Cellular mechanisms for Na(+) retention in portal hypertension are undefined, but epithelial Na(+) channels (ENaC) may be involved. Under high-salt diet, ENaC are absent from distal colon of rat but can be induced by mineralocorticoids such as aldosterone. Presence of rat ENaC was determined by amiloride inhibition of (22)Na(+) uptake in surface colonocytes 7 and 14 days after partial portal vein ligation (PVL) or sham surgery. At both times, uptake inhibition was significantly increased in PVL rats. Presence of mRNA transcripts, determined by RT-PCR, demonstrated that channel alpha- and gamma-subunits were similarly expressed in both groups but that beta-subunit mRNA was increased in PVL rats. This confirms that there was induction of rat ENaC and indicates that beta-subunit has a regulatory role. Urinary Na(+) was decreased for 3 days after PVL but was not different at other times, and serum aldosterone levels were elevated at 7 days, at a time when urinary Na(+) output was similar to that of sham-operated rats. We conclude that PVL leads to induction of ENaC in rat distal colon. An increase in aldosterone levels may prevent natiuresis and is probably one of several control mechanisms involved in Na(+) retention in portal hypertension.

The aim of the present work was to gain insight into a putative anticancer effect of dietary vitamin D3 (cholecalciferol) in a rat model of colon carcinogenesis. Male rats were assigned to three different dietary groups. The dietary regimens were based on a standard murine-defined diet (AIN-76A) or a stress diet containing 20% fat, reduced Ca2+ concentration, a high phosphorus-to-Ca2+ ratio, and either low or high vitamin D3 content. Colorectal cancer was induced by administration of the procarcinogen 1,2-dimethylhydrazine (DMH). Blood Ca2+, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and 25-hydroxyvitamin D3 [25(OH)D3] levels were measured in DMH-treated rats and in respective weight- and age-matched dietary control groups. Colonic epithelial proliferation was assessed by determining thymidine kinase (TK) activity, bromodeoxyuridine (BrdUrd) incorporation into crypt cell DNA, and the mean labeling index along the colonic crypt continuum. Maintenance of rats on the stress diet either unmodified or supplemented with vitamin D3 in the absence of carcinogen treatment provoked a time-dependent rise in colonic TK activity and hyperproliferation of colonic epithelium. DMH treatment of rats maintained on the standard diet caused a marked increase in the proliferative indexes of colonic epithelium and in expansion of the crypt proliferative compartment. TK activity and the crypt mitotic zone were significantly augmented in the animal group fed the stress diet. Supplementary vitamin D3 abrogated the stress diet-enhanced colonic responses to the carcinogenic insult. Colon tumor multiplicity was fourfold higher in animals fed the stress diet than in animals maintained on a standard diet. The marked rise in colonic tumor multiplicity and adenocarcinoma incidence in rats fed the stress diet was obliterated by supplemental dietary vitamin D3. Cumulatively, the present results indicate that dietary vitamin D3 impedes the neoplastic process in murine large intestine and strengthen the view that inappropriate changes in dietary components and micronutrients are contributory determinants of colorectal cancer.

Menopause and estrogen deficiency are associated with apparent intestinal resistance to vitamin D, which can be reversed by estrogen replacement. The in vivo influence of estrogens on duodenal vitamin D receptor (VDR) was studied in three groups of rats: ovariectomized (OVX), sham-operated, and ovariectomized rats treated daily with estrogen (40 microg/kg BW) for 2 weeks (OVX + E). Estrogen administration to OVX rats resulted in a 2-fold increase in VDR messenger RNA transcripts. 1,25(OH)2D3 was shown to bind specifically to one class of receptors in duodenal mucosal extracts, with a dissociation constant of 0.03 nM. Binding was significantly increased in duodenal extracts from OVX + E rats, compared with OVX rats (735 +/- 81 vs. 295 +/- 26 fmol/mg protein; P < 0.001); a comparable, 1.5- to 2-fold increase in VDR protein expression was observed in Western blot analyzes of the duodenal mucosa. Markers of VDR activity were increased in estrogen-exposed rats: calbindin-9k messenger RNA transcript content was 1.4- to 1.6-fold higher, and alkaline phosphatase activity was 1.4- to 3-fold higher in sham-operated and OVX + E, respectively, compared with OVX. 25(OH)D, 1,25(OH)2D, or PTH levels were not altered by estrogen treatment. Cumulatively, these findings suggest that estrogen up-regulates VDR expression in the duodenal mucosa and concurrently increases the responsiveness to endogenous 1,25(OH)2D. Modulation of intestinal VDR activity by estrogen, and subsequent influence on intestinal calcium absorption, could be one of the major protective mechanisms of estrogen against osteoporosis.

