Increased Calcium Influx and Ribosomal Content Correlate with Resistance to Endoplasmic Reticulum Stress-induced Cell Death in Mutant Leukemia Cell Lines

Cell clones were derived by treatment of HL-60 cells with stepwise increasing concentrations of econazole (Ec), an imidazole antifungal that blocks Ca2+ influx and induces endoplasmic reticulum (ER) stress-related cell death in multiple mammalian cell types. Clones exhibit 20- to more than 300-fold greater resistance to Ec. Unexpectedly, they also display stable cross-resistance to tunicamycin, thapsigargin, dithiothreitol, and cycloheximide but not doxorubicin, etoposide, or Fas ligand. Phenotypic analysis indicates that the cells display increased store-operated calcium influx and resistance to ER Ca2+ store depletion by Ec. E2R2, the most resistant clone, was observed to maintain protein synthesis levels after treatment with Ec or thapsigargin. Expression of GRP78, an ER-based chaperone, was induced by these ER stress treatments but to equal degrees in HL-60 and E2R2 cells. By using microarray analysis, at least 15 ribosomal protein genes were found to be overexpressed in E2R2 compared with HL-60 cells. We also found that ribosomal protein content was increased by 30% in E2R2 as well as other clones. The resistance phenotype was partially reversed by the ribosome-inactivating protein saporin. Therefore, increased store-operated calcium influx, resistance to ER Ca2+ store depletion, and overexpression of ribosomal proteins define a novel phenotype of ER stress-associated multidrug resistance. Cell clones were derived by treatment of HL-60 cells with stepwise increasing concentrations of econazole (Ec), an imidazole antifungal that blocks Ca2+ influx and induces endoplasmic reticulum (ER) stress-related cell death in multiple mammalian cell types. Clones exhibit 20- to more than 300-fold greater resistance to Ec. Unexpectedly, they also display stable cross-resistance to tunicamycin, thapsigargin, dithiothreitol, and cycloheximide but not doxorubicin, etoposide, or Fas ligand. Phenotypic analysis indicates that the cells display increased store-operated calcium influx and resistance to ER Ca2+ store depletion by Ec. E2R2, the most resistant clone, was observed to maintain protein synthesis levels after treatment with Ec or thapsigargin. Expression of GRP78, an ER-based chaperone, was induced by these ER stress treatments but to equal degrees in HL-60 and E2R2 cells. By using microarray analysis, at least 15 ribosomal protein genes were found to be overexpressed in E2R2 compared with HL-60 cells. We also found that ribosomal protein content was increased by 30% in E2R2 as well as other clones. The resistance phenotype was partially reversed by the ribosome-inactivating protein saporin. Therefore, increased store-operated calcium influx, resistance to ER Ca2+ store depletion, and overexpression of ribosomal proteins define a novel phenotype of ER stress-associated multidrug resistance. Calcium signals play a central role in many cellular activities including cell movement, secretion, proliferation, gene transcription, and cell death (1Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1773) Google Scholar, 2Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4451) Google Scholar). In non-excitable cells, the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; Ec, econazole; SOC, store-operated calcium; Tg, thapsigargin; DTT, dithiothreitol; FBS, fetal bovine serum; PBS, phosphate-buffered saline; WT, wild type; TRP, transient receptor potential. plays a key role in calcium signaling. Receptor-mediated activation of phospholipase C generates the second messenger inositol 1,4,5-triphosphate that diffuses rapidly through the cytosol to interact with inositol 1,4,5-triphosphate receptor on the ER membrane releasing calcium stored in the ER lumen. The resulting depletion of calcium within the ER triggers calcium entry from the extracellular milieu through the plasma membrane. This capacitive or store-operated calcium (SOC) influx (3Putney Jr., J.W. McKay R.R. BioEssays. 