guanosine-triphosphate and Adenocarcinoma

It is thought that KRAS oncoproteins are constitutively active because their guanosine triphosphatase (GTPase) activity is disabled. Consequently, drugs targeting the inactive or guanosine 5'-diphosphate-bound conformation are not expected to be effective. We describe a mechanism that enables such drugs to inhibit KRAS(G12C) signaling and cancer cell growth. Inhibition requires intact GTPase activity and occurs because drug-bound KRAS(G12C) is insusceptible to nucleotide exchange factors and thus trapped in its inactive state. Indeed, mutants completely lacking GTPase activity and those promoting exchange reduced the potency of the drug. Suppressing nucleotide exchange activity downstream of various tyrosine kinases enhanced KRAS(G12C) inhibition, whereas its potentiation had the opposite effect. These findings reveal that KRAS(G12C) undergoes nucleotide cycling in cancer cells and provide a basis for developing effective therapies to treat KRAS(G12C)-driven cancers.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Alleles; Antineoplastic Agents; Azetidines; Cell Line, Tumor; Cysteine; Cytidine Diphosphate; Enzyme Inhibitors; Glycine; Guanosine Triphosphate; HEK293 Cells; Humans; Lung Neoplasms; Molecular Targeted Therapy; Mutation; Piperazines; Protein Conformation; Proto-Oncogene Proteins p21(ras); Time Factors

RAS-like protein expressed in many tissues 1 (RIT1) is a disease-associated RAS subfamily small guanosine triphosphatase (GTPase). Recent studies revealed that germ-line and somatic RIT1 mutations can cause Noonan syndrome (NS), and drive proliferation of lung adenocarcinomas, respectively, akin to RAS mutations in these diseases. However, the locations of these RIT1 mutations differ significantly from those found in RAS, and do not affect the three mutational "hot spots" of RAS. Moreover, few studies have characterized the GTPase cycle of RIT1 and its disease-associated mutants. Here we developed a real-time NMR-based GTPase assay for RIT1 and investigated the effect of disease-associated mutations on GTPase cycle. RIT1 exhibits an intrinsic GTP hydrolysis rate similar to that of H-RAS, but its intrinsic nucleotide exchange rate is ∼4-fold faster, likely as a result of divergent residues near the nucleotide binding site. All of the disease-associated mutations investigated increased the GTP-loaded, activated state of RIT1 in vitro, but they could be classified into two groups with different intrinsic GTPase properties. The S35T, A57G, and Y89H mutants exhibited more rapid nucleotide exchange, whereas F82V and T83P impaired GTP hydrolysis. A RAS-binding domain pulldown assay indicated that RIT1 A57G and Y89H were highly activated in HEK293T cells, whereas T83P and F82V exhibited more modest activation. All five mutations are associated with NS, whereas two (A57G and F82V) have also been identified in urinary tract cancers and myeloid malignancies. Characterization of the effects on the GTPase cycle of RIT1 disease-associated mutations should enable better understanding of their role in disease processes.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Amino Acid Substitution; Cell Line; Guanosine Triphosphate; Humans; Hydrolysis; Lung Neoplasms; Mutation, Missense; Neoplasm Proteins; Noonan Syndrome; Protein Domains; ras Proteins; Urologic Neoplasms

For a solid tumor to grow, it must be able to support the compressive stress that is generated as it presses against the surrounding tissue. Although the literature suggests a role for the cytoskeleton in counteracting these stresses, there has been no systematic evaluation of which filaments are responsible or to what degree. Here, using a three-dimensional spheroid model, we show that cytoskeletal filaments do not actively support compressive loads in breast, ovarian, and prostate cancer. However, modulation of tonicity can induce alterations in spheroid size. We find that under compression, tumor cells actively efflux sodium to decrease their intracellular tonicity, and that this is reversible by blockade of sodium channel NHE1. Moreover, although polymerized actin does not actively support the compressive load, it is required for sodium efflux. Compression-induced cell death is increased by both sodium blockade and actin depolymerization, whereas increased actin polymerization offers protective effects and increases sodium efflux. Taken together, these results demonstrate that cancer cells modulate their tonicity to survive under compressive solid stress.

