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

Abstract

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.

Inhibition of Müller glial cell division blocks regeneration of the light‐damaged zebrafish retina

“…5(H,I)], indicating that at least a subset of Müller glia underwent cell death following inhibition of proliferation. This is consistent with cell culture data demonstrating PCNA knockdown resulted in cell cycle arrest and subsequent apoptosis (Takase et al, 1992;Elliott et al, 2002;Choi et al, 2005;Gehen et al, 2007;Yu et al, 2006).…”

“…First, the selective in vivo targeting of Müller glia to undergo apoptosis following PCNA knockdown is a novel finding. Cell culture studies have shown the potential for PCNA knockdown to lead to G1 arrest and cell death (Takase et al, 1992;Elliott et al, 2002;Choi et al, 2005;Gehen et al, 2007;Yu et al, 2006). Here we show an in vivo example of this proliferation arrest leading to cell death with the Müller glia.…”

Genetic transfer of PNAS‐4 induces apoptosis and enhances sensitivity to gemcitabine in lung cancer

“…The p53‐independent induction of p21 expression is associated with the induction of terminal growth arrest and differentiation (Dolnikov et al., 2000). E2F‐1, another target of p53, has an anti‐tumor efficacy in a variety of tumors by apoptosis through p53‐dependent and ‐independent mechanisms (Elliott et al., 2002). Therefore, the above two should be investigated further to elucidate the underlying mechanisms for combination therapy.…”

Transcriptional Regulation during p21/-induced Apoptosis in Human Ovarian Cancer Cells

“…WAF1⁄CIP1 enhances E2F-1-mediated apoptosis, independent of p53, in human colon cancer cells in vitro and in vivo (46,47). Nevertheless, other transcription factors are likely to be involved in transcription regulation as well, especially some apoptosis-associated transcription factors (Table V).…”