Amplification of genes encoding human myeloid membrane antigens after
DNA-mediated gene transfer
AT Look, SC Peiper, EC Douglass, JM Trent and CJ Sherr
Spontaneous amplification of genes encoding two different human myeloid
surface antigens was observed after DNA-mediated gene transfer of cellular
DNA from the human myeloid cell line HL-60 into NIH-3T3 mouse fibroblasts.
Transformed recipient cells with highly amplified expression of either of
two donor membrane polypeptides, gp150 or p67, were isolated with a
fluorescence-activated cell sorter (FACS), using monoclonal antibodies
specific for human myeloid cells. Immunoprecipitation of enzymatically
radioiodinated polypeptides from the surface of transformed NIH-3T3 cells
confirmed that expression of these proteins was amplified tenfold to
20-fold in comparison to their expression on human myeloid cell lines. The
cellular DNA of cloned secondary and tertiary transformants expressing high
levels of gp150 and p67 contained amplified sets of DNA restriction
fragments that hybridized with human repetitive DNA sequences. Cytogenetic
analysis of subclones overexpressing gp150 revealed extrachromosomal double
minutes (DMs), whose presence correlated with the unstable expression of
the membrane polypeptide. Human sequences in gp150-positive clones did not
localize to chromosomes, consistent with their association with
extrachromosomal DMs. By contrast, p67-positive subclones stably expressed
the antigen, and in situ hybridization to metaphase spreads demonstrated
that amplified human DNA sequences were integrated into a specific marker
chromosome. Cytogenetic analysis of the parental NIH- 3T3 subclone used in
these studies disclosed DMs in a low percentage of metaphases, suggesting
that the recipient cells have a propensity for amplifying donor DNA.
Volume 67,
Issue 3,
pp. 637-645,
03/01/1986
Copyright © 1986 by The American Society of Hematology
