scholarly journals DNA repair profile in gastrointestinal stromal tumors (gists) - novel perspectives for therapy

2014 ◽  
Vol 95 (6) ◽  
pp. 888-891
Author(s):  
S V Boichuk ◽  
B R Ramazanov ◽  
A R Galembikova ◽  
O R Galeev ◽  
I G Mustafin ◽  
...  
Keyword(s):  
Dna Repair ◽  
Cell Lines ◽  
Gastrointestinal Stromal Tumors ◽  
Dna Methyltransferase ◽  
Topoisomerase Ii ◽  
Human Fibroblasts ◽  
Intracellular Localization ◽  
Repair Pathway ◽  
Stromal Tumors ◽  
Recombination Pathway

Aim. To assess the expression of various types of DNA repair proteins in gastrointestinal stromal tumors (GISTs) to identify the possible defects in DNA repair pathways and therapeutic targets. Methods. The study was performed on the human fibroblasts, imatinib-sensitive vs imatinib-resistant GISTs and leiomyosarcomas (LMS) cell lines, as well. The cell lines indicated above were cultured in the corresponding culture medium supplemented with fetal bovine serum, L-glutamine and antibiotics (37 °C и 5% СО2). Protein expression level and its intracellular localization were assessed by Western blotting. Results. The reduced BRCA1 expression was observed in most of the GIST cell lines, which was associated with an up-regulation of Rad51, thereby indicating about the potential abnormalities of homologous recombination pathway in these cells. This phenomenon was typical for GISTs and was not observed in LMS cells lines. In contrast to LMS cell lines, all GIST cells showed an upregulation of O6-methylguanine DNA methyltransferase (MGMT), the key enzyme involved in alkylated DNA damage repair pathway. Most GIST cells exhibited high level of MSH6 known as a key member of mismatch repair pathway. Most notably, topoisomerases were over-expressed in all of GIST cell lines. Conclusions. We found several striking alterations in expression levels of DDR pathway enzymes in GISTs. For instance, an up-regulation of topoisomerases in all GISTs indicates that these cells might be sensitive to topoisomerase II inhibitors and could be potentially targeted therapeutically.

scholarly journals Inhibition of AKT-Signaling Sensitizes Soft Tissue Sarcomas (STS) and Gastrointestinal Stromal Tumors (GIST) to Doxorubicin via Targeting of Homology-Mediated DNA Repair

2020 ◽  
Vol 21 (22) ◽  
pp. 8842
Author(s):  
Sergei Boichuk ◽  
Firuza Bikinieva ◽  
Ilmira Nurgatina ◽  
Pavel Dunaev ◽  
Elena Valeeva ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Soft Tissue ◽  
Gastrointestinal Stromal Tumors ◽  
Topoisomerase Ii ◽  
Soft Tissue Sarcomas ◽  
Akt Signaling ◽  
Chemotherapeutic Agents ◽  
Tail Moment ◽  
Stromal Tumors

Activation of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway is well documented for a broad spectrum of human malignancies supporting their growth and progression. Accumulating evidence has also implicated AKT as a potent modulator of anti-cancer therapies via regulation of DNA damage response and repair (DDR) induced by certain chemotherapeutic agents and ionizing radiation (IR). In the present study, we examined the role of AKT signaling in regulating of Rad51 turnover and cytotoxic effects of topoisomerase II inhibitor, doxorubicin (Dox) in soft tissue sarcomas (STS) and gastrointestinal stromal tumors (GIST) in vitro. Blocking of AKT signaling (MK-2206) enhanced cytotoxic and pro-apoptotic effects of Dox in vast majority of STS and GIST cell lines. The phosphorylated form of Akt co-immunoprecipitates with Rad51 after Dox-induced DNA damage, whereas Akt inhibition interrupts this interaction and decreases Rad51 protein level by enhancing protein instability via proteasome-dependent degradation. Inhibition of Akt signaling in Dox-treated cells was associated with the increased number of γ-H2AX-positive cells, decrease of Rad51 foci formation and its colocalization with γ-H2AX foci, thereby revealing unsuccessful DDR events. This was also in consistency with an increase of tail moment (TM) and olive tail moment (OTM) in Dox-treated GIST and STS cells cultured in presence of Akt inhibitor after Dox washout. Altogether, our data illustrates that inhibition of AKT signaling is STS and GIST might potentiate the cytotoxic effect of topoisomerase II inhibitors via attenuating the homology-mediated DNA repair.

scholarly journals Frequency and prognostic value of mutations associated with the homologous recombination DNA repair pathway in a large pan cancer cohort

