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Transforming growth factor-beta (TGF-beta) regulates epithelial tissue homeostasis by activating processes that control cell cycle arrest, differentiation and apoptosis. Disruption of the TGF-beta signaling pathway often occurs in colorectal cancers. Earlier, we have shown that TGF-beta induces apoptosis through the transcription factor Smad3. Affymetrix oligonucleotide microarrays were used to identify TGF-beta/Smad3 target genes that regulate apoptosis in rat intestinal epithelial cells (RIE-1). We found that TGF-beta repressed the expression of the inhibitor of differentiation (Id) gene family. Knockdown of Id1 and Id2 gene expression induced apoptosis in RIE-1 cells, whereas overexpression of Id2 attenuated TGF-beta-induced apoptosis. TranSignal Protein/DNA arrays were used to identify the hypoxia-inducing factor-1 (HIF-1) as a downstream target of TGF-beta. HIF-1 is a basic helix-loop-helix protein, and overexpression of Id2 blocked HIF-1 activation by TGF-beta. Furthermore, knockdown of HIF-1 blocked TGF-beta-induced apoptosis. Thus, we have identified HIF-1 as a novel mediator downstream of Id2 in the pathway of TGF-beta-induced apoptosis. more...

Overexpression of cyclooxygenase-2 (COX-2), an inducible enzyme regulating prostaglandin release, is mechanistically linked to the development, growth, and spread of gastrointestinal (GI) cancers. GI peptide bombesin (BBS) was reported to stimulate COX-2 gene expression. Here we show that TGF-beta1 dramatically enhances the BBS-induced expression of COX-2 mRNA and protein, and the release of PGE2 in the model rat intestinal epithelial cell (RIE-1) line. The synergistic increase in COX-2 levels results from a combination of enhanced COX-2 transcription and reduced mRNA degradation. BBS, but not TGF-beta1, stimulated COX-2 promoter activity, and TGF-beta1 enhanced COX-2 mRNA stability through a p38(MAPK)-dependent pathway. The synergistic regulation of COX-2 expression by TGF-beta1 and BBS may contribute to the upregulation of COX-2 in GI cancers. more...

The current recommendation is that pancreatic resections be performed at hospitals doing10 pancreatic resections annually.To evaluate the extent of regionalization of pancreatic resection and the factors predicting resection at high-volume centers (10 cases/year) in Texas.Using the Texas Hospital Inpatient Discharge Public Use Data File, we evaluated trends in the percentage of patients undergoing pancreatic resection at high-volume centers (10 cases/year) from 1999 to 2004 and determined the factors that independently predicted resection at high-volume centers.A total of 3,189 pancreatic resections were performed in the state of Texas. The unadjusted in-hospital mortality was higher at low-volume centers (7.4%) compared to high-volume centers (3.0%). Patients resected at high-volume centers increased from 54.5% in 1999 to 63.3% in 2004 (P = 0.0004). This was the result of a decrease in resections performed at centers doing less than five resections/year (35.5% to 26.0%). In a multivariate analysis, patients who were75 (OR = 0.51), female (OR = 0.86), Hispanic (OR = 0.58), having emergent surgery (OR = 0.39), diagnosed with periampullary cancer (OR = 0.68), and living75 mi from a high-volume center (OR = 0.93 per 10-mi increase in distance, P0.05 for all OR) were less likely to be resected at high-volume centers. The odds of being resected at a high-volume center increased 6% per year.Whereas regionalization of pancreatic resection at high-volume centers in the state of Texas has improved slightly over time, 37% of patients continue to undergo pancreatic resection at low-volume centers, with more than 25% occurring at centers doing less than five per year. There are obvious demographic disparities in the regionalization of care, but additional unmeasured barriers need to be identified. more...

