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Biomedical subjects

C M Townsend

Publications and source records attributed to C M Townsend.

At least 73 records · Page 4Linked to original sources
PubMed1995

Role of calcium in the regulation of ornithine decarboxylase enzyme activity in mouse colon cancer cells.

Activity of ornithine decarboxylase (ODC), one of the rate-limiting enzymes in the pathway of polyamine biosynthesis, is regulated by various factors. In this study, we examined the role of Ca2+ in the regulation of ODC enzyme activity in mouse colon cancer cells (MC-26). KCl, a membrane-depolarizing agent that opens the voltage-dependent Ca(2+)-channel to increase intracellular Ca2+, decreased serum-induced ODC enzyme activity in MC-26 cells in a dose-dependent, reversible fashion. Both verapamil and nifedipine, inhibitors of the L-type voltage-dependent Ca(2+)-channel, decreased serum-induced ODC enzyme activity. W-7, a calmodulin inhibitor, decreased ODC enzyme activity in a dose-dependent, reversible fashion while trifluoperazine, another calmodulin inhibitor, failed to affect ODC enzyme activity in MC-26 cells. Our findings indicate that intracellular Ca2+ participates in the regulatory mechanism of ODC enzyme activity in MC-26 cells, although the exact role of Ca2+ is still unclear.

Animals↗
PubMed1995

Insulin-like growth factor-II expression in carcinoma in colon cell lines: implications for autocrine actions.

BACKGROUND: In the gastrointestinal tract, insulin-like growth factor-II (IGF-II) messenger RNA (mRNA) is localized mainly in mesenchymal cells, and is more abundant in the fetus than in the adult. The purposes of this study are to characterize the gene expression of IGF-II at the mRNA and protein level in seven different epithelial cell lines derived from colon carcinomas and to determine the action of IGF-II and IGF-receptors on a colon carcinoma cell line. STUDY DESIGN: Insulin-like growth factor-II mRNAs were examined by Northern analysis; conditioned media from colon carcinoma cells were concentrated, chromatographed, and examined by a specific IGF-II radioreceptor assay. Insulin-like growth factor receptors were examined by radioligand binding assays. The mitogenic role of IGF-II was determined by a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. RESULTS: Multiple sizes of IGF-II mRNAs were expressed in all colon carcinoma cell lines tested (six human cell lines: HCT116, COLO 205, COLO 320 DM, LoVo, DLD-1, and HT29, and one mouse cell line: MC-26). In the conditioned media of COLO 205 and HCT116 cells, 7.5 kilodaltons IGF-II and high molecular form (IGF-II and IGF binding protein complex) were detected. Both Type I and Type II IGF receptors were present on COLO 205 cells whose growth was stimulated by IGF-II. Addition of anti-IGF-I receptor and anti-IGF-II antibody in the cell culture significantly depressed growth of the COLO 205 cell line in the presence or absence of exogenous IGF-II. CONCLUSIONS: Insulin-like growth factor-II mRNAs are expressed in human and mouse colon carcinoma cell lines, which may induce production of a significant amount of biologically active IGF-II protein. The IGF-II secreted by COLO 205 cells may stimulate cell growth in an autocrine fashion through the Type I IGF receptors.

Affinity Labels↗
PubMed1994

Intratumor variability in prognostic indicators may be the cause of conflicting estimates of patient survival and response to therapy.

The DNA index, percentage of S-phase cells, proliferation fraction, and glutathione (GSH) content were determined at more than 1100 separate sites in 140 human tumors and 140 normal tissues. The study showed that the variability was so great from site to site within a tumor that there was only a 61% chance of identifying an aneuploid tumor clone (when present) if only a single site sample was analyzed for DNA content. Similar broad variability was observed in the percentage of S-phase cells, proliferation fraction, and glutathione content. Since these tumor characteristics are often used to predict the outcome of therapy and patient survival, the inaccuracy and underestimation of the test results may cause conflicting or erroneous predictions. The probability of finding an aneuploid clone or elevated percentage of S-phase cells proliferation fraction and GSH content increased dramatically as the number of sample sites studied per tumor was increased. Statistical analyses indicated that in order to achieve a 90% probability that the test results for these parameters were representative of the whole tumor: (a) all single site testing should be abandoned; (b) assays should be performed on samples taken from 3-7 different sites within each tumor; or (c) samples from each tumor should be pooled and the analyses run on a thoroughly mixed or homogenized aliquot of the multisite sample.

