In order to better understand colon cancer, a model system reflecting the heterogenous nature of this disease was developed and used in the development of new cytotoxic and non-cytotoxic therapeutic approaches. A large bank of colon carcinoma cell lines was established from primary human colon carcinomas and grouped based on their tumorigenicity in athymic mice, their growth rates in soft agarose and in tissue culture, and their secreted levels of carcinoembryonic antigen. These cell lines were later characterized based on cell surface proteins and antigens detected with antisera raised against a differentiated colon carcinoma cell line. Although these biochemical markers correlated with the biological classification of these cell lines, there was still extensive heterogeneity within each group in all properties examined. This colon carcinoma cell system was used to study natural vs. selected resistance to the anticancer drug mitomycin C (MMC). The differing IC50 values in vitro were reflected in the inhibition by MMC of xenograft growth in athymic mice. A new, more readily bioactivatable analogue of MMC was tried and shown to be more active in vitro and in vivo, suggesting that rapid efflux of the drug before activation may be important in examining causes of resistance to MMC. Another approach to the treatment of colon cancer is the use of non-cytotoxic agents such as growth factors and differentiation agents to restore normal growth to the malignant cells. We have isolated and characterized two types of polypeptides from colon carcinoma cells and conditioned medium from these cells. The first, transforming growth factors (TGF's) confer a transformed phenotype on non-transformed fibroblasts while the second, tumor inhibitory factors (TIF's), inhibits the anchorage independent growth of transformed cells. The fact that extracts of colon carcinoma cells contain both activities suggests that the heterogeneity of the cell lines could be due to different levels of TGF's and TIF's produced. The effectiveness of differentiation agents to restore normal growth control using a transformed mouse embryo cell line was examined. Treatment of these cells with differentiation agents restored normal growth control to these cells. An increased synthesis of TGF's resulted from these treatments. Therefore, differentiation agents may be useful in non-cytotoxic treatment. The use of this model system for human colon carcinoma will hopefully lead to more effective drugs for the treatment of colon cancer in man.

Protein C23 (Mr 110 000, pI = 5.5), a major phosphoprotein in the nucleolus of mammalian cells, has been shown to contain 1.3 mol% of NG,NG-dimethylarginine (DMA) [Lischwe, M.A., Roberts, K.D., Yeoman, L.C., & Busch, H. (1982) J. Biol. Chem. 257, 14600-14602]. A tryptic peptide from protein C23 that contains DMA has been isolated and sequenced. Its sequence is Gly-Glu-Gly-Gly-Phe-Gly-Gly-DMA-Gly-Gly-Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA- Gly-Gly- Gly-DMA-Gly-Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA-Gly-DMA-Gly-Gly-Phe-Gly-Gly- DMA-Gly-Gly-Phe-DMA-Gly-Gly-DMA-Gly-Gly-Gly-Gly-Asp-Phe-Lys. This peptide contains 34 glycine, 10 DMA, and 6 phenylalanine residues and has clusters of glycine and NG,NG-dimethylarginine interspersed with phenylalanine residues. A similar domain has been found at the amino terminus of a nucleolar protein of Mr 34,000, pI = 8.5. This sequence array may represent a conserved domain characteristic of a certain class of nuclear proteins. All of the methylated arginine residues in protein C23, the 34-kilodalton protein, and myelin basic protein [Carnegie, P.R. (1971) Biochem. J. 123, 57-67] have at least one adjacent glycine. Access of certain arginine methylases to arginine residues may be sterically possible because of the lack of a side chain on the adjacent glycine residue(s).

Abstract. A DNA-binding nonhistone protein, protein BA, was previously demonstrated to co-localize with U-snRNPs within discrete nuclear domains (Bennett, F. C., and L. C. Yeoman, 1985, Exp. CellRes., 157:379-386). To further define the association of protein BA and U-snRNPs within these discrete nuclear domains, cells were fractionated in situ and the localization of the antigens determined by double-labeled immunofluorescence. Protein BA was extracted from the nucleus with the 2.0 M NaC1 soluble chromatin fraction, while U-snRNPs were only partially extracted from the 2.0 M NaCl-resistant nuclear structures. U-snRNPs were extracted from the residual nuclear material by combined DNase I/RNase A digestions. Using an indirect immunoperoxidase technique and electron microscopy, protein BA was localized to interchromatinic regions of the cell nucleus.Protein BA was noted to share a number of chemical and physical properties with a family of cytoplasmic enzymes, the glutathione S-transferases. Comparison of the published amino acid composition of protein BA and glutathione S-transferases showed marked similarities. Nonhistone protein BA isolated from saline-EDTA nuclear extracts exhibited glutathione S-transferase activity with a variety of substrates. Substrate specificity and subunit analysis by SDS pop yacrylamide gel electrophoresis revealed that it was a mixture of several glutathione S-transferase isoenzymes. Protein BA isolated from rat liver chromatin was shown by immunoblotting and peptide mapping techniques to be two glutathione S-transferase isoenzymes composed of the Yb and Yb' subunits.Glutathione S-transferase Yb subunits were demonstrated to be both nuclear and cytoplasmic proteins by indirect immunolocalization on rat liver cryosections.

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