BACKGROUND: Epidemiologic studies have demonstrated an inverse correlation between dietary calcium and vitamin D intake and the incidence of colorectal carcinoma. Elevated serum levels of 25-hydroxyvitamin D3 (25-OH-D3) are associated with a major reduction in the incidence of this neoplasm. The reduction in tumor size and number induced by calcium supplements in an experimental carcinogenesis model was neutralized by vitamin D3 deficiency. To the authors' knowledge, vitamin D serum levels have never been determined previously in colorectal carcinoma patients. They compared serum 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 25-OH-D3, and parathyroid hormone (PTH) levels of colorectal carcinoma patients with those of healthy controls. METHODS: Serum 1,25(OH)2D3, 25-OH-D3, and PTH levels were determined in 84 colorectal carcinoma patients (10 with Stage I, 29 with Stage II, 25 with Stage III, and 20 with Stage IV) and 30 healthy controls, all of whom were normocalcemic and not taking calcium or vitamin D supplements. RESULTS: 25-OH-D3 serum levels were higher in cancer patients than controls, irrespective of stage. Serum 1,25(OH)2D3 decreased with advancing stage: 73 +/- 18, 48 +/- 16, 39 +/- 12, 34 +/- 13, and 75 +/- 20 pg/mL in Stages I, II, III, IV, and controls, respectively. There was a corresponding increase in serum PTH levels: 58.0 +/- 9.4, 73.7 +/- 14.4, 79.0 +/- 21.3, 100.4 +/- 30.9, and 51.2 +/- 3.9 pg/mL in Stages I, II, III, IV, and controls, respectively. Serum vitamin D metabolite levels did not correlate with gender, age, tumor localization, or histologic grade. CONCLUSIONS: An inverse correlation between serum levels of the active metabolite of vitamin D and colorectal carcinoma stage has been demonstrated for the first time, to the authors' knowledge, in colorectal carcinoma patients. Because 1,25(OH)2D3 has been shown to inhibit proliferation of colonic epithelial cells, decreased serum levels may facilitate the growth of colorectal carcinoma and influence its biologic behavior.

Epidemiological studies suggest that estrogen prevents neoplastic transformation in the intestinal mucosa. Estrogen was shown to increase the expression of vitamin D receptors (VDR) in a variety of tissues. 1,25-Dihydroxyvitamin D [1,25-(OH)2D] and several of its analogues are known as potent antineoplastic and prodifferentiative in many cell types, including colon-derived cells. The present study was designed to examine the effect of estradiol (E2) on dimethylhydrazine (DMH)-induced colon cancer in rats, and the possibility that E2 may exert its protective effect on the colon through modulation of the vitamin D-endocrine system. The in vivo effect of E2 on DMH-induced colorectal cancer was studied in four groups of ovariectomized female rats: (I) untreated control, (II) E2 treated, (III) DMH treated, and (IV) combined E2 and DMH treated. Significantly higher uterine weights and higher colonic estrogen receptor content confirmed the effectiveness of ovariectomy and E2 replacement. The number of malignant tumors in group IV was 2.3+/-1.1 (mean +/- SE) per rat, compared with 8.1+/-1.9 in group III (P < 0.001). Exposure to estrogen was associated with a marked increase in VDR mRNA content and VDR protein expression in the normal colonic mucosa. In tumor extracts VDR protein expression was considerably lower compared with normal mucosa. Estrogen treatment did not affect serum levels of 25(OH)D, 1,25(OH)2D, and PTH. Significant CpG island methylation in the VDR gene was observed in colonic tissue DNA harvested from rats treated with DMH, but not in colonic mucosae from rats treated with DMH + E2. The highest frequency of CpG methylation in the VDR gene was detected in DNA extracted from cancer tissue rims. In summary, the protective effect of estrogen against chemically induced colonic carcinogenesis is associated with reduced methylation of the VDR gene and with upregulation of both VDR gene transcription and protein expression. We suggest that estrogen may interfere with the process of CpG DNA methylation in the colonic mucosa to prevent silencing of the VDR gene. Increased VDR activity could be one of the mechanisms by which estrogen protects against neoplastic transformation in the colon.

