Jeffrey A. Moshier

Polyamine levels, ornithine decarboxylase (ODC) activity, and ODC-mRNA expression in normal and cancerous human colonocytes

Ornithine decarboxylase (ODC) and polyamines (putrescine, spermidine, and spermine) are crucial for cell proliferation. Recently, elevated ODC activity and polyamine levels have been suggested as biological markers for human colon cancer. In this study, we measured ODC activity and the levels of polyamines (putrescine, spermidine, spermine, and cadaverine) and acetyl-putrescine in human colonocytes isolated from cancerous areas compared to the adjacent normal colon tissue. In addition, ODC mRNA expression was compared between both groups. We found that colonocytes isolated from cancerous areas had significantly higher mean value of ODC activity, putrescine, spermidine, spermine, and cadaverine levels up to 1480%, 470%, 260%, 380%, and 510% respectively compared to colonocytes isolated from the adjacent normal colonic mucosa. No difference was found in acetyl-putrescine levels between cancerous and normal colonocytes. Steady-state levels of ODC mRNA were slightly elevated in cancerous colonocytes relative to normal colonocytes in two of three paired samples. However, the increase in ODC mRNA levels is not sufficient to account for the increase in ODC activity suggesting that colonocyte ODC activity is regulated post-transcriptionally.

Neuropeptide Y (NPY) enhances proliferation of human colonic lamina propria lymphocytes

Neuropeptide Y (NPY) is one member of a family of peptides with a wide range of physiological effects on the CNS, cardiovascular, and respiratory systems. NPY is widely distributed throughout the peripheral and central nervous systems. It has also been found within the colon, liver and gallbladder in close anatomic proximity to the mucosal immune system. In this study, we investigated the effect of NPY on human gut mucosal immune function. We examined colonic lamina propria lymphocyte (LPL) proliferation by measuring DNA synthesis, ornithine decarboxylase (ODC) activity, and polyamine biosynthesis. NPY enhanced ODC activity and polyamine biosynthesis in Con A-stimulated LPL, and enhanced thymidine incorporation into Con A-stimulated LPL but not into monocyte-depleted LPL. Moreover, exogenous IL1-beta partially restored NPYs stimulatory effect on monocyte-depleted LPL DNA synthesis. Our results demonstrate that NPY enhances human colonic LPL proliferation and that this effect is partially IL1-beta dependent. Our data also suggest that NPYs effect may be mediated via polyamine biosynthesis. We postulate that the NPY may have an important impact on human mucosal immune function.

Expression of protease genes in the gastric mucosa during aging

The current investigation examines the changes in the expression of pepsinogen C and cathepsin D and E genes in the gastric mucosa during aging and following physiological stimuli of fasting and refeeding. Northern blot analysis of gastric mucosal RNA, isolated from overnight fasted 6-, 12-, and 24-month-old male Fischer 344 rats, revealed that although steady-state mRNA levels of each of these protease remained essentially unchanged between 6 and 12 months of age, in 24-month-old rats the levels were decreased by about 60%, when compared with their younger counterparts. Interestingly, the relative concentration of beta-actin mRNA--but not 18s rRNA--in 12- and 24-month-old rats was also decreased by 23% and 37%, respectively, when compared with 6-month-old animals. In the next set of experiments, groups of young (3 month) and aged (24 month) rats were either fed throughout (controls) or fasted for 48 h and then fed for 6 h and 24 h. Gastric mucosal RNA from each group was assayed for steady-state mRNA levels of pepsinogen C and cathepsin D. Results showed that whereas in young rats fasting decreased pepsinogen C and cathepsin D mRNAs by 80-85%, in aged rats only pepsinogen mRNA was significantly decreased (45%), when compared with the corresponding initial fed controls. In both age groups, refeeding increased pepsinogen C mRNA concentration essentially to the respective initial fed levels. In contrast, cathepsin D mRNA levels in the gastric mucosa of aged rats was affected neither by fasting nor by refeeding. Our current data show that aging not only diminishes the expression of protease genes in the gastric mucosa, but also the expression of one of its structural genes, beta-actin. In addition, responsiveness of these protease genes to the physiological stimuli of fasting and refeeding is also attenuated by aging. We postulate that these age-related changes may in part be due to diminished differentiation of gastric mucosal cells.

Gastrin and epidermal growth factor induction of ornithine decarboxylase in rat colonic explants

An organ culture system was utilized to examine the effect of gastrin (G-17-I) and epidermal growth factor (EGF) on colonic mucosal ornithine decarboxylase (ODC) activity, and the expression of the ODC gene. Exposure of colonic mucosal explants to either gastrin or EGF (50-500 ng/ml) for only 4 h resulted in a profound stimulation (150-600%) in ODC activity over the basal level. These increases were essentially abolished by difluoromethylornithine (DFMO; 2 nmol/ml) or CaCl2 (2 umol/ml). Gastrin also activated the ODC gene in the colonic mucosa as evidenced by increased steady-state ODC mRNA levels in the colonic mucosal explants after 4 h exposure to the hormone, when compared with the controls. It is concluded that colonic mucosal ODC is responsive to both gastrin and EGF.

