BACKGROUNDBoth p16(INK4) and p21(Waf1) are tumor suppressors with similar biological functions in the regulation of cellular senescence. Previous reports showed that p16(INK4) could be activated by p21(Waf1) through transcriptional factor Sp1 in HeLa cells. This study was undertaken to determine the effects of p16(INK4) on the expression and functions of p21(Waf1).

METHODSHuman diploid fibroblast 2BS cells were stably transfected with sense (2BS/p16(INK4)), antisense p16(INK4) (2BS/asp16(INK4)) or empty vector (2BS/neo). Then they were assayed by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence activated cell sorting (FACS) and Western blot.

RESULTS2BS/p16(INK4) cells exhibited cell cycle arrest in both G1 and G2/M phases. Endogenous p21(Waf1) protein levels increased twofold in the 2BS/p16(INK4) cells, but not decreased in the 2BS/asp16(INK4) cells. p21(Waf1) mRNA levels were not affected in neither 2BS/p16(INK4) nor 2BS/asp16(INK4) cells.

CONCLUSIONp16(INK4) may play an important role in the regulation of cellular senescence by modulating the p21(Waf1) protein level via the posttranscriptional mechanism.

Cell Cycle ; Cells, Cultured ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p16 ; physiology ; Cyclin-Dependent Kinase Inhibitor p21 ; physiology ; Fibroblasts ; metabolism ; Humans ; Transcription, Genetic

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

Cell Cycle ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; analysis ; DNA Damage ; Dactinomycin ; pharmacology ; Humans ; Viral Nonstructural Proteins ; physiology

OBJECTIVETo investigate the biological function of RIG-G gene by establishing a cell line stably expressing RIG-G protein.

METHODSEctopic RIG-G gene was transfected into U937 cells by using Tet-off expression system. Changes before and after RIG-G expression were detected for cell growth, cell morphology, cell surface antigen and cell cycle regulating proteins.

RESULTSRIG-G protein arrested the cells at G0/G1 phase and inhibited cell growth by increasing the cell cycle inhibitors P21 and P27. As compared to that in control group, the proportion of cells at G0/G1 phase in RIG-G-expressing cell group increased from (43.9 +/- 5.6)% to (63.9 +/- 2.3)% (P < 0.01). The rate of growth inhibition was (68.7 +/- 0.2)%. In addition, an increase in CD11C expression [(61.3 +/- 1.1)% vs. (18.0 +/- 0.4)% (P < 0.01)] and in cells with morphologic features of partial differentiation (smaller cell size, reduced nucleus-cytoplasm ratio, notched nucleus and coarse chromatin) was also observed in RIG-G-expressing cells.

CONCLUSIONSRIG-G has potential abilities to inhibit cell proliferation and promote cell differentiation.

Cell Cycle ; genetics ; Cell Differentiation ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; physiology ; Plasmids ; genetics ; Transfection ; U937 Cells

OBJECTIVETo study the effects of arsenic trioxide (As(2)O(3)) on cell cycle and expression of cyclin dependent kinase inhibitors (CDKIs) in multiple myeloma (MM) cells, and explore its pharmacological mechanism.

METHODSThe DNA content of MM cells line HS-Sultan was analyzed by flow cytometry after exposure to As(2)O(3), the effects on expression of CDKI P15, P16 AND P21 were studied by reverse transcriptase PCR.

RESULTSDNA flow cytometric analysis showed that As(2)O(3) induced most of HS-Sultan cells, arrest at G(0)/G(1) phase and a small fraction at G(2)/M phase and apoptosis occurred mainly in S phase. There was no expression of P15 and P16 mRNA in untreated HS-Sultan cells and 1.0 micromol/L As(2)O(3) could make them expressed after exposed 24 or 48 hours respectively. Expression of P12 mRNA was obviously elevated by As(2)O(3) comparing with that of control.

CONCLUSIONOne of the pharmacological mechanisms of As(2)O(3) is to activate the expression of CDKI P15, P16 and P21, and consequently affect cell proliferation cycle.

