Associations between markers of colorectal cancer stem cells, mutation, microRNA and the clinical features of ulcerative colitis.

AIM: Several factors have been implicated in the pathogenesis of colorectal cancer (CRC) associated with ulcerative colitis (UC). We investigated markers of cancer cell pluripotency, including CD44 and CD166, microRNA-21 (miR-21) and microRNA-215 (miR-215), and APC, K-ras and DCC mutations in biopsy specimens from patients with UC to evaluate any correlations with clinical risk factors. METHOD: We observed 18 patients with UC and collected two biopsy specimens from each patient at diagnosis and at a follow-up end-point. We examined the expression of CD44, CD166, miR-21 and miR-215, and APC, K-ras and DCC mutations. We compared these markers at the two time points and assessed their associations with clinical characteristics, including the duration of colitis, histological alterations and the age of the patient at the onset of UC. RESULTS: Most (16/18) patients had alleviation of mucosal inflammation or remained stable during follow-up; one patient developed dysplasia and one had severe aggravation of the lesion during follow-up. Enhanced expression of CD44, CD166 and miR-21 with miR-215 was found in the specimens obtained at follow-up, despite alleviation of mucosal lesions. Coherence of cancer stem cell markers and miRNAs was seen in patients who had significant worsening of inflammation, dysplasia and a long duration of colitis. APC mutation occurred in only one patient; this patient had the longest duration of UC (23 years). CONCLUSION: Enhanced markers of CRC in follow-up colonic mucosal samples support the conclusion that the duration of UC plays the most important role in UC-related carcinogenesis.

Reduced expression of epidermal growth factor receptor related protein in gastric cancer.

OBJECTIVES: The recently cloned epidermal growth factor receptor related protein (ERRP) has been proposed to be a negative regulator of the epidermal growth factor receptor (EGFR). Because of the causal involvement of EGFR and its ligands in gastric cancer growth, we investigated expression of ERRP and cell proliferation in human gastric cancer. METHODS: We examined ERRP expression and localisation in surgical specimens of gastric cancers from 47 patients versus non-malignant gastric mucosa and determined their relationship to cell proliferation and differentiation. We also examined expression of ERRP by western blotting in three different gastric cancer cell lines. To further determine the functional properties of ERRP, we examined the effect of ERRP on epidermal growth factor (EGF) induced EGFR phosphorylation essential for its activation in MKN-28 gastric cancer cells. RESULTS: ERRP expression was dramatically reduced in gastric cancers (34% of all specimens positive) compared with non-malignant gastric mucosa (66% of specimens positive). Expression of ERRP in cancer cells inversely correlated with cell proliferation and grade of malignancy. Cell lines derived from metastatic gastric cancers had reduced ERRP expression compared with cell lines derived from a non-metastatic cancer. Exogenous ERRP protein markedly inhibited EGF induced EGFR phosphorylation in gastric cancer cells providing a novel molecular mechanism of its action. CONCLUSIONS: Our data indicate that downregulation of ERRP could play an important role in gastric cancer differentiation and progression. ERRP is a negative regulator of tumour cell proliferation and may exert its inhibitory effect, in part, by attenuating EGFR activation.

Experimental studies as an inspiration for clinical investigation.

This study represents an attempt of showing own author's example of using basic research data as an inspiration for the clinical studies. The project evaluates the role of gastrin in colorectal carcinogenesis as well as the differences of its action in proximal and distal colon. Colonocytes were isolated from Fischer-344 rats and incubated for 2 minutes with gastrin (10(-8)M). This treatment resulted with 60-70% rise in tyrosine kinase (Tyr-k) and 150-200% - in phospholipase C activity as regards to basal levels. In vivo infusion of gastrin for 5 days to Fischer-344 rats resulted with 90-150% increase in distal but not proximal colonic mucosal proliferative activity as well as tyrosine phosphorylation of several colonic mucosal proteins. In clinical study, the mean fasting gastrin level in the control group was significantly lower (p<0.01) than in patients with colorectal cancer before surgery. Mean plasma gastrin level in patients with distal tumor yielded 105,31 +/- 12,5 microU/l and was significantly higher than in patients with the proximal tumor site (42, 2 +/- 3,1 microU/l) (p<0,001). We conclude, that Tyr-k is involved in the mechanism of the trophic action of gastrin, particularly in distal colon. The differences in gastrin concentration in patients with distal and proximal tumors may probably contribute to the distinct pathogenesis and biological properties of those cancers.

