Induction of AML Preleukemic Fusion Genes in HSPCs and DNA Damage Response in Preleukemic Fusion Gene Positive Samples.

Preleukemic fusion genes (PFGs) occurring after DNA damage in hematopoietic stem progenitor cells (HSPCs) in utero often represent the initial event in the development of childhood leukemia. While the incidence of PFGs characteristic for acute lymphoblastic leukemia (ALL) was relatively well examined by several research groups and estimated to be 1-5% in umbilical cord blood (UCB) of healthy newborns, PFGs that are relevant to acute myeloid leukemia (AML) were poorly investigated. Therefore, this study is focused on the estimation of the incidence of the most frequent AML PFGs in newborns. For the first time, this study considered the inducibility of AML PFGs in different subsets of UCB HSPCs by low-dose γ-rays and also compared endogenous DNA damage, apoptosis, and reactive oxygen species (ROS) level between UCB samples containing or lacking AML PFGs. We found that: (i) the incidence of AML PFGs in UCB was 3.19% for RUNX1-RUNX1T1, 3.19% for PML-RARα, and 1.17% for KMT2A-MLLT3, (ii) 50 cGy of γ-rays did not induce RUNX1-RUNX1T1, PML-RARα, or KMT2A-MLLT3 PFGs in different subsets of sorted and expanded HSPCs, and (iii) the AML PFG+ samples accumulated the same level of endogenous DNA damage, as measured by the γH2AX/53BP1 focus formation, and also the same ROS level, and apoptosis as compared to PFG- controls. Our study provides critical insights into the prevalence of AML PFGs in UCB of newborns, without the evidence of a specific HSPC population more susceptible for PFG formation after irradiation to low-dose γ-rays or increased amount of ROS, apoptosis and DNA damage.

Backtracked analysis of preleukemic fusion genes and DNA repair foci in umbilical cord blood of children with acute leukemia.

The first event in origination of many childhood leukemias is a specific preleukemic fusion gene (PFG) that arises, often in utero, in hematopoietic stem/progenitor cells (HSPC) from misrepaired DNA double strand break (DSB). An immanently elevated level of DSB and impaired apoptosis may contribute to origination and persistence of PFG and donor cell-derived leukemia in recipients of allogeneic transplantation of umbilical cord blood (UCB). We investigated DSB, apoptosis and PFG in the backtracked UCB cells of leukemic patients. RNA from UCB of three patients with acute lymphoblastic leukemia, patient with acute megakaryoblastic leukemia and Down syndrome, and four healthy children was screened for common PFG by RT-qPCR. Presence of PFG was validated by sequencing. Endogenous γH2AX and 53BP1 DNA repair foci, cell populations, and apoptosis were analyzed in UCB CD34+/- cells with imaging and standard flow cytometry. We found MLL2-AF4 and BCR-ABL (p190) fusion genes in UCB of two out from four pediatric patients, apparently not detected at diagnosis, while UCB cells of TEL-AML1+ ALL patient were tested negative for this PFG and no PFG were detected in UCB cells of healthy children. No significant difference in DNA damage and apoptosis between UCB CD34+/- cells from healthy children and leukemic patients was observed, while Down syndrome trisomy increased DNA damage and resulted in distribution of cell populations resembling transient abnormal myelopoiesis. Our findings indicate increased genetic instability in UCB HSPC of leukemic patients and may be potentially used for diagnostics and exclusion of possibly affected UCB from transplantation.

Incidence of common preleukemic gene fusions in umbilical cord blood in Slovak population.

