A polyamine pathway-mediated mitogenic mechanism in enterochromaffin-like cells of Mastomys

M. Kidd, L. H. Tang, S. W. Schmid, K. Miu, and I. M. Modlin

Gastrointestinal Surgical Pathobiology Research Group, Yale University School of Medicine and the West Haven Veterans Affairs Medical Center, New Haven, Connecticut 06520-8062

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously demonstrated that in Mastomys species proliferation of gastric enterochromaffin-like (ECL) cells is predominantly regulated by gastrin and by transforming growth factor-alpha (TGF-alpha ) in the naive and neoplastic state, respectively. In this study we examined whether these intracellular mitogenic responses are mediated by polyamines and ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine biosynthesis. An ECL cell preparation of high purity was used to measure the effect of the polyamine derivatives putrescine, spermidine, and spermine on DNA synthesis by bromodeoxyuridine uptake. Both putrescine and spermidine augmented gastrin-stimulated, but not basal, DNA synthesis in naive cells. This proliferative response correlated with an increase in ODC activity that was partially inhibited (20%) by difluoromethylornithine (DFMO), an inhibitor of ODC (IC50, 30 pM). In contrast, all polyamines increased both basal and TGF-alpha -stimulated DNA synthesis as well as ODC activity in tumor ECL cells. DFMO completely inhibited the proliferative response of TGF-alpha (IC50, 3 pM). Thus polyamine biosynthesis is involved in proliferation of ECL cells and in particular the mitogenesis of tumor cells, suggesting a role for this pathway in the regulation of ECL cell transformation.

gastrin; ornithine decarboxylase; transforming growth factor-alpha

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE AFRICAN RODENT Mastomys is genetically susceptible to spontaneous gastric carcinoid (ECLoma) development and, depending on the breeding strain, between 40 and 50% of the animals have such lesions by 2 yr of age (16). Inhibition of parietal cell acid secretion via irreversible H2 receptor blockade (loxtidine) results in an elevation in serum gastrin levels which is sustained for the duration of the treatment. Enterochromaffin-like (ECL) cell hyperplasia is evident at 8 wk and neoplasia at 16 wk in ~80% of the animals (16, 17). Hypergastrinemia seems requisite for ECL cell hyperplasia, and cell transformation is related both to the level and the duration of hypergastrinemia (7, 13). However, cell growth subsequently becomes autonomous of the gastrin stimulation. We have previously characterized this gastrin-independent state as neoplasia or ECL cell transformation (16, 17). In addition, we have demonstrated that transforming growth factor-alpha (TGF-alpha ) becomes one of the regulatory mechanisms of proliferation during cell transformation via an autocrine pathway (27). This suggests that gastrin is important as an initiator of ECL cell replication, which is thereafter driven by a growth factor-mediated cascade.

In vitro studies with purified ECL cells isolated either from untreated or loxtidine-treated (8 wk) animals indicate that gastrin is the most potent activator of DNA synthesis in naive and hyperplastic cells. It has little effect in transformed cells, whereas TGF-alpha is most potent in transformed cells, with minimal effect in naive cells (27). TGF-alpha mediates the mitogenic effect via a process that involves protein tyrosine phosphorylation of the TGF-alpha epidermal growth factor receptor, whereas gastrin activates the cellular second messenger signal transduction system by activation of a receptor-linked GTP-binding protein and subsequent protein phosphorylation. The postreceptor events involved in the cascade of intracellular mechanisms by which either gastrin or TGF-alpha activates ECL cell DNA synthesis are unknown.

