ABSTRACT
We established a real-time quantitative PCR (RQ-PCR) with which to measure abundance of the asparagine synthetase (AS) mRNA. The level of AS mRNA paralleled AS enzyme activity, as well as the AS protein level detected by Western blotting and by in situ immunostaining. Cytotoxicity tests in vitro showed that the AS mRNA level also synchronized with cellular resistance to L-asparaginase in cell lines. Cellular levels of AS enzyme activity correlated with resistance to L-asparaginase. These results indicate that the AS mRNA level is an index of resistance to L-asparaginase. RQ-PCR is superior to enzyme assays, Western blotting, and immunostaining in the following ways: less labor and time, accurate and reproducible quantitativity, and broad dynamic range. In addition, RQ-PCR could evaluate differences in L-asparaginase sensitivity although immunostaining could not. And in clinical samples, we analyzed eight pediatric leukemia cases by this RQ-PCR to evaluate whether this method was applicable to clinical laboratories and the expression level of AS mRNA in each case were predictable for the effectiveness of L-asparaginase treatment. Consequently, this method was useful enough in defining candidates for selective therapy that targets an AS deficiency.
Subject(s)
Aspartate-Ammonia Ligase/genetics , Leukemia/diagnosis , Polymerase Chain Reaction/methods , Asparaginase/pharmacology , Aspartate-Ammonia Ligase/analysis , Aspartate-Ammonia Ligase/biosynthesis , Cell Line, Tumor , Cell Nucleus/immunology , Drug Resistance, Neoplasm/genetics , Gene Expression , Humans , Leukemia/therapy , RNA, Messenger/analysis , RNA, Messenger/biosynthesisABSTRACT
7,12-Dimethylbenz[a]anthracene (DMBA) elicits leukemia in Long-Evans rats (LE). This leukemia is mostly erythroblastic and 30% of leukemias have total and partial trisomy of #2 chromosome and the rest have diploid karyotype. The common duplication site is in 2q26-q34 and N-ras gene is located in 2q34. 7,8,12-Trimethylbenz[a]anthracene (TMBA) also induces similar leukemias. These leukemias reveal a highly specific mutation of N-ras gene as in human leukemias. N-ras mutation is induced 48h after DMBA treatment. Wild type N-ras allele is frequently lost in diploid leukemias but not in trisomy type. Therefore, a gene dosage problem related to the mutant N-ras gene is involved in development of leukemia. Some secondary genetic rearrangements involving abl and H-ras are also observed in cultured leukemia cells. DMBA-induced chromosome aberrations as well as leukemia are enhanced by erythropoietin and blocked by Sudan III given prior to DMBA treatment. This leukemia will provide an important tool for chemical carcinogenesis and leukemia studies.
Subject(s)
9,10-Dimethyl-1,2-benzanthracene/adverse effects , Disease Models, Animal , Leukemia/chemically induced , 9,10-Dimethyl-1,2-benzanthracene/administration & dosage , Animals , Bone Marrow Cells/pathology , Leukemia/etiology , Leukemia/genetics , Rats , Research/trends , ras Proteins/geneticsABSTRACT
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.