Dissociation between cell cycle arrest and apoptosis can occur in Li-Fraumeni cells heterozygous for p53 gene mutations

Abstract

The radiation response was investigated in two lymphoblastoid cell lines (LBC) derived from families with heterozygous germ-line missense mutations of p53 at codon 282 (LBC282) and 286 (LBC286), and compared to cells with wt/wt p53(LBC-N). By gel retardation assays, we show that p53-containing nuclear extracts from irradiated LBC282 and LBC286 markedly differ in their ability to bind to a p53 DNA consensus sequence, the former generating a shifted band whose intensity is 30 – 40% that of LBC-N, the latter generating an almost undetectable band. Unlike LBC286, which fail to arrest in G₁ after irradiation, LBC282 have an apparently normal G₁/S checkpoint, as they arrest in G₁, like LBC-N. While in LBC-N, accumulation of p53 and transactivation of p21^WAF1 increase rapidly and markedly by 3 h after exposure to γ-radiation, in LBC286 there is only a modest accumulation of p53 and a significantly delayed and quantitatively reduced transactivation of p21^WAF1. Instead, in LBC282 while p53 levels rise little after irradiation, p21^WAF1 levels increase rapidly and significantly as in normal LBC. Apoptotic cells present 48 h after irradiation account for 32% in LBC-N, 8 – 9% in LBC282 and 5 – 7% in LBC286, while the dose of γ-radiation required for killing 50% of cells (LD₅₀) is 400 rads, 1190 rads and 3190 rads, respectively, hence indicating that the heterozygous mutations of p53 at codon 282 affects radioresistance and survival, but not the G₁/S cell cycle control. In all LBC tested, radiation-induced apoptosis occurs in all phases of the cell cycle and appears not to directly involve changes in the levels of the apoptosis-associated proteins bcl-2, bax and mcl-1. Both basal as well as radiation-induced p53 and p21^WAF1 proteins are detected by Western blotting of FACS-purified G₁, S and G₂/M fractions from the three cell lines. p34^CDC2-Tyr15, the inactive form of p34^CDC2 kinase phosphorylated on Tyr15, is found in S and G₂/M fractions, but not in G₁. However, 24 h after irradiation, its levels in these fractions diminish appreciably in LBC-N but not in the radioresistant LBC286 and LBC282. Concomitantly, p34^CDC2 histone H1 kinase activity increases in the former, but not in the latter cell lines, hence suggesting a role for this protein in radiation-induced cell death. Altogether, this study shows that, in cells harbouring heterozygous mutations of p53, the G₁ checkpoint is not necessarily disrupted, and this may be related to the endogenous p53 heterocomplexes having lost or not the capacity to bind DNA (and therefore transactivate target genes). Radiation-induced cell death is not cell cycle phase specific, does not involve the regulation of bcl-2, bax or mcl-1, but is associated with changes in the phosphorylation state and activation of p34^CDC2 kinase.