Molecular Determinants of Bendamustine (BDM) Toxicity towards Hodgkin (H) and Reed-Sternberg (RS) Cell Lines From Hodgkin Lymphoma (HL).

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2763-2763
Author(s):  
Rosaria De Filippi ◽  
Stefania Crisci ◽  
Ferdinando Frigeri ◽  
Donatella Aldinucci ◽  
Domenico Galati ◽  
...  
Keyword(s):  
Cell Death ◽  
Dna Repair ◽  
Cell Lines ◽  
Cell Types ◽  
Cyclin B1 ◽  
Mitotic Catastrophe ◽  
Dna Repair Genes ◽  
Cell Death Pathways ◽  
Dose Dependent ◽  
Repair Genes

Abstract Abstract 2763 Patients with HL recurring after stem cell transplantation (SCT) are mostly incurable. Therefore development of new agents and strategies is an impellent medical need in this setting. BDM is a hybrid purine analogue/bi-functional alkylator active in B-cell tumors. Despite preliminary evidences of efficacy in refractory HL, the molecular mechanisms underlying the potential activity of BDM towards malignant H-RS cells were never explored. We investigated patterns of BDM cytotoxicity (12.5 to 100 mmol/L) in a panel of HL-derived cell lines (L1236, L428, KMH2, HDLM2, L540) and its time-dependent (8, 24, 48, 72 hrs) effects on genes involved in DNA-damage stress and repair response, apoptosis and cell cycle checkpoints by Q-RT-PCR. BDM induced a significant time- and dose-dependent inhibition of growth and survival in all cell types. L1236 cells displayed the highest sensitivity to the agent with an IC50, at 48 hrs, of 10.7 mmol/L, as opposed to KMH2, L428, L540 and HDLM2 cells with IC50 of 11.1, 12.4, 14.8 and 16.2 mmol/L, respectively. BDM elicited a dose-dependent increase of apoptosis (30% to 50% at 72 hrs) in all cell lines, as shown by Annexin-V/propidium iodide staining. The exquisite sensitivity to BDM of L1236 cells was confirmed by clonogenic assays, since these cells, after a 24 hr exposure, showed the lowest IC50 for secondary colony formation (0.7± 0.06 mmol/L) as compared to all other cell lines (IC50 range: 3.1 ± 0.28 to 15.0 ± 1.27 mmol/L). Most notably, however, BDM, within the same concentration range, activated different cell death subroutines among the various cell lines. Q-RT-PCR disclosed that BDM-induced cell death in L1236 was mainly dependent on triggering of apoptosis, as shown by the early (8–24 hrs) up regulation of the proapoptotic genes NOXA and p21, but not p27, without appreciable changes in expression levels of genes related to activation of the mitotic catastrophe process, i.e. PLK1, Aurora A kinase (AAK), and cyclin B1. In contrast, induction of mitotic catastrophe was a main determinant of BDM action on KMH2, L428 and L540 cells, as shown by early (8 hrs) and sustained (48 hrs) down-regulation of PLK1, AAK and cyclin B1 genes, without early changes in NOXA and p21 expression levels. This was confirmed by highly aberrant mitosis and further multinucleation. Interestingly, BDM induced the sequential activation of both these cell death pathways in HDLM2 cells only. In this cells, the early (8–24 hrs) down regulation of PLK1, AAK and cyclin B1 genes, was later (48–72 hrs) followed by a significant induction of NOXA, p21 and p27 genes. The highest sensitivity of L1236 cells to BDM correlated with the delayed (72 hrs) induction of the DNA-repair genes EXO-1 and ATR, but not ATM, as opposed to the earlier (24 hrs) and sustained up regulation of these DNA-repair genes, and of ATM, in all other cell types. Accordingly, only after 72 hrs from exposure to BDM, the proliferation-related genes C-MYC, E2F2 and cyclins (D1, D2) were up regulated in surviving L1236 cells, along with a >70% reduction in G0/G1 cells, a significant (48%) increase of cells in the G2 phase of the cell cycle and a 20% increment of those in S phase. Conversely, in the other cell lines, S phase accumulation (35% to 60.5% increase) and up regulation of proliferation-related genes in surviving cells occurred, as early as 24 hrs after exposure to BDM. Overall, these results indicate that BDM affects different cell death pathways among HL-derived cell lines. Specifically, in L1236 cells the agent induces the early up regulation of proapototic genes and G2 arrest, accompanied by a delayed activation of DNA repair genes. This changes may lead to cell death through apoptosis rather than mitotic catastrophe which, conversely, appeared the predominant cell death pathway activated by BDM in the other cell lines. Notably, while determinants of BDM toxicity in ‘bona fide’ HL cell lines consistently overlap with those described for tumor B-cells of non-Hodgkin lymphomas (mitotic catastrophe), L1236 cells display a different response pattern to the agent, more reminiscent of BDM action on myeloma tumor cells. Since only L1236 cells are clonotypically related to primary H-RS cells from which were derived, our results suggest that optimal BDM dosing and scheduling in HL may be different from those adopted for other B-cell tumors. In this sense, a lower BDM dosing through a weekly schedule is currently being tested at our institution in refractory HL. Disclosures: No relevant conflicts of interest to declare.

