guanosine-triphosphate and Colonic-Neoplasms

Transcription initiation factor 90 (TIF-90), an alternatively spliced variant of TIF-IA, differs by a 90 base pair deletion of exon 6. TIF-90 has been shown to regulate ribosomal RNA (rRNA) synthesis by interacting with polymerase I (Pol I) during the initiation of ribosomal DNA (rDNA) transcription in the nucleolus. Recently, we showed that TIF-90-mediated rRNA synthesis can play an important role in driving tumorigenesis in human colon cancer cells. Here we show that TIF-90 binds GTP at threonine 310, and that GTP binding is required for TIF-90-enhanced rRNA synthesis. Overexpression of activated AKT induces TIF-90 T310, but not a GTP-binding site (TIF-90 T310N) mutant, to translocate into the nucleolus and increase rRNA synthesis. Complementing this result, treatment with mycophenolic acid (MPA), an inhibitor of GTP production, dissociates TIF-90 from Pol I and hence abolishes AKT-increased rRNA synthesis by way of TIF-90 activation. Thus, TIF-90 requires bound GTP to fulfill its function as an enhancer of rRNA synthesis. Both TIF variants are highly expressed in colon cancer cells, and depletion of TIF-IA expression in these cells results in significant sensitivity to MPA-inhibited rRNA synthesis and reduced cell proliferation. Finally, a combination of MPA and AZD8055 (an inhibitor of both AKT and mTOR) synergistically inhibits rRNA synthesis, in vivo tumor growth, and other oncogenic activities of primary human colon cancer cells, suggesting a potential avenue for the development of therapeutic treatments by targeting the regulation of rRNA synthesis by TIF proteins.

Topics: Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; DNA, Ribosomal; Guanosine Triphosphate; HCT116 Cells; Humans; Ribosomes; RNA Polymerase I; RNA, Ribosomal; Signal Transduction; Transcription Factors; Transcription, Genetic

GTP-binding proteins play important roles in many essential biological processes, including cell signaling, trafficking, and protein synthesis. To assess quantitatively these proteins at the whole proteome level, we developed a high-throughput targeted proteomic method based on the use of isotope-coded GTP probes and multiple-reaction monitoring (MRM) analysis. Targeted proteins were labeled with desthiobiotin-GTP probes, digested with trypsin, and the ensuing desthiobiotin-conjugated peptides were enriched with streptavidin beads for LC-MS/MS analysis. We also established a Skyline MRM library based on shotgun proteomic data acquired for 12 different human cell lines. The library contained 605 tryptic peptides derived from 217 GTP-binding proteins, representing approximately 60% of the annotated human GTP-binding proteome. By using this library, in conjunction with isotope-coded GTP probes and scheduled LC-MRM analysis, we investigated the differential expression of GTP-binding proteins in a pair of primary/metastatic colon cancer cell lines (SW480 and SW620). We were able to quantify 97 GTP-binding proteins, and we further validated the differential expression of several GTP-binding proteins by Western blot analysis. Together, we developed a facile targeted quantitative proteomic method for the high-throughput analysis of GTP-binding proteins and applied the method for probing the altered expression of these proteins involved in colon cancer metastasis.

Topics: Cell Line, Tumor; Colonic Neoplasms; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Isotope Labeling; Neoplasm Metastasis; Proteomics

