George S. Johnson

Cyclic AMP levels in fibroblasts: Relationship to growth rate and contact inhibition of growth

Summary Cyclic AMP has been measured in transformed and untransformed fibroblasts. When many lines are compared during logarithmic growth, there is an inverse correlation between growth rate and cyclic AMP levels. In a contact-inhibited line (3T3-4) cyclic AMP increases at confluency, whereas in non-contact-inhibited lines, cyclic AMP decreases at confluency. Cyclic AMP probably regulates the growth rate and mediates contact inhibition of growth in cultured fibroblasts.

Cyclic AMP and the Transformation of Fibroblasts

Publisher Summary Cyclic AMP is found in a wide variety of organisms and cells. It is a chemical switch that regulates the activity of existing enzymes and also can induce the synthesis of new proteins. This chapter reviews the role of cyclic AMP in fibroblasts, especially the role of cyclic AMP in the transformation of these cells. The transformed fibroblasts grow faster than normal cells and have a greatly increased saturation density. The transformed cells look different, generally they are less spindly and more refractile in appearance; they are less adhesive to the substratum on which they are growing and will grow in soft agar, whereas normal cells will not; they have altered surface molecules and are readily agglutinated by plant lectins; they often secrete abnormal amounts of macromolecules; and have abnormal tRNA and increased aerobic glycolysis. Cyclic AMP has an important role in regulating many properties of normal fibroblasts. It is involved in controlling cell shape, adhesiveness to the substratum, motility, growth, agglutinability by lectins, and the synthesis of certain macromolecules. As a result of the transformation by chemical carcinogens, X-rays, oncogenic viruses, or spontaneous selection, cyclic AMP levels are decreased and this decrease produces some of the abnormal properties of transformed cells. Transformation by different viruses decreases adenylate cyclase activity by diverse mechanisms because a variety of transformation factors exist.

Control of DNA synthesis and mitosis in 3T3 cells by cyclic AMP

DNA synthesis and mitosis in synchronized Swiss 3T3-4 and Balb 3T3 cells is prevented by the addition of (but) 2cAMP during the early G1 phase of the cell cycle. Removal of (but) 2cAMP at up to eleven hours, or addition of (but) 2cAMP as early as three hours after planting fails to block DNA synthesis. However, in Swiss 3T3-4 cells, but not Balb 3T3 C1A31 cells, the addition or removal of (but) 2cAMP at three or six hours after planting causes DNA synthesis to reach a peak at an earlier time. Adding (but) 2cAMP in early S-phase does not inhibit DNA synthesis, but prevents subsequent mitosis. This indicates the presence of a cyclic AMP-dependent block in G2. Our results suggest that cyclic AMP has at least three regulatory functions during the cell cycle, two negative and one positive.

Studies on the levels of cyclic AMP in cells transformed by wild-type and temperature-sensitive Kirsten sarcoma virus

Abstract The concentration of cyclic AMP has been studied in the NRK line of rat cells transformed by wild-type and a temperature-sensitive derivative of the Kirsten strain of Murine sarcoma virus. The concentration of cyclic AMP is low in cells recently transformed by the Kirsten virus and in two clones derived from a recent infection. The differences in cyclic AMP concentrations are most evident in confluent cultures. Cyclic AMP rises markedly in the contact inhibited cells at confluency but remains low in the transformed cells. Cells transformed with a mutant of Kirsten sarcoma virus, temperature sensitive for the maintenance of transformation, show low levels of cyclic AMP at the permissive temperature and high levels at the nonpermissive temperature. When shifted from 39°C to 32°C it takes 24–48 hr for the cyclic AMP levels to fall in cells transformed by the mutant virus. Exogenous addition of N 6 2′-0-dibutyryl cyclic AMP to Kirsten transformed cells changes their morphology towards that of normal cells and decreases their growth rate, but contact inhibition of growth is not restored. Dibutyryl cyclic AMP and 8-Br-cyclic AMP both decrease the growth rate of NRK cells, but dibutyryl cyclic GMP and 8-Br-cyclic GMP have no effect on the growth rate, saturation density, or morphology of these cells.

N6-substituted adenines induce cell elongation irrespective of the intracellular cyclic AMP levels

Abstract The N 6 -substituted derivatives of adenine induce cell elongation in cultured fibroblasts within a few hours of addition to the growth medium. They also rapidly decrease cell motion and increase cell adhesion to the substratum. Similar effects on cell shape, motion, and adhesion to the substratum are observed during incubation with 1-methyl-3-isobutyl xanthine, a potent inhibitor of cyclic nucleotide phosphodiesterase which elevates the intracellular levels of cyclic AMP (cAMP) 7–8 fold. The N 6 derivatives of adenine inhibit cyclic nucleotide phosphodiesterase in cell homogenates; however, in the intact cell they do not elevate the levels of cAMP. Thus, their actions appear to be independent of the intracellular levels of cAMP. The relationship of these findings to the hypothesis that cAMP is the intracellular mediator of cell shape is discussed.

