The Wilms'-tumor gene WT1 may have a different function from a tumor-suppressor gene in some leukemias. Using the 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat leukemia system, we examined whether WT1 expression was involved during leukemogenesis, since this model enabled us to analyze cells altered by DMBA at various stages of leukemogenesis. By the semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) method, WT1 expression was detected in 15 (71%) of 21 DMBA-induced erythroblastic leukemias. Among 15 WT1-expressing leukemias, GATA-1, which is an erythroid-specific transcription factor and might regulate WT1 expression, was also expressed in 13 cases (p < 0.05). On the other hand, WT1 expression was not detected in any normal or early pre-leukemic rats and was detected in 1 of 8 rats in late pre-leukemic stages. These results showed that cells with a high expression level of WT1 tended to develop into leukemia and that WT1 contributed to leukemogenesis in the late stage, suggesting that the expression of WT1 plays an important role in cell proliferation and in maintaining the viability of some leukemia cells.
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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