Apoptosis in cells of different lineages is restrained by survival signals which depend upon cell-to-cell communication. The aim of this study was to determine whether colonic cells deprived of crypt ambient are doomed to die prior to their normal chronological demise. Apoptosis was studied in rat whole colonic tissue, in isolated intact crypts, and in colonic cell populations collected from the crypt axis at different stages of proliferation and differentiation. In a number of experiments, cell harvest was performed in the presence of either a tetrapeptide (YVAD-CMK) inhibitor of interleukin-1beta-converting enzyme (ICE), or tyrphostin A25, a protein tyrosine kinase inhibitor, or sodium-orthovanadate, a phosphatase inhibitor. DNA fragmentation was assessed by electrophoretic and nonisotopic-labeling procedures. The ultrastructure of colonic tissue specimens and isolated cells was examined by transmission electron microscopy. Apoptosis in whole colonic tissue and in isolated crypts was confined predominantly to cells resident in the upper crypt regions. In contrast, extensive apoptotic death was observed in isolated colonic cells, irrespective of their developmental stage and positional hierarchy within the crypt continuum at harvest time. An apoptotic gradient, however, was evident. Exposure to YVAD-CMK resulted in a marked decrease in the number of apoptotic cells. Treatment with tyrphostin A25 caused a sharp rise in the apoptotic index; conversely, vanadate significantly impeded apoptosis. Cumulatively, these results indicate that disordered intercellular communication provokes unscheduled ICE-mediated apoptosis of colonocytes, and that local signals along the crypt continuum control both the reprieve from death and the timely demise of distinct colonic cell populations. Attenuation of tyrosine phosphorylation may be a contributory event in the acquisition of the apoptotic phenotype.

Sodium butyrate causes alteration of colon cancer cell morphology and biology towards that of a more differentiated phenotype. The retinoblastoma gene encodes a nuclear phosphoprotein (pRb) present in a wide range of human cancer cell lines including colon cancer cell lines. pRB is synthesized throughout the cell cycle and phosphorylated in a phase specific manner: the predominant proteins in G0/G1 are the unphosphorylated species (110 kD) whereas phosphorylated pRb (112-114 kD) are in S and G2. 110 kD pRb binds transcription factors and prevents transcription of responsive genes such as the gene for thymidine kinase, which are expressed in late G1. The precise mechanisms controlling cell arrest are unknown, but recent data suggest that cyclin-dependent kinase inhibitors such as p16 may play a role. The aim of the present study was to assess the effect of sodium butyrate on cell cycle staging, thymidine kinase activity, phosphorylation of the pRb protein and expression of p16. We show that sodium butyrate treatment induces differentiation of LS174T colon cancer cells, inhibits thymidine kinase activity concomitantly with induction of pRb dephosphorylation, p16 transcription and cell cycle arrest at G0/G1. Initial dephosphorylation was observed 24 h after treatment of LS174T cells with sodium butyrate, whereas complete shift to the dephosphorylated form was observed 3 days after treatment. Induction of pRb dephosphorylation by sodium butyrate preceded inhibition of growth and the specific cell cycle arrest. RNase protection assay with a p16 specific riboprobe showed undetectable levels in proliferating cells to several fold increase in differentiated colonocytes. In conclusion, the results provide evidence for a specific cellular mechanism of butyrate induced growth arrest and differentiation of a colon cancer cell line.