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Daniel R. Principe ◽  
Matthew Narbutis ◽  
Regina Koch ◽  
Ajay Rana
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Synthetic Lethality ◽  
Parp Inhibitors ◽  
The Cancer Genome Atlas ◽  
Parp Inhibition ◽  
Repair Pathway ◽  
Wide Range ◽  
Recombination Pathway ◽  
Pan Cancer

AbstractPARP inhibitors have shown remarkable efficacy in the clinical management of several BRCA-mutated tumors. This approach is based on the long-standing hypothesis that PARP inhibition will impair the repair of single stranded breaks, causing synthetic lethality in tumors with loss of high-fidelity double-strand break homologous recombination. While this is now well accepted and has been the basis of several successful clinical trials, emerging evidence strongly suggests that mutation to several additional genes involved in homologous recombination may also have predictive value for PARP inhibitors. While this notion is supported by early clinical evidence, the mutation frequencies of these and other functionally related genes are largely unknown, particularly in cancers not classically associated with homologous recombination deficiency. We therefore evaluated the mutation status of 22 genes associated with the homologous recombination DNA repair pathway or PARP inhibitor sensitivity, first in a pan-cancer cohort of 55,586 patients, followed by a more focused analysis in The Cancer Genome Atlas cohort of 12,153 patients. In both groups we observed high rates of mutations in a variety of HR-associated genes largely unexplored in the setting of PARP inhibition, many of which were associated also with poor clinical outcomes. We then extended our study to determine which mutations have a known oncogenic role, as well as similar to known oncogenic mutations that may have a similar phenotype. Finally, we explored the individual cancer histologies in which these genomic alterations are most frequent. We concluded that the rates of deleterious mutations affecting genes associated with the homologous recombination pathway may be underrepresented in a wide range of human cancers, and several of these genes warrant further and more focused investigation, particularly in the setting of PARP inhibition and HR deficiency.

scholarly journals Inhibition of PARP Sensitizes Chondrosarcoma Cell Lines to Chemo- and Radiotherapy Irrespective of the IDH1 or IDH2 Mutation Status

Cancers ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1918 ◽  
Author(s):  
Sanne Venneker ◽  
Alwine B. Kruisselbrink ◽  
Inge H. Briaire-de Bruijn ◽  
Yvonne de Jong ◽  
Andre J. van Wijnen ◽  
...  
Keyword(s):  
Dna Repair ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Synthetic Lethality ◽  
Parp Inhibitor ◽  
Parp Inhibition ◽  
Idh Mutation ◽  
Mutation Status ◽  
Chondrosarcoma Cell ◽  
Idh Mutations

Chondrosarcomas are chemo- and radiotherapy resistant and frequently harbor mutations in isocitrate dehydrogenase (IDH1 or IDH2), causing increased levels of D-2-hydroxyglutarate (D-2-HG). DNA repair defects and synthetic lethality with poly(ADP-ribose) polymerase (PARP) inhibition occur in IDH mutant glioma and leukemia models. Here we evaluated DNA repair and PARP inhibition, alone or combined with chemo- or radiotherapy, in chondrosarcoma cell lines with or without endogenous IDH mutations. Chondrosarcoma cell lines treated with the PARP inhibitor talazoparib were examined for dose–response relationships, as well as underlying cell death mechanisms and DNA repair functionality. Talazoparib was combined with chemo- or radiotherapy to evaluate potential synergy. Cell lines treated long term with an inhibitor normalizing D-2-HG levels were investigated for synthetic lethality with talazoparib. We report that talazoparib sensitivity was variable and irrespective of IDH mutation status. All cell lines expressed Ataxia Telangiectasia Mutated (ATM), but a subset was impaired in poly(ADP-ribosyl)ation (PARylation) capacity, homologous recombination, and O-6-methylguanine-DNA methyltransferase (MGMT) expression. Talazoparib synergized with temozolomide or radiation, independent of IDH1 mutant inhibition. This study suggests that talazoparib combined with temozolomide or radiation are promising therapeutic strategies for chondrosarcoma, irrespective of IDH mutation status. A subset of chondrosarcomas may be deficient in nonclassical DNA repair pathways, suggesting that PARP inhibitor sensitivity is multifactorial in chondrosarcoma.

scholarly journals Establishment and characterization of novel cell lines and xenografts from patients with gastrointestinal stromal tumors

2013 ◽  
Vol 30 (1) ◽  
pp. 71-78 ◽  
Author(s):  
KAZUMASA FUKUDA ◽  
YOSHIRO SAIKAWA ◽  
HIROYUKI SAKO ◽  
YUMI YOSHIMURA ◽  
TSUNEHIRO TAKAHASHI ◽  
...  
Keyword(s):  
Cell Lines ◽  
Gastrointestinal Stromal Tumors ◽  
Stromal Tumors

scholarly journals Inducibility of the DNA repair gene encoding O6-methylguanine-DNA methyltransferase in mammalian cells by DNA-damaging treatments.

