Adenovirus-mediated gene transfer of P16INK4/CDKN2 into bax-negative colon cancer cells induces apoptosis and tumor regression in vivo

Abstract

The tumor -suppressor gene p16INK4 / CDKN2 ( p16 ) is a cyclin -dependent kinase ( cdk ) inhibitor and important cell cycle regulator. Here, we show that adenovirus -mediated gene transfer of p16 ( AdCMV.p16 ) into colon cancer cells induces uncoupling of S phase and mitosis and subsequently apoptosis. Flow cytometric analysis revealed that cells infected with AdCMV.p16 showed an initial G2 -like arrest followed by S phase without intervening mitosis ( DNA >4N ). Using microscopic analysis, deformed polyploid cells were detectable only in cells infected with AdCMV.p16 but not in control -infected cells. Subsequently, AdCMV.p16 -infected polyploid cells underwent apoptosis, as assessed by AnnexinV staining and DNA fragmentation, suggesting that cell cycle dysregulation is upstream of the onset of apoptosis. Treatment of mice with subcutaneously transplanted tumors of colorectal cancer cells with AdCMV.p16 but not AdCMV.p53 resulted in significantly reduced tumor volume and prolonged survival. Using an orthotopic model of liver metastasis, we observed both reduced local tumor growth and secondary intrahepatic metastasis after AdCMV.p16 treatment. Importantly, induction of apoptosis in vitro and reduction of tumor growth in vivo by p16 was p53 -as well as bax -independent because identical results were obtained using cancer cells, either wild type or mutant for p53 or bax. The studies suggest that an AdCMV.p16 -based treatment may be especially effective in patients with bax -negative colon cancer where overexpression of p53 appears not to be of therapeutic value.

The fate of pancreatic tumor cell lines following p16 overexpression depends on the modulation of CDK2 activity

“…17,18 Interestingly, ectopic expression of p16 also induced apoptosis in p53-deficient cell lines, implicating a p53-independent apoptotic pathway. 20 Taken together, these results indicate that the effects of p16 on tumor cells depend on the genetic alterations acquired during transformation and tumor progression. Given that apoptosis induction might yield higher therapeutic activity than induction of growth arrest, it is important to understand the molecular features that determine different cell fates after p16 reintroduction.…”

“…Although p16 does not induce cell cycle arrest, it has strong antiproliferative effects as a consequence of apoptosis induction in NP-18 cells. Other groups using similar approaches in different cells have also detected p16-induced cell death ( [17][18][19][20][21] , reviewed in Calbó and Mazo 16 ), but the molecular mechanisms remain unclear. It has been proposed that p16 expression results in p53-dependent apoptosis, 17,18 but lack of wt-p53 gene in NP-18 cells makes this unlikely.…”

“…However, p16-induced apoptosis has also been observed in a number of studies. p16-induced apoptosis resulted from the activation of p53-dependent apoptotic pathways ( [17][18][19][20][21] , reviewed in Calbó and Mazo 16 ), including bcl-2 downregulation and activation of caspases in non-small-cell lung cancer cells. 17,18 Interestingly, ectopic expression of p16 also induced apoptosis in p53-deficient cell lines, implicating a p53-independent apoptotic pathway.…”

p16Ink4a inhibits histologic progression and angiogenic signaling in min colon tumors

“…This result was somewhat surprising because p16 expression has been primarily associated with cell cycle arrest, rather than death. However, exogenous p16 expression has been found to cause cell death in some settings, 28,29 including colon carcinoma cells, 30 and the senescent state imposed by p16 can lead to death (see "Discussion"). 31 The overall proliferation index in p16-null tumors, as assessed by Ki67 staining, 32,33 was not higher than in p16-wt tumors.…”

Telomeres, stem cells, senescence, and cancer

“…While the molecular biology of this fate-decision is incompletely understood, the specific response appears to depend on many variables including cell type, genetic context, and proliferation state. Although a role for p16 INK4a in inducing apoptosis has been suggested (39)(40)(41)(42), the issue of whether p16 INK4a also has senescence-independent anticancer functions in vivo remains an area of active investigation.…”