Alternative end-joining originates stable chromosome aberrations induced by etoposide during targeted inhibition of DNA-PKcs in ATM-deficient tumor cells

ATM and DNA-PKcs coordinate the DNA damage response at multiple levels following the exposure to chemotherapy. The Topoisomerase II poison etoposide (ETO) is an effective chemotherapeutic agent that induces DNA double-strand breaks (DSB), but it is responsible from the chromosomal rearrangements frequently found in therapy-related secondary tumors. Targeted inhibition of DNA-PKcs in ATM-defective tumors combined with radio- or chemotherapy has been proposed as relevant therapies. Here, we explored the DNA repair mechanisms and the genetic consequences of targeting the non-oncogenic addiction to DNA-PKcs of ATM-defective tumor cells after exposure to ETO. We demonstrated that chemical inhibition of DNA-PKcs followed by treatment with ETO resulted in the accumulation of chromatid breaks and decreased mitotic index in both A-T cells and ATM-knocked-down (ATMkd) tumor cells. The HR repair process in DNA-PKcs-inhibited ATMkd cells amplified the RAD51 foci number, with no correlated increase in sister chromatid exchanges. The analysis of post-mitotic DNA lesions presented an augmented number of persistent unresolved DSB, without alterations in the cell cycle progression. Long-term examination of chromosome aberrations revealed a strikingly high number of chromatid and chromosome exchanges. By using genetic and pharmacological abrogation of PARP-1, we demonstrated that alternative end-joining (alt-EJ) repair pathway is responsible for those chromosome abnormalities generated by limiting c-NHEJ activities during directed inhibition of DNA-PKcs in ATM-deficient cells. Targeting the non-oncogenic addiction to DNA-PKcs of ATM-defective tumors stimulates the DSB repair by alt-EJ, which is liable for the origin of cells carrying stable chromosome aberrations that may eventually restrict the therapeutic strategy.Fil: de Campos Nebel, Ildefonso Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Palmitelli, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Perez Maturo, Josefina. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Gonzalez Cid, Marcela Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Measurement of drug-stabilized Topoisomerase II cleavage complexes by Flow Cytometry

The poisoning of Topoisomerase II (Top2) has been found to be useful as a therapeutic strategy for the treatment of several tumors. The mechanisms of these agents involves a drug-mediated stabilization of a Top2-DNA complex, termed Top2 cleavage complex (Top2cc), which maintains a 5´ end of DNA covalently bound to a tyrosine from Top2 through a phosphodiester group. Drug-stabilized Top2cc leads to Top2 linked-DNA breaks which are believed to mediate their cytotoxicity. Several time-consuming or cell type-limiting assays have been used in the past to study drug-stabilized Top2cc. Here, we describe a flow cytometry-based method that allows arapid assessment of drug-induced Top2cc, which is suitable for high throughput analysis in almost any kind of human cell. The analyses of the drug-induced Top2cc in the cell cycle context and the possibility to track its removal are additional benefits from this methodology.Fil: de Campos Nebel, Ildefonso Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Palmitelli, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Gonzalez Cid, Marcela Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Progression of chromosomal damage induced by etoposide in G2 phase in a DNA-PKcs deficient context

Etoposide (ETO), a drug used for the treatment of human tumors, is associated with the development of secondary malignancies. Recently, therapeutic strategies have incorporated chemosensitizing agents to improve the tumoral response to this drug. ETO creates DNA double strand breaks (DSB) via inhibition of DNA Topoisomerase II (Top2). To repair DSB, homologous recombination (HR) and non-homologous end-joining (NHEJ), involving D-NHEJ (dependent of DNA-PKcs) and B-NHEJ (backup repair pathway) are activated. We evaluated the progression of the DNA damage induced by the Top2 poison ETO in G2 HeLa human cells after chemical inhibition of DNA-PKcs. The inhibition by NU7026 together with ETO treatment resulted in a 2-fold higher rate of chromatid breaks and exchanges compared to ETO alone. Moreover, it was shown an increment in the percentage of micronuclei with H2AX positive signals in binucleated cells and a slight increase of dicentric chromosomes on second metaphases. It was also observed that in post-mitotic G1 phase, there is a closely association between unresolved DSB and MRE11 (Meiotic Recombination 11 homolog A) signals, demonstrating the contribution of MRE11 in the DSB repair by B-NHEJ. DNA-PKcs chemical inhibition impaired both D-NHEJ and HR repair pathways, altering the maintenance of chromosomal integrity and the cellular proliferative capacity. Thus, our results suggest that the chemosensitizing effectiveness of the DNA-PKcs inhibitor and the survival rate of aberrant cells may be determinants in the development of therapy-related tumors.Fil: Palmitelli, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: de Campos Nebel, Ildefonso Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Gonzalez Cid, Marcela Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Persistent genetic damage induced by topoisomerase II poisons in normal human fibroblasts: generation of chromoso me instability

