From single-strand breaks to double-strand breaks during S-phase: a new mode of action of the Escherichia coli Cytolethal Distending Toxin.

The Cytolethal Distending Toxin (CDT) is a genotoxin produced by several pathogenic bacteria. It is generally admitted that CDT induces double-strand breaks (DSB) and cell cycle arrest in G2/M-phase, in an ATM-dependent manner. Most of these results were obtained at high dose (over 1 µg ml(-1) ) of CDT and late after treatment (8-24 h). We provide here evidence that the Escherichia coli CDT (EcCDT) - at low dose (50 pg ml(-1) or LD50) and early after treatment (3-6 h) - progressively induces DNA DSB, mostly in S-phase. DSB formation is related to the single-strand breaks induction by CDT, converted into DSB during the S-phase. We also show that homologous recombination is mobilized to these S-phase-associated DSB. This model unveils a new mechanism for CDT genotoxicity that may play a role in cells partly deficient in homologous recombination.

Identifying the electronic character and role of the Mn states in the valence band of (Ga,Mn)As.

We report high-resolution hard x-ray photoemission spectroscopy results on (Ga,Mn)As films as a function of Mn doping. Supported by theoretical calculations we identify, for both low (1%) and high (13%) Mn doping values, the electronic character of the states near the top of the valence band. Magnetization and temperature-dependent core-level photoemission spectra reveal how the delocalized character of the Mn states enables the bulk ferromagnetic properties of (Ga,Mn)As.

Acute effect of resistance training volume on hormonal responses in trained men.

AIM: The purpose of this study was to investigate the acute hormonal response to resistance training sessions with different volumes in men. METHODS: Ten recreationally trained men (24.5±7.6 years; 76.2±9.2 kg; 175.6±1.5 cm; 24.5±5.5 kg/m(-2)) participated in the study. All subjects completed two experimental protocols with different volumes. The first protocol consisted of 3 sets at 80% of 6 RM and the second protocol was 3 sets at 80% of 12 RM with 2 minutes rest between sets and exercises, separated by seven days between them. The exercise order used was: barbell bench press, leg press, machine front lat-pull down, leg curl, shoulder abduction and leg extension. The blood variables analyzed were: testosterone, growth hormone (GH), cortisol and testosterone/cortisol ratio (T:C) before (Pre) and immediately after (Post) each exercise session. RESULTS: The intra-groups comparison for testosterone and hGH revealed a significant increase in 80%-6RM and 80%-12RM. Cortisol levels was significantly higher in 80%-12RM and T:C ratio in 80% 6 RM when compared Pre and Post values. Inter-groups comparison showed higher hGH and cortisol levels and lower T:C ratio for 80% 12 RM. There was no statistically significant different between 80%-6RM and 80%-12RM for testosterone. CONCLUSION: The present study confirms that the volume of resistance training can be an important factor in the modulation of acute hormonal responses.

Effects of resistance training on cytokines.

It is speculated that exercise training decreases resting levels of tumor necrosis factor alpha (TNF-alpha) and C-reactive protein (CRP); reduces body mass and leptin (LP); and increases adiponectin (AD) and insulin sensitivity. This systematic review analyzed the effectiveness of resistance training (RT) longitudinal clinical studies on AD, LP, CRP and TNF-alpha. Seventeen studies were included and the majority of randomized controlled trials support that RT produces increases in AD, and decreases in both LP and CRP. Greater responses in AD and LP were evident in overweight and obese individuals; while RT appeared to be effective in reducing CRP in obese individuals, and older adults. Additionally, women may be more responsive to RT effects on AD, LP and CRP. Training duration and intensity may affect the response of AD and CRP with greater responses shown with 16 weeks or more of training and/or with intensities greater than 80% of one repetition maximum. No response to RT of TNF-alpha levels was apparent. Although based on a limited number of studies, some of which are uncontrolled non-randomized in design, our review suggests some positive effects of RT programs on cytokine levels, but specifics of the responses in different populations need further elucidation.

Impaired telomerase activity in human cells expressing GFP-Ku86 fusion proteins.