Transforming growth factor beta1 (TGFbeta1) can act as a tumor suppressor or a tumor promoter depending on the characteristics of the malignant cell. We recently demonstrated that colon carcinoma cells transfected with oncogenic cellular K-rasV12, but not oncogenic cellular H-rasV12, switched from TGFbeta1-insensitive to TGFbeta1-growth-stimulated and also became more invasive (Yan, Z., Deng, X., and Friedman, E. (2001) J. Biol. Chem. 276, 1555-1563). We now demonstrate that TGFbeta1 growth stimulation of colon carcinoma cells is Ras-dependent and smad-independent. In U9 colon carcinoma cells, which are responsive to TGFbeta1 by growth stimulation, a truncating mutation at Gln-311 was found in the smad4 gene. Very little smad4 protein was detected in these cells. Loss of smad4 protein was confirmed by functional studies. In U9 cells co-transfected wild-type smad4, but not mutant smad4, mediated response of the 3TP-lux and pSBE promoter reporter constructs to TGFbeta1. Proliferation initiated by TGFbeta1 in U9 cells required Ras-mediated down-regulation of p21cip1 protein. Less p21cip1 was associated with cdk2 small middle dotcyclin complexes in TGFbeta1-treated U9 cells, and the cdk2 complexes had increased kinase activity. Elevation of p21cip1 levels diminished proliferative response to TGFbeta1. U9 cells expressing DN-N17ras neither proliferated in response to TGFbeta1 nor down-regulated the cdk inhibitor p21cip1, and TGFbeta1 activation of 3TP-lux in U9 cells was inhibited by DN-N17ras in a dose-dependent manner. TGFbeta1 also decreased p21cip1 levels and stimulated proliferation in SW480 cells, which express mutant K-Ras but no smad4 protein. TGFbeta1 did not activate or inhibit the p21cip1 promoter construct in U9 cells even in the presence of co-transfected smad4, or alter p21cip1 mRNA levels. Thus the decrease in p21cip1 levels was mediated by a TGFbeta-initiated Ras-dependent, but smad-independent post-transcriptional mechanism.

Topics: Blotting, Northern; Blotting, Western; CDC2-CDC28 Kinases; Cell Division; Colonic Neoplasms; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Mutation; Plasmids; Protein Binding; Protein Serine-Threonine Kinases; ras Proteins; RNA Processing, Post-Transcriptional; Signal Transduction; Smad4 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Previously we have shown that a positive correlation existed between the presence of beta1-6 branching of N-linked carbohydrate (detected as PHA-L reactivity) and the level of Ras activation in colon carcinoma cell lines. In these cell lines the major PHA-L-reactive species was found to be 180 kDa. Here we identified this species to be carcinoembryonic antigen (CEA) by demonstrating that: (a) CEA immunoreactivity and PHA-L reactivity colocalized on blots of crude cellular membranes from these cell lines, and that (b) immunoprecipitation of CEA resulted in quantitative coprecipitation of PHA-L reactivity at 180 kDa. Metabolic labeling of cell line HTB39 with [(3)H]mannose revealed that CEA was the predominantly labeled glycoprotein. This indicated that CEA was the major PHA-L-reactive species due its high level of expression. The amount of PHA-L reactivity present on CEA, expressed as the PHA-L/CEA ratio, was found to vary between cell lines. This ratio was found to correlate closely with the level of Ras activation in these cells. In cellular membrane isolated from primary colon carcinoma, the major PHA-L-reactive species was also 180 kDa. This reactivity colocalized with CEA immunoreactivity, indicating that the major beta1-6-branching glycoprotein in membranes from primary colon carcinoma was CEA. Similar to that seen in cell lines, the amount of PHA-L reactivity on CEA in human tumor samples varied, suggesting that a similar paradigm of Ras-induced expression of beta1-6 branching may occur in human colon carcinoma.

Topics: Blotting, Western; Carbohydrate Conformation; Carcinoembryonic Antigen; Cell Membrane; Colonic Neoplasms; Enzyme Activation; Glycoproteins; Guanosine Triphosphate; Humans; Mannose; Molecular Weight; Oncogene Protein p21(ras); Phytohemagglutinins; Precipitin Tests; Tumor Cells, Cultured

