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.

Clonal growth inhibition as a bioassay for thymosin alpha1: inactivation of Talpha1 by trifluroacetic acid.

Thymosin alpha1 (Talpha1) is an immune response modifying peptide isolated from thymus tissue. The synthetic peptide has been evaluated in clinical trials as an adjuvant to cancer chemotherapy, an enhancer of vaccine potency, and an anti-viral for both hepatitis B and C. Among its multiple in vitro activities is the inhibition of the clonal growth of hepatitis B transfected hepatoblastoma cells. This assay was used to define the relationship between bioactivity and immunoactivity of Talpha1. Talpha1 was treated with 50% trifluoroacetic acid (TFA) for 1 hr to inactivate the peptide. Talpha1 heated at 90 degrees C or at room temperature maintained its bioactivity but TFA completely eliminated the activity in the bioassay. The TFA inactivated Talpha1 had a retention time on reverse-phase chromatography identical to bioactive Talpha1 but reduced immunoreactivity. In addition to demonstrating the utility of clonal growth as a bioassay, these studies demonstrate that immunoreactivity rather than retention time on HPLC may be a better predictor of bioactivity of synthetic Talpha1.

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.

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.

Cytokine-mediated apoptosis and inhibition of virus production and anchorage independent growth of viral transfected hepatoblastoma cells.

Cytokine-mediated apoptotic destruction of viral-infected cells, downregulation of virus production and inhibition of anchorage dependent (clonal) cell growth were evaluated using virus-transfected human hepatoblastoma (HepG2) cells. The cytokines evaluated were interferon alpha (IFN-alpha), tumour necrosis factor alpha (TNF-alpha) and thymosin alpha 1 (T alpha 1), all of which have previously been implicated in control of various viral infections. The viruses evaluated were Hepatitis B (HBV) and the transforming virus, SV-40. TNF-alpha-induced apoptosis in the HBV-transfected cell line and the control HepG2 cells but not the HepG2 cells transfected with SV-40 virus. IFN-alpha and T alpha 1 had no effect on apoptosis. TNF-alpha also prevented the clonal growth of the HBV-HepG2 and control HepG2 but enhanced the growth of the SV-40-transfected HepG2 cells. IFN-alpha inhibited the clonal growth of all three cell lines in contrast to T alpha 1 which inhibited the clonal growth of only the HBV-transfected cells. Although TNF-alpha, IFN-alpha, and T alpha 1 when given alone did not significantly inhibit HBV-DNA production in the culture supernatant from HBV-HepG2 cells, the combination of T alpha 1 and IFN-alpha resulted in a statistically significant inhibition of virus production. These studies demonstrate that HepG2 cells transfected with HBV and SV-40 are useful for defining the mechanisms of cytokine activity. The HBV-transfected cells are especially useful in defining possible in vivo differences in responses to cytokines with respect to HBV production, apoptosis and clonal cell growth. Multiple mechanisms through which different cytokines can influence HBV infection and hepatoblastoma growth were identified and the importance of defining effective combinations to improve therapy in vivo demonstrated.

Human vaginal mucosal immune system: characterization and function.

OBJECTIVE: The mucosal immune system has been recognized as the first line of defense against foreign antigens. The limited information available on the mucosal immunity of the lower reproductive organs have restricted our ability to fight infections, especially, the sexually transmitted disease. The aim of this study was to characterize in-vitro the human vaginal lamina propria lymphocytes (VLPL), their cell surface phenotypes, and cellular function. METHODS: VLPL were isolated from human vaginal mucosa by enzymatic techniques. Cell surface characteristics were investigated by immunohistochemistry and flow cytometric analysis. Cellular immune function was evaluated by 3H-thymidine incorporation studies and ornithine decarboxylase (ODC) activity. RESULTS: Immunohistochemistry and flow cytometric analysis showed that the CD4/CD8 ratio of the human vaginal mucosa is reversed compared to the gut lamina propria lymphocytes (0.55 +/- 0.17). PHA and ConA mitogens enhanced VLPL thymidine incorporation, while PWM did not have any significant effect. Very high level of ODC activity was observed in VLPL after PHA stimulation. CONCLUSIONS: The human VLPL can be isolated, characterized, and respond to a mitogenic stimulus. We postulate that further analysis of the vaginal immune system will enhance our understanding of local defence mechanisms which will help in the development of new therapeutic modalities against vaginal infections.

