The TP53 tumour suppressor gene in colorectal carcinomas. II. Relation to DNA ploidy pattern and clinicopathological variables.

Heterozygous loss of the TP53 gene on chromosome arm 17p in colorectal carcinomas was strongly associated with DNA aneuploidy (P < 0.0001). This association was seen only in tumours with loss on both 17p and 17q (P < 0.001), but not for loss on 17p only. DNA near diploid (ND) carcinomas and DNA aneuploid (AN) tumours with DNA index > or = 1.1 and < 1.3 had similar frequencies of TP53 gene loss (49% and 42%, respectively), whereas AN tumours with DNA index > or = 1.3 had a significantly higher frequency of TP53 gene loss (85%) (P < 0.0001 and P < 0.0001, respectively). There was a significant association between loss of the TP53 gene and histological grade (P < 0.01), and there tended to be an association between loss of the TP53 gene and degree of cellular atypia (P < 0.05), with TP53 gene loss being most frequent in moderately differentiated carcinomas, and in carcinomas with severe cellular atypia, respectively. The proportion of tumours with loss of the TP53 gene increased significantly towards the distal part of the large bowel (P < 0.0001). These results indicate that different genetic mechanisms may be involved in the carcinogenesis in colon and rectum carcinomas, and in the two subsets of DNA aneuploid carcinomas. Furthermore, the data may suggest a role for the TP53 gene in the aneuploidisation process, possibly as a 'target' for a whole chromosome loss.

The TP53 tumour suppressor gene in colorectal carcinomas. I. Genetic alterations on chromosome 17.

In 231 colorectal carcinomas, allele variation at four restriction fragments length polymorphisms (RFLP) loci on chromosome 17 have been studied by Southern analysis. Heterozygous loss of the TP53 gene was found in 68% (129/189) of the carcinomas informative on both chromosome arms. In 41% (77/189) of the carcinomas the loss was found only on 17p. Two probes were used to detect alterations on 17p, pBHP53 and pYNZ22. When loss was demonstrated with pYNZ22, pBHP53 also always showed loss (n = 45), whereas when loss was demonstrated with pBHP53, only 45 of 54 (83%) showed loss with pYNZ22. Loss on 17q was found in 34% (64/189) of the carcinomas, and 6% (12/189) had loss on this chromosome arm, only. Loss on 17q was significantly associated with loss on 17p (P < 0.01). These data confirm that the TP53 gene is the target of loss on chromosome arm 17p in colorectal carcinomas, and demonstrate that loss of the TP53 gene is most frequently part of limited, subchromosomal loss. Furthermore, the results do not suggest any additional tumour suppressor gene(s) on chromosome 17 involved in colorectal carcinogenesis.

Genetic alterations within the retinoblastoma locus in colorectal carcinomas. Relation to DNA ploidy pattern studied by flow cytometric analysis.

Alterations within the retinoblastoma (Rb) gene, as detected by the VNTR probe p68RS2.0, and flow cytometric DNA pattern have been analysed in 255 colorectal carcinomas. A total of 35.3% of the tumours had alterations within the Rb gene. Amplification of one allele was demonstrated in 29.5% of the tumours, and loss of heterozygosity was found in 11.5%. No association was found between amplification within the Rb gene and clinicopathological characteristics of the patients. The high frequency of alterations demonstrated within the Rb gene, suggests that this gene is involved in colorectal carcinogenesis with amplification as by far the most abundant genetic alteration. This may imply that the Rb gene has an oncogene-like function in colorectal carcinomas, rather than acting as a tumour suppressor gene. Sixty-three per cent of the carcinomas were DNA aneuploid, and a significant association was demonstrated between amplification within the Rb gene and DNA aneuploidy (P less than 0.01). Two other chromosome loci were analysed, on chromosome 1p (probe pYNZ2) and on chromosome 2p (probe pYNH24), respectively. On chromosome 1p, heterozygous loss was found in 22.2% of the tumours, indicating an involvement of this chromosome in a subset of colorectal carcinomas.

Studies of the ADP/ATP carrier of mitochondria with fluorescent ADP analogue formycin diphosphate.

The ADP/ATP carrier was studied by a fluorescent substrate, formycin diphosphate which is the only fluorescent ADP analogue to bind. Its low quantum yield, short decay time and spectral overlap with tryptophan has as yet prevented its wider use. By incorporating fluorescent acceptors of formycin diphosphate fluorescence, anthracene-maleimide and vinylanthracene, into the membrane, these difficulties were circumvented. Only bound formycin diphosphate transfers energy to the probes so that the secondary emission of these probes is a measure for membrane-bound formycin diphosphate. The fluorescent transfer is inhibited by ADP, bongkrekate and carboxyatractylate whether added before or after incubation of formycin diphosphate showing that only binding to the adenine nucleotide carrier is measured. It also shows directly that the earlier demonstrated ADP fixation by bongkrekate is indeed a displacement into the matrix. The fluorescence decay time of the bound formycin diphosphate is measured as 1.95 ns compared to 0.95 ns of the free formycin diphosphate, indicating that formycin diphosphate is bound at the carrier in a non-polar environment. The depolarization decay time was found to be larger than 15 ns, indicating that carrier-bound formycin diphosphate is immobile within this time period.