Simian virus 40 rapidly lowers camp levels in mouse cells.

Abstract The addition of SV40 to contact inhibited Balb 3T3 cells causes a 2-fold decrease in intracellular cAMP levels. The levels reach a minimum 3 hours after virus addition, and after a few hours begin to rise toward normal. No significant changes in cAMP levels are observed after cells are exposed to UV-inactivated virus or are mock-infected. This is the earliest known effect of SV40 infection. We propose that SV40 induces host DNA synthesis by lowering cAMP levels.

Effects of sugars on the physiology of cultured fibroblasts

Abstract Pronounced, rapid, and reversible changes in cultured normal and transformed fibroblasts are observed following removal of sugars from the culture medium. Growth immediately ceases or greatly diminishes; it resumes at a normal rate without any appreciable delay following re-addition of glucose. The cell shape changes within 6 h to a more flattened and elongated form similar to that of cells treated with db-cAMP. The NAD + NADH ratio rises 4-fold and the ATP level falls about 25% in 2 h; however, the cAMP level remains constant. In transformed mouse SVT2 cells, other sugars will not replace glucose. A variant line of SVT2 (SVT2 SUG+) which will grow on numerous sugars has been isolated. Its growth rate, morphology and adhesion to the substratum, but not cAMP content, are dependent upon the sugar composition. These results suggest that the sugar composition of the growth medium has pronounced effects on cell physiology and that the effects are not mediated by cAMP.

Metabolic effects of glucose deprivation and of various sugars in normal and transformed fibroblast cell lines

Abstract The sugar composition of the growth medium influenced the NAD + NADH ratio, pyruvate and lactate production, and ATP levels in both normal and transformed fibroblast cell lines growing in tissue culture. Removal of glucose led to a rapid three- to fourfold rise in the NAD + NADH ratio, followed by a slower decline in the content of ATP. However, there was no change in the adenylate energy charge [ ( ATP + 1 2 ADP )/( ATP + ADP + AMP ) ] over a 2-h period. The NAD + NADH ratio was restored to the original level within 10 s of glucose readdition. The NAD + NADH ratios in cell lines growing on galactose were as high as for those incubated without sugars; growth on mannose or fructose produced intermediate ratios. There was an inverse relationship between the NAD + NADH ratio and pyruvate-lactate production for glucose, fructose and galactose. Thus, all cell lines had a high production of pyruvate and lactate but a low NAD + NADH ratio when grown on glucose. In contrast, when galactose served as the sugar source, acid production was low, while the ratio was high. All cell lines had comparable hexokinase activity, and glucose was the best substrate, mannose intermediate and fructose poorest. Hexokinase activity did not correlate with the relative degree of utilization of the sugars. These results suggest that the sugar composition of the growth medium affects the metabolic pattern of a cell line, including the NAD + NADH ratio, the ATP content and the production of pyruvate and lactate.

1-Methylnicotinamide and NAD metabolism in normal and transformed normal rat kidney cells

Abstract NAD, 1-methylnicotinamide, S-adenosylmethionine, and S-adenosylhomocysteine levels were analyzed in different clones of untransformed normal rat kidney cells and in cells transformed by different viruses. No consistent changes in the levels of these metabolites were apparent as a result of malignant transformation, and also differences in the levels of metabolites did not correlate with growth rate in the various cell lines. 3-Deazaadenosine prevented synthesis of 1-methylnicotinamide but not of NAD. The S-denosylmethionine/S-adenosylhomocysteine ratio did not change in serum-starved, growth-arrested cells although 1-methylnicotinamide synthesis increased about twofold. These results were used to consider possible physiological roles for 1-methylnicotinamide. Its intracellular levels did not correlate with growth rate and were not altered by transformation. No evidence was obtained that its synthesis is involved with maintenance of nicotinamide of S-adenosylmethionine levels. Thus the biological function for 1-methylnicotinamide remains a mystery.

Disproportionate accumulation of 18S and 28S ribosomal RNA in cultured normal rat kidney cells treated with picolinic acid or 5-methylnicotinamide.

Abstract Normal rat kidney cells treated with the pyridine derivative picolinic acid, or 5-methylnicotinamide, an inhibitor of ADP-ribosylation, are unable to process 28S rRNA and accumulate 60S ribosomal subunits in the cytoplasm. Synthesis of polyA(+) RNA, rRNA precursors, and the processing of 18S rRNA into 40S ribosomal subunits are almost unaffected. Serum starvation and treatment of cells with histidinol, cycloleucine, nicotinic acid, or 1,10-phenanthroline do not elicit this alteration in rRNA metabolism. Ribosomal subunits synthesized before picolinic acid addition have different stabilities after picolinic acid treatment. The 40S subunits are degraded while the 60S subunits are more stable, demonstrating that a compensatory mechanism exists to maintain preferentially existing subunits when they are no longer being synthesized. The results suggest that ADP-ribosylation is necessary for proper processing of 28S rRNA and therefore for formation of mature 60S ribosomal subunits.