Different Mechanisms of ras Proto-Oncogene Overexpression Detected by a Sib-Selection Tumorigenesis Assay
Different Mechanisms of ras Proto-Oncogene Overexpression Detected by a Sib-Selection Tumorigenesis Assay
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Date
1990
Authors
Bachurski, Cindy J.
Advisor
Hetrick, Frank M.
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DRUM DOI
Abstract
Human insulinoma and renal cell carcinoma DNAs were analysed by a sib-selection
tumorigenesis assay to detect the presence of dominant acting oncogenes. Although no
novel oncogenes were detected in the five tumor DNAs tested, the increased sensitivity of
the tumorigenesis assay allowed detection of overexpressed ras proto-oncogenes activated
during transfection. Tumorigenic overexpression of ras proteins occured by two different
mechanisms: gene amplification, and increased translation efficiency of a fusion protein.
Thirty to fifty fold amplification of a human N-ras gene was detected in primary and
secondary mouse tumors. All tumors containing amplified copies of N-ras overexpressed
p21 and the cloned gene had no coding sequence mutations. Since DNA from the original
human tumor and several metastases did not show N-ras gene amplification, it was
concluded that amplification had occured during the primary tumorigenesis assay.
In another series of tumors co-integration of the mouse c-H -ras gene during the
secondary transfection resulted in tumorigenic overexpression of ras protein without
elevation of mRNA levels. Three c-H-ras cDNA clones from secondary tumor RNA contained
no coding sequence mutations, but were divergent at the 3'end. Polymerase chain reaction
amplification of RNA showed novel alternative splicing at intron E of mouse c-H-ras mRNA in
both tumors and untransfected cells.
An upstream ATG was identified that potentially initiates translation of an open reading
frame (ORF) overlapping the H-ras p21 translation initiation site. A single base deletion
within exon -1 of the cDNA clones placed this upstream ATG in frame with the ras coding
sequence, creating a potential fusion protein. Translation of this mRNA in rabbit
reticulocyte extracts and Xenopus oocytes showed exclusive production of a p23 ras fusion
protein. When the upstream ATG was deleted, only p21 ras was translated in both systems.
Based on these results it is proposed that in vivo recognition of the upstream ATG and
translation of the ORF overlapping the p21 start site might serve to modulate the translation
of p21. The single base deletion and resultant ras fusion protein may constitute a novel
mechanism of ras overexpression by circumventing this translational regulation .