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BMC Cancer
2025 Sep 29;251:1434. doi: 10.1186/s12885-025-14932-0.
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Shared and non-overlapping functions of RECQL4 and BLM helicases in chemotherapeutics-induced glioma cell responses.
Wojnicki K
,
Wojtas B
,
Ciechomska IA
,
Kaza B
,
Guille M
,
Priebe W
,
Kaminska B
.
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OBJECTIVES: Human RECQL4 and BLM helicases participate in all DNA dependent processes, including replication stress, DNA damage repair. Both helicases are overexpressed in glioblastoma (GBM), a lethal primary brain tumour, characterised by resistance to radio- and chemotherapy. BLM-depleted glioma cells exhibit senescence-associated or polypoid phenotype when exposed to temozolomide (TMZ) and olaparib (OLA), a PARP inhibitor. This study aims to investigate how RECQL4 depletion influences the response of malignant gliomas to chemotherapeutics.
METHODS: We investigated the effect of RECQL4 depletion in glioma cells on cell growth, apoptosis, senescence and polyploidy in the response to combined TMZ and OLA treatment. We compared transcriptomes of RECQL4- and BLM-depleted LN18 and LN229 glioma cells. Drug-induced cytotoxicity, senescence-associated phenotypes, cell cycle alterations, and polyploidy were assessed using the MTT metabolic assay, β-galactosidase activity assay, and propidium iodide staining.
RESULTS: RECQL4 depletion modestly affected basal glioma cell viability and proliferation, similarly to knock out of the BLM protein. Deletion of RECQL4 in glioma cells (RQ4 KO) induced profound transcriptomic alterations, dissimilar to BLM depletion. RECQL4-depleted glioma cells treated with TMZ and OLA exhibited reduced viability and increased levels of apoptosis markers. The treatment induced cell cycle arrest, however, RQ4 KO cells did not show signs of senescence phenotype or polyploidisation, when compared to BLM KO glioma cells. Interestingly, both RQ4 KO and BLM KO cells were more resistant to WP744, a doxorubicin derivative, when compared to WT LN229 glioma cells.
CONCLUSION: Our results highlight the distinct roles of RecQ helicases in a response to chemotherapeutics and support a rationale for targeting RECQL4 as a therapeutic strategy in glioblastoma.
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41023983
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Fig. 1. RECQL4 or BLM depletion in glioma cells altered marginally cell viability and cell proliferation. A Representative immunoblot shows levels of RECQL4 and BLM proteins in WT and respective depleted glioma cells. B Cell viability determined by MTT metabolism assay of WT, RQ4 KO and BLM KO LN18 and LN229 glioma cells after 72 h post seeding (n = 5). C Cell proliferation measured by BrdU assay of WT, RQ4 KO and BLM KO of LN18 and LN229 glioma cells. Black solid line indicates values in control, WT cells. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s post-hoc test between WT and KO cells, (n = 5 in triplicates). Bars plotted with the mean ± SD, *p < 0.05
Fig. 2. Comparison of GO functional pathways resulting from transcriptomic changes in RECQL4 and BLM KO glioma cells. A RECQL4 and BLM expression in GBM cells (malignant annotation from Neftel et al. 2019 [20]), was depicted on the y/x axis, where each dot is a single cell. Positive correlation of RECQL4 and BLM genes was significant (correlation = 0.11, p-value < 2.2e-16). B-C KEGG analysis of differentially expressed genes (FDR corrected p < 0.05) displays deregulated pathways in LN18 (B) and LN229 BLM (C) KO cells when compared to respective WT controls
Fig. 3. RECQL4 deficiency in glioma cells does not sensitize glioma cells to TMZ and OLA treatment. A Viability of WT or RQ4 KO LN18 and LN229 cells after double, TMZ and OLA, treatments, determined by MTT metabolism test 72 h after the treatments. Cell viability of control cells set as 100% is represented by a black solid line. Grey triangles represent increasing doses of OLA (1 and 5 µM) with 250 µM TMZ. Statistical analysis was performed using linear contrast ANOVA analysis (#p < 0.001), mean ± SD, n = 4. Hedge’s ‘g’ stands for effect size. B Representative immunoblots showing upregulation of cleaved, apoptotic protein (c-PARP, c-casp7) levels in RQ4 KO LN18 and LN229 cells after TMZ and OLA in comparison to WT cells. GAPDH was used as a loading control. C Densitometric analysis of immunoblots from 3 experiments. Statistical significance was determined by one sample t-test on logarithmic raw data *p < 0.05, &p < 0.01, #p < 0.001), n = 3, mean ± SD. D, E Percentages cells in the cell cycle phases in cultures of RQ4 KO and WT LN18 (D) and LN229 (E) cells after the TMZ and OLA treatments, determined using the propidium iodide staining and flow cytometry, n = 3, ≥ 10000 events/sample, mean ± SD. F Representative images of F-actin staining of RQ4 KO LN18 and LN229 cells treated with 500 µM TMZ and 1 µM OLA (TMZ + OLA) for 48 h; DMSO served as a control. Nuclei were visualised using DAPI staining (total magnification 200x)
Fig. 4. RECQL4 deficiency does not affect drug induced cellular senescence in glioma cells. A, D Representative images of β-gal staining of RQ4 KO LN18 (A) and LN229 (D) cells after the TMZ and OLA treatments. Blue colour indicates the increased activity of β-galactosidase (examples marked by arrows) (B, E) Quantification of β-gal positive cells amongst control and treated RQL4 KO (B) LN18 and (E) LN229 cells. Statistical analysis was performed using a chi-squared test in comparison of treated versus control cells (above the bars, & p < 0.01, # p < 0.001), or between the WT and RQ4 KO cells (above the lines, *p < 0.05), n = 3, in duplicates, ± SD. C, F Cell granularity of TMZ and OLA-treated BLM KO and WT (C) LN18 and (F) LN229 cells determined by flow cytometry. Statistical analysis was performed using chi-square test in comparison of treated to control cells (CTRL) (above the bars, # p < 0.001) or between the WT and RecQL4 KO cells (above the lines, # p < 0.001), n = 3, ≥ 10,000 events/sample, ± SD. OR stands for odds ratio. OR = 1.1 CI95(1.05;1.13), OR = 3.5 CI95(3.36;3.64)
Fig. 5. Deficiency of RECQL4 or BLM helicases in glioma cells affects WP744-induced apoptotic cell death. A Cell viability determined by Presto Blue of WT, RQ4 KO and BLM KO of LN229 glioma cells 24 h after WP744 treatment (n = 3). Statistical analysis was performed using ANOVA contrast (*p < 0.05). B Representative images of WT, RQ4 KO and BLM KO LN229 glioma cells after 96 h in presence of WP744. Round, detached cells represent apoptosis phenotype (total magnification 100x)
