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Cyclic Nucleotide Modulation of in Vitro Morphological Transformation of Syrian Hamster Cells1

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Cyclic Nucleotide Modulation of in Vitro Morphological Transformation of Syrian Hamster Cells1 [CANCER RESEARCH 42, 1274-1278, April 1982] 0008-5472/82/0042-0000$02.00 Cyclic Nucleotide Modulation of in Vitro Morphological Transformation of Syrian Hamster Cells1 Stefan G. D. Mironescu,2 Sheldon M. Epstein,3 and Joseph A. DiPaolo Department of Pathology. University of South Carolina School of Medicine, Columbia, South Carolina 29208 ¡S.G. D. M., S. M. £.],and Laboratory of Biology, National Cancer Institute, Bethesda, Mary/and 20205 [J. D. P.] ABSTRACT tigating various parameters in which these compounds modu late malignant growth or cellular transformation may provide The cyclic nucleotides, cyclic adenosine 3':5'-monophos- insight into the carcinogenesis process. Alterations of cAMP4 phate (cAMP) and cyclic guanosine 3':5'-monophosphate metabolism often occur after viral transformation (2); further (cGMP) or their dibutyryl and monobrominated derivatives, may more, in some instances, the transformed morphology is re either increase or decrease morphological transformation of Syrian hamster embryo cells exposed to /v-methyl-A/'-nitro-A/'- versed by cAMP or Bt2cAMP (19). Moreover, these revertants are unstable and the transformed phenotype is reexpressed nitrosoguanidine (MNNG). The effect on transformation is pri when the cyclic nucleotide is removed. Exogenous cAMP pre marily a function of the parent cyclic nucleotide and the dura vents transformation by temperature-sensitive mutants of Rous tion of exposure to the nucleotides. At concentrations of 5 mw sarcoma virus (28); in contrast, cAMP (32, 33) and agents or larger for a minimum 24-hr exposure cAMP, cGMP, and capable of elevating the endogenous levels of cAMP (33) their congeners reduced the colony-forming ability of nontrans- enhance SV40, polyoma, and Moloney sarcoma virus transfor formed Syrian hamster embryo cells not exposed to MNNG; mation. These variable effects of the cyclic nucleotides, and however, cGMP and its derivatives caused less toxicity than their reversibility in some instances, further strengthen their cAMP or its analogs. cAMP and its monobrominated and di- value as possible probes for developing models to study se butyrylated derivatives decreased the transformation fre lected aspects of the phenomenon of carcinogenesis. quency associated with MNNG irrespective of whether the Recently, cAMP and cGMP have been implicated in chemical various adenylnucleotides were administered before or after carcinogenesis. The metabolic activation of parental carcino MNNG. The greatest inhibitory effect on MNNG-induced trans formation was obtained with A/6,O2-dibutyryl cyclic adenosine genic molecules (17), initiation (6) and promotion (7) of mouse 3':5'-monophosphate followed in order by 8-bromocyclic aden epidermal tumors, and the growth rate of several chemically osine 3':5'-monophosphate and cAMP. At equimolar doses, induced liver (3), mammary (5), and colonie (9) malignant and premalignant lesions are associated with various changes in the dibutyryl and brominated analogs of cGMP but not unsub- the intracellular amounts of cyclic nucleotides. The relationship stituted cGMP enhanced transformation when adminstered of these changes in cyclic nucleotide concentrations to the prior to exposure of the Syrian hamster embryo cells to MNNG neoplastic process requires elucidation since the enzyme(s) but reduced the transformation frequency when added after responsible for the. metabolism of cAMP and cGMP and the MNNG. The enhancing and inhibitory effects of the guanine levels of these nucleotides and their activities and kinetic cyclic nucleotide-induced alteration of MNNG-associated parameters in a variety of tissues and tumors vary widely (29, transformation frequencies were dose and time dependent and occurred in the order A/6,O2-dibutyryl cyclic guanosine 3':5'- 32). Because previous studies (13, 26) demonstrated that caf monophosphate > 8-bromocyclic guanosine 3':5'-monophos- feine and theophylline, oxypurines which inhibit the enzyme phate » cGMP. Butyric acid neither diminished nor increased cAMP phosphodiesterase (1 ), significantly affect transforma MNNG-induced transformation frequency. The latter suggests tion frequency of MNNG-exposed HEC, the role of cyclic that butyrate ions formed by metabolism of the cyclic nucleo nucleotides in modulating transformation of HEC by MNNG tide analogs were not a factor in the observed alterations of was determined. Further studies were believed to be warranted transformation frequency. since inhibition of phosphodiesterase activity could be ex pected to be associated with higher intracellular concentrations INTRODUCTION of cAMP. This would occur because reduction of phosphodi