1999; 21: 38-46Crossref PubMed Scopus (357) Google Scholar) serves to both expand the initial calcium signal and replenish the emptied ER store (4Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1291) Google Scholar, 5Barritt G.J. Biochem. J. 1999; 337: 153-169Crossref PubMed Scopus (279) Google Scholar). Although the nature of SOC channel remains unclear, the importance of SOC influx in maintaining cell viability has been demonstrated in different studies (6Kohn E.C. Sandeen M.A. Liotta L.A. Cancer Res. 1992; 52: 3208-3212PubMed Google Scholar, 7Estacion M. Mordan L.J. Cell Calcium. 1993; 14: 439-454Crossref PubMed Scopus (40) Google Scholar, 8Buckley N.E. Su Y. Milstien S. Spiegel S. Biochim. Biophys. Acta. 1995; 1256: 275-283Crossref PubMed Scopus (22) Google Scholar, 9Gommerman J.L. Berger S.A. Blood. 1998; 91: 1891-1900Crossref PubMed Google Scholar, 10Aktas H. Fluckiger R. Acosta J.A. Savage J.M. Palakurthi S.S. Halperin J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8280-8285Crossref PubMed Scopus (124) Google Scholar, 11Soboloff J. Berger S.A. J. Biol. Chem. 2002; 277: 13812-13820Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 12Soboloff J. Zhang Y. Minden M. Berger S.A. Exp. Hematol. 2002; 30: 1219-1226Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 13Zhang Y. Crump M. Berger S.A. Breast Cancer Res. Treat. 2002; 72: 265-278Crossref PubMed Scopus (19) Google Scholar). In addition to its key role in Ca2+ signaling, the ER is a site for protein synthesis, and these two functions intersect under conditions of ER stress. For instance, treatment of cells with thapsigargin (Tg), the inhibitor of the sarcoplasmic/endoplasmic Ca2+-ATPase responsible for transporting Ca2+ into the ER, rapidly depletes ER Ca2+ stores (14Golovina V.A. Blaustein M.P. Science. 1997; 275: 1643-1648Crossref PubMed Scopus (425) Google Scholar), inhibits the function of Ca2+-dependent chaperones, and triggers the unfolded protein response (15Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1934) Google Scholar). Econazole (Ec) is an antifungal imidazole that depletes Ca2+ from the ER of mammalian cells and blocks Ca2+ influx (16Gamberucci A. Fulceri R. Benedetti A. Bygrave F.L. Biochem. Biophys. Res. Commun. 1998; 248: 75-77Crossref PubMed Scopus (21) Google Scholar, 17Jan C.R. Ho C.M. Wu S.N. Tseng C.J. Biochim. Biophys. Acta. 1999; 1448: 533-542Crossref PubMed Scopus (46) Google Scholar). These effects result in sustained depletion of Ca2+ from ER stores and profound inhibition of protein synthesis leading to cell death (11Soboloff J. Berger S.A. J. Biol. Chem. 2002; 277: 13812-13820Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). We have shown that some transformed cells exhibit several orders of magnitude greater sensitivity to Ec than normal cells (12Soboloff J. Zhang Y. Minden M. Berger S.A. Exp. Hematol. 2002; 30: 1219-1226Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 13Zhang Y. Crump M. Berger S.A. Breast Cancer Res. Treat. 2002; 72: 265-278Crossref PubMed Scopus (19) Google Scholar). This enhanced sensitivity may be due to the activated nature of tumor cells since we have shown that concurrent stimulation of cells with growth factors such as steel factor (9Gommerman J.L. Berger S.A. Blood. 1998; 91: 1891-1900Crossref PubMed Google Scholar, 11Soboloff J. Berger S.A. J. Biol. Chem. 2002; 277: 13812-13820Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), epidermal growth factor, or bombesin (13Zhang Y. Crump M. Berger S.A. Breast Cancer Res. Treat. 2002; 72: 265-278Crossref PubMed Scopus (19) Google Scholar) enhances cell death; this is a process we refer to as activation-enhanced cell death. However, the nature of the differential sensitivity is not fully understood. To investigate further the molecular events associated with resistance and sensitivity to Ca2+ and ER stress-induced cell death, we developed mutant cell clones by selection of HL-60 leukemia cells with stepwise increasing concentrations of econazole. The resultant clones exhibit resistance to Ec and cross-resistance to other ER stress-inducing agents including Tg, tunicamycin (inhibitor of N-linked glycosylation), dithiothreitol (DTT; disulfide bond reducing agent), and cycloheximide (protein synthesis inhibitor). Phenotypic analysis indicates that these cells also exhibit resistance to Ec-induced ER Ca2+ depletion, display increased SOC influx, increased ribosomal protein content, and sustained protein synthesis levels in the presence of Ec and Tg. These studies therefore