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T Clair

Publications and source records attributed to T Clair.

At least 55 records · Page 3Linked to original sources
PubMed1989

Site-selective cyclic AMP analogs as new biological tools in growth control, differentiation, and proto-oncogene regulation.

The physiologic role of cyclic adenosine monophosphate (cAMP) in the growth control of a spectrum of human cancer lines, including leukemic lines, and v-rasH oncogene-transformed NIH/3T3 cells is demonstrated by the use of site-selective cAMP analogs. These cAMP analogs, which can select either of the two known cAMP binding sites of the cAMP receptor protein, induce potent growth inhibition, phenotypic change, and differentiation (leukemic cells) of cancer cells at micromolar concentrations with no sign of cytotoxicity. The growth inhibition parallels selective modulation of cAMP-dependent protein kinase isozymes, type I versus type II, and suppression of cellular proto-oncogene expression. Site-selective cAMP analogs thus provide new biological tools for investigating cell proliferation and differentiation and also for the improved management of human cancers.

Animals↗
PubMed1988

Synergistic inhibition of growth of breast and colon human cancer cell lines by site-selective cyclic AMP analogues.

Our past studies on the mechanism of cyclic AMP (cAMP)-mediated control of tumor growth, using the experimental rat mammary tumor models as well as human breast cancer cell lines, indicated that the action of cAMP is mediated by the RII cAMP receptor protein, the regulatory subunit of cAMP-dependent protein kinase type II (Y. S. Cho-Chung, J. Cyclic Nucleotide Res., 6: 163, 1980). We now shown that the site-selective cAMP analogues, which are manyfold more active in binding to the cAMP receptor protein than previously studied analogues, demonstrate a potent growth inhibition of seven breast and three colon human cancer cell lines. The cAMP receptor protein has two different cAMP binding sites, and cAMP analogues that selectively bind to either one of the two binding sites are known as either site 1 selective (C-8 analogues) or site 2 selective (C-6 analogues). Nineteen site-selective analogues, C-6 and C-8 monosubstituted and C-6,-8 disubstituted, were tested for their growth regulatory effect. The majority of these analogues demonstrated an appreciable growth inhibition, with no sign of toxicity in all 10 cancer lines at micromolar concentrations. The three most potent inhibitors were 8-Cl-, N6-benzyl-, and N6-phenyl-8-thio-p-chlorophenyl-cAMP, demonstrating 50% growth inhibition at 5-25 microM concentrations (IC50). Furthermore, N6-analogues, in combination with halogen or thio derivatives of C-8 analogues, demonstrated synergistic enhancement of growth inhibition. The growth inhibition paralleled a change in cell morphology, an augmentation of the RII cAMP receptor protein, and a reduction in p21 ras protein. The growth inhibition by 8-Cl-cAMP was not due to its metabolite, 8-Cl-adenosine, since: (a) the growth inhibition by 8-Cl-cAMP was released upon cessation of treatment, whereas that by 8-Cl-adenosine was not released; (b) 8-Cl-cAMP treatment did not affect cell cycle progression, whereas 8-Cl-adenosine brought about G1 synchronization; (c) 8-Cl-cAMP treatment caused reduction of p21 ras protein, whereas 8-Cl-adenosine did not affect p21 levels; and (d) 8-Cl-adenosine was not detected in either cell extracts or medium from the cells treated with 8-Cl-cAMP for 48-72 h. Site-selective cAMP analogues thus provide a new physiological means to control the growth of breast and colon human cancer cells.

Breast Neoplasms↗
PubMed1988

Reverse transformation of Harvey murine sarcoma virus-transformed NIH/3T3 cells by site-selective cyclic AMP analogs.