Glucocorticoids, such as dexamethasone, induce amiloride-sensitive Na+ conductances in rat distal colon epithelium. The activity of these conductances diminishes from the surface to the base of the crypt whereas cAMP-stimulated Cl- secretion decreases from the crypt base to the surface. These gradients are likely to be perturbed during carcinogenesis. We therefore determined the magnitude of Na+ and Cl- conductances in colonocytes isolated from normal and carcinogen-treated rats. Colon carcinogenesis was induced by injection of dimethylhydrazine (DMH) (18 mg/kg) for 5 weeks. Before sacrifice animals were treated for 3 days with dexamethasone. Colonocyte populations from the surface to the crypt base (C1-C5) were harvested from the distal colon by a Ca2+-chelating procedure. The activity of Na+ conductances was determined by uptake of 22Na+ by surface and crypt colonocyte populations and by membrane vesicles in the presence and absence of 10 microM amiloride. In control rats Na+ conductance was highest in surface colonocytes and absent in the crypt base. As early as 2 weeks after initiation of DMH treatment amiloride-inhibited Na+ uptake was virtually absent in the upper crypt. Transcriptional assessment of the alpha-, beta- and gamma-subunits that constitute the epithelial Na+ channel revealed that DMH treatment reduces the expression of beta-subunit mRNA. We then examined 36Cl- efflux from isolated colonocytes of normal and carcinogen-treated rats in response to forskolin (0.01 mM). Forskolin induced a marked rise in cAMP in lower crypt cells concomitant with a significant stimulation of 36Cl- efflux. Intracellular cAMP increased in upper crypt cells in response to forskolin without an increase in 36Cl- efflux. By contrast, upper crypt colonocytes from DMH-treated rats showed forskolin-stimulated efflux beginning 4 weeks after initiation of treatment. We conclude that induction of Na+ conductances by glucocorticoids is inhibited during the early stages of chemical carcinogenesis due to lack of induction of the beta-subunit of the channel. By contrast, Cl- transport is stimulated both in surface and lower crypt cell compartments during different stages of chemical carcinogenesis.

Rats injected with dimethylhydrazine for 5 weeks (DMH, 40 mg/kg body weight) invariably develop colonic cancer after a latency of some 10-14 weeks. Preliminary studies have suggested that Na+ absorption by surface colonic crypt cells is attenuated in the preneoplastic period (8-12 weeks after the first injection of DMH). The present study of glucocorticoid-treated (dexamethasone 6 mg/kg body weight, s.c. 3 days or triamcinolone 30 mg/kg body weight, s.c. 3 days) rats was undertaken to examine the ion transport properties of rat distal colon during this period in more detail. Ussing chamber studies of the distal colon and whole-cell patch-clamp measurements in surface cells, mid-crypt cells and crypt-base cells obtained from isolated crypts were performed. In Ussing chamber studies the equivalent short-circuit current inhibitable by amiloride (10 micromol/l) DMH-treated rats was about 40% of control. In addition, the hyperpolarizing effect of amiloride (10 micromol/l) on membrane voltage (Vm) was strongly attenuated in surface and mid-crypt cells of DMH-treated rats. Carbachol (CCH, 100 micromol/l), which predictably hyperpolarized surface, mid-crypt cells and crypt-base cells of control rats, had no significant effect on Vm in DMH-treated rats, but increased membrane conductance (Gm) significantly. This indicates that CCH probably activates both Cl- and K+ channels in all three colonic crypt compartments in the DMH-treated rats. Forskolin (5 micromol/l), which has the most pronounced effect in crypt-base cells in control rats, depolarized Vm and enhanced Gm in all three compartments in DMH-treated rats. These data indicate that DMH profoundly alters Na+ and Cl- transport in colonic crypts prior to the appearance of colonic adenocarcinoma and that these effects can be summarized as follows: (1) the Na+ conductance of surface cells is attenuated; (2) cells along the length of the crypt-lumen axis tend to lose their normal response to CCH and instead show simultaneous and comparable increases in K+and Cl- conductances; (3) the effect of forskolin is enhanced along the entire crypt axis. As a result colonic crypt transport is shifted to predominant Cl- secretion, findings which are characteristic of colonic carcinoma cell lines such as HT29 and T84 cells.

Our objective was to determine whether colorectal cancer tissue synthesizes and secretes biologically active gastrins resulting in a rise of gastrin levels in patients with adenocarcinoma of the colon. Blood samples for gastrin determination were taken from the artery feeding, and from the vein draining colon tumors, from a vein draining an uninvolved colon segment and from a peripheral vein. Tissue gastrin levels were measured in tumor tissues and normal mucosa taken by colonoscopic biopsy from colon cancer patients and healthy controls. The setting was a university hospital research laboratory. We had seventeen patients with colorectal cancer and 23 controls. No significant difference was found in peripheral venous blood gastrin levels between the cancer and the control groups. Serum gastrin concentration was not significantly different in the arterial blood which supplied the tumor area, the venous blood draining the tumor, the "uninvolved" mucosa or the control normal epithelium. Cancer tissue gastrin levels were lower than those measured in biopsies of uninvolved mucosa from cancer patients and normal controls. The present results show no rise of gastrin blood levels in patients with colon cancer, nor any evidence of gastrin-increased synthesis by the tumors.

