ALBERT

Description: Modern clinical treatments of childhood acute lymphoblastic leukemia (ALL) employ enzyme-based methods for depletion of blood asparagine in combination with standard chemotherapeutic agents. L-asparaginase (L-asp) therapy causes depletion of plasma asparagine followed by the loss of intracellular asparagine. Due to the lack of a rapid up-regulation of asparagine synthetase (ASNS) protein content in ALL cells, they are preferentially killed by L-asp. Elevated expression of ASNS within the leukemia cells causes decreased sensitivity to L-asp. The proof of ASNS deficiency in leukemia cells is considered to predictive for effectiveness of L-asp even in acute myeloid leukemia (AML) other than ALL patients. The establishment of quantitative estimation of ASNS protein content would be useful for the L-asp treatment in leukemia therapy. Objective: Our aim was to set up a flow cytometry system to check ASNS deficiency in leukemia cells and to investigate the sensitivity to L-Asp and the ASNS expression in AML leukemia cells. Methods: AML (KG-1, HL-60, U937) and ALL (MOLT-4, RS4;11) and CML (K562) cell lines were grown in RPMI1640 medium with 10% FCS. Primary leukemic cells from the peripheral blood or bone marrow of 20 AML patients were harvested on EDTA and isolated by Ficoll density gradient within 72h. ASNS expression was evaluated by cytosolic flow cytometry with Z5808 McAb (Hybridoma 31: 325-332.2012) and expressed as a ΔMFI(Difference of Mean Fluorescence Intensity(MFI) between by Z5808 and isotypic control) or MFI ratio(MFI by Z5808/MFI by isotypic control). When a sufficient amount of leukemic cells was available, sensitivity to L-asp (expressed as an IC50 - concentration inhibiting 50% of cell viability) was evaluated in vitro by incubating various concentrations of E. coliL-asp with the cells and by measuring the cell viability with a counting kit (WST1 viability assay) at day 3. Results: Determination of IC50 for the HL-60 (⊿MFI 48 ± 8.01, MFI ratio 1.77 ± 0.03) and U937 (⊿MFI 16.7 ± 0.47, MFI ratio 1.19 ± 0.02) demonstrated that these cells were equally sensitive to L-asp than the ALL cell line MOLT-4 in vitro (0.37 and 0.02IU/mL versus 0.15 IU/mL, respectively). K562 and KG-1 (⊿MFI 135.7 ± 5.66, MFI ratio 2.48 ± 0.09) cells with the highest ASNS expression exhibited resistance to L-asp (〉10 IU/ml). Both of ASNS Expression by ⊿MFI and MFI ratio was inversely correlated with L-asp sensitivity judging from cell line studies. Judging from cell line study, the threshold for ASNS protein expression effective for L-asp treatment was considered to be

Description: Fifty percent of Diamond-Blackfan anemia (DBA) patients possess mutations in genes coding for ribosomal proteins (RPs). To identify new mutations, we investigated large deletions in the RP genes RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26. We developed an easy method based on quantitative-PCR in which the threshold cycle correlates to gene copy number. Using this approach, we were able to diagnose 7 of 27 Japanese patients (25.9%) possessing mutations that were not detected by sequencing. Among these large deletions, similar results were obtained with 6 of 7 patients screened with a single nucleotide polymorphism array. We found an extensive intragenic deletion in RPS19, including exons 1-3. We also found 1 proband with an RPL5 deletion, 1 patient with an RPL35A deletion, 3 with RPS17 deletions, and 1 with an RPS19 deletion. In particular, the large deletions in the RPL5 and RPS17 alleles are novel. All patients with a large deletion had a growth retardation phenotype. Our data suggest that large deletions in RP genes comprise a sizable fraction of DBA patients in Japan. In addition, our novel approach may become a useful tool for screening gene copy numbers of known DBA genes.