"We figured that if there were going to be mutations that constitutively activated these enzymes, and that would thus be targetable by drugs, the kinase domains would be the ones to go for," said Vogelstein.
The researchers first identified the kinase domains of 138 tyrosine kinases and similar enzymes from the kinome database. They next extracted these same domains from 35 colorectal cancer cell lines, most of which had been generated by Markowitz and his colleagues. They then sequenced those domains for comparison.
Their studies revealed mutations within the kinase domains in 14 genes. The researchers then analyzed another 147 colorectal cancer cell lines in the same way for kinase-domain mutations in these genes, and then sequenced the entire coding region of all kinases that were mutated, in all discovering 46 new mutations.
The major difficulty, said Vogelstein, was distinguishing the "signal from the noise," in this case, the mutations that would trigger cancer from the huge number of harmless variants in kinase genes. "We saw hundreds of changes in genes in cancer cells that had not been identified before, and for each of these, HHMI research associate Alberto Bardelli had to perform comparisons with the normal tissues of the same patient, to see if the mutation was specific to the cancer," he said.
Analyses of mutations unique to the cancer cells strongly indicated that the mutations affect the function of the tyrosine kinases in the cells, constitutively activating them, said Vogelstein. "Most importantly, they were in positions of the kinase domains predicted to alter function, based on analogous mutations that have been seen before in other kinases," he said.
According to Vogelstein, these studies of the mutations indicate that about 30 percent of colorectal cancers have mutations in at least one ki
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Contact: Jim Keeley
keeleyj@hhmi.org
301-215-8858
Howard Hughes Medical Institute
8-May-2003