Regulation of gastrointestinal mucosal growth during aging.

The increase in the aging population has led to a growing interest in achieving a better understanding of the aging process and of diseases that are predominantly expressed during advancing age. Since the structural and, in turn, the functional integrity of the mucosa of the gastrointestinal tract (GI) are maintained by constant renewal of cells, a detailed knowledge of the events that initiate and regulate mucosal proliferative processes is essential for a better understanding of the normal aging process as well as age-associated dysfunctions, including malignancy that represent disorders of tissue growth. In Fischer-344 rats, aging is associated with increased mucosal proliferative activity in much of the GI tract. On the other hand, the functional properties are either decreased or remain unchanged during advancing age. Basal gastric acid and pepsin output decline during aging, as is gastrin secretion. In contrast, antral gastrin levels increase during this period, as is mucosal histidine decarboxylase activity. The age-related decline in gastrin secretion could partly be attributed to a higher ratio of somatostatin (D) to gastrin (G) cells in the antral mucosa. The age-related rise in GI mucosal proliferative activity could not be attributed to the trophic action of either gastrin or bombesin, since they caused no significant change in mucosal proliferation in aged rats. On the other hand, EGF and TGF-alpha appear to be involved in regulating mucosal proliferation during aging. Aging is associated with increased activation of EGF-receptor (EGFR), the common receptor for EGF and TGF-alpha. This could be due to (a) increased levels of membrane-bound precursor form(s) of TGF-alpha resulting in increased activation EGFR signaling processes through an autocrine/paracrine mechanism, (b) heightened sensitivity of mucosal EGFR to EGF and TGF-alpha such that comparatively lower levels of these peptides are required to activate EGFR in aged than in young animals and/or (c) loss of EGFR regulatory factor(s) such as ERRP (EGFR Related Protein), a "negative regulator" of EGFR.

Expression of ERRP in normal and neoplastic pancreata and its relationship to clinicopathologic parameters in pancreatic adenocarcinoma.

INTRODUCTION AND AIMS: Epidermal growth factor (EGF) and its receptor (EGFR) play crucial roles in cellular signaling in many malignancies, including pancreatic neoplasia. Attenuation of EGFR signaling has been considered novel strategy for the management of human malignancies in several ongoing clinical trials. We recently isolated a novel negative regulator of EGFR, termed EGF receptor related protein (ERRP), whose expression appears to attenuate EGFR activation. In the current study, the expression of ERRP in normal and neoplastic pancreas was investigated and correlated with the clinicopathologic parameters in pancreatic ductal adenocarcinoma (DA). METHODOLOGY: Using rabbit polyclonal antibody that specifically interacts with ERRP, immunohistochemical staining was performed on 45 benign pancreata and 106 cases of DA. The intensity and percentage of cells with cytoplasmic and membranous staining were scored as 0, 1, 2, or 3. A combined score was calculated as intensity x percent/3, and for comparative analysis, the data were arbitrarily divided into three groups: <20, 20-49, and > or =50. The expression of ERRP was correlated with patient age, gender, race, tumor size, stage, grade, and survival. RESULTS: ERRP was expressed in most benign ductal epithelium and islet cells, but not in normal acinar cells. In pancreatic ductal adenocarcinoma, ERRP expression frequency decreased progressively from well (WD) to moderate (MD) to poorly differentiated (PD) carcinoma (58%, 43%, and 15% respectively, < 0.001). ERRP expression was correlated with survival in DA showing decreased median survival with decreased ERRP score ( = 0.0035). Median survival of the lower intensity (0 or 1) group was less than that of the higher intensity (2 or 3) group (8 14 months, = 0.002). The higher expressing group (> or =50% of cells) had longer median survival (17 months) than the lower expressing (<50% of cells) group (10 months, = 0.003). Stepwise multiple regression analyses revealed that ERRP expression score and tumor grade are the significant predictors of survival in pancreatic ductal carcinomas ( < 0.03). CONCLUSION: ERRP is usually expressed in benign ductal epithelium, but not in ductal adenocarcinoma. Its expression decreases with decreasing tumor differentiation. Low levels of ERRP are associated with poor clinical outcome, suggesting that progressive loss of ERRP, a negative regulator of EGFR, may partly stimulate aggressive tumor cell growth in pancreatic adenocarcinoma.

