Gastrin stimulates expression of protooncogene c-myc through a process involving polyamines in IEC-6 cells

1995 ◽  
Vol 269 (6) ◽  
pp. C1474-C1481 ◽  
Author(s):  
J. Y. Wang ◽  
H. Wang ◽  
L. R. Johnson
Keyword(s):  
Specific Inhibitor ◽  
Cellular Protein ◽  
Mucosal Cell ◽  
Mrna Levels ◽  
Maximum Increase ◽  
Polyamine Biosynthesis ◽  
Small Intestinal ◽  
Rate Limiting ◽  
Mucosal Cell Proliferation ◽  
Rapid Appearance

The current study tested the hypothesis that the protooncogene c-myc is involved in the mechanism by which gastrin modulates mucosal cell proliferation. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Administration of gastrin resulted in the rapid appearance of c-myc mRNA in IEC-6 cells. The increased expression of c-myc began 1 h and peaked 4 h after exposure to gastrin. Maximum increase in c-myc mRNA levels was 7.5-fold the normal value. When cellular protein synthesis was inhibited by addition of cycloheximide, gastrin superinduced c-myc mRNA levels. Gastrin also significantly increased the mRNA levels for ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine biosynthesis, enzyme activity, and intracellular polyamines in IEC-6 cells. Treatment with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ODC, not only completely depleted intracellular polyamines but also significantly prevented the increased expression of c-myc in cells exposed to gastrin. These results show that 1) gastrin stimulates both polyamine biosynthesis and the expression of the c-myc protooncogene, and 2) depletion of intracellular polyamines by DFMO significantly prevented the increased expression of c-myc by gastrin.

scholarly journals Inhibition of polyamine synthesis induces p53 gene expression but not apoptosis

1999 ◽  
Vol 276 (4) ◽  
pp. C946-C954 ◽  
Author(s):  
Li Li ◽  
Ji Li ◽  
Jaladanki N. Rao ◽  
Minglin Li ◽  
Barbara L. Bass ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Inhibition ◽  
P53 Protein ◽  
Specific Inhibitor ◽  
P53 Gene ◽  
Mrna Levels ◽  
Intestinal Epithelial ◽  
Polyamine Biosynthesis ◽  
Fetal Bovine ◽  
P53 Mrna

The nuclear phosphoprotein p53 acts as a transcription factor and is involved in growth inhibition and apoptosis. The present study was designed to examine the effect of decreasing cellular polyamines on p53 gene expression and apoptosis in small intestinal epithelial (IEC-6) cells. Cells were grown in DMEM containing 5% dialyzed fetal bovine serum in the presence or absence of α-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, for 4, 6, and 12 days. The cellular polyamines putrescine, spermidine, and spermine in DFMO-treated cells decreased dramatically at 4 days and remained depleted thereafter. Polyamine depletion by DFMO was accompanied by a significant increase in expression of the p53 gene. The p53 mRNA levels increased 4 days after exposure to DFMO, and the maximum increases occurred at 6 and 12 days after exposure. Increased levels of p53 mRNA in DFMO-treated cells were paralleled by increases in p53 protein. Polyamines given together with DFMO completely prevented increased expression of the p53 gene. Increased expression of the p53 gene in DFMO-treated cells was associated with a significant increase in G1 phase growth arrest. In contrast, no features of programmmed cell death were identified after polyamine depletion: no internucleosomal DNA fragmentation was observed, and no morphological features of apoptosis were evident in cells exposed to DFMO for 4, 6, and 12 days. These results indicate that 1) decreasing cellular polyamines increases expression of the p53 gene and 2) activation of p53 gene expression after polyamine depletion does not induce apoptosis in intestinal crypt cells. These findings suggest that increased expression of the p53 gene may play an important role in growth inhibition caused by polyamine depletion.

