Elevated Calcium in Preneoplastic Cells Activates NF-κB and Confers Resistance to Apoptosis

Early preneoplastic cells (sup+) exhibit increased susceptibility to apoptosis, which is lost in late stage preneoplastic cells (sup−). Sup+ cells, which undergo apooptosis when cultured in low serum, show little or no DNA binding activity to nuclear factor (NF)-κB either in 10% or 0.2% serum. In contrast sup− cells, which are resistant to apoptosis in low serum, show a sustained constitutive activation of NF-κB. The constitutive activation of NF-κB observed in sup− cells is not due to loss of IκBα. We considered that the activation of NF-κB in sup− cells might be secondary to an increase in cytosolic Ca2+, since sup− cells have a cytosolic Ca2+ level that is double that in sup+ cells. In support of a role for Ca2+, lowering cytosolic Ca2+ in sup− cells by addition of the cell-permeable Ca2+ chelator 1,2 bis(O-aminophenoxy)ethane-N, N, N′, N′-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) reduced cytosolic Ca2+ by ∼31% relative to untreated sup− cells, concomitant with a 65% reduction in NF-κB DNA binding activity and a reduction in IκB kinase (IKK) activity. In sup− cells in low serum, addition of BAPTA-AM also resulted in a significant (∼50%) increase in caspase-3 activity. Raising extracellular Ca2+ in sup+ cells resulted in a slight activation of IκB kinase and in enhanced NF-κB DNA binding activity. Using proteasome and calpain inhibitors, we determined that the basal activity of NF-κB in sup− cells is largely proteasome-independent, but sensitive to calpain inhibitors. Taken together these data suggest that the elevated Ca2+ in sup− cells causes a modest activation of IKK, which likely contributes to the enhanced basal activation of NF-κB in sup− cells; however, the predominant effect of Ca2+ appears to be mediated by Ca2+-enhanced degradation by calpain.

It has been shown that signals that lead to proliferation frequently stimulate apoptosis unless they are counteracted by specific survival signals (1)(2)(3). Thus it is not surprising that a common feature of early preneoplastic cells is a susceptibility to apoptosis, which is overcome in later stage neoplastic cells by either stimulating survival signals or by inhibiting apoptotic signals (2,4). To better understand how cells overcome apoptosis during neoplastic progression, we have examined variants of Syrian hamster embryo cells that have been mutagenized to yield two immortalized cell lines representing different stages of neoplastic progression. It has been shown previously that early preneoplastic cells (supϩ) are more susceptible to apoptosis than late preneoplastic cells (supϪ) (4). The mechanism involved in this altered susceptibility to apoptosis could have important implications for carcinogenesis.
Many apoptotic stimuli activate NF-B 1 (8,9), and it was originally assumed that the increase in NF-B activity might be involved in stimulating apoptosis. However, recent studies have shown that if NF-B activation is inhibited, apoptosis is enhanced (5)(6)(7). Because of the importance of NF-B in apoptosis we were interested in investigating whether there were differences in NF-B activity in early versus late preneoplastic cells, as these cells have differing sensitivity to apoptosis.
NF-B is a transcription factor that is activated by numerous cytokines and stresses. NF-B is normally maintained in an inactive state in the cytoplasm, because it is bound to inhibitors of the IB family. Phosphorylation of IB targets it for degradation, thereby leading to activation of NF-B. We found a sustained basal activation of NF-B in late (supϪ), but not early stage (supϩ) preneoplastic cells. The sustained activation of NF-B in the supϪ cells correlates with their resistance to apoptosis following treatment with medium containing low serum, a condition that leads to apoptosis in the supϩ cells. We examined possible mechanisms for the sustained activation of NF-B in the supϪ cells and noted a correlation with elevated cytosolic calcium. The late preneoplastic supϪ cells have a basal cytosolic free Ca 2ϩ level that is more than double that observed in the early preneoplastic supϩ cells. An elevation in cytosolic Ca 2ϩ is commonly noted in transformed cells and has been proposed to be important in signaling cell proliferation (10,11). In this study, we present data showing that an elevation in cytosolic Ca 2ϩ can also provide survival signals by contributing to a sustained activation of NF-B.