Polyamine biosynthesis is a tightly regulated, ubiquitous eukaryotic cellular phenomenon, which is responsible for mediating growth responses to most agents stimulating cell growth, including gastrin and growth factors (24). The rate-limiting enzyme in polyamine synthesis is ornithine decarboxylase (ODC), which catalyzes the formation of putrescine from ornithine via decarboxylation. Thereafter, spermidine and spermine, respectively, are derived from putrescine by a series of aminopropyltransferase steps. Polyamine homeostasis is a dynamic, nonlinear process which comprises synthesis, interconversion, and uptake from extracellular sources. Decreases in intracellular polyamine concentrations by the competitive addition of difluoromethylornithine (DFMO) result both in a decrease in DNA synthesis as well as in an increase in polyamine uptake (12, 29). An increase in polyamine uptake often, but not always, results in a decrease in ODC activity (18). Polyamines are especially evident in rapidly proliferating cells and tissues such as developing embryos and tumors, which have higher putrescine and spermidine concentrations than nongrowing tissues (24). These positively charged amines alter DNA conformation and catalyze nucleosome condensation, effects considered to be of importance in establishing the conditions necessary for DNA synthesis (1, 2). In addition, increases in ODC activity and polyamine synthesis are evident in cells in the G1 phase of the cell division cycle (24), further supporting the critical role for ODC activity in cell proliferation.

In the gastrointestinal tract, previous immunocytochemical studies of gastric epithelia suggest that ODC is present in mucous neck cells and is colocalized with mucus (6, 11). In contradistinction, polyamine immunostaining indicates that the secretory granules of gastric chief cells are most strongly reactive for polyamine antiserum (8). The effect of gastrin on mucous cells appears to be partially mediated by the polyamine pathway (15), but the role of this pathway in mediating gastric endocrine ECL cell proliferation has not previously been identified. We hypothesized that polyamines and ODC activity might serve as a common intracellular mechanism in modulating the ECL cell proliferative response mediated by gastrin or TGF-alpha . To investigate this we examined the effect of polyamine derivatives on DNA synthesis and the correlation between ODC activity and cell proliferation in both naive or transformed ECL cells.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials

All chemicals, including polyamine derivatives and experimental buffers, were obtained from Sigma Chemical (St. Louis, MO) or were of the highest quality available. Loxtidine, an irreversible H2 blocker, was a kind gift of Glaxo (Ware, UK), human gastrin-17 and rat TGF-alpha were from Research Plus (Bayonne, NJ), bicinchoninic acid (BCA) protein assay reagent from Pierce (Rockford, IL), [14C]ornithine from NEN (Boston, MA; 43.8 mCi/mmol), 5-bromo-2'-deoxyuridine (BrdU) proliferation kit from Amersham (Arlington Heights, IL), collagen-I coated 96-well plates from Becton Dickinson (Bedford, MA), Nycodenz from Accurate Chemical and Scientific (Westbury, NY), and Pronase E from Boehringer Mannheim (Indianapolis, IN). DFMO was a kind gift of Dr. E. Bohme, Marrion Merrell Dow Research Institute (Cincinnati, OH).

Animals

All animals for these studies were bred from the Mastomys colony (Modlin/Yale) maintained at the West Haven Department of Veterans Affairs Medical Center. The animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals with equal sex distribution were randomly assigned to receive either water (control group) or loxtidine (1 mg · kg-1 · day-1) delivered in drinking water (2 g/l) for 16 wk (tumor group). All animals had free access to rodent chow (Purina, St. Louis, MO). Hypergastrinemia was confirmed by gastrin RIA.

Isolation of ECL Cells

Naive Mastomys ECL cells were prepared by a recalibration of the method of Prinz et al. (20), as previously reported by us (27). For each preparation, 8-10 nonfasting Mastomys (80-100 g) were used (n = 1). Mastomys tumor ECL cells were prepared as previously described (27). From each loxtidine-treated Mastomys (n = 1), macroscopic carcinoids, mainly free of other gastric mucosal cells, were isolated and minced by hand, and individual cells released by Pronase (1 mg/ml) and collagenase (1 mg/ml) digestion. Cells were either immediately used in ODC assays or cultured in growth medium (DMEM and F-12, 2% FCS, 0.5 mg/ml insulin-transferrin-sodium selenite, 10 nM hydrocortisone, 0.1 mg/100 ml gentamicin, pH 7.4) at a final concentration of 105 cells/ml, 100 µl/well.