1991 ◽  
Vol 11 (9) ◽  
pp. 4660-4668 ◽  
Author(s):  
G Fritz ◽  
K Tano ◽  
S Mitra ◽  
B Kaina
Keyword(s):  
Dna Repair ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Hepatoma Cells ◽  
X Rays ◽  
Gene Encoding ◽  
Mgmt Gene ◽  
Methylguanine Dna Methyltransferase ◽  
Rat Hepatoma ◽  
Rat Hepatoma Cells

The inducibility of the mammalian O6-methylguanine-DNA methyltransferase (MGMT) gene encoding the MGMT protein (EC 2.1.1.63) responsible for removal of the procarcinogenic and promutagenic lesion O6-alkylguanine from DNA was examined by an analysis of transcription of the MGMT gene following exposure of repair-competent (Mex+) and repair-deficient (Mex-) cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). While human and rodent Mex- cells (CHO-9, V79, HeLa MR) showed no detectable MGMT mRNA despite the presence of the gene in their genome, the amount of it in several Mex+ lines (NIH 3T3, HeLa S3, HepG2) paralleled their MGMT activity. However, none of these cell lines showed an increase in the MGMT mRNA level after treatment with various concentrations of MNNG. In contrast, MNNG-treated rat hepatoma cells, H4IIE and FTO-2B, both Mex+, had three- to fivefold more MGMT mRNA than the corresponding untreated controls as measured 12 to 72 h after alkylation. N-Methyl-N-nitrosourea, methyl methanesulfonate, N-hydroxyethyl-N-chloroethylnitrosourea, UV light, and X rays caused a similar accumulation of MGMT mRNA in rat hepatoma cells. Studies with inhibitors of RNA and protein synthesis indicate that the induced increase in the amount of MGMT mRNA was due to enhanced transcription of the gene. Furthermore, they revealed the turnover of the MGMT mRNA to be relatively low (half-life, greater than 7 h). Mutagen-induced increase of transcription of MGMT mRNA in H4IIE cells was accompanied by elevation of MGMT repair activity and resulted in reduction of mutation frequency after a challenge dose of MNNG. Although induction of MGMT mRNA transcription has been observed in two rodent hepatoma cell lines so far, this appears to be the first demonstration of inducibility of a mammalian gene encoding a clearly define DNA repair function. The transcription activation of the MGMT gene protects cells from the mutagenic effects of methylating agents.

scholarly journals Succinate Dehydrogenase-Deficient Gastrointestinal Stromal Tumors: Small Steps Toward Personalized Medicine?

2019 ◽  
Vol 12 ◽  
pp. 251686571984253 ◽  
Author(s):  
Gloria Ravegnini ◽  
Riccardo Ricci
Keyword(s):  
Succinate Dehydrogenase ◽  
Gastrointestinal Stromal Tumors ◽  
Dna Methyltransferase ◽  
Kinase Inhibitors ◽  
Alkylating Agents ◽  
Mgmt Methylation ◽  
Wild Type ◽  
Stromal Tumors ◽  
Methylguanine Dna Methyltransferase ◽  
Specific Alternative

Various molecular triggers define heterogeneous subsets of gastrointestinal stromal tumors (GISTs), differing in clinical behavior and drug sensitivity. KIT/PDGFRA-wild-type GISTs, including those succinate dehydrogenase (SDH)-deficient, are overall unresponsive to the tyrosine kinase inhibitors commonly used, fostering the development of specific alternative therapeutic strategies. Epigenetic inactivation of O6-methylguanine-DNA methyltransferase (MGMT) through promoter methylation leads to effectiveness of alkylating agents in several human cancers. SDH-deficient GISTs typically feature widespread DNA methylation. However, the actual occurrence of MGMT methylation in these tumors, potentially predisposing them to respond to alkylating drugs, has not been investigated so far. Here we discuss the recent findings concerning the occurrence of MGMT methylation in different GIST subgroups, including SDH-deficient ones, as a premise for a possible reappraisal of alkylating agents specifically targeting these small, otherwise overall chemorefractory, GIST subgroups.

