Dissociation between cell cycle arrest and apoptosis can occur in Li-Fraumeni cells heterozygous for p53 gene mutations.
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 p21WAF1 increase rapidly and markedly by 3 h after exposure to gamma-radiation, in LBC286 there is only a modest accumulation of p53 and a significantly delayed and quantitatively reduced transactivation of p21WAF1. Instead, in LBC282 while p53 levels rise little after irradiation, p21WAF1 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 gamma-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 p21WAF1 proteins are detected by Western blotting of FACS-purified G1, S and G2/M fractions from the three cell lines. p34CDC2-Tyr15, the inactive form of p34CDC2 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, p34CDC2 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 p34CDC2 kinase.
Delia
D
D
Istituto Nazionale Tumori, Divisione OSA, Milano, Italy.
Goi
K
K
Mizutani
S
S
Yamada
T
T
Aiello
A
A
Fontanella
E
E
Lamorte
G
G
Iwata
S
S
Ishioka
C
C
Krajewski
S
S
Reed
J C
JC
Pierotti
M A
MA
D017209:Apoptosis / Q000235:genetics*; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D002453:Cell Cycle / Q000235:genetics* / Q000528:radiation effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage / Q000235:genetics / Q000528:radiation effects; D016193:G1 Phase / Q000235:genetics; D016195:G2 Phase / Q000235:genetics / Q000528:radiation effects; D005720:Gamma Rays; D015972:Gene Expression Regulation, Neoplastic; D006579:Heterozygote; D006801:Humans; D016864:Li-Fraumeni Syndrome / Q000235:genetics* / Q000378:metabolism; D008214:Lymphocytes / Q000473:pathology / Q000528:radiation effects; D008938:Mitosis / Q000235:genetics / Q000528:radiation effects; D009154:Mutation; D010766:Phosphorylation; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism; D015854:Up-Regulation
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D016159:Tumor Suppressor Protein p53; D016203:CDC2 Protein Kinase