Expression of the ornithine decarboxylase gene in response to asparagine in intestinal epithelial cells

1996 ◽  
Vol 271 (1) ◽  
pp. G164-G171 ◽  
Author(s):  
J. Y. Wang ◽  
M. J. Viar ◽  
P. M. Blanner ◽  
L. R. Johnson
Keyword(s):  
Ornithine Decarboxylase ◽  
Half Life ◽  
Cellular Protein ◽  
Mrna Levels ◽  
Intestinal Epithelial ◽  
Normal Rat ◽  
Fetal Bovine ◽  
Mucosal Growth ◽  
Rate Limiting ◽  
In Cells

Refeeding fasted rats significantly stimulates mucosal growth and ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, but the exact mechanism responsible for induction of ODC at the molecular level is unknown. Of normal dietary constituents, the amino acid asparagine markedly increases ODC activity and mucosal growth when administered intragastrically. The current study examined the expression of the ODC gene in IEC-6 cells (a line of normal rat small intestinal crypt cells) after exposure to asparagine. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum. They were deprived of serum for 24 h before experiments. Exposure to asparagine at the dose of 10 mM resulted in the rapid increase in ODC mRNA levels. The increased expression of the ODC gene began 1 h after and peaked between 3 and 5 h after treatment with asparagine. Maximum increases in ODC mRNA levels were approximately fivefold the normal value. Increased levels of ODC mRNA in cells exposed to asparagine were paralleled by increases in ODC protein and enzyme activity and cellular polyamine levels. The half-life of mRNA for ODC in unstimulated IEC-6 cells was approximately 30 min and increased to > 2 h in cells exposed to 10 mM asparagine. The half-life of ODC activity also was increased in asparagine-treated cells. When cellular protein synthesis was inhibited by cycloheximide, asparagine superinduced ODC mRNA levels. Furthermore, asparagine also significantly stimulated DNA synthesis in IEC-6 cells. These results indicate that 1) asparagine stimulates ODC in IEC-6 cells through multiple pathways and 2) increased ODC mRNA levels result partly from a delay in the rate of degradation. These findings suggest that luminal amino acids stimulate gut mucosal growth in association with their ability to regulate ODC gene expression.

Polyamines and intestinal growth--increased polyamine biosynthesis after jejunectomy

1983 ◽  
Vol 245 (5) ◽  
pp. G656-G660 ◽  
Author(s):  
G. D. Luk ◽  
S. B. Baylin
Keyword(s):  
Cell Proliferation ◽  
Intestinal Adaptation ◽  
Intestinal Resection ◽  
Crypt Cell ◽  
Mucosal Cell ◽  
Ileal Mucosa ◽  
Cell Hyperplasia ◽  
Polyamine Biosynthesis ◽  
Crypt Cell Proliferation ◽  
Mucosal Cell Proliferation

Transient increases in the activities of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAM-DC), key enzymes in polyamine biosynthesis, may be critical to initiation of cell growth. We now report that such increases in ODC (X170) and SAM-DC (X83) activities, and their synthetic products putrescine (X4) and spermidine (X2), occur in rat ileal mucosa between days 1 and 4 after 50% intestinal resection. This is the time period of initiation of mucosal cell hyperplasia in intestinal adaptation after resection and is characterized by increased mucosal cell proliferation, as measured morphologically and biochemically. Intestinal weight increased by 76% and mucosal thickness by 48%. Mucosal DNA content increased by 67% and mucosal DNA synthesis by 104%. Increased intestinal crypt cell proliferation was manifested by a 120% increase in labeling per crypt and a 152% increase in crypt cell production rate (CCPR). The increase in ODC activity was closely associated with the increases in CCPR and rate of villus lengthening. Rates of mucosal cell proliferation, as measured by CCPR, and villus and crypt lengthening were significantly correlated with ODC activity (r = 0.97, 0.98, and 0.94, respectively; P less than 0.01 for all). Our results indicate that the increase in ODC activity, SAM-DC activity, and polyamine biosynthesis is closely associated with the process of adaptive postresectional crypt cell proliferation.

