Induction of growth arrest and cell death by overexpression of the cyclin-Cdk inhibitor p21 in hamster BHK21 cells

Sekiguchi, Toshio; Hunter, Tony

doi:10.1038/sj.onc.1201539

Cited by 49 publications

(45 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cells were then processed for FACScan analysis as described in Materials and methods. For tetracycline repressible vectors, tsBN462 cells were cultured either in the presence of Tetracycline (+) or in the absence of Tetracycline (7) after transfection as described previously (Sekiguchi and Hunter, 1998). Experiment II: tsBN462 cells were transiently transfected with an expression vector for human cyclin D1, E2F-1, SV40 large T antigen (SVLT), Cdk4, cyclin D1+Cdk4, CCG1/TAF II 250 or pCDEB control vector (vector) and an expression vector for CD20 and processed as described above transfected with vector DNA alone did not incorporate BUdR at 39.58C (Figure 1g), whereas tsBN462 cells incorporated BUdR at the permissive temperature of 33.58C (Figure 1f).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Overexpression of D-type cyclins, E2F-1, SV40 large T antigen and HPV16 E7 rescue cell cycle arrest of tsBN462 cells caused by the CCG1/TAFII250 mutation

Sekiguchi

Nishimoto

Hunter³

1999

Oncogene

Self Cite

View full text Add to dashboard Cite

Japan tsBN462 cells, which have a point mutation in CCG1/ TAF II 250, a component of TFIID complex, arrest in G1 at the nonpermissive temperature of 39.58C. Overexpression of D-type cyclins rescued the cell cycle arrest of tsBN462 cells, suggesting that the cell cycle arrest was through Rb. Consistent with this, overexpression of E2F-1, whose function is repressed by the hypophosphorylated form of Rb, also rescued the cell cycle arrest. Moreover, expression of the viral oncoproteins SV40 large T antigen and HPV16 E7, which both bind Rb and inactivate its function, rescued the cell cycle arrest, whereas HPV16 E6 did not. Mutation of the Rb-binding motif in E7 abrogated its ability to rescue the cell cycle arrest. Expression of exogenous cyclin D1, SV40 large T antigen or CCG1/TAF II 250 increased cyclin A expression at 39.58C. Coexpression of HPV16 E7 and adenovirus E1b19K, which blocks apoptosis, rescued the proliferation of tsBN462 cells at 38.58C. To investigate the mechanism underlying the lack of cyclin D1 expression, deletion analysis of cyclin D1 promoter was performed. The 0.15 kbp cyclin D1 core promoter region, which lacks any transcription factor binding motifs, still exhibited a temperature-sensitive phenotype in tsBN462 cells suggesting that CCG1/TAF II 250 is critical for the function of the cyclin D1 core promoter.

show abstract

Section: Resultsmentioning

confidence: 99%

“…The arrow indicates the position of the EES (early-early start sites) transcription start sites. b-galactosidase activities were measured as described previously (Sekiguchi and Hunter, 1998). This experiment was normalized for transfection e ciency with a cotransfected HSV tk luciferase reporter.…”

Section: Cell Lines and Cell Culture Conditionsmentioning

confidence: 99%

Overexpression of D-type cyclins, E2F-1, SV40 large T antigen and HPV16 E7 rescue cell cycle arrest of tsBN462 cells caused by the CCG1/TAFII250 mutation

Sekiguchi

Nishimoto

Hunter³

1999

Oncogene

Self Cite

View full text Add to dashboard Cite

show abstract

“…After trypsinization, a cell count was obtained using a Coulter Counter ZM and trypan blue exclusion assays were performed. Approximate IC 25 ( concentration that inhibits 25% of viable cell growth compared to controls ) values were calculated based on total viable cells and IC 25 values were used for the remainder of the experiments.…”

Section: Dose -Response Curvesmentioning

confidence: 99%

“…19 -24 In a reciprocal manner, E2F -1 can reverse the growth inhibitory effects of p21. 25,26 Interestingly, a recent study demonstrated that E2F -1 directly transactivates p21, and subsequently, E2F -1 transcriptional activity is inhibited. 27 Collectively, the evidence suggests a feedback loop mechanism between E2F -1 and p21 with apparently opposing activities that involve both direct and indirect interactions.…”

mentioning

confidence: 99%

C-terminal deletion mutant p21WAF1/CIP1 enhances E2F-1–mediated apoptosis in colon adenocarcinoma cells