define a novel phenotype of multidrug resistance associated with inducers of ER stress. Culture and Selection Conditions—Human HL-60 promyelocytic leukemia cells were grown in RPMI 1640 containing 10% fetal bovine serum (FBS) and antibiotics. In order to generate resistant cells, the cells were exposed to stepwise increasing concentrations of Ec that caused a reduction in the proportion of viable cells by at least 90% followed by a recovery phase. At the end of a series of such steps, cells were cloned by limiting dilution and expanded for further study. To maintain the phenotype of resistance, the Ec-resistant clones designated E2R2, E2R4, E2R6 were challenged with econazole once a month to ensure stability of the resistant phenotype. All the phenotypic characterizations were performed 1 week following drug treatment, and the cell viability was higher than 95%. Clonogenicity Assays—To measure clonogenicity, HL-60 or resistant clone cells (5 × 104) were incubated in 100 μl of RPMI 1640 with for in the presence or of different of were from For treatment with cells were incubated in for in RPMI with FBS, and for an cells were in containing in RPMI were after in at and E2R2 cells were as for the for At the end of the cells were and the were in containing were observed under and for were as of cells was Calcium of were performed by HL-60 or resistant clone cells were for in (5 bovine serum were incubated in in two and incubated on for 15 to for the of to were performed using a to at and The cell was to and cells using The of Ca2+ was to is the of the at and the and is the at and were from in of cells using in the and and presence and of is the for at M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and and were at the of × were with PBS, and in RPMI with bovine serum were with Ec 15 or 100 for 15 cells were with for in two in were with in followed by were in and the protein were in and using a used in the the that and and gene and for the of the be found at site HL-60 and E2R2 cells were grown under conditions at cell The cells were and from HL-60 or E2R2 were were with and through transcription, and to the gene The were and through an The was by and analysis of microarray were performed using developed by P. P. M. J.A. PubMed Google Scholar). of ribosomal protein genes was further by from equal cell from both wild and E2R2 cells were on an containing the were and were performed at in The for the ribosomal protein genes and were from and the for the gene was cloned The were by with The were also for the gene as a for For analysis of the was performed and by and × HL-60 or resistant clone cells under conditions were with PBS, and to the by M. M.J. Mol. Biol. PubMed Scopus (19) Google Scholar). The ribosomal was through of on of a 1 at × to the The were in μl of and by in on for were with and by on a with ribosomal proteins were by on a The was with to the protein for the of an ER stress were performed by were with different ER stress-inducing agents as for the of treatment, the cells were and in containing and inhibitor by followed by an The proteins in the × were by the were for 1 at in containing and incubated with or were in and incubated with to were in and using the as the To the presence of ribosomal proteins in we used a that the ribosomal protein μl of ribosomal proteins or of proteins from of the cells were on incubated with and with to used in the of of both wild and E2R2 cells were with Ec or for 1 were in containing inhibitor and inhibitor and protein were on as of was by using an Cell The were also for as a were by analysis of with by SOC calcium influx was by under the of calcium influx with resistance was by were by were as Selection of HL-60 order to the of of the ER stress-inducing Ec, we developed Ec-resistant of the promyelocytic leukemia cell HL-60 by the cells with stepwise increasing concentrations of the drug followed by recovery in the of drug a of We these cells by limiting dilution clones of Ec-resistant cells designated to E2R6 were and of the clones the levels of resistance were different degrees of resistance to the drug Ec. At the the of resistance for mutant clones from to more than compared with wild HL-60 cells. these mutant E2R2 cells the resistance to Ec by and the was the least resistant By the mutant cells