Decreased expression of protooncogenes c-fos, c-myc, and c-jun following polyamine depletion in IEC-6 cells

1993 ◽  
Vol 265 (2) ◽  
pp. G331-G338 ◽  
Author(s):  
J. Y. Wang ◽  
S. A. McCormack ◽  
M. J. Viar ◽  
H. Wang ◽  
C. Y. Tzen ◽  
...  
Keyword(s):  
Steady State ◽  
Specific Inhibitor ◽  
State Level ◽  
Cell Number ◽  
Mrna Levels ◽  
Direct Exposure ◽  
Mucosal Cells ◽  
Steady State Levels ◽  
Small Intestinal ◽  
Mucosal Growth

Direct exposure of small intestinal mucosal cells to luminal polyamines stimulates proliferation. This study tests the hypothesis that the protooncogenes c-fos, c-myc, c-jun, and junB are involved in the mechanism by which polyamines modulate mucosal growth. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS) in the presence of absence of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the rate-limiting enzyme for polyamine synthesis. Cellular polyamine levels, cell growth, and relative abundance of c-fos, c-myc, c-jun, and junB mRNAs, were measured at 1, 2, 4, 6, 8, and 12 days after initial plating. The intracellular polyamines, spermidine and spermine, and their precursor, putrescine, in DFMO-treated cells decreased significantly at 2 days and remained depleted thereafter. Although DFMO profoundly decreased growth and final cell number, both control and DFMO-treated cells entered a plateau phase by 6 days. In control cells, c-myc and c-jun mRNA levels significantly increased on days 4-6 and then returned to a basal level of expression, which was maintained thereafter. c-fos mRNA in quiescent cells after 24 h serum deprivation was significantly stimulated by 5% dFBS, although a steady-state level of c-fos mRNA was undetectable in control cells. Treatment with DFMO not only prevented increased expression of c-myc and c-jun protooncogenes at 4 days, but also significantly reduced steady-state levels of c-myc and c-jun mRNA between 6 and 12 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the transforming growth factor-β gene during growth inhibition following polyamine depletion

1998 ◽  
Vol 275 (2) ◽  
pp. C590-C598 ◽  
Author(s):  
Anami R. Patel ◽  
Ji Li ◽  
Barbara L. Bass ◽  
Jian-Ying Wang
Keyword(s):  
Growth Factor ◽  
Growth Inhibition ◽  
Half Life ◽  
Posttranscriptional Regulation ◽  
Transforming Growth Factor ◽  
Specific Inhibitor ◽  
Transforming Growth Factor Β ◽  
Cell Number ◽  
Mrna Levels ◽  
Polyamine Biosynthesis

Polyamine depletion and cytokine transforming growth factor-β (TGF-β) inhibit cell proliferation. The current study tests the hypothesis that polyamine depletion results in growth inhibition by altering expression of the TGF-β gene in intestinal epithelial cells. Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Cells were grown in DMEM in the presence or absence of α-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, for 6 and 12 days. Administration of DFMO not only depleted intracellular polyamines but also significantly increased the mRNA levels of TGF-β. Increased TGF-β mRNA in DFMO-treated cells was paralleled by an increase in TGF-β content. Depletion of intracellular polyamines by DFMO had no effect on the rate of TGF-β gene transcription, as measured by nuclear run-on assay. The half-life of mRNA for TGF-β in normal cells was ∼65 min and increased to >16 h in cells treated with DFMO for 6 or 12 days. Exogenous polyamine, when given together with DFMO, prevented the increased half-life of TGF-β mRNA in IEC-6 cells. TGF-β added to the culture medium significantly decreased the rate of DNA synthesis and final cell number in normal and polyamine-deficient cells. Furthermore, growth inhibition caused by polyamine depletion was partially but significantly blocked by addition of immunoneutralizing anti-TGF-β antibody. These results indicate that 1) depletion of intracellular polyamines induces the activation of the TGF-β gene through posttranscriptional regulation and 2) increased expression of the TGF-β gene plays an important role in the process of growth inhibition following polyamine depletion.

