Direct Involvement of Intracellular Ca2+ Transport ATPase in the Development of Thapsigargin Resistance by Chinese Hamster Lung Fibroblasts
1995; Elsevier BV; Volume: 270; Issue: 20 Linguagem: Inglês
10.1074/jbc.270.20.12140
ISSN1083-351X
AutoresArif Hussain, Christine Garnett, Michael G. Klein, Jyy-Jih Tsai-Wu, Martin F. Schneider, Giuseppe Inesi,
Tópico(s)RNA and protein synthesis mechanisms
ResumoThapsigargin (TG), a specific inhibitor of intracellular Ca2+ transport ATPases (SERCA), inhibits cell proliferation when added to culture media in the nanomolar concentration range. However, long term exposure to gradually increasing concentrations of TG induces resistance to TG inhibition in both the parental Chinese hamster lung fibroblast DC-3F and a subline derived from it via transfection and stable expression of a full-length cDNA encoding avian SERCA1 ATPase (DC-3F/Ca cells). TG resistance develops in parallel with selection of cells expressing higher levels of the endogenous SERCA2 as well as of the exogenous transfected SERCA1 ATPase, whose Ca2+ transport function can be studied in situ by imaging techniques and following isolation in microsomal fractions. Microsomes isolated from resistant cells contain two functionally distinct populations of ATPases: a population that is inhibited by stoichiometric titration with TG, and a population displaying resistance to inhibition even when TG exceeds the enzyme stoichiometry. It is apparent that resistance to TG develops in parallel with (a) selection of cells expressing high levels of SERCA ATPases, and (b) selection of an ATPase that is resistant to TG. Thapsigargin (TG), a specific inhibitor of intracellular Ca2+ transport ATPases (SERCA), inhibits cell proliferation when added to culture media in the nanomolar concentration range. However, long term exposure to gradually increasing concentrations of TG induces resistance to TG inhibition in both the parental Chinese hamster lung fibroblast DC-3F and a subline derived from it via transfection and stable expression of a full-length cDNA encoding avian SERCA1 ATPase (DC-3F/Ca cells). TG resistance develops in parallel with selection of cells expressing higher levels of the endogenous SERCA2 as well as of the exogenous transfected SERCA1 ATPase, whose Ca2+ transport function can be studied in situ by imaging techniques and following isolation in microsomal fractions. Microsomes isolated from resistant cells contain two functionally distinct populations of ATPases: a population that is inhibited by stoichiometric titration with TG, and a population displaying resistance to inhibition even when TG exceeds the enzyme stoichiometry. It is apparent that resistance to TG develops in parallel with (a) selection of cells expressing high levels of SERCA ATPases, and (b) selection of an ATPase that is resistant to TG. INTRODUCTIONThe Sarco-Endoplasmic Reticulum Ca2+ (SERCA)1 1The abbreviations are: SERCAsarco/endoplasmic reticulum Ca2+TGthapsigarginCHLChinese hamster lungMTXmethotrexateMOPS4-morpholinepropanesulfonic acid. ATPases are key enzymes in the regulation of cytosolic Ca2+ through the establishment and maintenance of intracellular Ca2+ stores. Specific inhibitors of the SERCA pumps have been identified, of which the sesquiterpene lactone thapsigargin (TG) is the most potent and specific (1Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (2986) Google Scholar, 2Sagara Y. Inesi G. J. Biol. Chem. 1991; 266: 13503-13506Abstract Full Text PDF PubMed Google Scholar, 3Kijima Y. Ogunbunmi E. Fleischer S. J. Biol. Chem. 1991; 266: 22912-22918Abstract Full Text PDF PubMed Google Scholar, 4Campbell A.M. Kessler P.D. Sagara Y. Inesi G. Fambrough D.M. J. Biol. Chem. 1991; 266: 16050-16055Abstract Full Text PDF PubMed Google Scholar, 5Lytton J. Westlin M. Hanley M.R. J. Biol. Chem. 1991; 266: 17067-17071Abstract Full Text PDF PubMed Google Scholar). Exposure of cell cultures to TG results in several effects, including alterations in intracellular Ca2+ homeostasis and inhibition of cell proliferation (6Gill D.L. Ghosh T.K. Bian J. Short A.D. Waldron R.T. Rybak S.L. Adv. Second Mess. Phosphoprotein Res. 1992; 26: 265-308PubMed Google Scholar, 7Short A.D. Bian J. Ghosh T.K. Waldron R.T. Rybak S.L. Gill D.