Cyclopentenyl cytosine (CPEC) is cytotoxic to several tumor cell lines. CPEC inhibits CTP synthesis resulting in depletion of cytidylate pools. The aim of this study was to examine CPEC's cytotoxic and antitumor activity in vitro and in vivo against human colon carcinoma HT-29, and to relate its action on CTP synthesis. CPEC exhibits potent cytotoxicity in vitro to HT-29 cells with an LC50 (concentration that is lethal to the survival of 50% cell colonies) of 2.4 microM and 0.46 microM following 2 h and 24 h exposure, respectively. Incubation of cells with CPEC for 2 h resulted in a dose-dependent decrease in cytidylate pools. The in vivo antitumor activity of CPEC in athymic mice transplanted subcutaneously (s.c.) with 3 million HT-29 cells was examined. Antitumor activity of CPEC was elucidated in early-staged tumor, wherein CPEC (1.5 mg/kg, QD x 9 or 3 mg/kg, QOD x 9) was administered intraperitoneally (i.p.) 24 h after tumor implantation and it resulted in a significant reduction in tumor weight to 48% of control. The effect of CPEC on established solid tumors in vivo was examined in athymic mice transplanted s.c. 14 days earlier with HT-29 cells and treated i.p. with 1.5 mg/kg CPEC, QD x 5 for 4 courses, with a 10 day-interval between courses. This treatment resulted in a significant reduction in tumor weight (72%) in the treated group. HPLC analysis of HT-29 tumor obtained from mice after treatment with CPEC showed a depletion of the CTP concentration reaching a nadir at 8 h. In conclusion, the present studies demonstrate potent antitumor activity of CPEC against freshly transplanted and established human colon carcinoma which can be corroborated with the drug's biochemical actions.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Cell Survival; Chromatography, High Pressure Liquid; Colonic Neoplasms; Cytidine; Cytidine Triphosphate; Dose-Response Relationship, Drug; Guanosine Triphosphate; HT29 Cells; Humans; Mice; Mice, Nude; Time Factors; Uridine Triphosphate

We hypothesized that unexplained increases in nucleoside triphosphates (NTP) observed by 31P magnetic resonance spectroscopy (MRS) after treatment of tumours by DNA-damaging agents were related to chemotherapy-induced up-regulation of the bcl-2 gene and DNA damage prevention and repair processes. To test this hypothesis, we treated HT-29 cells with 10(-4) M nitrogen mustard (HN2) and performed sequential perchloric acid extractions in replicate over 0-18 h. By reference to an internal standard (methylene diphosphonic acid), absolute changes in 31P-detectable high-energy phosphates in these extracts were determined and correlated with changes in bcl-2 protein levels, cell viability, cell cycle, apoptosis and total cellular glutathione (GSH) (an important defence against DNA damage from alkylating agents). After HN2 administration, bcl-2 protein levels in the HT-29 cell line rose at 2 h. Cell viability declined to 25% within 18 h, but apoptosis measured using fluorescence techniques remained in the 1-4% range. Increased cell division was noted at 4 h. Two high-energy interconvertible phosphates, NTP (P < or = 0.006) and phosphocreatine (PCr) (P < or = 0.0002), increased at 2 h concurrently with increased levels of bcl-2 protein and glutathione. This study demonstrates that bcl-2 and glutathione are up-regulated by HN2 and links this to a previously unexplained 31P MRS phenomenon: increased NTP after chemotherapy.

Topics: Adenosine Triphosphate; Ammonia; Antineoplastic Agents, Alkylating; Apoptosis; Cell Cycle; Chromatography, High Pressure Liquid; Colonic Neoplasms; Cytidine Triphosphate; Genes, bcl-2; Glutathione; Guanosine Triphosphate; HT29 Cells; Humans; Magnetic Resonance Spectroscopy; Mechlorethamine; Phosphocreatine; Purine Nucleotides; Up-Regulation; Uridine Triphosphate

Mice transplanted with a cachexia-inducing colonic adenocarcinoma (MAC16) show a progressive decrease in liver glycogen in direct proportion to the loss of body weight. Such tumours elaborate a lipid mobilizing factor (LMF), which produces a dose-dependent stimulation, not only of adipocyte adenylate cyclase, but also of hepatocyte adenylate cyclase in a GTP-dependent manner. These results suggest that LMF has the capacity to initiate hepatic glycogenolysis through an increase in cyclic AMP.