Cell Proliferation Assay by BrdU Incorporation

After 24 h in culture (96-well collagen-I coated plate, 104 cells/well), medium was aspirated and replaced with culture medium containing BrdU [prediluted labeling reagent (1:200), Amersham] and the indicated agent as previously described (27). After incubation for an additional 24 h, the medium was removed and the cells were fixed at 4°C in 90% ethanol-5% acetic acid-5% water and then washed in 0.1% Tween 20 in PBS (11.5 mg/ml Na2HPO4, 3 mg/ml NaH2PO4, 5.8 mg/ml NaCl, pH 7.4). The wells were incubated with anti-BrdU antibody with nuclease (1:400, Amersham) for 60 min at room temperature and, after three washes, incubated with rabbit anti-mouse IgG conjugated to horseradish peroxidase (1:800, Amersham) for 30 min at 20°C. After a further three washes, wells were incubated with substrate (30 mg o-phenylenediamine dihydrochloride in citrate-phosphate buffer, pH 4.12), and optical density was determined at 405 nm by spectrophotometry (Bio-Rad microplate reader model 450). Results are expressed as optical density units (405 nm), and final results are expressed as the ratio of means ± SE to control.

Evaluation of ODC Activity

Freshly isolated cells (105/100 µl culture medium) were incubated with or without gastrin (naive) or TGF-alpha (tumor) for 60 min at 37°C. ODC activity was then assayed by the modification of a previously described methodology (10). Briefly, after stimulation, cells were homogenized in cold Tris · HCl buffer (2.4 mg/ml Tris · HCl, 0.03 mg/ml EDTA, 0.3 mg/ml dithiothreitol, pH 7.5) and centrifuged, and an aliquot of the cytosolic extract was removed and later assayed for protein content using the BCA method. A second aliquot of the cytosolic extract was immediately incubated in the reaction buffer containing radiolabeled [14C]ornithine (200 nCi) for 60 min at 37°C. The reaction was terminated by addition of HCl (37%), and the liberated product was extracted in Betablend over 30 min at 37°C and counted. Final results (dpm · µg protein-1 · 60 min-1) were expressed as ratio to control.

Experimental Design

Naive cells. An isolated, purified preparation of naive Mastomys ECL cells was used in all experiments (27). To evaluate an effect on DNA synthesis, we incubated cells with either gastrin, putrescine, spermidine, or spermine alone (10-12-10-6 M), or with a combination of each polyamine (10-12-10-6 M) and a maximal concentration of gastrin (10-8 M) for 24 h. To examine whether the combined effect of polyamines and gastrin was mediated via ODC, we next investigated the effect of the irreversible ODC inhibitor DFMO on gastrin (10-8 M)-stimulated DNA synthesis. Thereafter we determined the effect of gastrin (10-10-10-6 M) on the activity of this enzyme using a radiometric technique to measure the formation of 14CO2 from [14C]ornithine.

Tumor cells. An isolated, purified preparation of Mastomys tumor ECL cells was used in all experiments (27). To evaluate an effect on DNA synthesis, we incubated cells with either TGF-alpha , putrescine, spermidine, or spermine alone (10-12-10-6 M) or with a combination of each polyamine (10-12-10-6 M) and a maximal concentration of TGF-alpha (10-9 M) for 24 h. To examine whether the effect of polyamines and TGF-alpha was mediated via ODC, we next evaluated the effect of DFMO on TGF-alpha (10-9 M)-stimulated DNA synthesis. Thereafter we evaluated the effect of TGF-alpha (10-13-10-9 M) on the activity of this enzyme using a radiometric technique. In addition, to examine whether gastrin had an effect on tumor cells we evaluated the ability of this agent (10-11-10-7 M) to alter ODC activity.