Selected Resistance to Topoisomerase II Inhibitors Correlates with the Over Expression of FANCF In a Fanconi Anemia/BRCA DNA Repair Pathway Independent Manner.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3372-3372
Author(s):  
Kenneth H. Shain ◽  
Liang Nong ◽  
Danielle Yarde ◽  
Vasco Oliveira ◽  
William S. Dalton
Keyword(s):  
Dna Repair ◽  
Mrna Expression ◽  
Cell Lines ◽  
Fanconi Anemia ◽  
Repair Pathway ◽  
Core Complex ◽  
Doxorubicin Resistance ◽  
Over Expression ◽  
The Core ◽  
Enhanced Expression

Abstract Abstract 3372 Enhanced expression of the Fanconi Anemia (FA)/BRCA DNA repair pathway correlates with melphalan-resistance in multiple myeloma (MM) cell lines. Continued investigation demonstrated a bortezomib sensitive RelB/p50-mediated regulation of the FA/BRCA pathway contributed to the observed melphalan resistance.(Yarde et al 2009) The FA/BRCA pathway represents a co-dependent DNA damage response pathway involving thirteen loss of function complementation groups cloned from FA patients. The key functional event of this pathway is the interdependent mono-ubiquitination (Ub) of FANCD2 and FANCI (ID complex) by the E3 Ub-ligase activity of the FA core complex a multimer consisting of 8 FA (FANCA, B, C, E, F, G, L and M) and three non-FA proteins (FAAP100, FAAP24 and HES1). Formation of the core complex and mono-Ub of the ID complex appears to revolve around the flexible adapter protein FANCF. Nuclear localization of the core complex components requires binding of FANCA/G and FANCC/E subcomplexes to the C- terminal domain (CTD) and NTD domains of FANCF, respectively. This complex associates with FANCM:FAAP24 at sites of interstand crosslinks (ICL) via the FANCM-binding domain of FANCF, culminating in ID complex mono-Ub, recruitment of BRCA1, BRCA2/FANCD1, FANCJ and FANCN, and homologous recombination (HR) repair. Reduced function of this pathway has been associated with increased genomic instability, cancer susceptibility, and increased sensitivity to DNA cross-linking agents in FA. However, as predicted by the role of the FA/BRCA pathway in DNA repair, enhanced expression of the FA/BRCA pathway has been shown to play an important role in resistance to agents requiring HR for ICL repair. We next examined expression of this pathway in models of resistance to DNA damaging agents not predicted to utilize FA/BRCA activity. We screened 8226/Dox40 doxorubicin resistant and 8226/MR20 mitoxantrone resistant MM cell lines for expression of the 12 FA/BRCA pathway members with quantitative PCR (qPCR) using customized micro-fluidic cards. Interestingly, in these models of topoisomerase (topo) II inhibitor resistance qPCR demonstrated a 2.6 (p<0.05) and 1.7 (p<0.05) fold over expression of FANCF mRNA relative to drug sensitive RPMI8226 cells. Importantly, mRNA expression of other the eleven FA/BRCA pathway constituents was not increased relative to sensitive cells. To further characterize the relationship between FANCF and doxorubicin resistance, we examined mRNA and protein expression of FANCF in RMPI8226, 8226/Dox6 and 8226/Dox40 MM cell lines (representing progressive levels of doxorubicin resistance). FANCF qPCR demonstrated a 2 and 4.7 fold increased in mRNA expression in the 8226/Dox6 and 8226/Dox40 cell lines, respectively (p= 0.103 and p= 0.034) suggesting that increasing expression of FANCF correlated with increasing dox resistance. A similar doxorubicin resistance- dependent increase in FANCF protein was demonstrated by Western blot analysis of these cell lines. Consistent with mRNA results, FANCD2 or FANCG protein levels remained unchanged in the doxorubicin resistant versus sensitive cell lines These observations suggest that FANCF may contribute to topoII inhibitor-mediated DNA double strand break repair, a process that primarily thought to involve non-homologous end joining (NHEJ) independent of the FA/BRCA pathway. To determine if FANCF expression alone could facilitate doxorubicin resistance, pQCXIP-control or pQCXIP-FANCF constructs were expressed in RPMI8226 sensitive MM cells. MTT assays demonstrated a greater than 2 fold resistance to doxorubicin in FANCF over expressing cells at 48 and 96 hours (IC50: 1.33 ×10−6 and 5.3×10−9M) as compared to control cells (3.26×10−6 and 1.13×10−8M). Taken together, these results indicate that the flexible adaptor protein FANCF may participate in doxorubicin resistance independently of other FA/BRCA members. However, future studies will be needed to elucidate the nature of FANCF in doxorubicin resistance. Disclosures: No relevant conflicts of interest to declare.