Topics: Actins; Adenocarcinoma; Azides; Biomechanical Phenomena; Breast Neoplasms; Cation Transport Proteins; Cell Culture Techniques; Cell Line, Tumor; Cell Survival; Cytoskeleton; Female; Guanosine Triphosphate; Humans; Models, Biological; Osmosis; Sodium; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Tissue Scaffolds

The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Shape; Female; Focal Adhesions; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Humans; Integrin beta1; Male; Microtubules; Neoplasm Invasiveness; Neoplasm Proteins; Prostatic Neoplasms; Pseudopodia; rac1 GTP-Binding Protein; Recombinant Fusion Proteins; Rho Guanine Nucleotide Exchange Factors; rhoB GTP-Binding Protein; RNA, Small Interfering

The cancer stem cell (CSC) theory predicts that a small fraction of cancer cells possess unique self-renewal activity and mediate tumor initiation and propagation. However, the molecular mechanisms involved in CSC regulation remains unclear, impinging on effective targeting of CSCs in cancer therapy. Here we have investigated the hypothesis that Rac1, a Rho GTPase implicated in cancer cell proliferation and invasion, is critical for tumor initiation and metastasis of human non-small cell lung adenocarcinoma (NSCLA). Rac1 knockdown by shRNA suppressed the tumorigenic activities of human NSCLA cell lines and primary patient NSCLA specimens, including effects on invasion, proliferation, anchorage-independent growth, sphere formation and lung colonization. Isolated side population (SP) cells representing putative CSCs from human NSCLA cells contained elevated levels of Rac1-GTP, enhanced in vitro migration, invasion, increased in vivo tumor initiating and lung colonizing activities in xenografted mice. However, CSC activity was also detected within the non-SP population, suggesting the importance of therapeutic targeting of all cells within a tumor. Further, pharmacological or shRNA targeting of Rac1 inhibited the tumorigenic activities of both SP and non-SP NSCLA cells. These studies indicate that Rac1 represents a useful target in NSCLA, and its blockade may have therapeutic value in suppressing CSC proliferation and metastasis.

Topics: Adenocarcinoma; Animals; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Knockdown Techniques; Guanosine Triphosphate; Humans; Lung; Lung Neoplasms; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplastic Stem Cells; rac1 GTP-Binding Protein; RNA, Small Interfering; Side-Population Cells

Green tea polyphenols (GTP) have been reported to inhibit DNA methylation in cultured cells. Here, we tested whether oral consumption of GTPs affects normal or cancer-specific DNA methylation in vivo, using mice. Wild-type (WT) and transgenic adenocarcinoma of mouse prostate (TRAMP) mice were given 0.3% GTPs in drinking water beginning at 4 weeks of age. To monitor DNA methylation, we measured 5-methyl-deoxycytidine (5mdC) levels, methylation of the B1 repetitive element, and methylation of the Mage-a8 gene. Each of these parameters were unchanged in prostate, gut, and liver from WT mice at both 12 and 24 weeks of age, with the single exception of a decrease of 5mdC in the liver at 12 weeks. In GTP-treated TRAMP mice, 5mdC levels and the methylation status of four loci hypermethylated during tumor progression were unaltered in TRAMP prostates at 12 or 24 weeks. Quite surprisingly, GTP treatment did not inhibit tumor progression in TRAMP mice, although known pharmacodynamic markers of GTPs were altered in both WT and TRAMP prostates. We also administered 0.1%, 0.3%, or 0.6% GTPs to TRAMP mice for 12 weeks and measured 5mdC levels and methylation of B1 and Mage-a8 in prostate, gut, and liver tissues. No dose-dependent alterations in DNA methylation status were observed. Genome-wide DNA methylation profiling using the HpaII tiny fragment enrichment by ligation-mediated PCR assay also revealed no significant hypomethylating effect of GTP. These data indicate that oral administration of GTPs does not affect normal or cancer-specific DNA methylation in the murine prostate.