Statistical Evaluation

Results are expressed as means ± SE. The number of ECL cell preparations is indicated by n. Statistical analysis was performed using the two-tailed Student's t-test for paired values as appropriate, and P < 0.05 were considered significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Naive ECL Cells

Effect of polyamines on DNA synthesis. We initially undertook to measure the effect of putrescine, spermidine, and spermine on basal and stimulated ECL cell DNA synthesis. None of the polyamines alone (up to 1 µM) significantly altered basal DNA synthesis over the 24-h experimental period (Fig. 1). However, both putrescine and spermidine augmented the stimulatory effect of gastrin (10-8 M), with estimated EC50 values of 8 × 10-10 M and 2 × 10-9 M, respectively (Fig. 1). In contrast, spermine had no detectable effect.


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Fig. 1.   Stimulation of DNA synthesis by polyamines. DNA synthesis is normalized to control. Although gastrin stimulated DNA synthesis as expected, none of the polyamines alone significantly altered basal DNA synthesis. However, both putrescine and spermidine augmented stimulatory effect of gastrin, with effective concentrations between 10-8 M and 10-6 M, respectively. In contrast, spermine had no detectable effect (n = 4). BrdU, 5-bromo-2'-deoxyuridine. , Gastrin (10-8 M); bullet , putrescine plus gastrin; triangle , spermidine plus gastrin; black-diamond , spermine plus gastrin; open circle , putrescine alone; black-triangle, spermidine alone; star , spermine alone. All points are means ± SE.

Effect of DFMO on DNA synthesis. We next measured the effect of the irreversible ("suicide") ODC inhibitor DFMO on basal and stimulated DNA synthesis. As in previous experiments, incubation with gastrin (10-8 M) significantly (P < 0.01) stimulated DNA synthesis. DFMO alone did not significantly alter basal DNA synthesis at concentrations up to 1 µM (Fig. 2). Pretreatment of the cells for 30 min with this agent resulted in a partial inhibition (~20%) of gastrin-stimulated DNA synthesis. This effect, albeit biphasic, was significant between concentrations of 10-9 and 10-6 M (P < 0.05).


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Fig. 2.   Effect of difluoromethylornithine (DFMO) on DNA synthesis. As in previous experiments, incubation with gastrin (10-8 M) significantly (P < 0.01) stimulated DNA synthesis. DFMO alone, however, did not significantly alter basal DNA synthesis but pretreatment of cells for 30 min with this agent dose dependently inhibited gastrin-stimulated DNA synthesis with IC50 of ~3 × 10-11 M and maximal inhibitory concentration of 10-9 M (~20% inhibition, P < 0.05, n = 9). , Gastrin (10-8 M); bullet , gastrin plus DFMO; open circle , DFMO alone.

Gastrin stimulation of DNA synthesis and ODC activity. To evaluate the relationship between gastrin-altered ECL cell DNA synthesis and ODC activity we measured the effect of this mitogen both in cultured cells and in acutely isolated naive ECL cells. Gastrin stimulated DNA synthesis over 24 h, with an estimated EC50 of 5 × 10-11 M and a maximal response of 2.6 ± 0.4 of the control value. Similarly, gastrin stimulated ODC activity with an estimated EC50 of 3 × 10-10 M and a maximal response of 1.55 ± 0.03 of the control value (P < 0.01; Fig. 3). The specificity of the gastrin-stimulated ODC activity response was determined when preincubation of cells with DFMO (10-8 M) for 30 min reversed the stimulatory effect of this agent (10-9 M; data not shown).


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Fig. 3.   Stimulation of DNA synthesis and ornithine decarboxylase (ODC) activity by gastrin. Gastrin stimulated DNA synthesis over 24 h with estimated EC50 of 5 × 10-11 M and maximal concentration of 10-10 M (2.6 ± 0.4, P < 0.01, n = 7). In contrast, gastrin stimulated ODC activity (measured by 14CO2 release from [14C]ornithine) with estimated EC50 of 3 × 10-10 M and maximal activity of 10-8 M (1.55 ± 0.03, P < 0.01, n = 4). , BrdU uptake; bullet , ODC activity.

