Modulation of serum-stimulated DNA synthesis in cultured human fibroblasts by cAMP

Density-dependent inhibition of growth of cultured human fibroblasts was associated with a 3- to 4-fold rise in the intracellular concentration of cyclic AMP (cAMP). Serum lowered cAMP levels in 2–5 min, with the low levels persisting for several hours. When quiescent fibroblast cultures were treated with 10% serum, the incorporation of [³H]TdR into DNA increased after a 10–16 h lag, reaching a peak by 20–24 h. Dibutyryl cyclic AMP (db-cAMP), when present throughout serum treatment, produced a dose-dependent inhibition of [³H]TdR incorporation. Half-maximal inhibition was seen with 0.1 mM db-cAMP. When db-cAMP or another cyclic nucleotide phosphodiesterase inhibitor, l-methyl-3-isobutylxanthine (SC-2964), was added together with serum to maintain elevated cAMP levels and after 4 h was replaced with fresh serum-containing medium, the wave of DNA synthesis induced by serum was not delayed. This implied that stimulation by serum could occur without an initial decrease in cAMP concentration. In contrast, db-cAMP added 8 h later than serum and not removed, inhibited [³H]TdR incorporation at the peak to the same extent as db-cAMP added together with serum. The inhibition decreased progressively when db-cAMP was added more than 8 h after serum. These results suggested that a cAMP-sensitive step occurred approx. 8 h after the addition of serum in mid-G1 of the cell cycle. Results obtained using fibroblasts synchronized at the G1/S boundary with hydroxyurea or exposed to db-cAMP for 24 h suggested that db-cAMP also inhibited TdR incorporation at the G1/S interphase or during S phase. Thus, whereas reduced cAMP concentrations did not appear to serve as an initial trigger for serum-stimulated DNA synthesis in human fibroblasts, db-cAMP and SC-2964, presumably by elevating cAMP levels, appeared to act in mid-G1 and possibly at the G1/S boundary or within S phase to inhibit thymidine incorporation.     

Electron microscope analysis of amplifying ribosomal DNA from Xenopus laevis.

The inhibition of human prostatic epithelial cell (MA-160) replication by cAMP and certain analogs was explored in tissue cultures. When untreated fetal bovine serum was used to supplement the culture medium, cyclic AMP (cAMP) markedly inhibited cell growth. The inhibition was reversed by equimolar concentrations of uridine. Inhibition by 8-methyl-thio-cAMP (MES) was somewhat less effective and was not reversed by uridine. After heat treatment of the fetal bovine serum, which inactivated the cAMP phosphodiesterases, cAMP became less effective in cell growth inhibition, whereas the activity of MES remained unaltered. Dibutyryl cAMP (db-cAMP) had no effect on cell growth, however, when combined with the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), significant retardation of cell replication was observed. Cells treated for 24 h with 0.5 mM MES took up and incorporated significantly less [³H]TdR and [³H]uridine than control cells. Treatment of cells with 0.5 mM cAMP for 24 h, on the other hand, resulted in both substantially increased [³H]TdR uptake and increased [³H]uridine incorporation into RNA. The effects of similar treatment with db-cAMP plus MIX closely paralleled those of MES with marked inhibition of the uptake and incorporation of both thymidine and uridine.     

Drug-induced alterations in morphology and level of cAMP in cultured human glioma cells

Human glioma cells (138 MG) in serum-free medium within 1–3 h obtained a stellate astrocyte-like morphology after exposure to adrenalin (0.1 mM) or isoproterenol (0.1 mM). The changes, which were preceded by an increase in the cAMP content of the cells, could be antagonized by sotalol. The induced morphological alterations reversed on prolonged incubation (24 h). Two phosphodiesterase inhibitors, papaverine (0.2 mM) and RO 20 1974 had similar effects. Prostaglandin e₁ caused the highest increase in the cAMP level, followed by morphological changes which persisted for at least 72 h. A positive correlation between the level of cAMP and the duration of the astrocyte-like morphology was found. The N⁶-substituted adenine derivatives, zeatin and dimethylaminopurine induced a persistent stellate morphology without affecting the cAMP content. These substances possibly act as cAMP agonists.     

Distinct effects of calcium- and cyclic AMP-enhancing factors on cytoskeletal synthesis and assembly in mouse osteoblastic cells.