EXPERIMENTAL PROCEDURES
Cell Culture-Two cell lines (supϩ and supϪ), originally immortalized via asbestos mutagenesis of Syrian hamster embryo cells, were used in these studies (12). Cells were maintained in Dulbecco's modified IBR medium containing 10% fetal calf serum (FCS), 100 units/ml penicillin, and 100 g/ml streptomycin. Cultures were maintained in a 37°C incubator with 10% CO 2 /90% air. Low serum conditions were either 0.2 or 0.8% fetal calf serum, depending upon the age of the serum. As the serum aged, later experiments required the higher percentage of fetal calf serum.
Measurement of NF-B DNA Binding Activity-Extracts of nuclear proteins were prepared using a modification of the method of Dignam et al. (13). Syrian hamster embryo cells were plated at a density of 1.1 ϫ 10 6 cells/150-mm diameter plate in 20 ml of IBR medium with 10% FCS for 24 h, then switched to various treatments for 16 -18 h. Cells were scraped into medium plus 20 ml of cold calcium-and magnesium-free phosphate-buffered saline (CMF-PBS), centrifuged at 480 ϫ g, and then washed in 10 ml of cold CMF-PBS. Following centrifugation, cell pellets were resuspended in 500 l of lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 0.1% IGEPAL, 0.5 mM dithiothreitol, 1.0 mM orthovanadate, 0.4 mM phenylmethylsulfonyl fluoride, and 2 g/ml each of aprotinin, leupeptin, and pepstatin), vortexed, and the nuclei pelleted by centrifugation at 10,000 ϫ g for 5 min. Nuclear pellets were resuspended in 30 l of extraction buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 25% glycerol, 0.5 mM dithiothreitol, 1.0 mM orthovanadate, 0.4 mM phenylmethylsulfonyl fluoride, and 2 g/ml each of aprotinin, leupeptin, and pepstatin) and centrifuged at 16,000 ϫ g for 15 min. Supernatants were collected and protein content quantified using the Pierce BCA assay.
Binding reactions for NF-B/DNA electrophoretic mobility shift assays were performed for 20 min at room temperature in binding buffer (15 mM HEPES, pH 7.9, 50 mM NaCl, 0.5 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 0.5 mM PMSF) using 10 g of nuclear protein, 169 pg of 32 P-labeled oligonucleotide probe (5Ј-AGTTGAGGGGACTTTCCCAGG-C-3Ј), and 1.5 g of poly(dI-dC)⅐poly(dI-dC) in a total volume of 18 l. Bound complexes were resolved on 6% polyacrylamide nondenaturing gels, and densitometry of autoradiographs was performed using the software package NIH Image.
Calcium Measurements-Cytosolic calcium was measured as described previously (14). Briefly, cells were plated at a density of 6 ϫ 10 4 cells/plate in IBR medium with 10% serum in 30-mm plates containing 22-mm diameter round glass coverslips. Fura-2 AM (2 M) was introduced to each plate 30 min prior to measurements. Following the loading period, coverslips were washed twice with CMF-PBS and placed in a custom-built holder. The entire unit was placed on the stage of a Nikon inverted epifluorescence microscope coupled to a PTI Deltascan dual excitation wavelength spectrofluorometer. Measurements were taken in a window containing two to six cells. Cells were excited at 340 and 380 nm, and emitted fluorescence was measured at 510 nm. Background fluorescence, measured from a field not containing cells, was subtracted.