In summary, in naive ECL cells, 1) polyamines potentiated gastrin-stimulated cell proliferation; 2) gastrin-stimulated DNA synthesis was partially inhibited by the ODC inhibitor DFMO; and 3) gastrin-stimulated DNA synthesis exhibited similar potency with gastrin-stimulated ODC activity.

Tumor ECL Cells

Effect of polyamines on DNA synthesis. We initially undertook to measure the effect of putrescine, spermidine, and spermine on basal and stimulated ECL cell DNA synthesis. All of the polyamines significantly increased (~30%) basal DNA synthesis over the 24-h experimental period (Fig. 4A). The estimated EC50 values were 3 × 10-12 M for putrescine and 2 × 10-10 M for spermidine and spermine, respectively. In addition, all of the polyamines significantly augmented (~40%) the stimulatory effect of TGF-alpha (10-9 M; Fig. 4B). The estimated EC50 values were 2 × 10-11 M for putrescine, 2 × 10-10 M for spermidine, and 3 × 10-10 M for spermine, respectively


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Fig. 4.   Stimulatory effect of polyamines on tumor cell DNA synthesis. All polyamines significantly increased (~30%) basal DNA synthesis over 24-h experimental period (A). open circle , Putrescine; triangle , spermidine; , spermine. There was no specific difference among any polyamines. In addition, all polyamines significantly augmented (~40%) stimulatory effect of transforming growth factor-alpha (TGF-alpha , 10-9 M, B). Effective concentrations for polyamines were 10-8 M to 10-6 M (n = 6). black-lozenge , TGF-alpha (10-9 M); bullet , putrescine plus TGF-alpha ; black-triangle, spermidine plus TGF-alpha ; , spermine plus TGF-alpha .

Effect of DFMO on DNA synthesis. We next measured the effect of DFMO on basal and stimulated DNA synthesis. As in previous experiments incubation with TGF-alpha (10-9 M) significantly (1.58 ± 0.02, P < 0.01) stimulated DNA synthesis (Fig. 5). DFMO alone did not have a substantial inhibitory effect on basal DNA synthesis (Fig. 5), but pretreatment of the cells for 30 min with this agent dose dependently inhibited TGF-alpha (10-9 M)-stimulated DNA synthesis with an IC50 of ~3 × 10-12 M and a maximal inhibition of ~90% at 10-9 M (1.07 ± 0.15, P < 0.05 vs. TGF-alpha ).


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Fig. 5.   Inhibitory effect of DFMO on DNA synthesis. Incubation with TGF-alpha (10-9 M) significantly (1.58 ± 0.02, P < 0.01) stimulated DNA synthesis. DFMO alone did not have substantial inhibitory effect on basal DNA synthesis, but pretreatment of cells for 30 min with this agent dose dependently inhibited TGF-alpha (10-9 M)-stimulated DNA synthesis with IC50 of 3 × 10-12 M and maximal inhibitory concentration of 10-9 M (~90%, 1.07 ± 0.15, P < 0.05 vs. TGF-alpha , n = 5). , TGF-alpha (10-9 M); bullet , TGF-alpha plus DFMO; open circle , DFMO alone.

TGF-alpha stimulation of DNA synthesis and ODC activity. We then measured the effect of TGF-alpha on DNA synthesis and ODC activity in ECL tumor cells. TGF-alpha stimulated DNA synthesis over 24 h with an estimated EC50 of 5 × 10-12 M and a maximal concentration of 10-10 M (1.92 ± 0.14, P < 0.01, Fig. 6). TGF-alpha stimulated ODC activity with an estimated EC50 of 3 × 10-12 M and a maximal concentration of 10-11 M (1.5 ± 0.08, P < 0.01). Pretreatment of the cells for 30 min with DFMO (10-8 M) reversed the stimulatory effect of TGF-alpha (10-11 M) on ODC activity (1.17 ± 0.07, P < 0.05 vs. TGF-alpha alone; data not shown). Gastrin had no effect on tumor ECL cell ODC activity (data not shown). This is consistent with our previous observation that the effect of gastrin on tumor cell DNA synthesis is negligible (27).


