Molecular genetic studies of tumor suppressor gene regions on chromosomes 13 and 17 in colorectal tumors.
BACKGROUND
In the majority of colorectal carcinomas, both copies of the tumor suppressor gene TP53 (tumor protein 53) are known to be inactivated. In contrast to a loss of tumor suppressor function, it has been suggested that an increased copy number of the RB1 gene is involved in the progression of these tumors.


OBJECTIVE
To determine genetic alterations at chromosomes 13 and 17 in colorectal tumors, we have studied several loci on these chromosomes, with special focus on the RB1 and TP53 genes at both the level of DNA sequence and the level of gene expression.


METHODS
Restriction fragment length polymorphism analysis was performed after alkaline Southern blotting of the DNA fragments and hybridization (in 7% sodium dodecyl sulfate and 0.5 M NaPO4) of the nylon membranes with multiprimed, radioactively labeled probes. Total RNA was extracted from tissue biopsy specimens by homogenization of the samples in guanidinium thiocyanate followed by separation in a CsCl gradient. By use of an image-processing system, x-ray film signals were measured densitometrically. Point mutations within the TP53 gene were detected by use of polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis. Direct sequencing of PCR products revealed the exact nature of the mutations. Protein expression of TP53 was seen by immunostaining of sections from paraffin-embedded material using a mouse monoclonal antibody. The two-sided Fisher's Exact Test was used for statistical analysis.


RESULTS
An increase in allelic copy number at 13q loci was seen in 10 (32%) of 31 tumors. In the majority of the cases, this increase probably reflected a change in the diploid status of chromosome 13; in some cases, however, only part of the 13q seemed to be involved. The RB1 gene showed an elevated level of RNA compared with the beta-actin signal. Fourteen (48%) of 29 tumors showed loss of heterozygosity at loci on 17p, and base mutations within the TP53 gene were seen in 14 (42%) of 33 tumors. RNA and protein analyses of TP53 revealed an increased level of expression in the tumors compared with normal mucosa. Allelic variations seen at 13q and 17p were not associated (P = .7).


CONCLUSIONS
Our results suggest that, in addition to aneuploidy, gain of specific chromosome 13 sequences is involved in the tumorigenesis of the colon and rectum. In addition, they confirm the importance of TP53 mutations for the progression of such tumors and support the view that accumulation of events is more important than the order of events. The genetic changes observed at chromosome arms 13q and 17p seem to be independent of each other.
Lothe
R A
RA
Department of Genetics, Norwegian Radium Hospital, Montebello, Oslo.
Fossli
T
T
Danielsen
H E
HE
Stenwig
A E
AE
Nesland
J M
JM
Gallie
B
B
Børresen
A L
AL
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D015152:Blotting, Northern; D002882:Chromosomes, Human, Pair 13 / Q000502:physiology*; D002886:Chromosomes, Human, Pair 17 / Q000502:physiology*; D003062:Codon / Q000235:genetics; D015179:Colorectal Neoplasms / Q000235:genetics*; D015342:DNA Probes; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics; D004586:Electrophoresis; D005260:Female; D015870:Gene Expression / Q000235:genetics; D016147:Genes, Tumor Suppressor / Q000235:genetics*; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D012334:RNA, Neoplasm / Q000032:analysis; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D003062:Codon; D015342:DNA Probes; D004273:DNA, Neoplasm; D012334:RNA, Neoplasm; D016159:Tumor Suppressor Protein p53