{Whole-cell patch-clamp studies were performed in isolated colonic crypts of rats pretreated with dexamethasone (6 mg/kg subcutaneously on 3 days consecutively prior to the experiment). The cells were divided into three categories according to their position along the crypt axis: surface cells (s.c.); mid-crypt cells (m.c.) and crypt base cells (b.c.). The zero-current membrane voltage (Vm) was -56 +/- 2 mV in s.c (n = 34); -76 +/- 2 mV in m.c. (n = 47); and -87 +/- 1 mV in b.c. (n = 87). The whole-cell conductance (Gm) was similar (8-12 nS) in all three types of cells. A fractional K+ conductance accounting for 29-67% of Gm was present in all cell types. A Na+ conductance was demonstrable in s.c. by the hyperpolarizing effect on Vm of a low-Na+ (5 mmol/l) solution. In m.c. and b.c. the hyperpolarizing effect was much smaller, albeit significant. Amiloride had a concentration-dependent hyperpolarizing effect on Vm in m.c. and even more so in s.c.. It reduced Gm by approximately 12%. The dissociation constant (KD) was around 0.2 micromol/l. Triamterene had a comparable but not additive effect (KD = 30 micromol/l

Transforming growth factor beta 1 (TGF-beta 1) has been implicated in both the stimulation of angiogenesis in vivo and in the inhibition of endothelial cell proliferation in vitro systems. In this study we present evidence showing that under certain experimental conditions TGF-beta 1 may inhibit neovascularization in vivo. TGF-beta 1 was incorporated into ethylene vinyl acetate copolymer (Elvax 40) matrixes which provide a valuable vehicle for the controlled and sustained delivery of bioactive peptides. The biological effectiveness of TGF-beta 1 sequestered in polymer matrices was assessed by measuring the inhibition of [3H]-thymidine incorporation into the DNA of cultured mink lung epithelial cells. Neovascularization was induced in both corneas of albino rabbits by one deep-seated limbal silk suture. Elvax 40 matrixes loaded with TGF-beta 1 (release rate, 1.66 ng/24 h) were implanted in rabbit corneal stroma. "Empty" polymers in the contralateral eye served as controls. Aliquots of aqueous fluid were withdrawn, and the presence activity of phagocytic cells was assessed by the production of superoxide anion (O2) which was measured by the superoxide dismutase-inhibitable reduction of ferricytochrome C. Polymer-enclosed TGF-beta 1 implanted in rabbit corneas significantly suppressed angiogenesis (2.65 +/- 0.4 mm compared to 3.05 +/- 0.3 mm in contralateral controls p < 0.05). Superoxide production in 100 microliters aliquots of aqueous fluid was 0.95 +/- 0.18 and 0.6 +/- 0.18 nmol O2/10 min in control eyes and in the eyes bearing sequestered TGF-beta 1, respectively (p < 0.02). These results indicate that under the experimental conditions selected in this study, TGF-beta 1 significantly suppressed in vivo angiogenesis.

In a previous study [Lohrmann et al: Pflügers Arch Eur J Physiol 1995;429:517-530] we have shown that chromanol K⁺ channel blockers inhibit Cl^– secretion in rabbit colon. Their effect was easily demonstrable after stimulation by hormones acting through increases of cytosolic cAMP. The present study was undertaken to test in more detail the mechanism of action of one of these compounds (293 B). Two types of studies were performed: Ussing chamber experiments in rabbit distal colon and whole cell patch clamp studies in the isolated in vitro perfused rat colonic crypts. Carbachol (CCH, 100 µmol/l) enhanced Cl^– secretion, quantified as equivalent short circuit current (I_sc), in rabbit colon significantly more than did prosta-glandin E₂ (PGE₂). In whole cell patch clamp studies in rat colonic crypt cells from the base, CCH hyperpolarized the membrane voltage (V_m) and enhanced whole cell conductance (G_m). In agreement with previous impalement studies, 10 µmol/l 293 B depolarized V_m in forskolin-treated rat colonic crypt base cells even further and reduced G_m. In Ussing chamber experiments in rabbit colon, 293 B abolished the I_sc induced by PGE2. CCH, in the continued presence of 293 B, still induced a large I_sc. These data indicate that 293 B specifically inhibits the forskolin- but not the CCH-induced Cl^– secretion, and supports our previous conclusion that this class of substances inhibits a cAMP-regulated K⁺ conductance.