Effective clinical therapy to augment intestinal absorption of water and electrolytes does not exist; the gut hormone, peptide YY (PYY), is a potent proabsorptive agent in animal models. The purpose of our study was to evaluate the effects of two novel PYY analogs, BIM-43073D and BIM-43004C, on intestinal absorption. Dogs with ileal Thiry-Vella fistulae (TVF) were treated with either PYY, BIM-43073D, or BIM-43004C. Administration of BIM-43073D significantly increased water and sodium absorption over baseline and maintained this level of increased absorption for a longer duration than an equimolar dose of PYY. Administration of BIM-43004C significantly increased sodium and water absorption over baseline at a level equal to that of PYY. The novel PYY analogs, BIM-43073D and BIM-43004C, are effective proabsorptive agents with BIM-43073D producing more sustained effects than PYY. These compounds may be clinically useful in the treatment of gut malabsorption in conditions such as cholera, Crohn's disease, and the short-bowel syndrome. more...

Growth inhibition of human cancers (e.g., endocrine tumors) by interferons (IFNs) has been demonstrated, but the exact cellular mechanisms remain largely undefined. IFNs and other cytokines can activate novel Stat (Signal transducers and activators of transcription) proteins, which translocate to the nucleus to activate target genes. The purpose of this study was to determine the effect of IFN-alpha and IFN-gamma on the Stat pathway using a unique human pancreatic carcinoid tumor, BON, established in our laboratory.BON cells were treated with IFN-alpha (500 U/mL) or IFN-gamma (500 U/mL); nuclear protein was extracted at selected intervals. Steady state levels of Stat proteins 1, 3, and 5 were measured by Western blot; protein binding was assessed by electrophoretic mobility shift assay (EMSA) using probes containing either the Stat1/Stat3 or Stat5 binding sites.Treatment with IFN-alpha increased predominantly Stat3 and Stat5 protein levels and binding activities. IFN-gamma increased Stat1, 3, and 5 protein levels, with maximal elevations occurring at 24 to 48 hours after addition; Stat3 and 5 binding activities were also increased.We have shown that both IFN-alpha and IFN-gamma can induce Stat protein binding (particularly Stat3 and Stat5) to their cognate DNA consensus sites and increase Stat protein steady state levels in BON cells. Delineating the signaling pathways altered by IFN treatment will provide a better understanding of downstream gene targets and mechanisms for IFN-mediated growth inhibition of endocrine tumors. more...

Gastrin-secreting tumors have been identified inectopic locations including the ovary; the mechanismsregulating gastrin gene expression, its distribution,and signaling pathways in these ectopic tissues are not known. The purpose of our present studywas to determine: (1) whether the gastrin gene andpeptide could be detected in ovarian cancer cell lines,(2) if functional gastrin releasing peptide receptors (GRP-R) are present, and (3) whether gastringene expression is altered by GRP. Five ovarian cancercell lines (SW626, OVCA 420, OVCA 429, OVCA 432, andOVCA 433) were analyzed. We identified gastrin gene and peptide expression in the SW626 cell linebut not in the OVCA lines. SW626 cells express afunctional GRP-R that is correctly coupled to theCa2+ signaling pathway. Treatment of SW626cells with bombesin, the amphibian equivalent of GRP, inhibitedexpression of the gastrin gene in a time- anddose-dependent fashion. The SW626 ovarian cancer cellline will provide a useful model to further defineregulation and expression of both the gastrin gene andpeptide in ectopic (nongastrointestinal)tissues. more...

The growth of the human gastrinoma model (PT) in athymic nude mice is stimulated by bombesin (BBS), an amphibian peptide homologous to both human gastrin-releasing peptide (GRP) and neuromedin B (NMB). The mechanism is not known, and a potent and specific GRP-R antagonist BIM26226, which has low affinity for NMB-R, was used in vivo in athymic nude mice bearing gastrinoma subcutaneously. Both the BBS and BIM26226 stimulated the growth of PT, and the growth stimulation was even greater when given together. RT-PCR study of gastrinoma revealed the presence of both GRP-R and NMB-R mRNA, but much more abundant NMB-R mRNA. We conclude that BBS-stimulated growth of gastrinoma involves both GRP-R and NMB-R, and our findings suggest that GRP-R mediates negative and NMB-R produces positive growth effects on gastrinoma. more...