Antineoplastic Agents ; pharmacology ; Arsenicals ; pharmacology ; Cell Cycle ; drug effects ; physiology ; Cyclin-Dependent Kinase Inhibitor p15 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Humans ; Multiple Myeloma ; drug therapy ; metabolism ; pathology ; Oxides ; pharmacology ; RNA, Messenger ; genetics ; Tumor Cells, Cultured

OBJECTIVECytotoxin-associated protein (CagA) of H. pylori has been confirmed to be closely associated with gastric inflammation and tumorigenesis, but the mechanism behind it is little understood. In this study, we try to determine roles of CagA(+) strain in activating PI3K/Akt1 signaling pathway, and affecting expression of p21(WAF1/CIP1) and p27(KIP1), and also in releasing IL-8 in host cells.

METHODSAkt1 phosphorylation and IL-8 levels of CagA(+) and CagA⁻ strain infected AGS cells were detected by ELISAs. Two quantitative RT-PCRs were established to measure p21(WAF1/CIP1) and p27(KIP1) mRNA levels in the CagA(+) and CagA⁻ strain infected cells. LY294002, an inhibitor of PI3K/Akt pathway, was used to define effect of the pathway in IL-8 release.

RESULTSCagA(+) strain could induce an obvious elevation of Akt1 phosphorylation in the infected AGS cells while CagA? strain failed to do so. The CagA(+) H. pylori strain infected AGS cells showed significant drops both in p21(WAF1/CIP1) and p27(KIP1) mRNA levels, whereas the CagA⁻ H. pylori strain caused a remarkable increase in p21(WAF1/CIP1) mRNA without affecting p27(KIP1) gene transcription in the AGS cells. Both the CagA(+) and CagA⁻ H. pylori strains enabled AGS cells to produce close elevated levels of IL-8, and the LY294002 block resulted in unexpected elevations of IL-8 levels.

CONCLUSIONSCagA can activate PI3K/Akt1 pathway that plays an inhibitory role in IL-8 release in H. pylori infected AGS cells. Activation of PI3K/Akt1 pathway and subsequent negative regulation of p21(WAF1/CIP1) and p27(KIP1) expression might be involved in CagA-associated carcinogenesis.

Antigens, Bacterial ; genetics ; physiology ; Bacterial Proteins ; genetics ; physiology ; Cell Line ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; Cyclin-Dependent Kinase Inhibitor p27 ; Gastric Mucosa ; cytology ; enzymology ; microbiology ; Helicobacter pylori ; metabolism ; pathogenicity ; physiology ; Humans ; Interleukin-8 ; secretion ; Intracellular Signaling Peptides and Proteins ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Transcription, Genetic ; Virulence

OBJECTIVETo reveal the role of Phosphatidylinositol 3-kinases (PI3Ks) in regulating human diploid fibroblast (2BS cell) senescence as well as the possible mechanisms involved.

METHODSUsing a PI3Ks specific inhibitor, LY294002, cell cycle, apoptosis, proliferation, senescence association beta-galactosidase staining as well as senescence association CKIs, p16(INK4) and p21(Cip1) protein expressions were all measured in the low passages of 2BS cells.

RESULTSBoth 25 micro mol/L and 50 micro mol/L concentrations of LY294002 could cause a significant decrease in cells entering into S phase, and this cell cycle of G(1) phase arrest was dose-dependent. Meanwhile, LY294002 contributed to apoptosis, caused 2BS cell growth arrest, and activated senescence association beta-galactosidase (P < 0.05). In addition, LY294002 could induce time-course expressions of p16(INK4) and p21(Cip1) in 2BS cell lines.

CONCLUSIONSPI3Ks inhibitor LY294002 could induce senescence-like changes in 2BS cell lines. Two senescence associated CKIs, p16(INK4) and p21(Cip1), might be involved in this senescence phenotype proceeding in 2BS cell lines.

Cells, Cultured ; Cellular Senescence ; drug effects ; Chromones ; pharmacology ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; analysis ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Fibroblasts ; drug effects ; physiology ; G1 Phase ; drug effects ; Humans ; Morpholines ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors

In cancer gene therapy, restriction of antitumor transgene expression in a radiation field by use of ionizing radiation-inducible promoters is one of the promising approaches for tumor-specific gene delivery. Although tumor suppressor protein p53 is induced by low doses (<1 Gy) of radiation, there have been only a few reports indicating potential utilization of a p53-target gene promoter, such as that of the p21 gene. This is mainly because the transiently transfected promoter of p53-target genes is not much sensitive to radiation. We examined the response of the p21 gene promoter to low-dose radiation when transduced into a human breast cancer cell line MCF-7 by use of recombinant adeno-associated virus (rAAV) vectors. It was shown that the p21 gene promoter transduced by rAAV vectors was more highly radiation-responsive than that transiently transfected by electroporation. A significant induction of the p21 gene promoter by radiation of low doses down to 0.2 Gy was observed. When cells were transduced with the p21 gene promoter-driven HSVtk gene by rAAV vector, they were significantly sensitized to repetitive treatment with low dose radiation (1 Gy) in the presence of the prodrug ganciclovir. It was therefore considered that the p21 gene promoter in combination with a rAAV vector is potentially usable for the development of a low-dose radiation-inducible vector for cancer gene therapy.
X-Rays ; Tumor Cells, Cultured ; Transgenes/*radiation effects ; Transduction, Genetic ; Promoter Regions (Genetics)/*radiation effects ; Humans ; Genetic Vectors/*radiation effects ; Gene Therapy/methods ; Electroporation/methods ; Dose-Response Relationship, Radiation ; Cyclin-Dependent Kinase Inhibitor p21/*genetics ; Adenoviridae ; 3' Untranslated Regions/physiology

The objective of this study was to explore the effect of p21(WAF1) on the sensitivity to chemotherapeutic agent VP-16, etoposide, in leukemia cell line K562. A p21(WAF1) retroviral expression vector was constructed, and mediated by FuGENE(TM)6, it was transfected into K562 cells, which without p21(WAF1) expression. The ecotropic expression of p21(WAF1) in K562 cells was identified by RT-PCR and Western blot, and named K562-p21(WAF1) cell. The K562-p21(WAF1) cells were exposed to VP-16 at different concentrations and different times, then, the sensitivity to VP-16 was examined by cell viability and MTT assay, the apoptosis induced by VP-16 was examined by DNA fragments electrophoresis and flow cytometric Annexin V-PI dual labeling technique. The results showed that the ecotropic expression of p21(WAF1) decreased the sensitivity of K562 cells to VP-16. After treatment of VP-16, the DNA ladder was examined in control K562-neo cells at 20 microgram/ml, but in K562-p21(WAF1) cells at 80 microgram/ml. With FCM, the number of apoptotic K562-neo cells was 14.9% and 25.4% respectively after treated with 20 microgram/ml VP-16 for 12 and 24 hours, and it was 6.94% and 10.96% in K562-p21(WAF1) cells. Our results suggest that the expression of p21(WAF1) could reduce the sensitivity of K562 cells to VP-16 and inhibit the apoptosis of K562 cells
Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Division ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; genetics ; physiology ; Drug Screening Assays, Antitumor ; Etoposide ; pharmacology ; Humans ; K562 Cells ; Transfection ; Tumor Cells, Cultured

OBJECTIVETo study effects of the expression of transforming growth factor (TGF)-beta1 on the growth of Smad4-null pancreatic cancer cells.

METHODSTGF-beta1 eukaryotic expression vector was transfected into pancreatic cancer cell line BxPC3. Effects of the expressison of TGF-beta1 was studied by growth curve analysis and flow cytometry. Cell motility was monitored by wound-healing assay. Western blot was used to estimate the expression level of p21(WAF/CLIP1), a cyclin-dependent kinase inhibitor.

RESULTSTransfection of TGF-beta1 changed the morphology of BxPC3 into spindle shaped cells. The growth rate of BxPC3 began to decrease after the fourth day of TGF-beta1 transfection, compared with the control groups. Flow cytometry showed that the percentages of cells in the S phase were (27.53 +/- 0.02)%, (26.32 +/- 0.01)% and (17.01 +/- 0.03)% in naïve BxPC3, vector-control group and TGF-beta1 transfection group respectively. Lesser cells entered the S phase after TGF-beta1 transfection (P < 0.01), but no difference was seen between the BxPC3 and vector groups (P > 0.05). The expression of p21(WAF/CLIP1) increased upon the expression of TGF-beta1.

CONCLUSIONThe Smad4-independent pathway of TGF-beta1 not only induces epithelial-mesenchymal transition in pancreatic cancer BxPC3, but also inhibits its growth through the up-regulation of p21(WAF/CLIP1).

Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Gene Deletion ; Genetic Vectors ; Humans ; Pancreatic Neoplasms ; genetics ; metabolism ; pathology ; S Phase ; Smad4 Protein ; genetics ; Transfection ; Transforming Growth Factor beta1 ; biosynthesis ; genetics ; physiology ; Up-Regulation