The intriguing observation has been made that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] receptors are present in tissues not involved in calcium homeostasis and that 1,25(OH)2D3 exerts an antiproliferative, differentiation-promoting action in a variety of cancer cell lines, including cells of the large intestine. It was therefore deemed of interest to study 1,25(OH)2D3 expression and biological activity in a murine model of colon carcinogenesis. Colon carcinogenesis was induced in male rats by the sequential administration of 1,2-dimethylhydrazine dihydrochloride (DMH). Levels and binding characteristics of 1,25(OH)2D3 receptors were assessed in control and DMH-treated rat colonic mucosal high-speed supernatants. In concurrent studies, 1,25(OH)2D3 was administered (s.c., 400 ng/rat) prior to, together with and after DMH challenge and the activity of ornithine decarboxylase (ODC), a growth-related DMH-induced enzyme, was determined in colonic cytosols. Serum Ca2+ levels were measured concurrently. Rats submitted to identical treatment schedules were killed 10 weeks after termination of DMH administration and the whole colon was opened and examined for tumors. The results show that (i) rat colonic mucosa possesses a single class of high-affinity 1,25(OH)2D3 receptors; (ii) DMH administration provokes a marked reduction (50%) in 1,25(OH)2D3 binding sites without affecting Kd values; (iii) DMH administered concurrently with 1,25(OH)2D3 suppressed the vitamin D-induced hypercalcemia and restored serum Ca2+ concentrations to basal levels; and (iv) 1,25(OH)2D3 delivered prior to DMH challenge obliterated the typical DMH-induced early colonic ODC activity peak and markedly reduced (50%) the number of colon adenocarcinomas. The present findings indicate that a colon-specific potent carcinogen interferes with the biological expression of 1,25(OH)2D3 and that vitamin D administered prior to a carcinogenic insult is able to reduce significantly the incidence of colon tumors, presumably acting as an antiproliferative or differentiation-promoting agent.

SUMMARY The activity of cAMP-dependent and cAMP-independent protein kinases, a class of enzymes involved in the regulation of cell proliferation was measured in rat colonic epithelium. Sequential cell populations harvested by a stepwise scraping technique from colonic crypt regions were identified by histology and incorporation of [3HJ-thymidine into DNA. cAMP-independent phosphorylation of casein, in the presence of [y-32PJATP, was markedly suppressed by quercetin, a bioflavonoid known to inhibit G-type casein kinase, protein kinase-C and tyrosine protein kinase. Conversely, the cyclic nucleotide regulatable form requiring histone as substrate was responsive to the action of the heat stable protein kinase inhibitor. The protein kinase species were characterised and partially purified by DEAE-cellulose chromatography. The activity ofcAMP-dependent protein kinase in colonic cytosols (pmol 32P/min/mg protein, means (SE)) increased from 129.4 (15.9) in superficial ceUl populations to 238-5 (31.4) in lower crypt cell fractions (p<0 01). Colonic cAMPindependent protein kinase activity increased from 87-3 (15-6) in surface cell preparations to 17841 (300) in lower crypt cell populations (p<002). A comparable activity gradient was observed in membrane fractions. The activity gradient persisted when the results were expressed as a function of celular DNA. These findings indicate that protein kinases display a defined topological segregation along the colonic crypt regions and that during migration to the lumen colonic cells attenuate enzyme signals supposedly related to tissue growth.The colonic epithelium provides a unique model of cells at different stages of differentiation aligned in an orderly pattem along the crypt-lumen axis.'2 Differentiation of colonic cells is accompanied by congruent morphological and biochemical changes.'2 Synthesis of nucleic acids is restricted to mitotically active young cells in the crypt regions: during the migration toward the lumen, the maturing cells acquire the typical absorptive and secretory functions and limited life expectancy.'2 Senescent colonocytes are shed into the bowel lumen and replaced by cells migrating from the proliferative zones. The sizes of the proliferative and functional compartments along the crypt surface axis are maintained within precise boundaries by multiple homoeostatic mechanisms.' 2

Cl- efflux, CFTR transcription and forskolin-stimulated cAMP activity occur in both crypt and villus epithelial cells in duodenum. Possible interaction between CFTR and Na+ channels is apparently limited to parts of the colonic crypt. Lack of duodenal beta-subunit expression makes ENaC activity unlikely.

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