Topics: Adenocarcinoma; Adenylyl Cyclases; Adipocytes; Adipose Tissue; Animals; Cachexia; Cell Membrane; Cells, Cultured; Colonic Neoplasms; Epididymis; Guanosine Triphosphate; Kinetics; Lipid Mobilization; Liver; Liver Glycogen; Male; Mice; Mice, Inbred Strains; Peptides; Weight Loss

Fifty percent of human colon carcinomas contain activating mutations in the K-ras gene. However, whether these alterations in K-ras affect the function of Ras proteins in growth factor (GF) signal transduction is now known. Here we have characterized a previously defined human colon carcinoma cell model system for K-ras gene mutations and for altered levels of Ras protein expression and have examined whether these alterations affect Ras function in GF signal transduction. Sequence analysis of PCR-amplified K-ras gene fragments indicated that among the more aggressive cell lines, four had a normal K-ras sequence, whereas 3 others (isolated from the same human tumor) contained a mutation at codon 13. In contrast, all 7 of the less aggressive cell lines contained a mutation at either codon 12 or 13. In addition to the presence of a K-ras mutation, one cell line expressed higher levels of the K-Ras protein and displayed elevated Ras-GTP loading (in the absence of GF addition) compared with the other cell lines examined. Despite these alterations, the mitogenic GF combination epidermal growth factor + insulin + transferrin resulted in an activation of Ras and extracellular signal-regulated kinase 2. Collectively, our results indicate that the malignant phenotype of the cell lines was not correlated with the presence of K-ras mutations or with higher levels of Ras protein expression. Furthermore, K-ras mutations, high levels of K-Ras protein expression, and elevated Ras-GTP loading, as they occur naturally in human colon carcinomas, do not abolish the function of Ras in GF signaling.

Topics: Base Sequence; Blotting, Western; Cell Line; Codon; Colonic Neoplasms; DNA Primers; Epidermal Growth Factor; Exons; Genes, ras; Growth Substances; Guanosine Triphosphate; Humans; Insulin; Kinetics; Molecular Sequence Data; Point Mutation; Polymerase Chain Reaction; ras Proteins; Signal Transduction; Transferrin; Tumor Cells, Cultured

The objective of the present study was to examine the effect of modifying the fatty acid composition of membranes on cell growth and phosphoinositide specific phospholipase C (PLC) activity in HT-29 colon cancer cells. Cells were seeded at a density of 12 x 10(3) cells/cm2 and supplemented with 30 microM of either 18:0, 18:2 (n6) or 18:3 (n3) complexed to bovine serum albumin (BSA) in DMEM medium. Cell growth was followed for 12 days. The 18:0 supplemented cells (control) reached maximum growth at day nine which was greater than either 18:2 (n6) or 18:3 (n3) supplemented cells. There was no difference between the latter two groups in their growth. To investigate the fatty acid incorporation of the supplemented fatty acid and how they may influence composition in the cell membrane, we examined the fatty acid composition of each phospholipid (PL) species. Both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were significantly influenced by the type of fatty acid supplemented. Supplementation with 18:0 resulted in HT-29 cell membranes having more monounsaturated fatty acids than the cells grown in the other fatty acids. Polyunsaturated fatty acid (PUFA) supplementation (both 18:2 and 18:3) resulted in the enrichment of PUFA in the PL fractions. Cells supplemented with 18:3 (n3) had the highest unsaturation index in membrane PE as compared to the other phospholipid species. PLC activity of the membranes was measured using PIP2 as a substrate in the presence of 15 micrograms alamethicin and 42 microM free calcium. The contribution of G protein to the activity of the enzyme was assessed using GTP gamma(S). PLC activity of HT-29 cells was 16% higher in the presence of GTP gamma(S) response. GTP gamma(S)-activated PLC activity of 18:3 (n3) supplemented cells was 81% of those supplemented with either 18:0 or 18:2 (n6) cells. It is concluded that the decrease in cell proliferation with supplementation with 18:3 (n3) may be mediated through its inhibitory effect on PLC, which provides the second messengers for protein kinase C (PKC) activation. PLC may be influenced by an increased unsaturation index of the PE fraction of the HT-29 tumor cell membranes.