Topics: Adenocarcinoma; Animals; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Flavonoids; Guanosine Triphosphate; Immunoenzyme Techniques; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phenols; Polyphenols; Prostate; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tea; Tissue Distribution

Animal, epidemiological and clinical studies have demonstrated the anti-tumor activity of pharmacological proteasome inhibitors and the cancer-preventive effects of green tea consumption. Previously, one of our laboratories reported that natural ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG] and (-)-gallocatechin-3-gallate [(-)-GCG], are potent and specific proteasome inhibitors. Another of our groups, for the first time, was able to enantioselectively synthesize (-)-EGCG as well as other analogs of this natural GTP. Our interest in designing and developing novel synthetic GTPs as proteasome inhibitors and potential cancer-preventive agents prompted our current study.. GTP analogs, (+)-EGCG, (+)-GCG, and a fully benzyl-protected (+)-EGCG [Bn-(+)-EGCG], were prepared by enantioselective synthesis. Inhibition of the proteasome or calpain (as a control) activities under cell-free conditions were measured by fluorogenic substrate assay. Inhibition of intact tumor cell proteasome activity was measured by accumulation of some proteasome target proteins (p27, I kappa B-alpha and Bax) using Western blot analysis. Inhibition of tumor cell proliferation and induction of apoptosis by synthetic GTPs were determined by G(1) arrest and caspase activation, respectively. Finally, inhibition of the transforming activity of human prostate cancer cells by synthetic GTPs was measured by a colony formation assay.. (+)-EGCG and (+)-GCG potently and specifically inhibit the chymotrypsin-like activity of purified 20S proteasome and the 26S proteasome in tumor cell lysates, while Bn-(+)-EGCG does not. Treatment of leukemic Jurkat T or prostate cancer LNCaP cells with either (+)-EGCG or (+)-GCG accumulated p27 and IkappaB-alpha proteins, associated with an increased G(1) population. (+)-EGCG treatment also accumulated the pro-apoptotic Bax protein and induced apoptosis in LNCaP cells expressing high basal levels of Bax, but not prostate cancer DU-145 cells with low Bax expression. Finally, synthetic GTPs significantly inhibited colony formation by LNCaP cancer cells.. Enantiomeric analogs of natural GTPs, (+)-EGCG and (+)-GCG, are able to potently and specifically inhibit the proteasome both, in vitro and in vivo, while protection of the hydroxyl groups on (+)-EGCG renders the compound completely inactive.

Topics: Adenocarcinoma; Apoptosis; bcl-2-Associated X Protein; Calpain; Carcinoma; Carrier Proteins; Caspases; Cell Line; Cysteine Endopeptidases; G1 Phase; Guanosine Triphosphate; Humans; Intracellular Signaling Peptides and Proteins; Jurkat Cells; Male; Microfilament Proteins; Multienzyme Complexes; Muscle Proteins; Phenols; Polymers; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Stereoisomerism; Tea; Transcriptional Elongation Factors

Prostate cancer cells derived from transgenic mice with adenocarcinoma of the prostate (TRAMP cells) were treated with the HMG-CoA reductase inhibitor, lovastatin. This caused inactivation of the small GTPase RhoA, actin stress fiber disassembly, cell rounding, growth arrest in the G1 phase of the cell cycle, cell detachment and apoptosis. Addition of geranylgeraniol (GGOL) in the presence of lovastatin, to stimulate protein geranylgeranylation, prevented lovastatin's effects. That is, RhoA was activated, actin stress fibers were assembled, the cells assumed a flat morphology and cell growth resumed. The following observations support an essential role for RhoA in TRAMP cell growth: (1) TRAMP cells expressing dominant-negative RhoA (T19N) mutant protein displayed few actin stress fibers and grew at a slower rate than controls (35 h doubling time for cells expressing RhoA (T19N) vs 20 h for untransfected cells); (2) TRAMP cells expressing constitutively active RhoA (Q63L) mutant protein displayed a contractile phenotype and grew faster than controls (13 h doubling time). Interestingly, addition of farnesol (FOL) with lovastatin, to stimulate protein farnesylation, prevented lovastatin-induced cell rounding, cell detachment and apoptosis, and stimulated cell spreading to a spindle shaped morphology. However, RhoA remained inactive and growth arrest persisted. The morphological effects of FOL addition were prevented in TRAMP cells expressing dominant-negative H-Ras (T17N) mutant protein. Thus, it appears that H-Ras is capable of inducing cell spreading, but incapable of supporting cell proliferation, in the absence of geranylgeranylated proteins like RhoA.

