Cells lacking CIP1/WAF1 genes exhibit preferential sensitivity to cisplatin and nitrogen mustard

Abstract

We have previously shown that p53 disruption sensitizes certain cancer cell types to cisplatin (CDDP) (Fan et al., 1995). In the present study we investigated the role of the p53 downstream eector, p21(p21), in this sensitization. Studies were performed in human colon cancer HCT-116 cells and murine embryonic ®broblasts (MEF) with intact versus disrupted p21 genes. For comparison, HCT-116 cells lacking p53 function were also prepared through stable transfection with the human papillomavirus type-16 E6 gene. HCT-116/E6 cells were found to be more sensitive than control transfectants to CDDP and another DNA crosslinking agent, nitrogen mustard (HN2). HCT-116 cells with disrupted p21 genes also exhibited greater CDDP and HN2-sensitivity than parental HCT-116 cells. In contrast, the clonogenic survival of HCT-116 cells exposed to ionizing radiation, adriamycin, taxol or vincristine was not aected by p53 or p21 disruption. Sensitization of HCT-116/p21 7/7 cells to CDDP and HN2 was not limited to the HCT-116 cell background since MEF from p21 knockout mice were also more sensitive to these DNA crosslinking agents. Investigations into a possible cause of this enhanced sensitivity revealed that HCT-116 cells lacking p53 or p21 function exhibited a reduced ability to repair cisplatin-damaged CAT-reporter plasmids transfected into the cells. In addition, we found that HCT-116/ p21 7/7 cells were much more susceptible to HN2-induced cell cycle delay than parental cells. Our results suggest that p21 disruption preferentially sensitizes at least some cell types to DNA crosslinking agents.

Mutated p21WAF1/CIP1/SDI1 lacking CDK-inhibitory activity fails to prevent apoptosis in human colorectal carcinoma cells

“…DLD-1 cells were cultured with (300 mM) or without mimosine after 3 h adriamycin treatment (2 mg/ml) (c), or cultured after X-ray irradiation (14 Gy) (d) and mimosine or butyrolactone I induces endogenous p21 in DLD-1 cells (data not shown). Our results suggest that inhibition of CDK activity by p21 is required for G1-phase arrest and the G1-phase arrest is required for prevention of ADR-or X-ray-induced apoptosis probably because DNA repair would be carried out during the G1-phase (MacDonald et al, 1996;Fan et al, 1997). p21 is thought to be a potential tumor suppressor gene because the other CDK-inhibiting genes, p15 and p16, often are mutated in human tumors; whereas, only a few p21 mutations have been reported (Bhatia et al, 1995;Balbin et al, 1996;Li et al, 1995).…”

Ultraviolet-irradiation-induced apoptosis is mediated via ligand independent activation of tumor necrosis factor receptor 1

“…UV, cisplatin and melphalan-induced DNA damage is repaired via the nucleotide excision repair pathway (Friedberg et al, 1995;Fan et al, 1995). Cisplatin (Kondo et al, 1995;Sanchez-Perez et al, 1998) and melphalan (this study) can also induce apoptosis and although the underlying molecular mechanisms remain elusive, excessive DNA damage has been implicated as a trigger (Kondo et al, 1995;Sanchez-Perez et al, 1998).…”

“…We next sought to investigate this issue. We reasoned that if UV activation of TNFR1 occurs in response to a signal that originates as a consequence of damaged nuclear DNA then ionizing radiation (IR) inducing double strand breaks and/or the UV-mimetic agents inducing qualitatively UV-type DNA damage (Fan et al, 1995) should also activate the apoptotic axis of TNFR1 and FADD-DN should be able to block those e ects.…”

Scatter factor protects epithelial and carcinoma cells against apoptosis induced by DNA-damaging agents

“…Western immunoblotting was performed as described previously (Fan et al, 1995). Brie¯y, cells were centrifuged, washed with PBS, and lysed at 08C for 30 min in 100 ± 200 ml/100 mm dish of lysis bu er (1% NP-40, in PBS containing 2 mM 4-(2-aminoethylbenzensulfonyl¯uoride, 10 mg/ml leupeptin, 10 mg/ml aptotinin, 10 mM NaF, 1 mM Na orthovandate and 5 mM Na pyrophosphate).…”