The onset and extent of genomic instability in sporadic colorectal tumor progression - PubMed (original) (raw)

The onset and extent of genomic instability in sporadic colorectal tumor progression

D L Stoler et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Cancer cell genomes contain alterations beyond known etiologic events, but their total number has been unknown at even the order of magnitude level. By sampling colorectal premalignant polyp and carcinoma cell genomes through use of the technique inter-(simple sequence repeat) PCR, we have found genomic alterations to be considerably more abundant than expected, with the mean number of genomic events per carcinoma cell totaling approximately 11,000. Colonic polyps early in the tumor progression pathway showed similar numbers of events. These results indicate that, as with certain hereditary cancer syndromes, genomic destabilization is an early step in sporadic tumor development. Together these results support the model of genomic instability being a cause rather than an effect of malignancy, facilitating vastly accelerated somatic cell evolution, with the observed orderly steps of the colon cancer progression pathway reflecting the consequences of natural selection.

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Figures

Figure 1

Figure 1

Inter-SSR PCR analyses of colorectal cancers (14). (CA)8 was anchored with either RG or RY, where R is an equal mixture of the purines adenosine and guanine, and Y is an equal mixture of the pyrimidines cytosine and thymidine. (CG)4 was anchored with RY. Patients 3131 and 3213 each were examined with the (CA)- and (CG)-based primers, and products amplified from tumor DNA were compared with those from normal colonic tissue DNA from the same patient. Dots placed alongside lanes indicate bands found at significantly greater (>2 fold) intensity in the tumor or normal tissue DNA. For patient 3213 one new band from the tumor migrated close to the position of a band that disappeared from the normal tissue, and thus may represent a small deletion, indicated by >.

Figure 2

Figure 2

The size distribution of altered bands is similar to the size distribution of total inter-SSR PCR products. (a) Size distribution of inter-SSR PCR products for 58 human colorectal cancers. The number of inter-SSR PCR products for (CA)8RY-primed (black bars) and (CA)8(RG)-primed (gray bars) reactions was determined for each 100-bp size interval. (b) The number of new or lost inter-SSR PCR bands was determined for each indicated 100-bp size interval. New bands or bands with increased intensity in the tumor are shown by bars above the x axis; bars below the axis indicate lost bands or bands with decreased intensity in the tumor.

Figure 3

Figure 3

Characterization and verification of an altered inter-SSR PCR product amplified from tumor DNA. (A) Tumor (T) and normal (N) DNA from patient 3153 was used as a template in independent PCRs primed with (CA)8RG alone or with the _Eco_RI linker-tagged primer GGAATTC (CA)8RG. (B) Clones were prepared in vector pCR 2.1 (Invitrogen) by using their TA cloning kit. The insert size of the clones was the same as that of the original inter-SSR PCR product. (C) Clones were sequenced in both directions by using the T7 primer (forward) and the M13 primer (reverse). Sequence data are shown for a 78/74-bp region that contained the tumor-specific difference in the 923/919-bp sequences of the cloned inter-SSR PCR products. (D) Nested tumor specific primers G19–1P1 and G19–1P2 were used to prime three independent reactions from the original tumor and normal DNA, confirming the presence of a four-base insertion in the tumor DNA. Control PCRs used the clone 9T- and clone 23N-containing plasmids as templates.

Figure 4

Figure 4

Inter-SSR PCR analyses of a colorectal carcinoma and synchronous adenomatous polyp from patient 3022. Three band alterations are present in the polyp and four in the carcinoma, with two bands common to the benign and malignant tumors.

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