Calcitonin stimulation of cyclic adenosine 3':5'-monophosphate production with growth inhibition in human renal adenocarcinoma cell lines

Responsiveness of cyclic adenosine 3':5'-monophosphate (cAMP) to parathyroid hormone, calcitonin, and vasopressin was studied in six human renal adenocarcinoma cell lines. Four of six renal adenocarcinoma cell lines showed increased cAMP content in response to calcitonin while the other two did not. Neither parathyroid hormone nor vasopressin increased the concentration of cAMP in each of these cell lines. The growth rate of KU-2 cells, which responded to calcitonin with an increase of cAMP content, was inhibited by calcitonin. On the other hand the growth rate of calcitonin-nonsensitive KH-39 cells was unaltered. The growth inhibitory effect of the hormone on KU-2 cells could be considered to be mediated by the increased cAMP levels from the following results: (a) there was positive correlation between the cellular cAMP content and growth inhibition after various amounts of calcitonin addition; (b) KU-2 growth was also suppressed by N6,O2'-dibutyryl cAMP; and (c) a group of KU-2 cells which had become resistant to calcitonin-induced growth inhibition showed a diminished cAMP increase in response to calcitonin.     

Cyclic adenosine 3',5'-monophosphate binding proteins in human colorectal cancer and mucosa.

Cyclic AMP Binding Proteins (cAMP-BP) levels have been measured by means of a competitive binding assay in the cytosols of 50 human colorectal cancers. These levels have been related to those in mucosa both adjacent to and distant from the tumour in the same patients. Cyclic AMP-BP were higher in tumour than in either adjacent (P less than 0.000001) or distant mucosa (P less than 0.00001). Binding of cAMP in adjacent mucosa was lower than that in distant mucosa (P less than 0.0001). There was no significant difference in the level of binding between tumours arising from different sites in the colon and binding was not related to age or sex of the patient. However, binding was higher in Dukes' B than Dukes' C cancers (P less than 0.005). There was also a trend for cAMP binding levels to be higher in moderately differentiated than in poorly differentiated cancers (P = 0.07). Thus cAMP-BP appear to be over-expressed in human colorectal cancers and levels are related to the stage and grade.     

Cholecystokinin inhibition of tumor growth and gastrin-stimulated cyclic adenosine 3':5'-monophosphate metabolism in human gastric carcinoma in nude mice

This study deals with the effect of four types of COOH-terminal cholecystokinin (CCK) fragments on the growth of xenotransplantable human gastric cancer (SC-6-JCK, a poorly differentiated adenocarcinoma) whose growth has been promoted by pentagastrin. The growth of the tumor was inhibited using daily s.c. injections of CCK-octapeptide (CCK-8) and glutaryl-CCK-8 at a dose of 500 micrograms/kg body weight. After 30 days of treatment with CCK-8 or glutaryl-CCK-8, a significant decrease was observed in the tumor weight (P less than 0.05) and the tumor size P less than 0.01) in comparison with those of the control. But treatment with CCK-12 and pyroglutamyl-CCK-8 did not produce inhibition of tumor growth. Furthermore the correlation between the effect of CCK-8 on the normal rise in tumor cyclic adenosine 3':5'-monophosphate (cAMP) levels caused by pentagastrin injection and tumor growth was studied. The increase of cAMP by a single i.p. injection of pentagastrin at a dose of 20 micrograms/mouse was significantly inhibited by pretreatment with CCK-8 at concentrations equimolar to pentagastrin (P less than 0.05), while cAMP in the tumor was slightly elevated by a single i.p. injection of CCK-8 alone. Also in the in vitro study, CCK-8 inhibited the increase of cAMP and the activation of cAMP-dependent protein kinase which was stimulated by pentagastrin. These results suggest that proliferation of gastrin-dependent human gastric cancers may be suppressed by CCK in competition with gastrin.     

Cyclic adenosine 3':5'-monophosphate phosphodiesterase activity in malignant and cyclic adenosine 3':5'-monophosphate-induced "differentiated" neuroblastoma cells.

The regulation of cyclic adenosine 3:5-monophosphate (cyclic AMP) phosphodiesterase activity in homogenates of malignant and cyclic AMP-induced "differentiated" neuroblastoma cells was studied. Neuroblastoma cells of at least three mouse and one human clone had both the low (2 to 4 muM) and the high (66 to 106 muM) Km phosphodiesterase. In cyclic AMP-induced differentiated cells the values of Km were decreased, whereas the values of Vmax appeared to be slightly increased. Magnesium and manganese stimulated phosphodiesterase activity. Calcium, zinc, copper, mercury, ethylenediaminetetraacetic acid, and imidazole completely inhibited phosphodiesterase activity in malignant cells, whereas the above agents, except ethylenediaminetetraacetic acid, only partially inhibited enzyme activity in differentiated cells. Ethylenediaminetetraacetic acid completely reduced phosphodiesterase activity in differentiated cells. The pH optimum for phosphodiesterase activity was about 8 in both malignant and differentiated cells. The present studies show that the values of Km and Vmax and the sensitivity of phosphodiesterase activity to divalent ions change in cyclic AMP-induced differentiated neuroblastoma cells, and therefore we propose that the reverse may be true during malignant transformation of nerve cells.     