The Ku heterodimer is a DNA end-binding protein that promotes the non-homologous end joining (NHEJ) pathway of DNA double strand break (DSB) repair by recruiting the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). Ku is also a normal component of telomeres where it is required for telomere maintenance, interacting not only with the DNA but also with various telomere proteins including telomerase. The way in which Ku simultaneously plays such distinct roles, end-joining at DSBs and end-maintenance at telomeres, is unclear. One way to address this is to study cells in which the NHEJ and telomeric roles of Ku have been separated. Here we describe human cells that express fusions between the large human Ku subunit (Ku86) and a fluorescent protein tag. These cells have reduced telomerase activity and increased sensitivity to ionizing radiation (IR) but no change in their DNA-PK activity or in the DNA end-binding of endogenous Ku. Cells with particularly large amounts of one fusion protein undergo progressive telomere shortening and cellular senescence. These data are consistent with models in which Ku recruits telomerase to telomeres or activates recruited telomerase and suggest that the Ku86 fusion proteins specifically block this role.

Rat feeding trials: A comprehensive assessment of contaminants in both genetically modified maize and resulting pellets.

We analyzed a comprehensive set of contaminants in MON810 and NK603 genetically modified (GM) maize, and their non-GM counterparts, used in a rat feeding study (the GMO90 + project). Both the maize grains and the manufactured pellets were characterized. Only minor differences in contaminant levels between GM and corresponding non-GM harvests were evidenced. Fumonisin and deoxynivalenol mycotoxins were the pollutants present in the highest amounts, with concentrations that were however largely below acceptance reference values. Our data reporting slightly lower levels of fumonisin in MON810 compared to its non-GM counterpart corroborate the lower susceptibility of insect resistant Bt maize to fumonisin-producing fungi. Traces of glyphosate (0.016 mg/kg) were evidenced in grains from NK603 treated crops. Regarding the pellets, analysis of more than 650 potentially toxic substances revealed low amounts of various mycotoxins, pesticides and heavy metals. Concentrations of contaminants quantified in the pellets were however far below the maximum level of residues values set by regulatory agencies, and no substantial differences in contaminants between GM and non-GM pellets were observed. Moreover, when comparing the contamination status of grains and pellets, we demonstrate yet again that characterizing the grains is actually not sufficient to foresee the quality of the produced pellets.

Analysis of the correlation between knee extension torque and patellar tendon elastic property.

PURPOSE: Quadriceps strength and patellar tendon (PT) are directly linked and intimately related to daily activities and lower limb function. However, the correlation between knee extension torque (KT) and PT Young's modulus (E) measured directly is still unknown. METHOD: We used supersonic shearwave imaging (SSI) to evaluate the elastic property of PT in healthy young men and analysed its correlation with KT. Twenty-two healthy young males were included and both knees were examined. The E of the PT in the dominant and non-dominant legs was assessed by SSI elastography. KT in maximal voluntary isometric contraction was measured with an isokinetic dynamometer. RESULT: No correlations between KT and PT E were observed in dominant or non-dominant side (P = 0·458 and 0·126, respectively). No significant differences in KT or PT E were observed between both legs (P = 0·096 and 0·722, respectively). Intra-day ICC was rated good (D1 - 0·886, P<0·001 and 0·88, P<0·001) and excellent (D2 - 0·928, P<0·001 and 0·900, P<0·001) for both legs. Inter-day ICC was rated moderate for both legs (0·651, P = 0·016 and 0·630, P = 0·018, respectively). CONCLUSION: No significant correlations were found between KT and PT E, suggesting that quadriceps strength is not an accurate predictor for PT mechanical properties in subjects with no specific training engagement. Habitual loading pattern can play a determinant role in PT mechanical properties, regardless of quadriceps strength. Further investigation on SSI acquisition protocols should be conducted to guarantee higher inter-day ICC values.

DNA replication but not nucleotide excision repair is required for UVC-induced replication protein A phosphorylation in mammalian cells.

Exposure of mammalian cells to short-wavelength light (UVC) triggers a global response which can either counteract the deleterious effect of DNA damage by enabling DNA repair or lead to apoptosis. Several stress-activated protein kinases participate in this response, making phosphorylation a strong candidate for being involved in regulating the cellular damage response. One factor that is phosphorylated in a UVC-dependent manner is the 32-kDa subunit of the single-stranded DNA-binding replication protein A (RPA32). RPA is required for major cellular processes like DNA replication, and removal of DNA damage by nucleotide excision repair (NER). In this study we examined the signal which triggers RPA32 hyperphosphorylation following UVC irradiation in human cells. Hyperphosphorylation of RPA was observed in cells from patients with either NER or transcription-coupled repair (TCR) deficiency (A, C, and G complementation groups of xeroderma pigmentosum and A and B groups of Cockayne syndrome, respectively). This exclude both NER intermediates and TCR as essential signals for RPA hyperphosphorylation. However, we have observed that UV-sensitive cells deficient in NER and TCR require lower doses of UV irradiation to induce RPA32 hyperphosphorylation than normal cells, indicating that persistent unrepaired lesions contribute to RPA phosphorylation. Finally, the results of UVC irradiation experiments on nonreplicating cells and S-phase-synchronized cells emphasize a major role for DNA replication arrest in the presence of UVC lesions in RPA UVC-induced hyperphosphorylation in mammalian cells.

