BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Antineoplastic Agents↗

PubMedDate not supplied

A functional in vitro model to examine signaling mechanisms in gastrin-mediated human cell growth.

Gastrin (G-17) is a trophic hormone with a high affinity for the cholecystokinin-B receptor (CCK-BR); the mechanisms linking receptor binding and activation of downstream events to cell growth are not known, and these studies have been hampered by the lack of a cell model. We have established a pancreatic carcinoid cell line, BON, which produces a number of gut hormones; however, these cells lack native CCK-BR. The purpose of our study was to develop a model cell line containing the CCK-BR and to characterize the cellular mechanisms involved in gastrin regulation of human cell growth. BON cells were transfected with an expression plasmid containing the human CCK-BR, and stable clones were selected using G418. Functional CCK-BR was confirmed by reverse transcriptase-polymerase chain reaction, 125I-gastrin binding, and mobilization of intracellular calcium ([Ca2+]i) in response to G-17. Stable transfectants were treated with G-17 (+/-) the CCK-BR antagonist, L365,260 (L-60); growth was assessed using a Coulter counter. G-17 stimulated the growth of the stable clones, whereas the selective CCK-BR antagonist, L-60, abolished this G-17-mediated trophic effect. We have shown that G-17, acting through the CCK-BR, mobilizes [Ca2+]i as a second messenger and stimulates cell growth. Our unique BON cell line, stably transfected with the human CCK-BR, provides a novel paradigm to further delineate signaling mechanisms in gastrin regulation of human cell growth.

Cell Division↗

PubMedDate not supplied

The effect of aging on the growth of colon cancer.

Reports on the effect of patient age on the prognosis for colon cancer vary. We have tested the effect of aging using a model of murine colon adenocarcinoma in groups of mice of different ages. In experiment A, Balb/c mice of age groups 3-4 weeks, 10-12 weeks, 24-32 weeks and 40-48 weeks (13 mice/group) were injected with 5 X 10(4) MC-26 cells subcutaneously in the right flank. Tumors were measured twice weekly, and the rate of occurrence of tumor, mortality rate, and growth rate were calculated. In experiment B, the same plan as experiment A was used, except mice of age groups I (14 days), II (3-4 weeks), and III (20-22 months) were used with tumor doses of 1 X 10(4) cells and 5 X 10(5) cells (9-15 mice/group). In both experiments, the rate of growth of tumor, mortality rate, and sizes of tumors obtained were the same. In experiment A, the rate of occurrence of the tumors was the same in all groups, but in experiment B the occurrence of the tumor varied. A palpable tumor appeared earliest in the weanling mice (14 days), next in old mice (20-22 months), and last in the young adult group (3-4 weeks). Tumor doubling time was longest in the young adult mice (7 days), intermediate in the old mice (6.1 days), and shortest in the weanling mice (5.5 days). Established tumors grew at similar rates (as assessed by doubling time), independent of host age. Mortality rates were similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗

PubMedDate not supplied

Pentagastrin-stimulated DNA synthesis in mouse gut is influenced by the circadian system.

Gastrin is trophic for rodent gut mucosa. Proglumide, a competitive inhibitor of gastrin, can exert an antitrophic effect and can block pentagastrin-stimulated DNA synthesis. We have examined the influence of the circadian system on pentagastrin-stimulated DNA synthesis in the murine stomach (glandular and nonglandular stomach) and colon. We studied 224 male CD2F1 mice divided into four groups. Group A was ad lib fed (controls). Groups B, C, and D received 6-9 intraperitoneal injections of either NaCl, pentagastrin or pentagastrin + proglumide, at 8-h intervals prior to sacrifice. Mice from each group (A-D) were killed (by cervical dislocation) at 3-h intervals for 24 h. Incorporation of tritiated thymidine (DNA synthesis) was measured, and significant (p less than 0.001) circadian rhythms were found, which were not eliminated after treatment with either pentagastrin or pentagastrin + proglumide. DNA synthesis in the glandular stomach increased significantly after treatment with pentagastrin , but only during the span of time when DNA synthesis was increasing also in control mice; it had no effect at other times. Proglumide blocked the effect of pentagastrin only during the time of increasing DNA synthesis; it had no effect at other times. The identical regimen given at different times in the circadian cycle yielded significantly different results. In the intact animal, studies on the effects of various stimulators or inhibitors of DNA synthesis should be time-qualified.

Animals↗

PubMedDate not supplied

Age-related changes in polyamine biosynthesis after fasting and refeeding.