Topoisomerase II (Top2) poisons idarubicin (IDA) and etoposide (ETO) are anticancer drugs that target Top2 stabilizing DNA-enzymecomplexes and generating double-strand breaks (DSB). These DNA lesions are dangerous because they lead to genomic instability and promotetumorigenesis. These drugs are associated with the development of leukemias characterized by translocations of the MLL gene in treated patients.Our aim was to analyze the residual genetic damage induced by IDA and ETO in normal human fibroblasts at different times. Cells were treatedwith sublethal concentrations of IDA and ETO for 2 h and persistent DSB were evaluated in interphase nuclei and chromosomal aberrations (CA)in metaphase at 26 h post-treatment. In addition, micronuclei and MLL gene rearrangements were determined in interphase nuclei at 30 h posttreatment.Unrepaired persistent DSB induced by IDA and ETO turned into chromatid and chromosome breaks and improper repair in chromatidand chromosome exchanges. Simultaneously with increased CA, there was a marked reduction of the mitotic index, principally in cultures treatedwith IDA, due to the accumulation of cells in G2/M phase of cell cycle. This chromosomal damage progressed to the following interphase causingan increase in the micronucleated cells and in the rearrangements of MLL gene. The persistent DNA damage produced by IDA and ETO in normalhuman cells plays an important role in the possible induction of Top2 poisons-mediated secondary malignancies.Topoisomerase II (Top2) poisons idarubicin (IDA) and etoposide (ETO) are anticancer drugs that target Top2 stabilizing DNA-enzyme complexes and generating double-strand breaks (DSB). These DNA lesions are dangerous because they lead to genomic instability and promote tumorigenesis. These drugs are associated with the development of leukemias characterized by translocations of the MLL gene in treated patients. Our aim was to analyze the residual genetic damage induced by IDA and ETO in normal human fibroblasts at different times. Cells were treated with sublethal concentrations of IDA and ETO for 2 h and persistent DSB were evaluated in interphase nuclei and chromosomal aberrations (CA) in metaphase at 26 h post-treatment. In addition, micronuclei and MLL gene rearrangements were determined in interphase nuclei at 30 h posttreatment. Unrepaired persistent DSB induced by IDA and ETO turned into chromatid and chromosome breaks and improper repair in chromatid and chromosome exchanges. Simultaneously with increased CA, there was a marked reduction of the mitotic index, principally in cultures treated with IDA, due to the accumulation of cells in G2/M phase of cell cycle. This chromosomal damage progressed to the following interphase causing an increase in the micronucleated cells and in the rearrangements of MLL gene. The persistent DNA damage produced by IDA and ETO in normal human cells plays an important role in the possible induction of Top2 poisons-mediated secondary malignancies.Fil: de Campos Nebel, Ildefonso Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Palmitelli, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Acevedo, S.. Instituto de Investigaciones Hematológicas; ArgentinaFil: Gonzalez Cid, Marcela Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Tyrosyl-DNA-phosphodiesterase I (TDP1) participates in the removal and repair of stabilized-Top2α cleavage complexes in human cells

Tyrosyl-DNA-phosphodiesterase 1 (TDP1) is a DNA repair enzyme that removes irreversible protein-linked 3′ DNA complexes, 3′ phosphoglycolates, alkylation damage-induced DNA breaks, and 3′ deoxyribose nucleosides. In addition to its extended spectrum of substrates, TDP1 interacts with several DNA damage response factors. To determine whether TDP1 participates in the repair of topoisomerase II (Top2) induced DNA lesions, we generated TDP1 depleted (TDP1kd) human tumoral cells. We found that TDP1kd cells are hypersensitive to etoposide (ETO). Moreover, we established in a chromatin context that following treatment with ETO, TDP1kd cells accumulate increased amounts of Top2α cleavage complexes, removing them with an altered kinetics. We also showed that TDP1 depleted cells accumulate increased γH2AX and pS296Chk1 signals following treatment with ETO. Similarly, cytogenetics analyses following Top2 poisoning revealed increased amounts of chromatid and chromosome breaks and exchanges on TDP1kd cells in the presence or not of the DNA-PKcs inhibitor NU7026. However, the levels of sister chromatid exchanges were similar in both TDP1kd and control non-silenced cell lines. This suggests a role of TDP1 in both canonical non-homologous end joining and alternative end joining, but not in the homologous recombination repair pathway. Finally, micronucleus analyses following ETO treatment revealed a higher frequency of micronucleus containing γH2AX signals on TDP1kd cells. Together, our results highlight an active role of TDP1 in the repair of Top2-induced DNA damage and its relevance on the genome stability maintenance in human cells.Fil: Borda, Miguel Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Palmitelli, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Verón, Gustavo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Gonzalez Cid, Marcela Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: de Campos Nebel, Ildefonso Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Analysis of basal chromosome instability in patients with chronic lymphocytic leukaemia