TATA binding protein discriminates between different lesions on DNA, resulting in a transcription decrease.

DNA damage recognition by basal transcription factors follows different mechanisms. Using transcription-competition, nitrocellulose filter binding, and DNase I footprinting assays, we show that, although the general transcription factor TFIIH is able to target any kind of lesion which can be repaired by the nucleotide excision repair pathway, TATA binding protein (TBP)-TFIID is more selective in damage recognition. Only genotoxic agents which are able to induce kinked DNA structures similar to the one for the TATA box in its TBP complex are recognized. Indeed, DNase I footprinting patterns reveal that TBP protects equally 4 nucleotides upstream and 6 nucleotides downstream from the A-T (at position -29 of the noncoding strand) of the adenovirus major late promoter and from the G-G of a cisplatin-induced 1,2-d(GpG) cross-link. Together, our results may partially explain differences in transcription inhibition rates following DNA damage.

Potentiation of chlorambucil cytotoxicity in B-cell chronic lymphocytic leukemia by inhibition of DNA-dependent protein kinase activity using wortmannin.

In this study, we examined the ability of wortmannin to modulate chlorambucil (CLB) cytotoxicity in lymphocyte samples from patients with B-cell chronic lymphocytic leukemia (B-CLL). It has been suggested previously that enhanced cross-link repair is a primary mechanism of resistance to nitrogen mustards (NMs) in B-CLL. DNA-dependent protein kinase (DNA-PK) is involved in the repair of double-strand breaks and in rejoining steps in recombination mechanisms. Mutants defective in this process are hypersensitive to alkylating agents. We have recently demonstrated that the activity of DNA-PK is a determinant in the cellular response of B-CLL to CLB. The DNA-PK gene has homology to the P110 phosphatidylinositol 3-kinase (PI 3-K). Wortmannin, an inhibitor of P110 PI 3-K, also inhibits DNA-PK activity in vitro. We investigated the effect of wortmannin on DNA-PK activity and CLB toxicity in the lymphocytes from 11 patients with B-CLL. Our results demonstrate that DNA-PK activity is decreased after exposure to wortmannin in a dose-dependent manner. Wortmannin, at nontoxic concentrations, synergistically sensitized B-CLL lymphocytes to the effects of CLB. Moreover, we observed a significant correlation when we compared the fold decrease in DNA-PK activity and the synergistic value (I), obtained when wortmannin was used at 0.1 microM. In the resistant B-CLL lymphocyte samples, there was a highly significant correlation between the ability of wortmannin at 0.1 and 0.25 microM to decrease the level of DNA-PK activity and to increase CLB sensitivity. In a model of primary human tumor cells, our findings suggest that the inhibition of DNA-PK activity may be a powerful way to overcome resistance to NMs such as CLB and point to new possibilities to improve the effectiveness of NM therapy.

A DNA double-strand break defective fibroblast cell line (180BR) derived from a radiosensitive patient represents a new mutant phenotype.

The 180BR cell line was derived from an acute lymphoblastic leukemia patient who overresponded to radiation therapy and died following radiation morbidity. 180BR cells are hypersensitive to the lethal effects of ionizing radiation and are defective in the repair of DNA double-strand breaks (DSBs). The levels and activity of the proteins of the DNA-dependent protein kinase complex are normal in 180BR cells. To facilitate a measurement of V(D)J recombination, we have characterized 180BRM, a SV40-transformed line derived from 180BR. 180BRM retains the radiosensitivity and defect in DSB repair characteristic of 180BR. The activities associated with DNA-dependent protein kinase are also normal in 180BRM cells. The ability to carry out V(D)J recombination is comparable in 180BRM and a reference control transformed human cell line, MRC5V1. These results show that 180BR and 180BRM differ from the rodent mutants belonging to ionizing radiation complementation groups 4, 5, 6, and 7 and, therefore, represent a new mutant phenotype, in which a defect in DNA DSB rejoining is not associated with defective V(D)J recombination. Furthermore, we have shown that 180BR can arrest at the G1-S and G2-M cell cycle checkpoints after irradiation. These results confirm that 180BR can be distinguished from ataxia telangiectasia.