The first event in origination of many childhood leukemias is likely the presence of preleukemic clone (transformed hematopoietic stem/progenitor cells with preleukemic gene fusions (PGF)) in newborn. Thus, the screening of umbilical cord blood (UCB) for PGF may be of high importance for developing strategies for childhood leukemia prevention and treatment. However, the data on incidence of PGF in UCB are contradictive. We have compared multiplex polymerase chain reaction (PCR) and real-time quantitative PCR (RT qPCR) in neonates from Slovak National Birth Cohort. According to multiplex PCR, all 135 screened samples were negative for the most frequent PGF of B-lineage acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). To explore the prevalence of prognostically important TEL-AML1, MLL-AF4 and BCR-ABL (p190), 200 UCB were screened using RT qPCR. The initial screening showed an unexpectedly high incidence of studied PGF. The validation of selected samples in two laboratories confirmed approximately » of UCB positive, resulting in ∼4% incidence of TEL-AML1, ∼6.25% incidence of BCR-ABL1 p190, and ∼0.75% frequency of MLL-AF4. In most cases, the PGF presented at very low level, about 1-5 copies per 105 cells. We hypothesize that low PGF numbers reflect their relatively late origin and are likely to be eliminated in further development while higher number of PGF reflects earlier origination and may represent higher risk for leukemia.

DNA damage response in CD133 + stem/progenitor cells from umbilical cord blood: low level of endogenous foci and high recruitment of 53BP1.

UNLABELLED: Abstract Purpose: Human hematopoietic stem cells (HSC) are thought to be a major target of radiation-induced leukemogenesis and also provide a relevant cellular model for assessing cancer risk. Cluster of designation 133+ (CD133+) is a marker found in human progenitor and hematopoietic stem cells. Our study examined the repair of radiation-induced DNA double-strand breaks (DSB) in CD133 + umbilical cord blood cells (UCBC). MATERIALS AND METHODS: After γ-irradiation, endogenous and induced DSB were evaluated in CD133 + UCBC, CD133 - UCBC and peripheral blood lymphocytes (PBL) in terms of phosphorylated histone 2A family member X (γH2AX) and tumor suppressor p53 binding protein 1 (53BP1) foci. RESULTS: We found that repair signaling in CD133 + UCBC is different from CD133 - UCBC and PBL. These differences include lower endogenous DSB levels and higher 53BP1 recruitment. CONCLUSIONS: Our data, together with a recent report on radiation-induced γH2AX and 53BP1 foci in CD34 + cells, indicate enhanced DNA repair capacity in HSC as compared to mature lymphocytes.

Umbilical cord blood cells CD133+/CD133- cultivation in neural proliferation media differentiates towards neural cell lineages.

BACKGROUND AND AIMS: Umbilical cord blood (UCB) has been identified as a good source of hematopoietic and nonhematopoietic stem cells that can be easily isolated. In the present study we investigated the possibility of whether stem cells in mononuclear UCB grown under defined conditions can produce progeny with neural phenotype. METHODS: A combination of antigen-driven magnetic cell sorting (MACs) method and defined culture conditions specific for cells of neural lineages were used for isolation, expansion and differentiation of CD133+/- cells from UCB. Both UCB-derived fractions were expanded by exposure to growth factors (EGF, bFGF). Differentiation was induced by replacing them with fetal bovine serum. Using immunocytochemistry, the cell markers for neural (MAP2, GFAP, RIP) and non-neural lineages (S-100, von Willebrand factor) were detected. RESULTS: The analysis revealed occurrence of fully mature neural and non-neural lineages, which showed qualitative and quantitative differences between population of CD133+ and CD133- cells. The expression levels of MAP2 and RIP in CD133+ were significantly higher than in CD133-, more GFAP positive cells were found in the CD133-. At the same time, S-100 was expressed by 32.47 ± 6.24% of CD133- cells and 29.42 ± 1.32% of CD133- cell expressed a von Willebrand factor antigen. CONCLUSIONS: Our results indicate that stem cells derived from umbilical cord blood are easy to obtain, proliferate and are able to differentiate towards the cells of neural lineages, which represents a promising way for their utilization in cell-based therapies for CNS injuries and diseases.

Comparative study of four fluorescent probes for evaluation of natural killer cell cytotoxicity assays.