Polyamines are necessary for cell migration by a small intestinal crypt cell line

1993 ◽  
Vol 264 (2) ◽  
pp. G367-G374 ◽  
Author(s):  
S. A. McCormack ◽  
M. J. Viar ◽  
L. R. Johnson
Keyword(s):  
Cell Migration ◽  
Concomitant Treatment ◽  
Polyamine Biosynthesis ◽  
Migration Period ◽  
Culture Model ◽  
Ulcer Model ◽  
Small Intestinal ◽  
Rate Limiting ◽  
Intestinal Crypt Cell

Studies from our laboratory have shown that polyamines are essential for the normal repair of duodenal erosions induced in vivo in a rat stress-ulcer model. In that model, the inhibition of ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, with alpha-difluoromethylornithine (DFMO) almost entirely prevented healing. Healing could be restored by oral polyamines. In this paper, we have investigated whether the polyamines are required for the early stages of epithelial restitution using an IEC-6 cell culture model of cell migration. Treatment of the cells with DFMO for 4 days reduced cell migration 80%. Migration could be restored to normal by concomitant treatment with putrescine (PUT), spermidine (SPD), or spermine (SPM), but not by their addition during the migration period (6 h) only. If DFMO treatment was not begun until the migration period, it still reduced cell migration 20%, and this deficit could not be restored by concomitant addition of the polyamines. Intracellular polyamine levels at these times, i.e., 6 h or 4 days, were an important factor in these results. Only PUT was undetectable after 6 h of DFMO. SPD and SPM were still at normal levels at 6 h. SPD was undetectable at 4 days, but SPM was still at 40% of normal. These data give added importance to PUT because its absence reduced cell migration after only 6 h, while SPD and SPM were still present in normal amounts. Perhaps exogenous SPD and SPM restored cell migration when present with DFMO for 4 days treatment primarily because they contributed to intracellular PUT through the acetyltransferases.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals A Yes-Associated Protein (YAP) and Insulin-Like Growth Factor 1 Receptor (IGF-1R) Signaling Loop Is Involved in Sorafenib Resistance in Hepatocellular Carcinoma

Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3812
Author(s):  
Mai-Huong T. Ngo ◽  
Sue-Wei Peng ◽  
Yung-Che Kuo ◽  
Chun-Yen Lin ◽  
Ming-Heng Wu ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Combination Index ◽  
Specific Inhibitor ◽  
In Vivo Model ◽  
Mrna Levels ◽  
Margin Distribution ◽  
Related Proteins ◽  
Emt Markers

The role of a YAP-IGF-1R signaling loop in HCC resistance to sorafenib remains unknown. Method: Sorafenib-resistant cells were generated by treating naïve cells (HepG2215 and Hep3B) with sorafenib. Different cancer cell lines from databases were analyzed through the ONCOMINE web server. BIOSTORM–LIHC patient tissues (46 nonresponders and 21 responders to sorafenib) were used to compare YAP mRNA levels. The HepG2215_R-derived xenograft in SCID mice was used as an in vivo model. HCC tissues from a patient with sorafenib failure were used to examine differences in YAP and IGF-R signaling. Results: Positive associations exist among the levels of YAP, IGF-1R, and EMT markers in HCC tissues and the levels of these proteins increased with sorafenib failure, with a trend of tumor-margin distribution in vivo. Blocking YAP downregulated IGF-1R signaling-related proteins, while IGF-1/2 treatment enhanced the nuclear translocation of YAP in HCC cells through PI3K-mTOR regulation. The combination of YAP-specific inhibitor verteporfin (VP) and sorafenib effectively decreased cell viability in a synergistic manner, evidenced by the combination index (CI). Conclusion: A YAP-IGF-1R signaling loop may play a role in HCC sorafenib resistance and could provide novel potential targets for combination therapy with sorafenib to overcome drug resistance in HCC.