with Ec once a we have the resistant phenotype for more than Clones to ER from that Ec induces the depletion of Ca2+ from the ER resulting in profound inhibition of protein synthesis and cell death. We therefore the sensitivity of resistant clone cells to other ER stress-inducing By using a we found that the mutant clones were more resistant to Tg, tunicamycin, and In in sensitivity to such as and was observed in these cell not E2R2 cells the resistance to stress-inducing agents and were found to be the most they were for further analysis of HL-60 cells for with the agents of such as and In with this we have of and in cells with Ec, Tg, and Zhang and S. A. In E2R2 cells levels of in response to these agents for drug concentrations that to more than of ER stress-related in HL-60 cells, of the were to E2R2 cells The cross-resistance to these agents was not observed with inducers of cell death both cell sensitivity to and Fas agents that not to the These therefore that E2R2 cells for resistance to Ec exhibit cross-resistance to ER stress-associated in Calcium in Ec is to ER Ca2+ depletion and influx, we the Ca2+ content of the mutant cells. calcium concentrations were in HL-60 and resistant clone cells by In the for HL-60 and E2R2 cells was and found to be and 100 is an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ and of Ca2+ from the ER with capacitive influx (14Golovina V.A. Blaustein M.P. Science. 1997; 275: 1643-1648Crossref PubMed Scopus (425) Google Scholar). shown in stimulation with thapsigargin to a of was in both cell of a but magnitude followed by a and further of influx through SOC Ca2+ the and magnitude of the Ca2+ signal were greater in the resistant clone compared with cells. The of the for two cells were in E2R2 cells and in HL-60 cells Ca2+ content and Ca2+ influx The abbreviations used as under the SOC were derived by under the in the presence of from under the in its SOC were derived by under the in the presence of from under the in its in a In order to and influx, cells were with in the presence of a Ca2+ influx to influx In this the in HL-60 in E2R2 cells, and Ca2+ were that ER Ca2+ content was the in both cell but that the mutant cells greater store-operated In order to this Ca2+ influx was by Ca2+ to the following treatment in shown in both the influx and under the were in E2R2 compared with cells. We therefore that E2R2 cells exhibit Ca2+ levels and ER Ca2+ content compared with However, capacitive Ca2+ influx is higher in E2R2 cells in response to than By using the calcium we found that the other resistant clones and E2R6 also increased calcium influx ER calcium content as cells The increased calcium influx observed in these resistant clones was found to with the resistance resistance, to Ec the that increased Ca2+ influx with ER stress resistance. ER Calcium by with a (11Soboloff J. Berger S.A. J. Biol. Chem. 2002; 277: 13812-13820Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), we that Ec protein synthesis of cells through sustained depletion of ER calcium In order to investigate E2R2 cells were also resistant to ER calcium depletion, the cells were incubated with levels of Ec. E2R2 and cells were observed to levels of Ca2+ from stores The of Ca2+ into the is with from stores in the of However, the two cell were incubated with econazole in for at and Ca2+ content in the ER store was as shown in the ER Ca2+ store in cells was the mutant cells levels of ER These that Ec from the ER and influx in E2R2 cells, E2R2 cells of ER and the cells studies from other shown that overexpression of the to resistance to ER stress H. J.L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. R.J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, H. S. S. J. Cell. Biochem. 