Caspase-3 Activity Assay-Cells were seeded in 100-mm plates at a density of 5 ϫ 10 5 cells/plate and grown for 24 h. Cells were washed with CMF-PBS, and 10 ml of the appropriate treatment medium was added. After 16 -18 h, cells (adherent and floating cells) were harvested by scraping into treatment medium followed by centrifugation at 480 ϫ g for 5 min at 4°C. The cells were resuspended in 5 ml of cold CMF-PBS, centrifuged as above, and the resulting pellets lysed in 40 l of hypotonic lysis buffer (25 mM HEPES, pH 7.5, 5 mM MgCl 2 , 5 mM EDTA, 5 mM dithiothreitol, 2 mM PMSF, 10 g/ml pepstatin A, 10 g/ml leupeptin) followed by four cycles of freezing and thawing in liquid nitrogen. The lysate was centrifuged in a 4°C microcentrifuge for 20 min at 16,000 ϫ g. Supernatants were collected and protein content quantified using the Pierce BCA assay. The caspase-3 activity assay was performed according to the protocol in the CaspACE Assay System (Promega). Data are expressed as caspase activity (change in arbitrary fluorescence units) corrected for background caspase activity.
IKK Activity Assays-Activity of IB kinase (IKK) was determined by immunoprecipitation followed by a phosphorylation assay. Cells were seeded in 100-mm diameter plates at a density of 5 ϫ 10 5 cells/plate and grown for 24 -40 h before treatments. Whole cell lysates were prepared by scraping the cells into medium, centrifuging at 480 ϫ g, and washing the pellets twice with 5 ml of cold CMF-PBS. Pellets were lysed in 100 l of lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0, 1% Nonidet P-40, 10 mM p-nitrophenyl phosphate, 10 mM ␤-glycerophosphate, 1 mM benzamidine, 1 mM PMSF, 10 g/ml aprotinin, 1 g/ml pepstatin). Lysates were then centrifuged at 14,000 rpm in a microcentrifuge for 30 min at 4°C and the supernatants taken for immunoprecipitation, which was performed in 500 l of lysis buffer for 2-3 h at 4°C using 200 g of cell lysate and an agarose-conjugated antibody to IKK-␣ (Santa Cruz Biotechnology catalog number sc-7182AC). Immunoprecipitates were washed four times with lysis buffer and once with kinase buffer (Cell Signaling Technology catalog number 9802), supplemented with 10 mM p-nitrophenyl phosphate, 2 mM MnCl 2, 1 mM benzamidine, 1 mM PMSF, 10 g/ml aprotinin, and 1 g/ml leupeptin. Kinase reactions were carried out for 30 min at 30°C in 30 l of kinase buffer containing 2 g of GST-IB-␣ substrate (Santa Cruz Biotechnology catalog number 4094), 10 Ci of [␥-32 P]ATP, and 10 M ATP. Reactions were terminated with 5ϫ Laemmli sample buffer and boiled 5 min at 95°C before performing SDS-polyacrylamide gel electrophoresis using 8% Tris-glycine gels. Densitometry of autoradiographs was performed using NIH image.
Western Blots-Cells were plated at a density of 1.1 ϫ 10 6 cells/ 150-mm diameter plate in 20 ml of IBR medium with 10% serum for 24 h, then switched to various treatments for 16 -18 h. Whole cell lysates were prepared by scraping the cells into medium plus 20 ml of cold CMF-PBS, centrifuging at 480 ϫ g, and then washing two times in 5 ml of cold CMF-PBS. Pellets were then lysed in 500 l of lysis buffer (1% Nonidet P-40, 0.5% SDS, 150 mM NaCl, 50 mM Tris, pH 7.4) supplemented with protease and phosphatase inhibitors (2 g/ml leupeptin, 2 g/ml pepstatin A, 2 g/ml aprotinin, 400 M PMSF, 1 mM sodium orthovanadate). Protein concentrations were determined using the Pierce BCA assay, and 25 g of total protein per lane was loaded on a 10% acrylamide SDS-polyacrylamide gel electrophoresis gel. Following Western blotting to a nitrocellulose membrane, IB-␣ and IB-␤ were detected using Santa Cruz antibodies sc-371 and sc-945, respectively, and Bcl-2 was detected using Transduction Laboratories antibody number B46620.
