Imatinib Increases Cytotoxicity of Melphalan and Their Combination Allows An Efficient Killing of CML Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4254-4254
Author(s):  
Cesarina Giallongo ◽  
Piera La Cava ◽  
Daniele Tibullo ◽  
Nunziatina Parrinello ◽  
Provvidenza Guagliardo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Drug Resistance ◽  
Dna Repair ◽  
Conflicts Of Interest ◽  
Leukemic Cells ◽  
Proliferative Potential ◽  
Cell Accumulation ◽  
M Phase ◽  
Pre Treatment ◽  
Human Leukemic Cells

Abstract Abstract 4254 BCR/ABL-positive cells are relatively resistant to chemotherapy and, in order to evaluate the effect of Imatinib (IM) in reverting drug-resistance, we evaluated on K562 the toxicity of 1 h exposure to cytosine arabinoside (ARA-C) 20 μM, hydroxyurea (HU) 100 μM, and melphalan (MEL) 20 μM, after a pre-treatment of 24 h with 1 μM IM. The doses of the drugs were similar to that achieved in the plasma after standard chemoterapeutic treatment. Cell viability was evaluated by ATP-lite at 24, 48 and 72 hs from beginning of drug-free condition. The combinations of IM plus MEL induced the highest cytotoxicity (P<0,001 at 24, 48 and 72 hs vs MEL alone) indicating that pre-treatment with IM increased K562 exposition to the genotoxic damage of MEL. We next analyzed effects on cell cycle and DNA damage by alkaline comet assay induced by this drug combination and we observed that DNA damage peaked at 48 h with IM/MEL combination. In addition, flow cytometry analysis showed that IM/MEL combination reduced the cell accumulation in G2/M phase induced by MEL (P<0.001 vs MEL), thus reducing the ability to DNA repair and recovery. These data indicate that inhibition of BCR/ABL activity by IM increased cell cytotoxicity of MEL by reducing the effectiveness of the DNA-repair pathways and decreasing the time for DNA repair at the G2/M checkpoint.. To ascertain that these results were linked to BCR/ABL inhibition, TonB.210, a cell line where the BCR-ABL expression is inducible by doxycycline (DOX), were treated in the same conditions. Only TonB.210 cultured with DOX were insensitive to MEL while IM/MEL combination reverted these drug resistance (P<0,001). In the final step we studied the sequential association IM/MEL on the proliferative potential of myeloid progenitors of 6 CML patients at diagnosis. IM/MEL combination increased the reduction of the overall number of colonies in comparison to IM alone (P<0.05 vs IM). In addition, the analysis on CFU-GM and BFU-E colonies by qRT-PCR demonstrated that the IM/MEL combination led to the highest reduction in the number of BCR/ABL positive colonies (P<0.01 vs IM). Therefore, our data indicate that the pre-inhibition of BCR/ABL activity by IM increases the toxicity of MEL and allows an efficient killing of human leukemic cells, thus suggesting new therapeutic combinations for CML patients Disclosures: No relevant conflicts of interest to declare.

Impaired Expression of the DNA Repair Factor DDB2 in Human Myeloid Leukemias.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2046-2046
Author(s):  
Rishu Takimoto ◽  
Junji Kato ◽  
Koichi Takada ◽  
Takuya Matsunaga ◽  
Yoshiro Niitsu
Keyword(s):  
Dna Repair ◽  
Excision Repair ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Leukemic Cells ◽  
Repair System ◽  
Myeloid Leukemias ◽  
Repair Factor ◽  
Human Leukemic Cells ◽  
Damaged Dna

Abstract Recent studies have suggested that chromosomal deletions might represent a mechanism of inactivation of DNA repair system in various hematological malignancies, including myeloid leukemias. However, the precise mechanisms remain unclear. Damaged DNA binding protein-2 (DDB2), a DNA repair factor induced by tumor suppressor p53, plays an important role in the nucleotide excision repair of UV-damaged DNA. Despite frequent mutations of p53 in human leukemic cells, the role of DDB2 on the leukemogenesis is unkown. In this study, we examined expression of DDB2 mRNA in four human myeloid leukemia cell lines (K562, KG1, HL60, and MEG01) and in fresh leukemic cells obtained from 4 patients with myeloid leukemias. In all leukemia cells, expression of DDB2 mRNA was remarkably decreased as compared to that of CD34 cells We then assayed DDB2-dependent DNA repairing activity in the leukemic cells using specific antibodies against photoproducts, and found that DNA repairing activity was reduced. When a plasmid encoding DDB2 gene (pCV-DDB2) was transduced into K562 cells, the DNA repairing activity was significantly restored. Finally we tested the expression of DDB2 mRNA in five myeloid leukemias obtained from patients, and found loss of DDB2 expression in four patients. These results suggested that in human myeloid leukemias, suppression of DDB2 expression may contribute to accumulation of gene mutation through the dysfunction of DNA repair.