Topics: Alamethicin; Calcium; Carbachol; Cell Division; Colonic Neoplasms; Deoxycholic Acid; Enzyme Activation; Fatty Acids; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Membrane Lipids; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositols; Phosphatidylserines; Phospholipids; Sphingomyelins; Tumor Cells, Cultured; Type C Phospholipases

LU103793 (NSC D-669356) is a new synthetic derivative of Dolastatin 15, an antiproliferative compound which was isolated from the mollusk Dolabella auricularia. Like Dolastatin 15, LU103793 is highly cytotoxic in vitro (IC50 = 0.1 nM). To investigate the mechanism of action of LU103793, we used a combination of biochemical and cellular methods. Turbidity assays with bovine brain microtubules demonstrated that LU103793 inhibits microtubule polymerization in a concentration-dependent manner (IC50 = 7 microM). Treatment with this compound also induced depolymerization of preassembled microtubules. Cell cycle analysis of tumor cell lines treated with LU103793 indicated a block in the G2-M phase. At the cellular level, it induced depolymerization of microtubules in interphase cells and development of abnormal spindles and chromosome distribution in mitotic cells. Although these effects are very similar to the cellular alterations caused by vinblastine, LU103793 does not inhibit vinblastine binding to unpolymerized tubulin in vitro. Our results suggest that LU103793 exerts its cytotoxic activity primarily through disruption of microtubule organization.

Topics: Adenocarcinoma; Amino Acid Sequence; Antineoplastic Agents; Breast Neoplasms; Cell Cycle; Cell Division; Chromosomes, Human; Colonic Neoplasms; Drug Interactions; Guanosine Triphosphate; HeLa Cells; Humans; Hydrolysis; Microtubules; Mitosis; Molecular Sequence Data; Neoplasms; Oligopeptides; Tubulin; Tumor Cells, Cultured; Vinblastine

Here the K-ras genotypes of nine colon carcinoma cell lines are compared to the protein glycosylation patterns found in these cells. By a variety of methodologies utilizing lectins to probe carbohydrate structure, we find evidence that five out of six cell lines having K-ras mutations have elevated amounts of beta 1-6 branching at the trimannosyl core of N-linked carbohydrate. None of the three K-ras wild type cell lines assayed have evidence of elevated beta 1-6 branching. In five out of five cell lines examined, the amount of beta 1-6 branching correlates with the extent of cellular ras-GTP elevation and supports the hypothesis that expression of beta 1-6 branching in colon carcinoma cell lines is quantitatively linked to K-ras activation. These results are discussed in the context of the ras-signalling pathway.

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Carbohydrates; Cell Membrane; Colonic Neoplasms; Genes, ras; Genotype; Glycoproteins; Glycosylation; Guanosine Triphosphate; Humans; Lectins; Molecular Sequence Data; Mutation; Neoplasm Proteins; Phenotype; Tumor Cells, Cultured

Small GTP-binding proteins have been implicated in the regulation of many dynamic cellular processes. The carboxyl termini of parietal cell small GTP-binding proteins were cloned using a 3'-rapid amplification of cDNA ends (RACE) technique with a degenerate oligonucleotide primer based on the WDTAGQE consensus GTP-binding sequence. Six out of 53 clones demonstrated a novel Rab sequence, now designated Rab25. The complete sequence was obtained using 3'-RACE and revealed a deduced amino acid sequence having 63% identity with Rab11. The deduced amino acid sequence demonstrated a carboxyl-terminal CCQNI and also a novel GTP-binding site sequence of WDTAGLE. Nevertheless, recombinant Rab25 was able to bind GTP on blot. A major 1.2 kilobase Rab25 message was detected throughout the gastrointestinal mucosa and in lung and kidney tissue. No message was detected in brain, heart, liver, or skeletal muscle. In gastric tissue, Rab25 was absent in the bowel wall; among mucosal cells, it was highly enriched in parietal cells compared to chief cells. Rab25 mRNA was also detected in several colon carcinoma lines including LIM1215 and HT-29. The results indicate that Rab25 represents a novel member of the Rab family with an epithelial distribution.