Androgen regulation of the human ornithine decarboxylase promoter in prostate cancer cells.

We studied the response of the human ornithine decarboxylase (ODC) promoter to androgen in human prostate cancer cell lines. In the well-differentiated, androgen-sensitive human prostate cancer line LNCaP, a genomic ODC promoter fragment that includes putative androgen response elements was suppressed by androgen. In contrast, the androgen-regulated probasin promoter was induced by androgens. The ODC promoter was also induced by cotransfected androgen receptor in the poorly differentiated, androgen-insensitive human prostate cancer cell line PPC-1. We examined the effects of cotransfected mutant androgen receptors containing the LNCaP mutation or DNA-binding mutations. All cotransfected androgen receptors switched the ODC androgen response from suppression to induction in LNCaP cells. Gel-shift and DNA footprint assays demonstrated androgen receptor binding to an ODC sequence that does not contain a consensus androgen response element. Deletion of the sequence abolished androgen suppression of the ODC promoter. We propose a model of pleiotropic gene regulation by androgen that requires a regulatory balance between androgen receptor and a transcription factor binding to the nonconsensus androgen response element.

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.

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.

Effect of thyroxine on pancreatic digestive enzymes and ornithine decarboxylase gene expression in neonatal rats.

BACKGROUND: Thyroxine has been shown to play a role in the development of exocrine pancreatic enzymes in neonatal rats. METHODS: To further evaluate the regulatory mechanisms for thyroxine in pancreatic development, we examined the changes in the expression of pancreatic enzymes (amylase and trypsinogen) and ornithine decarboxylase (ODC) genes following daily injection of thyroxine for 5 and 10 days to neonatal rats (5 days old). RESULTS: Total pancreatic proteins and DNA contents as well as the activity of ODC and exocrine enzymes were significantly increased after 5 and 10 days of thyroxine treatment. These increases were associated with parallel alterations (to three to fourfold rise) in steady-state mRNA levels of both amylase and trypsinogen. In contrast, thyroxine only produced a 57-68% increase in steady-state ODC mRNA levels. CONCLUSIONS: These data suggest that thyroxine stimulated the express of amylase and trypsinogen genes partly due to increased transcriptional rate and/ or decreased mRNA turnover. Thyroxine also stimulated ODC gene expression. However, the stimulatory mechanisms may involve transnational or posttranslational regulation of ODC and are independent of thyroxine effects.

Thymosin-alpha 1, but not interferon-alpha, specifically inhibits anchorage-independent growth of hepatitis B viral transfected HepG2 cells.

BACKGROUND: Thymosin-alpha 1 is a biological response modifier that has been used clinically, alone and in combination with interferon-alpha for the treatment of chronic hepatitis B viral infection. Both immunomodulatory and immediate intracellular 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-alpha 1 and interferon-alpha, individually and combined, as 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-alpha 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, 10000 units/ml of interferon-alpha inhibited parental HepG2, HepG2-N4Z and HepG2P9T2 by 33%, 41% and 87%, respectively. The combination of thymosin-alpha 1 and interferon-alpha consistently inhibited HepG2-Nu2 clonal growth more effectively than either treatment alone, reaching maximum inhibition levels of 51%. CONCLUSIONS: Thymosin-alpha 1 specifically inhibits the tumorigenic growth of HBV-transfected HepG2 cells in contrast to the general inhibition displayed by interferon-alpha. 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.

Thymosin alpha 1 does not promote growth or oncogenic transformation.