et al. 2002

View full text Add to dashboard Cite

The present study was designed to investigate the efficacy of combination gene therapy using adenoviral vectors expressing gene products shown to possess apoptotic activity: E2F -1 ( Ad -E2F -1 ) and a C -terminal deletion mutant of p21 WAF1 / cIP1 ( Ad -p21 -PCNA ), on growth inhibition and apoptosis of human colon cancer cells in vitro and in vivo. Marked E2F -1 and p21 -PCNA overexpression in response to adenovirus infection was evident by Western blot analysis. IC 25 concentrations of each virus were used for each treatment in vitro to detect cooperative effects on cell death. Coexpression of E2F -1 and p21 -PCNA resulted in an additive effect on cell death compared to infection with either virus alone. Cell cycle analysis, poly( ADP -ribose ) polymerase ( PARP ) cleavage and analysis of cell morphology also revealed that coinfection with both Ad -E2F -1 and Ad -p21 -PCNA enhanced cellular apoptosis compared to either virus alone. Interestingly, E2F -1 protein expression was markedly enhanced in the E2F -1 / p21 -PCNA adenovirus combination compared to Ad -E2F -1 infection alone. However, these same effects were not evident in cells coinfected with Ad -E2F -1 and an adenovirus expressing wild -type human p21 WAF1 / CIP1 ( Ad -p21 WT ). The increase in E2F -1 expression with coexpression of E2F -1 and p21 -PCNA was not a result of increased E2F -1 protein stability, but was related to increased transcriptional activity from the CMV promoter. Cell cycle analysis revealed G1 arrest 72 hours following single -gene therapy with either the wild -type or mutant p21, whereas increased accumulation of cells in G2 / M phase was demonstrated in the E2F -1 -overexpressing cells. In the combined therapies, E2F -1 / p21 -PCNA treatment still resulted in G1 arrest, but E2F -1 was able to counteract the G1 arrest when coinfected with p21 WT . These results provide further evidence of the importance of the p21:PCNA -binding domain in mediating the complex cell cycle interaction between E2F -1 and p21. Simultaneous intratumoral injection of Ad -E2F -1 and Ad -p21 -PCNA dramatically reduced tumor burden of SW620 xenografts compared to either treatment alone in our in vivo model but not in HT -29 colon cancer xenografts. When combined with Ad -p21 -PCNA , E2F -1 adenovirus therapy resulted in approximately 95% decrease in tumor volume of SW620 tumor xenografts compared with controls ( P < .05 ). In conclusion, although simultaneous delivery of E2F -1 and p21 -PCNA transgenes results in increased E2F -1 expression and enhanced apoptosis of both SW620 and HT -29 colon cancer cells in vitro, this combination was only effective in the treatment of SW620 metastatic colon cancer in vivo. This may represent a potentially useful combination gene therapy strategy for metastatic colon cancer.

show abstract

“…Primary cells can survive in culture for long periods of time without proliferation, a property that is used in applications such as the co-cultivation of growth arrested primary fibroblast feeder cells with stem cells. This has motivated us to investigate endogenous growthcontrol mechanism rather than using sub-optimal cell culture conditions or overexpression of inhibitory molecules that may impair cell viability (Sekiguchi and Hunter 1998;Schroeder et al 2002). It has been demonstrated that the doxycycline induction system is non-toxic and is compatible with self-regulatory growth-control during fermentation (Mazur et al 1999).…”

Section: Discussionmentioning

confidence: 99%

Application of a Reversible Immortalization System for the Generation of Proliferation-Controlled Cell Lines

et al. 2004

View full text Add to dashboard Cite

To employ physiological mechanisms to control cell growth primary cells were reversibly immortalized using the SV40 TAg. The cells showed a fibroblast-like morphology. When the expression of the TAg was turned off, the cells arrested in the G0/G1 cell cycle phase. The cell culture could be kept for over 1 week in the proliferation-controlled state while the growth arrest remained fully reversible. The regulation was highly efficacious in that the arrested cell population did not spontaneously resume growth, suggesting that in the absence of the immortalizing gene expression endogenous growth-control mechanisms can keep these cells in a viable state for a prolonged time. Recombinant protein expression increased in growth-controlled cells when compared to conventionally cultured cells. Analysis of a secreted pharmaceutical protein revealed high product integrity without any signs of degradation. Therefore, it is feasible to apply genetic regulation of cell immortalization to obtain proliferation-controlled cell lines and this technique may be of interest to generate novel biotechnological producer cells.Abbreviations: DMEM -Dulbecco's modified Eagle's medium; Dox -doxycycline; eGFP -enhanced green fluorescent protein; EPO -recombinant human erythropoietin; FCS -fetal calf serum; IRES -internal ribosomal binding site; LTR -long terminal repeat; MEF -murine embryonic fibroblast cell; neoNeomycin phosphotransferase; TAg -SV40 virus large T-antigen; w.t. -wild type

show abstract

Induction of growth arrest and cell death by overexpression of the cyclin-Cdk inhibitor p21 in hamster BHK21 cells

Cited by 49 publications

References 43 publications

Overexpression of D-type cyclins, E2F-1, SV40 large T antigen and HPV16 E7 rescue cell cycle arrest of tsBN462 cells caused by the CCG1/TAFII250 mutation

Overexpression of D-type cyclins, E2F-1, SV40 large T antigen and HPV16 E7 rescue cell cycle arrest of tsBN462 cells caused by the CCG1/TAFII250 mutation

C-terminal deletion mutant p21WAF1/CIP1 enhances E2F-1–mediated apoptosis in colon adenocarcinoma cells

Application of a Reversible Immortalization System for the Generation of Proliferation-Controlled Cell Lines

Contact Info

Product

Resources

About