Materials-The NF-B oligonucleotide probe was purchased from Promega, proteinase K from Boerhinger-Mannheim, and RNase A from 5 Prime 3 3 Prime. BAY 11-7082, lactacystin, proteasome inhibitor-I, EST, and calpain inhibitor-V were obtained from Calbiochem. All other chemicals were acquired from Sigma/Aldrich. IBR medium was obtained from Life Technologies Inc., and fetal calf serum was obtained from Summit Technologies.
Statistics-Results are expressed as mean Ϯ S.E. For comparison between two groups, statistical significance was determined by Student's t test. For multiparameter comparisons, statistical significance was determined by analysis of variance, adjusting for multiple comparisons using Tukey's post hoc test. A value of p Ͻ 0.05 was considered to be significant.

RESULTS
Previous studies have shown that early stage preneoplastic supϩ cells have enhanced susceptibility to apoptosis under antiproliferative conditions such as treatment with low serum, whereas later stage preneoplastic supϪ cells do not (4). We were interested in determining the basis for this difference in susceptibility to apoptosis. We examined whether there were differences in Bcl-2 expression levels between supϩ and supϪ cells. As shown in Fig. 1, there is no significant difference in Bcl-2 levels in supϩ and supϪ cells.
Because NF-B activation has been shown to be anti-apoptotic, we next investigated whether differences in NF-B activation between the two cell lines might explain the difference in apoptotic susceptibility. To determine whether NF-B activation differs in supϩ versus supϪ cells, we cultured each cell line in medium supplemented with 10% FCS for 24 h. The medium containing 10% FCS was then replaced with medium containing either 10% or low serum for an additional 18 h prior to cell harvest. Extracts enriched for nuclear proteins were prepared and analyzed by electrophoretic mobility shift assay (EMSA). Supϩ cells showed relatively little DNA binding activity of NF-B, either in 10% or low serum (Fig. 2, lanes 1 and  3). In contrast, supϪ cells showed constitutive NF-B activation in 10% FCS (ϳ5.3 times supϩ levels in 10% serum) and even stronger activation when switched to low serum (ϳ15.8 times supϩ levels in low serum).
Because supϪ cells have constitutive activation of NF-B and decreased susceptibility to apoptosis in low serum, we tested whether pharmacological lowering of NF-B in supϪ cells would render them more susceptible to apoptosis. BAY 11-7082 has been reported to inhibit phosphorylation of IB and thus to decrease NF-B activation (15,16). SupϪ cells were cultured for 24 h and then treated for 18 h with 1 or 2 M BAY 11-7082. NF-B activation was decreased with BAY 11-7082 treatment (Fig. 3A). BAY 11-7082 treatment also inhibited IKK activity (Fig. 3B). Similarly treated supϪ cells were assayed for apoptotic induction using caspase-3 activity as a measure of apoptosis after 18 h treatment in low FCS, with and without BAY 11-7082. As shown in Fig. 3C, caspase-3 activity was significantly higher in low serum-treated supϪ cells in the presence of BAY 11-7082, compared with supϪ cells in low serum alone. Fig. 3C further shows that caspase-3 activity in supϪ cells in low serum plus BAY 11-7082 was similar to that in supϩ cells in low serum.
We were interested in the mechanism responsible for the constitutive activation of NF-B in the supϪ cells. Constitutive activation of NF-B is observed in many tumor and viralinfected cells. Some tumor cells have constitutive activation of NF-B due to mutation or loss of IB␣ (17,18). However, loss of IB␣ does not account for the constitutive activation of NF-B in supϪ cells, because we found slightly increased levels of IB␣ in supϪ versus supϩ cells (see Fig. 4A). An increase in IB␣ in supϪ cells is consistent with constitutive activation of NF-B in these cells, because expression of IB␣ has been shown to be up-regulated by NF-B (19,20). Constitutive activation of NF-B can also occur because of hypophosphorylation of IB␤ (21). However, as shown in Fig. 4B, IB␤ has a similar electrophoretic mobility in supϩ and supϪ cells, suggesting a similar level of phosphorylation.