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4986-4990Crossref PubMed Scopus (247) Google Scholar). However, resistance to TG inhibition of cell proliferation can be developed (8Gutheil J.C. Hart S.R. Belani C.P. Melera P.W. Hussain A. J. Biol. Chem. 1994; 269: 7976-7981Abstract Full Text PDF PubMed Google Scholar) and is associated with TG-resistant ATP-dependent calcium transport into intracellular stores, as shown in situ(9Waldron R.T. Short A.D. Hussain A. Gill D.L. FASEB J. 1994; 8: A1416Google Scholar). To facilitate characterization of the TG-resistant enzyme, we have used a cell line transfected and selected for stable expression of exogenous SERCA1 ATPase (DC-3F/Ca cells), in addition to a parent line (DC-3F cells) sustaining only expression of endogenous ATPase. We describe here the functional properties of the SERCA ATPases affected by the development of resistance to TG, both in situ and following isolation.EXPERIMENTAL PROCEDURESCell LinesDC-3F is a cell line that was originally established from Chinese hamster lung (CHL) fibroblasts (10Biedler J.L. Riehm H. Cancer Res. 1970; 30: 1174-1184PubMed Google Scholar). As described previously, DC-3F/Ca was derived from DC-3F by transfection of the dual promoter expression vector pHβFCaA3 containing a mutant methotrexate (MTX)-resistant dihydrofolate reductase (DHFR) cDNA (under the control of a SV4O promoter) and an avian SERCA1-encoding cDNA (under the control of a β-actin promoter), followed by selection in MTX (11Hussain A. Lewis D. Sumbilla C. Lai L.C. Melera P.W. Inesi G. Arch. Biochem. Biophys. 1992; 296: 539-546Crossref PubMed Scopus (10) Google Scholar). TG-resistant cell lines (DC-3F/TG and DC-3F/CaTG) were developed via stepwise selection of DC-3F or DC-3F/Ca cells in gradually increasing concentrations of TG (LC Laboratories) over the course of several months, with the final maintenance concentration being 2 μM TG for each line (8Gutheil J.C. Hart S.R. Belani C.P. Melera P.W. Hussain A. J. Biol. Chem. 1994; 269: 7976-7981Abstract Full Text PDF PubMed Google Scholar). All these cell lines were maintained in α-minimum essential medium media (Sigma) supplemented with 7% fetal calf serum (Life Technologies, Inc.) plus appropriate concentrations of drug where indicated. Thus, the cells were maintained in the following: DC-3F/Ca in 25 μg/ml MTX, DC-3F/TG in 2 μM TG, DC-3F/CaTG in 25 μg/ml MTX plus 2 μM TG. Parental DC-3F cells were cultured in the absence of any drug. In some of the experiments with DC-3F/CaTG cells where TG was removed from the culture media for various lengths of time, the cells were still kept on 25 μg/ml MTX.Cell Growth AssaysCells were plated at 10,000-15,000 cells/plate in 35-mm plates and incubated overnight at 37°C. TG was added the next day, and the cells were incubated for an additional 72 h at 37°C, then trypsinized and counted.Microsome PreparationThe procedure was described by Sumbilla et al.(12Sumbilla C. Lu L. Lewis D.G. Inesi G. Ishii T. Takeyasu K. Feng Y. Fambrough D.M. J. Biol. Chem. 1993; 268: 21185-21192Abstract Full Text PDF PubMed Google Scholar).Immunodetection StudiesImmunofluorescent detection of transfected SERCA1 in the cells was carried out as described by Karin et al.(13Karin N.J. Kaprielian Z. Fambrough D.M. Mol. Cell. Biol. 1989; 9: 1978-1986Crossref PubMed Scopus (70) Google Scholar).Determination of SERCA protein in microsomes was obtained via SDS-polyacrylamide gel electrophoresis gels (7.5% polyacrylamide), run as per the method of Laemmli (14Laemmli U.K Nature. 