Mutagenesis in S49 mouse lymphoma cells: induction of resistance to ouabain, 6-thioguanine, and dibutyryl cyclic AMP.

PC12 cells, derived from a rat pheochromocytoma, were mutagenized and selected in media containing agents known to elevate intracellular concentrations of cyclic AMP (cAMP). More than 40 clones were isolated by selection with cholera toxin or 2-chloroadenosine or both. The variants that were deficient in accumulating cAMP were obtained by using a protocol in which 1 ,IM 8-bromo-cAMP was included in addition to the agonist. Certain of these variants were partially characterized with respect to the site of altered cAMP metabolism. The profiles of adenylate cyclase activity responsiveness of certain variants to guanosine-5'-(0,-y- The use of variants of cloned cells clearly has advanced the understanding of the regulation of cyclic AMP (cAMP) concentration and illuminated the means by which it exerts its effects (13, 23). Phenomena involving cAMP in a particular cell type may not occur generally, however, as exemplified by the diverse relationships of cAMP concentration to proliferation of cultured cells (4).The PC12 cell line, derived from a rat pheochromocytoma, has been studied extensively as a model for growth and differentiation of cells derived from the neural crest. In response to nerve growth factor, the tumor cells undergo striking morphological and functional changes and develop into cells resembling neurons (34). The cells synthesize cAMP in response to the activation of an adenosine receptor coupled to adenylate cyclase (17), but the effect of nerve growth factor on cAMP content is inconsistent and of small magnitude (34). The cyclic nucleotide has been proposed to serve as a permissive or synergistic factor rather than to function as an obligatory intermediate (16), but other evidence supports dissociation of effects of cAMP and of nerve growth factor (14,20,30).Variants of PC12 cells deficient in cAMP accumulation or refractory to its actions should be useful in defining more precisely the role of cAMP in these processes, and additionally they might provide clues linking the cyclic nucleotide to other functions of cells derived from the neural crest. We have applied the rationale initially used in studies of the S49 lymphoma system to the PC12 line, and we have employed procedures closely similar to those of Bothwell et al. (3), who obtained PC12 variants with altered response to nerve growth factor. High intracellular concentrations of cAMP can be obtained in these cells by treating with 2-chloroadenosine, which acts on adenosine receptors in the plasma membrane, or by treating with cholera toxin, which irreversibly activates cyclase. In either case, the treated cells stop dividing, extend processes, and exhibit cytotoxicity with time. In this paper we describe the procedures that have been used to obtain more than 40 different clones and the * Corresponding author. cAMP phenotypes, which appear separable into at least two general categories with respect to forskolin stimulation. Revertants to a wild-type cyclic nucleotide phenotype have been isolated and are also described briefly. Cell culture and ...

Section: Methodsmentioning

confidence: 99%

Mutants of PC12 cells with altered cyclic AMP responses.

Block¹,

Kon²,

Breckenridge³

1984

“…As reported previously (12), mutant extracts require 20-to 30-foldhigher concentrations of cAMP to activate cA-PK than do wild-type extracts (Fig. 5).…”

Section: Resultsmentioning

confidence: 49%

“…This mutant is distinguished from wild-type cells on the basis of (i) increased effective concentration of Bt2cAMP for 50%o growth inhibition (EC50) (17); (ii) increased apparent activation constant (Ka) of cA-PK for cAMP in vitro (12); (iii) increased thermal lability of cA-PK activity; and (iv) altered electrophoretic mobility of the regulatory (R) subunit of cA-PK in the charge dimension of two-dimensional gels (29). Together, these traits suggest that the lesion accounting for this phenotype is a base substitu-tion in an R subunit structural gene.…”

mentioning

confidence: 99%

“…Biochemical analysis of the variants has revealed them almost exclusively to contain lesions affecting the function or expression of cAMP-dependent protein kinase (cA-PK) (12), the intracellular mediator of cAMP effects (24). Similar findings have been reported in neuroblastoma (27), Y1 adrenocortical (14), macrophage-like (25), and Chinese hamster ovary (28) cell lines.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Revertants of an S49 cell mutant that expresses altered cyclic AMP-dependent protein kinase.