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Fig. 6.   Stimulation of DNA synthesis and ODC activity by TGF-alpha . TGF-alpha stimulated DNA synthesis over 24 h with estimated EC50 of 5 × 10-12 M and maximal concentration of 10-10 M (1.92 ± 0.14, P < 0.01). In contrast TGF-alpha stimulated ODC activity with estimated EC50 of 3 × 10-12 M and maximal concentration of 10-11 M (1.5 ± 0.08, P < 0.01, n = 5). , BrdU uptake; bullet , ODC activity.

In summary, in tumor ECL cells 1) polyamines stimulated both basal and TGF-alpha -stimulated cell proliferation; 2) TGF-alpha -stimulated DNA synthesis was completely inhibited by ODC inhibitor DFMO; and 3) TGF-alpha -stimulated DNA synthesis exhibited similar potency with TGF-alpha -stimulated ODC activity.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The gastric endocrine ECL cell plays a critical role in the regulation of gastric exocrine function. Of particular pathological significance is the self-replicative nature of ECL cells under long-term hypergastrinemia in Mastomys and rats (27). Gastrin as a physiological hormone promotes cell proliferation, and sustained hypergastrinemia may culminate in ECL cell hyperplasia and neoplasia (7, 27). The loss of gastrin "control" is associated with the involvement of alternative growth regulatory mechanisms, such as a TGF-alpha autocrine pathway (27). Although gastrin and TGF-alpha activate different postreceptor second messenger systems, it is not known whether they share a common downstream intracellular denominator which is involved in activation of DNA synthesis process.

Consistent with our hypothesis, we have demonstrated for the first time that polyamine biosynthesis is involved in both gastrin and TGF-alpha -mediated cell proliferative response in a purified gastric endocrine ECL cell population, and in addition we have delineated the relationship between DNA synthesis and ODC activity and these stimuli. Previous investigations attempted to measure ODC activity in whole mucosal homogenates to stimuli which activate ECL cells (omeprazole, antrectomy, gastrin infusion). Unfortunately these studies cannot identify the precise cell source (3, 15). Indeed, because the ECL cell is only ±1% of the total mucosal mass, it is possible that these studies would not reliably detect changes in this cell population.

Addition of polyamines to a population of purified naive ECL cells had no effect on DNA synthesis. Naive ECL cells have a relatively long dividing time in vivo, ~60 days in the mouse (28), and in vitro, gastrin typically activates only ±20% of cells to take up and incorporate BrdU over a 24-h period (20). The combination of these observations suggests that there are relatively few mitogenic-sensitive cells in the late G1 phase. The lack of effect of polyamines on basal DNA synthesis may reflect either no uptake of these agents or a minimal mitogenic effect which is not detectable by the method of BrdU uptake. In contrast, cells stimulated by gastrin were responsive to polyamines. Thus both putrescine and spermidine amplified the effect of gastrin on BrdU incorporation by ~40%. The lack of effect of spermine may reflect the fact that it is an intracellular end-derivative of polyamines and poorly taken up by cells (21). We then explored the relationship between ODC activity and DNA synthesis in ECL cells. As might be predicted gastrin stimulated ODC activity as well as DNA synthesis in naive ECL cells. The effect of gastrin on BrdU uptake was biphasic, as has been previously demonstrated (20, 27). The nonparallel relationship between DNA synthesis and ODC activation suggests that additional mechanisms are, however, involved in gastrin-stimulated DNA synthesis. The partial inhibition of gastrin-stimulated DNA synthesis by DFMO, an ODC inhibitor, would seem to confirm that the trophic effect of this agent is only partly mediated via a polyamine-regulated pathway. However, it is possible that at high concentrations, DFMO perturbs other cellular events or, alternatively, that compensatory polyamine uptake occurs which may account for the partial inhibitory effect of this agent in naive cells.