Thymosin alpha 1 (T alpha 1) is an immune modulatory peptide which has been evaluated in a variety of clinical trials. Although no in vivo adverse effects, including enhancement of tumor growth, have been noted, in vitro studies suggesting a role for T alpha 1 in cell growth have been reported. The studies presented in this report evaluated both exogenously added T alpha 1 and endogenously expressed T alpha 1 as factors which could either promote growth of tumor cells or induce transformation. No effect of exogenous T alpha 1 on cell growth was found. NIH-3T3 cells transfected with cDNA for the precursor ProThymosin alpha (Pro T alpha) expressed elevated levels of authentic T alpha 1 but did not demonstrate either enhanced proliferation in liquid culture or transformation as defined by the loss of contact inhibition or anchorage independent growth in soft agar. Thus these studies argue against the hypothesis that T alpha 1 is either an intracellular or extracellular growth promoter.

Regulation of ornithine decarboxylase gene expression by the Wilms' tumor suppressor WT1.

The importance of ornithine decarboxylase (ODC) to cell proliferation is underscored by the complex array of cell-specific mechanisms invoked to regulate its synthesis and activity. Misregulation of ODC has severe negative consequences on normal cell function, including the acquisition of tumorigenic growth properties by cells overexpressing ODC. We hypothesize that ODC gene expression is a candidate target for the anti-proliferative function of certain tumor suppressors. Here we show that the Wilms' tumor suppressor WT1 binds to multiple sites within the human ODC promoter, as determined by DNase I protection and methylation interference assays. The expression of WT1 in transfected HCT 116, NIH/3T3 and HepG2 cells represses activity of the ODC promoter controlling expression of a luciferase reporter gene. In contrast WT1 expression enhances ODC promoter activity in SV40-transfected HepG2 cells. Both the extent of modulation of ODC gene expression and the mediating WT1 binding elements are cell specific. Constructs expressing WT1 deletion mutants implicate two regions required for repressor function, as well as an intrinsic activation domain. Understanding the regulation of ODC gene expression by WT1 may provide valuable insights into the roles of both WT1 and ODC in development and tumorigenesis.

Ornithine decarboxylase transformation of NIH/3T3 cells is mediated by altered epidermal growth factor receptor activity.

Ornithine decarboxylase (ODC) has been shown to be oncogenic in transfected NIH/3T3 cells overexpressing the enzyme from a heterologous promoter. These cells, designated as NODC-2 cells, acquire proliferative properties associated with tumorigenic transformation such as loss of contact inhibition, decreased population doubling time, anchorage-independent growth, and tumor production in nude mice. At least one of these parameters, loss of contact inhibition, remains dependent on elevated ODC levels. We have used these cells to investigate the molecular mechanisms by which ODC overexpression drives cell transformation and to examine the involvement of other proto-oncogene products in this process. An interaction between ODC overexpression and the epidermal growth factor receptor (EGF-R) was suggested initially by the elevation of both basal (300%) and ligand-induced (457%) EGF-R tyrosine kinase activities in NODC-2 cells compared to similarly treated control NLK cells. Disruption of EGF-R mediated signal transduction in NODC-2 cells both by treatment with tyrphostin-25 or by transfection with a vector expressing a dominant negative EGF-R mutant resulted in reacquisition of contact-inhibited growth and suppression of anchorage-independent, clonogenic growth in soft agar. We conclude that ODC-induced transformation of NIH/3T3 cells is mediated, at least partly, by alterations in EGF-R signal transduction activity.

Ornithine decarboxylase and tyrosine kinase activity in juvenile polyps of childhood.

Juvenile polyps (JP) are the most common colonic tumor in children. Although considered benign, malignant transformation has been reported in JP. Ornithine decarboxylase (ODC) and tyrosine kinase (TyK) enzymes are markers for a rapid cell proliferation index. DNA aneuploidy score and p53 gene expression are late malignant changes seen in patients with colon cancer. In this study, we investigated ODC and TyK activities as well as DNA aneuploidy score and p53 expression in juvenile polyps compared with the adjacent normal colonic mucosa. Results showed that ODC was significantly increased in JP compared with the adjacent normal colonic mucosa. TyK activity was increased in 3/5 polyps and decreased in 2/5 polyps compared with the mucosa. Mean TyK activity was higher in JP compared with normal mucosa but did not reach significance (707 and 632 pmol/mg pmol, respectively). Moreover, changes in phosphorylization of TyK proteins was also observed in JP but not in normal mucosa. JP had a normal DNA aneuploidy score and showed no expression of p53 gene. We conclude that JP do not express p53 gene and aneuploidy but had higher activity of ODC and TyK enzymes, suggesting a higher stage of cell proliferation.

