We report the first evidence of increased levels of the retinoblastoma (Rb) message in a majority of colorectal cancers when compared with normal mucosa. Southern blot analysis showed an increase in Rb gene copy number in at least 28% of colorectal carcinomas relative to normal mucosa. These results plus previous reports of nonrandom chromosome 13 gains in approximately 50% of colorectal cancers suggest that an increase in Rb gene copy number occurs frequently in these tumors. Possible mechanisms pertaining to overexpression of the Rb gene are discussed in relation to its role as a recessive cancer gene.

A cDNA for chicken avidin was identified in a chicken oviduct cDNA library by screening with antibodies and synthetic oligodeoxyribonucleotides. Four recombinant clones were characterized and each contained the sequence of the oligonucleotide probes used in screening. They were capable also of expressing an antigen recognizable by a polyclonal or a mixture of monoclonal antibodies raised against avidin. The longest clone, lambda cAV4, contained the entire coding sequence of avidin along with a signal peptide of 24 amino acids. An avidin mRNA, approximately 700 nucleotides in length, was induced by a single injection of progesterone over a period of twenty four hours. The avidin mRNA was distributed in a tissue-specific manner, since detectable concentration of the mRNA appeared only in the oviduct after stimulation with progesterone alone or with a combination of progesterone and estrogen. No avidin mRNA was detected in the liver or kidney under these conditions. Preliminary results on the genomic complexity of avidin suggest a single copy gene. Isolation of the natural gene for avidin and studies on its regulation now can be initiated using the cDNA probe.

In an effort to understand the possible role of Rb in cellular growth control, we have investigated the abundance and the state of phosphorylation of Rb protein (pRb) in normal and colon tumor cell lines as well as in matched colon tumors, adenomas and adjoining normal colonic mucosa. Resting normal human fibroblast cell lines were found to have only unphosphorylated pRb and phosphorylation of pRb occurred when the cells entered G1-S phase. In general, the colon tumor tissues had at least 1.5-2.0 fold increase in the abundance of pRb and 1.5-2.5 fold increase in the percentage of its phosphorylation as compared to the corresponding normal colonic mucosa. Whereas, the adenomas had similar pRb level and its phosphorylation status as observed in the normal colonic mucosa. The actively growing tumor cell lines had approximately two fold higher total pRb than normal cell lines. Although, the percentage of phosphorylated form in growing tumor cell lines as well as normal cell lines were almost equal, it was still considerably higher than normal colonic mucosa. Moreover, DNA binding assay revealed reduced binding affinity of pRb from colon tumor cell line SW480 as compared to the normal cell line WI38. These results suggest that the abundance of pRb and its phosphorylation level may have a role in the cellular growth control in human colonic epithelium.

We have compared the expression of the retinoblastoma (Rb) and p53 genes in normal human fibroblasts, colon carcinoma cell lines, matched pairs of colorectal tumor tissues and adjacent normal mucosa and in synchronized human diploid fibroblast cell line WI38. The increased expression of Rb and p53 RNA was observed in a majority of colorectal cancers in comparison to adjacent normal mucosa and is accompanied by proportional increase in the expression of histone H3 gene. The Rb and p53 RNA levels varied significantly between the various colon carcinoma cell lines. However, we found that the expression of Rb and p53 RNA is regulated differently in cell cycle synchronized normal human fibroblasts. The Rb mRNA level did not change with the position in the cell cycle and did not differ significantly whether the cells were serum deprived or in 10% serum. But p53 mRNA expression follows the same pattern as histone H3 mRNA.

Tumor-specific alterations at the p53 gene locus in 30 human vestibular schwannomas (VS) comprising 10 confirmed NF2 cases and 20 sporadic cases were analyzed. We found loss of heterozygosity (LOH) at the first intron of the p53 gene locus in 54% of the informative cases. This is the first report showing LOH at the p53 gene locus in a significant number of human VS and both sporadic and NF2 cases show the LOH event. Increased levels of normal size p53 mRNA and p53 protein were found in all the tumors analyzed. Thus p53 appears to be deregulated in all the tumors suggesting that p53 alterations may be associated with tumor progression in VS. There was a negative significant correlation of patients' age and percentage of Ser 392 phosphorylated p53 protein. The tumor samples obtained from younger patients of 35 yr and below showed higher percentage of Ser 392 phosphorylated p53 protein compared to the tumors of older patients. The increased percentage of Ser 392 phosphorylated p53 protein indicates that it could be involved in the acceleration of tumor growth in the younger patients. Our results suggest that age dependent phosphorylation of p53 protein and deregulation of p53 gene has a role in the development of human vestibular schwannomas.