Cloning and characterization of the {\it DR -nm23\/} gene and its associated product, a novel member of the {\it nm23\/} gene family

Robert Vincent P Martinez, Thomas Jefferson University

Abstract

Chronic myelogenous leukemia (CML) evolves in two distinct stages: a chronic and a blast crisis phase. The molecular changes associated with the blast crisis transition are largely unknown. The primary objective of this work was the identification of a gene or genes which could be involved in the fatal transition to blast crisis. To this end we identified a novel member of the nm23 gene family which we termed DR-nm23. We have isolated both the DR-nm23 cDNA and gene with its 5$\sp\prime$-flanking region. The DR-nm23 cDNA was isolated on the basis of its differential overexpression in a blast-crisis cDNA library relative to the T-lymyhoblastoid cell line, CCRF-CEM. The DR-nm23 cDNA has $\sim$70% sequence similarity to the putative metastatic suppressor gene, nm23-H1 and its associated homologue nm23-H2. The deduced amino acid sequence similarity to the proteins encoded by these two latter genes is $\sim$65% and includes domains and amino acid residues (the leucine zipper-like and the RGD domain, a serine and a histidine residue in the NH$\sb{2\sp-}$ and in the COOH-terminal portion of the protein, respectively) postulated to be important for nm23 function. DR-NM23 mRNA is preferentially expressed at early stages of myeloid differentiation of highly purified CD34$\sp+$ cells. Its constitutive expression in the myeloid precursor 32cl3 cell line, which is growth-factor dependent for both proliferation and differentiation, results in inhibition of granulocytic differentiation induced by granuolcyte colony-stimulating factor and causes apoptotic cell death. The results are consistent with a role for DR-nm23 in normal hematopoiesis and raise the possibility that its overexpression contributes to differentiation arrest, a feature of blastic transformation in chronic myelogenous leukemia.^ We have now found the expression of DR-nm23 is not restricted to hematopoietic cells but is also detected in an array of solid tumor cell lines ranging from carcinoma of the breast, colon, and prostate, as well as in the glioblastoma cell line T98G. We have also isolated both the gene and its 5$\sp\prime$-flanking region, and found that DR-nm23 localizes on chromosome 16q13. The gene consists of 6 exons and 5 introns. When fused in-frame to the nucleotide sequence for the green fluorescent protein and transfected in SAOS-2 cells, it generates a protein of the predicted size that localizes to the cytoplasm. The 5$\sp\prime$-flanking region of DR-nm23 does not contain a canonical TATA-box or a CAAT-box, but it is G + C-rich and contains two binding sites for the developmentally regulated transcription factor AP-2. Transient expression assays of DR-nm23 promoter-CAT constructs demonstrated that the segment from nucleotides $-$1028 to +123 has the highest activity in hematopoietic K562 cells and in TK-ts13 hamster fibroblasts. Moreover, AP-2 induced a 3-fold transactivation of the DR-nm23 5$\sp\prime$-flanking segment from nucleotides $-$1676 to +123 and specifically interacted with oligomers containing putative AP-7 binding sites ($-$936 to $-$909, and $-$548 to $-$519) as indicated by electrophoretic mobility shift assay. Furthermore, nuclear run-on assays from high and low DR-nm23-expressing cells (K562 and CCRF-CEM, respectively), revealed similar transcription rates. Therefore, the regulation of the DR-nm23 gene expression might involve other mechanisms. ^

Subject Area

Biology, Molecular|Biology, Cell|Health Sciences, Oncology

Recommended Citation

Martinez, Robert Vincent P, "Cloning and characterization of the {\it DR -nm23\/} gene and its associated product, a novel member of the {\it nm23\/} gene family" (1997). ETD Collection for Thomas Jefferson University. AAI9727332.
http://jdc.jefferson.edu/dissertations/AAI9727332

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