In this work, we used immunohistochemical and western analysis to show that RECQ1 is highly expressed in human brain glioblastoma and it is confined in the nuclei of the tumor cells, suggesting that this protein plays an important role in glioblastoma tumor growth. This conclusion was validated on a tissue microarray containing a total of primary 63 glioblastoma and 19 perilesional tissues and indicated that the higher expression of RECQ1 in the tumors is not related to the sex or the age of the individuals. RECQ1 expression is also not related to the methylation status of the MGMT
gene which identifies a subgroup of glioblastoma more susceptible to the cytotoxic effect of temozolomide treatment. Interestingly, we see that RECQ1 is also highly expressed in other types of tumors, in agreement with previous findings [40
]. However, only in the case of brain tumors, the high expression of RECQ1 is paralleled to a reduced expression of the same protein in perilesional and/or normal tissues, possibly due to the low degree of proliferation of brain cells. Hence, we believe that RECQ1 would be an ideal target for chemotherapy especially in the case of brain tumors since its depletion by RNAi, or its inhibition by selective compounds, would primarily affect tumoral cells.
An essential role of RECQ1 in tumor growth and proliferation is confirmed by our clonogenic assays where we noticed a significant reduction in the number of colonies when T98G and U87 cells were inhibited for the expression of RECQ1. This notion is supported by our previous observation that RECQ1 plays an important role during DNA replication, which is distinct from that of other human RecQ helicases [29
]. In addition, we previously showed that RECQ1, BLM, and WRN are characterized by different substrate specificities [42
]. In agreement with these findings, Sharma et al. showed that RECQ1 is important for HeLa cell proliferation and plays a unique role in the maintenance of genome integrity [36
]. Consistently, we find that the depletion of RECQ1 results in spontaneous γ-H2AX foci formation and HU hypersensitivity in T98G cells, suggesting that RECQ1 plays an important and unique role in DNA repair during DNA replication. Concerning the specific role of RECQ1 in genome maintenance, Sharma et al. suggested that RECQ1 might be involved in the regulation of the homologous recombination pathway of DNA double-strand break repair [36
]. Interestingly, our immunofluorescence experiments indicate that RECQ1 loss results in fewer RAD51 foci compared to γ-H2AX foci indicating that RECQ1 might not play a major role in homologous recombination (HR). Consistently, we do not observe any significant defect in HR frequency in RECQ1 siRNA-inhibited cells (indeed, HR frequency was slightly increased upon RECQ1 depletion) (data not shown). The hyper-recombination phenotype of RECQ1-depleted cells suggests either that RECQ1 is involved in the suppression of some illegitimate recombination events, as already proposed for other helicases of the same family [44
], or that loss of RECQ1 results in the accumulation of some form of DNA lesion or strand breaks, other than DSBs, that might subsequently lead to repair by HR if not properly repaired.
The resistance of glioma cells to TMZ is mainly associated with levels of the DNA repair protein O6
-alkylguanine alkyltransferase (AGT), which removes alkyl groups at the O6
position of guanine. In fact, O6-benzylguanine (O6-BG), an inhibitor for AGT, reduces resistance to TMZ [45
]. Moreover, it has been demonstrated that chemosensitivity of tumor cells to TMZ correlates with the inhibition of telomerase activity [46
]. Our studies using malignant glioma cell lines with low (U87-MG) and high levels of AGT (T98G), show that RECQ1 suppression by RNA interference increases the sensitivity of these cells to TMZ, independently of the AGT expression levels. Our data show also that the hypersensitivity of the RECQ1 depleted cells to TMZ is not linked to the methylation status of the MGMT
gene which is methylated in both T98G and U87 cells or to the presence of stereotypical mutations in the IDH1
genes which are absent in these cells. The increased sensitivity of RECQ1 depleted glioblastoma cells to TMZ support the notion that RECQ1 plays a unique role in DNA repair during DNA replication in malignant cells.
Interestingly, a recent study showed that RECQ1 silencing in cancer cells induces a cell specific mitotic catastrophe not observed in normal cells [19
]. The absence of RECQ1 might promote the accumulation of DNA damage in the M-phase arrested cells due to the deficient G1 and G2 checkpoint functions of cancer cells. These events would then lead to this cell type specific mitotic death. Under normal conditions, the accumulation of DNA damage and mitotic cell death is avoided during the S and G2 phases by the upregulation of different repair enzymes, such as RECQ1. Thus, cancer cells might maintain a greater copy number of DNA repair enzymes to restore DNA damage in a short time since the normal time of cell cycle arrest needed for DNA repair during the S and G2 phases is unavailable due to defects in the checkpoint activity.
Defects in the genes of three of the five human DNA helicases, BLM, WRN and RECQ4, are responsible for distinct genetic disorders associated with cancer predisposition. Moreover, allelic losses or deletion of chromosome 12p12, where the RECQ1
gene is located, is a frequent event in a wide range of solid tumors [47
], and a single-nucleotide polymorphism of the RECQ1
gene has been associated with a reduced survival of pancreatic cancer patients [9
]. In this regard, RecQ helicases might be considered as "tumor suppressors" that prevent neoplastic transformation through the control of chromosomal stability. The fact that RecQ helicases, such as BLM and RECQ1, are upregulated in tumors and provide growth advantage to cancer cells might appear incompatible with the proposed tumor suppression function of these proteins. Recent studies indicated that WRN also supports oncogenic proliferation [17
]. Indeed, the expression of several RecQ helicases is also increased upon cellular transformation by EBV and SV40 antigen [41
]. A possible explanation for this paradox is that, in somatic cells, DNA repair defects affecting genome integrity due to a RecQ helicase deficiency may lead to cancer predisposition. Conversely, increased RecQ helicase expression might be required in transformed or actively proliferating cells to resolve and repair the elevated load of DNA intermediates that are generated during active replication.