Vitamin A and retinoic acids immunomodulation on human gut lymphocytes.

Epidemiological studies have suggested an important immunomodulatory role for vitamin A and other related vitamin A compounds in adults and children. Although vitamin A is absorbed via the gastrointestinal tract, its affect on the gut mucosal immune cells has not been adequately investigated. We investigated the in-vitro effect of vitamin A (retinol) and its retinoid acid (RA) compounds (13-cis- and all trans-retinoic acids) on the human gut mucosal immune system as represented by colonic lamina propria lymphocyte (LPL) proliferation, and ornithine decarboxylase (ODC) activity. Results showed that retinol suppressed and trans-retinoic acid enhanced thymidine incorporation into LPL. 13-cis retinoic acid did not significantly affect LPL DNA synthesis. Similarly, retinol (0.025 microgram/ml and 10 micrograms/ml) and 13-cis retinoic acid (conc. 10 micrograms/m) suppressed, while all trans-retinoic acid (conc. 10 micrograms/ml) enhanced ODC activity in PHA-stimulated LPL. Interestingly, the effects of retinol and all trans-RA were abolished when LPL were previously depleted of macrophages. Addition of monocyte-associated lymphokines, IL-1 and IL-6, showed that IL-1 partially replaced the enhancing effect of all trans-RA previously observed on LPL thymidine incorporation. IL-6 did not affect LPL DNA synthesis irrespective of the vitamin A compound used. We conclude that retinol and retinoid acids (13-cis, all trans-) may alter the human colonic immune system possibly via IL-1 cytokine, but not via IL-6. The data suggest that vitamin A and its retinoid compounds may participate in the modulation of the gut immune system.

Thymosin-α1, but not interferon-α, specifically inhibits anchorage-independent growth of hepatitis B viral transfected HepG2 cells

Abstract Background: Thymosin-α 1 is a biological response modifier that has been used clinically, alone and in combination with interferon-α for the treatment of chronic hepatitis B viral infection. Both immunomodulatory and immediate intracelluilar mechanisms have been postulated to explain the effect of these two agents on HBV-infected hepatocytes. Methods: In this study, hepatitis B transfected HepG2 hepatoblastoma cells (HepG2-Nu2), derived from 2.2.15 cells, were used as an in vitro model to determine the efficacy of thymosin-α 1 and interferon-α, individually and combined, a proliferation inhibitors of HBV-infected cells. For comparison, parental HepG2 cells and an SV40-transfected HepG2 cell line (HepG2P9T2) were also evaluated. Results: In a clonogenic soft agar assay, thymosin-α 1 inhibited the anchorage-independent growth of the HepG2-Nu2 cells by 40% compared with untreated controls, but did not inhibit parental HepG2 or HepG2P9T2 clonal growth. The response was dose dependent over concentrations spanning three log units. In comparison, 10 000 units/ml of interferon-α inhibited parental HepG2, HepG2-N4Z and HepG2P9T2 by 33%, 41% and 87%, respectively. The combination of thymosin-α 1 and interferon-α consistently inhibited HepG2-Nu2 clonal growth more effectively than either treatment alone, reaching maximum inhibition levels of 51%. Conclusions: Thymosin-α 1 specifically inhibits the tumorigenic growth of HBV-transfected HepG2 cells in contrast to the general inhibition displayed by interferon-α. This panel of cell lines may be an important resource for dissecting the mechanism by which thymosin, alone or in combination with other drugs, influences HBV-infected hepatocytes and/or HBV-associated carcinoma.

Expression of the amylase gene in the rat exocrine pancreas during postnatal development: effect of dexamethasone.

Pancreatic amylase enzyme activity starts to increase rapidly around weaning (17-21 days) and reaches the adult level during postnatal development in the rat. To see whether the maturation of amylase involves changes in amylase gene expression, total pancreatic RNA was prepared from rats of various ages (term-fetus, 5, 10, 15, 20, 28 days and adult). Northern blots of these RNAs were probed with amylase cDNA. Levels of amylase mRNA peaked around 10 days i.e., about 1 week prior to peak amylase enzyme activity. The role of glucocorticoid in pancreatic amylase development was studied by giving rat pups at ages 5, 10 and 30 days a single injection (i.p.) of dexamethasone (DX). They and their littermates (controls) were killed 24 and 48 h after the injection. Increases in amylase mRNA levels were seen in the DX treated 5- and 10-day-old groups with corresponding increases in amylase enzyme activities. A slight decrease in amylase mRNA level was, however, observed in the DX treated 30-day-old pups which also had a slight decrease in amylase enzyme activity suggesting an age dependent differential responsiveness to DX. A time sequence study with 10-day-old pups killed after a single injection of DX at 6, 12, 24 and 48 h showed a rapid increase in mRNA levels which peaked around 12 h. Amylase enzyme activity, however, did not peak until 24 h after DX injection. These results suggest that pancreatic amylase is regulated at the level of gene expression in both normal- and DX-induced maturation. Regulation appears to occur at the transcription level as both increases to amylase activity and mRNA were blocked by actinomycin D.