Aging is associated with increased proliferation and decreased apoptosis in the colonic mucosa.

Although the incidence of colon cancer increases with advancing age, reasons for this increase are not fully understood. Earlier studies have demonstrated that in Fischer-344 rats, aging is associated with increased crypt cell production in the colon, an event considered to be central to the initiation of carcinogenesis. Apoptosis also plays a critical role in the development and progression of colon cancer. Therefore, we have examined the age-related changes in proliferation and apoptosis in the colonic mucosa of 4-5, 12-14, and 22-24 month-old Fischer-344 rats. We have observed that proliferative activity in the colon, as assessed by proliferating cell nuclear antigen immunoreactivity, is higher (50-80%) in 12-14 and 22-24 month-old rats than in their 4-6 month-old counterparts. In contrast, the number of apoptotic cells, (as determined by TdT-mediated dUTP nick-end labeling assay) in the colonic mucosa of 12-14 and 22-24 month-old rats are considerably lower (50-60%) than in 4-6 month-old animals. These changes are accompanied by a concomitant reduction (75%) in pro-apoptotic Bak and stimulation (200%) of anti-apoptotic Bcl-xL levels. Since activation of caspases is associated with initiation and maintenance of apoptosis, we also analyzed the levels of pro and active forms of caspase-3, 8 and 9. The levels of active forms of caspase-3, 8 and 9 are found to be considerably (60-80%) lower in the colonic mucosa of 22-24 month-old rats, compared to their younger counterparts. This is accompanied by decreased cleavage of poly(ADP-ribose) polymerase, a substrate for caspases. In conclusion, our data show that aging enhances proliferation, but attenuates apoptosis in the colonic mucosa. These changes may partly be responsible for the age-related rise in colorectal cancer.

Curcumin induced modulation of cell cycle and apoptosis in gastric and colon cancer cells.

We have investigated the chemopreventive role of curcumin in gastrointestinal cancers by studying the regulation of proliferation and apoptosis in gastric (KATO-III) and colon (HCT-116) cancer cells. Curcumin inhibited cell proliferation and induced G2/M arrest in HCT-116 cells. Investigation of the levels of cyclins E, D and B by immunoblot analysis showed cyclin B level was unaffected, whereas cyclin D and E levels declined with curcumin in both cell lines. Investigation of cyclin-dependent kinases, Cdk2 and Cdc2, showed activity of Cdc2, but not Cdk2, increased markedly in response to curcumin. In both cell lines, immunoblot analysis indicated that curcumin caused induction of apoptosis as evidenced by cleavage of PARP, caspase-3, and reduction in Bcl-XL levels. Curcumin also stimulated the activity of caspase-8, which initiates Fas signalling pathway of apoptosis. Curcumin therefore appears to exert its anticarcinogenic properties by inhibiting proliferation and inducing apoptosis in certain gastric and colon cancer cells.

Increased in vitro activation of EGFR by membrane-bound TGF-alpha from gastric and colonic mucosa of aged rats.

Although aging is associated with increased epidermal growth factor receptor (EGFR) tyrosine kinase activity in Fischer 344 rat gastric and colonic mucosa, the regulatory mechanisms for the age-related rise in EGFR tyrosine kinase are poorly understood. Transmembrane transforming growth factor-alpha (TGF-alpha) may modulate EGFR function through an autocrine/juxtacrine mechanism. The present study aimed to determine the contribution of membrane-bound precursors of TGF-alpha in enhancing EGFR activation in the gastric and colonic mucosa during aging. The extent of EGFR tyrosine phosphorylation, a measure of EGFR activation, was substantially higher (300--350%) in the gastric and colonic mucosa of 23- (aged) vs. 4-mo-old (young) Fischer 344 rats. This was accompanied by an increase (200--1,000%) in the relative concentration of 18- to 20-kDa membrane-bound precursor forms of TGF-alpha. The amount of TGF-alpha bound to EGFR was also higher (150-250%) in the gastric and colonic mucosa of aged vs. young rats. In vitro studies revealed that exposure of HCT 116 cells (a colon cancer cell line) to TGF-alpha from gastric and colonic mucosal membranes of aged rats caused a 200--250% higher activation of EGFR and extracellular signal-related kinases (p42/44) compared with young rats. Our data suggest that the membrane-bound precursor form(s) of TGF-alpha may partly be responsible for enhancing EGFR activation in the gastric and colonic mucosa of aged rats, probably though an autocrine/juxtacrine mechanism(s).