Cytotoxicity is one of the major defence mechanisms against both virus-infected and tumor cells. Radioactive (51)chromium ((51)Cr) release assay is a "gold standard" for assessment of natural killer (NK) cytolytic activity in vitro. Several disadvantages of this assay led us to design alternative tools based on flow cytometry analysis. Four different fluorescent dyes, calcein acetoxymethyl ester (CAM), carboxyfluorescein succinimidyl ester (CFSE), Vybrant DiO (DiO) and MitoTracker Green (MTG) were tested for labeling of NK target K-562 cells. Target staining stability, spontaneous release of fluorochromes and subsequent accumulation in bystander unstained cells were measured using fluorimetry and flow cytometry. Healthy donor peripheral blood mononuclear cells and affinity column purified NK cells were used as effectors coincubated with target K-562 cells at different E:T ratios for 3h and 90 min, respectively. Fluorescent probe 7-amino-actinomycin D was used for live and dead cell discrimination. Bland-Altman statistical method was applied to measure true agreement for all CAM-(51)Cr, CFSE-(51)Cr, DiO-(51)Cr and MTG-(51)Cr pairs analyzed. Based on the data, none of the four proposed methods can be stated equivalent to the standard (51)Cr release assay. Considering linear relationships between data obtained with four fluorochromes and (51)Cr release assay as well as linear regression analysis with R(2)=0.9393 value for CAM-(51)Cr pair, we found the CAM assay to be the most closely related to the (51)Cr assay.

Autologous biograft and mesenchymal stem cells in treatment of the diabetic foot.

OBJECTIVES: This study was performed to test a new technique for treatment of chronic non-healing wound (diabetic ulcer) using autologous biograft composed of autologous skin fibroblasts on biodegradable collagen membrane (Coladerm) in combination with autologous mesenchymal stem cells (MSC) derived from the patient's bone marrow. DESIGN: The bone marrow aspirate of the patient with diabetic foot was applied directly to the wound and injected into the edges of the wound, finally covered with prepared autologous biograft. The patient received two additional treatments with cultured MSC on day 7 and 17. RESULTS: The wound showed a steady overall decrease in wound size and an increase in the vascularity of the dermis and in the dermal thickness of the wound bed after 29 days of combined treatment. CONCLUSIONS: Closing and healing of the non-healing diabetic ulcer was achieved by using the given combined therapy.

Extracellular acidosis elevates carbonic anhydrase IX in human glioblastoma cells via transcriptional modulation that does not depend on hypoxia.

Most solid tumors display extracellular acidosis, which only partially overlaps with hypoxia and induces distinct adaptive changes leading to aggressive phenotype. Although acidosis is mainly attributable to excessive production of lactic acid, it also involves carbonic anhydrase (CA) IX-mediated conversion of CO(2) to an extracellular proton and a bicarbonate ion transported to cytoplasm. CA IX is pre-dominantly expressed in tumors with poor prognosis and its transcription and activity are induced by hypoxia. Here we investigated whether low extracellular pH in absence of hypoxia can influence CA IX expression in cell lines derived from glioblastoma, a tumor type particularly linked with acidosis. Our data show that extracellular acidosis increased the level of CA IX protein, mRNA and the activity of minimal CA9 promoter that contains binding sites for HIF-1 and SP-1 transcription factors. Mutation within each of these two biding sites reduced the promoter activity, but did not eliminate the increase by acidosis. Transfection of HIF-1alpha cDNA produced additive inducing effect with acidosis. Normoxic acidosis was accompanied by HIF-1alpha protein accumulation and transiently increased phosphorylation of ERK1/2. Expression of a dominant-negative mutant of ERK2 reduced the CA9 promoter activity in both standard and acidic conditions. Similar result was obtained by inhibitors of MAPK and PI3K pathways, whose combination completely suppressed CA IX expression and abolished induction by acidosis. Altogether, our results suggest that acidosis increases the CA IX expression via a hypoxia-independent mechanism that operates through modulation of the basic CA9 transcriptional machinery.