Extracellular ATP in concentrations of 5-50 gLM displayed very little mitogenic activity by itself but it caused synergistic stimulation of [3H]thymidine incorporation in the presence of phorbol 12-tetradecanoate 13-acetate, epidermal growth factor, platelet-derived growth factor, insulin, adenosine, or 5'-(N-ethyl) These findings raise the possibility that exogenous ATP acts as a growth factor, and this was implied in two recent papers (23, 24), where ATP was compared to "more conventional mitogens." However, it is premature to call ATP a mitogen without more evidence than has been available. One must be careful to rule out the possibility that ATP is converted to adenosine by ubiquitous hydrolytic enzymes on the cell surface (ectoenzymes) (32). It was demonstrated some years ago in Rozengurt's laboratory (11-13) and by others (33) that adenosine is a potent mitogen for Swiss 3T3 cells, and this is also true for certain adenosine analogues such as NECA.In this paper, we show that exogenous ATP is a mitogen for 3T3, 3T6, A431, HFF (34), and DDT1-MF2 (35) cells. We confirm that adenosine is also mitogenic, but several lines of evidence show that ATP acts independently, in its own right, and not by being first converted to adenosine by ectoenzymes.
MATERIALS AND METHODS
Materials. [3H]Thymidine was purchased from Amersham.Culture media and fetal bovine serum (FBS) were from GIBCO. ATP, adenosine, epidermal growth factor (EGF), adenosine 5'-[8,y-imido]triphosphate (AdoPP[NH]P), and all other nucleotides were from Boehringer Mannheim. Bovine insulin, NECA, and phorbol 12-tetradecanoate 13-acetate (PTA) were from Sigma. Platelet-derived growth factor (PDGF) was obtained from Amgen Biologicals. All other chemicals were of the highest purity available.Cell Culture. Swiss 3T3 and 3T6 mouse fibroblasts, human A431 epidermoid carcinoma cells (14), and DDT1-MF2 cells (35), derived from a vas deferens tumor of the hamster, were maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO) containing 10 mM Hepes, 44 mM NaHCO3, penicillin (110 units/ml), streptomycin (100 ,ug/ml), and 0.5% (vol/vol) (3T6), 5% (A431, DDT1-MF2), or 10% (3T3) FBS.Cells were grown at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. Two days after seeding, the 3T3 cells were given fresh medium supplemented as before and then allowed to become confluent and quiescent for 5 days. The A431, DDT1-MF2, and 3T6 cultures were used 4, 5, or
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