Overexpression of Human Topoisomerase I in Baby Hamster Kidney Cells: Hypersensitivity of Clonal Isolates to Camptothecin1
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(CANCER RESEARCH 52, 525-532, Februar) l. 19921 Overexpression of Human Topoisomerase I in Baby Hamster Kidney Cells: Hypersensitivity of Clonal Isolates to Camptothecin1 Knut R. Madden and James J. Champoux2 Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195 ABSTRACT conserved regions in all three sequences (8). These highly conserved regions suggest that the eukaryotic topo I may be The 3645-base pair human topoisomerase I complementary DNA (cDNA) clone isolated by D'Arpa et al. (Proc. Nati. Acad. Sci. USA, «5: subdivided into distinct functional domains. One such domain is the conserved carboxy-terminal 70 amino acids, which con 2543-2547,1988) and a mutated version of the cDNA encoding a protein tain the active-site tyrosine identified in the yeast species (8, 9). with phenylalanine instead of tyrosine at position 723 have been overex- pressed 2- to 5-fold in stably transfected baby hamster kidney cells. The Based on the homology with the yeast proteins, the active site overexpressed proteins are the same size as the topoisomerase I present residue in the human topo I is predicted to be the tyrosine in Hela cells, indicating that the cDNA clone contains the complete located at position 723 of the amino acid sequence. topoisomerase I coding sequence. Some human colon carcinoma cells CTT is a cytotoxic alkaloid which has been shown in yeast have increased levels of topoisomerase I and are hypersensitive to the (14, 15) and mammalian cells (16-18) to exert its effect by drug camptothecin. The overexpressed wild-type topoisomerase I does specifically interacting with topo I. The topo I-DNA covalent not affect the cell growth or morphology of the baby hamster kidney intermediate or a cleavable complex appears to be the target cells, suggesting that elevated levels of topoisomerase I alone are not for CTT (19, 20), and recent studies suggest that the drug acts sufficient to cause cell transformation. However, the overexpressed wild- by prolonging topo I-induced single-strand breaks (20, 21). The type protein is active, as shown by the hypersensitivity of clonal cell lines cytotoxic effects of the drug are thought to result from long- to camptothecin. The mutant form of topoisomerase I is enzymatically lived topo I-associated breaks in chromosomal DNA that inter inactive by two criteria. First, extracts of Escherichia coli cells carrying the mutant cDNA contain no activity capable of relaxing superhelical fere with critical cellular processes such as transcription and DNA under conditions where activity is easily detectable in extracts from replication (22-26). Consistent with this hypothesis is the ob cells containing the wild-type cDNA. Second, baby hamster kidney cells servation in S. cerevisiae that the cytotoxic effects of CTT stably transfected by the mutant cDNA are no more sensitive to camp depend upon the presence of an active topo I in the cell (14, tothecin than control untransfected cells. These results indicate that 15, 27). Recent results suggest that CTT might be an effective tyrosine 723 is essential for enzyme activity and are consistent with chemotherapeutic agent against those human colon carcinomas predictions based on homology comparisons with the yeast enzymes, that that contain elevated levels of topo I (28). this is the active-site tyrosine in the human topoisomerase I. A human topo I cDNA clone has been isolated that is 3645 base pairs in length and contains a long open reading frame INTRODUCTION extending from the beginning of the clone to position 2506 (13). The first in-frame ATG codon occurs at position 212, and DNA topoisomerases catalyze the breakage and rejoining of translation from this start codon would yield a M, ~91,000 DNA strands to permit topologica! changes in DNA. Topo I3 protein containing 765 amino acids. The sequence surrounding breaks and rejoins one strand of duplex DNA, whereas type II this ATG codon is very similar to the context associated with topoisomerase breaks both DNA strands. Upon breakage of the eukaryotic start codons (29) and, like other mammalian genes DNA, type I topoisomerase becomes covalently attached to the that do not display tissue-specific expression (30), the GC broken strand at the site of the nick; topo I from Escherichia content of the putative 5' untranslated region is significantly coli attaches to the 5' end at the break, whereas topo I from higher than that of the coding region. However, since this eukaryotes attaches to the 3' end (for recent reviews see Refs. reading frame remains open up to the 5' end of the clone, this 1-3). The covalent intermediate can be trapped in vitro by particular cDNA clone could be missing the 5'-most coding denaturing the enzyme with SDS or alkali (4, 5). The topo I region of the topo I gene. If so, the human topo I protein would attaches to DNA through a phosphodiester