The main aim of this study was to determine whether changes in mucin production/secretion during the growth phases of a human adenocarcinoma cell line, HT-29, are associated with a different tumorigenic potential. HT-29 cells cultured in DMEM supplemented with 10% FCS were harvested in the exponential and post-confluent phases of growth. Metabolic labeling of the cells was performed using [3H]-glucosamine. Following a 24 hr-incubation period, radioactivity was measured in 1 ml-aliquots of cell cytosol and culture medium. Concurrently, mucin synthesis was assessed by size exclusion chromatography of [3H]-glucosamine-labeled glycoprotein in a Sepharose CL-4B column. Clonigenic assay in soft agar of pre- and post-confluent HT-29 cells was determined by scoring viable colonies according to size and number using phase-contrast microscopy. To assess in vivo tumorigenicity, pre-and post-confluent HT-29 cells (4 x 10(6)) in 0.2 ml DMEM were inoculated into nude mice. Tumor size and volume were recorded for 31 days. H-29 cells grew as multilayers of unpolarized, undifferentiated cells. Colony forming efficiency was similar at all growth stages. A significant increase in mucin synthesis was noted in HT-29 cells harvested in the exponential phase of growth compared to the stationary phase (148.3 +/- 41.9 versus 49.1 +/- 5.0, mean +/- SE, dpm/4 x 10(3) cells, p < 0.05). Mucin secretion was not significantly changed. Tumorigenicity in nude mice was consistently higher when the cells were injected in the exponential phase of growth. On day 31 after cell inoculation the average tumor volume/site was 332.7 mm3 in mice injected with HT-29 cells in log phase compared to 142.7 mm3 (p < 0.01) in animals which received post-confluent cells. Tumor weights were 0.36 g and 0.22 g respectively (p < 0.05). The present results indicate a definitive correlation between growth phases of HT-29 cells and mucin synthesis: mucin production was significantly higher in exponentially growing cells. The cloning efficiency in soft agar, a marker of in vitro tumorigenicity, was similar in HT-29 cells irrespective of the growth stage. A main finding of the present study was the increased in vivo tumorigenicity of colon cancer cells inoculated into athymic mice in the log phase of growth compared to cells harvested at post-confluence. These results are consistent with the view that mucin plays an important contributory role in determining tumorigenicity.

In the present study we have determined membrane, cytosolic, and cytoskeleton-associated tyrosine protein kinase (TPK) activity in human colon cancer cell lines exposed to (i) the differentiation-promoting agents sodium butyrate and 8-chloro-cyclic-adenosine 3',5'-monophosphate (8-Cl-cAMP), (ii) tyrphostins, specific TPK inhibitors, or (iii) differentiation-inducing culture manipulations. Treatment of human colon cancer cell lines, LS 174T, COLO 205, and SW620, with sodium butyrate and 8-Cl-cAMP or tyrphostins AG-30 and AG-34, significantly attenuated TPK activity concomitantly with an increase in the activity of alkaline phosphatase, an enzymatic marker of intestinal cell differentiation. The differentiated phenotype induced in Caco-2 and HT-29 colon cancer cells by culture manipulation was associated with a significant decrease in cytoskeleton-associated TPK activity and marked activity of alkaline phosphatase (AP). Electron microscopy and freeze-fracturing analysis of HT-29 cells showed that the gradual transition from the undifferentiated to the differentiated phenotype resulted in the acquisition of a distinct polarized morphology. Immunocytochemical phosphotyrosine analysis of cultured SW620 cells showed positive staining mostly localized in zones of focal contacts. A marked reduction in phosphotyrosine staining with notable changes in cell morphology was observed in SW620 cells exposed to tyrphostins. Cumulatively, the present results indicate that the induction of the differentiated phenotype in colon cancer cells is associated with a marked decrease in TPK activity and tyrosine phosphorylation.