Previous studies have indicated that the effects of parathyroid hormone (PTH) on osteoblastic function involve alteration of cytoskeletal assembly. We have reported that after a transitory cell retraction, PTH induces respreading with stimulation of actin, vimentin and tubulins synthesis in mouse bone cells and that this effect is not mediated by cAMP. In order to further elucidate the role of intracellular cAMP and calcium on PTH action on bone cell shape and cytoskeleton we have compared the effects of calcium- and cAMP-enhancing factors on actin, tubulin and vimentin synthesis in relation with mouse bone cell morphology, DNA synthesis and alkaline phosphatase activity as a marker of differentiation. Confluent mouse osteoblastic cells were treated with 0.1 mM isobutylmethylxanthine (IBMX) for 24 h. This treatment caused an increase in the levels of cytoskeletal subunits associated with an elevation of cAMP. Under these conditions, PTH (20 nM) and forskolin (0.1 microM) produced persistent cytoplasmic retraction. PTH and forskolin treatment in presence of IBMX (24 h) induced inhibitory effects on actin and tubulin synthesis evaluated by [35S]methionine incorporation into cytoskeletal proteins identified on two-dimensional gel electrophoresis. Under these culture conditions PTH and forskolin also caused disassembly of microfilament and microtubules as shown by the marked reduction in Triton X soluble-actin and alpha- and beta-tubulins. In contrast, incubation of mouse bone cells with 1 microM calcium ionophore A23187 (24 h) resulted in increased monomeric and polymeric forms of actin and tubulin while not affecting intracellular cAMP. Alkaline phosphatase activity was increased in all conditions while DNA synthesis evaluated by [3H]thymidine incorporation into DNA was stimulated by PTH combined with forskolin and inhibited by the calcium ionophore. These data indicate that persistent elevation of cAMP levels induced by PTH and forskolin with IBMX cause cell retraction with actin and tubulin disassembly whereas rising cell calcium induces cytoskeletal protein assembly and synthesis in mouse osteoblasts. The results point to a distinct involvement of calcium and cAMP in both cytoskeletal assembly and DNA synthesis in mouse bone cells.     

Effect of cAMP on nucleoside metabolism. I. Effect on thymidine transport and incorporation in monkey cells (CV-1)

The effect of cAMP on a monkey kidney cell line (CV-1) noninfected and infected by SV40 was studied. No effect was found on either growth rate or cell morphology when concentrations up to 1 mM of 3′5′ cAMP were used. However, cAMP was found to increase the incorporation of ³H-thymidine into both cellular and viral DNA without a net increase in DNA synthesis. This increased incorporation was found to be related to an enhanced uptake of thymidine into the nucleotide pool which is reflected in an increase of phosphorylated nucleotides. This, coupled with a lack of effect of cAMP on endogenous deoxyribonucleotide production, produces an increased specific activity of the deoxyribonucleotide triphosphates, with a resultant increase in specific activity of DNA.     

Cell cycle synchronization of porcine granulosa cells in G1 stage with mimosine

The success of somatic cell nuclear transfer depends critically on the cell cycle stage of the donor nucleus and the recipient cytoplast. Karyoplasts in the G0 or G1 stages are considered to be the most suitable for nuclear transfer. In the present study, we used a reversible cell cycle inhibitor, mimosine, to synchronize porcine granulosa cells (GCs) in G1 phase of the cell cycle. Porcine GCs were obtained from 3 to 5mm ovarian follicles of slaughtered gilts. The effect of mimosine on the proliferation, DNA synthesis and cell cycle stage of cultured cells was examined by incorporation of radiochemical 3H-thymidine, immunocytochemical detection of incorporated thymidine analogue 5-bromo-2-deoxyuridine (BrdU) and flow cytometry analyses. Mimosine treatment of pig GCs for 24h resulted in proliferation arrest in vitro. Treatment with 0.5mM mimosine significantly (P<0.05) inhibited 3H-thymidine incorporation after 24h of culture (4.6% +/- 0.1) and after 24h of culture in serum deprived medium (41.3% +/- 3.8), in comparison to controls (100%). Inhibition of DNA synthesis was further confirmed by immunocytochemical and flow cytometry analyses. Compared with controls (78.2%), mimosine treatment for 24h increased the proportion of G0/G1 cells in the culture (85.7%) more effectively than serum starvation (SS; 81.2%). Mimosine-caused G1 arrest of porcine GCs was fully reversible and cells continued to proliferate after removing the drug, especially when they were stimulated by EGF.     