Breast Neoplasms↗
PubMed1994

BON cells display the intestinal pattern of neurotensin/neuromedin N precursor processing.

Antisera towards the bioactive peptides, neurotensin (NT, 13 residues) and neuromedin N (NMN, 6 residues), as well as towards three regions of their 147-residue canine precursor were used to identify and to quantitate precursor-derived peptides in extracts of human BON cells. This cell-line, which was obtained from a human pancreatic carcinoid tumor, constitutively expresses NT/NMN mRNA and secretes NT. Quantitation of seven precursor-derived peptides led us to conclude that BON cells display the intestinal pattern of NT/NMN precursor processing, which is primarily characterized by the production of a large molecular (125 amino acid) form of NMN. Four large molecular components, identified by immunochemical analyses and Western blotting, displayed physico-chemical properties which, for the most part, were consistent with the structures predicted from the partially-known human mRNA sequence. However, as shown previously for these peptides in canine gut, the empirically determined M(r) and pI values were slightly higher than those predicted solely from the amino acid content, perhaps due to the presence of additional substituents. These results suggest that BON cells may provide a good in vitro model in which to study the regulation of intestinal NT/NMN precursor processing and the nature of the enzyme(s) involved.

Amino Acid Sequence↗
PubMed1994

Growth-regulatory effect of gastrin on human colon cancer cell lines is determined by protein kinase a isoform content.

Cell growth is regulated by various peptide growth factors through receptor-linked multiple intracellular signal-transduction pathways, such as the cyclic adenosine monophosphate (cAMP) pathway. cAMP activates cAMP-dependent protein kinase A (PKA) either to stimulate or inhibit cell growth. The effect on growth is determined by the presence of two isoforms of the regulatory (R) subunit of PKA; activation of RI alpha-type PKA leads to stimulation of growth, activation of RII beta-type inhibits cell growth. We determined whether the effect of gastrin on the growth of human colon cancer cells is determined by cell-specific content of PKA. We utilized two human colon cancer cell lines: LoVo, growth of which is stimulated by gastrin, and HCT116, growth of which is inhibited by gastrin. Activation of both types of PKA with 8-Br-cAMP mimicked the regulation of growth by gastrin; preferential activation of RII beta-type PKA with 8-Cl-cAMP inhibited growth of both cell lines. LoVo cells possess the predominantly RI alpha isoform of PKA at the mRNA and protein level; HCT116 cells possess predominantly the RII beta-type PKA. The cAMP-mediated regulation of growth (either stimulatory or inhibitory) by gastrin on these human colon cancer cells was determined by the predominant isoform of PKA.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Gastrin stimulates growth of human colon cancer cells via a receptor other than CCK-A or CCK-B.

Two receptors for cholecystokinin (CCK) have been isolated which also bind gastrin: CCK-A type and CCK-B type, both are coupled to phospholipase C (PLC) activation. However, identification of the "true" gastrin receptor remains controversial. We determined which CCK receptor mediated the trophic effect of gastrin on human colon cancer cells (LoVo). LoVo cells lack mRNA for either CCK receptor by Northern hybridization. Gastrin stimulated cyclic AMP production, not PLC activity, in LoVo cells. The trophic effect was not blocked by receptor antagonists for CCK-A (L364,718) or CCK-B (L365,260). The gastrin receptor pharmacology on LoVo cells and the lack of appropriate transcripts suggest that gastrin stimulated growth of these cells by a receptor other than CCK-A or CCK-B type and there likely exists another receptor for gastrin.

Cell Division↗
PubMed1994

Effects of gastrin on 3',5'-cyclic adenosine monophosphate, intracellular calcium, and phosphatidylinositol hydrolysis in human colon cancer cells.

Gastrin is a trophic factor for some human colon cancer cells. However, the signal-transduction pathways by which gastrin regulates growth are still unknown. We examined the effect of synthetic human gastrin-17 (G-17) on signal-transduction pathways and cell growth using 4 different human colon cancer cell lines (LoVo, COLO 320, HT-29, and HCT116). G-17 stimulated the production of cyclic AMP in LoVo, COLO 320, and HCT116 cells, while G-17 stimulated phosphatidylinositol hydrolysis and mobilization of intracellular calcium in HT-29 cells. The growth-regulatory effect of G-17 on these colon cancer cells (stimulatory on LoVo, COLO 320, and HT-29 cells; inhibitory on HCT116 cells) was well correlated with the effect of G-17 on the signal-transduction pathway in each cell line. We further examined the effect of a selective cholecystokinin-B type receptor antagonist, JMV 320, on G-17-induced signal-transduction pathways and G-17-regulated growth. In each cell line, the effect of JMV 320 on G-17-induced signal-transduction pathways was well correlated with that on G-17-regulated growth. G-17 appears to regulate, at least to some extent, growth of human colon cancer cells through gastrin receptor-linked signal-transduction pathways that are cell-specific.

