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Abstract
A camptothecin-resistant (DC3F/C-10) Chinese hamster cell line that contains a catalytically altered and camptothecin (CPT)-resistant DNA topoisomerase I (top 1) (Tanizawa, A., and Pommier, Y. (1992) Cancer Res. 52, 1848-1854) and the parent cell line (DC3F) were used to compare top 1 mRNAs and cDNAs. Northern blot analysis showed a single 4.1-kilobase band without quantitative reduction between the two cell lines. We have cloned and sequenced top 1 cDNAs. DC3F and DC3F/C-10 top 1 c-DNA are 3591 and 3626 base pair long, respectively, and encode 767 amino acids. The homology of deduced amino acid sequences between Chinese hamster and mouse or human top 1 are 98.1 and 96.7, respectively. cDNAs from DC3F/C-10 and DC3F cells differ by a single base point mutation (G to A) which results in an amino acid change from Gly505 to Ser (Gly505-->Ser). G505 corresponds to Gly503 of human top 1 cDNA and is located 220 amino acids away from the presumed catalytic Tyr725. The point mutation in the Chinese hamster top 1 is located in a region that is highly conserved among all cloned top 1 cDNAs (plant ATH, vaccinia virus, Shope fibroma virus, Drosophila, Saccharomyces cerevisiae, Schizosaccharomyces pombe, mouse, and Human). A mutation of Asp533 to Gly in this same region has been shown to confer CPT resistance for human top 1. Chinese hamster top 1 protein with a Gly505-->Ser mutation that was expressed in bacteria was resistant to CPT, indicating that this single base mutation is involved in CPT resistance. Our results suggest that the highly conserved region around Gly505 plays an important role in the interactions among top 1, DNA, and CPT.
Highlights
Ster cell line that contains a catalytically altered and inhibitor [1,2,3,4], and itsderivatives are among the most promcamptothecin (CPT)-resistant DNA topoisomerase I ising anti-cancer agents currently cilninical trials ( 5 ) .Despite(Tanizawa, A., and Pommier, Y. (1992)Cancer the experimental evidence that CPT binds to top 1.DNA
Our results show that of libraries from DC3F/C-10 and DC3F cells, respectively
Chinese hamster top 1 cDNA has two additional amino acids ( a ) .Shaded boxes represent amino acid coding Sequences.The positions of catalytic tyrosine ( Y )and mutated amino acids which were found in human CPT-11-resistant cell lines
Summary
This oligonucleotide is derived from the Tetrahymena ribosomal DNA sequence containing a strong top 1cleavage site (A) [24], in which the +1base has been mutated to a G(italic letters) to enhance CPT cleavage [4, 7].3’-End labeling of the upper strand was performed by using terminal deoxynucleotidyl transferase and 32P-. An additional 1477- bases at the3‘-end (5’-AACGG-3’)when compared with that bp clone, which contains an open reading frame These changes did not affect the a 1126-bp clone (WTS12) were obtained from the second set amino acid sequence. Clrev plasmid indicated that the observed catalytic activity in the cell lysates from PE/W1 and PE/C1cultures was due to expressed Chinese hamstetrop 1proteins (data not shown). Chinese hamster top 1 cDNA has two additional amino acids ( a ) .Shaded boxes represent amino acid coding Sequences.The positions of catalytic tyrosine ( Y )and mutated amino acids which were found in human CPT-11-resistant cell lines Chinese hamster DC3F and DC3F/C-10 cells (reduced catalytic activity and CPTresistance) and suggest that the GlYo6 +Ser mutationin DC3F/C-lO top 1is responsible for reduced specific catalytic activity and CPT resistance
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