Cloning of a novel EGFR-related peptide: a putative negative regulator of EGFR.

Although epidermal growth factor receptor (EGFR) plays a key role in regulating cell proliferation, differentiation, and transformation in many tissues, little is known about the factor(s) that may modulate its function. We have isolated a cDNA clone from the rat gastroduodenal mucosa whose full length revealed 1,958 bp that contained 227 bp of 5'-untranslated region (UTR) and an open-reading frame encoding 479 amino acids, followed by 290 bp of 3'-UTR. It showed ~85% nucleotide homology to the external domain of the rat EGFR. We refer to the product of the newly isolated cDNA as EGFR-related protein (ERRP). In Northern blot analysis with poly(A)(+) RNA from different rat tissues, ERRP cDNA hybridized to several mRNA transcripts with the strongest reaction noted with a transcript of approximately 2 kb. Maximal expression of the 2-kb mRNA transcript was observed in the small intestine, followed by colon, liver, gastric mucosa, and other tissues. Transfection of ERRP cDNA into a colon cancer cell line, HCT116, resulted in a marked reduction in proliferation in monolayer and colony formation in soft agar compared with the vector-transfected controls. In another colon cancer cell line, Caco-2, with a tetracycline-regulated promoter system, induction of ERRP expression in the absence of doxycycline was associated with a marked reduction in EGFR activation and proliferation. We conclude that the ERRP cDNA may represent a new member of the EGFR gene family and that ERRP plays a role in regulating cell proliferation by modulating the function of EGFR.

Aging enhances G(1) phase in the colonic mucosa of rats.

Although in Fischer-344 rats, aging has been shown to be associated with increased crypt cell production in the colonic mucosa, no information is available about the responsible intracellular mechanisms for the age-related rise in colonic mucosal cell proliferation. To determine whether cell cycling events are affected by aging, the present investigation examines the age-related changes in Cdk2 activity and the regulation of this process in the colonic mucosa. Colonic mucosae from 4-, 13- and 24-month-old Fischer-344 rats were assayed for Cdk2 activity and protein expression of Cdk2, cyclin D1 and E, as well as p21(Waf1/Cip1) (total and the fraction bound to Cdk2), p53 and phosphorylated Rb. Kinase activity and protein levels of Cdk2, as well as cyclin D1 concentration in the colonic mucosa, rose steadily with advancing age. However, the levels of cyclin E in the colonic mucosa were found to be higher in 24-month-old than 13-month-old rats, compared to their 4-month-old counterparts. On the other hand, levels of mucosal p21(Waf1/Cip1) (total and the fraction bound to Cdk2), one of the universal inhibitors of Cdks, were found to be lower in aged than in young rats. This was accompanied by a parallel decrease in mucosal p53, a tumor suppressor protein that is known to regulate p21(Waf1/Cip1). Additionally, we observed that the levels of phosphorylated Rb protein, a form which is involved in regulating progression of cells through the S phase, are increased in the colonic mucosa of 24-month-old rats, but not in 13-month-old animals, when compared with their 4-month-old counterparts. Our data suggest that, G(1) to S phase transition, as well as progression through the S phase of the cell cycle are accelerated in the colonic mucosa of aged rats.

Induction of transcriptional activity of AP-1 and NF-kappaB in the gastric mucosa during aging.