Calcium↗
PubMed1994

Bombesin stimulates the in vitro growth of a human gastric cancer cell line.

Bombesin (BBS) and its mammalian equivalent, gastrin-releasing peptide (GRP), exhibit diverse biological functions, including that of a neurotransmitter, a regulator of gastrointestinal hormone release, and a trophic factor for various normal and neoplastic tissues. Bombesin stimulates the growth of normal cells of the stomach, pancreas, and bronchial epithelium as well as cells in breast cancer, gastrinoma, and small cell lung cancer. The purpose of this study was to determine whether BBS regulates the growth of a human gastric cancer cell line (SIIA) in vitro, and if so, to examine the mechanisms of signal-transduction that are involved. We found that BBS stimulated the growth of SIIA cells in vitro. The GRP receptor antagonists, BIM 26189 and BIM 26226, had no effect on growth of SIIA cells. Although these antagonists blocked the BBS-induced increase of [Ca2+]i, they failed to block the growth-stimulatory effect of BBS. BBS stimulated intracellular tyrosine phosphorylation of multiple proteins, with a predominant protein of apparent molecular weight of 125 kDa. Inhibition of intracellular tyrosine kinases by tyrphostin blocked the growth-stimulatory effect of BBS on SIIA cells. These results indicate that BBS exerts its trophic effect on SIIA cells through a receptor(s) linked to tyrosine kinase pathway, but not to the phospholipase C (PLC) pathway.

Adenocarcinoma↗
PubMed1994

Gastrointestinal hormones and cell proliferation.

There is no question that gut peptides are trophic for normal gut mucosa. Gut peptides can function in an endocrine, paracrine or autocrine fashion. We examined the effects of gut peptides on the growth of animal and human cancers of the gastrointestinal (GI) tract and pancreas in vivo and in vitro. We also examined the role of growth factors and bioamines in the regulation of growth of human endocrine tumors. Our studies have shown that gut peptides (gastrin, VIP, neurotensin, and bombesin) regulate growth of some cancers of the GI tract and pancreas. We have found that peptide action is mediated through specific receptors and that cell-specific differences in receptor expression occur. We have also begun to examine the intracellular signal-transduction pathways involved in endocrine and autocrine actions of these peptides on growth of GI cancers. We have found that cell-type-specific differences exist among the various signal-transduction pathways (cyclic AMP, phosphatidylinositol hydrolysis (PI), intracellular calcium ([Ca2+]i) mobilization, and tyrosine phosphorylation) and that different receptors for the same hormone may be linked to different signal-transduction pathways depending upon cell type. We have also found that autocrine growth regulation of human pancreatic carcinoid occurs through specific receptor-mediated signal-transduction pathways. We will discuss the mechanisms of action and potential therapeutic uses of manipulation of gut hormone levels or hormone antagonists to inhibit the growth of GI tract cancers.

Animals↗
PubMed1994

Transforming growth factor-beta inhibits rat intestinal cell growth by regulating cell cycle specific gene expression.

Transforming growth factor-beta 1 (TGF-beta 1) inhibits the growth of intestinal cells, but the mechanisms involved are unknown. Using a rat intestinal crypt cell line (IEC-6), we determined the site of action in the cell cycle that TGF-beta 1 acts to suppress proliferation. We also examined the effect of TGF-beta 1 on the expression of proliferation-associated "immediate early" genes (zif268, jun-B, c-myc) during the early G1 phase and the cdc2 gene during the transition from the G1 phase to the S phase of the cell cycle. Cell cycle progression was determined by incorporation of 3H-thymidine, and gene expression was analyzed by Northern blot analysis. We found that TGF-beta 1 acts to inhibit proliferation of rat intestinal crypt cells by blocking cell cycle progression at the middle G1 phase. The genes activated during G1 can be divided into TGF-beta 1 insensitive (zif268, jun-B, and c-myc) and TGF-beta 1 sensitive (the cdc2 gene). TGF-beta 1 suppresses the induction of the cdc2 gene during the G1/S transition without inhibiting the activation of immediate early genes during the early G1 phase.

Animals↗
PubMed1994

Cytocidal effect of high energy shock wave on tumour cells enhanced with larger dose and multiple exposures.