Eighteen site-selective cAMP analogs modified at either the C-8 position or the C-6 position were tested for their growth regulatory effects on the Harvey murine sarcoma virus-transformed NIH/3T3 clone 13-3B-4 cells grown in a serum-free defined medium. All 18 analogs, when tested individually, exhibited an appreciable growth inhibitory effect at micromolar concentrations. The most potent growth inhibitory analogs contained a thio moiety at the C-8 position. In general, C-6 analogs required 5-10-fold greater concentrations than C-8 analogs to produce the same degree of growth inhibition. The growth inhibition induced by these analogs was accompanied by a change in cell morphology; cells treated with the analogs exhibited the morphology characteristic of untransformed fibroblasts, while untreated cells retained a transformed phenotype. The regulatory subunit of cAMP-dependent protein kinase, the cAMP receptor protein, has two different intrachain cAMP binding sites, and cAMP analogs modified at the C-8 position (C-8 analogs) are generally selective for Site 1, while analogs modified at the C-6 position (C-6 analogs) are generally selective for Site 2. Thus, C-8 and C-6 analogs were tested in combination to enhance the growth regulatory effect. Both growth inhibition and morphological change were enhanced synergistically by a combination of the C-6 and C-8 analogs. Two C-6 analogs or two C-8 analogs added together did not cause synergism. For both growth inhibition and phenotypic change, C-8 thio analogs acted far more synergistically than C-8 amino analogs when cells were treated in combination with C-6 analogs, suggesting a response of the RII rather than the RI cAMP receptor protein. DEAE-cellulose chromatography revealed that the growth inhibition, in fact, correlates with an increase of the RII cAMP receptor protein and a decrease of the RI receptor protein. The growth inhibitory effect of the site-selective analogs was not due to the cytotoxic effect of adenosine metabolites as shown by the different behavior of 8-Cl-cAMP compared with 8-Cl-adenosine in 1) cell cycle effects and 2) release from growth inhibition. It is concluded that the observed growth inhibition and phenotypic reversion of 13-3B-4 cells is most likely mediated through the cellular effector, the RII cAMP receptor protein.

Animals↗
PubMed1988

Selective modulation of protein kinase isozymes by the site-selective analog 8-chloroadenosine 3',5'-cyclic monophosphate provides a biological means for control of human colon cancer cell growth.

Differential expression of type I and type II cAMP-dependent protein kinase isozymes has been linked to growth regulation and differentiation. We examined the expression of protein kinase isozymes in the LS 174T human colon cancer cell line during 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cAMP)-induced growth inhibition. Two species of RII (the regulatory subunit of protein kinase type II) with apparent Mr 52,000 (RII52) and Mr 56,000 (RII56) and a single species of RI (the regulatory subunit of protein kinase type I) with Mr 48,000 were identified in the cancer cells. RI and both forms of RII were covalently labeled with 8-azidoadenosine 3',5'-cyclic [32P]monophosphate, and two anti-RII antibodies that exclusively recognize either RII52 or RII56 resolved two forms of the RII receptors. 8-Cl-cAMP treatment induced a decrease of RI and an increase of both RII52 and RII56 in the cytosols of cancer cells and rapid translocation (within 10 min) of RII52 from the cytosol to nucleus. 8-Cl-cAMP caused transcriptional activation of the RII52 receptor gene and inactivation of the RI receptor gene. It also exhibited high-affinity site-1-selective binding to the purified preparations of both RII receptor proteins. Thus, differential regulation of various forms of cAMP receptor proteins is involved in 8-Cl-cAMP-induced regulation of cancer cell growth, and nuclear translocation of RII52 receptor protein appears to be an early event in such differential regulation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Site-selective cAMP analogs at micromolar concentrations induce growth arrest and differentiation of acute promyelocytic, chronic myelocytic, and acute lymphocytic human leukemia cell lines.

Cyclic AMP (cAMP)-dependent protein kinase may play a role in the functional and morphological differentiation of leukemic cells. In this study, we showed that the cAMP analogs, potent activators of protein kinase recently shown to be selective for either site 1 or site 2 cAMP binding sites of protein kinase, demonstrate potent growth inhibition of acute promyelocytic, chronic myelocytic, and acute lymphocytic leukemic cell lines with no sign of toxicity. The growth inhibition accompanied monocytic differentiation in HL-60 cells and a loss of nuclear terminal deoxynucleotidyl transferase activity in Molt-4 leukemic cells. The growth inhibition also paralleled a decrease in c-myc protein and RI cAMP receptor protein. Thus, cAMP analogs selective for either site 1 or site 2 of the protein kinase appear to restore a coupling of proliferation and maturation in leukemic cells.

Cell Differentiation↗
PubMed1987

Site-selective cAMP analogs induce nuclear translocation of the RII cAMP receptor protein in Ha-MuSV-transformed NIH/3T3 cells.