Clinical outcome prediction in esophageal adenocarcinoma based on tumor and germ-line single nucleotide polymorphisms (SNP) in DNA-repair pathways

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 15144-15144 ◽  
Author(s):  
H. Yoon ◽  
K. M. Murphy ◽  
M. K. Gibson
Keyword(s):  
Dna Repair ◽  
Tumor Cells ◽  
Esophageal Adenocarcinoma ◽  
Cell Lines ◽  
Normal Tissue ◽  
Germ Line ◽  
Normal Mucosa ◽  
Snp Analysis ◽  
Dna Repair Genes ◽  
Repair Genes

15144 Background: Germ-line SNPs in DNA repair enzymes are studied as predictive factors in various cancers. More rarely studied, however, is the presence of SNPs in tumor cells and how they relate to both germ-line SNPs as well as outcome. We explored the presence of and relationship between germ-line and tumor SNPs in esophageal adenocarcinoma using two systems: (1) Cell lines, to determine whether loss of heterozygosity (LOH) occurs near DNA repair genes, and for genotyping; (2) Patient samples, to determine whether SNPs differ between normal and tumor mucosa. Methods: (1) For LOH analysis, we examined three short tandem repeat (STR) loci on 19q13.2- 13.3 (near DNA-repair genes XPD, ERCC1, and XRCC1) in four esophageal adenocarcinoma cell lines. (The STR markers have a false positive rate of <10-3 for LOH when all three demonstrate homozygosity.) Then, using a real-time PCR allelic discrimination TaqMan assay (AB), we analyzed two SNPs of interest in these cell lines. (2) We performed SNP analysis on tumor and adjacent normal mucosa from paraffin-embedded esophageal specimens taken at resection in patients with T3N0–1 esophageal adenocarcinoma who received preoperative cisplatin, paclitaxel, gefitinib and radiotherapy followed by transhiatal resection. Results: (1) Cell lines: SEG1 and BiC1 were consistent with LOH, showing a single-allele pattern at XPD 751 (C allele) and XPD 312 (G allele). TE7 and SKGT4 did not have LOH. (2) Tumor and normal tissue: We obtained data on two patients for XPD 751. Genotypes in normal mucosa were heterozygous for one patient and homozygous at the minor allele (Q/Q) for the second patient. Genotypes in tumor were identical to those in normal tissue. Conclusions: Our cell line data shows that LOH occurs in esophageal tumor cells at DNA-repair genes of interest. Our data in two patients with esophageal adenocarcinoma did not demonstrate a difference at XPD 751 between tumor and normal tissue. Given the technical success and encouraging data from this work, we plan to evaluate tissue from ∼90 patients who underwent preoperative cisplatin-based chemoradiotherapy followed by surgery (as part of completed ECOG trial E1201). [Table: see text]