Cultured LLC-WRC256 (Walker rat carcinoma) cells were exposed to different doses of high energy shock waves (HESW). The immediate viabilities were 98% in the control cells, and 74%, 53% and 18% following 400, 800, and 1500 HESW treatment, respectively. Surviving cells in the 400 and 800-treated HESW demonstrated delayed upward growth rate curves, and the 1500 HESW-treated a downward curve. Agar clonogenic efficiencies for surviving cells were 36% (control), 20% (400 HESW), 15% (800 HESW) and 3% (1500 HESW). LLC-WRC256 tumours in Wistar rats were treated once every other day with 1500 HESW on a total of three occasions. Tumours treated with HESW grew more slowly (4.9 cm3) than those in the control (13.5 cm3). HESW fragmented cells and destroyed cell membranes and intracellular organelles. A histological examination of tumours treated with HESW demonstrated local haemorrhage with necrosis in the HESW focus area. Damage to the surrounding skin and soft tissue was slight and transient. These findings suggest that the growth of tumour cells can be suppressed in vitro and in vivo by treatment with HESW.

Animals↗
PubMed1994

Analysis of multiple molecular changes in human endocrine tumours.

To define the molecular changes occurring in endocrine tumours, we have analysed three human endocrine tumours established in our laboratory: BON, a functioning carcinoid tumour from the pancreas; SIM, a nonfunctioning carcinoid of the ileum; and STAN, a pheochromocytoma. A homozygous point mutation of the N-ras gene was identified at codon 61 in BON cells in conjunction with overexpression of N-ras mRNA and protein. BON cells also exhibited increased expression of c-myc and cdc2 kinase mRNA and protein; TGF-beta 1, p53 and retinoblastoma (RB) mRNA and protein levels were decreased. In addition, increased expression of the mdm2 oncogene and both the truncated and the wild-type RB protein were noted in BON. SIM cells exhibited moderately increased N-ras and c-myc mRNA levels along with decreased levels of RB mRNA and protein. Similar to BON and SIM, analysis of STAN showed increased N-ras and c-myc levels. Our data show multiple molecular changes in the three human endocrine tumours with the BON cell line exhibiting the most dramatic changes. Furthermore, our data suggest the existence of different molecular pathways in the pathogenesis of endocrine tumours. These cell lines will provide unique in vitro models to further analyse the significance of these molecular alterations.

Aged↗
PubMed1994

Organ physiology of aging.

With improvements in medical care over the last several decades, individuals are living longer and, as a result, more surgical procedures will be performed in the geriatric patient. Normal physiologic aging is characterized by a gradual loss of reserve capacity. The effects of the aging process on various organ systems do not usually affect function in the normal state; however, during periods of stress (such as with a surgical procedure or illness), the elderly patient may not be able to meet the increased metabolic demand. This loss of reserve capacity is the single most important factor that decreases the elderly patient's ability to tolerate operations. It is imperative that the surgeon identify the elderly patient who is at increased risk for complications. Specific consideration must be given to proper management of fluid and electrolyte replacement, respiratory management to prevent atelectasis and pneumonia, and monitoring for possible cardiac complications.

Aging↗
PubMed1994

Gastrinomas demonstrate amplification of the HER-2/neu proto-oncogene.

OBJECTIVE: This study determined whether genomic amplification of HER-2/neu or mutations of the p53 and ras genes were present in gastrinomas. SUMMARY BACKGROUND DATA: Amplification of HER-2/neu, a proto-oncogene related to the epidermal growth factor receptor, and mutation of the ras proto-oncogene and p53 tumor suppressor gene appear to play a role in the pathogenesis of some human cancers. Little is known about possible molecular alterations in gastrinomas, tumors that may be particularly virulent because of gastrin overproduction, resulting in the severe ulcer diathesis, the Zollinger-Ellison syndrome. METHODS: The differential polymerase chain reaction (PCR) procedure was used to detect amplification of the HER-2/neu gene in DNA samples from the novel human gastrinoma cell line (PT) and from paraffin-embedded samples of gastrinomas. Sequencing techniques were used to determine whether mutations of the p53 or ras (Ha-ras, N-ras, Ki-ras) genes were present. RESULTS: Amplification (> twofold) occurred in all gastrinoma tumor samples. Compared with normal pancreas or ileum, a 4- to 12-fold amplification of HER-2/neu was found in 3 gastrinomas, 3 to 3.3-fold in four samples and 2.1- to 2.4-fold in the remaining five tumors. A heterozygous point mutation in the p53 gene (codon 273) was found in a single sample; none of the gastrinomas contained a mutation of the ras genes. CONCLUSIONS: Amplification of the HER-2/neu gene, but not alterations of either p53 or ras, may be involved in the pathogenesis of gastrinomas. The unique PT cell line will be a useful model to further elucidate the molecular mechanisms that contribute to gastrinoma formation and growth.