Site-selective cAMP analogs, depending on the position of their substituents on the adenine ring, selectively bind to either site 1 or site 2 of the known cAMP binding sites of protein kinase. Treatment of Harvey murine sarcoma virus-transformed NIH/3T3 cells with such site-selective analogs results in growth inhibition and phenotypic reversion, and the combination of a C-8 thio or halogen analog (site 1 selective) with an N6 analog (site 2 selective) produces a synergistic effect. We report here that the growth inhibitory effect of the analogs correlates with the nuclear translocation of the RII cAMP receptor protein, the regulatory subunit of protein kinase type II. The transformed NIH/3T3 cells contained no detectable level of RII in the nucleus, whereas nontransformed NIH/3T3 cells exhibited a high level of nuclear RII. Within 30 min after treatment of the transformed cells with the site-selective analogs, immunofluorescence against the RII protein markedly increased in the cell nucleus. The nuclear translocation of the RII cAMP receptor protein is an early event in the reverse transformation of the fibroblasts treated with site-selective cAMP analogs.

Affinity Labels↗
PubMed1987

Site-selective cyclic AMP analogs provide a new approach in the control of cancer cell growth.

Site-selective cyclic AMP analogs bind to site 1 or site 2 of the known cAMP-binding sites depending on the position of substituents on the purine ring, either at C-2 and C-8 (site 1) or at C-6 (site 2). The growth inhibitory effect of such site-selective cAMP analogs used in this investigation with 15 human cancer cell lines surpassed that of analogs previously tested. The most potent analogs were 8-chloro, N6-benzyl and N6-phenyl-8-p-chlorophenylthio-cAMP. The combination of a C-8 with an N6 analog had synergistic effects. The 24 site-selective analogs tested produced growth inhibition ranging from 30 to 80% at micromolar concentrations with no sign of toxic effects. Growth inhibition was not due to a block in a specific phase of the cell cycle but paralleled a change in cell morphology, an increase of the RII cAMP receptor protein and a decrease of p21 ras protein. Since the adenosine counterpart of the 8-chloro analog produced G1 synchronization without affecting the RII and p21 ras protein levels, it is unlikely that an adenosine metabolite is involved in the analog effect. Site-selective cAMP analogs thus provide a new biological tool for control of cancer growth.

Breast Neoplasms↗
PubMed1987

Prognostic significance of the expression of a ras protein with a molecular weight of 21,000 by human breast cancer.

Expression of the cellular ras Mr 21,000 protein (p21) has been measured in tumors from breast cancer patients who at time of presentation had no evidence of metastatic disease. Western blotting analysis revealed that 37 of 54 (69%) tumors contained p21 levels 2- to 10-fold greater than those of control breast tissues. An excessive increase of p21 (5- to 10-fold over the control value) occurred more frequently in tumors of T3 and T4 stages [15 of 25 (60%)] than in tumors at T2 stage [6 of 29 (21%)], suggesting a correlation between advancement of disease and high p21 levels. p21 levels were positively related to the involvement of axillary lymph nodes at the time of primary treatment. As no correlations were detected between p21 levels and a gross pathological parameter, tumor grade, it is possible that p21 levels may reflect the degree of cellular malignancy. This is supported by data on tumor recurrence; 13 of 16 patients (81%) with tumors expressing low p21 levels were disease free for greater than or equal to 4 years after primary treatment, whereas only 5 of 9 patients (56%) with high p21 tumors remained disease free. These results suggested that a quantitative enhancement of p21 oncogene protein is associated with both the progression and prognosis of breast cancer.

Breast Neoplasms↗
PubMed1986

Cyclic AMP suppresses expression of v-rasH oncogene linked to the mouse mammary tumor virus promoter.

Clone 433.3 of NIH 3T3 cells is a stable carrier of the MMTV LTR:v-rasH chimeric DNA. Only in the presence of dexamethasone (a synthetic glucocorticoid), 433.3 cells exhibit an induced level of p21 transforming protein and phenotypic transformation. N6,O2'-dibutyryl cAMP (DBcAMP) antagonized the effect of dexamethasone in a time - and concentration - dependent manner. DBcAMP (5 X 10(-4)M) added 18 hr prior to the addition of dexamethasone (10(-7)M) almost completely blocked the hormone effect: cells contained levels of p21 20% of that in the cells treated with dexamethasone alone, and formed flat, contact inhibited monolayers. On the basis of these results together with our previous data on mammary carcinomas in vivo, we postulate that cAMP may be an intracellular suppressor acting at a regulatory locus of both cellular and viral ras genes.

Animals↗
PubMed1985

Two classes of cAMP analogs synergistically inhibit p21 ras protein synthesis and phenotypic transformation of NIH/3T3 cells transfected with Ha-MuSV DNA.

