The naturally occurring polyamine spermine induces haemoglobin synthesis in murine erythroleukaemia (MEL) cells. Haemoglobin production was accompanied by accumulation of cytoplasmic beta-globin mRNA and growth inhibition, but not by cell-cycle block or changes in cell volume. Hexamethylene-bisacetamide (HMBA), a well known differentiating agent, also induces haemoglobin production, but causes a G1 block and decreases cell volume. These findings indicate that HMBA and spermine affect MEL cells differently, even though both induce haemoglobin production.

Mucinous colorectal cancer often presents at an advanced stage. We have previously observed that mucin production by human colon-cancer cells correlates with their ability to colonize the liver in experimental animal models. The present study was undertaken in order to further elucidate the mechanisms by which production of mucin by colon-cancer cells affects metastasis. Cell lines showing high mucin production (HMP) (HM 7, HM 3 and LS LiM 6) demonstrated increased adherence to basement membrane proteins and invaded a reconstituted basement membrane to a greater extent than their counter-part cell lines showing low mucin production (LMP) (LS174T and LM 12). Adherence of the LMP parental cell line LS174T to various matrix proteins was potentiated by the addition of purified human colon-cancer mucin in a dose-dependent fashion. HMP cell lines secreted more proteolytically active type-IV collagenase than LMP lines, and collagenase activity was further stimulated by purified mucin in a dose-dependent manner. Specific inhibition of mucin O-glycosylation by benzyl-alpha-N-acetylgalactosamine significantly affected each of the metastasis-related events, with the greatest effect on the HMP cell lines. The present data further indicate that mucin may play an important role in the metastatic process.

Tyrosine protein kinase (TPK) co-isolated with subcellular components derived from human colonic epithelium. The highest TPK activity, measured in the Triton X-100-insoluble cytoskeletal pellet, was directly related to the degree of malignancy of colonic tissue. TPK activity was assayed by measuring the incorporation of [gamma-32P] from [gamma-32]ATP into the synthetic polymer [Glu80Tyr20]n substrate. Lineweaver-Burk plots yielded an apparent Km of 167 micrograms/ml for [Glu80Tyr20]n and of 19 microM for ATP: Vmax for the phosphate donor was 0.9 nmol/min/mg protein. TPK activity was markedly stimulated by the metal ions Mg2+ and Mn2+ and significantly suppressed by tyrphostins, potent specific TPK inhibitors, shown to interfere with TPK-dependent growth processes. This is first report to present evidence for TPK activity associated with cytoskeleton-enriched subcellular preparations harvested from human colonic epithelium.

The activities of the growth-related enzymes ornithine decarboxylase (ODC) and casein kinase II (CK-II) were assayed along the colon crypt axis in a precise temporal sequence following administration of 1,2-dimethylhydrazine (DMH) to male rats. The time course of events monitored in colonic cell populations sequentially harvested by a scraping procedure shows that the potent carcinogenic insult induces an early and late ODC activity peak: the distinct biphasic response of the decarboxylase was observed in all colonic crypt compartments. The activity gradient of CK-II was markedly altered in DMH-treated cell populations: brisk activity of the kinase was observed in the upper crypt zone, the preserve of the mature, non-dividing colonocyte. The enhanced responses of ODC and of CK-II to DMH proceeded the actual polyp and tumor formation. The polycations spermine and spermidine, bioactive molecules formed in the ODC-controlled polyamine pathway, were shown to markedly activate colonic CK-II. This observation suggests that ODC and CK-II, enzymes with different catalytic purposes, crosstalk within the colonic crypt continuum. The present findings indicate that the differentiation arrest of colonic cells and their misplacement in forbidden zones of the crypt axis during DMH-induced carcinogenesis is accompanied by early alterations in the activity and topology of disparate enzymes which are part of the orderly growth program of the normal colonic cell.