Topics: Actin Cytoskeleton; Adenocarcinoma; Alkyl and Aryl Transferases; Animals; Antineoplastic Agents; Apoptosis; Cell Adhesion; Cell Division; Cell Size; Diterpenes; Drug Interactions; Enzyme Activation; Farnesol; G1 Phase; Genes, ras; GTP-Binding Proteins; Guanosine Triphosphate; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Male; Mevalonic Acid; Mice; Mice, Transgenic; Polyisoprenyl Phosphates; Prostatic Neoplasms; Protein Prenylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins p21(ras); rac GTP-Binding Proteins; rhoA GTP-Binding Protein; Sesquiterpenes; Tumor Cells, Cultured

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

The effects of 7-hydroxycoumarin, genistein and quercetin on two ras-oncogene-driven tumour cells (rat breast adenocarcinoma and human bladder carcinoma) were investigated using cellular (proliferation and migration) and molecular targets (p21ras GTPase activity and intracellular amount of p21ras protein). All three compounds inhibited the growth of both cell lines. Genistein was the most effective substance. Furthermore, 7-hydroxycoumarin and genistein affected the motile machinery of both cell lines because major fractions of the cells were slowed down or stopped locomotion. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), a well-known tumour promoter, increased the locomotion behaviour of the cells; the time of migration, the velocity and the distance of migration increased under the control of PMA. 7-Hydroxycoumarin decreased the relative amount of intracellular p21ras, and concomitantly a PMA-induced decrease of p21ras GTPase activity could be partially antagonized by 7-hydroxycoumarin. Because of the low toxicity and the mode of action evaluated, it is likely that the best role for these substances may be adjuvant therapy of some malignancies following surgery. Profiles directed to migration and proliferation inhibition make these drugs exceptional candidates for chemopreventive strategies in tumours diagnosed as having increased ras oncogene levels.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Cell Division; Cell Movement; Collagen; Culture Media; Genes, ras; Genistein; Guanosine Triphosphate; Humans; Indicators and Reagents; Isoflavones; Mammary Neoplasms, Experimental; Oxidation-Reduction; Proto-Oncogene Proteins p21(ras); Quercetin; Rats; Tetrazolium Salts; Tumor Cells, Cultured; Umbelliferones; Urinary Bladder Neoplasms

Mycophenolic acid (MPA), a potent and specific inhibitor of IMP dehydrogenase, exerts its anti-mitotic action by a rapid depletion of the cellular content of guanine nucleotides. Although MPA is a potent inhibitor of GTP synthesis in the HT29 line of human colorectal adenocarcinoma cells in short-term culture, its ability to depress the cloning efficiency of these cells was found to be markedly less than against the mouse mammary carcinoma line, EMT6. In vivo, MPA is efficiently converted to the biologically inactive O-glucuronide derivative thereby limiting its effectiveness as an anti-tumor agent. Investigation of the fate of MPA incubated with monolayer cultures of HT29 and EMT6 cells revealed that the compound is rapidly converted to the O-glucuronide derivative by HT29 cells, but not by EMT6 cells. Confirmation of the identity of the glucuronide formed by HT29 cells was obtained by its conversion to MPA after incubation with beta-glucuronidase and by comparison of the mass spectrum of its HPLC peak with that of synthetic MPA O-glucuronide. Cultures of two other lines of human colorectal adenocarcinoma cells, Colo-205 and LoVo, also depleted their culture media of MPA although we have not yet established whether these cells also synthesize the glucuronide. The intrinsic partial resistance of HT29 cells to MPA appears to be associated with the ability of these cells to convert MPA to the biologically inactive glucuronide. These results, in conjunction with other reports of the capacity of colorectal cancer cells for Phase I and II metabolism of xenobiotics, may have implications for the design of drugs intended for the treatment of colorectal cancer.

Topics: Adenocarcinoma; Animals; Antibiotics, Antineoplastic; Cell Division; Colorectal Neoplasms; Drug Resistance, Neoplasm; Female; Glucuronates; Glucuronosyltransferase; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Kinetics; Mammary Neoplasms, Experimental; Mice; Mycophenolic Acid; Tumor Cells, Cultured

