplasia (9), high-grade dysplasia (4), carcinoma in situ (CIS, 11) and invasive carcinoma (10) based on the degree of cytologic and structural atypia. p53 overexpressions were found in 6 (17.6%, 3 in CIS, 3 in invasive carcinoma) and more frequently observed in the advanced histologic stages (p<0.05). Loss of p16 staining was found only in 2 (6%) of low-grade dysplasia specimen. Loss of p14 staining was found in 21 (61.7%, 7 in low-grade dysplasia, 2 in high-grade dysplasia, 8 in CIS, and 4 in invasive carcinoma) and was frequently observed in low-grade and high-grade dysplasia compared to p53 (p<0.05). CONCLUSIONS: In mucin-hypersecreting bile duct tumor, p14 and p53 may play a role in the early and advanced stage of carcinogenesis, respectively. Further study regarding genetic and epigenetic alterations in p14 and p53 gene may be needed.
Adult ; Aged ; Bile Duct Neoplasms/*genetics/secretion ; Carcinoma/*genetics ; Cyclin-Dependent Kinase Inhibitor p16/*genetics/secretion ; English Abstract ; Female ; *Genes, p16 ; *Genes, p53 ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Mucins/*secretion ; *Mutation ; Tumor Suppressor Protein p14ARF/*genetics

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*