Topics: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Colonic Neoplasms; DNA; Gene Expression; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Intestinal Mucosa; Kidney; Lung; Molecular Sequence Data; rab GTP-Binding Proteins; Rabbits; Recombinant Proteins; RNA, Messenger; Sequence Analysis, DNA; Tumor Cells, Cultured

In our attempts to evaluate the influence of chemopreventive agents on intermediate biomarkers of colon cancer, we have investigated the effect of D,L-alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase and piroxicam, a non-steroidal anti-inflammatory drug (NSAID) on the expression levels of biochemically active p21ras, the protein product of cellular ras protooncogenes during the development of azoxymethane (AOM) induced colon carcinogenesis in male F344 rats in order to explore the plausibility of using p21ras as an intermediate biochemical marker of colon cancer. Groups of male F344 rats were fed the modified AIN-76A diets containing 0 or 150 p.p.m. piroxicam or 4000 p.p.m. DFMO and administered s.c. AOM dissolved in normal saline at a dose of 15 mg/kg body wt/week, once weekly, for 4 weeks. Vehicle control groups received s.c. equal vol of normal saline. Groups of animals were then killed at 0, 4, 16, 24 and 32 weeks after the last injection of AOM or saline and their colonic mucosa and tumors analyzed for biochemically active p21ras levels. AOM treatment significantly increased the expression of biochemically active p21ras. The AOM-induced expression of biochemically active p21ras was significantly suppressed by dietary DFMO and piroxicam. DFMO exerted a more pronounced inhibitory effect on AOM-induced colon tumor development as well as the expression of biochemically active p21ras. These results indicate that the determination of biochemically active p21ras may be effectively used in clinical chemoprevention trials as an intermediate end-point to monitor the colon carcinogenesis.

Topics: Affinity Labels; Animals; Azoxymethane; Biomarkers, Tumor; Blotting, Western; Colonic Neoplasms; Eflornithine; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Guanosine Triphosphate; Male; Piroxicam; Proto-Oncogene Proteins p21(ras); Rats

In the regulation of GTP biosynthesis, complex interactions are observed. A major factor is the behavior of the activity of IMPDH, the rate-limiting enzyme of de novo GTP biosynthesis, and the activity of GPRT, the salvage enzyme of guanylate production. The activities of GMP synthase, GMP kinase and nucleoside-diphosphate kinase are also relevant. In neoplastic transformation, the activities and amounts of all these biosynthetic enzymes are elevated as shown by kinetic assays and by immunotitration for IMPDH. In cancer cells, the up-regulation of guanylate biosynthesis is amplified by the concurrent decrease in activities of the catabolic enzymes, nucleotidase, nucleoside phosphorylase, and the rate-limiting purine catabolic enzyme, xanthine oxidase. The up-regulation of the capacity for GTP biosynthesis is also manifested in the stepped-up capacity of the overall pathways of de novo and salvage guanylate production. The linking with neoplasia is also seen in the elevation of the activities of IMPDH and GMP synthase and de novo and salvage pathways as the proliferative program is expressed as cancer cells enter log phase in tissue culture. The activity of GMP reductase showed no linkage with neoplastic or normal cell proliferation; however, in induced differentiation in HL-60 cells the activity increased concurrently with the decline in the activity of IMPDH. This reciprocal regulation of the two enzymes is observed in differentiation induced by retinoic acid, DMSO or TPA in HL-60 cells. In support of enzyme-pattern-targeted chemotherapy, evidence was provided for synergistic chemotherapy with tiazofurin (inhibitor of IMPDH) and hypoxanthine (competitive inhibitor of GPRT and guanine salvage activity) in patients and in tissue culture cell lines. These investigations should contribute to the clarification of the controlling factors of GMP biosynthesis, the role of the various enzymes, the behavior of GMP reductase in mammalian cells and the application of the approaches of enzyme-pattern-targeted chemotherapy in patients.