1970; 227: 680-685Crossref PubMed Scopus (205964) Google Scholar) and blotted onto nitrocellulose paper. The filters were blocked with 3% gelatin in Tris-buffered saline solution for 1 h at room temperature, and then probed with either the SERCA1 CaF3-5C3 antibody or a mouse SERCA2 antibody that recognizes both isoforms of SERCA2 (IID8 F6) (15Jorgensen A.O. Arnold W. Pepper D.R. Kahl S.D. Mandel F. Campbell K.P. Cell Motil. Cytoskel. 1988; 9: 164-174Crossref PubMed Scopus (76) Google Scholar). Bound antibody was detected with a secondary antibody (goat anti-mouse IgG conjugated to horseradish peroxidase) and a commercially available ECL kit (Amersham Corp.). Densitometry was carried out using an LKB Ultroscan XL enhanced laser densitometer.High Resolution Ca2+ ImagingThe procedures for subcellular imaging of Ca2+ stores in permeabilized cells were as described by Short et al.(16Short A.D. Klein M.G. Schneider M.F. Gill D.L. J. Biol. Chem. 1993; 268: 25887-25893Abstract Full Text PDF PubMed Google Scholar), who demonstrated that under appropriate permeabilization conditions, fura-2 remains trapped within intracellular organelles where it reports the local [Ca2+]. Ca2+ uptake into organelles was initiated by adding ICM plus 3 mM ATP and 1 or 10 μM free Ca2+. TG was added to the appropriate solution as indicated, from a 1000-fold concentrated stock solution dissolved in Me2SO. Changes produced by Ca2+ binding to fura-2 within the organelles were determined by fluorescence microscopy, using dual excitation fluorescence at 380 and 358 nm, nearest neighbor deblurring of images at 2-μm focal planes for each wavelength and conversion to the pseudo-color images using image processing procedures described by Short et al.(16Short A.D. Klein M.G. Schneider M.F. Gill D.L. J. Biol. Chem. 1993; 268: 25887-25893Abstract Full Text PDF PubMed Google Scholar).Ca2+ Transport StudiesCa2+ transport was assayed by incubating 5 μg/ml DC-3F/Ca or DC-3F/CaTG microsomes, or 10 μg/ml DC-3F/TG microsomes, in a reaction mixture containing 20 mM MOPS, pH 7.0, 80 mM KCl, 5 mM MgCl2, 0.2 mM CaCl2 supplemented with 45Ca2+ (specific activity of 3000 dpm/nmol), 5 mM K+ oxalate, and 0.2 mM EGTA at 37°C. For the Ca2+ affinity experiments, free Ca2+ concentrations were calculated from total Ca2+ and EGTA according to Fabiato and Fabiato (17Fabiato A. Fabiato F. J. Physiol. 1978; 276: 233-255Crossref PubMed Scopus (827) Google Scholar). The reaction was initiated by the addition of 5 mM ATP. At sequential times (up to 60-90 min), 1-ml samples were filtered through a 0.45-μm Millipore filter and quenched with 20 ml of chilled lanthanum chloride/MOPS, pH 7.0, solution. Ca2+ uptake was determined by quantitating the radioactivity on the filters via liquid scintillation counting and was expressed per mg of microsomal protein.RESULTSCharacterization of the TG-resistant Cell LinesFig. 1 demonstrates that the DC-3F/TG and the DC-3F/CaTG cell lines, developed by exposure to increasing concentrations of TG, are highly resistant to inhibition by TG. That is, DC-3F/TG and the DC-3F/CaTG cells are nearly 3 orders of magnitude more resistant to TG than parental DC-3F and DC-3F/Ca cells.It can be also shown by immunofluorescence staining that resistance to TG develops in parallel with selection of cells expressing high levels of SERCA ATPase. In fact, it is shown in Fig. 2(compare A and C) that in the DC-3F/Ca population used in these studies, more than 50% of the cells have lost expression of the transfected SERCA1 when compared to the original DC-3F/Ca cell line previously described (11Hussain A. Lewis D. Sumbilla C. Lai L.C. Melera P.W. Inesi G. Arch. Biochem. Biophys. 