Factors that control cellular proliferation might do so by regulating quantitative expression of viral or cellular oncogenes. Since the growth regulatory effect of cAMP is well-known, the effect of cAMP on ras gene expression was examined on Ha-MuSV-transformed 13-3B-4 cells (NIH-3T3) grown in chemically defined serum-free medium. Treatment of cells with two classes of cAMP analogs which are selective for the two different cAMP-binding sites of type II protein kinase, in combination, synergistically inhibited both p21 ras protein synthesis and phenotypic transformation. The inhibition was also demonstrated with these analogs singly but at higher concentrations. The decrease in p21 synthesis was inversely correlated with an increase in the RII cAMP receptor protein, the regulatory subunit of type II protein kinase. These results suggest a role for cAMP and its receptor protein in the regulation of v-rasH oncogene expression.

Animals↗
PubMed1983

Arrest of hormone-dependent mammary cancer growth in vivo and in vitro by cholera toxin.

Growth of 7,12-dimethylbenz(alpha)anthracene-induced mammary carcinoma in rat was arrested by daily s.c. injections of cholera toxin. At a dose of 2 micrograms/200-g rat, tumors regressed to 50% of their initial size within 2 weeks, and 85% of tumors regressed completely within 4 to 5 weeks. The same response to cholera toxin was observed with another hormone-dependent mammary tumor, MTW9, but not with the hormone-independent tumors, DMBA No. 1 and MT 13762. The latter result was consistent with the lack of response of these hormone-independent tumors to hormone removal (ovariectomy) or N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate treatment. The growth-inhibitory effect of cholera toxin was dose dependent, and upon cessation of treatment tumors resumed growth; after complete regression, however, tumors did not reappear until 6 months after termination of the treatment. An amount of cholera toxin as high as 10 micrograms/day/200-g rat s.c. injected over a 6-week period showed no systemic toxicity in the animals. The growth of human breast cancer cells (MCF-7) in culture was also inhibited by a daily supplement of cholera toxin. At a concentration of 100 ng/ml, the cell replication ceased completely within 2 days. The growth inhibitions, both in vivo and in vitro, were accompanied by marked increases in the cellular cyclic adenosine 3':5'-monophosphate content and type II cyclic adenosine 3':5'-monophosphate-dependent protein kinase activity as well as a decrease of estrogen binding activity. Thus, extinction of mammary cancer can be achieved by cholera toxin, an agent that stimulates the intracellular cyclic adenosine 3':5'-monophosphate system.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1983

Anticarcinogenic effect of N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate on 7,12-dimethylbenz(a)anthracene mammary tumor induction in the rat and its relationship to cyclic adenosine 3':5'-monophosphate metabolism and protein kinase.

A single intubation of 7,12-dimethylbenz(a)anthracene (DMBA) (20 mg in 1 ml sesame oil) to female Sprague-Dawley rats at 50 days of age produces primary mammary carcinomas in 80% of rats at 100 to 150 days of age. Administration of N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (DBcAMP) p.o. beginning at 1 day prior to DMBA intubation resulted in marked delay and reduction of tumor production: only 15% as many DBcAMP-treated rats had tumors as in the control group (DMBA only) with 60 days of delay in the first tumor appearance. DMBA-induced tumor production was preceded by changes in the cyclic adenosine 3':5'-monophosphate (cAMP) metabolism and protein kinase of the mammary gland. Within 24 hr post-DMBA intubation, the intracellular cAMP level and adenylate cyclase activity increased with an increase in type I isozyme of cAMP-dependent protein kinase, a form which has been associated with increased proliferative activity and a less differentiated cellular state in other tissues. The increases in cAMP level, adenylate cyclase activity, and the protein kinase activity were transient, and the values decreased to below the control values by Day 10 post-DMBA intubation. In mammary glands of rats that had received DBcAMP, the cAMP level and protein kinase isozyme pattern were similar to those of older rats that are no longer susceptible to the carcinogen. The inhibitory effect on DMBA-induced carcinogenesis may be related to the modifications that DBcAMP induces on cAMP level, adenylate cyclase activity, and cAMP-dependent protein kinase of the mammary gland.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1981

Growth arrest and morphological change of human breast cancer cells by dibutyryl cyclic AMP and L-arginine.

The growth in vitro of human breast cancer cells, line MCF-7, was inhibited by a daily supplement of L-arginine (1 milligram per milliliter). Arginine acted synergistically with dibutyryl adenosine 3',5'-monophosphate (cyclic AMP) (10(-6) molar) to enhance the growth inhibitory effect: the cell replication ceased completely within 2 days after treatment. The growth arrest accompanied a change in cell morphology and was preceded by increases in the cellular concentration of cyclic AMP, adenylate cyclase, and type II cyclic AMP-dependent protein kinase activities as well as a decrease of estrogen binding activity. The results suggest that growth of human breast cancer cells is subject to cyclic AMP-mediated regulation and that arginine may play a specific role in this process.