Microsatellite Instability (MSI) In High Risk Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukaemia (AML) Cells Promotes Frameshift Mutations In DNA Repair Genes: Indications for PARP Inhibitor Therapy.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1194-1194
Author(s):  
Terry J Gaymes ◽  
Azim Mohamedali ◽  
Austin G Kulasekararaj ◽  
Sydney Shall ◽  
Ghulam J. Mufti
Keyword(s):  
Dna Repair ◽  
High Risk ◽  
Cell Lines ◽  
Chromosomal Abnormalities ◽  
Gene Transcript ◽  
Dna Repair Genes ◽  
Monosomy 7 ◽  
Frameshift Mutations ◽  
Group 3 ◽  
Repair Genes

Abstract Abstract 1194 Despite major advances in the biology and pathogenesis of myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) identification of the most effective and safest form of treatment continue to present a formidable challenge particularly in older patients. Older patients (>70 years) that constitute the majority of MDS/AML patients are often resistant to chemotherapy, achieve short lived remission and are not candidates for stem cell transplantation. Therefore the emphasis is to prolong survival or improve the quality of life. Currently, a number of therapeutic strategies are being evaluated and these include treatment with DNA methyltransferase or histone deacetylase inhibitors. Seminal work in breast cancer have shown that inhibitors of poly ADP ribose polymerase (PARP) activity can selectively target tumour cells through exploitation of inherent DNA repair defects. MDS/AML are characterized by genomic instability (GI) and single nucleotide polymorphism arrays (SNPA) karyotyping show that loss of heterozygosity (LOH) and uniparental disomy (UPD) are common in MDS/AML and it has been suggested that the underlying cause of this GI is a defect in double strand DNA repair. We have demonstrated that non homologous end joining, a major pathway for the repair of double strand DNA breaks is overactive and associated with extensive joining errors in primary AML cells. Hence, potentially MDS/AML patients are candidates for PI therapy. We have also shown more recently, that 15% of MDS/AML primary patient cells and cell lines are sensitive to PARP inhibitors (PI) through exploitation of homologous recombination DNA repair defects. To further elucidate the mechanisms that underlie PI sensitivity in MDS/AML we tested for microsatellite instability (MSI) in MDS/AML cell lines and high risk MDS patients and the presence of frameshift mutations in specific DNA repair genes that confer PI sensitivity. MSI is a change in length of a microsatellite allele caused by insertion or deletion of nucleotides that are misincorporated during DNA replication and not removed by the mismatch repair pathway. Using fluorescent PCR analysis, PI sensitive cell lines, P39, KG-1 and Molm-13 showed MSI-high (instability at ≥ 2 loci) at 5 mononucleotide microsatellites, in contrast to 12 PI insensitive cell lines that showed no MSI at these loci. We also show using fluorescent PCR and DNA sequencing that these MSI positive cell lines demonstrate MSI (monoallelic 1–2 base pair [bp] deletion) in the coding region microsatellites of DNA repair genes, Ataxia telancgiectasia mutated gene (ATM), CTiP, and MRE11. Monoallelic 1–2 bp base pair deletions at these loci produced frameshift mutations that induced aberrant gene splicing transcripts in ATM and MRE11 and a markedly truncated CTiP gene transcript. No MSI was detected in DNA repair genes CHK1, RAD50, PTEN, BLM and ATR in these cell lines and no mutations were observed at any DNA repair gene microsatellite in the 13 PI insensitive cell lines. We then determined MSI in high risk MDS patients with or without monosomy 7 (-7/del7q). 13 of 63 (21%) high risk MDS patients showed MSI (9 MSI-low and 4 MSI-high). Of the 13 MSI positive patients, 7 (4 MSI-high, 3 MSI-low) had monosomy 7 and other complex chromosomal abnormalities (Group 1, 54%), 2 (MSI-low) patients had isolated monosomy 7 (Group 2, 15%) whilst 4 patients (MSI-low) had normal cytogenetics (Group 3, 30%). Constitutional DNA from these patients did not show MSI at these loci. Significantly, however, Group 3 with MSI and normal cytogenetics all had widespread UPD and cryptic chromosome changes determined by SNPA. Strikingly, thus all 13 patients with MSI possessed chromosomal abnormalities, both gross and cryptic. Furthermore, 12 patients (19%) found to be cytogenetically normal and lacking UPD and genomic aberrations by SNPA did not show MSI. We have also identified that 3 patients with MSI-high (Group 1) and 1 patient with MSI-low (Group 3) had a monoallelic 1 bp deletion in the CTiP exon coding microsatellite. 1 bp deletion within the coding exon of CTiP resulted in an abbreviated CTiP gene transcript. In conclusion, we have made the important correlation between MSI and subsequent frameshift mutations in specific DNA repair genes with the gross and cryptic chromosomal changes observed in MDS/AML. Identification of a cohort of MDS/AML patients with MSI would herald a significant advancement for the selection of candidates for PI therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Adaptive upregulation of DNA repair genes following benzo(a)pyrene diol epoxide protects against cell death at the expense of mutations