1992; 296: 539-546Crossref PubMed Scopus (10) Google Scholar). On the other hand, it is clear that 100% of the cells express SERCA1 ATPase following selection of DC-3F/Ca in TG (compare B and D). Furthermore, the overall expression of SERCA1 within each cell in the TG-resistant population appears to be increased, as judged by fluorescence intensity. Western blotting of microsomal fractions reveals that SERCA expression is greatly increased following the acquisition of resistance to TG, with expression of the exogenous SERCA1 being 20-fold higher and that of the endogenous SERCA2 being 2-fold higher in DC-3F/CaTG when compared to DC-3F/Ca cells (Fig. 3, Table I).Figure 2:Immunofluorescent visualization of SERCA1 in DC-3F/Ca (panels A and C) and DC-3F/CaTG (i.e. TG-resistant) cells (B and D). Panels A and B show phase contrast images of a field of cells. Panels C and D show the corresponding immunofluorescence images. Cells were fixed and stained with a primary antibody to SERCA1, then a secondary FITC-conjugated goat anti-mouse IgG, as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3:Western blotting. Microsomal fractions were obtained from DC-3F/Ca and DC-3F/CaTG and analyzed with SERCA isoform-specific antibodies. Top row, blot probed with an antibody (CaF3-5C3) directed against the exogenously transfected and expressed avian SERCA1. Lane 1, DC-3F/Ca (5 μg of microsomal protein); lanes 2-4, DC-3F/CaTG (0.5, 1.0, and 5.0 μg of microsomal protein, respectively). Bottom row, a separate blot was probed with an antibody (IID8 F6) that recognizes the endogenous SERCA2 (IID8 F6 cannot distinguish between SERCA2a and SERCA2b). Lane 1, DC-3F/Ca (5 μg); lane 2, DC-3F/CaTG (5 μg). Relative SERCA1 content is 20-fold greater and SERCA2 content 2-fold greater in DC-3F/CaTG than in DC-3F/Ca.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table ICharacteristics of DC-3F/CaTG microsomes isolated from cells harvested after removal of TG from the culture media Open table in a new tab Evaluation of Ca2+ Storing Capacity in SituDirect in situ imaging of Ca2+ within the organelles of DC-3F/Ca and DC-3F/CaTG cells was undertaken following the loading of organelles with fura-2 and permeabilization of the plasma membrane with saponin. In these studies, the ratio of fura-2 fluorescence at excitation wavelengths of 380 and 358 nm provided a relative estimate of the Ca2+ concentration within the organelles, represented by pseudo-color images of the perme-abilized cells in Fig. 4. Fig. 4B (compared to Fig. 4A) shows that in permeabilized DC-3F/Ca cells, exposure to ICM plus 1 μM free Ca2+ and 3 mM ATP resulted in uptake of Ca2+ into the organelles. Ca2+ uptake was dependent on the presence of both Ca2+ and ATP (not shown) and was completely blocked by 2 μM TG (Fig. 4, C and D). No additional uptake was observed in the presence of ICM containing ATP plus 10 μM Ca2+ (not shown).Figure 4:Pseudo-color Ca2+ images of permeabilized DC-3F cells. Within each pair of images, left panel shows the organellar Ca2+ image just before, and right panel after exposure of the cells to ICM containing 1 μM Ca2+ and 3 mM ATP. Each pair of images shows a different cell. The Ca2+ calibration is given as the calculated [Ca2+] normalized to the K of the indicator (see text). The 10-μm bar reflects relative cell size. A and B, DC-3F/Ca cells. C and D, DC-3F/Ca cells in the presence of 2 μM TG. E and F, DC-3F/CaTG cells. G and H, DC-3F/CaTG cells in the presence of 4 μM TG. I and J, parental DC-3F cells. Note that DC-3F/Ca cells take up Ca2+ to levels about 2-fold higher than DC-3F/CaTG cells; the parental line takes up relatively little Ca2+.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4 (E and F) shows that the organelles of DC-3F/CaTG cells are able to sustain ATP-dependent Ca2+ uptake, which is not inhibited by 4 μM TG (Fig. 4, G and H). Ca2+ uptake within the DC-3F/CaTG cells, however, was lower than in DC-3F/Ca cells by about one-half, even though the immunofluorescence results in Fig. 2 suggest that DC-3F/CaTG cells have significantly higher SERCA1 expression than the DC-3F/Ca cells. The reduced Ca2+ uptake in the DC-3F/CaTG cells may in part be a consequence of partial inhibition of the SERCA ATPases by the TG