We have examined age-related changes in the stimulatory effects of distal small bowel resection on the growth of the pancreas and on polyamine biosynthesis. Two groups of male Fischer 344 rats (young; 4-month-old, aged; 25-month-old) underwent either 70% distal small bowel resection or transection of distal small bowel (control). Rats from each treatment group of each age were sacrificed on the 10th or 20th postoperative day (POD), and pancreas was weighed and assayed for DNA, RNA, and protein contents as well as polyamines. We found that young rats with distal small bowel resection exhibited significant increases in pancreatic weight, RNA, protein, putrescine, and spermine contents on the 10th POD, but aged rats that had undergone resection, showed only increased spermine content. On the 20th POD distal small bowel resection increased pancreatic weight, DNA, RNA, and protein as well as spermidine and spermine contents in young rats; aged rats had increased pancreatic weight only. These findings suggest that the trophic responses of the pancreas to distal small bowel resection are altered in aged rats, and that the polyamine biosynthetic pathway appears to be involved in this impaired response. more...

Two groups of male Fisher 344 rats (young: 4 months old; aged: 25 months old) underwent either 70% distal small bowel resection or sham operation (small bowel transection). Rats from each treatment group of each age were sacrificed on the 10th (N = 15: young rats; N = 13: aged rats) or 20th (N = 15: young; N = 13: aged) postoperative day (POD), and the duodenal mucosa was weighed and assayed for DNA, RNA, and protein contents, as well as for specific activities of the disaccharidase, sucrase, maltase, and lactase. Compared to the sham operation, distal small bowel resection significantly increased DNA by 48%, RNA by 122%, and protein by 75% in young rats and DNA by 40%, RNA by 92%, and protein by 71% in aged rats on the 20th POD. Both young and aged rats showed similar adaptive hyperplasia on the 10th POD. On the 20th POD after distal small bowel resection, specific activities of all tested enzymes were significantly increased in young rats (sucrase +86%, maltase +110% and lactase +64%), but showed no significant changes in aged rats. These findings suggest that the duodenum of aged rats may have sufficient proliferative potential to respond to distal small bowel resection, but that the mechanisms governing return of function in response to distal small bowel resection are inhibited in aged rats, compared to those mechanisms in the young. more...

Exogenous administration of epidermal growth factor (EGF) enhances tumor growth of H2T, a hamster pancreatic cancer that is inoculated in the cheek pouch. The effect of endogenous EGF on tumor growth, however, is not known. The main source of EGF in the body is the submandibular glands. This study examined the influence of submandibular gland resection (SMx) on H2T tumor growth in the cheek pouch and compared it to the growth in SC tissue. Bilateral SMx or sham operation was performed on male Syrian golden hamsters. In 30 hamsters (n = 15 for each operation group), 5 x 10(4) and 5 x 10(5) H2T cells were inoculated in the bilateral cheek pouches and intrascapular SC tissue, respectively (Exp. 1). In another 30 hamsters (n = 15 for each operation group), 5 x 10(5) and 1 x 10(6) H2T cells were inoculated at the same sites (Exp. 2). Two longest perpendicular diameters of the tumor were measured once a week for 12 wk (Exp. 1) or 8 wk (Exp. 2), and the tumor area was calculated. The tumor area in the cheek pouch became significantly smaller in the SMx group than the sham-operated group after the 8th wk (Exp. 1) or the 7th wk (Exp. 2). On the other hand, the tumor area in the SC tissue did not show any difference between groups through the experiments.(ABSTRACT TRUNCATED AT 250 WORDS) more...

A clone of human gastric cancer cells (AGS-6) and the parental line (AGS-P) from which it was isolated were used in cell survival studies to determine whether pretreatment for 24, 48 or 72h with alpha-difluoromethylornithine (DFMO, 5mM) would increase the cell's sensitivity to 5-Fluorouracil (5FU), Adriamycin (Adria), 1-(2-chloroethyl)-3-(4-methyl cyclohexyl)-1-nitrosourea (MeCCNU), or Bleomycin (Bleo). Generally, the AGS parental cells were most sensitive to the anticancer agents after exposures to DFMO. However, there was no way to predict in advance from DFMO-induced changes in ornithine decarboxylase (ODC), polyamine or cell kinetics values, how long an exposure to DFMO was required before sensitization to an anticancer agent occurred. The degree of potentiation for a single drug was variable from time to time during exposure to DFMO, and broad differences in the sensitizations were demonstrated among the four anticancer drugs. The AGS-6 clone exhibited little or no increased sensitivity as a result of pretreatment with DFMO, even though the DFMO-induced reductions in ODC and polyamine values in these cells were similar to those produced in the more sensitive parental line. more...