Interestingly, as shown in Fig. 5, supϪ cells, which have constitutive activation of NF-B, have a 2-fold increase in cytosolic Ca 2ϩ compared with supϩ cells, which lack basal NF-B activation. Cytosolic free Ca 2ϩ in supϪ cells was 220 Ϯ 26 nM, a value double that measured in the supϩ cells (97 Ϯ 18 nM). An elevation in Ca 2ϩ has been suggested to activate NF-B (22)(23)(24)(25)(26). To determine whether the constitutive activation of NF-B in supϪ cells is related to the observed increase in cytosolic free Ca 2ϩ , we treated supϪ cells in medium plus 10% serum with the cell-permeable Ca 2ϩ chelator BAPTA-AM. Cells were grown for 24 h and then treated with 10 M BAPTA-AM for 1 or 2 h. Base-line NF-B binding activity decreased to ϳ34% of control at 1 and 2 h after treatment with BAPTA-AM (Fig. 6). To determine whether BAPTA-AM treatment in fact lowered cytosolic Ca 2ϩ levels, intracellular Ca 2ϩ was measured spectrofluorometrically in fura-2-loaded cells. Cells were treated with BAPTA-AM for 2 h, loaded with fura-2 AM for 30 min, washed in PBS, and fluorescence was measured. Cytosolic Ca 2ϩ levels of BAPTA-AM-treated supϪ cells decreased by ϳ31% compared with untreated supϪ cells (Fig.  7). Thus, lowering cytosolic Ca 2ϩ levels with BAPTA-AM also lowers NF-B binding activity. Furthermore, as shown in Fig.  8, lowering cytosolic Ca 2ϩ with BAPTA-AM resulted in a significant increase in caspase-3 activity measured at 4 h after treatment of supϪ cells in low serum. SupϪ cells placed in low serum showed only minimal activation of caspase-3. However,

FIG. 2. NF-B activation is increased in sup؊ cells compared with sup؉ cells.
EMSA was performed to assess NF-B DNA binding activity. Nuclear extracts were prepared from cells treated for 18 h in either 10% or low serum. Specificity of the NF-B band was determined by electrophoretic mobility supershift (data not shown). The gel is representative of eight similar experiments.

Calcium, NF-B, and Apoptosis
lowering cytosolic Ca 2ϩ with BAPTA-AM resulted in a 48% increase in caspase-3 activity (Fig. 8). Caspase-3 activity was measured at 4 h to ensure that measurements were made at a time point comparable with the NF-B and cytosolic Ca 2ϩ measurements, as well as to ensure that BAPTA-AM was still present in the cells. These data suggest that the lowering cytosolic Ca 2ϩ in the supϪ cells causes a decrease in NF-B activation and an increased susceptibility to apoptosis. Thus, reducing NF-B in supϪ cells increases apoptosis, as would be expected if activation of NF-B in supϪ cells is responsible for their observed resistance to apoptosis.
We also tested whether elevating calcium in supϩ cells by raising extracellular calcium would alter NF-B. As shown in Fig. 9, raising extracellular Ca 2ϩ to 3 mM also resulted in a slight but significant activation of NF-B. We also investigated whether altering cell calcium altered the activity of IKK. As shown in Fig. 10, raising extracellular calcium also caused a significant, but modest, 39% increase in IKK activity in supϩ cells. In contrast, lowering cytosolic calcium with addition of BAPTA-AM significantly decreased (by 66%) IKK activity in supϪ cells.