Immunosuppressive effect of budesonide on human lamina propria lymphocytes

Budesonide, a beta-adreno-receptor agonist, is comparable to corticosteroid in the treatment of patients with inflammatory bowel disease with the advantage of minimal side effect. Although the immunomodulatory effects of budesonide on the circulatory and respiratory mucosal immune system have been reported, its effect on the human gut immune system has not been published. In this study, the effect of budesonide on the human gut immune system was compared to methyl-prednisolone. The cellular immune function was measured in-vitro by DNA synthesis, ornithine decarboxylase (ODC) activity and TNFalpha secretion. We found that both drugs have a comparable inhibitory effect on DNA synthesis, ODC activity and suppression of TNFalpha secretion. Exogenous addition of IL-2, did not restore the antiproliferative effect of both drugs. We conclude that budesonide has a comparative suppressive effect to methyl-prednisolone on the gut immune system which is not related to IL-2 secretion. The antiproliferative response may explain the therapeutic effect of budesonide on patients with inflammatory bowel disease.

Effect of gastric mucosal injury on ornithine decarboxylase in young and aged rats

The present investigation examines the changes in ornithine decarboxylase (ODC) activity, level of the enzyme, and the expression of its gene in gastric mucosa of young (4-month) and aged (24-month) Fischer-344 male rats 6 h after intragastric administration of either 2 M NaCl (1 ml/130 g b.w.) or an equivalent volume of water (controls). In addition, electronmicroscopy was performed to evaluate the ultrastructural changes in the gastric mucosa. Although administration of 2 M NaCl virtually eliminated the surface epithelium in both young and aged rats, the extent to injury in older animals extended beyond the surface epithelium. In aged rats, epithelial cells in the deeper parts of the gastric glands demonstrated severe swelling with vacuolization and disintegration of the cell organelles, with dying and dead cells. Basal gastric mucosal ODC activity (data from the controls) in aged rats was found to be 118% (p less than 0.001) above the young animals. This was also associated with similar increases in the concentration of ODC (as determined by Western-blot analysis) and a steady-state rise in ODC mRNA. Intragastric administration of 2 M NaCl (which caused gastric mucosal injury) resulted in a 625% increase in mucosal ODC activity in young rats, but in aged rats it produced a 112% increase when compared with the corresponding controls. In young rats, the increase in gastric mucosal ODC activity after injury was also associated with about a 2-fold rise in the enzyme protein concentration and a 4-fold increase in steady-state ODC mRNA levels. In contrast, gastric mucosal injury in aged rats, which resulted in a 112% increase in ODC activity, produced about a 30% reduction in the concentration of ODC and a 15-20% reduction in steady-state mRNA levels, when compared with the respective controls. The current data demonstrate that aging is associated with decreased responsiveness of gastric mucosal ODC to injury which may in part be responsible for diminished regenerative capacity of the gastric mucosa in aged animals. Furthermore, in aged rats the injury-induced stimulation of mucosal ODC activity is not associated with increased activation of its gene.

TYROSINE KINASE AND ORNITHINE DECARBOXYLASE ACTIVATION IN CHILDREN WITH HELICOBACTER PYLORI GASTRITIS

H. pylori infection has been considered a risk factor for the development of gastric malignancy. Ornithine decarboxylase and tyrosine kinases activities are increased in patients with colon or esophageal cancer. In this study we compared the ODC and tyrosine kinases activities in the gastric mucosa of children with H. pylori infection and normal mucosa. Gastric biopsies were prospectively collected from children during routine upper endoscopic procedure. H. pylori infection was determined histologically. Biopsies were analyzed for ODC activity, total tyrosine kinases activities, and for the activity of protooncogene tyrosine kinase pp60c-src. The mean ODC activity (pmol14CO2/mg. protein/hr) and total tyrosine kinases activity (pmol32P/mg. protein) were 186 and 5877 for H. pylori infected mucosa; and 229 and 4300, for normal mucosa, respectively (p > 0.05). Tyrosine kinase pp60c-src protein levels were similar between H. pylori infected mucosa and normal mucosa (3.12 and 2.15 pmol32P/mg. protein, respectively; p > 0.05). There was no correlation between gastric inflammation and the level of ODC or tyrosine kinase activities. ODC and tyrosine kinase activities in the gastric mucosa are similar in children with H. pylori infection compared to normal mucosa. The data suggest that these enzymes cannot be used as markers for future cancer development in children.