INHIBITION OF CELL GROWTH IN THE G1 PHASE BY ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE

Incorporation of tritiated thymidine into acid-precipitable material was used to measure the rate of DNA synthesis in secondary cultures of human diploid fibroblasts. Confluent cultures of human diploid fibroblasts, which are synchronized in the G₁ phase due to contact inhibition, were released from growth inhibition either by the addition of fresh medium to the cultures or by trypsinization and replating at nonconfluent densities. Either treatment resulted in a synchronous wave of DNA synthesis beginning 10–15 h after treatment and peaking at 20–25 h. In confluent cultures stimulated by fresh medium, either the addition of 0.25 mM N⁶, O²-dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP) to the medium in the interval 4–8 h after stimulation or the replacement of the fresh medium in that same 4 h interval with the depleted medium present on the cells for the 2 day period before stimulation delayed the synchronous onset of DNA synthesis in the cultures by about 4 h. In nonconfluent cultures freshly seeded from trypsinized confluent cultures, this same depleted medium obtained after a 2 day incubation of fresh medium on confluent cultures is shown to support the progress of the cells into S phase; however, the addition of 0.25 mM db-cAMP to the medium 3½ h after replating still partially prevented the initiation of DNA synthesis in the cultures. The results are discussed in terms of the role of serum and cAMP in the control of cell growth in fibroblast cultures.     

Proliferation of PHA-stimulated lymphocytes measured by combined autoradiography and sister chromatid differential staining

Lymphocyte proliferation in culture was studied by combined [³H]TdR incorporation and sister chromatid differential staining. The majority of 1st division metaphases in a 72 h culture commenced DNA synthesis after 48 h and had a cell cycle of less than 24 h. A small proportion of cells from some donors commenced DNA synthesis between 24–30 h and had cell cycle times of up to 48 h. Although many cells entered DNA synthesis at the same time, they showed marked asynchrony in the length of their cell cycle, with some completing one, some two and others three cell cycles in the 72 h culture period. The time taken for cells to enter S following stimulation with PHA ranged from 24 to 48 h and there was considerable variation between donors in the number of fast and slow responding cells.     

In vitro studies of the effect of intermittent compressive forces on cartilage cell proliferation.

Intermittent compressive (IC) forces (96 mm Hg, 0.3 Hz) inhibit by 35–60% the serum stimulated increase in ornithine decarboxylase activity (ODC) in chick embryo epiphyseal cartilage cells and rat chondrosarcoma cells. IC had no effect on mouse fibroblast L-cells ODC. The dose-response pattern of the IC effect indicated an all-or-none response with a threshold at 80 mm Hg, a pressure roughly equivalent to the in vivo weight bearing force. The k_m of the cartilage cell ODC, measured at four hours, was about 0.1 mM and was not affected by IC. The V_max, on the other hand, was significantly reduced by IC which is consistent with less enzyme or non-competitive inhibition. IC also produced a significant increase in cAMP levels in both cartilage explants and isolated cells in the presence and absence of serum and a significant reduction in ³H-thymidine incorporation into DNA. The findings show that cellular cAMP, on one hand, and ODC and DNA synthesis, on the other hand, change in opposite directions following exposure to serum and/or IC. Investigation of the IC effect on DNA synthesis in serum-deprived synchronized cartilage cells revealed that IC reduced the number of cells going into S but did not lengthen the G₁ phase. Exposure to IC early in G₁ (0–13 hours) produced the full effect, whereas IC application between 13 to 24 hours (pre S) had no effect. IC had no effect on ³H-thymidine incorporation in L-cells.     

Cyclic AMP-dependent regulation of ornithine decarboxylase activity in Chinese hamster ovary cells maintained with a salts/glucose medium.

Ornithine decarboxylase activity (ODC) increased about 7 fold 6--8 h following 10mM asparagine (ASN) addition to confluent cultures that had been previously serum deprived and then placed in a salts/glucose medium. Optimal concentrations of dibutyryl cAMP (dB cAMP) when incubated with the ASN caused up to a 50 fold increase in the activity of this enzyme after 7--8 h. The enhancement of ODC activity by ASN and dB cAMP was not sensitive to continuous (0--7 h) treatment with actinomycin D but similar treatment with cycloheximide depressed enzyme activity 40--60%. The synergistic stimulation of ODC activity by dB cAMP added with ASN was dose dependent and the dB cAMP stimulation of ODC activity displayed an absolute requirement for ASN when cells were maintained in the salts/glucose medium. The addition of dB cAMP always further enhanced ODC activity above the levels produced by addition of various levels of ASN (1 to 40mM) to the salts/glucose medium. Other agents which elevated cAMP levels such as 1-methyl-3-isobutylxanthine (IBMX) also enhanced ODC activity when administered with ASN. Additionally, treatment with sodium butyrate at concentrations ranging from 0.001mM to 5.0mM did not elevate ODC activity above the activity obtained with ASN alone. Addition of dB cAMP at various times after placing cells in salts/glucose medium with ASN further stimulated ODC activity only when added during the first 3-4 h. These results demonstrate the involvement of cAMP in the ASN mediated stimulation of ODC activity using cells maintained in a salts/glucose medium.     

Stimulation of DNA synthesis,cell multiplication,and ornithine decarboxylase in 3T3 cells by multiplication stimulating activity (MSA)

Multiplication stimulating activity (MSA) has been purified from the conditioned media of rat liver cells in culture by a modification of the procedure of Dulak and Temin. Purified MSA stimulates [³H] thymidine incorporation into DNA in subconfluent, serum starved 3T3 cells. Cell cycle analysis by the flow microfluorometer shows that the [³H] thymidine incorporation data reflects DNA synthesis. MSA also stimulates the multiplication of serum starved subconfluent 3T3 cells. MSA is approximately 10-fold less active in 3T3 cells than in chick embryo fibroblasts in stimulating [³H] thymidine incorporation into DNA. MSA causes a 2–10-fold increase in ornithine decarboxylase (ODC) activity in 3T3 cells and the dose response curve parallels the dose response curve for [³H] thymidine incorporation into DNA. The Km of ODC for ornithine is 0.12 mM. There is a 30% decrease in the activity of ornithine transaminase (OTA) during the time period in which MSA causes an increase in ODC activity. Insulin also stimulates [³H] thymidine incorporation into DNA, cell multiplication and ODC activity over the same concentration range as shown for MSA, however, the extent of stimulation by insulin is less than that observed following MSA addition.     

Ionic changes associated with lead stimulation of DNA synthesis in Balb/c3T3 cells

Lead at slightly subtoxic concentrations markedly stimulated the rate of DNA synthesis in cultured animal cells. This stimulation was closely correlated with formation of a precipitate that was adsorbed and taken up by the cells under certain medium conditions. Data suggest that a precipitate-induced perturbation of the surface membrane leads to intracellular changes responsible for stimulation of DNA synthesis. Maximum stimulation of³H-thymidine incorporation by optimum concentrations of lead is delayed about 8 h compared to that in serum stimulation. In cells stimulated significantly by lead, but not in unstimu-lated cells, a reproducible rise of about 13% in intracellular magnesium occurred over a 24 h period, with an 8 h lag in the increase compared to that observed in serum stimulation. In view of the increases in intracellular magnesium consistently associated with and preceding stimulation of DNA synthesis by several different mitogens including serum and insulin, the present time-coordinated positive correlation between magnesium and DNA synthesis provides evidence for the primary involvement of this divalent cation in growth stimulation produced by lead.     

The effect of the tumor promoter, phorbol myristate acetate (PMA), on hyaluronic acid (HA) synthesis by chicken embryo fibroblasts

Treatment of chicken embryo frbroblasts (CEF) with the tumor promoter, phorbol myristate accetate (PMA), resulted in a rapid increase in their ability to synthesize the glycosaminoglycan, hyaluronic acid (HA), whereas the parent compound, phorbol, had no effect. CEF cultures incubated with PMA (100 ng/ml) for 6 h resulted in a 15-fold increase in HA synthetase activity compared with phorbol-treated control cultures. The incorporation of [³H]acetate into HA and chemical determination of this polymer also demonstrated increased synthesis of HA by cells treated with PMA. We have previously shown that CEF infected with a temperature-sensitive mutant of Rous sarcoma virus, LA24, exhibit increased synthesis of HA upon transformation. PMA treatment of cells transformed with RSV-LA24 results in a further 1.5-fold stimulation of HA synthesis. These data indicate that PMA causes an increased synthesis of HA in CEF which is analogous to the increased synthesis of HA found in virally transformed CEF.     

Increased collagen synthesis in Kirsten sarcoma virus-transformed BALB 3T3 cells grown in the presence of dibutyryl cyclic AMP

Growth of Kirsten sarcoma virus-transformed BALB 3T3 (Ki-3T3) cells in the presence of dibutyryl cyclic AMP (dbcAMP) resulted in alteration of morphology, inhibition of growth, and increased collagen synthesis as measured by incorporation of ¹⁴C-proline into collagenase-digestible protein. There was an increase in incorporation of ¹⁴C-proline into collagen when expressed not only as dpm per μg DNA or protein, but also as the relative rate of collagen synthesis compared to total cellular protein synthesis, which suggests that an alteration in amino acid transport cannot totally account for the increased incorporation into collagen. The three properties studied were all affected over a concentration range of 0.10 to 1.0 mM dbcAMP, but each had a slightly different dose-response curve. At 0.5 mM dbcGMP or sodium butyrate, there was no affect on growth, morphology, or the relative rate of collagen synthesis indicating specificity for the dibutyryl analog of cAMP. Growth of the parent line, BALB 3T3, was inhibited by 0.5 mM dbcAMP, but the relative rate of collagen synthesis did not increase. These results suggest that although growth, morphology, and collagen synthesis are altered in transformed cells so that they more closely resemble those of the parent line, each property may be regulated independently.     

Decreased DNA synthesis in mammalian cells : After exposure to deoxyadenosine

The effect of a pre-treatment with 2 mM deoxyadenosine on DNA synthesis in bovine liver cells in vitro was investigated. The incorporation of ³H-deoxythymidine was strongly depressed after 1 h treatment and recovered only gradually during the following 4 to 6 h. Addition of 50 μM deoxyguanosine during this time effectively counteracted the depression. An autoradiographic study revealed that the depression was due to a decrease in thymidine incorporation per cell rather than a decrease of the proportion of thymidine-incorporating cells. It is concluded from these results that the rate of DNA synthesis is decreased during the initial hours after removal of the deoxy-adenosine because of a continued deficiency of dGTP. This conclusion is supported by the appearance of a high proportion of ³H-label in replicating intermediates after 1 h of incorporation which is otherwise only observed after pulse labelling for 5 to 10 min.     

Contributions of differential p53 expression in the spontaneous immortalization of a chicken embryo fibroblast cell line

Background

The present study was carried out to determine whether the p53 pathway played a role in the spontaneous immortalization of the SC-2 chicken embryo fibroblast (CEF) cell line that has been in continuous culture for over three years.

Results

The SC-2 cell line emerged from an extended crisis period with a considerably slower growth rate than primary CEF cells. The phenotype of the SC-2 cells changed dramatically at about passage 80, appearing smaller than at earlier passages (e.g., passage 43) and possessing a small, compact morphology. This morphological change coincided with an increase in growth rate. Passage 43 SC-2 cells expressed undetectable levels of p53 mRNA, but by passage 95, the levels were elevated compared to primary passage 6 CEF cells and similar to levels in senescent CEF cells. However, the high level of p53 mRNA detected in passage 95 SC-2 cells did not correlate to functional protein activity. The expression levels of the p53-regulated p21^WAF1 gene were significantly decreased in all SC-2 passages that were analyzed. Examination of the Rb pathway revealed that E2F-1 and p15^INK4b expression fluctuated with increasing passages, with levels higher in passage 95 SC-2 cells compared to primary passage 6 CEF cells.

Conclusion

The present study suggests that altered expression of genes involved in the p53 and Rb pathways, specifically, p53 and p21^WAF1, may have contributed to the immortalization of the SC-2 CEF cell line.     

Differences in inhibition by IDF45 (an inhibitory diffusible factor) of early RNA synthesis stimulation induced by pp60 v-src and various mitogens.

Factors inhibiting cell growth have been isolated from different cell types. However, little information is available concerning their mode of action. A novel growth inhibitory factor of 45 kDa (IDF45) was recently purified to homogeneity from medium conditioned by 3T3 cells. This molecule was able to inhibit DNA synthesis and the growth of chick embryo fibroblasts (CEF) in a reversible manner. By contrast, DNA synthesis stimulated by v-src expression in CEF was poorly inhibited by IDF45. In order to gain further insight into the IDF45 mode of action in normal and transformed CEF, we compared the effects of IDF45 on early stimulation of RNA synthesis induced in CEF by different mitogenic factors and by v-src gene expression. Stimulation, by serum, of RNA synthesis was inhibited by IDF45; however, inhibition increased when cells were preincubated with IDF45 before addition of serum and cell labeling for 2 h. IDF45 was also able to inhibit partially the stimulation of RNA synthesis induced by PMA and PDGF but was unable to inhibit stimulation of RNA synthesis induced by insulin and v-src expression. By contrast, stimulation of RNA synthesis induced by IGF-I was rapidly 100% inhibited by IDF45. The effect of IDF45 on DNA synthesis stimulated by the different mitogens was also determined and was correlated with the effect of IDF45 on RNA synthesis. These results suggest that the modes of action of IDF45 on stimulation of RNA synthesis by v-src and by insulin are similar. Our present results agree with others showing the bifunctional activity of IDF45 as an IGF-binding protein and as an inhibitory molecule in DNA stimulation induced by serum.     

Effects of removing PHA from cultures of PHA-stimulated lymphocytes

The proliferative capacity of PHA-stimulated lymphocytes following removal of PHA from the cultures was investigated. Lymphocytes were incubated with different PHA concentrations for 3 or 24 h and were then cultured in fresh medium with or without PHA in the original concentration. Cell proliferation was measured by incorporation of ³H-TdR. The effect of removing PHA was found to vary with the PHA concentration used for stimulation. Thus removal of PHA at 3 and 24 h from cells stimulated with half the optimal and at 3 h from cells stimulated with optimal PHA concentrations inhibited thymidine incorporation almost completely. Removal at 24 h from the latter cells resulted in a moderately decreased thymidine incorporation, whereas no decrease was seen after the removal of PHA from cells stimulated with twice the optimal concentration. When the cells were stimulated with very high PHA concentrations (20 × optimal), removal of PHA even resulted in an increased thymidine incorporation, a phenomenon that most probably has to do with the utilization of exogenous thymidine being inhibited by high PHA concentrations.The decreased thymidine incorporation after removal of low PHA concentrations was due to a reduction in the number of cells entering the proliferation cycle as well as to a decreased multiplication of cells already in DNA synthesis. This shows that PHA stimulates the cells even after they have initiated DNA synthesis. Various explanations for the results are discussed.     

Rat epiphyseal cells in culture: Responsiveness to bone-seeking hormones

Summary Cell cultures derived from young rat epiphyseal cartilage were grown for approximately 2 wk in BGJ _b medium supplemented with 10% fetal bovine serum to reach confluence. These cells were identified as chondrocytes as checked by morphology, the presence of alkaline phosphatase, and a positive type II collagen antibody reaction. The cells also responded to different hormonal treatment. Parathyroid hormone (PTH) increased cyclic AMP production by 50% within 15 min of treatment, whereas prostaglandin E₂ (PGE₂) caused an increase of 160%. Calcitonin (CT) did not affect cAMP production in these cells. DNA synthesis 24 h after hormonal treatment was increased by PTH (2.5-fold) and PGE₂ (2-fold), but not by CT. Among the vitamin D metabolites, 24,25(OH)₂D₃ increased significantly the [³H]thymidine incorporation into DNA, whereas 1,25(OH)₂D₃ effect was minimal. These results provide evidence for the use of these cell cultures as a model for cartilage in vitro when studying biological and hormonal responsiveness.     

Cyclic AMP and theophylline enhance DNA synthesis in L cells stimulated with anti-actin IgG and [(IgG)2protein A]2 complex by recruiting cells into S-phase

Surface binding of anti-actin IgG alone or in a M_r = 716 000 [(IgG)₂Protein A]₂ complex results in a stimulation of DNA synthesis and cell growth in L cells. Cyclic-AMP (0.01–1.0 mM) added to such cell cultures augmented DNA synthesis as measured by incorporation of [³H]thymidine into DNA. Theophylline (0.1–1.0 mM), a phosphodiesterase inhibitor which prevents enzymatic breakdown of cAMP, had similar effects, but cGMP (0.01–1.0 μM) reversed the effects of cAMP and theophylline upon DNA synthesis. Analysis of the cell cycle by flow cytometry revealed that antibody produced a shift (7%) of cells from the G₁-phase to the S-phase (DNA-synthetic) of the cell cycle at 72 hr of incubation. Addition of cAMP (0.5 mM) to cell cultures, however, produced significant shifts of antibody stimulated cells from G₁-phase to S-phase at all time points measured, i.e., 24 (12%),48 (22%),72 hr (23%). Thus, antibody recruited cells into S-phase of the cell cycle and cAMP greatly augmented the effect. These observations suggest that the mechanism of activation of L cell growth by antibody to surface antigens involves a recruitment of cells into the DNA-synthetic phase and that the effect may be mediated by cAMP.