Topics: Animals; Cell Differentiation; Cell Division; Colonic Neoplasms; Evaluation Studies as Topic; GMP Reductase; Guanosine Monophosphate; Guanosine Triphosphate; Humans; Hypoxanthine; Hypoxanthines; IMP Dehydrogenase; Inosine Monophosphate; Leukemia, Promyelocytic, Acute; Liver Neoplasms, Experimental; NADH, NADPH Oxidoreductases; Ribavirin; Tumor Cells, Cultured

Tiazofurin is effective in treating end-stage leukemic patients (Tricot et al., Cancer Res 49:3696-3701, 1989). In sensitive tumors, the active metabolite of tiazofurin, TAD, potently inhibits IMP dehydrogenase activity, resulting in reduced guanylate pools. To elucidate tiazofurin activity in human solid tumors, we examined its activity in human colon carcinoma HT-29. Tiazofurin exhibited an LC50 of 35 microM in cultured HT-29 cells. Incubation of HT-29 cells with 100 microM tiazofurin for 2 h resulted in TAD formation (9.3 nmol/g cells) and in a 64% decrease in GTP pools. For biochemical and chemotherapy studies, athymic nude mice were transplanted s.c. with HT-29 cells. Twenty-four days later, mice were injected i.p. with tiazofurin (500 mg/kg); 6 h later, tumors were removed and analyzed. These tumors formed 17 nmol/g of TAD with decreased GTP pools (56%). To study oncolytic activity, transplanted mice were treated 24 h later with tiazofurin (500 mg/kg, once a day for 10 days). To examine the effectiveness of tiazofurin in established tumors, the drug was administered to mice 14 days after tumor implantation (500 mg/kg, once a day for 5 days, course repeated 4 times with a 10-day rest). Both treatment schedules resulted in significant antitumor activity. This study illustrates the potential usefulness of tiazofurin in treating human colon carcinoma.

Topics: Adenine Nucleotides; Animals; Antineoplastic Agents; Colonic Neoplasms; Drug Screening Assays, Antitumor; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Male; Mice; Mice, Nude; Ribavirin; Tumor Cells, Cultured

alpha 2-Adrenergic agonist preincubation resulted in a leftward shift in the subsequent concentration-response curve to forskolin-stimulated adenylate cyclase activity in membranes from HT29 cells, a human colon adenocarcinoma cell line. This effect was much less pronounced than the effect seen in the intact cell cyclic AMP production assays. Removal of GTP from the assay caused a further slight leftward shift in the concentration-response curve. In [3H]forskolin binding experiments, alpha 2-adrenergic agonist preincubation caused a doubling of the maximal number of binding sites (80 vs 31 fmol/mg protein) compared to control. The addition of MgCl2 and NaF to the assay buffer increased control binding 5-fold. With agonist preincubation, there was a further increase in binding in the presence of MgCl2 and NaF which was not significantly different from the appropriate control. Pertussis toxin pretreatment blocked both the leftward shift in the forskolin concentration-response curve and the increase in maximal number of binding sites, indicating that a pertussis toxin sensitive protein is involved in these changes. Activation of cyclic AMP production in the intact cell by cholera toxin followed by norepinephrine preincubation and then stimulation by forskolin resulted in a degree of sensitization similar to that seen in the membrane adenylate cyclase and binding assays. Pertussis toxin also blocked this sensitization. It appears that if the cyclase system is highly activated, then the degree of sensitization is similar in the membrane and intact cell assay.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclases; Adrenergic alpha-Agonists; Cell Membrane; Colforsin; Colonic Neoplasms; Cyclic AMP; Guanosine Triphosphate; Humans; Pertussis Toxin; Tumor Cells, Cultured; Virulence Factors, Bordetella