2016 ◽  
Vol 44 (22) ◽  
pp. 10727-10743 ◽  
Author(s):  
Markus Christmann ◽  
Catherine Boisseau ◽  
Rebekka Kitzinger ◽  
Christian Berac ◽  
Sebastian Allmann ◽  
...  
Keyword(s):  
Cell Death ◽  
Dna Repair ◽  
Dna Repair Genes ◽  
Diol Epoxide ◽  
Repair Genes

scholarly journals Restoration of deficient DNA Repair Genes Mitigates Genome Instability and Increases Productivity of Chinese Hamster Ovary Cells

2021 ◽  
Author(s):  
Philipp N. Spahn ◽  
Xiaolin Zhang ◽  
Qing Hu ◽  
Nathaniel K. Hamaker ◽  
Hooman Hefzi ◽  
...  
Keyword(s):  
Dna Repair ◽  
Cell Line ◽  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Genome Instability ◽  
Therapeutic Proteins ◽  
Chinese Hamster ◽  
Dna Repair Genes ◽  
Cho Cell ◽  
Repair Genes

AbstractChinese Hamster Ovary (CHO) cells are the primary host used for manufacturing of therapeutic proteins. However, production instability of high-titer cell lines is a major problem and is associated with genome instability, as chromosomal aberrations reduce transgene copy number and decrease protein titer. We analyzed whole-genome sequencing data from 11 CHO cell lines and found deleterious single-nucleotide polymorphisms (SNPs) in DNA repair genes. Comparison with other mammalian cells confirmed DNA repair is compromised in CHO. Restoration of key DNA repair genes by SNP reversal or expression of intact cDNAs improved DNA repair and genome stability. Moreover, the restoration of LIG4 and XRCC6 in a CHO cell line expressing secreted alkaline phosphatase mitigated transgene copy loss and improved protein titer retention. These results show for the first time that correction of key DNA repair genes yields considerable improvements in stability and protein expression in CHO, and provide new opportunities for cell line development and a more efficient and sustainable production of therapeutic proteins.

scholarly journals DNA Repair Genes as Drug Candidates for Early Breast Cancer Onset in Latin America: A Systematic Review

2021 ◽  
Vol 22 (23) ◽  
pp. 13030
Author(s):  
Laura Keren Urbina-Jara ◽  
Emmanuel Martinez-Ledesma ◽  
Augusto Rojas-Martinez ◽  
Francisco Ricardo Rodriguez-Recio ◽  
Rocio Ortiz-Lopez
Keyword(s):  
Breast Cancer ◽  
Dna Repair ◽  
Cell Lines ◽  
Latin American ◽  
Young Women ◽  
Dna Repair Genes ◽  
Gene Repair ◽  
Drug Candidates ◽  
Large Patient ◽  
Repair Genes

