Important note: Information in this article was accurate in 1993. The state of the art may have changed since the publication date.
Deoxycytidine kinase and drug resistance in cancer and AIDS (Meeting abstract).
Proc Annu Meet Am Assoc Cancer Res; 34:565-6 1993. Unique Identifier : AIDSLINE ICDB/93694303 Mitchell BS; Ayscue LH; Dept. of Pharmacology, Univ. of North Carolina, Chapel Hill, NC; 27599
Abstract:
Deoxycytidine kinase (dCK) catalyzes the phosphorylation of 2'-deoxyadenosine, 2'-deoxyguanosine, and 2'-deoxycytidine to their corresponding 2'-deoxynucleoside monophosphates and is also required for the phosphorylation of a variety of clinically useful nucleoside analogs including cytosine arabinoside (araC), 2',3'-dideoxycytidine, 2-chlorodeoxyadenosine, and 2-fluoro-araA to their active nucleotide forms. Deficiency of dCK activity has been associated with resistance to the cytotoxic effects of each of these agents in cultured cell lines in vitro and may play a role in mediating drug resistance in vivo. To investigate the molecular basis of such resistance, we purified the dCK protein from Molt-4 T lymphoblasts, isolated the corresponding cDNA clone, and characterized the dCK gene. The dCK protein consists of two identical 30.5-kD subunits and is encoded by a 2.4-kb mRNA. The cDNA contains a glycine-rich ATP binding site that is characteristic of nucleoside kinases and has been found to have regions of sequences similar to the herpes virus thymidine kinase gene, suggesting a common origin of these two proteins. The gene for dCK consists of seven exons spread over a minimum of 34 kb of the genome and is located on chromosome 4 (q13-21). It contains a core promoter within a 700-bp 5' Hind III fragment that has 4 potential Sp1 binding sites and a 9-bp region of sequence identity to the E2F transcription factor binding site. Human CEM cell lines selected for resistance to araC (A) or 2',3'-dideoxycytidine (B) were analyzed for dCK mRNA levels and found to contain moderately (A) or markedly (B) reduced levels on Northern blot analysis. The cDNAs corresponding to these mRNAs were subsequently characterized. Cell line A contained a G to A point mutation within the ATP binding site and a 115-bp deletion that corresponded to the fifth exon of the gene, most likely a result of defective RNA splicing due to a point mutation at the 5' splice donor site. Each mutation resulted in complete loss of dCK enzymatic activity when the cDNA was expressed in an E coli system. Cell line B contained a single point mutation resulting in a Gln to Arg substitution at amino acid 156; the corresponding expressed protein had markedly reduced, but measurable, catalytic activity. The very low levels of dCK mRNA in this cell line made it likely that the second dCK allele was not expressed. We have subsequently studied an HL-60 cell line that was selected for araC resistance in the absence of mutagenesis. This line has a complete deficiency of dCK mRNA by PCR analysis, and our preliminary results indicate an interstitial deletion of chromosome 4 in the q21 region that is responsible for the loss of a portion of the dCK gene. Although the promoter of the dCK gene is highly G/C rich, we have found no evidence that methylation of cytosine residues is involved in the regulation of dCK expression. Mechanisms involved in the complete loss of allelic expression in the absence of a structural gene mutation remain under investigation, as does the role of dCK deficiency in mediating drug resistance in clinical disease. (5 Refs)
Keywords: Adenosine Triphosphate/METABOLISM Alleles Amino Acid Sequence Chromosome Deletion Cytarabine/THERAPEUTIC USE Deoxyadenosines/*GENETICS/METABOLISM Deoxycytidine Kinase/*GENETICS/METABOLISM Deoxyguanosine/*GENETICS/METABOLISM Drug Resistance/*GENETICS DNA/METABOLISM Leukemia, Promyelocytic, Acute/DRUG THERAPY Mutation RNA Splicing RNA, Messenger/GENETICS ABSTRACT 930930
M9391187
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Important note: Information in this article was accurate in 1993. The state of the art may have changed since the publication date.