originally present in the culture medium. It should be pointed out that Ca2+ uptake within the DC-3F/CaTG cells is still much higher than that obtained with the parental line DC-3F (Fig. 4, compare H with J).Owing to uncertainties in the estimation of the affinity of fura-2 for Ca2+ within the organelles, we have expressed the calibration of fluorescence ratio as [Ca2+] normalized to the Kdof the indicator. In spite of this approximation, the images shown in Fig. 4, which are examples derived from observations made in the course of 11 independent experiments, give a clear indication of the occurrence of TG sensitive and TG-resistant Ca2+ uptake by ER stores in situ. A more quantitative evaluation was obtained by measuring directly the ATP-dependent Ca2+ uptake by microsomal vesicles isolated from the cells (see below).It is noteworthy that the uptake studies in permeabilized cells were carried out in the presence of FCCP, which inhibits uptake of Ca2+ by mitochondria. Therefore, the contribution of mitochondrial Ca2+ in our imaging studies was negligible.Ca2+ Transport in Isolated MicrosomesThese studies were best performed with microsomes isolated from DC-3F/Ca cells due to the relatively high expression of SERCA1 and, hence, the relatively high rates of Ca2+ transport in such cells. The activities of microsomes prepared from DC-3F/Ca and DC-3F/CaTG (TG-resistant) cells are shown in Fig. 5A. The average initial rates of Ca2+ transport are 21.2 ± 2.3 nmol of Ca2+ uptake/mg of protein/min for DC-3F/Ca microsomes and 14.2 ± 2.6 nmol of Ca2+ uptake/mg of protein/min for the DC-3F/CaTG microsomes (Table I). Therefore, the microsomal membranes of the TG-resistant cells retain approximately half of their overall capacity for Ca2+ transport as compared with controls (i.e. DC-3F/Ca cells), which have not been exposed to TG. Since the SERCA1 content is approximately 20-fold greater and SERCA2 content is 2-fold greater in the microsomes derived from the TG-resistant cells (Fig. 3 and), the percentage of functional SERCA ATPase in the DC-3F/CaTG cells is apparently less than 5%, due to the inhibition produced by the TG present in the culture media until cell harvest. Nevertheless, this residual activity is much higher than that recovered from (nontransfected) DC-3F or DC-3F/TG cells (Fig. 5A).Figure 5:Ca2+ transport activity of microsomes. A, microsomal vesicles were obtained from DC-3F/Ca (■), DC-3F/CaTG (●), DC-3F (◊), and DC-3F/TG cells (▼). The DC-3F/CaTG and DC-3F/TG cells were maintained on TG until the day of microsomal harvest, but the reaction medium for Ca2+ uptake contained no TG. B, Ca2+ uptake rates were determined in the presence of various concentrations of TG added to the reaction medium for Ca2+ uptake. Rates of Ca2+ transport (expressed as fractional values of the maximal rate obtained in the absence of TG) are plotted as a function of TG concentration for microsomes obtained from DC-3F/Ca (■), DC-3F/CaTG (●), and DC-3F/TG cells (▼).View Large Image Figure ViewerDownload Hi-res image Download (PPT)When Ca2+ uptake by isolated microsomes is measured as a function of the concentration of TG added to the reaction mixture for Ca2+ uptake, 50% inhibition of the maximal rates of uptake requires 100 nM TG for the microsomes obtained from DC-3F/CaTG cells, as compared to 0.1 nM TG for the microsomes obtained from DC-3F/Ca cells (Fig. 5B). Most importantly, the low (Fig. 5A) activity recovered from (nontransfected) DC-3F/TG cells requires TG concentrations in the micromolar range for inhibition (Fig. 5B). In fact, this low level of highly resistant ATPase may be present even in the DC-3F/CaTG microsomes as a small percentage of the total ATPase, whose inhibition spans a wide range of TG concentrations. Note that in Fig. 5B the activities are normalized; in fact, in DC-3F/TG it is only a small fraction of the DC-3F/CaTG activity (Fig. 5A).Removal of TG from Culture Media