FK-506 and cyclosporine A (CsA) immunomodulation of the human gut mucosal immune system.

FK-506 and cyclosporine A (CsA) are two immunosuppressive drugs used in the treatment of patients after liver and small intestine transplantation. A clinical advantage of FK-506 over CsA has been observed in these patients. Although the immunomodulation of both drugs has been well documented in the circulatory immune system, their effect on the mucosal immune system is not well established. In this study, the effect of FK-506 on the human gut mucosal immune system was compared to CsA. Proliferation of human colonic lamina propria lymphocytes (LPL) was measured by DNA synthesis and ornithine decarboxylase (ODC) activity. Results show that FK-506 and CsA suppress LPL DNA proliferation in a dose-dependent manner. FK-506 had a stronger antiproliferative effect compared to CsA. Moreover, the antiproliferative effect of both drugs was not dependent on monocytes or monocyte-associated factors (IL-1 beta, IL-6). In addition, exogenous addition of IL-2 did not restore the suppressive effect of either drug on LPL DNA synthesis. We conclude that: (1) both drugs have an antiproliferative effect on the human mucosal immune system; and (2) the stronger effect of FK-506 on human LPL compared to CsA may explain its superior clinical response observed in patients after liver/small intestine transplantation.

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 NPY's 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 NPY's effect may be mediated via polyamine biosynthesis. We postulate that the NPY may have an important impact on human mucosal immune function.

Transformation of NIH/3T3 cells by ornithine decarboxylase overexpression.

Ornithine decarboxylase (ODC) plays a rate-limiting role in polyamine biosynthesis and is intimately associated with cell proliferation and function. Although elevated levels of ODC mRNA, protein, and enzyme activity are consistently detected in transformed cells and tumors, the question remains as to whether ODC gene overexpression has a causative role in tumorigenesis. We have stably transfected NIH/3T3 fibroblasts with an expression construct containing human ODC complementary DNA under transcriptional control of the human beta-actin promoter. Cells transfected with the beta-actin/ODC DNA construct, designated NODC cells, and control transfectants, termed NLK cells, were analyzed for ODC gene expression and cell growth characteristics. ODC activity and mRNA levels were elevated 3-6-fold in NODC cells relative to NLK cells. NODC cells, in contrast to NLK control cells, are not contact inhibited, exhibit anchorage-independent growth, cycle more rapidly, and induce tumors in nude mice more efficiently and rapidly. These results directly establish a causative role for the misregulation of ODC gene expression in the acquisition of a transformation phenotype and provide a model to examine the interaction of ODC and other gene products in neoplastic development.

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.

Multiple promoter elements govern expression of the human ornithine decarboxylase gene in colon carcinoma cells.

Overexpression of the ornithine decarboxylase (ODC) gene may be important to the development and maintenance of colonic neoplasms, as well as tumors in general. In this study, we examined the promoter elements governing constitutive expression of the human ODC gene in HCT 116 human colon carcinoma cells and, for comparison, K562 human erythro-leukemia cells. It was determined by functional analysis that the promoter elements responsible reside within the 378 bp immediately upstream from the transcription start site. Within this sequence, there are at least three regions that modulate the efficiency of the ODC promoter cooperatively. Both DNA bandshift and footprint assays demonstrated all three regions to be rich in sites that bind to nuclear proteins isolated from HCT 116 and K562 cells; the protein binding pattern of non-transformed, diploid fibroblasts was found to be much less complex. Several of the protein binding sequences have little or no homology to common regulatory elements. We suggest that the constitutive activity of the ODC gene in HCT 116 colon carcinoma cells, and perhaps transformed cells in general, involves a complex interaction of multiple regulatory sequences and their associated nuclear proteins. Finally, the saturation of the promoter in these transformed cell lines suggests that high levels of protein binding in the ODC promoter may contribute to elevated constitutive expression of this gene.