The prevalence of breast cancer in young women (YWBC) has increased alarmingly. Significant efforts are being made to elucidate the biological mechanisms concerning the development, prognosis, and pathological response in early-onset breast cancer (BC) patients. Dysfunctional DNA repair proteins are implied in BC predisposition, progression, and therapy response, underscoring the need for further analyses on DNA repair genes. Public databases of large patient datasets such as METABRIC, TCGA, COSMIC, and cancer cell lines allow the identification of variants in DNA repair genes and possible precision drug candidates. This study aimed at identifying variants and drug candidates that may benefit Latin American (LA) YWBC. We analyzed pathogenic variants in 90 genes involved in DNA repair in public BC datasets from METABRIC, TCGA, COSMIC, CCLE, and COSMIC Cell Lines Project. Results showed that reported DNA repair germline variants in the LA dataset are underrepresented in large databases, in contrast to other populations. Additionally, only six gene repair variants in women under 50 years old from the study population were reported in BC cell lines. Therefore, there is a need for new approaches to study DNA repair variants reported in young women from LA.

scholarly journals Alterations of DNA repair genes in the NCI-60 cell lines and their predictive value for anticancer drug activity

DNA Repair ◽  
2015 ◽  
Vol 28 ◽  
pp. 107-115 ◽  
Author(s):  
Fabricio G. Sousa ◽  
Renata Matuo ◽  
Sai-Wen Tang ◽  
Vinodh N. Rajapakse ◽  
Augustin Luna ◽  
...  
Keyword(s):  
Dna Repair ◽  
Cell Lines ◽  
Anticancer Drug ◽  
Predictive Value ◽  
Dna Repair Genes ◽  
Drug Activity ◽  
Repair Genes

scholarly journals Tumor Infiltrating Lymphocytes and Macrophages Improve Survival in Microsatellite Unstable Colorectal Cancer

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sumana Narayanan ◽  
Tsutomu Kawaguchi ◽  
Xuan Peng ◽  
Qianya Qi ◽  
Song Liu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Repair ◽  
Immune Cell ◽  
Tumor Infiltrating Lymphocytes ◽  
Cell Types ◽  
The Cancer Genome Atlas ◽  
Dna Repair Genes ◽  
M1 Macrophage ◽  
Infiltrating Lymphocytes ◽  
Repair Genes

Abstract Due to the loss of DNA repair mechanisms in colorectal cancer (CRC) with microsatellite instability (MSI), somatic mutations accumulate within DNA; making them more prone to attack by tumor infiltrating lymphocytes (TIL) and macrophages. We hypothesize that MSI-High (MSI-H) patients have favorable survival due to increased tumor immunogenicity. The Cancer Genome Atlas (TCGA) was used to evaluate gene expression from 283 patients with CRC, comparing MSI-H and microsatellite stable (MSS) patients. CIBERSORT algorithm estimated the fraction of immune cell types. We found that low expression of DNA repair genes (MLH1, MLH3, PMS1, PMS2, ATR, PRKDC, ATM, BRCA2) associated with MSI-H. MSI-H was directly associated with Helper T-cells (p = 0.034) and M1 macrophages (p < 0.0001). MSI-H tumors associated with diminished intra-tumoral heterogeneity as well as higher expression of checkpoint molecules PD-1, PD-L1, CTLA4, LAG3 and TIM3 (p < 0.0001). Improved OS was seen in patients with low ATM, PMS2 and MLH3. In the TCGA CRC cohort, decreased expression of DNA repair genes associated with MSI-H. MSI-H patients had improved survival, likely due to higher TIL and M1 macrophage infiltration as well as lower intra-tumoral heterogeneity. MSI-H also associates with expression of immune checkpoint molecules with potential for development of therapeutic targets.

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