following Acquisition of Resistance by DC-3F/CaTG CellsSince stepwise selection in TG results in the overexpression of TG-resistant SERCA pumps in the DC-3F/CaTG cell line, we explored the consequences of removing TG selection on the functional properties of SERCA in these resistant lines. Thus, in some experiments, TG was removed from the culture media and the microsomes subsequently harvested for analysis after 2, 6, or 12 days following the TG removal.Western blot analysis reveals that SERCA1 expression gradually decreases upon removal of TG, so that 12 days after TG removal the overall expression of SERCA1 is reduced to one-third of that seen when cells are continuously maintained on TG (Fig. 6, Table I). Expression of the endogenous SERCA2 also decreases in DC-3F/CaTG as these cells are kept off TG (Fig. 6).Figure 6:Analysis of DC-3F/CaTG cells taken off TG. Western blotting of microsomal fractions obtained from DC-3F/CaTG at various times following removal of TG from the culture medium. SERCA1: lane 1, DC-3F (5 μg); lane 2, DC-3F/Ca (10 μg); lane 3, microsomes from DC-3F/CaTG cells maintained on TG until day of harvest (2 μg); lane 4, microsomes from DC-3F/CaTG harvested 2 days following removal of TG from culture medium (2 μg); lane 5, 6 days following TG removal (2 μg); lane 6, 12 days following TG removal (2 μg). SERCA2: lane 1, DC-3F (40 μg); lane 2, DC-3F/TG (40 μg); lane 3, DC-3F/Ca (40 μg); lane 4, microsomes from DC-3F/CaTG cells maintained on TG until day of harvest (40 μg); lane 5, microsomes from DC-3F/CaTG cells harvested 2 days following removal of TG from the culture medium (40 μg); lane 6, 6 days (40 μg); lane 7, 12 days (40 μg).View Large Image Figure ViewerDownload Hi-res image Download (PPT)When the microsomes obtained serially from DC-3F/CaTG cells following withdrawal of TG were tested for their Ca2+ transport function, we found a 4-fold increase in the overall Ca2+ transport activity after only 2 days following TG withdrawal, as compared to DC-3F/CaTG cells that had been continuously maintained on TG (Table I). This overall increase in activity tapered off in several days following withdrawal of TG (Table I). Considering the overall expression of SERCA protein, and assuming no changes in ATPase turnover, our experiments indicate that the percentage of functional enzyme was reduced to 5% as a consequence of continuous exposure to TG, and then increased gradually by the 12th day following removal of TG. Furthermore, SERCA-dependent Ca2+ transport became progressively more sensitive to TG inhibition as microsomes were harvested following withdrawal of TG from the cell culture media (Table I, Fig. 7). It is noteworthy that even though a minimal change in overall SERCA1 and SERCA2 expression occurred within the first 2 days following withdrawal of TG (Fig. 6), the ED50 value for TG inhibition of Ca2+ transport by isolated microsomes decreased by more than 16-fold (Fig. 7, Table I). This suggests that a small fraction of the SERCA pump is TG-insensitive and operative primarily in the TG-resistant cells, while operation of a TG-sensitive enzyme becomes more prominent following withdrawal of TG.Figure 7:Rates of Ca2+ transport (expressed as fractional values of maximal rate of transport obtained in the absence of TG) are plotted as a function of TG concentration. ●, microsomes from DC-3F/CaTG cells maintained on TG until day of harvest; □, microsomes from DC-3F/CaTG harvested 2 days following removal of TG from the culture medium; +, 6 days; ▲, 12 days; ■, DC-3F/Ca, i.e. no exposure to TG in the culture medium. Experimental points are the average of three measurements for two different microsomal preparations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Ca2+ Transport as a Function of Ca2+ ConcentrationAs the SERCA enzymes require Ca2+ for activation of their function, characterization of these ATPases in the TG-resistant line DC-3F/CaTG was extended