Suppression of apoptosis and clonogenic survival in irradiated human lymphoblasts with different TP53 status.
The influence of radiation-induced apoptosis on radiosensitivity was studied in a set of closely related human lymphoblastoid cell lines differing in TP53 status. The clonogenic survival of irradiated TK6 cells (expressing wild-type TP53), WTK1 cells (overexpressing mutant TP53), and TK6E6 cells (negative for TP53 owing to transfection with HPV16 E6) was assessed in relation to the induction of apoptosis and its suppression by caspase inhibition or treatment with PMA as well as after treatment with caffeine. Measurements using the alkaline comet assay and pulsed-field electrophoresis of the induction and repair of DNA strand breaks showed similar kinetics of the processing of early DNA damage in these cell lines. The cytochalasin B micronucleus assay revealed identical levels of residual damage in the first postirradiation mitosis of these cells. Abrogation of TP53-dependent apoptosis in TK6E6 cells resulted in a distinct increase in radioresistance. Further suppression of apoptosis as observed in WTK1 cells overexpressing mutant TP53 apparently was not responsible for the high radioresistance of WTK1 cells, since other means of highly efficient suppression of apoptosis (caspase inhibition or PMA treatment) increased the clonogenic survival of irradiated TK6 cells only to levels similar to those of TK6E6 cells with abrogated TP53-dependent apoptosis. Considering the similar levels of residual chromosomal damage in TK6E6 cells and WTK1 cells, a hitherto unknown mechanism of tolerance needs to be inferred for these TP53 mutant cells. This residual damage tolerance, however, appears to require an intact G2/M-phase checkpoint function since the relative radioresistance of the WTK1 cells was completely lost upon caffeine treatment, which also resulted in a failure of the TK6 and TK6E6 cells to execute apoptosis. In this situation, the cellular response seems to be dominated entirely by TP53-independent mitotic failure.
Schäfer
Jörg
J
Radiobiology Section, Department of Radiotherapy, University of Heidelberg, INF 400, 69120 Heidelberg, Germany.
Bachtler
Jens
J
Engling
Andre
A
Little
John B
JB
Weber
Klaus-Josef
KJ
Wenz
Frederik
F
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D002110:Caffeine / Q000494:pharmacology; D002273:Carcinogens; D002470:Cell Survival / Q000528:radiation effects; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D020552:Comet Assay; D003571:Cytochalasin B / Q000494:pharmacology; D004249:DNA Damage; D004307:Dose-Response Relationship, Radiation; D016158:Genes, p53 / Q000235:genetics*; D006801:Humans; D008214:Lymphocytes / Q000528:radiation effects*; D015162:Micronucleus Tests; D013755:Tetradecanoylphorbol Acetate; D013997:Time Factors
D000590:Amino Acid Chloromethyl Ketones; D002273:Carcinogens; D000697:Central Nervous System Stimulants; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D003571:Cytochalasin B; D002110:Caffeine; D013755:Tetradecanoylphorbol Acetate