Cyclic adenosine 3':5'-monophosphate and methotrexate transport in L1210 cells.

Treatment of L1210 cells with glucose, 1-methyl-3-isobutylxanthine, theophylline, and prostaglandins E1 and E2 was used to increase to varying degrees the endogenous level of cyclic adenosine 3':5'-monophosphate (cAMP). In an inverse manner these agents decreased the rate of methotrexate (MTX) transport into the cells. Azide, conversely, lowered the cAMP level and enhanced MTX transport. Quantitative information about this relationship was obtained by plotting the reciprocal rates of MTX transport as a function of cAMP levels. These data suggest that MTX transport is regulated by the level of cAMP.     

Effects of cyclic adenosine 3':5'-monophosphate upon glycoprotein and carcinoembryonic antigen synthesis and release by human colon cancer cells

The role of cyclic adenosine 3':5'-monophosphate (cAMP) in the regulation of the synthesis and release of glycoproteins and of carcinoembryonic antigen by colon cancer cells was studied using LS174T cells in vitro. Adenylate cyclase and cAMP phosphodiesterase activities were assessed by measuring cellular cAMP in response to forskolin and cholera toxin (adenylate cyclase activators) and to 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor). Each agent increased cAMP levels significantly. Dibutyryl-cAMP (1 mM) stimulated glycoprotein synthesis and release when [3H]fucose was used as a precursor. The synthesis and release of carcinoembryonic antigen, a membrane-associated glycoprotein antigen, was also significantly increased by these test agents. A close dose-response relationship existed for forskolin and for cholera toxin between cAMP generation and carcinoembryonic antigen release. cAMP may play a role in regulating the synthesis and release of glycoprotein antigens by colon cancer cells.     

Growth inhibition of human glioma cells induced by 8-chloroadenosine, an active metabolite of 8-chloro cyclic adenosine 3':5'-monophosphate.

8-Chloroadenosine 3':5'-monophosphate (8ClcAMP) inhibits the growth of human glioma cell lines at much lower concentrations than more commonly used cyclic AMP analogues, without inducing morphological differentiation. The mechanism by which 8ClcAMP exerts this effect is not fully understood. We examined whether the growth-inhibitory effect of this compound is due to an active metabolite, using a sulforhodamine protein stain assay to determine the proliferation rate of the WF human glioma cell line. 8-Chloroadenosine, one of the metabolites, inhibited the proliferation of WF human glioma cells more potently than 8ClcAMP. In the presence of adenosine deaminase, which converts 8-chloroadenosine into 8-chloroinosine, 8-chloroadenosine no longer inhibited human glioma cell growth. Addition of adenosine deaminase also largely reduced the growth-inhibitory effect of 8ClcAMP, but not of 8-(4-chlorophenylthio)cAMP. High performance liquid chromatography analysis revealed that at least part of the 8ClcAMP in the culture medium is converted into 8-chloroadenosine. We concluded that 8ClcAMP exerts its growth-inhibitory effect through its active metabolite 8-chloroadenosine. Adenylate cyclase assays showed that 8-chloroadenosine does not affect the intracellular cAMP production through adenosine A1 or A2 receptor activation, which makes it unlikely that 8-chloroadenosine inhibits glioma cell growth by increasing the intracellular cyclic AMP concentration.     

Urinary excretion of cyclic guanosine 3':5'-monophosphate and cyclic adenosine 3':5'-monophosphate in rats bearing transplantable liver and kidney tumors.

Cyclic guanosine 3':5'-monophosphate (cyclic GMP) and cyclic adenosine 3':5-monophosphate were measured in the urine of normal rats and those bearing transplantable liver and kidney tumors. The level of cyclic GMP ranged from 1.4 to 1.6 mumoles/g urinary creatinine in several strains of rats without tumors. Rats bearing Morris hepatomas 20, 21, 9618A and 9633F and kidney tumor MK2 had urine levels of cyclic GMP from 1.3 to 3.6 mumoles/g creatinine. Rats bearing the fast growing hepatomas 9618A2 and 3924A and Morris kidney tumor MK3 had urinary values of 5.6. 41.9, and 32.7 mumoles cyclic GMP per g creatinine, respectively. Urine levels of cyclic adenosine 3':5'-monophosphate ranged from 10.1 to 19.7 mumoles/g creatinine in all normal and tumor-bearing rats and were not significantly different in any of the groups examined.     

