ANALYSIS OF PLASMA-MEMBRANE CA2-ATPASE EXPRESSION IN CONTROL AND SV40-TRANSFORMED HUMAN FIBROBLASTS()

It has been long known that neoplastic transformation is accompanied b y a lowered requirement for extracellular Ca2+ for growth. The studies presented here demonstrate that human fibroblastic cell lines produce the two commonly found 'housekeeping' isoforms of the plasma membrane Ca2+-ATPase (PMCA), PMCA1b and 4b, and that the expression of both is demonstrably lower in cell lines neoplastically transformed by SV40 t han in the corresponding parental cell lines. Western blot analyses of lysates from control (GM00037) and SV40-transformed (GM00637) skin fi broblasts revealed a 138 kDa PMCA whose level was significantly lower in the SV40-transformed cells relative to either total cellular protei n or a-tubulin. Similar analyses of plasma membrane preparations from control (WI-38) and SV40-transformed (WI-38VA13) lung fibroblasts reve aled 3-4-fold lower levels of PMCA in the SV40-transformed cells. Comp etitive ELISAs performed on detergent solubilized plasma membrane prep arations indicated at least 3-4-fold lower levels of PMCA in the SV40- transformed cell lines compared to controls. Reverse transcriptase cou pled-PCR analyses showed that PMCA1b and PMCA4b were the only isoforms expressed in all four cell lines. The PMCA4b mRNA level detected by N orthern analysis also was substantially lower in SV40 transformed skin fibroblasts than in non-transformed fibroblasts. Quantitative RT-PCR analyses showed levels of PMCA1b and 4b mRNAs to be 5 and 10-fold lowe r, respectively, in GM00637 than in GM00037 when the levels of PCR pro ducts were normalized to glyceraldehyde-3-phosphate dehydrogenase (G3P DH) mRNA. These results demonstrate that the expression of these disti nct PMCA genes is substantially lower in SV40 transformed human skin a nd lung fibroblasts and may be coordinately regulated in these cells.