to determine the Ca2+ concentration dependence of their activation. Fig. 8 compares the rates of Ca2+ transport as a function of Ca2+ concentration for microsomes obtained from DC-3F/CaTG and from the same cell line harvested 12 days after withdrawal of TG. These experiments show hardly any difference in the Ca2+ concentration range required for activation of microsomes obtained from cells exposed to TG until harvest or cells harvested 12 days following removal of TG. Therefore, it is apparent that TG resistance does not impair the ability of the SERCA pumps to function within the Ca2+ concentration range required for homeostasis.Figure 8:Ca2+ dependence of Ca2+ transport by various microsomes. Microsomes obtained from DC-3F/CaTG maintained on TG until day of harvest (□) or harvested 12 days following removal of TG from the culture medium (▲). The experimental points are averages from three experiments. In each experiment the velocity was obtained from six time points taken over a 40-min incubation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONInterference with various cell functions and signaling pathways as a consequence of specific inhibition of SERCA ATPases by TG has been reported in several studies (for recent review, see Ref. 18Inesi G. Sagara Y. J. Membr. Biol. 1994; 141: 1-6Crossref PubMed Scopus (159) Google Scholar). An important effect of TG is the inhibition of cell proliferation in parallel with depletion of intracellular Ca2+ stores (6Gill D.L. Ghosh T.K. Bian J. Short A.D. Waldron R.T. Rybak S.L. Adv. Second Mess. Phosphoprotein Res. 1992; 26: 265-308PubMed Google Scholar). It is of interest that DC-3F cells develop resistance to TG and continue to proliferate following gradual exposure to increasing concentrations of the inhibitor (8Gutheil J.C. Hart S.R. Belani C.P. Melera P.W. Hussain A. J. Biol. Chem. 1994; 269: 7976-7981Abstract Full Text PDF PubMed Google Scholar). Although a 2-3-fold overexpression of the multidrug resistance transporter p-glycoprotein occurs in association with the development of resistance to TG, this modest overexpression of the drug transporter does not account for the very large (more than 2 orders of magnitude) increase in resistance of DC-3F cells to TG (8Gutheil J.C. Hart S.R. Belani C.P. Melera P.W. Hussain A. J. Biol. Chem. 1994; 269: 7976-7981Abstract Full Text PDF PubMed Google Scholar). Therefore, with the experiments described in this report we attempted to clarify whether SERCA ATPases are directly involved in the mechanism of TG resistance. A most important finding is that microsomes obtained from cells exposed for a long time to inhibitory concentrations of TG retain residual Ca2+ transport activity. This demonstrates that, under our experimental conditions, resistance to TG is due to factors related directly to the ATPase.It has been clearly demonstrated that, due to its extremely high affinity, the amount of TG required to inhibit isolated wild-type ATPase is stoichiometrically equivalent to the target protein, even when the ATPase is present at subnanomolar concentrations (2Sagara Y. Inesi G. J. Biol. Chem. 1991; 266: 13503-13506Abstract Full Text PDF PubMed Google Scholar). For this reason, the higher TG concentration required to obtain inhibition of microsomes obtained from the DC-3F/CaTG cells (Fig. 5B) may be related to the higher ATPase content of these microsomes, in addition to the presence of TG-resistant ATPase within these cells. On the other hand, the disproportionately high concentrations of TG (Fig. 5B) required to inhibit the very low amount of ATPase (Figs. 5A and 6) recovered from the (nontransfected) DC-3F/TG cells must be attributed to a distinct, TG-resistant SERCA ATPase, which may correspond to an endogenous isoform selected during the induction of TG resistance. Therefore, the TG-resistant cells developed in our experiments contain two ATPase populations: a population which is