scholarly journals Effects of bicarbonate on intercompartmental reducing-equivalent translocation in isolated parenchymal cells from rat liver

1974 ◽  
Vol 140 (3) ◽  
pp. 355-361 ◽  
Author(s):  
Michael N. Berry ◽  
Harold V. Werner ◽  
Ernest Kun
Keyword(s):  
Malate Dehydrogenase ◽  
Inhibitory Action ◽  
Ethanol Oxidation ◽  
Specific Inhibitor ◽  
Isolated Liver Cells ◽  
Glycerol Uptake ◽  
Parenchymal Cells ◽  
Rate Limiting ◽  
Isolated Liver ◽  
Phosphate Medium

1. Incubation of isolated liver cells in a medium containing bicarbonate raises malate concentrations almost sixfold compared with values obtained in a bicarbonate-free phosphate medium. The malate concentration of about 0.3mm in bicarbonate medium is of the same order as the Km for malate dehydrogenase. 2. The utilization of ethanol, glyercol and sorbitol was increased by 20–35% in bicarbonate medium. 3. Fluoromalate, a specific inhibitor of malate dehydrogenase and the malate carrier, inhibited or ethanol oxidation by 23%, glycerol uptake by 20% and sorbitol uptake by 42% in bicarbonate medium, but had a much smaller inhibitory action in phosphate medium. In consequence fluoromalate almost abolished the stimulatory effects of bicarbonate on substrate utilization. 4. Difluoro-oxaloacetate, a specific inhibitor of aspartate aminotransferase, had about one-half the inhibitory activity of fluoromalate. The two inhibitors in combination were less effective than fluoromalate by itself. 5. It is concluded that bicarbonate stimulates the utilization of reduced substrates, which are oxidized in the cytoplasmic compartment of the liver cell, by increasing the activity of rate-limiting malate dehydrogenase-dependent intercompartmental hydrogen shuttles. Both malate–oxaloacetate and malate–aspartate systems are involved in these hydrogen-translocation processes.

scholarly journals Mitochondrial gene expression in small intestinal epithelial cells

1995 ◽  
Vol 308 (2) ◽  
pp. 665-671 ◽  
Author(s):  
T P Mayall ◽  
I Bjarnason ◽  
U Y Khoo ◽  
T J Peters ◽  
A J S Macpherson
Keyword(s):  
Epithelial Cells ◽  
Cytochrome Oxidase ◽  
Cytochrome B ◽  
Intestinal Epithelial Cells ◽  
Mitochondrial Gene ◽  
Cytochrome Oxidase I ◽  
Mrna Levels ◽  
Intestinal Epithelial ◽  
Mitochondrial Transcription Factor ◽  
Small Intestinal

Most mitochondrial genes are transcribed as a single large transcript from the heavy strand of mitochondrial DNA, and are subsequently processed into the proximal mitochondrial (mt) 12 S and 16 S rRNAs, and the more distal tRNAs and mRNAs. We have shown that in intestinal epithelial biopsies the steady-state levels of mt 12 S and 16 S rRNA are an order of magnitude greater than those of mt mRNAs. Fractionation of rat small intestinal epithelial cells on the basis of their maturity has shown that the greatest ratios of 12 S mt rRNA/cytochrome b mt mRNA or 12 S mt rRNA/cytochrome oxidase I mt mRNA are found in the surface mature enterocytes, with a progressive decrease towards the crypt immature enteroblasts. Cytochrome b and cytochrome oxidase I mt mRNA levels are relatively uniform along the crypt-villus axis, but fractionation experiments showed increased levels in the crypt base. The levels of human mitochondrial transcription factor A are also greater in immature crypt enteroblasts compared with mature villus enterocytes. These results show that the relative levels of mt rRNA and mRNA are distinctly regulated in intestinal epithelial cells according to the crypt-villus position and differentiation status of the cells, and that there are higher mt mRNA and mt TFA levels in the crypts, consistent with increased transcriptional activity during mitochondrial biogenesis in the immature enteroblasts.

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