Base Sequence↗
PubMed1994

Bombesin improves survival from methotrexate-induced enterocolitis.

OBJECTIVE: The authors determined whether bombesin could improve survival from methotrexate (MTX)-induced enterocolitis. SUMMARY BACKGROUND DATA: Bombesin prevents gut mucosal atrophy, which is produced by feeding rats an elemental diet. Administration of MTX produces a lethal enterocolitis in rats fed an elemental diet. METHODS: On treatment day 0, 60 rats were divided randomly into three groups and fed an elemental diet (Vivonex TEN, Sandoz, Minneapolis, MN) as the only source of nutrition. Groups were subdivided further to receive either saline or bombesin (10 micrograms/kg, subcutaneously, three times a day) beginning either on day 0 or day 14. Methotrexate (20 mg/kg, intraperitoneally) was given to all rats 14 days after the start of an elemental diet. RESULTS: Bombesin prevented the mucosal atrophy in the ileum produced by the elemental diet and significantly decreased mortality in rats given MTX (whether given as a pretreatment or at the time of MTX administration). CONCLUSION: Bombesin significantly improved survival in a lethal model of MTX-induced enterocolitis, possibly by maintaining gut mucosal structure. Administration of bombesin to patients receiving chemotherapy may be clinically useful in preventing the severe enterocolitis induced by various chemotherapeutic agents.

Animals↗
PubMed1994

Characterization of a human pancreatic carcinoid in vitro: morphology, amine and peptide storage, and secretion.

The study of functioning human endocrine tumors has been hampered by a lack of suitable in vitro models. We have established the first permanent cell line of a human pancreatic carcinoid tumor (BON) in culture. BON cells grow in monolayer culture and form colonies in soft agar. Injection of BON cells into nude mice produces transplantable tumors in a dose-dependent fashion. The histology of tumors in athymic mice from injection of dispersed, cultured BON cells is similar to the original histology of the resected tumor. Significant amounts of neurotensin, pancreastatin, and serotonin (5-HT) are demonstrated in the cells by radioimmunoassay (RIA) and the presence of chromogranin A, bombesin, and 5-HT is confirmed by immunocytochemistry. Numerous round and pleomorphic dense-core neurosecretory granules are present on electron microscopy. Functional receptors for acetylcholine, 5-HT, isoproterenol, and somatostatin are present on cultured cells. BON cells possess a specific transport system for uptake of 5-HT from the medium; this uptake system may be a route for regulation of autocrine effects of 5-HT on carcinoid cells. This unique human carcinoid tumor cell line should provide the opportunity for new insight into the biology of carcinoid tumors and of specific intracellular mechanisms for secretagogue action in the release of amines and peptides.

Adult↗
PubMed1994

Inhibitory effect of calcium channel blockers on growth of pancreatic cancer cells.

Calcium, which binds to calmodulin inside the cells, is an important mediator of various intracellular processes, including cell proliferation. We speculated that blockade of Ca2+ influx into the cells by Ca(2+)-channel blockers, such as phenytoin and verapamil, might affect the Ca(2+)-calmodulin pathway leading to suppression of cell growth. In this study, we examined the effect of phenytoin and verapamil on growth of two human pancreatic cancer cell lines, MIA PaCa-2 and CAV, in vitro and in vivo. Both phenytoin and verapamil inhibited growth of the two cell lines in a dose-dependent fashion. Phenytoin and verapamil each significantly prolonged doubling time of MIA PaCa-2 and the combination of the two drugs acted synergistically. The activity of ornithine decarboxylase, which is a rate-limiting enzyme of the polyamine pathway that is closely related to cell proliferation, was significantly inhibited by both drugs in a time-dependent fashion. Phenytoin, but not verapamil, inhibited growth of MIA PaCa-2 tumors xenotransplanted into nude mice, whereas both phenytoin and verapamil inhibited the growth of CAV tumors. Since phenytoin and verapamil are known to have fewer side effects than conventional antineoplastic drugs, these results suggest their possible use in novel therapeutic strategies.

Calcium Channel Blockers↗