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Insulin-like growth factor-I (IGF-I) increases extracellular acidification rate (ECAR), a measure correlated with proliferation for nonautocrine cells. To evaluate the effect of autocrine IGF-I secretion on cell responsiveness, a cell line that secretes IGF-I was tested. SV40-lGF-I cells also registered concentration-dependent increases in ECAR; however, unlike the parental cell line, signal attenuation upon repeat challenges was not evident. Furthermore, SV40-IGF-I cells did not proliferate in response to IGF-I. We investigated if lack of proliferation was due to differences in the protocols of the assays ([3H]thymidine incorporation and microphysiometry). We identified three key differences in the protocols: surface substrate, cell density, and fluid residence time. We found no increase in [3H]thymidine incorporation for cells on either tissue-culture plastic or polycarbonate transwells. Control levels of [3H]thymidine incorporation were cell-density-dependent, but IGF-I did not increase proliferation at any density studied. Finally, we investigated IGF-I stimulation for cells under microphysiometer flow conditions and found no proliferative response to IGF-I. We found that the cells do respond to IGF-I with increased amino acid uptake. These data suggest that IGF-I signaling is operational in the SV40-IGF-I cells, but the transduction pathway for IGF-I-induced proliferation is compromised, despite the fact that these cells respond to fetal bovine serum with increased growth. Ongoing studies are focused on identifying which elements in the signaling cascade are altered by autocrine secretion of IGF-I.

Binding of growth factors by cell-surface receptors is an essential means by which cells regulate normal tissue growth and differentiation. Exposure to growth factors is often transient, and our goal was to determine whether short-term exposure to insulin-like growth factor-1 (IGF-1) would lead to activation, assayed as cell proliferation, of mammary epithelial cells. The MAC-T cell line is an immortalized bovine mammary epithelial cell line, chosen as our model mammary cell line because of its known sensitivity to IGF-1. Using the Cytosensor Microphysiometer System, a biosensor capable of measuring extracellular acidification, we were able to measure activation of the cells owing to IGF-1 addition in real time and found that peak acidification occurred in only 14 min. We show that this rapid response to IGF-1 is dose dependent and specific for IGF-1. A significant increase in [3H]thymidine incorporation by cells after a similar short-term exposure to IGF-1 suggests that the measured increase in extracellular acidification following IGF-1 addition is physiologically relevant. This technology offers a new, novel, and rapid means for the study of IGF-1 activity, as well as the screening of IGF-1 inhibitors, in mammary epithelial cells.