esterase activity would reduce the rate of hydrolysis of cAMP The demonstration that cyclic nucleotides may regulate cell and therefore higher amounts of the cyclic nucleotide would multiplication (19), cell morphology (16, 30, 35), and synthesis be present in cells. Therefore, logarithmic cultures of HEC of some macromolecules (15, 16, 30, 34) suggests that inves- were exposed to cAMP, cGMP, their dibutyryl and brominated derivatives, or BA, for intervals up to 48 hr. Cultures were ' This study was supported in part by Grants IN-107C and IN-107E from the exposed to these compounds either before or after incubation American Cancer Society and 1-SO7RR05815-01 from NIH. A preliminary report was presented at the 67th Annual Meeting of the American Association for with MNNG. BA was included, in part, because it is a metabolic Cancer Research, May 4 to 8, 1976, Toronto. Canada (24). 2 Dr. Mironescu died during the preparation of this manuscript; however, 4 The abbreviations used are: cAMP, cyclic adenosine 3':5'-monophosphate; reprint requests addressed to Dr. S. G. D. Mironescu in Columbia, S. C., will still Bt2cAMP, /v^.C^-dibutyryl cyclic adenosine 3':5'-monophosphate; cGMP, cyclic be honored. guanosine 3':5'-monophosphate; MNNG, N-methyl-N'-nitro-W-nitrosoguanidine; 3 Present address: Department of Pathology; New Jersey School of Ostéo HEC, Syrian hamster embryo cells; BA, butyric acid; SBrcAMP, 8-bromocyclic pathie Medicine; College of Medicine and Dentistry of New Jersey, 300 Broad adenosine 3':5'-monophosphate; SBrcGMP, 8-bromocyclic guanosine 3':5'- way, Camden, N. J. 08103. monophosphate; Bt2cGMP, W.O2-dibutyryl cyclic guanosine 3':5'-monophos- Received April 6, 1981; accepted December 18, 1981. phate. 1274 CANCER RESEARCH VOL. 42 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1982 American Association for Cancer Research. Cyclic Nucleotides Modulate Transformation derivative of Bt2cAMP (15). Cell survival was ascertained for RESULTS each experimental condition by determining the number of cells capable of colony formation. Transformed cells were Cytotoxicity Studies. Except for BA, the relative cloning quantitated by the focus transformation procedure (25, 26). efficiency of HEC is not affected by any compound at a con centration of 1.0 HIM or less (Chart 1). At concentrations MATERIALS AND METHODS greater than 1.0 mM, however, the cyclic nucleotides and derivatives induced an increased but not predictably dose- Cells. Primary fibroblast cultures of HEC in plastic Petri dishes or dependent inhibition of colony formation. The greatest inhibi flasks, initiated by trypsinization of eviscerated 13 to 14 days of tion of cloning efficiency (50%) occurred with 10 mM Bt2cAMP gestation Syrian hamster (Mesocricetus auratus) embryos (LVK:LAK; Lakeview Hamster Colony, Newfield, N. J.), were grown in Dulbecco's (Chart 1/0; Bt2cGMP (Chart 1B) was slightly less inhibitory. At modified Eagle's minimal essential medium with Earle's salts and high 10 HIM concentrations, cAMP and cGMP were the least inhib glucose (4.5 g/liter) concentration supplemented with nonessential itory compounds. Between 0.1 and 10 mM BA, the survival amino acids (2%, v/v), minimal essential medium vitamins (1%, v/v), decreased with the log of the dose (Chart 1/0; 2, 5, and 10 mM penicillin (100 Ill/ml), streptomycin (100 /ig/ml), and 10% fetal bovine inhibited 80, 90, and 100% of the growth, respectively. In the serum (Microbiological Associates, Inc., Bethesda, Md.). Cultures were transformation experiments, nontoxic doses of cyclic nucleo maintained as in earlier studies (25, 26). tides and BA were selected; furthermore, no additional lethality Survival Studies. Survival of logarithmically growing tertiary cultures resulted when combinations with MNNG were made. of HEC, as well as cultures treated with MNNG, cyclic nucleotides, To evaluate the cell-killing effect of the carcinogen, toxicity their derivatives, or BA, was determined. Cultures were dispersed of MNNG was also determined (Chart 2). Thus, cell density either after a 24-hr period of exposure to the test compounds or in the could be compensated to ensure that the same number of case of MNNG after 180 min with trypsin:versene (1:250) and diluted with complete medium (25, 26). Aliquots of 5 x 102 cells in 0.5 ml viable cells was plated for other studies. Cell survival, as medium were inoculated into 25-sq cm plastic flasks containing 5 ml of indicated by cloning efficiency, decreased linearly (semilog prewarmed conditioned:fresh medium (1:2, v/v). Conditioned medium plot) with 0.25 to 0.75 fig MNNG per ml. The shoulder observed from nontreated cultures of HEC was centrifuged and sterilized by in this chart with concentrations less than 0.25 jug/ml could be suction filtration (26). After 10 to 14 days of incubation at 37° in a the result of repair of sublethal damage. humidified atmosphere with
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