inactivated by stoichiometric titration with TG (prevalent in the transfected DC-3F/CaTG cells) and a population which is resistant to TG and derives in DC-3F/CaTG from exogenous and/or endogenous ATPase, and in DC-3F/TG from an endogenous isoform.The overexpression of the TG sensitive SERCA, in addition to the TG-resistant SERCA, indicates that the experimental protocol used here favors the selection of cells with high expression of SERCA ATPases. Resistance to TG then develops in parallel with (a) selection of cells expressing high level of SERCA ATPases and (b) selection of a TG-resistant SERCA ATPase isoform. It is apparent that overexpression of SERCA1 and SERCA2 ATPases does not completely account for the TG resistance acquired by the DC-3F/CaTG and DC-3F/TG cells. Ultimately, selection of a TG-resistant SERCA ATPase is required to provide sufficient function to sustain cytosolic Ca2+ housekeeping and proliferation of TG-resistant cells. The TG-resistant ATPase could derive from a “latent” inherently resistant endogenous SERCA isoform which may be present in at least a few of the DC-3F cells. The latter suggestion is consistent with the known presence of TG-resistant SERCA ATPase isoforms in platelets (19Tao J. Haynes D.H. J. Biol. Chem. 1992; 267: 24972-24982Abstract Full Text PDF PubMed Google Scholar) and in association with the inositol 1,4,5-trisphophate-insensitive Ca2+ stores (20Bian J. Ghosh T.K. Wang J.C. Gill D.L. J. Biol. Chem. 1991; 266: 8801-8806Abstract Full Text PDF PubMed Google Scholar). INTRODUCTIONThe Sarco-Endoplasmic Reticulum Ca2+ (SERCA)1 1The abbreviations are: SERCAsarco/endoplasmic reticulum Ca2+TGthapsigarginCHLChinese hamster lungMTXmethotrexateMOPS4-morpholinepropanesulfonic acid. ATPases are key enzymes in the regulation of cytosolic Ca2+ through the establishment and maintenance of intracellular Ca2+ stores. Specific inhibitors of the SERCA pumps have been identified, of which the sesquiterpene lactone thapsigargin (TG) is the most potent and specific (1Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (2986) Google Scholar, 2Sagara Y. Inesi G. J. Biol. Chem. 1991; 266: 13503-13506Abstract Full Text PDF PubMed Google Scholar, 3Kijima Y. Ogunbunmi E. Fleischer S. J. Biol. Chem. 1991; 266: 22912-22918Abstract Full Text PDF PubMed Google Scholar, 4Campbell A.M. Kessler P.D. Sagara Y. Inesi G. Fambrough D.M. J. Biol. Chem. 1991; 266: 16050-16055Abstract Full Text PDF PubMed Google Scholar, 5Lytton J. Westlin M. Hanley M.R. J. Biol. Chem. 1991; 266: 17067-17071Abstract Full Text PDF PubMed Google Scholar). Exposure of cell cultures to TG results in several effects, including alterations in intracellular Ca2+ homeostasis and inhibition of cell proliferation (6Gill D.L. Ghosh T.K. Bian J. Short A.D. Waldron R.T. Rybak S.L. Adv. Second Mess. Phosphoprotein Res. 1992; 26: 265-308PubMed Google Scholar, 7Short A.D. Bian J. Ghosh T.K. Waldron R.T. Rybak S.L. Gill D.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4986-4990Crossref PubMed Scopus (247) Google Scholar). However, resistance to TG inhibition of cell proliferation can be developed (8Gutheil J.C. Hart S.R. Belani C.P. Melera P.W. Hussain A. J. Biol. Chem. 1994; 269: 7976-7981Abstract Full Text PDF PubMed Google Scholar) and is associated with TG-resistant ATP-dependent calcium transport into intracellular stores, as shown in situ(9Waldron R.T. Short A.D. Hussain A. Gill D.L. FASEB J. 1994; 8: A1416Google Scholar). To facilitate characterization of the TG-resistant enzyme, we have used a cell line transfected and selected for stable expression of exogenous SERCA1 ATPase (DC-3F/Ca cells), in addition to a parent line (DC-3F cells) sustaining only expression of endogenous ATPase. We describe here the functional properties of the SERCA ATPases affected by the development of resistance to TG, both in situ and following isolation.
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