Although aging enhances expression and tyrosine kinase activity of epidermal growth factor receptor (EGFR) in the gastric mucosa, there is no information about EGFR signaling cascades. We examined the age-related changes in mitogen-activated protein kinases (MAPKs) [extracellular signal-related kinases (ERKs), c-Jun NH(2)-terminal kinases (JNKs), and p38], an EGFR-induced signaling cascade, and activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) transcriptional activity in the gastric mucosa of 4- to 6-, 12- to 14-, and 22- to 24-mo-old Fischer 344 rats. AP-1 and NF-kappaB transcriptional activity in the gastric mucosa rose steadily with advancing age. This can be further induced by transforming growth factor-alpha. The age-related activation of AP-1 and NF-kappaB in the gastric mucosa was associated with increased levels of c-Jun, c-Fos, and p52, but not p50 or p65. Total and phosphorylated IkappaBalpha levels in the gastric mucosa were unaffected by aging. Aging was also associated with marked activation of ERKs (p42/p44) and JNK1. In contrast, aging decreased p38 MAPK activity in the gastric mucosa. Our observation of increased activation of ERKs and JNK1 in the gastric mucosa of aged rats suggests a role for these MAPKs in regulating AP-1 and NF-kappaB transcriptional activity. These events may be responsible for the age-related rise in gastric mucosal proliferative activity.

Aging alters gastric mucosal responses to epidermal growth factor and transforming growth factor-alpha.

Administration of pharmacological doses of epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) in young rats stimulates gastric mucosal proliferation, but, in aged rats, the same treatment inhibits proliferation. This may be due to enhanced ligand-induced internalization of EGF receptor (EGFR). In support of this, we demonstrated that although a single injection of EGF (10 microg/kg) or TGF-alpha (5 microg/kg) in young (4-6 mo old) rats greatly increased membrane-associated EGFR tyrosine kinase activity, the same treatment slightly inhibited the enzyme activity in aged (24 mo old) rats. This treatment also produced a greater abundance of punctate cytoplasmic EGFR staining in gastric epithelium of aged rats, consistent with EGFR internalization. In vitro analyses demonstrated that exposure of isolated gastric mucosal cells from aged but not young rats to 100 pM TGF-alpha resulted in marked increases in intracellular EGFR tyrosine kinase activity and that induction of EGFR tyrosine kinase activity in mucosal membranes from aged rats occurred at doses 1,000-fold less than those required in young rats. Our data suggest that aging enhances sensitivity of the gastric mucosa to EGFR ligands. This may partly explain EGFR-mediated inhibition of gastric mucosal proliferation in aged rats.

TGFalpha is required for full expression of the transformed growth phenotype of NIH 3T3 cells overexpressing ornithine decarboxylase.

Ornithine decarboxylase (ODC) overexpressed from a heterologous promoter drives the tumorigenic transformation of NIH 3T3 cells and provides a model to investigate the underlying molecular mechanisms. These transformed cells, designated NODC cells, exhibit elevated levels of epidermal growth factor receptor (EGFR) tyrosine kinase (Tyr-k) activity relative to control transfected cells and inhibition of EGFR Tyr-k activation suppresses the transformed growth phenotype of these cells. Thus, ODC-induced transformation of NIH 3T3 cells appears to be mediated, at least in part, by enhanced signaling through the EGFR pathway. Here we extend these studies by evaluating: (i) the effects on growth regulation of overexpressing ODC in EGFR-deficient NIH 3T3 cells; (ii) the potential role of TGFalpha in mediating the EGFR-dependent transformation of NIH 3T3 cells by ODC. Disruption of EGFR-TGFalpha interactions either by deleting EGFR, by treatment with anti-TGFalpha neutralizing antibody or by transfection with a TGFalpha antisense expression vector suppressed acquisition of the full transformed growth phenotype. Specifically, the loss of contact inhibition and the capacity for clonogenic growth appear more dependent on EGFR-TGFalpha interactions than anchorage-independent growth in ODC-overexpressing cells. ODC overexpression does not alter the amount, localization or secretion of TGFalpha. Thus, TGFalpha is not the ODC-responsive component of the EGFR signaling pathway but appears to be critically involved in development of the transformed phenotype of NODC cells.

Hemorrhage and renal ischemia-reperfusion upregulates the epidermal growth factor receptor in rabbit duodenum.

