Overexpression of ras in mucus-secreting human colon carcinoma cells of low tumorigenicity.

We have investigated the expression of the protooncogenes of the myc and ras family in HT29 cells and in three differentiated clonal cell lines derived from this colon carcinoma cell line. In contrast to the decrease in myc expression seen when leukemia cells are induced to differentiate, we have found no changes in expression of the myc gene family in differentiated colon carcinoma cells. However, a greater than 5-fold increase in expression of sequences which hybridize to Ha-ras was observed in cells which secrete mucin, with a smaller increase seen in expression of Ki-ras in the same cells. This increase was not seen in cells which exhibit vectorial transport of water and ions, and which are not mucus-secreting. All differentiated lines were less tumorigenic in nude mice than the parental HT29 cells, irrespective of the level of ras expression. These results are consistent with the reports that ras expression is highest in the most differentiated cells of the colon and is substantially decreased in metastatic human colon tumors as compared to primary colon tumors. The data also suggest that a high level of ras gene expression is a marker for a particular differentiated state in colon cells rather than being directly equated with transformation or tumorigenicity. Hence, the results may reflect on some of the discrepancies concerning ras gene expression in human colon and other tumors which appear in the literature.

Effects of 6-mercaptopurine on developing muscle cells in vitro.

Treating differentiating muscle cells in vitro with 6-MP has resulted in a number of myopathic changes, some of which resemble the changes seen in 6-MP-treated neonatal rats. 6-MP treatment was cytotoxic to myotubes, but not myoblasts. The degenerative changes observed in 6-MP-treated myotubes were quite similar to those described in the neonatal rats by Alleva and his colleagues (1981). The results of this investigation demonstrate that differentiating muscle grown in vitro can be used to investigate tissue-specific toxicity, although the mechanism by which 6-MP results in selective toxicity in myotubes remains to be elucidated.

Amplification and elevated expression of c-myc in a chemically induced mouse colon tumor.

The amplification and level of expression of four cellular oncogenes was investigated in dimethylhydrazine-induced Mouse Colon Tumor 36 of Corbett et al. [Corbett, T. H. Griswold, D. P., Jr., Roberts, B. J., Peckham, J. C., and Schabel, F. M., Jr. Cancer (Phila.), 40:2660-2680, 1977], which has been used to evaluate potential chemotherapeutic agents. A 30- to 40-fold amplification of c-myc was observed in the tumor DNA as compared to the DNA of normal colonic mucosa and liver. No changes were observed in the number of copies of c-mos, erbB, or bas. Digestion with three different restriction enzymes demonstrated that there was no major alteration in the genomic structure of c-myc in the amplified DNA. The level of c-myc RNA was 10-fold higher in both the total and polyadenylated RNA from the tumor when compared to RNA from normal colonic mucosa. Probes specific for the three myc exons were used in hybridization experiments and demonstrated that all three exons were present in the amplified DNA and in the mRNA.

Investigating the myopathic effects of 6-mercaptopurine on developing skeletal muscle cells in vitro.

6-Mercaptopurine (6-MP) is an adenine antagonist which has been shown to cause skeletal muscle atrophy in neonatal rats. To investigate the effects of 6-MP on developing muscles, pure populations of myoblasts and myotubes in vitro were treated with 6-MP (5-50 micrograms/ml). The viability and protein content of myotubes, but not myoblasts, was decreased by 6-MP. Microscopic examination of the 6-MP-treated (50 micrograms/ml) myotubes demonstrated that severe degenerative changes had occurred, including extensive necrosis, inhibition of myotube formation, and the appearance of intracellular vacuoles. However, 6-MP treatment decreased the incorporation of radiolabeled leucine and thymidine by myoblasts, but not myotubes. These findings indicate that 6-MP exerted a selective toxicity on myotubes but not myoblasts.

A scanning electron microscopic study of the development of the shoulder, visceral arches, and the region ventral to the cervical somites of the chick embryo.

Scanning electron microscopy (SEM) was used to investigate the development of the shoulder in the chick embryo. Initially the wing grows on an axis perpendicular to the dorso-ventral and cranial-caudal axes of the embryo, but soon begins to grow in a ventral and partially caudal direction. The change in axis of outgrowth occurs while the shoulder forms at the cranial proximal portion of the wing. Analysis of SEM observations, together with an analysis of serially sectioned embryos and photographs of live embryos in ovo has demonstrated that the shoulder continues to grow out on an axis perpendicular to the dorso-ventral axis of the embryo, while the caudal and distal portions of the wing grow ventrally. The change in axis of outgrowth seems to be due to 1) the formation of the viscera under the wing, 2) the closing of the membrana reuniens to form a continuous sheet covering the viscera under the wing. 3) caudal movement of the duct of Cuvier and the cranial margin of the pleural coelom, and 4)ventral movement of the lateral body fold caudal to the wing. Although the visceral arches undergo major morphogenetic changes during this period, the visceral arches do not appear to have an influence on shoulder development.