We have previously reported that the in vitro growth of MC-26 mouse colon cancer and H2T hamster pancreatic cancer cells are inhibited by cyclosporine (CsA) and alpha-difluoromethylornithine (DFMO). The present study was designed to investigate the effects of these two drugs on the two experimental tumors (MC-26 and H2T) growing in vivo. Forty-eight male Balb/c mice or Syrian golden hamsters were inoculated with MC-26 (250,000) or H2T (500,000) cells, respectively, and then were randomized into four groups of 12 each: group I was control; group II received CsA; group III received DFMO; group IV received a combination of CsA and DFMO. MC-26 tumors were significantly more sensitive than H2T tumors to the effects of CsA and DFMO. MC-26 tumor growth and tumor weight, as well as the tumor content of DNA, RNA, and protein were all significantly more reduced by CsA and DFMO than were the H2T tumors. Our present study shows that both CsA and DFMO are potent inhibitors of MC-26 colon carcinoma growth in vivo, though DFMO is more than twice as effective as CsA. DFMO also produced greater reductions in the tumor content of DNA, RNA, and protein than did CsA. DFMO significantly decreased the concentrations of polyamines in both H2T and MC-26 tumors; the MC-26 tumors were affected to a greater degree. more...

The effects of alpha Difluoromethylornithine (DFMO)2 on survival of human gastric cancer clones were studied in vitro. The responses were dose and time dependent. Treatments which lasted for less than 12 h were cytotoxic at only the highest doses used. The greatest effects on survival were observed only when cells were treated for 48 and 72 h. The effects on the clones produced by such prolonged treatment durations were heterogeneous, with survival values differing by as much as 460%. By contrast, the clonal survival responses to short DFMO treatments (12 h) were very uniform (dose differential of only 19%); however, this uniformity in response could be achieved only by using non-pharmacological doses of DFMO. The heterogeneity in survival responses in the clones might be slightly associated with their levels of intracellular spermidine. Clones with the smallest amounts of intracellular spermidine at the start of treatment were most sensitive to DFMO. However, this association may not hold up with further testing in other gastric cancer clones or when studied in other cancer lines in vitro. more...

Treatment of human gastric cancer clones in vitro with low doses of DFMO (5 mM) produced elongation of the cell population doubling times and lowering of the saturation densities. By contrast, DFMO treatment of normal human skin fibroblasts altered only the saturation density. The lack of an effect of 5 mM DFMO on the doubling time of normal fibroblasts may be directly related to baseline intracellular putrescine levels, which were about 2.5 times higher than in the cancer cells. The same dose of DFMO caused a rapid decrease in intracellular polyamine levels in the tumor clones. The effects on the doubling time and saturation density were almost totally abolished by the addition of 50 microM putrescine to the growth medium during the first 24 h of treatment with DFMO. Exposure to 5 mM DFMO for 24 h caused the human gastric cancer cells to become blocked in G1 phase only, and this led to a reduction in the fraction of cells in S phase. The G1 block was reversible and this cohort of cells eventually passed through S phase and then through G2 and M. A higher 100 mM dose of DFMO and longer exposure times for both doses produced cell cycle changes and death of more than 90% of the cell population. These data suggest that cell kinetics changes observed under these experimental conditions may reflect polyamine-related alterations in the biochemical events of cell cycle progression kinetics; but may also be the result of DFMO-induced loss of cell viability. more...