To study the role of EGF-R in small intestinal adaptation to hemorrhage and I/R, anesthetized rabbits were implanted aseptically with arterial and venous catheters and bilateral renal artery Doppler flow probes and silastic occluders and allowed to recover. Rabbits were then randomly assigned to one of six groups: time control; hemorrhage (22.5 mL/kg) and 2.5 hours of renal occlusion (hemorrhage plus I/R); hemorrhage plus I/R and 2:1 LRS resuscitation; hemorrhage plus I/R and 3:1 LRS resuscitation; hemorrhage alone; or I/R alone. Rabbits were killed 48 hours after hemorrhage, and a section of duodenum was collected for analysis. Hemorrhage plus I/R induced a 2.5-fold increase in EGF-R tyrosine kinase activity compared with that found in the control group (P < .05), and this effect was not modified by either LRS resuscitation regimen. This increased activity was associated with similar Increases in EGF-R protein concentrations and approximately a 50% increase in EGF-R messenger (m)RNA levels compared with levels found in the control group. Further analysis of possible regulatory mechanisms for the increased EGF-R expression after hemorrhage plus I/R detected higher levels of EGF-R phosphorylation compared with those found in the control group but no significant increases in transforming growth factor-alpha mRNA levels. These data, coupled with a significant increase in duodenal thlobarbituric acid-reactive substance concentrations from rabbits in the hemorrhage plus I/R group, support the hypothesis that tyrosine kinase signal transduction pathways involving the EGF-R are activated in the small intestine after hemorrhage, renal I/R, or both, and this process may be mediated, at least in part, by oxidant stress.

Folic acid inhibition of EGFR-mediated proliferation in human colon cancer cell lines.

Although accumulating evidence suggests a chemopreventive role for folic acid in colon cancer, the regulation of this process in unknown. We hypothesize that supplemental folic acid exerts its chemopreventive role by inhibiting mucosal hyperproliferation, an event considered to be central to the initiation of carcinogenesis in the gastrointestinal tract. The present investigation examines the effect of supplemental folic acid on proliferation of Caco-2 and HCT-116 colon cancer cell lines. Furthermore, because certain tyrosine kinases, particularly epidermal growth factor receptor (EGFR), play a role in regulating cell proliferation, we also examined the folic acid-induced changes in tyrosine kinase activity and expression of EGFR. In Caco-2 and HCT-116 cells, maintained in RPMI 1640 medium containing 1 microg/ml folic acid, we observed that the supplemental folic acid inhibited proliferation in a dose-dependent manner. Pretreatment of HCT-116 and Caco-2 cell lines with supplemental folic acid (1.25 microg/ml) completely abrogated transforming growth factor-alpha (TGF-alpha)-induced proliferation in both cell lines. Tyrosine kinase activity and the relative concentration of EGFR were markedly diminished in both cell lines following a 24-h exposure to supplemental folic acid. The folic acid-induced inhibition of EGFR tyrosine kinase activity in colon cancer cell lines was also associated with a concomitant reduction in the relative concentration of the 14-kDa membrane-bound precursor form of TGF-alpha. In conclusion, our data suggest that supplemental folic acid is effective in reducing proliferation in two unrelated colon cancer cell lines and that EGFR tyrosine kinase appears to be involved in regulating this process.

Induction of G(1) checkpoint in the gastric mucosa of aged rats.

Although in Fischer 344 rats aging is found to be associated with increased gastric mucosal proliferative activity, little is known about specific changes in the regulatory mechanisms of this process. To determine whether changes in cell cycling events could partly contribute to the age-related rise in gastric mucosal proliferative activity, the present investigation examines changes in cyclin-dependent kinase (Cdk2) activity and the regulation of this process in the gastric mucosa of Fischer 344 rats aged 4 (young), 13 (middle aged), and 24 (old) mo. We observed that aging is associated with a progressive rise in activity and protein levels of Cdk2 in the gastric mucosa. This is also found to be accompanied by a concomitant increase in cyclin E but not cyclin D1 levels. On the other hand, the levels of p21(Waf1/Cip1) (total as well as the fraction associated with Cdk2), a nuclear protein that is known to inhibit different cyclin-Cdk complexes, are found to decline in the gastric mucosa with advancing age. In contrast, with aging, there was a steady rise in p53 levels in the gastric mucosa. We have also observed that the levels of phosphorylated retinoblastoma protein, a form that participates in regulating progression through the S phase, are markedly elevated in the gastric mucosa of aged rats. In conclusion, our data suggest that, in the gastric mucosa, aging enhances transition of G(1) to S phase as well as progression through the S phase of the cell cycle. However, the age-related decline in p21(Waf1/Cip1) in the gastric mucosa appears to be independent of p53 status.

