Purpose: The human genetic disorder ataxia-telangiectasia (AT) is a multisy
stem disease characterized by extreme radiosensitivity. Although ionizing r
adiation was known to induce c-fos transcription and cellular protein kinas
e C (PKC) induces the expression of this immediate response gene, little is
known about how mutated AT (ATM) or PKC-mediated signal transduction pathw
ay modulates the c-fos gene transcription and gene expression, Here we have
studied the effect of PKC inhibitor (PKCI) on radiation sensitivity and c-
fos transcription in normal and AT cells, and also studied whether PKCI eff
ect on c-fos occurs in Ras-dependent pathway.
Methods and Materials: Normal (LM217) and AT (AT5BIVA) cells were transfect
ed with PKCI expression plasmid and integration and overexpression of PKCI
was evaluated by polymerase chain reaction and northern blotting, respectiv
ely. Cells were irradiated at a dose of 5 Gy/min with Cs-137 irradiator and
harvested 48 h after irradiation and investigated apoptosis with TUNEL met
hod. The c-fos transcription activity was studied by performing compute ass
isted tomography (CAT) assay of reporter gene after transfection of c-fos C
AT plasmid into LM and AT cells. Overexpression of Ras protein in transfect
ed cells was shown by western blotting.
Results: Our results demonstrated for the first time a role of PKCI on the
radiation sensitivity and c-fos transcription in LM and AT cells. PKCI incr
eased radiation induced apoptosis in LM cells (5% to 20%) but reduced apopt
osis slightly in AT cells. The basal c-fos transcription activity is 70 tim
es lower in AT cells than in LM cells. This c-fos transcription activity wa
s repressed by overexpression of PKCI in LM cells but not in AT cells. Afte
r induction of c-fos by Ras protein, overexpression of PKCI repressed c-fos
transcription in LM cells but not in AT cells.
Conclusions: Overexpression of PKCI increased radiation sensitivity and rep
ressed c-fos transcription in LR I cells but not in AT cells, and this is r
elated with Ras, These results suggest that the effect of PKCI on c-fos tra
nscription activity is related with Ras dependent signal transduction pathw
ays and these mechanisms are different between normal fibroblasts, LM and A
TM mutated, AT cells, The data obtained by this study provided evidence for
novel transcriptional difference between LM and AT cells and this may be a
reason for increased radiation sensitivity of AT cells. (C) 2001 Elsevier
Science Inc.