MAPPING THE APC GENE REGION
ISOLATION OF THE APC GENE
The first candidate gene for APC was cloned by Kinzler et al (1991a). Using DNA probes known to be around the APC gene, a panel of sporadic colorectal carcinomas was studied. One probe which was named cosmid 5.71 detected a rearranged restriction fragment in the DNA of one tumour. Further studies indicated that the sequences within 5.71 were highly conserved in rodent DNA. A gene "MCC" (Mutated in Colorectal Cancer) was identified from these sequences and it was found that it encoded a 829 amino acid protein with a region of similarity to a G-protein. It was established that the rearrangement found in the tumour disrupted the gene's coding region and furthermore two other tumours displayed point mutations in MCC. At this time, MCC had not been tested in the germline of FAP patients so it was not certain whether MCC was indeed APC. Bourne (1991) suggested two hypotheses for the action of the MCC protein and how it might lead to colon cancer. The first was that it binds to and inhibits G-proteins through the calcium/phosphonositide pathway, which is mitogenic in many cells. The second, and preferred, hypothesis was that of the coiled coil. Here, the protein would form a structure where long regions of alpha helix wrap around each other (hence-a coiled coil). Coiled coils, in general, tend to serve as spacers between other proteins which bind to the globular domains at either end. Cell proliferation is frequently regulated by cell shape thus one can imagine a filamentous cellular structure disrupted by a coiled coil may somehow derepress a mitogenic signal.
Three further candidate genes around the APC region were isolated by Joslyn et al
(1991) using nested deletions. The first had a sequence identical to "SRP19" a gene coding for part of a ribosomal signal recognition particle. The second was termed DPI (deleted in polyposis 1), this was transcribed in the same orientation as MCC. Two other stretches of DNA were found and termed DP2 and DP3, however these were found to overlap and hence be a single gene DP2.5 transcribed in the same orientation as SRP19. The small nested deletions (100-260kb) in two patients with FPC encompassed these 3 genes and it was established that MCC was outside this deleted region. All three genes were sequenced.
Discovery that MCC was outside the deleted region shed doubt on its role in FPC despite the fact that its 3' end had not been isolated. It was postulated that the deletion mutations could alter or remove the regulatory elements affecting MCC expression. Alternatively, MCC alterations could be a second step in tumorigenesis. It is, of course, possible that these alterations play no part in tumour formations and are just coincidental. In an accompanying paper, Groden et al (1991) identified that it was the DP2.5 that was indeed the APC gene. Single strand conformation polymorphism (SSCP) studies identified four alterations of DP2.5 exons unique to FPC patients. Two of these mutations were base substitutions leading to a change from amino acid to a stop codon, the other two were small deletions leading to frameshifts.
In tandem with the above two papers Kinzler et al (1991b) and Nishisho et al (1991) (accompanying papers) also identified the APC locus. A portion of the region closely linked to APC (between the markers YN5.48 and YN5.64) was saturated with yeast artificial chromosome (YAC) cloned contiguous stretches of DNA (contigs). Six contigs were isolated in total spanning approximately 5.5Mb of DNA. Three of these encompassing ~4Mb were contained within the central portion of this region. In the first, the gene "FER" was found and in the second, the gene "TB1" (identified by cross hybridisation between human and rodent DNA's). The third contig was the one
of most interest, it was initiated from the MCC gene and hence contained it. It contained nine overlapping YACs (also one cosmid centromeric of the MCC gene called "L5.79") and within this region, four genes were identified, the first was MCC. The second "TB2" was identified, it showed no similarity to any existing sequence and there was no evidence linking it to colorectal tumorigenesis in any way. The third "SRP19" was an already cloned and sequenced gene (see above). The final gene was found to be mutated in the germ line of FAP patients and in sporadic colorectal cancers and was hence identified as the APC gene.
DNA from colorectal tumours and normal colonic mucosa was examined with clones from the genes FER, TB1, TB2, SRP19, MCC and APC (Nishisho et al., 1991). Only MCC and APC seemed to be implicated in colorectal neoplasia. Then the germ line DNA of FPC and GS patients was examined for subtle alterations in MCC and APC. Unique point mutations of the APC gene only were found in five out of 103 FAP patients-four of which were a change from an amino acid to a stop codon, the fifth an Arg-Cys change. APC and MCC are very close (~150kb apart). This, and the fact that both are implicated in colorectal tumorigenesis seems to suggest an interaction between the two. Furthermore the protein encoded by the APC gene (consisting of 2,843 amino acids), like that of the the MCC gene (829 amino acids), is predicted to have coiled coil regions (Kinzler et al., 1991b).