Laminin, a major basement membrane-specific glycoprotein, promotes the attachment, migration, and invasion of a variety of tumor cells. Since laminin is present in the perisinusoidal matrix of the liver, we studied its effects on liver colonization by human colon cancer cells (HM7, LiM6) previously shown to have liver-metastasizing ability in athymic mice. These malignant cells expressed high levels of a 32-kDa laminin-binding protein on Western blot analysis when compared to the low metastatic parental cell line. Coinjection of laminin alpha chain-derived peptides which contain the amino acid sequence Ile-Lys-Val-Ala-Val (IKVAV) significantly stimulated liver colonization as determined by liver weight (P < 0.005) and number of tumor nodules (P < 0.02) 3 weeks after splenic-portal inoculation into nude mice. No stimulation was seen with a control peptide containing the same amino acids but in a scrambled sequence. In contrast, the Tyr-Ile-Gly-Ser-Arg peptide from the laminin beta 1 chain significantly inhibited HM7 liver colonization. These differences were not due to alterations in the number of cells initially reaching the liver as determined by injection of [125I]iododeoxyuridine-labeled tumor cells, but retention in the liver was stimulated by the IKVAV-containing peptides. Flow analysis indicated that the IKVAV peptide may act, in part, by stimulating homotypic adhesion of tumor cells. These data suggest that interactions of colon cancer cells with the IKVAV site on laminin may play a role in the formation of metastatic foci in the liver through cell-cell or cell-substratum interactions which promote metastasis.

Ornithine decarboxylase (ODC) is a rate-determining enzyme of the polyamine-biosynthetic pathway. We sought to produce cells with impaired ODC function in order to study the biological functions of polyamines. Saccharomyces cerevisiae strains were obtained by one-step gene replacement of a 900 bp fragment of the yeast ODC gene (SPE1) with the yeast URA3 gene. Spores derived from SPE1/spe1 cells germinated at reduced efficiency relative to SPE1/SPE1. Sustained growth of spe1 haploid mutants in polyamine-free medium led to intracellular polyamine depletion, reduction in budding index, G1 arrest and cessation of growth, and cells that were large and misshapen. All of these effects were completely reversed by adding polyamines to the medium, even after 5 days of polyamine starvation. A diploid yeast strain bearing two copies of disrupted spe1 lost heterozygosity at the mating-type locus more often when grown in the absence of polyamines than when grown in their presence, indicating that polyamine deficiency leads to either chromosome loss or to mitotic recombination.

In the present study we used monoclonal antibodies to investigate the expression of phosphotyrosine, c-myc and c-Ha-ras proteins along the crypt continuum of normal and transformed rat colon tissue. Colon cancer was induced by administration of dimethylhydrazine. Particular attention was focused on the immunohistochemical pattern of murine colon mucosa during preneoplastic stages so as to permit the identification of putative changes in the expression/location of the oncoproteins prior to frank neoplasia. The immunohistochemical analysis of tyrosinephosphorylated proteins in the normal rat indicated that positive staining was mostly restricted to the lower colonic crypt zones. The carcinogenetic insult altered the magnitude and positional profile of phosphotyrosine along the colon crypt axis during the preneoplastic period. An intense positive reaction was observed in the upper crypt regions. Four weeks following the last DHM administration, viz. before tumor appearance, positive staining was evident in invasive adenocarcinoma tissue. In contrast to phosphotyrosine, the feeble c-myc immunohistochemical staining of normal rat colonic did not exhibit a focal topology. However, following DMH administration and prior to frank neoplasia, a substantial increase in the staining intensity for c-myc was noted, confined mostly to the supranuclear region of luminal cells. Invasive adenocarcinomas displayed intense cytoplasmic c-myc immunoreactivity. p21 c-Ha-ras expression and location along the colon crypt axis showed a different pattern when compared to p62 c-myc and phosphotyrosine. The p21 c-Ha-ras protein was prominently expressed in surface epithelium of normal and DMH-treated rats. Midcrypt colonocytes exhibited moderate p21 ras staining; in contrast, proliferating colonic cells resident in the lower crypt regions were consistently negative. These results suggest that c-Ha-ras gene product plays an important contributory role in determining the differentiated phenotype of the colonic cell.