We have shown in earlier studies that repeated weekly exposures of a human astrocytoma clone (AST 3-4) to MeCCNU (10 micrograms/ml for 1 h per week) produced a linear decrease in survival over the first 3 weekly treatments. But, after that time these cells became progressively more resistant to the 10 micrograms/ml concentration of the agent. In the studies reported here we show that these previously treated cells were also less responsive to other doses ranging from 1 to 30 micrograms MeCCNU/ml. This change in sensitivity to MeCCNU was accompanied by collateral changes in response to other agents: resistance to BCNU and Galactitol, increased sensitivity to Adriamycin, and no change to ionizing radiation. These experiments suggest that once repeated treatments with a single agent cause a tumor cell population to become more resistant, sensitivity to other agents may also change unpredictably. more...

The effects of alpha-difluoromethylornithine (DFMO) on survival, cell kinetics and polyamine levels were studied on two clones of human astrocytoma in vitro. The survival responses were dose and time dependent; and treatments with DFMO which lasted for 72 h resulted in heterogeneous responses with one clone being up to 6 times more sensitive than the other. Shorter treatments produced more uniform killing in the clones. A continuous exposure of the cells to 5 mM DFMO resulted in a rapid decrease in putrescine values in both clones, followed by decreases in the spermidine levels. These effects were closely followed by 148% to 170% increases in cell population doubling times, and a lowering of saturation densities. No clear correlations could be established among baseline polyamine levels and cell kinetics or survival responses to DFMO treatments. more...

The purpose of this study was to characterize the effects of aging on gastric acid secretion and on serum and antral concentrations of gastrin in rats. Young and old Fischer 344 rats were prepared with gastric fistulas. Twenty-four hours after surgery, graded doses of human synthetic gastrin-17 (SHG-17) (2, 5, 10, 20, and 40 μg/kg) were given intravenously in random order. Gastric secretions were collected for gastric acid measurement before and at 15-min intervals after each dose of gastrin. In a separate study, blood was collected and the stomachs were removed for antral gastrin extraction from fed young and old rats. Serum and antral gastrin was measured by radioimmunoassay. The basal and gastrin-stimulated acid secretions were significantly decreased in aged rats compared to the young rats. The basal acid output was 0.4±0.2 μeq/15 min in the aged rats and 1.5±0.5 μeq/15 min in the young. The maximal acid output stimulated by gastrin was 11.1±1.8 μeq/15 min in the aged rats and 24.2±2.8 μeq/15 min in the young. Both serum and antral concentrations of gastrin were significantly decreased in aged rats. Serum gastrin concentration was 114.8±7.4 pg/ml in the aged rats and 192.0±14.4 pg/ml in the young. Antral gastrin concentration was 3.9±0.5 μg/g tissue in the aged rats, which was significantly less than the concentration in the young (6.5±0.4 μg/g tissue). Antral gastrin content did not change with aging. Gastric acid secretion in aged rats is significantly decreased compared to the young in both the basal condition and in response to fixed doses of exogenous gastrin. Diminished concentrations of circulating gastrin may well be responsible, at least in part, for the diminished acid secretion in the aged rats. more...

alpha-Difluoromethylornithine (DFMO) is a known irreversible inhibitor of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. Cyclosporine (CsA) has been reported to inhibit ODC activity in vitro. In the present study, we compared the effects of DFMO and CsA on growth, survival, and polyamine levels in mouse colon cancer (MC-26) and hamster pancreatic cancer (H2T) cells in vitro. The growth and survival of MC-26 and H2T cells were inhibited by both DFMO and CsA. However, H2T cells were observed to be significantly more sensitive than MC-26 cells to both CsA and DFMO. The inhibitory effects of CsA were blocked by the addition of the polyamine, putrescine, in both MC-26 and H2T cells. Polyamine levels were altered significantly in both MC-26 and H2T cells treated with CsA and DFMO. However, the profile of these alterations differed between MC-26 and H2T cell lines. Putrescine and spermidine levels in MC-26 cells were more sensitive to DFMO inhibition than were H2T cells. Spermine levels were consistently elevated in MC-26 cells exposed to CsA or DFMO, while the level of spermine in H2T cells decreased significantly in response to the same drugs. These results suggest that CsA and DFMO exhibit different effects on colon and pancreatic cancer growth in vitro. In addition, the differences in the sensitivity of pancreatic and colon cancer to CsA and DFMO indicate potentially important differences in polyamine metabolism between the two cell lines. more...