Transforming growth factor beta (TGF-beta is a multifunctional regulator of cell growth. It has become increasingly clear that TGF-beta action depends on the responsive cell types. Thus TGF-beta stimulates proliferation of mesenchymal cells and, in contrast, inhibits the growth of a large variety of epithelial cells. Recent studies, albeit controversial, support the notion that a gradient in mRNA transcripts encoding TGF-beta is maintained along the crypt-villus continuum of the small intestine in close correlation with the stage of differentiation of the enterocyte. Exogenous TGF-beta has been shown to inhibit the proliferation of a non-transformed rat jejunal crypt cell line. Additional salient findings have indicated that colon carcinoma cell lines recalcitrant to the restraining action of TGF-beta on proliferation are poorly differentiated, and that moderately differentiated colon tumor lines do retain, at least partly, their responsiveness to TGF-beta. To the best of our knowledge, no evidence is available pertaining to a contributory role of TGF-beta in the signalling mechanisms regulating growth and differentiation of normal colonic epithelial cells. In addition, we are ignorant of whether the interesting findings related to a functional relationship between TGF-beta and colon carcinoma cells lines (vide supra) are applicable to colonic preneoplastic and tumor cells in their natural habitat. In this review, we dwell on the available facts and missing observations, and present conceivable hypotheses pertaining to a putative role of TGF-beta in colon differentiation and neoplasia.

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 [3H]-thymidine into DNA. cAMP-independent phosphorylation of casein, in the presence of [gamma-32P]ATP, 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 of cAMP-dependent protein kinase in colonic cytosols (pmol 32P/min/mg protein, means (SE)) increased from 129.4 (15.9) in superficial cell populations to 238.5 (31.4) in lower crypt cell fractions (p less than 0.01). Colonic cAMP-independent protein kinase activity increased from 87.3 (15.6) in surface cell preparations to 178.1 (30.0) in lower crypt cell populations (p less than 0.02). A comparable activity gradient was observed in membrane fractions. The activity gradient persisted when the results were expressed as a function of cellular 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.

Rat antral mucosae maintained for 6 h in organ culture responded to carbamylcholine with a significant increase in endogenous cyclic GMP production and gastrin secretion. The acetylcholine analogue exerted a stimulatory action within a defined concentration range: exposure of antral explants to carbachol concentrations greater than the optimal stimulatory dose was accompanied by a marked decrease in both cyclic GMP production and gastrin release. Exogenous 8-Br-cyclic GMP (1 mM) significantly augmented gastrin secretion into the culture media during 6-12 h culture periods. Cycloheximide (0.1 mM) and the Ca2+ channel-blocker verapamil (5 microM) prevented 8-Br-cyclic GMP from acting as a gastrin secretagogue. Addition of cyclic somatostatin-14 (0.1 mM) to culture media was attended by complete inhibition of 8-Br-cyclic GMP-stimulable gastrin secretion. These results provide evidence that cyclic GMP may play a mediatory role in the coupling of gastrin secretory processes to agonist stimulation. It would seem that the secretagogue action of 8-Br-cyclic GMP requires unabated Ca2+ transmembrane fluxes and protein biosynthesis. Since somatostatin-14 abrogates the stimulatory effect of 8-Br-cyclic GMP on antral gastrin secretion, it is surmised that the inhibitory tetradecapeptide acts at a locus (or loci) distal to domains involved in the actual generation of the cyclic nucleotide.

Acid secretion was monitored in five duodenal ulcer patients using intragastric glass pH electrodes located in the gastric body and antrum under basal conditions and after the administration of cimetidine. It was shown that differences exist between the body and antral pH in the basal state and in the response to cimetidine. The basal hydrogen ion concentration (mean +/- SEM, mmol/l) in the body, 25.97 +/- 5.03, exceeded that in the antrum, 10.59 +/- 6.44 (p less than 0.05). After cimetidine, the pH rose to 3.5 at both electrodes, this stage being shorter in the antrum (16 min) than body (54 min) (p less than 0.0125). During the next stage the pH rose above 3.5 and the hydrogen ion concentration (mean +/- SEM, mmol/l) was very low in both the body, 0.05 +/- 0.01 (p less than 0.0005 compared to basal) and antrum, 0.08 +/- 0.05 (p less than 0.05 compared to basal). Recovery to basal pH levels occurred more quickly in the body than antrum. Intragastric pH-metry offers a reliable method for studying the mode of action of pharmacological agents on the stomach and contributes information not made available by routine gastric analysis.