HB-EGF is associated with DNA damage and Mcl-1 turnover in human glioma cell lines treated by Temozolomide.

Temozolomide (TMZ) is the main chemotherapeutic agent used for treating newly diagnosed Glioblastoma Multiforme (GBM), the most frequent malignant brain tumors in adults. This alkylating agent induces DNA double strand breaks (DSBs) which in turn lead to apoptosis by activating the Bcl-2 controlled mitochondrial pathway. However, GBM invariably recur as tumors become resistant to TMZ. We investigated the implication of EGFR ligands in this resistance and we found that the pro Heparin Binding Epidermal Growth Factor (proHB-EGF) expression is linked to the early response to TMZ in human glioma cell lines. However, HB-EGF does not affect apoptosis per se although its expression is associated with the degradation of Mcl-1. HB-EGF is implicated in DSBs repair as silencing of HB-EGF increased γH2AX foci half-life as well as USP9X expression, two features that could be linked to the observed effect on Mcl-1. Our data demonstrate a new role for HB-EGF in TMZ treated cell lines.

Differentiation-related response to DNA breaks in human mesenchymal stem cells.

We have recently shown that the in vitro differentiation of human mesenchymal stem cells (hMSCs) was accompanied by an increased sensitivity toward apoptosis; however, the mechanism responsible for this shift is not known. Here, we show that the repair of DNA double-strand breaks (DSBs) was more rapid in undifferentiated hMSCs than in differentiated osteoblasts by quantification of the disappearance of γ-H2AX foci in the nuclei after γ-irradiation-induced DNA damage. In addition, there was a marked and prolonged increase in the level of nuclear Ku70 and an increased phosphorylation of DNA-PKcs. This was accompanied by an augmentation in the phosphorylation of ATM in hMSCs post-irradiation suggesting the nonhomologous end joining repair mechanism. However, when hMSCs were induced to differentiate along the osteogenic or adipogenic pathways; irradiation of these cells caused an expeditious and robust cell death, which was primarily apoptotic. This was in sharp contrast to undifferentiated hMSCs, which were highly resistant to irradiation and/or temozolomide-induced DSBs. In addition, we observed a 95% recovery from DSB in these cells. Our results suggest that apoptosis and DNA repair are major safeguard mechanisms in the control of hMSCs differentiation after DNA damage.

Identification of a transient state during the acquisition of temozolomide resistance in glioblastoma.

Drug resistance limits the therapeutic efficacy in cancers and leads to tumor recurrence through ill-defined mechanisms. Glioblastoma (GBM) are the deadliest brain tumors in adults. GBM, at diagnosis or after treatment, are resistant to temozolomide (TMZ), the standard chemotherapy. To better understand the acquisition of this resistance, we performed a longitudinal study, using a combination of mathematical models, RNA sequencing, single cell analyses, functional and drug assays in a human glioma cell line (U251). After an initial response characterized by cell death induction, cells entered a transient state defined by slow growth, a distinct morphology and a shift of metabolism. Specific genes expression associated to this population revealed chromatin remodeling. Indeed, the histone deacetylase inhibitor trichostatin (TSA), specifically eliminated this population and thus prevented the appearance of fast growing TMZ-resistant cells. In conclusion, we have identified in glioblastoma a population with tolerant-like features, which could constitute a therapeutic target.

Bak and Mcl-1 are essential for Temozolomide induced cell death in human glioma.

Temozolomide (TMZ) is an alkylating agent used for the treatment of glioblastoma multiforme (GBM), the main form of human brain tumours in adults. It has been reported that TMZ induced DNA lesions that subsequently trigger cell death but the actual mechanisms involved in the process are still unclear. We investigated the implication of major proteins of the Bcl-2 family in TMZ-induced cell death in GBM cell lines at concentrations closed to that reached in the brain during the treatments. We did not observe modulation of autophagy at these concentrations but we found an induction of apoptosis. Using RNA interference, we showed that TMZ induced apoptosis is dependent on the pro-apoptotic protein Bak but independent of the pro-apoptotic protein Bax. Apoptosis was not enhanced by ABT-737, an inhibitor of Bcl-2/Bcl-Xl/Bcl-W but not Mcl-1. The knock-down of Mcl-1 expression increased TMZ induced apoptosis. Our results identify a Mcl-1/Bak axis for TMZ induced apoptosis in GBM and thus unravel a target to overcome therapeutic resistance toward TMZ.

DNMT3L interacts with transcription factors to target DNMT3L/DNMT3B to specific DNA sequences: role of the DNMT3L/DNMT3B/p65-NFκB complex in the (de-)methylation of TRAF1.

DNMT3L i.e. DNA (cytosine-5)-methyltransferase 3-like protein, is devoid of cytosine methyltransferase activity, despite clear homology to DNMT3A and DNMT3B, due to the mutation of key catalytic residues. However, DNMT3L participates in de novo methylation reactions through its direct interaction with DNMT3A and DNMT3B. In the present study, we investigated if DNMT3L interacts also directly with transcription factors (TFs). Using TF arrays, we identified 73 TFs that interacted with DNMT3L, 13 of which (ASH2L, ATF1, ATF3, BLZF1, CDX2, CERM, E2F3, E2F4, GCNF, GTF2I, GTF3C5, NFkB-p65 and RXRα) interacted only with DNMT3L, but not with DNMT3A/B. By focusing on the interaction with NFkB-p65, we demonstrate that DNMT3L forms a complex with DNMT3B and NFkB-p65 and that this complex is required for the control of DNA methylation at the TRAF1 promoter in the T98G glioma cell line. In addition, our experiments describe the DNA methylation at TRAF1 as being dynamic with a demethylation phase involving TET3. Thus, our data suggests that DNMT3L can address DNMT3A/B to specific sites by directly interacting with TFs that do not directly interact with DNMT3A/B. In summary, our data provide a new avenue for the direction of site-specific de novo DNA methylation catalyzed by DNMT3A/B.