Tryosine kinase and ornithine decarboxylase activation in children with Helicobactor 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 pp60(c-src). The mean ODC activity (pmol 14CO2/mg. protein/hr) and total tyrosine kinases activity (pmol 32P/mg. protein) were 186 and 5877 for H. pylori infected mucosa; and 229 and 4300, for normal mucosa, respectively (p> 0.05). Tyrosine kinase pp60(c-src) protein levels were similar between H. pylori infected mucosa and normal mucosa (3.12 and 2.15 pmol 32P/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.

Induction of EGFR tyrosine kinase in the gastric mucosa of diabetic rats.

Diabetes mellitus is associated with spontaneous gastric mucosal injury and enhanced susceptibility of the mucosa to damaging agents. Little information is available about the biochemical changes that occur in the gastric mucosa of diabetes mellitus. Evidence is accumulating that tyrosine kinases, particularly the EGF-receptor (EGFR), are involved in regulating a variety of structural and functional properties of the gastric mucosa. The primary objectives of this investigation were to determine whether diabetes induces morphological changes in the gastric mucosa, and if so, whether these changes are associated with alterations in EGFR tyrosine kinase. Diabetes-induced changes in gastric mucosal morphology were also examined. Diabetes was induced in 3- to 4-month-old male Fischer-344 rats by streptozotocin (STZ; 45 mg/kg; i.v.). Four weeks after induction of diabetes mellitus, the gastric mucosa of overnight-fasted rats was found to be slightly atrophic. A reduction in gastric mucosal thickness with deposition of fibrous tissue above the muscularis layer was observed in the stomach of overnight-fasted diabetic rats. These changes were associated with a marked stimulation in tyrosine kinase activity and protein expression of EGFR. The relative concentrations of several precursor forms of TGF-alpha in both membrane and cytosolic fractions from the gastric mucosa of overnight-fasted diabetic rats were also found to be significantly above the corresponding controls. This suggests that endogenous TGF-alpha may play a critical role in regulating mucosal EGFR tyrosine kinase through a juxtacrine/paracrine mechanism.

Aging and cancer of the stomach and colon.

Although the incidence of most human malignancies including cancer of the gastrointestinal tract increases dramatically with advancing age, the precise role of aging in that increase remains a matter of continued controversy. Many probable explanations for the age-related rise in cancer incidence have been offered including altered carcinogen metabolism and the cumulative effects of protracted exposure to cancer-causing agents. Neoplasia of the stomach or colon is a multi-stage process with hyperproliferation being central to the initiation of carcinogenesis. Since aging is associated with increased gastrointestinal mucosal cell proliferation, the possibility that aging itself may render target cells more susceptible to carcinogenic transformation continues to be an area of intense interest and study. This review will examine the evidence for age-related alterations in the structural and functional properties of the gastric and colonic mucosa in an effort to further elucidate the potential mechanisms of carcinogenesis which may be involved during the aging process.

Differential expression of EGFR during early reparative phase of the gastric mucosa between young and aged rats.

Aging is associated with decreased reparative ability of the gastric mucosa. Our recent data suggest a role for epidermal growth factor receptor (EGFR) in the mucosal reparative processes. Thus we examined changes in EGFR tyrosine kinase activity as well as expression and subcellular localization of EGFR and its ligand transforming growth factor-alpha (TGF-alpha) in the gastric mucosa of young (4-mo-old) and aged (24-mo-old) Fischer 344 male rats during the early reparative phase after acute injury induced by 2 M NaCl. Within 240 min of injury, significant epithelial restitution was observed in the gastric mucosa of young but not of aged rats. Expansion of the neck region and initiation of foveolar cell migration could be seen within 45 min of injury in young rats but not until 90 min in aged rats. In young rats mucosal EGFR tyrosine kinase activity increased at 45 min after injury and subsequently fell to basal levels. Mucosal EGFR mRNA increased throughout the reparative phase as did content of the EGFR ligand TGF-alpha. In contrast, although the basal tyrosine kinase activity and levels of EGFR mRNA and TGF-alpha were elevated in the gastric mucosa of aged rats, injury did not cause increases in these parameters. Immunofluorescent localization suggests that internalization and/or degradation of EGFR may be higher in aged than in young rats. We suggest that diminished induction of EGFR tyrosine kinase activity and increased EGFR internalization after injury may in part be responsible for the age-related decrease in the reparative capacity of the gastric mucosa.