Cyclin-dependent Kinase Inhibitors Uncouple Cell Cycle Progression from Mitochondrial Apoptotic Functions in DNA-damaged Cancer Cells

Andy J Minn,Joan Massagué,Hong-Van Le

doi:10.1074/jbc.m504689200

Abstract

DNA damage results in transcriptional induction of p53 target genes, including the cyclin-dependent kinase (CDK) inhibitor p21(Cip1) (CDKN1A) and the proapoptotic Bcl-2 family member p53 up-regulated modulator of apoptosis (PUMA). Depending on the cellular context, p21(Cip1) and PUMA mediate cell cycle arrest and apoptosis, respectively. By imposing cell cycle arrest at the expense of apoptosis, p21(Cip1) can sharply reduce the effectiveness of DNA-damaging anticancer agents in colorectal cancer cells. We investigated the link between cell cycle progression and the onset of apoptosis in DNA-damaged cells by analyzing the activation of the apoptotic cascade in p21(Cip1)-deficient HCT116 colorectal cancer cells. DNA damage induced a similar level of p53 activation and PUMA induction in p21(Cip1)-deficient cells compared with wild-type isogenic counterparts. p21(Cip1) did not act as a direct blocker of PUMA. However, only p21(Cip1)-deficient cells showed extensive cytochrome c release, mitochondrial membrane depolarization, and caspase activation. An increase in caspase activation occurred as these cells reached M-phase and incurred polyploidy. When ectopically expressed in p21(Cip1)-deficient HCT116 cells, p21(Cip1), its family member p27(Kip1), and the structurally unrelated CDK inhibitor p16(Ink4a) were similarly effective at causing cell cycle arrest and inhibiting DNA damage-induced apoptotic events such as cytochrome c release, mitochondrial membrane depolarization, and activation of the caspase cascade. These observations suggest that by blocking dysregulated cell cycle progression, CDK inhibitors can influence the sensitivity of the mitochondria to proapoptotic signals in DNA damage-induced cancer cells.

Highlights

Many tumor-specific treatments are based on inducing DNA damage by radiation or chemotherapeutic agents
DNA damage induced a similar level of p53 activation and p53 up-regulated modulator of apoptosis (PUMA) induction in p21Cip1-deficient cells compared with wild-type isogenic counterparts. p21Cip1 did not act as a direct blocker of PUMA
HCT116 cells that are rendered deficient in p21Cip1 by deletion of the CDKN1A locus become hypersensitive to apoptosis induced by DNA damage [16]

Summary

MATERIALS AND METHODS

Cell Culture—DLD1 cells conditionally expressing PUMA and HCT116 wild-type and p21Ϫ/Ϫ cells were a kind gift from Drs Vogelstein and Kinzler. The membrane was probed with antibodies specific for PUMA (a kind gift from Drs Vogelstein, Kinzler, and Yu), p53 (sc-98; Santa Cruz), Bcl-xL (BD Biosciences), or p21Cip (ab-11; Neomarkers), developed with the ECL system (Amersham Biosciences), and stripped with RestoreTM Western blot stripping buffer (Pierce) shaking at 37 °C for 20 min. The fixed cells were washed and resuspended in 0.3– 0.5 ml of DNA staining buffer (3.8 mM sodium citrate, 20 ␮g/ml propidium iodide, 0.125 ␮g/ml RNase A). To stain cells with FITC-VAD-fmk (CaspACETM; Promega) and propidium iodide, cells were cultured for the indicated times with 5 ␮M FITC-VAD-fmk and 70 ng/ml adriamycin. Both floating and adherent cells were pooled, washed, and fixed with BD Cytofix/CytopermTM (BD Biosciences) solutions according to the manufacturer’s specifications. Cells were washed in PBS, resuspended in FACS buffer, and Rh123 fluorescence was measured by flow cytometry

RESULTS

CDK Inhibitors Uncouple Cell Cycle Progression

Findings

DISCUSSION