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

Abstract

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...

Interactions between Effector Cell Activity and Lymphokines: Implications for Recovery from Herpesvirus Infections

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The content and metabolism of cyclic adenosine 3', 5'-monophosphate and cyclic guanosine 3', 5'-monophosphate in adenocarcinoma of the human colon.

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