2000; 77: PubMed Scopus Google Scholar). the cross-resistance phenotype of the E2R2 cells, we therefore the levels of shown in was found to be overexpressed in both E2R2 and HL-60 cells following treatment with ER stress-inducing However, E2R2 cells not exhibit higher levels of than HL-60 cells. Therefore, overexpression not to to the cross-resistance phenotype in E2R2 cells. as is a factor that is in response to cellular stress including ER stress Jr., M. J. Mol. Cell. Biol. PubMed Scopus Google Scholar, M. Mol. Cell. Biol. 1993; 13: PubMed Scopus Google Scholar, J.W. H. A. L.J. R. Mol. Cell. Biol. PubMed Scopus Google Scholar). of in response to ER stress death, we levels of following to different ER stress-inducing HL-60 cells cells were induced to protein that in not associated with the resistance phenotype. of Genes in E2R2 order to further investigate the mutant and cells, we used microarray analysis to the gene of E2R2 cells with that of the wild HL-60 cells. The two cell were grown under conditions and to cell in order to associated with growth with we observed genes was increased in E2R2 cells compared with HL-60 cells. we observed that at least 15 of the ribosomal protein genes were overexpressed in E2R2 cells at levels from to genes ribosomal proteins of the of the and and ribosomal proteins of the of the and ribosomal protein genes by microarray protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein in a To further overexpression of ribosomal protein genes observed by the of ribosomal protein genes was by in wild and clones. shown in ribosomal protein genes and were found to be in E2R2 cells compared with HL-60 cells. with the microarray of the levels of the ribosomal protein genes on in these two cell in E2R2 cells In order to investigate these in gene were into we ribosomal protein content in the wild and resistant clone cell were by through a The presence of ribosomal protein in this was by with ribosomal shown in and of ribosomal proteins were from from 1 × E2R2 or HL-60 cells, increased ribosomal protein was by and was of the ribosomal protein genes in the microarray ribosomal content was also observed by followed by analysis ribosomal in E2R2 cells was also observed of in and E2R6 clones also increased ribosomal protein levels in these cells We therefore that the increased of ribosomal protein genes into increased ribosomal protein and content in resistant cells. E2R2 to of increased ribosomal content with that Ec inhibits protein synthesis to investigate protein synthesis in E2R2 and cells. shown in protein synthesis levels in cells were in the mutant and cells. observed in cells, of HL-60 cells to Ec or protein We found that or Ec of of Tg, and 30% of of Ec, In E2R2 cells protein synthesis at of protein synthesis in response to ER stress is through of at Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). We therefore the of in response to Ec and Tg. shown in and as observed in cells (11Soboloff J. Berger S.A. J. Biol. Chem. 2002; 277: 13812-13820Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), HL-60 cells challenged with these increased of In challenged E2R2 cells levels compared with WT, in with to protein synthesis in the presence of Ec and Tg. The to protein synthesis in or challenged cells that these cells may be resistant to protein synthesis shown in E2R2 cells exhibit profound resistance to the protein synthesis E2R2 cells were at least more resistant HL-60 cells to cycloheximide at the We that E2R2 cells also exhibit cross-resistance to the protein synthesis inhibitor of by the role of increased ribosomal protein content in resistance, we the of a protein from the on protein synthesis and by the of a bond of protein synthesis Biochim. Biophys. Acta. 1993; PubMed Scopus Google Scholar, A. M. M.J. M. J. PubMed Scopus Google Scholar). of HL-60 cells with protein synthesis by resistant clones were not the resistant cells were exposed to with Ec or Tg, protein synthesis was by cell viability as by that Ec and resistance was partially reversed in clones to degrees with the that of function partially resistance. In this we have and a of resistant clones from HL-60 cells by selection with increasing concentrations of Ec. These mutant cells exhibit resistance to Ec as well as cross-resistance to a of other ER stress-inducing but not other agents such as etoposide, doxorubicin, or Fas ligand. Phenotypic analysis that the mutant cells have normal levels of and ER Ca2+ levels but display increased capacitive Ca2+ influx, resistance to ER depletion by Ec, increased ribosomal content, maintain protein synthesis and following to Ec or Tg. The increased calcium influx in the mutant clones is with the resistance to Ec, and of resistance is induced with the protein saporin. these define a novel phenotype of ER stress-associated multidrug resistance. Although the