Extracellular nucleotides stimulate proliferation in MCF-7 breast cancer cells via P-2-purinoceptcrs

Nucleotides such as ATP can act as extracellular effector molecules by interaction with specific cellular receptors known as P-2(-) purinoceptors. Recently, we cloned the human P-2U purinoceptor from osteoclastoma and demonstrated its expression in skeletal tissues. In the current study we have investigated the expression of P-2U purinoceptors in human breast tumour cell lines and examined functional effects of extracellular nucleotides on these cells. By reverse transcription-linked polymerase chain reaction (RT-PCR) the expression of mRNA for P-2U purinoceptors was demonstrated in four human breast cancer cell lines, Hs578T, MCF-7, SK-Br3 and T47-D. In MCF-7 cells, extracellular ATP (1-100 mu M) elevated intracellular free calcium concentration [Ca2+](i), indicating that these cells express functional P-2-purinoceptors. UTP elevated [Ca2+](i) in an identical manner to ATP, whereas 2-methylthioATP was completely ineffective, and ADP only partially effective. This pharmacological profile suggests that the P-2 subtype may be the only P-2-purinoceptor expressed by these cells. The functional significance of P-2U purinoceptor expression by MCF-7 cells was investigated by analysing the effects of extracellular ATP on cell proliferation. The slowly hydrolysed analogue of ATP, ATP gamma S (which was also shown to elevate [Ca2+](i)), induced proliferation of MCF-7 cells when added daily to serum-free cultures over a period of 3 days. ASP gamma S-induced proliferation was demonstrated by three separate methods, detection by scintillation counting of [H-3]thymidine incorporation, immunocytochemical detection of 5-bromo-2-deoxyuridine incorporation and direct counting or cell numbers. These data suggest that ATP, possibly released at sites of tissue injury or inflammation, may be capable of growth factor action in promotion of tumour proliferation or progression.