We have characterized the alpha 2-adrenergic receptor in membranes from the human colonic adenocarcinoma cell line, HT29, using the recently introduced alpha 2-agonist 5-bromo-6-[2-imidazolin-2-yl-amino]quinoxaline [( 3H]UK-14,304), two other radioligands, and a series of adrenergic agonists and antagonists. We also investigated alpha 2-agonist inhibition of HT29 cell adenylate cyclase and reversal of inhibition by alpha-adrenergic antagonists. [3H] Yohimbine saturation experiments indicated a single class of sites with a KD of 0.61 nM which agreed with the kinetically determined KD of 0.62 nM. Computer analysis of kinetic and saturation experiments with [3H]UK-14,304 revealed two classes of sites. From the saturation data, one site had high affinity for the radioligand (0.14 nM) and comprised 33% of the total number of sites, whereas the other site had lower affinity (6.1 nM). The total number of sites labeled by [3H]UK-14,304 (360 fmol/mg of protein) was approximately equal to the number of sites labeled by [3H]yohimbine (330 fmol/mg), whereas [3H]para-aminoclonidine labeled fewer sites of a single class. Rank order potencies of adrenergic agonists and antagonists obtained from competition binding assays indicated that: the same receptors were labeled by the three radioligands, and the receptors were of the alpha 2 subtype. UK-14,304 and epinephrine inhibited forskolin- and vasoactive intestinal peptide-stimulated adenylate cyclase in a dose-dependent manner up to 32%. Inhibition of the enzyme was reversed by yohimbine and, less potently, by phentolamine and prazosin in a dose-dependent manner. The HT29 cell line appears to be a useful model system for the investigation of the regulation and mechanism of action of alpha 2-adrenergic receptors in human tissues.

Topics: Adenocarcinoma; Adenylyl Cyclase Inhibitors; Adrenergic alpha-Agonists; Brimonidine Tartrate; Cell Line; Clonidine; Colforsin; Colonic Neoplasms; Dose-Response Relationship, Drug; Guanosine Triphosphate; Humans; Kinetics; Magnesium; Quinoxalines; Radioligand Assay; Receptors, Adrenergic, alpha; Vasoactive Intestinal Peptide; Yohimbine

Sodium and GppNHp, a GTP analogue, decrease the specific binding of the alpha 2-adrenergic agonist [3H]clonidine on membranes from the HT29 cells; affinity of the ligand for the receptor is also decreased. The effects of the two agents are additive. By contrast, combination of sodium and GppNHp increases the number of binding sites for the alpha 2-adrenergic antagonist [3H]yohimbine. Sodium and GppNHp also decrease the potency of epinephrine and clonidine to displace [3H]yohimbine from the alpha 2-adrenoceptors of the HT29 cells

Topics: Adenocarcinoma; Binding, Competitive; Cell Line; Clonidine; Colonic Neoplasms; Epinephrine; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Receptors, Adrenergic, alpha; Sodium; Yohimbine

An unusual isozyme of lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27), LDHk, has been described in cells transformed by the Kirsten murine sarcoma virus (KiMSV). This isozyme appears to contain one or more subunits encoded by the transforming gene of KiMSV and is readily distinguished from other isozymes of LDH. Specifically, it is more basic than other LDH isozymes, has an apparent subunit structure of (35,000)4(22,000)1, is essentially inactive if assayed under a normal atmosphere, and is strongly inhibited by GTP and various related compounds. We have examined human cancer and normal tissue controls for expression of an activity like LDHk. In 11 out of 16 human carcinomas, LDHk activity was increased 10- to 500-fold over the level seen in adjoining nontumor tissue. In contrast, other LDH isozymes were increased by only 2- to 5-fold.

Topics: Aerobiosis; Breast Neoplasms; Colonic Neoplasms; Female; Guanosine Triphosphate; HeLa Cells; Humans; Isoenzymes; L-Lactate Dehydrogenase; Neoplasms; Reference Values