Regulation of DNA-dependent protein kinase activity in leukemic cells.

The DNA-dependent protein kinase (DNA-PK) complex is composed of a catalytic (DNA-PKcs), and a regulatory subunit (Ku70/Ku86 heterodimer). The expression and function of DNA-PK subunits was investigated in purified blood lymphocytes obtained from patients with chronic lymphocytic leukemia (CLL) either refractory to chemotherapy or untreated. Variations in DNA-PK activity were found amongst CLL samples by comparison to human cell lines. It was noticeable that the low DNA-PK activity was associated with samples from untreated patients that exhibited a sensitivity phenotype, determined in vitro, to the radiomimetic agent neocarcinostatin by comparison to samples from refractory patients. The regulation in DNA-PK activity was associated with Ku heterodimer expression while DNA-PKcs was unaffected. Moreover, the presence of an altered form of the Ku86 subunit was identified in samples with low DNA-PK activity. These results suggest a regulation process of the DNA-PK activity in fresh human cells.

Human normal peripheral blood B-lymphocytes are deficient in DNA-dependent protein kinase activity due to the expression of a variant form of the Ku86 protein.

The heterodimeric Ku protein, which comprises a 86 kDa (Ku86) amd a 70 kDa (Ku70) subunits, is an abundant nuclear DNA-binding protein which binds in vitro to DNA termini without sequence specificity. Ku is the DNA-targeting component of the large catalytic sub-unit of the DNA-dependent protein kinase complex (DNA-PK[CS]), that plays a critical role in mammalian double-strand break repair and lymphoid V(D)J recombination. By using electrophoretic mobility shift assays, we demonstrated that in addition to the major Ku x DNA complex usually detected in cell line extracts, a second complex with faster electrophoretic mobility was observed in normal peripheral blood lymphocytes (PBL) extracts. The presence of this faster migrating complex was restricted to B cells among the circulating lymphocyte population. Western blot analysis revealed that B cells express a variant form of the Ku86 protein with an apparent molecular weight of 69 kDa, and not the 86 kDa- full-length protein. Although the heterodimer Ku70/variant-Ku86 binds to DNA-ends, this altered form of the Ku heterodimer has a decreased ability to recruit the catalytic component of the complex, DNA-PK(CS), which contributes to an absence of detectable DNA-PK activity in B cells. These data provide a molecular basis for the increased sensitivity of B cells to ionizing radiation and identify a new mechanism of regulation of DNA-PK activity that operates in vivo.

Inhibition of Ku heterodimer DNA end binding activity during granulocytic differentiation of human promyelocytic cell lines.

The heterodimeric Ku protein (composed of the Ku 86 and Ku 70 sub-units) is a nuclear protein which binds to DNA termini without sequence specificity. Ku is the DNA-targeting component of the large catalytic sub-unit of the DNA-dependent protein kinase complex that is required for the repair of DNA double-strand breaks in mammalian cells. We studied the expression and function of Ku/DNA-PK during granulocytic differentiation of two human promyelocytic cell lines, HL60 and NB4, a process associated to decreased radiation resistance. After 3 days exposure to differentiating agents (either all-trans-retinoic acid or DMSO), Ku binding to double stranded (ds)-DNA ends declined dramatically whereas Ku protein levels remain unchanged. The nuclear, but not cytoplasmic, fraction of differentiated HL60 cells extracts exhibited a heat-sensitive inhibitory activity towards DNA binding of recombinant Ku heterodimer. We further demonstrate that immunoprecipitation of Ku is impaired in extracts from differentiated cells by using two antibodies that recognize epitopes within the C-terminus DNA binding domains of Ku 70 and Ku 86 proteins. These results favor the hypothesis of a protein interacting with Ku that would prevent DNA binding of heterodimerized Ku protein by steric hindrance.

Mutator phenotype of BCR--ABL transfected Ba/F3 cell lines and its association with enhanced expression of DNA polymerase beta.