Bortezomib Enhances Melphalan Response by Altering Fanconi Anemia (FA)/BRCA Pathway Expression and Function.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 840-840 ◽  
Author(s):  
Danielle N. Yarde ◽  
Lori A. Hazlehurst ◽  
Vasco A. Oliveira ◽  
Qing Chen ◽  
William S. Dalton
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Dna Repair ◽  
Low Dose ◽  
Dna Damage Repair ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Multiple Myeloma Cell ◽  
Damage Repair ◽  
Pre Treatment

Abstract The FA/BRCA pathway is involved in DNA damage repair and its importance in oncogenesis has only recently been implicated. Briefly, 8 FA/BRCA pathway family members facilitate the monoubiquitination of FANCD2. Upon monoubiquitination, FANCD2 translocates to the DNA repair foci where it interacts with other proteins to initiate DNA repair. Previously, we reported that the FA/BRCA pathway is upregulated in multiple myeloma cell lines selected for resistance to melphalan (Chen, et al, Blood 2005). Further, reducing FANCF in the melphalan resistant 8226/LR5 myeloma cell line partially reversed resistance, whereas overexpressing FANCF in the drug sensitive 8226/S myeloma line conferred resistance to melphalan. Others have reported, and we have also verified, that bortezomib enhances melphalan response in myeloma cells; however, the mechanism of enhanced melphalan activity in combination with bortezomib has not been reported. Based on our observation that the FA/BRCA pathway confers melphalan resistance, we hypothesized that bortezomib enhances melphalan response by targeting FA/BRCA DNA damage repair pathway genes. To investigate this hypothesis, we first analyzed FA/BRCA gene expression in 8226/S and 8226/LR5 cells treated with bortezomib, using a customized microfluidic card (to detect BRCA1, BRCA2, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, RAD51 and RAD51C) and q-PCR. Interestingly, we found that low dose (5nM) bortezomib decreased many FA/BRCA pathway genes as early as 2 hours, with maximal decreases seen at 24 hours. Specifically, 1.5- to 2.5-fold decreases in FANCA, FANCC, FANCD2, FANCE and RAD51C were seen 24 hours post bortezomib exposure. Moreover, pre-treatment of myeloma cells with low dose bortezomib followed by melphalan treatment revealed a greater than 2-fold reduction in FANCD2 gene expression levels. We also found that melphalan treatment alone enhanced FANCD2 protein expression and activation (monoubiquitination), whereas the combination treatment of bortezomib followed by melphalan decreased activation and overall expression of FANCD2 protein. Taken together, these results suggest that bortezomib enhances melphalan response in myeloma by targeting the FA/BRCA pathway. Further understanding of the role of the FA/BRCA pathway in determining melphalan response may allow for more customized and effective treatment of myeloma.

scholarly journals Reduced Chemosensitivity of FLT3/ITD Mutated Cells to Cytarabine and Quizartinib Under Hypoxic Conditions

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1579-1579
Author(s):  
Heiko Konig ◽  
Adriana L Rogozea ◽  
Garrett H Kinnebrew ◽  
Mircea Ivan
Keyword(s):  
Drug Resistance ◽  
Growth Inhibition ◽  
Oxygen Tension ◽  
Glucose Transporter ◽  
Conflicts Of Interest ◽  
Unmet Need ◽  
Leukemic Cells ◽  
Primary Cells ◽  
Glucose Transporter 1 ◽  
Hypoxic Conditions

Abstract Background: FLT3/ITD mutated AML is characterized by short remission duration and high relapse rates due to the survival of a small fraction of leukemic cells (LCs) that outlive initial therapy. There is compelling evidence that the hypoxic niche in the bone marrow (BM) provides a sanctuary where subpopulations of LCs evade cytotoxic therapy and acquire drug resistance. In order to define the mechanisms involved in this process, multiple studies have focused on the interactions between LCs and the BM microenvironment but less is known about to the role of hypoxia as a modulator of drug resistance. Hence, the effects of standard and investigational therapies under hypoxic conditions are largely unknown. Here, we investigated the cytotoxic response of FLT3/ITD-mutated cells to conventional and targeted therapy under normoxic (21% O2) and hypoxic (1% O2) conditions. Methods: Molm14 (M14) cells and primary cells from relapsed/refractory FLT3/ITD mutated AML patients were incubated in culture medium under normoxic and hypoxic conditions. Cytarabine (Cy) or Quizartinib (Quiz) were added as single agents at the indicated concentrations. After 48 hours proliferation and apoptosis assays were performed using MTT assays, annexin V/PI staining and FACS analysis (M14). Cells were also assessed for FLT3 protein and Hypoxia inducible factor (HIF) target gene expression by western blotting and PCR arrays (RT2 ProfilerTM PCR Array, Human Hypoxia Signaling Pathway Plus, Qiagen) (M14 and primary cells), respectively. Results: We found that M14 cells were significantly less susceptible to treatment under hypoxic conditions, both when exposed to Cy and targeted FLT3 inhibition with Quiz (Growth inhibition, 0.25nM Quiz: 8.5±4.2% (1% O2) vs. 22±2.5% (21% O2), p<.05; 49.6±2.5% (1% O2) vs. 58.1±2.3% (21% O2), n=8, p<.05; 0.25µM Cy: 3.7±2.5% (1% O2) vs. 14.4±1.5% (21% O2), p<.01; 1 µM Cy: 26.3±4.1% (1% O2) vs. 53.3±3.8% (21% O2), n=10-12, p<.001). In line with these findings, the apoptotic response of M14 cells to Cy treatment was significantly blunted under hypoxic conditions (Fold increase in apoptotic cells vs. untreated control, 1 µM: 5.7±0.8 fold [21% O2] vs. 2.7±0.5 fold [1% O2], n=4, p<.05). While not reaching the significance threshold, the trend was similar for the Quiz-treated cells. This effect was extended to primary, relapsed/refractory FLT3/ITD-mutated cells derived from patients (n=2) treated at our institution, where growth inhibition was consistently decreased in hypoxia at all doses of Cy and Quiz tested. Not unexpectedly, these primary cells required significantly higher doses compared to the established M14 cell line (1-10µM for Cy, and 50-200nM for Quiz). Importantly, Quiz appeared to be similarly effective in inactivating FLT3 activity under low and high oxygen tension. Therefore identifying tractable pro-survival genes induced by the low oxygen microenvironment should represent a viable strategy to increase the effectiveness of anti-leukemic agents. In order to identify such target candidates, we surveyed the effects of Quiz and Cy on a panel of 84 hypoxia-regulated genes using PCR arrays. Our preliminary studies revealed that while HIF pathway remains largely functional in the presence of Quiz or Cy, these agents severely blunt induction of several hypoxia genes (including Glucose transporter 1 [Glut1] and Carbonic Anhydrase 9 [CA9]) in both primary cells and M14 cells. Conclusions: 1.Hypoxia limits the cytotoxic effects of conventional and targeted therapeutic agents in primary and established FLT3/ITD mutated AML cells. 2. Quizartinib effectively abrogates FLT3 signaling under both normoxic and hypoxic conditions, indicating that other signaling pathways are critical for leukemic cell survival under reduced oxygen tension. 3. The induction of several potentially druggable HIF targets is disrupted by anti-AML agents, warranting the further investigation of combinational approaches between standard anti-AML agents and HIF targeted strategies. 4. Such therapeutic combinations are likely to be particularly effective undermicro-environmental stress conditions (e.g. severe hypoxia) thus addressing an unmet need in AML therapy. Disclosures No relevant conflicts of interest to declare.

Abstract 780: Atorvastatin Activates A Novel Nbs1-dependent Mechanism Of Accelerating Dna Repair In Atherosclerosis

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Melli M Mahmoudi ◽  
Isabelle C Gorenne ◽  
John R Mercer ◽  
Nichola L Figg ◽  
Martin R Bennett
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Oxidant Stress ◽  
Dna Breaks ◽  
Western Blots ◽  
Dna Damage And Repair ◽  
Pre Treatment ◽  
Reactive Oxidant ◽  
Sirna Knockdown ◽  
Dependent Mechanism

There is increasing evidence that reactive oxidant species (ROS) and DNA damage promote the development and complications of atherosclerosis. Although statin therapy reduces both ROS and DNA damage in atherosclerosis, the mechanism of this effect is unknown. We first examined expression of DNA damage and repair markers in vascular smooth muscle cells (VSMCs) of human atherosclerotic plaques. With increasing disease severity, there was increased VSMC expression of the DNA repair markers P-ATM/ATR substrate and P-H2AX from 2.7%±2.2 and 0.5±0.71 [mean±SEM] (AHA Grade I/II), to 21%±3.5 and 36.5±2.1 (Grade III) lesions, and 86.5%±0.7 and 69.3±7.6 (Grade IV/V). Cultured plaque VSMCs also showed a 1.5 fold increased oxidant stress; a 4.4 fold increased double-stranded DNA breaks, and expression of P-H2AX by Western blots. ROS analogues induced a robust DNA damage response in VSMCs, characterised by lengthening of tails on COMET assay, and activation of ATM and P-H2AX, with completion of repair by 6 hours. Atorvastatin pre-treatment accelerated DNA repair by approximately 2 hours without inhibiting ROS induction or DNA damage, and markedly accelerated the kinetics of nibrin (NBS-1) and P-H2AX activation, both proteins recruited to sites of DNA damage, by preventing degradation of NBS-1. Atorvastatin induced phosphorylation of HDM2, an E3 ligase and putative regulator of NBS-1 stability, and siRNA knockdown of HDM2 replicated the effect of atorvastatin on NBS-1. The ability of atorvastatin to accelerate repair was completely dependent upon NBS-1, as atorvastatin was ineffective in cells either null or expressing constitutively active NBS-I. In summary, we have demonstrated a novel NBS-1-dependent mechanism by which statins accelerate DNA repair in atherosclerosis, through HDM2 phosphorylation and stabilisation of NBS-1. We believe that both NBS-1 and HDM2 are critical to DNA repair in atherosclerosis.

ERG Overexpression Induces Cell Adhesion Mediated Drug Resistance in Acute Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1389-1389
Author(s):  
Liliana H. Mochmann ◽  
Martin Neumann ◽  
Verena Nowak ◽  
Monika Dejewska ◽  
Jutta Ortiz-Tanchez ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Adhesion ◽  
Acute Leukemia ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
K562 Cells ◽  
Cellular Adhesion ◽  
Leukemic Cells ◽  
Morphological Transformation ◽  
A Genome

Abstract Abstract 1389 Overexpression of the ETS transcription factor ERG in subtypes of acute lymphoblastic and myeloid leukemias has been correlated with a poor prognosis. The underlying ERG mediated chemotherapy resistance of the leukemic cells may explain the link of poor outcome to ERG expression. We recently observed that ERG overexpression in K562 cells induced adhesion and morphological changes with elongated with bi-directional protrusions. This morphological transformation was mediated by WNT11, a direct target of ERG in acute leukemia. Herein, we determined the ERG transcriptional program responsible for cell adhesion and drug resistance potential. A genome wide transcriptional profiling was performed to identify candidate genes responsible for the observed cell shape changes. mRNA from doxycycline induced K562 cells harbouring tet-on inducible ERG expression constructs (pTRE-Tight-BI-DsRed-ERG) were analyzed with Affymetrix GeneChip (U133 2.0 plus). Non induced clones were used as a control. Genes significantly upregulated after ERG induction included WNT11 and genes associated with biological adhesion included CD44, ITGA10, FLT4, SELP, CD24, TYROBP and SHANK3. The upregulation (≥ 2-fold) of these potentially novel targets of ERG were also validated by RT-PCR. Interestingly, cell cultures of ERG induced cells incubated with a combination of both WNT11 and CD44 antibodies to block cell adhesion showed inhibition of the ERG induced morphological transformation. Furthermore, FACS analysis of ERG induced cells stained for Annexin V showed an increase in apoptosis (24%) by the addition of CD44 and WNT11 antibodies (dilutions 1: 250) whereas the no antibody control was measured at <3%. Non induced cultures were unaffected by the addition of both antibodies compared to the no antibody control. Based on these experiments, we further investigated the contribution of ERG induced cell adhesion to stroma given that leukemia cells interact with the bone marrow microenvironment. Co-cultures of HS5 stroma cells and ERG inducible K562 cells unexpectedly augmented the cell count of ERG induced cells by 2–3-fold on a stromal monolayer versus a non stromal monolayer control. Interestingly, ERG induced cells distinctively had a high induction of apoptosis (> 60%) with the addition of CD44 and WNT11 (dilutions 1: 250) blocking antibodies to co-culture assays. Cell culture of ERG induced K562 cells with a stromal monolayer bed resulted in resistance to cytarabine (Ara-C) and to midostaurin (PKC412). Apoptosis evaluation of DsReD ERG/Annexin V double positive cells treated with either Ara-C (10–30μg/ml) or PKC412 (10–30μM) were measured by FACS at only 14% and 20%, respectively. In contrast, non induced K562 cultures (absent of ERG) had a significantly higher apoptosis induction range: 46% to 55% after exposure to Ara-C and 56% to 65% after exposure to PKC412. We conclude that direct contact and cell adhesion with stromal cells promote an ERG-dependent survival mechanism and mediate resistance to Ara-C or PKC412. In addition, the observed cell death effects by WNT11 and CD44 antibodies in cultures with ERG induced cells strongly indicate their involvement in ERG-dependent cellular adhesion. We propose that, in acute leukemia patients with aberrant ERG overexpression, ERG may mediate genetic and morphological transformation (i.e. cell adhesion) of leukemic cells resulting in an escape mechanism and the development of resistance to chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Somatic Mutations and Loss-Of-Heterozygosity Impair The DNA Repair Functions Of CUX1 in Myelodysplastic Syndromes (MDS)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1246-1246
Author(s):  
Naoko Hosono ◽  
Zubaidah M Ramdzan ◽  
Ranjana Pal ◽  
Hideki Makishima ◽  
Bartlomiej P Przychodzen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Loss Of Heterozygosity ◽  
Research Funding ◽  
Oxidative Dna Damage ◽  
Somatic Mutations ◽  
Excision Repair ◽  
Chromosome 7 ◽  
Chromosomal Translocations ◽  
Leukemic Cells

Abstract Chromosome 7 lesions are common and are associated almost uniformly with a deleterious outcome in MDS and related myeloid neoplasms. We analyzed a large cohort of these patients (pts) (n=1595) and identified those with loss of heterozygosity (LOH) of chromosome 7, including del(7q), monosomy7 (-7) and UPD(7q) in 171 cases. Using single nucleotide polymorphism (SNP)-array karyotyping, 3 commonly deleted regions (CDR) have been isolated, including 7q22, 7q34, and 7q35-36. CDR 7q22 was involved in 119 cases and spanned among others PLOD3, RBL5 and CUX1 genes, which could play a role in del(7q) pathogenesis. To further investigate the molecular pathogenesis of -7/del(7q), we performed whole exome new generation sequencing (NGS) in 428 pts with MDS and related conditions; this cohort included 72 -7/del(7q) or UPD7. When we studied the mutational status of genes in 7q22, we noted 5 cases of CUX1mutations, including 2 with heterozygous and 3 with homozygous mutations (associated with UPD7q). These mutations were validated by Sanger sequencing and targeted deep sequencing of DNA from both tumor and normal cells. All CUX1 mutations revealed critical structural and functional determinants such as nonsense mutations resulting in premature stop codons (n=3), nonsense mutation located in the DNA-binding Cut homeodomain (p.R1296K, n=1) and splice site mutation (n=1). CUX1 mutation occurred in MDS (n=2) and MDS/MPN (n=3), and concomitant TET2 mutations were seen in 4 cases. No hemizygous CUX1mutations were found in cases with del(7q). CUX1 is haploinsufficient in cases with -7/del(7q). In total 166 cases (10.9%) had -7/del(7q) involving CUX1, in addition to pts in whom CUX1 was affected by likely hypomorphic/inactivating mutations. Pts with decreased expression of CUX1 had poor survival compared to pts without CUX1 (p<.01, HR=1.99), suggesting that deficient function (deletion or mutation) of CUX1affects disease progression. In addition to somatic mutations, LOH of CUX1 has been reported in several cancers. A role of CUX1 as a haploinsufficient tumor suppressor cannot be explained by the known functions of CUX1 in stimulating cell proliferation, motility and resistance to apoptosis. We recently identified a novel molecular function of CUX1 in DNA repair that may explain how haplo-deficient expression of CUX1 contributes to leukemic transformation. We used single cell gel electrophoresis (comet assay) to show that Cux1-/- mouse embryo fibroblasts (MEFs) are deficient, while Cux1+/- MEFs are haploinsufficient, in the repair of oxidative DNA damage. Using an inverse-PCR assay, following etoposide exposure, the frequency of chromosomal translocations involving the mixed lineage leukemia (Mll) gene is significantly and progressively increased in Cux1+/- and Cux1-/- MEFs compared to Cux1+/+ MEFs. We then performed comet assays using primary leukemic cells that harbor a frameshift mutation predicted to inactivate CUX1 by producing a C-terminally truncated protein devoid of a nuclear localization signal. Repair of oxidative DNA damage was delayed in leukemic cells compared to bone marrow cells from a healthy donor. Using an in vitrobase excision repair assay, we show that repair of 8-oxoguanine is reduced in a cell line displaying LOH of CUX1. Similarly, repair of 8-oxoguanine is reduced following siRNA-mediated CUX1 knockdown, but is rescued by the addition of a purified CUX1 protein. Together these results demonstrate that CUX1 plays a direct role in DNA repair and that inactivation of one CUX1 allele reduces the DNA repair capability of cells. In conclusion, novel somatic mutations of CUX1 as a candidate gene are associated with poor prognosis in MDS pts with -7/del(7q) and UPD7. Somatic events constitute loss of function of CUX1, resulting in insufficiency of DNA repair mechanisms, which is associated with leukemogenesis and could be considered as a new therapeutic target. CUX1 mutations may affect base excision repair, and dysfunction of CUX1 could theoretically predispose to chromosomal translocations and complex karyotype, seen in conjunction with del(7q) cases. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Abrin induced oxidative stress mediated DNA damage in human leukemic cells and its reversal by N-acetylcysteine

2008 ◽  
Vol 22 (8) ◽  
pp. 1902-1908 ◽  
Author(s):  
A.S.B. Bhaskar ◽  
Utsab Deb ◽  
Om Kumar ◽  
P.V. Lakshmana Rao
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Leukemic Cells ◽  
Human Leukemic Cells

scholarly journals Fusion Tyrosine Kinases Induce Drug Resistance by Stimulation of Homology-Dependent Recombination Repair, Prolongation of G2/M Phase, and Protection from Apoptosis

2002 ◽  
Vol 22 (12) ◽  
pp. 4189-4201 ◽  
Author(s):  
Artur Slupianek ◽  
Grazyna Hoser ◽  
Ireneusz Majsterek ◽  
Agnieszka Bronisz ◽  
Maciej Malecki ◽  
...  
Keyword(s):  
Dna Damage ◽  
Drug Resistance ◽  
Tyrosine Kinases ◽  
Transformed Cells ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Recombination Repair ◽  
M Phase ◽  
Induced Apoptosis ◽  
In Cells

ABSTRACT Fusion tyrosine kinases (FTKs) such as BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFβR, TEL/TRKC(L), and NPM/ALK arise from reciprocal chromosomal translocations and cause acute and chronic leukemias and non-Hodgkin's lymphoma. FTK-transformed cells displayed drug resistance against the cytostatic drugs cisplatin and mitomycin C. These cells were not protected from drug-mediated DNA damage, implicating activation of the mechanisms preventing DNA damage-induced apoptosis. Various FTKs, except TEL/TRKC(L), can activate STAT5, which may be required to induce drug resistance. We show that STAT5 is essential for FTK-dependent upregulation of RAD51, which plays a central role in homology-dependent recombinational repair (HRR) of DNA double-strand breaks (DSBs). Elevated levels of Rad51 contributed to the induction of drug resistance and facilitation of the HRR in FTK-transformed cells. In addition, expression of antiapoptotic protein Bcl-xL was enhanced in cells transformed by the FTKs able to activate STAT5. Moreover, cells transformed by all examined FTKs displayed G2/M delay upon drug treatment. Individually, elevated levels of Rad51, Bcl-xL, or G2/M delay were responsible for induction of a modest drug resistance. Interestingly, combination of these three factors in nontransformed cells induced drug resistance of a magnitude similar to that observed in cells expressing FTKs activating STAT5. Thus, we postulate that RAD51-dependent facilitation of DSB repair, antiapoptotic activity of Bcl-xL, and delay in progression through the G2/M phase work in concert to induce drug resistance in FTK-positive leukemias and lymphomas.

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