Wetters¹,

Coffino

1982

Self Cite

Dibutyryl adenosine 3',5'-phosphate (Bt2cAMP)-sensitive (Bt2cAMP1) revertants were isolated from a resistant S49 cell mutant carrying a structural gene lesion in the regulatory subunit of cAMP-dependent protein kinase (cA-PK). This was accomplished with a counter-selection in which, first, Bt2cAMP was used to reversibly arrest revertants, and then a sequence of treatments with bromodeoxyuridine, 33258 Hoechst dye, and white light was used to kill cycling mutant cells. Reversion rates in nonmutagenized cultures could not be accurately measured, but spontaneous revertants do occur and with frequencies of less than 10-7 to i0-5. The mutagens ethyl methane sulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and ICR191 increased the reversion frequency. In all cases, reversion to Bt2cAMP sensitivity was associated with restoration of wildtype levels and apparent activation constant for cAMP of cA-PK. MNNG induced revertants whose cell extracts contained cA-PK activity distinguishable from that of wild type by thermal lability. EMS did not. The counter-selection effectively isolates rare phenotypes and is therefore a useful tool in further somatic genetic experiments. The association of reversion with alterations in cA-PK function supports all previous data from this and other laboratories implicating cA-PK as the intracellular mediator of cAMP effects. Reversion is probably the result of a mutational event. Induction of reversion by ICR191 suggests the existence of a novel mechanism for generating revertants in somatic cells.

“…(36). By using dibutyryl cAMP-mediated cytolysis of S49 mouse lymphoma cells for selection, a number of mutations affecting RI structure and function have been collected (10,34). When characterized by assays of endogenous protein kinase activity, these mutants require 5-to 20-fold higher concentrations of cAMP than do wild-type cells for halfmaximal kinase stimulation (10).…”

mentioning

confidence: 99%

Fine-structure mapping of charge-shift mutations in regulatory subunit of type I cyclic AMP-dependent protein kinase.

Steinberg¹

1984

A variety of structural mutations that alter functional properties of regulatory subunit (R) of type I cyclic AMP-dependent protein kinase are available in the cultured S49 mouse lymphoma cell system. Many of these mutations also alter the electrostatic charge of R by about 1 or 2 units. By a novel peptide mapping procedure, a number of these "charge-shift" structural mutations were localized to small regions within the R polypeptide. The procedure employed two-dimensional polyacrylamide gel electrophoresis to separate large overlapping fragments generated from denatured, affinity-purified R by limited digestion with papain. Mutations were mapped to intervals between the endpoints of these fragments. The position of one mutation was confirmed by mapping a new site for cleavage by Staphylococcus aureus V8 protease. Six different Ka mutations, which increase the concentrations of cyclic AMP required for kinase activation, mapped to three clusters in the carboxy-terminal half of R. Second-site mutations that cause phenotypic reversion of a single Ka mutant strain mapped to either side of the original mutation. By using charge-shift mutations for calibration, a map of charge density distribution was constructed for the R polypeptide. This map allowed tentative assignment of mutational lesions to portions of the R amino acid sequence implicated in cyclic AMP binding.Cyclic AMP (cAMP)-dependent protein kinases (EC 2.7.1.37), the intracellular transducers for responses involving cAMP elevation, have tetrameric structures consisting of two catalytic subunits and a regulatory subunit (R) dimer (16, 17). Animals have two major classes of cAMP-dependent protein kinase, designated as types I and II, that differ primarily in R (12, 17, 41). Kinase activation proceeds by a concerted reaction in which cAMP binds to the inactive holoenzyme and promotes dissociation of the catalytic subunit from R (4, 14, 17). Each R monomer has two binding sites for cAMP (3,7,40). These sites are distinguishable by differences in dissociation rates of bound cAMP and by differences in relative affinities for analogs of cAMP (15,24,25). Site 1 prefers cAMP derivatives modified in the C-8 position, and site 2 prefers derivatives modified in the N6 position. Both sites are thought to be important for kinase activation (20,26). Binding of cAMP induces conformational changes in R that can be detected by changes in reactivity of cysteine residues (1) or by changes in circular dichroism (27). These structural changes presumably contribute to cooperativity in cAMP binding (25)