SHORT REPORT Eect of p21 waf1/cip1 transgene on radiation induced apoptosis in T cells Rati Fotedar* 1 , Howard Brickner 2,3 , Neshat Saadatmandi 3 , Tristan Rousselle 1 , Ludger Diederich 1 , Anil Munshi 2 , Barbara Jung 2 , John C Reed 4 and Arun Fotedar* ,2,3 1 Institut de Biologie Structurale JP Ebel, 41 avenue des Martyrs, 38027 Grenoble Cedex 1, France; 2 Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, California 92121, USA; 3 Division of Molecular Biology, La Jolla Institute for Allergy and Immunology, 11149 North Torrey Pines Road, La Jolla, California 92037, USA; 4 Burnham Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, USA The cyclin kinase inhibitor p21 WAF1/Cip1 is upregulated by the tumor suppressor p53. While p21 is central for the G-1 arrest mediated by p53, it is still unclear if p21 also functions as a downstream eector of p53 dependent apoptosis. Apoptosis induced by DNA damage but not dexamethasone is p53 dependent in thymocytes. To investigate the physiological role of p21 in apoptosis, we have generated transgenic mice in which the p21 transgene is targeted for restricted expression in the T cell lineage. Thymocytes from p21 transgenic mice were hypersensitive to cell death induced by DNA damaging agents such as ionizing radiation and UV, but not be dexamethasone. Irradiated p21 transgenic thymocytes had approximately twofold more apoptotic cells as compared to irradiated age matched littermate control mice. Radiation induced death is comparable in thymocytes from p21+Bcl2+double transgenic mice and age matched littermate controls, indicating that the Bcl2 transgene rescues the radiation hypersensitivity imposed by p21. However, thymocytes from p537/7 mice even when they expressed the p21 transgene, were resistant to death induced by radiation. Together these results show that thymocytes from p21 transgenic mice are hypersensitive to radiation induced programmed cell death and suggest that the radiation hypersensitivity of p21 transgenic thymocytes involves p53 dependent path- way and signals in addition to p21. Keywords: p21 waf1 ; transgenic mice; radiation; apopto- sis; p53 In mammalian cells, progression through the cell cycle is regulated by the cyclin dependent kinase (CDK) family of protein kinases. CDK-cyclin kinase activity during the cell cycle is regulated by phosphorylation/ de-phosphorylation of the CDK subunit, regulated destruction of the cyclin subunit, and by proteins which bind and inhibit CDK-cyclin kinases. In mammalian cells two distinct families of cyclin-CDK kinase inhibitors, p21 and p16, have been described. The p21 family includes the structurally related proteins, p21 WAF-1/Cip1 (el-Deiry et al., 1993; Harper et al., 1993; Xiong et al., 1993), p27 Kip1 (Polyak et al., 1994; Toyoshima and Hunter, 1994), and p57 Kip2 (Lee et al., 1995; Matsuoka et al., 1995) all of which inhibit a variety of CDK-cyclin kinases by binding to both cyclin and CDK subunits of the activated cyclin-CDK complex (Fotedar et al., 1996). Members of the p16 family, which includes p16 (Hannon and Beach, 1994) p14/p15, p18 and p19 preferentially inhibit CDK4- cyclin D and CDK6-cyclin D kinases (reviewed in Sherr and Roberts, 1995). The N-terminal residues of p21, p27 and p57 share signi®cant homology (Lee et al., 1995; Matsuoka et al., 1995), however, they dier substantially in their C terminal regions. The C- terminal region of p21 inhibits DNA elongation in vitro by binding PCNA (Chen et al., 1996; Luo et al., 1995). p21 expression is implicated in cell cycle arrest (Brugarolas et al., 1995; Deng et al., 1995), in withdrawal of cells from the cell cycle during dierentiation (Halevy et al., 1995) and in cell senescence (Brown et al., 1997). Targeted expression of p21 in hepatocytes inhibits cell proliferation, interferes with postnatal liver development and prevents liver regeneration (Wu et al., 1996). Cells with a null mutation in the p21 gene have provided direct evidence of a role for p21 in cell cycle arrest following DNA damage. Fibroblasts from p21 null mice are partially de®cient in DNA damage induced cell cycle arrest (Brugarolas et al., 1995; Deng et al., 1995) and the lack of both p21 alleles in a cancer cell line completely overcomes the G-1 arrest (Wald- man et al., 1996). p21 expression is upregulated by the p53 tumor suppressor protein. Transcriptional activa- tion of speci®c target genes is an important part of p53 function. Of the known transcriptional targets of p53, p21 which is induced upon DNA damage in a p53 dependent manner (Dulic et al., 1994), is most likely to control cell cycle arrest (Brugarolas et al., 1995; Deng et al., 1995). In addition, p53 is essential for apoptosis triggered by agents which induce DNA damage in thymocytes (Clarke et al., 1993; Lowe et al., 1993). In contrast to the requirement of p53 for radiation induced apoptosis, lack of p21 has been reported to have no eect on p53 dependent apoptosis of thymocytes in p21 null mice (Brugarolas et al., 1995; Deng et al., 1995). In order to determine the role of p21 in apoptosis of thymocytes, we generated transgenic vectors which express p21 in a tissue restricted manner in the T cell lineage. The human p21 cDNA (Fotedar et al., 1996) was cloned into the p1017 vector (Garvin et al., 1990) where the expression of the p21 transgene is under the control of the proximal Lck promoter (Figure 1a). The proximal Lck promoter has previously been used to *Correspondence: A Fotedar and R Fotedar Received 3 September 1998; revised 14 January 1999; accepted 14 January 1999 Oncogene (1999) 18, 3652 ± 3658 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc