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

Otten, Jacques; Johnson, George S.; Pastan, Ira

doi:10.1016/s0006-291x(71)80212-7

Cited by 425 publications

(125 citation statements)

References 4 publications

Supporting

Mentioning

110

Contrasting

Unclassified

Order By: Relevance

“…Our results showing that prostaglandins El and EZ raise cyclic AMP in py 3T3 cells are at variance with a previous paper on prostaglandin unresponsive adenylate cyclase in such cells [27]. In addition, the present cells had higher levels of cyclic AMP (but grew to higher cell densities) than their parent 3T3 cells (unpublished observations) whereas other transformed cells often have lower cyclic AMP levels than their non-transformed counterparts [12]. Possibly, the polyoma virus genome of 3T3 cells can be integrated in different ways into the host cell DNA as has been shown for 3T3 cell lines transformed with simian virus 40 [28].…”

Section: Discussioncontrasting

confidence: 89%

Prostaglandin E₂ Production in 3T3 Cells Transformed by Polyoma Virus Raises the Intracellular Adenosine 3′: 5′‐Monophosphate Levels

Claesson

Lindgren

Hammarström

1977

European Journal of Biochemistry

View full text Add to dashboard Cite

High cellular adenosine 3' : 5 '-monophosphate (cyclic AMP) levels were found in a polyoma-virustransformed 3T3 fibroblast line, which produces comparatively high prostaglandin E2 concentrations. Prostaglandin E2 and cyclic AMP increased with time during 6 days of growth. Both effects were prevented by three different cyclo-oxygenase inhibitors. Addition of prostaglandin Ez, at a concentration which would have been synthesized in the absence of inhibitor, reversed the effect of indomethacin, one of the cyclo-oxygenase inhibitors, on cyclic AMP. It is concluded that endogenous prostaglandin E2 production in these transformed cells influences their cellular cyclic AMP levels.E-type prostaglandins can raise cyclic AMP levels in many tissues and cultured cells by stimulating adenylate cyclase [l]. A number of reports indicate that animal cells in culture produce prostaglandins (see e.g. [2-11 3). However, it has not been clear if this prostaglandin production has effects on cellular cyclic AMP levels since the amounts of prostaglandins produced are often orders of magnitude lower than the amounts added to raise cyclic AMP. The question if endogenous prostaglandin production can influence cyclic AMP levels in cell cultures is an important one since many reports indicate that cyclic AMP has essential roles in the regulation of cell proliferation and differentiation . The present report shows that prostaglandin E2 production by polyoma-virustransformed 3T3 cells raises their cellular cyclic AMP concentrations. MATERIALS AND METHODSDulbecco's modified Eagle's medium, calf serum, trypsin solution (2.5 %), and penicillin/streptomycin solution (10000 I.U./ml-10 mg/ml) were pur- Cell CulturesA polyoma-virus-transformed Balb/c 3T3 mouse embryo fibroblast line, originally cloned by Dr G. Todaro, was kindly given to us by Dr M. M. Burger (University of Basel, Switzerland). This cell line will be referred to as py 3T3. The cells were passaged with 0.25 % trypsin and planted at 2 x lo5 cells per 90-mm petri dish in 10 ml of Dulbecco's modified Eagle's medium containing 10% calf serum, 100 I.U./ ml of penicillin G and 0.1 mg/ml of streptomycin. Cultures were incubated at 37 "C in an atmosphere of 10% C02 in air and a relative humidity of 80%. The cells were free of mycoplasma contamination as judged by absence of cytoplasmatic incorporation of 3H-labeled thymidine [20]. All experiments were carried out in culture media containing 10% calf serum.

show abstract

Section: Discussioncontrasting

confidence: 89%

Prostaglandin E₂ Production in 3T3 Cells Transformed by Polyoma Virus Raises the Intracellular Adenosine 3′: 5′‐Monophosphate Levels

Claesson

Lindgren

Hammarström

1977

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…The ability of high concentrations of cAMP to suppress the agglutinability of transformed cells (11)(12)(13)(14) and the appearance of agglutinability in normal cells after lowering cAMP levels (14), suggests that the low cAMP levels in transformed cells (3,4) are at least in part responsible for their agglutinability.…”

Section: Discussionmentioning

confidence: 99%

“…Adenosine 3': 5'-monophosphate (cAMP) regulates numerous functions of cultured fibroblastic cells. Among these are growth rate (1)(2)(3)(4)(5), cell shape (2,(6)(7)(8), adhesiveness (9), motility (10), and agglutination by plant lectins such as concanavalin A (Con A) (11)(12)(13)(14). Indeed, when transformed cells are treated with cAMP analogues such as dibutyryl cAMP (Bt2cAMP), their agglutinability is markedly diminished (11)(12)(13)(14).…”

mentioning

confidence: 99%

Cyclic AMP modulates microvillus formation and agglutinability in transformed and normal mouse fibroblasts.

Pastan¹

1975

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We have utilized dark field microscopy to observe the surface microstructure of living cultured cells. Using this method, we have found that dibutyryl cAMP treatment causes regression of the numerous, long cell surface microvilli present on L929 cells. Thirty minutes after removal of dibutyryl cAMP, microvilli reappear. An inhibitor of phosphodiesterase (methylisobutylxanthine) and a stimulator of adenylate cyclase (prostaglandin El), both of which raise cAMP levels, cause regression of microvilli in 15 min. Untransformed 3T3 cells show very few microvilli when viewed still attached to their substratum or after removal with EDTA. Treatment of these cells with trypsin causes the formation of numerous microvilli on their surface.. When clumps of cells agglutinated by concanavalin A are examined by thin section electron microscopy, the cells are seen to be held together by a "forest" of interdigitating microvilli and only rarely is there apposition of the areas of membrane between microvilli. At the same time the distribution of surfacebound concanavalin A was examined using immunofluorescent light microscopy, and concanavalin A was found to be uniformly distributed over the cell surface. We propose that agglutinability of mouse and rat fibroblasts is regulated through the modulation of cell surface microvilli by cAMP, and that transformed cells are highly agglutinable because their low cAMP levels result in the formation of numerous surface microvilli. Adenosine 3': 5'-monophosphate (cAMP) regulates numerous functions of cultured fibroblastic cells. Among these are growth rate (1-5), cell shape (2, 6-8), adhesiveness (9), motility (10), and agglutination by plant lectins such as concanavalin A (Con A) (11)(12)(13)(14). Indeed, when transformed cells are treated with cAMP analogues such as dibutyryl cAMP (Bt2cAMP), their agglutinability is markedly diminished (11)(12)(13)(14) MATERIALS AND METHODS Examination of Cells by Light Microscopic Methods. L929or 3T3-4 cells were grown on 22 X 40 mm sterile glass coverslips in 20 cm2 plastic dishes in Dulbecco-Vogt's modified Eagle's medium supplemented with 10% calf serum and penicillin-streptomycin (50 units/ml each) at 370 in 95% air-5% CO2. Preparation and addition of Bt2cAMP, 1-methyl-3-isobutylxanthine, and prostaglandin El were performed as previously described (8). Cells on coverslips were viewed either under phase contrast microscopy or dark field, visible light microscopy on a microscope stage in a plastic enclosure containing 95% air, 5% CO2. The plastic enclosure was kept in a constant temperature room at 37°. Coverslips were removed from their dishes and overlaid with a second sterile coverslip. This sandwich with cells in between was placed on the stage of a Zeiss RA or inverted phase contrast microscope. For dark field observation, a dark field oil condenser (Zeiss ultracondenser) and a 40 X oil immersion lens with an integral iris diaphragm were used. The light source for this observation was a dc-powered mercury vapor light source (HBO 200W/4) u...

show abstract

“…This evidence derives primarily from experiments in which the division of cells grown in culture has been shown to be inhibited by exogenous cyclic AMP or by exposure to agents that induce increases in intracellular cyclic AMP concentration. This view of the antiproliferative action of cyclic AMP has been clarified by the recent observations of Otten et al (25) and Sheppard (26) that indicate that high concentrations of intracellular cyclic AMP are associated with the limitation of "normal" proliferation and acquisition of contact inhibition, whereas low cellular concentrations are related to "malignant" transformation and uncontrolled proliferation without contact inhibition. These studies imply that biochemical events involving high intracellular concentration of cyclic AMP promote cellular differentiation and that lowering of the concentration of cyclic AMP allows antagonistic events associated with cell proliferation to proceed.…”

Section: Methodsmentioning

confidence: 99%

Guanosine 3′:5′-Cyclic Monophosphate: A Possible Intracellular Mediator of Mitogenic Influences in Lymphocytes

Hadden

Haddox

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

342

114

View full text Add to dashboard Cite

Adenosine 3':5'-cyclic monophosphate (cyclic AMP) has been shown to have an antimitotic role in various cell types, and it has been hypothesized that'a decrease of cyclic AMP concentration in the cell initiates or permits cell division. This hypothesis has been evaluated with respect to clonal proliferation of lymphocytes. Two potent mitogenic agents, phytohemagglutinin and concanavalin A, which induce thymic-dependent lymphocytes to undergo clonal' proliferation, were examined for their ability to initiate proliferation and to alter the concentrations of cyclic AMP and guanosine 3':5'-cyclic monophosphate (cyclic GMP) in purified human peripheral blood-lymphocytes. Optimal mitogenic concentrations of phytohemagglutinin and concanavalin A produced 10-to 50-fold 'increases in the concentration of lymphocyte cyclic GMP within the first 20 min of'exposure to the mitogens. No changes were seen in the concentration of cyclic AMP after stimulation with either mitogen in purified form. Increases of less than 2-fold in 'the concentration of lymphocyte cyclic AMP observed with a less purified preparation of phytohemagglutinin could be attributed to the agglutinating rather than the mitogenic properties of the mitogen. A revised hypothesis' is presented in which a temporally discrete rise in lymphocyte cyclic GMP concentration is viewed as an active signal to induce proliferation, while the elevation of cyclic AMP concentration in these cells is viewed as a regulatory influence that limits or inhibits mitogenic action.The lymphocyte, a cell whose normal functions include, clonal proliferation after stimulation by antigen, provides a model for studying the biochemical events related to initiation of cellular division. Mitogens, such as phytohemagglutinin (PHA) and concanavalin A (Con A), have been used to induce clonal proliferation in lymphocytes, particularly those derived from the thymus. Several experiments (1-6) have been performed in which the relationship between cyclic AMP concentration and mitogen-induced lymphocyte division has been investigated. The results indicate (a) that cyclic AMP itself does not significantly stimulate division of lymphocytes in the resting phase ("Go" or "G1" phase) of the cell cycle (1), (b) that increases in cyclic AMP concentration resulting from stimulation by hormones and polynucleotides do not induce lymphocytes to divide (2, 3), and (c) that agents that promote increases in Iymphocyte cyclic AMP concentration inhibit mitogen-induced clonal proliferation (4-6).Abbreviations: PHA, phytohemagglutinin; Con A, concanavalin A.t To whom reprint requests should be addressed: Box 494 Mayo, University of Minnesota Hospitals, Minneapolis, Minn. 55455.In contrast to the foregoing is the observation that high concentrations of PHA can induce small increases in the amounts of cyclic AMP in lymphocytes (4). These observations have been interpreted as support for the concept that PHA exerts its mitogenic effects through an increase in cellular cyclic AMP concentration. Support for the o...

show abstract

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

Cited by 425 publications

References 4 publications

Prostaglandin E₂ Production in 3T3 Cells Transformed by Polyoma Virus Raises the Intracellular Adenosine 3′: 5′‐Monophosphate Levels

Prostaglandin E₂ Production in 3T3 Cells Transformed by Polyoma Virus Raises the Intracellular Adenosine 3′: 5′‐Monophosphate Levels

Cyclic AMP modulates microvillus formation and agglutinability in transformed and normal mouse fibroblasts.

Guanosine 3′:5′-Cyclic Monophosphate: A Possible Intracellular Mediator of Mitogenic Influences in Lymphocytes

Contact Info

Product

Resources

About

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

Cited by 425 publications

References 4 publications

Prostaglandin E2 Production in 3T3 Cells Transformed by Polyoma Virus Raises the Intracellular Adenosine 3′: 5′‐Monophosphate Levels

Prostaglandin E2 Production in 3T3 Cells Transformed by Polyoma Virus Raises the Intracellular Adenosine 3′: 5′‐Monophosphate Levels

Cyclic AMP modulates microvillus formation and agglutinability in transformed and normal mouse fibroblasts.

Guanosine 3′:5′-Cyclic Monophosphate: A Possible Intracellular Mediator of Mitogenic Influences in Lymphocytes

Contact Info

Product

Resources

About

Prostaglandin E₂ Production in 3T3 Cells Transformed by Polyoma Virus Raises the Intracellular Adenosine 3′: 5′‐Monophosphate Levels

Prostaglandin E₂ Production in 3T3 Cells Transformed by Polyoma Virus Raises the Intracellular Adenosine 3′: 5′‐Monophosphate Levels