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome resulting from the translocation t(9-22) producing the chimeric 190 and 210 kDa BCR-ABL fusion proteins. Evolution of the CML to the more agressive acute myelogenous leukemia (AML) is accompanied by increased cellular proliferation and genomic instability at the cytogenetic level. We hypothezised that genomic instability at the nucleotide level and spontaneous error in DNA replication may also contribute to the evolution of CML to AML. Murine Ba/F3 cell line was transfected with the p190 and p210-encoding BCR-ABL oncogenes, and spontaneous mutation frequency at the Na-K-ATPase and the hypoxanthine guanine phosphoribosyl transferase (HPRT) loci were measured. A significant 3-5-fold increase in mutation frequency for the transfected cells relative to the untransfected control cells was found. Furthermore, we observed that BCR-ABL transfection induced an overexpression of DNA polymerase beta, the most inaccurate of the mammalian DNA polymerases, as well as an increase in its activity, suggesting that inaccuracy of DNA replication may account for the observed mutator phenotype. These data suggest that the Philadelphia abnormality confers a mutator phenotype and may have implications for the potential role of DNA polymerase beta in this process.

Elderly patients with aggressive non-Hodgkin's lymphoma: disease presentation, response to treatment, and survival--a Groupe d'Etude des Lymphomes de l'Adulte study on 453 patients older than 69 years.

PURPOSE: To clarify disease characteristics and optimal treatment for elderly patients with non-Hodgkin's lymphoma (NHL), we performed a randomized trial in 453 patients older than 69 years with aggressive lymphoma. PATIENTS AND METHODS: Two hundred twenty patients received cyclophosphamide 750 mg/m2, teniposide (VM-26) 75 mg/m2, and prednisone 40 mg/m2/d for 5 days (CVP) and 233 patients received CVP plus pirarubicin (THP-doxorubicin) 50 mg/m2 (CTVP), each for six courses every 3 weeks. RESULTS: The median age was 75 years. Most patients had clinically aggressive disease; 30% had one and 53% two or three adverse prognostic parameters as defined by the International Prognostic Index. More patients on the CTVP arm had an elevated lactic dehydrogenase (LDH) level, but the two groups were otherwise well balanced. CTVP treatment was more frequently associated with leukopenia, thrombocytopenia, and infectious complications. Death during chemotherapy occurred in 16% and 21% of patients on the CVP and CTVP arms, respectively (not significant). Forty percent of patients achieved a complete response (CR): 47% on CTVP and 32% on CVP (chi2 = 20.98, P = .0001). The median time to treatment failure (TTF) was 7 months for CTVP versus 5 months for CVP (log-rank test, P < .05). The median survival time was 13 months in both groups; however, the 5-year survival rate was 26% with CTVP versus 19% with CVP (chi2 = 4.68, P < .05). Lymphoma progression was the primary cause of death. CONCLUSION: Elderly patients with aggressive lymphoma have an aggressive disease with adverse prognostic parameters at the time of diagnosis. Slightly longer survival was observed for patients treated with an anthracycline-containing regimen.

Ku70/Ku80 protein complex inhibits the binding of nucleotide excision repair proteins on linear DNA in vitro.

We have previously reported that the incision efficiency of the nucleotide excision repair (NER) reaction measured in vitro with cell-free human protein extracts was reduced by up to 80% on a linearized damaged plasmid DNA substrate when compared to supercoiled damaged DNA. The inhibition stemed from the presence of the DNA-end binding Ku70/Ku80 heterodimer which is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). Here, the origin of the repair inhibition was assessed by a new in vitro assay in which circular or linear plasmid DNA, damaged or undamaged, was quantitatively adsorbed on sensitized microplate wells. The binding of two NER proteins, XPA and p62-TFIIH, indispensable for the incision step of the reaction, was quantified either directly in an ELISA-like reaction in the wells with specific antibodies or in Western blotting experiments on the DNA-bound fraction. We report a dramatic inhibition of XPA and p62-TFIIH association with UVC photoproducts on linear DNA. XPA and p62-TFIIH binding to DNA damage was regained when the reaction was performed with extracts lacking Ku activity (extracts from xrs6 rodent cells) whereas addition of purified human Ku complex to these extracts restored the inhibition. Despite the fact that DNA-PK was active during the NER reaction, the mechanism of inhibition relied on the sole Ku complex, since mutant protein extracts lacking the catalytic DNA-PK subunit (extracts from the human M059J glioma cells) exhibited a strong binding inhibition of XPA and p62-TFIIH proteins on linear damaged DNA, identical to the inhibition observed with the DNA-PK+ control extracts (from M059K cells).