Raising extracellular Ca 2ϩ in supϩ has a very modest effect of NF-B activation and IKK activity. These data contrast with the robust Ca 2ϩ dependence of IKK and NF-B activity in supϪ cells. Fig. 11 shows the relationship of cytosolic Ca 2ϩ to NF-B activation and to IKK activity, standardized to their activities in supϩ cells in 10% FCS. In supϩ cells, increased cytosolic Ca 2ϩ results in a proportionately similar increase in IKK and NF-B. SupϪ cells show a Ca 2ϩ -related increase in IKK, which is similar to that observed in supϩ cells; however, in supϪ cells increased Ca 2ϩ correlates with a much larger percentage increase in NF-B activity versus IKK activity. These data suggest that a large proportion of the enhancement of NF-B activation attributable to Ca 2ϩ is not mediated by IKK.
To better understand the mechanism involved in the Ca 2ϩinduced activation of NF-B, we examined the effect of proteasome and calpain inhibition on the basal activity of NF-B in supϪ cells. Fig. 12A shows that neither the proteasome inhibitor lactacystin nor proteasome inhibitor-I reduced the level of basal NF-B activity. Fig. 12A further demonstrates that the inability of proteasome inhibitors to block basal NF-B activation is not due to lack of an effective concentration, because both inhibitors significantly inhibited the LPS-induced activation of NF-B. Since the basal NF-B activity in supϪ cells is Ca 2ϩ -dependent, but proteasome-independent, we examined whether calpain inhibition would affect basal NF-B activity. As shown in Fig. 12B, the calpain inhibitors EST and calpain inhibitor-V significantly reduced basal NF-B activity. Taken together these data suggest that the elevated Ca 2ϩ in supϪ cells causes a modest activation of IKK, which likely contributes to the enhanced basal activation of NF-B in supϪ cells; however, the predominant effect of Ca 2ϩ appears to be mediated by Ca 2ϩ -enhanced degradation by calpain. DISCUSSION Two preneoplastic Syrian hamster embryo cell lines representing different stages of neoplastic progression have been used for these studies. Early stage preneoplastic cells (supϩ) have been shown to suppress tumorogenicity when hybridized with tumor cells, whereas supϪ cells do not suppress tumorogenicity in cell hybrids. The RB and p53 genes are wild type in both cell types. Previous studies have shown that the supϩ cells are more susceptible to apoptosis than the later stage preneoplastic supϪ cells (4,27).
In this paper we report that late stage preneoplastic cells that are resistant to apoptosis have a high basal level of NF-B activation. As NF-B has been reported to be anti-apoptotic, we examined whether pharmacologically reducing NF-B in supϪ cells would enhance apoptosis. As shown in Figs. 3 and 8, . Note that the gels and densitometry in A and B are from different gels and therefore cannot be compared directly. In a separate experiment we measured IKK activity in supϩ and supϪ cells (10%) with the same exposure on the same gel and found that supϩ cells had an activity that was ϳ53% of that observed in supϪ cells. *, indicates significantly different than pooled controls (p Ͻ 0.05).
reducing the level of NF-B in the supϪ cells, with either BAY 11-7082 or BAPTA-AM, enhanced their susceptibility to apoptosis in low serum. A common feature of neoplastic progression is a reduced susceptibility to apoptosis, although early preneoplastic cells often exhibit enhanced apoptosis (28). It has been shown that activation of cell proliferation can lead to apoptosis unless cell survival pathways such as NF-B are also activated (1)(2)(3). This study suggests that increased activation of NF-B is involved in the reduced apoptotic susceptibility of the late stage preneoplastic supϪ cells. To gain insight into how cells activate NF-B as part of the neoplastic progression, we investigated the mechanism by which NF-B is activated under basal conditions in supϪ cells. Loss or mutation of IB␣ has been reported to activate NF-B in some tumor cells (17,18). However, loss of IB␣ does not account for the enhanced NF-B levels in supϪ cells. In fact we find enhanced levels of IB␣ in supϪ cells, consistent with NF-B regulation of the promoter for IB␣ (19,20). Hypophosphorylated IB␤ has also been reported to lead to constitutive activation of NF-B (21). However, we find no evidence for hypophosphorylation of IB␤.