Time-dependent gastrin-stimulated ODC activity has previously been reported both in the pancreatic cell line AR42J (22, 26) and colonic mucosal explants (14). Activation of the gastrin receptor in these cells is coupled to ODC activity via protein kinase C (26). In colonic cells, the effect of gastrin on ODC was preceded by an initial increase in tyrosine kinase activity which could be abolished by genistein, a protein tyrosine kinase inhibitor (30). These results indicate that gastrin activation of ODC is mediated via upstream protein phosphorylation. The mechanism by which gastrin mediates ODC activity in ECL cells is unknown but may involve at least the tyrosine kinase pathway, given that genistein has been demonstrated to abolish the effect of gastrin on DNA synthesis (23).

Polyamines are typically overexpressed by most types of cancer (24), and alterations in polyamine homeostasis (by the addition of DFMO) have previously been demonstrated to result in growth inhibition of a pancreatic "carcinoid" cell line (BON) (4). Because addition of polyamines to carcinoid tumor ECL cells resulted in stimulation of basal DNA synthesis (~30%) it seems probable that polyamines have a direct mitogenic effect on tumor ECL cells. Interestingly, in contrast to naive cells, spermine stimulated tumor cell DNA synthesis, suggesting that either direct uptake of this agent occurs in tumor cells or an alternative biochemical pathway is operative. Of particular interest was the observation that the mitogenic effect of TGF-alpha was significantly augmented by all tested polyamines. This is consistent with previous observations that growth factors increase both polyamine uptake, as well as intracellular polyamine concentrations and DNA synthesis (5, 19, 25). The effect of polyamines both on stimulated naive cells and tumor cells appears to be comparable. In contrast to naive cells, preincubation with DFMO completely inhibited TGF-alpha -stimulated DNA synthesis. This suggests that the TGF-alpha -mediated pathway is largely driven via ODC activity and polyamine biosynthesis and may be a reflection that tumor cells, because of their high proliferative rate, are more dependent on polyamine biosynthesis than their normal counterparts. This is consistent with our observations that inhibition of ODC activity by DFMO culminates in virtual abolition of DNA synthesis in transformed ECL cells, but only a partial inhibition of nontransforming ECL cell proliferation. This observation is similar to reports that quiescent fibroblasts, fibroblast growth factor, and platelet-derived growth factor all stimulate ODC activity through at least one pathway involving protein kinase C (9). It was unexpected that DFMO had no effect over 24 h on basal DNA synthesis. This may reflect the fact that ECL cells are well differentiated and are not rapidly proliferating.

In addition, TGF-alpha stimulated tumor ECL cell ODC activity with an EC50 of 3 × 10-12 M, a value consistent with that of the effect on tumor cell DNA synthesis (5 × 10-12 M). This supports the conclusion that the proliferative effect of TGF-alpha is mediated almost exclusively by this pathway in ECL tumor cells. This proposal is further strengthened by the observation that DFMO completely inhibits TGF-alpha -stimulated DNA synthesis.

In summary, we have demonstrated that the activity of the dominant trophic factor of the naive ECL cell, gastrin, appears to be only partially mediated by polyamine biosynthesis. In contrast, TGF-alpha -regulated proliferation of the transformed ECL cells appears to be significantly mediated via the ODC pathway. Although the primary mechanism by which gastrin mediates cell proliferation requires further investigation, the observation that polyamine homeostasis is substantially involved in tumor ECL cell proliferation, while not being unexpected, suggests a major role for growth factor-dependent tyrosine kinases in the regulation of ECL cell transformation.

    ACKNOWLEDGEMENTS

This work was supported by Veterans Administration Merit Award to I. M. Modlin and the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48820 to L. H. Tang.

    FOOTNOTES

Address for reprint requests: I. M. Modlin, Yale Univ. School of Medicine, 333 Cedar St., PO Box 208062, New Haven, CT 06520-8062.

Received 25 August 1997; accepted in final form 14 April 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(2):G370-G376
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