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

Cancer cells have an increased ability to synthesize GTP (guanosine triphosphate) because of increased activity of IMP DH (inosine 5'-phosphate dehydrogenase, EC 1.1.1.205). Because IMP DH activity is rate limiting for de novo biosynthesis of GTP, this enzyme was suggested as a sensitive target for chemotherapy. Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) is converted in the cells into the active metabolite, TAD, (thiazole-4-carboxamide adenine dinucleotide) which potently inhibits IMP DH activity. By adding TAD to tissue extracts one can determine the extent of inhibition of IMP DH. We applied the IMP DH assay method to extracts of normal ovaries (N = 11) and epithelial ovarian carcinomas (N = 10). The IMP DH activity (mean +/- SE) in ovarian carcinoma was 21.1 +/- 5.8 which was markedly higher than that observed in normal ovaries (2.9 +/- 0.7 nmol/hr/mg protein) (P < 0.05%). The inhibition by TAD of IMP DH activity in ovarian carcinomas (N = 4) was 81%. The results indicate that IMP DH activity is elevated sevenfold in ovarian carcinomas as compared to normal ovary and can be inhibited by exposure to tiazofurin (TAD). Similar high IMP DH activity and inhibition of the activity by TAD was observed in patients with chronic granulocytic leukemia in blast crisis among whom 70 to 80% remissions were reported. Since there is increased IMP DH activity in human ovarian carcinomas and in OVCAR-5 cells and tiazofurin and TAD inhibit IMP DH activity of these cells and the proliferation of human ovarian carcinoma xenografts in the mouse, tiazofurin may merit serious consideration for a Phase II trial for patients with recurrent/refractory epithelial ovarian carcinoma.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Female; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Middle Aged; Ovarian Neoplasms; Ovary; Ribavirin; Tissue Extracts

Ornithine decarboxylase (ODC) activity was measured in colon adenocarcinomas and adjacent normal-appearing colon mucosa from a total of 40 patients undergoing surgical resections. The enzyme activity was measured in the presence and absence of GTP, since recent work has demonstrated a GTP-activatable form of ODC in some murine and human tumors. In general, ODC specific activity was higher in adenocarcinomas than in adjacent normal-appearing mucosa. Of greater interest, however, was the finding that 13 of 40 tumors and 3 of 40 mucosae contained a GTP-activatable form of ODC. These are minimal estimates of the proportion of tissues positive for this enzyme form, since a multiple sampling protocol indicated that expression of a GTP-activatable ODC was not uniform throughout a given tumor. Chromatographic analyses of tumor extracts revealed the presence in some tumors of multiple size forms of ODC, only some of which were activated by GTP. Enzyme kinetic data indicated that the multiple forms of ODC can have different affinities for L-ornithine and that GTP can "normalize" the aberrant kinetic properties of these forms. While there was no statistically significant correlation of the presence of a GTP-activatable ODC with stage of disease, analysis of our data revealed a positive association of a GTP-activatable ODC with tumor site; a much higher percentage of tumors of the cecum contained this ODC isoform than tumors of other colonic segments (64% versus less than or equal to 25% for other sites). These results demonstrate (a) the presence of a functionally distinct form of ODC in some human colon adenocarcinomas and (b) a distinct regional distribution of this ODC form within the colon. We suggest this alteration in a key enzyme in the growth-associated pathway of polyamine biosynthesis may play a role in colon tumor progression.

Topics: Adenocarcinoma; Colorectal Neoplasms; Enzyme Activation; Guanosine Triphosphate; Humans; Intestinal Mucosa; Kinetics; Ornithine Decarboxylase

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

The addition of molybdate to intact or homogenized cells of the endometrial adenocarcinoma line HEC-1 during incubation with [3H]estradiol ([3H]E2) at 4 C causes substantial increases in cytoplasmic E2 binding levels. A similar effect can be observed in homogenates of normal human endometrium. These effects of molybdate appear to involve activation of E2-binding sites. Fractionation of the homogenates and recombination of different fractions revealed that activation of specific E2-binding sites by by MoO4= requires cytosolic factors as well as factors associated with the cell membrane. In homogenates of neoplastic cells (HEC-1) and normal endometrium, the addition of ATP, GTP, or cGMP was also found to increase E2 binding to levels as high as those obtained by the addition of MoO4=. In contrast, the addition of cAMP was found to lower specific E2 binding levels and to counteract the effects of MoO4=, ATP, GTP, and cGMP. Levels of intracellular cAMP and cGMP can change rapidly in cells in culture. Since cGMP causes E2 binding levels to increase while cAMP causes them to decrease, changes in the levels of these two cyclic nucleotides may explain the fluctuation in concentrations of specific estrogen binders that we have previously reported to occur in cultured endometrial cells.

Topics: Adenocarcinoma; Adenosine Triphosphate; Cell Line; Cell Membrane; Cyclic AMP; Cyclic GMP; Cytosol; Endometrium; Estradiol; Female; Guanosine Triphosphate; Humans; Molybdenum; Uterine Neoplasms