Atsushi Oda

Novel point mutations in the αIIb subunit (Phe289 → Ser, Glu324 → Lys and Gln747 → Pro) causing thrombasthenic phenotypes in four Japanese patients

We analysed the molecular basis of Glanzmann thrombasthenia (GT) in four Japanese patients with type I or type II disease. Polymerase chain reaction (PCR) and subsequent direct sequencing of platelet RNA and genomic DNA revealed three single nucleotide substitutions of the αIIb gene, which were confirmed by allele‐specific PCR or restriction analysis. One patient with type I GT had a T to C base substitution in exon 11 resulting in a Phe (TTT)‐289 to Ser (TCT) mutation (F289S) of the subunit. Another type I patient had a G to A base substitution in exon 12 resulting in a Glu (GAA)‐324 to Lys (AAA) mutation (E324K). Interestingly, two unrelated patients with type II GT shared an A to C base substitution in exon 23, a region previously not associated with GT, resulting in a Gln (CAA)‐747 to Pro (CCA) mutation (Q747P). To analyse the effects of these mutations on αIIbβ3 surface expression, the wild‐type αIIb cDNA or mutant αIIb cDNAs were transfected into Chinese hamster ovary (CHO) cells together with a wild‐type β3 cDNA. Flow cytometric analysis using an anti‐αIIbβ3 complex antibody revealed that 50.6% of CHO cells with wild‐type αIIbβ3 expressed complexes, whereas only 1.6%, 7.7% and 31.3% of cells, with αIIb(F289S)β3, αIIb(E324K)β3 and αIIb(Q747P)β3 expressed complexes, respectively. Our data indicate that these three novel point mutations in the αIIb subunit may hamper surface expression of the αIIbβ3 complex, thus resulting in the quantitative GT phenotypes of platelets from these patients.

STI571-resistant KT-1 cells are sensitive to interferon-α accompanied by the loss of T-cell protein tyrosine phosphatase and prolonged phosphorylation of Stat1

OBJECTIVE The high incidence of acquired drug resistance to STI571 during treatment of chronic myelogenous leukemia (CML) patients in blast crisis has become a problem. We studied the effects of interferon-alpha (IFN-alpha) on a novel STI571-resistant CML cell line and its molecular mechanisms in vitro. MATERIALS AND METHODS KT-1 is a unique CML cell line that remains sensitive to the therapeutic IFN-alpha concentration. We developed novel STI571-resistant KT-1 cells (designated KTR cells) by gradually increasing the concentration of STI571. RESULTS All seven KTR clones became more sensitive to IFN-alpha than KT-1 cells. IFN-alpha induced more prolonged phosphorylation of Stat1 for 24 hours in all seven KTR clones than in KT-1cells. Tyrosine phosphorylation of Jak1 in KTR cells was not prolonged compared to KT-1cells. T-cell protein tyrosine phosphatase (TC-PTP) was down-regulated in all KTR clones, and SH-PTP1 phosphatase also was down-regulated in some KTR clones. The transient transduction of TC-PTP cDNA into the KTR subline prevented the IFN-alpha-induced prolonged phosphorylation of Stat1 and recovered the sensitivity against IFN-alpha. These results indicated that the loss of TC-PTP is involved in the IFN-alpha-induced prolonged phosphorylation of Stat1 and in the higher sensitivity to IFN-alpha in KTR cells. CONCLUSION We demonstrated that STI571-resistance does not confer cross-resistance to IFN-alphain KT-1 cells. The loss of TC-PTP contributed to the IFN-alpha-induced prolonged phosphorylation of Stat1 and the higher sensitivity to IFN-alpha in KTR cells.

Serum thrombopoietin and erythropoietin levels in patients with acute promyelocytic leukaemia during all-trans retinoic acid treatment

Endogenous serum thrombopoietin (TPO) and various cytokines including erythropoietin (EPO), interleukin (IL)‐3, IL‐6, IL‐11, granulocyte‐colony stimulating factor (G‐CSF), granulocyte‐macrophage‐colony stimulating factor (GM‐CSF) and stem cell factor (SCF) levels were measured in five patients with acute promyelocytic leukaemia (APL) during all‐trans retinoic acid (RA) treatment. During differentiation‐inducing therapy, platelet counts slowly increased and reached a peak between days 29 and 46 (median day 35). Serum TPO levels increased parallel to the increasing platelet counts and reached a maximum level during the first 10–20 d of all‐trans RA treatment. The circulating TPO levels then decreased in inverse correlation to the platelet counts. These unique changes in serum TPO levels revealed that TPO levels were not regulated by platelet or megakaryocyte mass in patients with APL during differentiation‐inducing therapy, and it would appear that TPO levels are directly regulated by all‐trans RA during the first 10–20 d of treatment. In addition, the change in circulating EPO levels and reticulocyte counts were similar to that of the TPO levels and platelet counts during all‐trans RA treatment, suggesting a close relationship between TPO and EPO signalling.