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Get an Invitation. Bookmarked by hnnng 23 Feb Bookmarked by hnnng 10 Feb Abnormalities in the DNA repair pathways might be responsible for the inability of the currently used chemotherapeutics to eliminate the GSC subpopulation. In this work, we compared the expression of sixty DNA repair related genes between primary glioblastoma cell cultures and the glioblastoma enriched stem cell primary cultures.
MTT test was used to analyze the effect of selected drugs and immunofluorescence to evaluate the load of DNA damage. In addition, the other genes that we found to be up-regulated in GSC versus glioblastoma primary cells should be further investigated as glioblastoma theranostics.
Glioblastoma GBM is the most common and aggressive malignancy of the central nervous system. Despite major progress in cancer treatment in the last decade, GBM remains fatal with 12—15 months median survival after diagnosis [ 1 , 2 ]. Standard therapy currently consists of minimal resection, followed by radiotherapy alone or in combination with temozolomide.
The main cause of mortality in GBM patients is the recurrence of the tumor. According to the cancer stem cell theory, recurrence is a consequence of therapeutics failing to completely eliminate a subpopulation of cancer cells with stem cell characteristic, called cancer stem cells, that have been first described in GBM by Singh et al.
Besides in GBM [ 5 , 6 ], the presence of these cells has been confirmed in many tumor types, which has been correlated with worse prognosis in breast, head and neck and oropharyngeal cancer as well as in glioma [ 7 — 10 ]. Several approaches have been suggested to eradicate cancer stem cells, such as induction of differentiation, immunotherapy or genetic manipulation that would block their proliferation or sensitize them to radio- or chemo-therapy [ 11 ].
DNA repair pathways were extensively studied in carcinogenesis [ 12 ], because defects in these pathways enable tumor cells to accumulate mutations that enhance their proliferation and survival in the complex host tissue microenvironment. At the same time, these DNA repair deficiencies provide the rationale for exploring DNA-damaging drugs for cancer treatment, because treatment with these drugs would cause cell-cycle arrest and consequent cell death in DNA repair defective cancer cells.
Interestingly, increased resistance of GBM cells to radiotherapy was suggested to be due to DNA damage response preferentially activated in GBM stem cells in comparison to their non-stem cells counterparts [ 13 , 14 ]. However, the relevance of intrinsic DNA repair efficacy as a resistance mechanism of glioma stem cells remains elusive. In the present study, we identified a panel of genes that are up-regulated in GSC vs GBM cells, isolated from the same patients.
After surgery tumors were histopathologically reviewed by two independent pathologists and in accordance WHO classification and recommendations.
All samples used in this study were IDH wild type. Alternatively, the cells were grown under the culture conditions promoting selective growth of glioblastoma stem cells GSC as neurospheres. Neurospheres were analyzed after minimum three passages. Quality was examined spectrophotometrically and on agarose gel; 0. Reaction conditions were the same as for the array. Quantification was performed using ImageJ software.
NCHk neurospheres or primary GSC spheres were broken to single cell suspension before transfection. Optimal downregulation was achieved by double transfection with 50 nM siRNA and was stable between 48 and h after transfection. Cell viability was measured using MTT assay. Stock solutions were prepared in DMSO and then diluted in cell media to the final concentrations.
Highest final concentration of DMSO was 0. Cisplatin Sigma stock solution was prepared in physiological solution and diluted to applied final concentration. U or NCHk cells or primary GSC cells were plated on well plates at a density of cell per well and treated with indicated concentrations of replication stress inducing drugs for 72 h.
Plates were centrifuged, the violet formazan crystals were dissolved in DMSO and the absorbance was measured at the wavelength nm. Cells were then fixed in 3. Cells were counterstained with Toto3 , and mounted in Vectashield. The average number of foci was obtained from three independent experiments analyzing at least 30 cells per sample. Among these 60 genes, we included genes encoding for factors involved in key double-strand break and single-strand break DNA repair pathways, comprising factors involved in homologous recombination, non-homologous end joining, mismatch repair, nucleotide excision repair and DNA damage response.
We compared seven primary glioma cell cultures with five GSC-enriched primary cultures, out of those pairs three were coming from same patient.
Gene expression was normalized to the 18S ribosomal RNA. Based on this analysis, we found 12 genes significantly up-regulated in GSC Table 1. Because of the known heterogeneity of GBM tumors [ 17 ], we have also compared the three matched samples—primary and GSC-enriched cultures obtained from the same patient. The results confirmed the significant 1. The latter two cell types are possibly present in GBM parenchyma and in the tumor microenvironment.
We selected a panel of replication stress inducing drugs, namely camptothecin CPT and etoposide that are inhibitors of the topoisomerase 1 Top1 and topoisomerase 2 Top2 , respectively. CPT primarily causes single strand break formation whereas etoposide induces double-strand break accumulation. We used also the cross-linking agent cisplatin CisPt , the alkylating agents temozolomide and methyl methanesulfonate MMS that form different kind of DNA adducts, as well as hydrogen peroxide H 2 O 2 that induces both single and double strand breaks.
We found that in non-treated cells there was only a minor and statistically non-significant increase in the number of foci per cell—in fact, almost all cells were negative for yH2AX. In particular, MMS treatment resulted in the formation of 4.
Cells were stained for yH2AX red and counterstained with Toto3 blue. Below quantification of pChk1 normalized to tubulin. The enzyme was shown to be increased in locally advanced prostate cancer [ 22 ]. Moreover, Emmons et al. A possible explanation is that we have used low concentrations of drugs with the idea to focus on concentrations that would be potentially useful also under physiological conditions. In case of CPT, we have used concentrations up to 25 nM that have an effect only on replicating cells but not on postmitotic G1 or quiescent cells [ 33 ] and therefore do not induce significant cell death.
GSC differentiation was recently suggested as a promising treatment modality, due to impairing the known resistance mechanisms of stem cells. It also neither blocked nor enhanced ATRA-induced differentiation.
MSH2 is a protein involved in mismatch repair but its role in GBM drug sensitivity has never been investigated in detail. High levels of MSH2 correlated with glioma malignancy [ 39 ] and were detected in multi-resistant malignant gliomas [ 40 ]. However, a minor downregulation of MSH2 was found to cause drastic increase in the resistance to temozolomide [ 41 ].
It was however found to have prognostic value in some other types of cancer, such as colon [ 42 ]. These studies would be important because the proteins coded by these genes might also contribute to the drug-resistant phenotype of GBM. By the same token, this enzyme can also be target for therapy so called theranostic , which alone or in combination with other targets, would reduce the GBM stemness, allowing the use of lower doses of chemotherapeutics to eliminate the tumor.
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The utility and limitations of neurosphere assay, CD immunophenotyping and side population assay in glioma stem cell research. Brain Pathol. Zurich Switz. CAS Google Scholar. Tysnes BB, Bjerkvig R. Cancer initiation and progression: involvement of stem cells and the microenvironment. Biochim Biophys Acta. Clinicopathological significance and prognostic value of CD expression in triple-negative breast carcinoma.
Cancer Sci. Frequency of cells expressing CD44, a head and neck cancer stem cell marker: correlation with tumor aggressiveness. Head Neck. Cancer stem cell enrichment marker CD a prognostic factor for survival in patients with human papillomavirus-positive oropharyngeal cancer. Eur J Cancer Oxf Engl Prognostic value of glioma cancer stem cell isolation in survival of primary glioblastoma patients.
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