Differential growth inhibition in two human carcinoma cell lines by cyclic adenosine 5'-monophosphate analogs.

The inhibition of cell replication in two human carcinoma cell lines by various cyclic AMP analogs was explored. In all instances, the addition of the cyclic nucleotide phosphodiesterase inhibitor 1-methyl-3-isoburylxanthine resulted in synergistic growth inhibition by the analogs. A correlation was found between an analog's ability to inhibit growth and its ability to activate protein kinase. A differential effect of the breakdown product 8-bromo-AMP (8-BrAMP) on cell replication in the two cell lines was observed; i.e., one cell type was extremely sensitive to inhibition by 8-BrAMP and the growth inhibition could not be reversed by uridine, whereas the other cell type was less sensitive to 8-BrAMP and the growth inhibition was significantly reversed by uridine.     

Changes in lymphoid cyclic adenosine 3':5'-monophosphate metabolism during murine leukemogenesis.

Cyclic adenosine 3':5'-monophosphate (cyclic AMP) levels were slightly increased in preleukemic AKR mouse thymus cells, compared with nonleukemic thymus cells, but were markedly reduced in leukemic cells. Adenylate cyclase activity rose during the preleukemic and leukemic phases of leukemogenesis. Although the drop of epinephrine-induced stimulation of thymus adenylate cyclase in the preleukemic phase was probably age related, there was an additional decrease of adenylate cyclase activation by epinephrine in leukemic cells. Cyclic AMP phosphodiesterase activity was slightly higher in preleukemic cells and more than fourfold AKR thymus. These observations suggest that cyclic AMP phosphodiesterase is largely responsible for the low levels of cyclic AMP in leukemic cells. Significant changes in cyclic AMP metabolism are already detectable before neoplastic cells may be found in the thymus.     

Croton oil- and benzo(a)pyrene-induced changes in cyclic adenosine 3':5'-monophosphate and cyclic guanosine 3':5'-monophosphate phosphodiesterase activities in mouse epidermis.

The topical application of croton oil, benzo(a)pyrene, acetic acid, and 12-O-tetradecanoyl-phorbol-13-acetate to mouse skin caused an increase in the activity of epidermal low-affinity cyclic adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase. The increase was most pronounced with croton oil, began between 4 and 6 hr after application of this material, and was maintained for at least 48 hr. The activity of cyclic guanosine 3':5'-monophosphate phosphodiesterase was also increased by treatment with croton oil or 12-O-tetradecanoyl-phorbol-13-acetate, but detailed time courses were not obtained. Increased activity was observed in both the soluble fractions and the washed particulate fractions of epidermis. Fractionation of soluble extracts from acetone-treated epidermis on DEAE-cellulose columns showed the presence of enzymes with specificity for both cyclic AMP and cyclic guanosine 3':5'-monophosphate, together with a peak catalyzing the hydrolysis of both cyclic AMP and cyclic guanosine 3':5'-monophosphate. The activity of this latter nonspecific activity was selectively increased following treatment with croton oil. The increase in cyclic AMP phosphodiesterase activity was partially abolished by multiple injections of cycloheximide, suggesting that new protein synthesis was involved. Injection of the alpha-receptor antagonist phentolamine abolished a croton oil-induced rise in epidermal cyclic AMP levels and decreased the induction of cyclic AMP phosphodiesterase activity. From these results it was concluded that the increase in enzyme activity was induced by cyclic AMP.     

Inhibition of DNA synthesis and alteration of cyclic adenosine 3',5'-monophosphate levels in RSa cells by human leukocyte interferon.

Human leukocyte interferon inhibits the proliferation of the virus-transformed human embryonic cells RSa. Incorporation of [3H]thymidine (TdR) into an intracellular pool and the activity of TdR kinase were reduced in the interferon-treated RSa cell culture. High-degree (90%) inhibition of [3H]TdR incorporation was associated with concentrations of added interferon that produced more than a twofold increase in the intracellular cyclic AMP (cAMP) level, and low-degree inhibition was associated with smaller increases in cAMP. In the IFr cell culture, which is relatively resistant to the anticellular action of interferon, considerably less inhibition of TdR incorporation and a slight increase in cAMP were observed. Extracellularly added dibutyryl-cAMP inhibited the proliferation of both RSa and IFr cells to almost the same degree. A decrease in cAMP level and the initiation of DNA synthesis of G1-phase-arrested RSa cells by serum addition were prevented when cells were pretreated with interferon. These results indicated that intracellular cAMP may mediate the inhibitory effect of interferon on DNA synthesis and cell growth.