Characterization of pyrazofurin-resistant HeLa cells with amplification of UMP synthase gene

Abstract

Three different phenotypes have been characterized in HeLa cells that have been selected for resistance to pyrazofurin, a potent inhibitor of the de novo pyrimidine biosynthetic enzyme UMP synthase. All of the resistant cell lines had a coordinate increase in UMP synthase activity, UMP synthase-specific mRNA, and UMP synthase gene sequences. In one of the resistant cell lines, the amplification of the UMP synthase gene is associated with a stable phenotype. There is no decrease in UMP synthase gene copy number or UMP synthase activity when these cells are grown for over six months in the absence of pyrazofurin. Another resistant cell line that has a higher level of gene amplification when grown in the presence of pyrazofurin loses its elevated UMP synthase activity and amplified DNA sequences with growth in the absence of the drug. A third cell line that possessed a moderate level of UMP synthase gene amplification is tenfold more resistant to pyrazofurin than the cell line with the highest level of amplification. The extraordinary level of resistance is due to a decreased level of activity for the enzyme adenosine kinase that is required for the conversion of pyrazofurin to its inhibitory monophosphate form.

DNA amplification is rare in normal human cells.

“…In selections with MTX the frequency of colonies that exhibited some growth after isolation was 9 X 10-7. These frequencies are relative to starting cell numbers and (40)(41)(42)(43)(44), for amplification of the gene encoding the a-subunit of the Na, K-ATPase with oubain (45,46), for amplification of an adenine deaminase gene with adenine nucleosides (47), and for amplification of the UMP synthase gene with pyrazofurin (48 (25), ouabain (49,50), azaguanine (51), 6-thioguanine (50), and diphtheria toxin (52) and selection of keratinocytes with 6-thioguanine (53 (7,54) but not in one other (55). The …”

Unstable and stable CAD gene amplification: importance of flanking sequences and nuclear environment in gene amplification.

“…6) were isolated from a single parental single-cell clone. In previous work (22,41) the drug-resistant lines analyzed were derived from uncloned populations and therefore were not likely to be derived from the same parental clone. Second, we analyzed the stability of the amplified CAD genes at the first selective step and not after long periods of growth under selective conditions.…”

Molecular cloning and nucleotide sequence for the complete coding region of human UMP synthase.

“…These findings indicate that the 3'-noncoding sequence of pHUSc39 is contiguous with the coding region of UMP synthase in the gene structure. This 3'-end fragment also hybridized to mRNA from a human cell line that has amplified the UMP synthase gene -60-fold and shows a 40-fold increase in UMP synthase mRNA (17). The 3' fragment hybridizes only slightly to the mature 2.0-kb UMP synthase mRNA but hybridizes more strongly to higher molecular weight forms of the mRNA.…”

“…The enzyme activity in deficient cells can be increased to near normal levels if the cells are grown in the presence of certain drugs or nucleotide analogues (13)(14)(15)(16). This increased enzyme activity is caused by an increase in the amount of immunoprecipitable UMP synthase protein in cell extracts (16 AgtlO cloning vector by using human T-lymphoblast (HPB-ALL, human peripheral blood acute lymphoblastic leukemia) mRNA, as described (17,18). The plaques were screened on duplicate filters by the method of Benton and Davis (19) as described by Maniatis et al (20).…”

“…The human cDNA library was screened with a UMP synthase-specific probe, pUMPS2, that had been isolated by differential hybridization from a rat hepatoma cDNA library (26). The initial screening of the cDNA library resulted in the isolation of a single plaque, AHUSc22, from which a 1.45-kilobase (kb) EcoRI insert was subcloned into pBR322 to produce pHUSc22 (17). The library was rescreened with the pHUSc22 insert as the hybridization probe; eight additional positive plaques were identified, and the isolated inserts were used to produce clones pHUSc32 to pHUSc39.…”