Molecular Genetic Studies of Tumor suppressor Gene Regions on chromosomes 13 and 17 in Colorectal Tumors

• Published in: JNCI : Journal of the National Cancer Institute 1992-07, Vol.84 (14), p.1100-1108
• Main Authors: Lothe, Ragnhild A., Fossli, Tellef, Danielsen, Håvard E., Stenwig, Anna Elisabeth, Nesland, Jahn M., Gallie, Brenda, Børressen, Anne-Lise
• Format: Article
• Language: English
• Subjects: Adolescent; Adult; Aged; Aged, 80 and over; Base Sequence; Biological and medical sciences; Blotting, Northern; Chromosomes, Human, Pair 13 - physiology; Chromosomes, Human, Pair 17 - physiology; Codon - genetics; Colorectal Neoplasms - genetics; DNA Probes; DNA, Neoplasm - analysis; DNA, Neoplasm - genetics; Electrophoresis; Female; Gastroenterology. Liver. Pancreas. Abdomen; Gene Expression - genetics; Genes, p53 - genetics; Genes, Tumor Suppressor - genetics; Humans; Male; Medical sciences; Middle Aged; Molecular Sequence Data; Mutation; Other diseases. Semiology; Polymerase Chain Reaction; RNA, Neoplasm - analysis; Stomach. Duodenum. Small intestine. Colon. Rectum. Anus; Tumor Suppressor Protein p53 - genetics
• ISSN: 0027-8874; 1460-2105
• DOI: 10.1093/jnci/84.14.1100

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 RBI gene is involved in the progression of these tumors. Purpose: 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 RBI 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 multi-primed, 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 immuno-staining 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 RBI gene showed an elevated level of RNA compared with the β-actin signal. Four-teen (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