Interactions of the transcription/DNA repair factor TFIIH and XP repair proteins with DNA lesions in a cell-free repair assay.

We have studied the interactions between DNA damage and human proteins involved in the early steps of nucleotide excision repair (NER) reaction under in vitro conditions with human protein extracts. By using a new assay, we have detected a long-lived DNA/protein complex involving XPA and TFIIH in the course of the NER process. The formation of this complex is exclusively limited to DNA lesions that are substrates of the human excinuclease. We show that, while XPA binding to damaged DNA is ATP-independent, stable association of TFIIH with DNA lesions is promoted by ATP hydrolysis and is dependent on the integrity of XPA and XPC proteins in the cell extract. In addition, XPC is necessary to promote a stable binding of XPA to UV-irradiated DNA. Finally, the co-binding of XPA and TFIIH to DNA damage is correlated to a dose-dependent titration of TFIIH and not XPA from the free protein fraction.

Late autologous transplantation in chronic myelogenous leukemia with peripheral blood progenitor cells mobilized by G-CSF and interferon-alpha.

In chronic myelogenous leukemia (CML), autologous stem cell transplantation could be a promising new approach for patients with no cytogenetic response after interferon alpha (IFN-alpha) therapy. We report data on 28 CML patients autotransplanted in chronic phase with peripheral blood progenitor cells mobilized with G-CSF (5 microg/kg/day x 5 days) given subcutaneously while continuing IFN-alpha therapy. At mobilization, 23 patients (82%) were in complete hematological remission (CHR), 16 (57%) achieved a minor cytogenetic response (mcr). We obtained, after stimulation, a median of 37.4 x 10(9)/l (6.9-108) white blood cells, 7.2 x 10(8)/kg (2.2-16.6) mononuclear cells, 39 x 10(4)/kg (4.8-403.5) CFU-GM and 4.2 x 10(6)/kg (0-58.6) CD34+ cells. Six patients received GM-CSF after transplantation. All patients engrafted, with no significant influence stemming from the Sokal index score and pretransplantation IFN-alpha therapy duration. The first cytogenetic evaluation after transplantation showed 11 (39%) major cytogenetic response (Mcr), and nine (32%) mcr with no significant correlation between these responses, the Sokal index score, and pretransplantation IFN-alpha therapy duration, although there was a significant impact from GM-CSF administration (P=0.01). After transplantation, 26 patients received IFN-alpha alone or associated with hydroxyurea. The median follow-up was 12 months after transplantation and 57 months after diagnosis. At the time of follow-up, nine patients were in CHR, six remained stable in chronic phase, three presented an mcr and one remained in Mcr. At the last follow-up, 22 patients were alive. We conclude that the results of this strategy are encouraging in poor IFN-alpha responders but that other prospective studies that try to maintain the cytogenetic responses obtained immediately after transplantation are needed.

Report of 34 patients with clonal chromosomal abnormalities in Philadelphia-negative cells during imatinib treatment of Philadelphia-positive chronic myeloid leukemia.

Imatinib mesylate (Gleevec), an inhibitor of the BCR-ABL tyrosine kinase, was introduced recently into the therapy of chronic myeloid leukemia (CML). Several cases of emergence of clonal chromosomal abnormalities after therapy with imatinib have been reported, but their incidence, etiology and prognosis remain to be clarified. We report here a large series of 34 CML patients treated with imatinib who developed Philadelphia (Ph)-negative clones. Among 1001 patients with Ph-positive CML treated with imatinib, 34 (3.4%) developed clonal chromosomal abnormalities in Ph-negative cells. Three patients were treated with imatinib up-front. The most common cytogenetic abnormalities were trisomy 8 and monosomy 7 in twelve and seven patients, respectively. In 15 patients, fluorescent in situ hybridization with specific probes was performed in materials archived before the initiation of imatinib. The Ph-negative clone was related to previous therapy in three patients, and represented a minor pre-existing clone that expanded after the eradication of Ph-positive cells with imatinib in two others. However, in 11 patients, the new clonal chromosomal abnormalities were not detected and imatinib may have had a direct effect. No myelodysplasia was found in our cohort. With a median follow-up of 24 months, one patient showed CML acceleration and two relapsed.