scholarly journals HOXA10 is associated with temozolomide resistance through regulation of the homologous recombinant DNA repair pathway in glioblastoma cell lines

2014 ◽  
Vol 5 (5-6) ◽  
pp. 165-174 ◽  
Author(s):  
Jin Wook Kim ◽  
Ji Young Kim ◽  
Ja Eun Kim ◽  
Seung-Ki Kim ◽  
Hyun-Tai Chung ◽  
...  
Keyword(s):  
Dna Repair ◽  
Cell Lines ◽  
Recombinant Dna ◽  
Glioblastoma Cell ◽  
Repair Pathway ◽  
Glioblastoma Cell Lines

The Hsp90 Inhibitor, 17-AAG, and Topoisomerase ll Inhibitor, Etoposide, Synergistically Inhibit Leukemias with FLT3 Mutations through Inhibition of DNA Repair Proteins, Chk1 and Rad51.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2461-2461
Author(s):  
Qing Yao ◽  
Brenda Weigel ◽  
John Kersey
Keyword(s):  
Dna Repair ◽  
Cell Lines ◽  
Topoisomerase Ii ◽  
Leukemia Cells ◽  
G1 Arrest ◽  
Wild Type ◽  
Inhibitory Effects ◽  
Inhibition Of Proliferation ◽  
Dna Repair Proteins ◽  
Flt3 Mutations

Abstract 17-AAG, an analogue of gelganamycin is an inhibitor of the molecular chaperone Hsp90, that results in apoptosis and inhibition of proliferation of myeloid or mixed lineage (MLL fusion gene induced) leukemia cells. The sensitivity to 17-AAG is highest in leukemia with FLT3 mutations, intermediate in wild type FLT3 and lowest in FLT3 negative leukemia cells. 17-AAG results in reduction of total levels of protein kinases FLT3, RAF, AKT and Chk1, a protein involved in DNA repair. Etoposide is a clinically effective agent in myeloid or mixed lineage leukemia therapy. Etoposide inhibits topoisomerase II and results in production of DNA double-strand breaks (DSBs). Normally these DSBs can be repaired by Rad51 via homologous recombination (HR). In DNA repair, Chk1 is required for HR through the interaction with Rad51. The effects of 17-AAG on Rad51 are unknown. Our current study evaluated the single and combined effects of 17-AAG and etoposide, and the mechanism of these effects in human leukemia cell lines with or without FLT3 mutations. Cell growth experiments using the MTT assay showed that the cell lines with MLL fusion genes and internal tandem duplication of FLT3 (ITD-FLT3) (Molm13 and MV4;11) were sensitive to both 17-AAG and etoposide. The IC50s for 17-AAG were 31 nM and 40 nM, respectively; the IC50s for etoposide were 37.7 nM and 45.6 nM, respectively. Wild-type FLT3 cells (HPB-Null and RS4;11) were less sensitive to both 17-AAG and etoposide; the IC50s for 17-AAG were 470 nM and 700 nM, respectively, and the IC50s for etoposide were 72.2 nM and 101.5 nM, respectively. The combination effects of 17-AAG and etoposide on cell proliferation were analyzed using Combination Index method (CalcuSyn software). Importantly, we observed synergistic inhibitory effects in FLT3-ITD cells (MV4;11 and Molm13) but only additive effects in wild type FLT3 cells (HPB-Null and RS4;11). Cell cycle analysis of MV4;11 and Molm13 cells showed that 17-AAG increased cells in G0/G1 phase after a 24 hrs treatment, while etoposide induced G2/M arrest only. Combined treatment with 17-AAG and etoposide results in Go/G1 arrest before the cells enter S phase, as with 17-AAG alone. Western-blotting showed that 17-AAG inhibited FLT3, Chk1 and in novel results Rad51 in ITD-FLT3 leukemia cells. To address the importance of FLT3 mutations on DNA repair proteins, Chk1 and Rad51, we used small interfering RNA (siRNA) targeted to FLT3. The results showed that Chk1 and Rad51 are dependent on constitutively activated FLT3 expression in ITD-FLT3 cells. In conclusion, the combination of 17-AAG with etoposide results in synergistic cellular inhibitory effects in ITD-FLT3 leukemia cells. The mechanism of the synergistic effects was found to be in part the result of inhibitory actions of 17-AAG on FLT3 dependent DNA repair proteins, Chk1 and in new findings Rad51 which are required for the repair of DNA damage induced by etoposide. 17-AAG, which is currently in clinical trials, combined with a topoisomerase II inhibitor, such as etoposide, has the potential to enhance therapeutic efficacy, particularly in high risk myeloid or mixed lineage leukemias with FLT3 mutation.

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