ER calcium depletion to SOC activation remains is that SOC calcium influx, from the Ca2+ signal induced by also plays an role in the ER store G.J. Biochem. J. 1999; 337: 153-169Crossref PubMed Scopus (279) Google Scholar, Jr., J.W. J. Cell Sci. PubMed Google Scholar). We observed that a with Ec the ER store in HL-60 cells but not in E2R2 cells This of Ec has on but higher concentrations of the ER store in both cell with the that E2R2 cells have levels of Ca2+ in ER and increased SOC influx we that the increased SOC associated with E2R2 cells is responsible for the of calcium into the ER for the ER calcium store depletion caused by Ec. Although resistance to ER depletion by Ec may be to increased capacitive influx, not this increased capacitive influx also to the cross-resistance to other ER stress E2R2 cells to the effects of Tg, profound resistance to its analysis of the role of Ca2+ influx in associated with these agents is in studies channel proteins responsible for SOC influx have on the transient receptor gene of and have been of the to and from the have been shown to function as SOC in different cells Nat. Cell Biol. 2002; PubMed Scopus Google Scholar, C.R. A. 2002; PubMed Scopus Google Scholar). by using we were to of of the genes in HL-60 or E2R2 cells not is that a channel protein is associated with the increased influx observed in E2R2 cells. is to the channel or in resistant cells. of is observed in response to ER and overexpression has been shown to resistance to by ER stress or other events H. J.L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. R.J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, H. S. S. J. Cell. Biochem. 2000; 77: PubMed Scopus Google Scholar, P. R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Although we observed increased levels of in response to ER stress a response was in both HL-60 and E2R2 cells, both by and by microarray These that overexpression is not associated with the increased resistance in E2R2 cells. a calcium cell was some that resistance to antifungal and also a in the of ER stress induced by other such as tunicamycin M. J. 2002; 21: PubMed Scopus Google Scholar). However, this was as to and we have observed that E2R2 resistance is by that the resistance in E2R2 cells is to the calcium cell that multiple ribosomal genes were overexpressed in E2R2 cells to ribosomal content and protein We observed that ribosomal content in E2R2 cells was increased by ribosomal content was also observed in resistant and E2R6 cells. Although protein synthesis were the in E2R2 cells compared with HL-60 cells, the increased of may have to the sustained protein synthesis observed in the presence of Ec and Tg. overexpression may also have to the observed resistance to that the ribosome-inactivating protein partially resistance in resistant clones is with a role for ribosomal protein overexpression in resistance to ER stress. We that a of function may be to maintain normal levels of protein synthesis and cell concentrations of not protein synthesis in resistant clones containing increased content, the of with Ec or protein synthesis the for cell The that resistant clones also exhibit other phenotypic such as increased SOC influx may the resistance not be In with that E2R2 cells protein synthesis in the presence of Ec and Tg, we also observed levels in these cells with these is the for protein synthesis levels in response to ER stress Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), is that in may to resistance. may stress due to the of E2R2 cells to maintain Ca2+ in the These under protein genes of the of by P. Mol. Biol. 1997; PubMed Scopus Google Scholar). Although of has been well J. Biochem. 2000; PubMed Scopus Google Scholar), is of the of these analysis may of for this is as increased Ca2+ influx with ribosomal protein we that these two in E2R2 cells events to resistance. In and of mutant leukemia cell resistant to Ec have Ca2+ influx and ribosomal content as two phenotypic with resistance. of cross-resistance of these cells to other ER stress-associated agents a novel phenotype of multidrug resistance associated with ER stress. of the importance of these and other into normal and transformed cells ER stress. We for with calcium and for

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