The effect of aging on ornithine decarboxylase (ODC) activity and polyamine biosynthesis in the proximal small intestine was studied in two groups of male Fisher 344 rats (young [4-month old] and aged [26- to 27-month old]) using a fasting and refeeding model. In control (nonfasted) rats, levels of polyamines (putrescine, spermidine and spermine) and ODC activity were significantly higher in aged compared with young rats. In aged rats, fasting significantly reduced the levels of putrescine by 41%, spermidine by 23%, and spermine by 11%; however, fasting had no effect on polyamine levels in young rats. ODC activity was decreased 75% in young and 50% in aged rats after fasting compared with the respective age-matched controls. Conversely, 2 h after reinstituting a chow diet increased ODC activity by 17-fold in young rats but only 8-fold in aged rats. Putrescine levels were also increased in both age groups after refeeding; however, similar to ODC activity, these increases were much less in aged rats. In addition, spermidine and spermine levels remained significantly depressed in the aged groups even after 24 h of refeeding. These findings suggest that the normal rigid control of gut polyamine biosynthesis and proliferation noted in young rats is markedly altered with aging.

Aging↗

PubMedDate not supplied

The role of Src family kinases in the normal and neoplastic gastrointestinal tract.

Src family kinases are a group of non-receptor tyrosine kinases that mediate signal transduction pathways involved in the growth and differentiation of normal tissues. Considerable evidence exists for a role of these proteins in neoplastic progression in various organ systems including the nervous, hematopoietic and skeletal systems. In addition, the role of the Src kinase family has been characterized for colon cancer, but only limited progress has been made in delineating the role of Src kinases in the normal gastrointestinal (GI) tract and extracolonic GI cancers. In this review, we provide an up-to-date assessment of the Src family kinases in the normal and neoplastic GI tract.

Biomarkers, Tumor↗

PubMedDate not supplied

Indirect inhibitory effect of a neurotensin receptor antagonist on human colon cancer (LoVo) growth.

The effect of the nonpeptide neurotensin (NT) receptor antagonist SR48692 on NT-mediated growth of xenografted human colon cancers (LoVo) was determined. Sixty-four athymic nude mice, inoculated with LoVo cells at a single site, were randomized into four groups of 16 mice each to receive either vehicle NT (600 microg/kg), SR48692 (2 mg/kg) or NT+SR48692 administered s.c., t.i.d., for 25 days. Treatment with NT significantly stimulated LoVo tumour growth, weight, DNA and protein content; SR48692 blocked this NT-mediated effect but had no effect when administered as a single agent. In addition, normal jejunum and ileum were removed and assessed. Similar to the effects on LoVo tumours, NT stimulated jejunal and ileal growth; SR48692 blocked this NT-mediated effect. In contrast to our in vivo findings, NT had no effect on LoVo cell growth in vitro. Also, Northern blot analysis demonstrated no expression for the NT receptor in either LoVo tumour cells or xenografted tumours. Our findings suggest that the trophic effect of NT on LoVo may be through an indirect effect; one possibility is that administration of NT may stimulate the release of other trophic factors which then stimulate tumour growth. The nonpeptide NT receptor antagonist SR48692 will be a useful agent to delineate the specific effects of NT on neoplastic as well as normal gastrointestinal tissues.

Analysis of Variance↗

PubMedDate not supplied

Prevention of mucosal atrophy: role of glutamine and caspases in apoptosis in intestinal epithelial cells.

Glutamine starvation induces apoptosis in enterocytes; therefore glutamine is important in the maintenance of gut mucosal homeostasis. However, the molecular mechanisms are unknown. The caspase family of proteases constitutes the molecular machinery that drives apoptosis. Caspases are selectively activated in a stimulus-specific and tissue-specific fashion. The aims of this study were to (1) identify specific caspases activated by glutamine starvation and (2) determine whether a general caspase inhibitor blocks glutamine starvation-induced apoptosis in intestinal epithelial cells. Rat intestinal epithelial (RIE-1) cells were deprived of glutamine. Specific caspase activation was measured using fluorogenic substrate assay. Apoptosis was quantified by DNA fragmentation and Hoechst nuclear staining. Glutamine starvation of RIE-1 cells resulted in the time-dependent activation of caspases 3 (10 hours) and 2 (18 hours), and the induction of DNA fragmentation (12 hours). Caspases 1 and 8 remained inactive ZVAD-fluoromethyl ketone, a general caspase inhibitor, completely blocked glutamine starvation-induced caspase activation, DNA fragmentation, and nuclear condensation. These results indicate that glutamine starvation selectively activates specific caspases, which leads to the induction of apoptosis in RIE-1 cells. Furthermore, inhibition of caspase activity blocked the induction of apoptosis, suggesting that caspases are potential molecular targets to attenuate apoptotic responses in the gut.

Amino Acid Chloromethyl Ketones↗