Adaptive upregulation of DNA repair genes following benzo(a)pyrene diol epoxide protects against cell death at the expense of mutations.

A coordinated and faithful DNA damage response is of central importance for maintaining genomic integrity and survival. Here, we show that exposure of human cells to benzo(a)pyrene 9,10-diol-7,8-epoxide (BPDE), the active metabolite of benzo(a)pyrene (B(a)P), which represents a most important carcinogen formed during food preparation at high temperature, smoking and by incomplete combustion processes, causes a prompt and sustained upregulation of the DNA repair genes DDB2, XPC, XPF, XPG and POLH. Induction of these repair factors on RNA and protein level enhanced the removal of BPDE adducts from DNA and protected cells against subsequent BPDE exposure. However, through the induction of POLH the mutation frequency in the surviving cells was enhanced. Activation of these adaptive DNA repair genes was also observed upon B(a)P treatment of MCF7 cells and in buccal cells of human volunteers after cigarette smoking. Our data provide a rational basis for an adaptive response to polycyclic aromatic hydrocarbons, which occurs however at the expense of mutations that may drive cancer formation.

Nuclear RAC1 is a modulator of the doxorubicin-induced DNA damage response.

Rho GTPases like RAC1 are localized on the inner side of the outer cell membrane where they act as molecular switches that can trigger signal transduction pathways in response to various extracellular stimuli. Nuclear functions of RAC1 were identified that are related to mitosis, cell cycle arrest and apoptosis. Previously, we showed that RAC1 plays a role in the doxorubicin (Dox)-induced DNA damage response (DDR). In this context it is still unknown whether cytosolic RAC1 modulates the Dox-induced DDR or if a nuclear fraction of RAC1 is involved. Here, we silenced RAC1 in mouse embryonic fibroblasts (MEF) pharmacologically with EHT1864 or by using siRNA against Rac1. Additionally, we transfected MEF with RAC1 mutants (wild-type, dominant-negative, constitutively active) containing a nuclear localization sequence (NLS). Afterwards, we analysed the Dox-induced DDR by evaluation of fluorescent nuclear γH2AX and 53BP1 foci formation, as well as by detection of activated proteins of the DDR by western blot to elucidate the role of nuclear RAC1 in the DDR. Treatment with EHT1864 as well as Rac1 knock-down reduced the Dox-induced DSB-formation to a similar extent. Enhanced nuclear localization of dominant-negative as well as constitutively active RAC1 mimicked these effects. Expression of the RAC1 mutants altered the Dox-induced amount of pP53 and pKAP1 protein. The observed effects were independent of S1981 ATM phosphorylation. We conclude that RAC1 is required for a substantial activation of the Dox-induced DDR and balanced levels of active/inactive RAC1 inside the nucleus are a prerequisite for this response.

Apoptosis induced by temozolomide and nimustine in glioblastoma cells is supported by JNK/c-Jun-mediated induction of the BH3-only protein BIM.

The outcome of cancer therapy strongly depends on the complex network of cell signaling pathways, including transcription factor activation following drug exposure. Here we assessed whether and how the MAP kinase (MAPK) cascade and its downstream target, the transcription factor AP-1, influence the sensitivity of malignant glioma cells to the anticancer drugs temozolomide (TMZ) and nimustine (ACNU). Both drugs induce apoptosis in glioma cells at late times following treatment. Activation of the MAPK cascade precedes apoptosis, as shown by phosphorylation of Jun kinase (JNK) and c-Jun, a main component of AP-1. Pharmacological inhibition and siRNA mediated knockdown of JNK and c-Jun reduced the level of apoptosis in LN-229 glioma cells treated with TMZ or ACNU. Analyzing the underlying molecular mechanism, we identified the pro-apoptotic gene BIM as a critical target of AP-1, which is upregulated following TMZ and ACNU. Importantly, shRNA mediated downregulation of BIM in the malignant glioma cell lines LN-229 and U87MG led to an attenuated cleavage of caspase-9 and, consequently, reduced the level of apoptosis following TMZ and ACNU treatment. Overall, we identified JNK/c-Jun activation and BIM induction as a late pro-apoptotic response of glioma cells treated with alkylating anticancer drugs.