| Abstract |
A series of hydroxyurea (HU) resistant human cell lines were derived from
K-562 by selection in increasing concentrations of HU. The most resistant
line, KH-450, was capable of growth in 6 mM HU. The resistant cell lines
exhibited increasing size and doubling times with increasing resistance.
Ribonucleotide reductase (RR) isolated from these cell lines exhibited
increased activity and altered response to HU. The KH-300 line, selected
in 4 mM HU, had a 6.6-fold increase in activity and a 3.5 fold increase in
the IC50 for HU. The KH-450 line had 4-fold increase in activity and a 18-
fold increase in the IC50 for HU.
Northern and Southern blot analysis indicated that the M1 subunit was
unchanged. Blot analysis of the M2 subunit indicated an increase in gene
copy number and expression. Maximal gene copy number was noted was a 4-
fold increase in the KH-300 line. The KH-450 line had a 2-fold increase in
gene copy number. The expression of M2 correlated with the gene copy
number. The scenario for the development of the KH-450 line was initial
amplification of a normal M2 allele during selection of the intermediate
resistant cell lines. A mutation occurred in the M2 subunit at an
undetermined step. Final selection in 6 mM HU allowed the cell to deamplify
the normal M2 allele, presumably by outgrowth of the low gene copy
number sub-population, containing an altered M2 subunit.
Blot analysis also revealed that the genes coding for ornithine
decarboxylase (ODC) and P58 (unknown function) were co-amplified with the
M2 gene. The increased levels of ODC conferred cross-resistance to
difloromethylornithine in the HU-resistant cells. Attempts to isolate M2
genomic clones from a KH-450 genomic DNA lambda phage library and a
chromosome 2 specific library were unsuccessful. A partial genomic clone
of the M1 gene was isolated.
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