There are recent data suggesting that Ca 2ϩ can modulate NF-B activity (22-26, 29 -34). Since we find a greater than 2-fold higher basal cytosolic Ca 2ϩ concentration in supϪ cells compared with supϩ cells, we considered the possibility that the elevation in cytosolic Ca 2ϩ might be involved in the increased NF-B activation observed in the supϪ cells. In support of this hypothesis, we report that lowering cytosolic Ca 2ϩ by addition of the cell-permeant Ca 2ϩ chelator BAPTA-AM to supϪ cells reduced NF-B activation and significantly reduced IKK activity. Furthermore, the BAPTA-AM-dependent lowering of NF-B binding also rendered the supϪ cells susceptible to apoptosis in low serum. These data suggest that the increased NF-B binding in the supϪ cells is secondary to the increase in Ca 2ϩ and is responsible for the reduced apoptosis observed in the supϪ cells.
An elevation in cytosolic Ca 2ϩ is a common observation in transformed cells (10). It has been shown that lowering extracellular Ca 2ϩ results in a reversible block of the cell cycle in normal cells, whereas SV40-transformed cells continue to proliferate in low Ca 2ϩ media (11). The data in this manuscript are consistent with the hypothesis that high cytosolic Ca 2ϩ in transformed cells may provide the cell with a reduced susceptibility to undergo apoptosis by activating NF-B, an antiapoptotic factor.
The mechanism by which Ca 2ϩ regulates NF-B is unclear, and there are data to suggest that multiple pathways may be involved (22)(23)(24)(25)(26)(31)(32)(33)(34). Ca 2ϩ is reported to activate IKK (via calcineurin, see Refs. 23 and 32), and Ca 2ϩ is also reported to activate calpain-dependent degradation of IB (31,33). Lowering Ca 2ϩ in the supϪ cells causes a large reduction in NF-B, consistent with the hypothesis that the higher Ca 2ϩ in supϪ cells is necessary for the higher basal NF-B. However, raising Ca 2ϩ in the supϩ cells only slightly elevates NF-B and is not sufficient to elevate NF-B to the same level as found in supϪ cells. Thus the data suggest that high Ca 2ϩ in the supϪ cells is required for activation of NF-B, but raising Ca 2ϩ in the supϩ cells is not sufficient. Furthermore, the data in Fig. 11 suggest that the constitutive activation of NF-B in supϪ cells is Ca 2ϩdependent, but largely IKK-independent. These data are consistent with Ca 2ϩ activation of NF-B in supϪ cells being primarily mediated by calpain. Calpain is a Ca 2ϩ -activated protease, which has been reported to play a role in the degradation of IB␣ (31), and calpain 3 deficiency is associated with increased apoptosis in limb-girdle muscular dystrophy type 2A (33). Also, these data are similar to those observed in primary B cells that have constitutive activation of NF-B. Fields et al. (30) have reported that the constitutive activation of NF-B in B lymphocytes is calcium-dependent, but proteasome-independent.
In summary, many transformed cells exhibit an increase in cytosolic Ca 2ϩ . We demonstrate that lowering cytosolic Ca 2ϩ reduces the activation of NF-B and confers susceptibility to FIG. 11. Relationship of NF-B binding activity and IKK activity to cytosolic Ca 2؉ levels. NF-B binding activity, IKK activity, and calcium are expressed as a percentage of the respective supϩ 10% FCS controls. Calcium levels for supϩ cells were measured in 10% FCS or in 10% FCS supplemented with 3 mM Ca 2ϩ . Calcium levels for supϪ cells were measured in 10% FCS or in 10% FCS plus 10 M BAPTA-AM (4-h treatment). apoptosis in supϪ cells. We further show that the constitutive activation of NF-B observed in supϪ cells is proteasomeindependent. Taken together these data suggest that an elevation in Ca 2ϩ , which is common in tumor cells, may provide these cells with resistance to apoptosis via activation of NF-B.