313-323DNA ligand Hoechst-33342 significantly
enhances UV induced cytotoxicity in human glioma cell lines (BMG-1 & U-87) with
supra additive increase in cell death, cytogenetic damage, cell cycle delay,
apoptosis and inhibition of PLDR. Cytotoxicity of Hoechst-33342 arises due to
its interference in the breakage-rejoining reaction of DNA topoisomerases by stabilization
of cleavable complexes. Since topoisomerases have also been implicated in the
generation of potentially lethal DNA breaks by interaction with various types
of DNA damage including UV induced DNA lesions, we investigated in present
studies the role of functional topoisomerases in the synergistic cytotoxicity
of Hoechst-33342 and UV in a human glioma cell line (BMG-1). Topoisomerase I activity
analyzed by the plasmid relaxation assay, was significantly enhanced upon UV
irradiation, implying a possible role of this enzyme in the processing of UV
induced lesions. However, this increase in the activity was reduced by >50%
in cells incubated with Hoechst-33342 for 1 hr prior to irradiation. Imunoflowcytometric
analysis of the chromatin bound topoisomerases I and II levels (cleavable complex)
using topoisomerases I and II anti-antibodies showed a good correlation between
the induction of apoptosis by Hoechst-33342 and UV and enhancement in the level
of topoisomerase II mediated cleavable complexes. Induction of apoptosis was associated
with a decline in the level of Bcl2. Taken together; these studies show
that supra additive cytotoxic effects of UV-C and Hoechst-33342 in BMG-1 cells
are consequences of enhanced stabilization of topo II mediated cleavable complexes
and alterations in specific signal transduction pathways of apoptosis, besides the
inhibition of topoisomerase mediated repair processes