We have developed a restriction map of the chromosome 21 breakpoint region involved in t(8;21)(q22;q22.3) acute myelogenous leukemia (AML) and have isolated a genomic junction clone containing chromosome 8 and 21 material. Using probes from these regions, rearrangements have been identified in each of nine cases of t(8;21) AML examined. In addition, we have isolated cDNA clones from a t(8;21) AML cDNA library that contain fused sequences from chromosome 8 and 21. The chromosome 8 component, referred to as ETO (for eight twenty-one), is encoded over a large genomic region, as suggested by the analysis of corresponding yeast artificial chromosomes (YACs). The DNA sequence of the chromosome 21 portion of the fusion transcript is derived from the normal AML1 gene. A striking similarity (67% identity over 387 bp, with a corresponding 69% amino acid identity) was detected between AML1 and the Drosophila segmentation gene, runt. The critical consequence of the translocation is the juxtaposition of 5′ sequences of AML1 to 3′ sequences of ETO, oriented telomere to centromere on the der(8) chromosome.
We have molecularly cloned the human myeloperoxidase (MPO) gene from the lambda gt11 expression library by screening with an affinity- purified MPO antibody. The cDNA clone of the MPO gene was used to study MPO gene expression in leukemic cells. The amino acid sequence predicted from the nucleotide sequence of the cDNA clone pMP401 matched exactly the 23 amino acid sequence of the NH2-terminal of the 60,000 MPO subunit. We found that MPO cDNA hybridized to a single EcoRI genomic band of 19 kb, indicating that the MPO gene represents a single gene in the human genome. Northern blot analysis of RNA isolated from leukemic cell lines and acute myelogenous leukemia (AML) patients' samples shows that MPO gene expression correlated with myeloid lineage. The intensity of MPO mRNA expression on Northern blot correlated with the level of MPO expression by cytochemical staining. Multiple species of MPO mRNA were found. This indicates that a single MPO gene may encode different RNA species through a mechanism of posttranscriptional processing or that multiple transcriptional start/termination sites exist in the MPO gene.
The Philadelphia (Ph1) chromosome in chronic myelogenous leukemia (CML) involves reciprocal translocation of the bcr gene and the c-abl oncogene. The fused bcr/abl gene is transcribed into two types of chimeric mRNA. By means of a combined method of S1 nuclease protection and polymerase chain reaction, we amplified sequences representative of the chimeric bcr/abl transcripts. Only 5 micrograms of total cellular RNA is needed and the chimeric bcr/abl message can be detected at a dilution of 1:100,000. We also detected residual chimeric bcr/abl transcripts in the remission samples from two Ph1-positive CML patients. This technique has the potential to identify a subpopulation of Ph1-positive CML patients in remission who are at high risk of relapse.
We have developed a restriction map of the chromosome 21 breakpoint region involved in t(8;21)(q22;q22.3) acute myelogenous leukemia (AML) and have isolated a genomic junction clone containing chromosome 8 and 21 material. Using probes from these regions, rearrangements have been identified in each of nine cases of t(8;21) AML examined. In addition, we have isolated cDNA clones from a t(8;21) AML cDNA library that contain fused sequences from chromosome 8 and 21. The chromosome 8 component, referred to as ETO (for eight twenty-one), is encoded over a large genomic region, as suggested by the analysis of corresponding yeast artificial chromosomes (YACs). The DNA sequence of the chromosome 21 portion of the fusion transcript is derived from the normal AML1 gene. A striking similarity (67% identity over 387 bp, with a corresponding 69% amino acid identity) was detected between AML1 and the Drosophila segmentation gene, runt. The critical consequence of the translocation is the juxtaposition of 5′ sequences of AML1 to 3′ sequences of ETO, oriented telomere to centromere on the der(8) chromosome.
Philadelphia chromosome-positive (Ph1) acute leukemia is a heterogeneous subset of acute leukemia with a poor prognosis. We studied five patients to determine the potential for phenotypic and molecular heterogeneity. Cellular characterization studies included light myeloperoxidase (L-MPO), terminal deoxynucleotidyl transferase (TdT), ultrastructural MPO (U-MPO), and immunophenotyping by flow cytometry using T11, T3, T4, T8, Leu 1, B1, Leu 12, HLA-DR (la), CALLA (J5), OKM1, My4, My7, My8, My9, and My10. DNA was analyzed for rearrangements of the breakpoint cluster region (bcr), immunoglobulin heavy chain, joining region (JH), immunoglobulin kappa light chain constant region (C kappa), and T cell receptor (TcR beta). RNA dot blots were hybridized by using molecular probes for MPO and TdT. We found that four of five cases were acute mixed-lineage leukemia (AMLL). One patient had acute unclassifiable leukemia. Of the four patients classified as having AMLL, three showed myeloid and lymphoid features, with one patient showing myeloid, T cell, and B cell features. The last case showed T cell and B cell features only. In one patient MPO/RNA was positive in spite of insufficient L-MPO or U-MPO to diagnose acute myelogenous leukemia (AML), thereby suggesting significant MPO gene expression before the production of sufficient MPO protein to meet the French-American-British criteria for AML. Three of the five patients showed rearrangement of bcr (cases 1, 2, and 5). Studies of these five patients support the concepts of molecular and phenotypic heterogeneity in Ph1 acute leukemia, demonstrate a high incidence of AMLL in this subset of acute leukemia, and support the use of lineage-associated molecular probes to define lineage at an earlier stage than previously possible.
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