Genomic instability is a hallmark of cancer, contributing to tumour development and transformation, being chromosome instability (CIN) the most common form in human cancer. Chronic lymphocytic leukaemia (CLL) is the most frequent adult leukaemia in the Western world. In this study, we have evaluated basal CIN in untreated patients with CLL by measuring chromosome aberrations (CAs) and micronucleus (MN) frequency and their association with different prognostic factors. Seventy-two patients and 21 normal controls were analysed. Cytogenetic and fluorescence in situ hybridisation (FISH) studies were performed. IGHV (immunoglobulin heavy chain variable region) mutational status was evaluated by reverse transcription polymerase chain reaction and sequencing. An increased number of CA in patients compared with controls ( P = 0.0001) was observed. Cases with abnormal karyotypes showed increased CA rate than those with normal karyotypes ( P = 0.0026), with a particularly highest frequency in cases with complex karyotypes. Among FISH risk groups, a significant low frequency of CA was found in patients with no FISH alterations compared to those with del13q14 and ≥2 FISH alterations ( P = 0.0074). When mean CA value (6.7%) was considered, significant differences in the distribution of low and high CA frequency between cases with normal and abnormal karyotypes ( P = 0.002) were observed. By MN analysis, higher frequency in patients compared to controls ( P = 0.0001) was also found, as well as between cases with ≥2 FISH abnormalities and those with no FISH alterations ( P = 0.026). Similarly, significant differences were observed when patients were divided according to mean MN frequency (2.2%; P ≤ 0.04). Interestingly, patients with high MN frequency had shorter time to first treatment than those with low frequency ( P = 0.024). Cases with mutated and unmutated IGHV status showed increased CA and MN frequencies compared to controls ( P ≤ 0.0007), but no differences between both groups were found. Our results support the strong interaction between CIN and genomic complexity as well as their influence on poor outcome in this pathology.Fil: Palmitelli, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Stanganelli, Carmen Graciela. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Stella, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Krywinski, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Bezares, Raimundo F.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Dr. Teodoro Álvarez"; ArgentinaFil: Gonzalez Cid, Marcela Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Inverse PCR to perform long-distance haplotyping: main applications to improve preimplantation genetic diagnosis in hemophilia

Among other applications of long-distance haplotype phasing in clinical genetics, determination of linked DNA markers as surrogate for problematic structural variants (e.g., repeat-mediated rearrangements) is essential to perform diagnosis from low-quality DNA samples. We describe a next-of-kin-independent (physical) phasing approach based on inverse-PCR (iPCR) paired-end amplification (PI). This method enables typing the multialleles of the short tandem repeat (STR) F8Int21[CA]n at the F8-intron 21, as a surrogate DNA marker for the F8-intron 22 inversion (Inv22), the hemophilia A-causative hotspot, within the transmitted haplotype in informative carriers. We provide proof-of-concept by blindly validating the PI approach in 15 carrier mother/affected-son duos. Every F8Int21[CA]n STR allele determined in phase with the Inv22 allele in the female carriers from the informative duos was confirmed in the hemizygous proband (P = 0.00003). A second surrogate STR locus at the F8-IVS22 was obtained by the PI approach improving severe-HA preimplantation genetic diagnosis by augmenting heterozygosity in Inv22 carriers bypassing the requirement for family linkage analysis. The ability of the PI-assay to combine other marker pairs was demonstrated by haplotyping a SNV (F8:c.6118T > C) with a >28kb-distant F8-IVS22 STR. The PI approach has proven flexibility to target different marker pairs and has potential for multiplex characterization of iPCR products by massively parallel sequencing.Fil: Abelleyro, Miguel Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Marchione, Vanina Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Palmitelli, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Radic, Claudia Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Neme, Daniela. Fundación de la Hemofilia Alfredo Pavlovsky; ArgentinaFil: Larripa, Irene Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Medina Acosta, Enrique. Universidade Estadual Do Norte Fluminense Darcy Ribeiro; BrasilFil: de Brasi, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Rossetti, Liliana Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin