Deregulation of p53/p21Cip1/Waf1 pathway contributes to polyploidy and apoptosis of E1A+cHa-ras transformed cells after γ-irradiation

Abstract

The p53/p21 Cip1/Waf1 -dependent checkpoint control of G1/S and G2/M phases of the cell cycle in response to DNA damage is an important mechanism of genome stability maintenance in normal cells. In many tumor cells, due to frequent point mutations and deletions of p53, the stringent control of the cell cycle and apoptosis is compromised. We have examined the cell cycle control and cell death of the rat embryo ®broblast cells (REF) transformed by E1A+cHa-ras oncogenes and expressing wild type p53. Gamma-irradiation at a dosage of 6 Gy has been used to analyse the p53-dependent transactivation of the target p21 cip1/waf1 gene and the levels of activity of cyclin-dependent kinases. Our results show that the cell cycle inhibitors p21 Cip1/Waf1 and p27 KIP accumulate in response to irradiation both in REF and E1A+cHa-ras cells. In contrast to normal REF cells, the accumulation of p21 Cip1/Waf1 and p27 KIP inhibitors, however, does not lead to inhibition of Cdk2 and cyclins E, A-associated kinase activities and to a G1/S block in E1A+cHa-ras cells. It is unlikely that the lack of inhibitory function of p21 Cip1/Waf1 can be explained by its inability to bind Cdk2 and Cdk4 kinases or PCNA. Moreover, the p21 Cip1/Waf1 -associated kinase activity is increased upon g-irradiation of E1A+cHa-ras cells. We suggest that inactivation of p21 Cip1/Waf1 may be accounted for by its interaction with E1A oncoproducts as the inhibitor is detected in immunoprecipitates using E1A-speci®c antibodies. During a temporary G2/M delay induced by g-irradiation, E1A+cHa-ras transformants continue DNA replication, which leads to accumulation of polyploid cells with lobulated nuclei and micronuclei. Thus, DNA damage of E1A+cHa-ras transformed cells, with a combination of functionally active wild type p53 and inactive p21 Cip1/Waf1 , contributes to formation of polyploid cells which then die due to apoptosis.

Switch from p53 to MDM2 as differentiating human keratinocytes lose their proliferative potential and increase in cellular size

“…Our observations strongly support this model when considered alongside other lines of evidence: (a) the deregulation of the p53/p21 Cip1 pathway has been found to produce a defective cell cycle and polyploidy in leukemic cells and E1A/ras transformed embryo ®broblasts (Peled et al, 1996;Bulavin et al, 1999); (b) loss of p53 allows endoreplication after mitotic spindle inhibition in a variety of systems, whereas its presence is able to suppress it (Minn et al, 1996;Di Leonardo et al, 1997;Notterman et al, 1998;Casenghi et al, 1999); (c) MDM2 expression in transgenic mammary gland produced enlarged polyploid cells (Lundgren et al, 1997;Reinke et al, 1999); (d) overexpression of p53 target p21 Cip1 has been reported to block cell cycle in G2/M and cause endoreplication and polyploidy when Rb was not functional in a variety of mouse and human cells, including a human epidermoid carcinoma (Niculescu et al, 1998). Consistently, we and others have found that p21 Cip1 is upregulated and Rb down-regulated during keratinocyte di erentiation (Figure 1c; Harvat et al, 1998;Hauser et al, 1997;unpublished results) and MDM2 has the capacity to inhibit Rb (Xiao et al, 1995).…”

Caspase independence of radio-induced cell death

“…Reportedly, radio-induced nuclear alterations that announce cell death include apoptosis but also micro-and multinucleation (Bush and McMillan, 1993;Muller et al, 1995;Mariya et al, 1997;Olive and Durand, 1997;Guo et al, 1998;Bulavin et al, 1999) (Figure 3a). To determine the relationship between caspase activation and nuclear change, we doublestained g-irradiated cells with the chromatin dye Hoechst 33342 and an antibody that recognizes the large subunit of mature, proteolytically active caspase-3 (Casp-3a).…”

“…This type of cell death occurs during or shortly after a dysregulated or failed mitosis and can be accompanied by morphological alterations such as micronuclei (MN) (which often are chromosomes or chromosome fragments that have not been distributed evenly between the daughter nuclei and are encapsulated in nuclear membranes) and multinucleation (the presence of two or more nuclei with similar or heterogeneous sizes, resulting from deficient separation during cytokinesis) (Roninson et al, 2001;Castedo et al, 2004a;Okada and Mak, 2004). Ionizing radiation can indeed induce the development of micronucleated cells (Muller et al, 1995) as well as that of giant, multinuclear cells (Olive and Durand, 1997;Bulavin et al, 1999). Although several authors have reported a good inverse correlation between the formation of MN and clonogenic survival (Muller et al, 1995;Mariya et al, 1997), others have failed to show such a correlation (Bush and McMillan, 1993;Guo et al, 1998).…”

Inactivation of the Wip1 phosphatase inhibits mammary tumorigenesis through p38 MAPK–mediated activation of the p16Ink4a-p19Arf pathway

“…CAT assays were carried out as described 11 ; an arrow marks the position of acetylated chloramphenicol. The levels of p53 in nuclear extracts 35 were determined using western immunoblotting. (b) Levels of the indicated proteins involved in cell cycle control were analyzed in wild-type (WT) and Ppm1d-null MEFs expressing E1A and Hras1.…”