bond to a tyrosine be predicted to have at least 70 additional amino acids, and the residue in the active site of the enzyme (6). The active-site corresponding molecular weight would be larger by at least tyrosine residue has been identified in the enzymes from E. coli 8,000. Since the molecular weights for the human and other and from the yeasts Saccharomyces cerevisiae and Schizosac- eukaryotic type I topoisomerases have been reported to range charomyces pombe (7-9). from 90,000 to 110,000 (31), one cannot rule out this latter Although there is virtually no sequence similarity between possibility by comparing the predicted with the observed mo the E. coli and eukaryotic topo I genes (10), there is a strong lecular weights. Evidence presented in this paper supports the homology among the eukaryotic enzymes, including yeast and hypothesis that this cDNA clone contains the entire coding humans (8, 9, 11-13). Alignment of the predicted amino acid sequence for human topo I, and therefore, by inference, the sequences for humans, S. cerevisiae, and S. pombe reveals two start codon must be located at position 212. The same conclu sion has been reached by Kunze et al. (32), using a different Received 7/9/91; accepted 11/11/91. The costs of publication of this article were defrayed in part by the payment experimental approach. of page charges. This article must therefore be hereby marked advertisement in A number of observations suggest that the eukaryotic topo I accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by Grant DMB-8917877 from the National Science Foundation. is involved in both transcription and DNA replication and that 2To whom requests for reprints should be addressed, at Department of mechanisms exist to target the enzyme to its proper locus for Microbiology SC-42, University of Washington, Seattle, WA 98195. activity. Topo I is localized to actively transcribed regions of 3The abbreviations used are: topo I, type I topoisomcrase; cDNA, comple the genome (33-36) and has been reported to be associated mentary DNA; CTT, camptothecin; BHK, baby hamster kidney; MTX, metho- trexate; SDS, sodium dodecyl sulfate; TD, 8 g NaCI, 0.38 g KCI, l g Na2HPO4/ with RNA polymerase I (37). It has also been found to be liter (pH 7.4); TTBS, 0.05% Tween 20, 20 min Tris (pH 7.5), 0.5 M NaCI. associated with replicating SV40 DNA (24, 38-40) and, inter- 525 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1992 American Association for Cancer Research. OVEREXPRESSION OF HUMAN TOPOJSOMERASE estingly, the mapping of specific topo I break sites on SV40 replicative intermediates has revealed a strand preference that correlates with the direction of replication (40). It is presumed that specific domains of the topo I protein are recognized by other cellular proteins to mediate the proper intranuclear tar geting of the enzyme. In the present work, we have overexpressed the wild-type and H. Topo I Expression Construct an enzymatically inactive mutant form of human topo I in MlIK (p«M16)9.5kbp cells with two goals in mind. First, we wanted to test the possibility that overexpression of topo I would be sufficient to cause cell transformation (28). Second, by overexpressing an inactive form of the protein, we wanted to test whether a dominant negative phenotype (41) would result due to compe tition by the inactive protein for the normal nuclear targets of topo I. The differential sensitivity of the overexpressing cell lines to camptothecin was used to determine whether the over Fig. 1. Human topo I expression construct. Plasmid pKM16 was generated by produced topo I was active. In the course of these experiments inserting an EcoRl fragment containing the human topo I cDNA clone (shaded sequences) into the Smal site of the pNUT vector. In this construct, the topo I it was necessary to establish that the cDNA clone contains all coding sequences are in the correct orientation for transcription from the mouse of the coding sequences for human topo I. metallothionein promotor (MTp). The pNUT vector contains a mutant dihydro folate reducÃasegene (DHFR) that is driven by the SV40 early promotor (SVp) and confers resistance to MTX. Plasmid pKM17 contains the topo I cDNA MATERIALS AND METHODS inserted in the opposite orientation. Plasmid pKM18 has the topo I cDNA inserted in the correct orientation, but the tyrosine codon at position 723 is replaced with a phenylalanine codon (723pl" mutant). Reagents. Restriction enzymes and T4 ligase were purchased from New England Biolabs, Inc., or Bethesda Research Labs, Inc. Klenow fragment, T4 polynucleotide kinase, and the Sequenase kit were pur tween plasmid pKM16 and pKM18 are the two base pair changes chased from U.S. Biochemical Corporation. Calf intestinal alkaline introduced with the oligonucleotide was performed by the sequence phosphatase was obtained from Boehringer Mannheim, and [a-32P] analysis (49, 50) and fragment replacement strategy discussed in "Re ATP and [3H]thymidine were from DuPont-New England Nuclear.