Myeloproliferative neoplasms (MPN) are multiple disease entities characterized by clonal expansion of one or more of the myeloid lineages (i.e. granulocytic, erythroid, megakaryocytic and mast cell). JAK2 mutations, such as the common V617F substitution and the less common exon 12 mutations, are frequently detected in such tumor cells and have been incorporated into the diagnostic criteria published by the World Health Organization since 2008. However, the mechanism by which these mutations contribute to MPN development is poorly understood. We examined gene expression profiles of MPN patients focusing on genes in the JAK–STAT signaling pathway using low-density real-time PCR arrays. We identified the following 2 upregulated genes in MPN patients: a known target of the JAK–STAT axis, SOCS3, and a potentially novel target, SPI1, encoding PU.1. Induction of PU.1 expression by JAK2 V617F in JAK2-wildtype K562 cells and its downregulation by JAK2 siRNA transfection in JAK2 V617F-positive HEL cells supported this possibility. We also found that the ABL1 kinase inhibitor imatinib was very effective in suppressing PU.1 expression in BCR-ABL1-positive K562 cells but not in HEL cells. This suggests that PU.1 expression is regulated by both JAK2 and ABL1. The contribution of the two kinases in driving PU.1 expression was dominant for JAK2 and ABL1 in HEL and K562 cells, respectively. Therefore, PU.1 may be a common transcription factor upregulated in MPN. PU.1 is a transcription factor required for myeloid differentiation and is implicated in erythroid leukemia. Therefore, expression of PU.1 downstream of activated JAK2 may explain why JAK2 mutations are frequently observed in MPN patients.
The authors report 2 male patients with dyskeratosis congenita (DC) in a Japanese kindred. Sequencing of the complementary DNA of the dyskerin gene (DKC1) revealed a T-to-C transition at nucleotide 1285 in exon 12 that resulted in a novel missense mutation L398P. Despite harboring the same mutation in the DKC1 gene, one patient had significantly milder hematological symptoms than the other, indicating that there may be other factors that determine the severity of DC.
We established a real-time quantitative PCR (RQ-PCRAsparagine is not an essential amino acid obtained from outside the body because it is synthesized by using the hydrolysis energy of ATP from aspartic acid and glutamine via asparagine synthetase (AS). Even when the asparagine supply is reduced, normal cells can compensate by synthesizing L-asparagine. However, lymphoblastic cells require external asparagine for growth as they lack sufficient AS activity. 1-3 Thus, L-asparaginase is effective against childhood acute lymphoblastic leukemia (ALL) during the induction of remission or the intensification phases of treatment. 4,5 Asparagine in the blood, cerebrospinal fluid and bone marrow is depleted by L-asparaginase. A reduction of asparagine leads to cell death, since exposure to L-asparaginase in vitro induces the fragmentation of DNA and morphological changes typical of apoptosis in a mouse lymphoma cell line 6 and in NIH3T3 cells. 7 An asparagine deficiency can be evoked by the intracellular depletion of glutamate and glutamine. 7 The apoptosis of leukemia cells induced by L-asparaginase may be associated with cell cycle arrest in the G1 phase because DNA strand breaks can be seen in G1 phase cells as early as 8 hours after L-asparaginase exposure. 8 L-asparaginase activates AS expression and the overexpression of human AS protein can induce the L-asparaginase-resistance phenotype. 9 The expression of AS and sensitivity to L-asparaginase were correlated not only in leukemic, but also in ovarian cancer cells. 10 These observations indicate the importance of monitoring AS activity as a marker for clinical decisions regarding L-asparaginase therapy. The present study established real-time quantitative PCR (RQ-PCR) as a method of measuring AS expression. We discuss the clinical application of this method compared with immunohistochemistry, Western blotting, and enzyme activity.
Materials and Methods
Cell Lines and Culture ConditionsK562, HL60, U937, and MOLT4 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). All cells were suspension-cultured in RPMI 1640 medium (Invitrogen Corp., Carlsbad, CA) containing 10% fetal bovine serum (FBS; Sigma, St. Louis, MO) and penicillin and streptomycin under a 5% CO 2 atmosphere. MOLT4/R 11 and U937/R 1 were established by sequential
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