Arginine↗
PubMed1981

Cyclic adenosine 3':5-monophosphate receptor proteins in hormone-dependent and -independent rat mammary tumors.

The molecular species of cyclic adenosine 3':5'-monophosphate (cAMP) receptor proteins (high-affinity-binding proteins) present in hormone-dependent and -independent rat mammary carcinomas were identified and characterized. Three major types of cAMP receptor proteins, with molecular weights of 39,000, 48,000, and 56,000, specifically incorporated the photoaffinity label, 8-azido-cyclic adenosine 3':5'-[32P]monophosphate and were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the tumor cytosols. The M.W. 48,000 and 56,000 receptor proteins appeared to be the regulatory subunits of cAMP-dependent protein kinase types I and II, respectively, and the M.W. 39,000 receptor protein was the proteolytic fragment of the M.W. 56,000 receptor protein. The relative amounts of these cAMP receptor proteins varied from one tumor type to another and showed no correlation with respect to the hormone dependency of tumors. Under two-dimensional gel electrophoresis, however, the M.W. 56,000 receptor protein from hormone-dependent tumors migrated as a doublet and shifted to either a more acidic or more basic charge than that of the receptor protein of hormone-dependent tumors. The alteration of the charge of the receptor did not affect the affinity for cAMP binding, because both hormone-dependent and hormone-independent tumor cytosols exhibited the dissociation constant for cAMP of approximately 10(-8) M. The M.W. 56,000 cAMP receptor protein from hormone-dependent tumors exhibited self-phosphorylation, but that from hormone-independent tumors did not. The diethylaminoethyl cellulose elution profiles of cAMP receptor proteins also differed between hormone-dependent and -independent tumors; cAMP binding activity from hormone-dependent tumors coeluted with cAMP-dependent protein kinase activity, whereas most of the cAMP binding activity from hormone-independent tumors eluted at a higher ionic strength than did cAMP-dependent protein kinase activity. These results suggest that the charge alteration of cAMP receptor proteins, which appears to occur at a site remote from that of cAMP binding, may be associated with the hormone independency of mammary tumors.

Affinity Labels↗
PubMed1980

Relationship of hormone dependency to estrogen receptor and adenosine 3',5'-cyclic monophosphate-binding proteins in rat mammary tumors.

Estrogen-binding activity and the binding activity of adenosine 3',5'-cyclic monophosphate (cAMP) were assayed in the cytosol of biopsy specimens from 70 rat mammary tumors. The response of these tumors to host ovariectomy was followed. The hormone dependency of the tumors was closely related to the relative concentrations of estrogen receptor (ER) and cAMP-binding protein (CR) in the tumor cytosol. When ER and CR were expressed as the ER/CR ratio, 36 of 38 (95%) hormone-dependent tumors had ratios of 35 X 10(-3) or more, whereas 31 of 32 (97%) hormone-independent tumors had ratios of less than 35 X 10(-3). When ER alone was measured, hormone dependency could be correctly predicted in only 60% of the tumors because the ER range for individual tumors of both types greatly overlapped. Ratios of ER/CR were also elevated in normal estrogen target tissues as compared to those in normal estrogen nontarget tissues. Thus determination of the ER/CR ratio in tumor cytosols appeared to be a more reliable method of predicting hormone dependency than was determination of ER alone.

Animals↗
PubMed1979

Cyclic AMP receptor triggers nuclear protein phosphorylation in a hormone-dependent mammary tumor cell-free system.

Adenosine 3',5'-monophosphate (cyclic AMP) receptor protein of 56,000 daltons increases markedly in mammary tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) after incubation of tumor slices with cyclic AMP, benzamide, and arginine. Incubation of cytosol from these tumor slices with nuclei from unincubated tumors results in nuclear uptake of the 56,000-dalton cyclic AMP receptor and in phosphorylation of the 76,000-dalton nuclear protein. Binding of the 56,000-dalton receptor and phosphorylation of the 76,000-dalton protein also occur in DMBA tumor nuclei when protein kinase type II of bovine heart is used. The results suggest that cyclic AMP receptor is involved in the nuclear events of a hormone-dependent mammary tumor.

9,10-Dimethyl-1,2-benzanthracene↗