Here, we investigated the influence of Rac family
small GTPases on mechanisms of the DNA damage response (DDR) stimulated by
topoisomerase II poisons. To this end, we examined the influence of the Rac-specific small molecule inhibitor
EHT1864 on Ser139 phosphorylation of
histone H2AX, a widely used marker of the DDR triggered by
DNA double-strand breaks.
EHT1864 attenuated the
doxorubicin-stimulated DDR in a subset of cell lines tested, including HepG2
hepatoma cells.
EHT1864 reduced the level of
DNA strand breaks and increased viability following treatment of HepG2 cells with
topo II
poisons. Protection by
EHT1864 was observed in both p53 wildtype (HepG2) and p53 deficient (Hep3B) human
hepatoma cells and, furthermore, remained unaffected upon pharmacological inhibition of p53 in HepG2. Apparently, the impact of Rac on the DDR is independent of p53. Protection from
doxorubicin-induced DNA damage by
EHT1864 comprises both S and G2 phase cells. The inhibitory effect of
EHT1864 on
doxorubicin-stimulated DDR was mimicked by pharmacological inhibition of various
protein kinases, including JNK, ERK, PI3K, PAK and CK1.
EHT1864 and
protein kinase inhibitors also attenuated the formation of the
topo II-
DNA cleavable complex. Moreover,
EHT1864 mitigated the constitutive phosphorylation of topoisomerase IIα at positions S1106, S1213 and S1247.
Doxorubicin transport, nuclear import/export of
topoisomerase II and Hsp90-related mechanisms are likely not of relevance for
doxorubicin-stimulated DDR impaired by
EHT1864. We suggest that multiple
kinase-dependent but p53- and
heat shock protein-independent Rac-regulated nuclear mechanisms are required for activation of the DDR following treatment with
topo II
poisons.