Leukemia (2001) 15, 1537–1543 2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu The Hsp90 inhibitor geldanamycin selectively sensitizes Bcr-Abl-expressing leukemia cells to cytotoxic chemotherapy MV Blagosklonny1, T Fojo1, KN Bhalla3, J-S Kim1, JB Trepel1, WD Figg1, Y Rivera2 and LM Neckers2 Departments of 1Developmental Therapeutics and 2Cell and Cancer Biology, Medicine Branch, National Cancer Institute, NIH, Bethesda and Rockville, MD, USA; and 3Moffitt Cancer Center, Tampa, FL, USA The Bcr-Abl fusion protein drives leukemogenesis and can ren- Materials and methods der leukemia cells resistant to conventional chemotherapy. Geldanamycin (GA), a drug which destabilizes Hsp90- associated proteins, depletes cells of Bcr-Abl, an Hsp90 client, Cell lines and reagents but not of Abl. Both HL60 cells transfected with Bcr-Abl and naturally Ph1-positive K562 leukemia cells are resistant to most HL60 and Jurkat, human leukemia cell lines, were obtained cytotoxic drugs, but were found to be sensitive to GA. Further- from American Type Culture Collection (Manassas, VA, USA). more, GA sensitized Bcr-Abl-expressing cells to doxorubicin (DOX) and paclitaxel (PTX). In contrast, in parental HL60 cells, HL60-Bcr-Abl, a Bcr-Abl stable transfected HL-60 cells, were 8,16 90 nM GA inhibited PARP cleavage, nuclear fragmentation, and described previously. Paclitaxel (Taxol), was Bristol-Myers cell death caused by 500 ng/ml DOX. Like GA, STI 571 (an (Princeton, NJ, USA) product. Adriamycin (doxorubicin) was inhibitor of the Abl kinase) sensitized Bcr-Abl-expressing cells obtained from Sigma (St Louis, MO, USA) and dissolved in to DOX. Unlike GA, STI 571 did not antagonize the cytotoxic DMSO as a 2 mg/ml stock solution. STI 571 was kindly pro- effects of DOX in parental HL60 cells. These results indicate vided by Novartis Pharma (East Hanover, NJ, USA). The pro- that sensitization of Bcr-Abl-expressing cells, but not desensi- tization of HL60 cells, depends on inhibition of Bcr-Abl. Thus, teasome inhibitor PS-341 was a kind gift from Dr Peter Elliott, GA differentially affects leukemia cells depending on their Bcr- Proscript, Inc. (Cambridge, MA, USA). Abl expression and selectively increases apoptosis in Bcr-Abl- expressing cells. Leukemia (2001) 15, 1537–1543. Keywords: oncogenes; Bcr-Abl; chemotherapy; cytoprotection; gendanamycin; HSP90 Immunoblot analysis Cells were lysed and soluble proteins were harvested in TNES buffer (50 mm Tris HCl pH 7.5, 100 mm NaCl, 2 mm EDTA, Introduction 1mm sodium orthovanadate, 1% (v/v) NP40) containing pro- tease inhibitors (20 ␮g/ml aprotinin, 20 ␮g/ml leupeptin, 1 mm Chronic myelogenous leukemia (CML) is characterized by a PMSF). Proteins were resolved with SDS-PAGE as previously reciprocal t(9;22) chromosomal translocation, known as the described.15,17 Immunoblotting was performed using rabbit Philadelphia chromosome, that fuses the truncated Bcr gene polyclonal anti-human PARP (Upstate Biotechnology, Lake to the truncated c-Abl.1,2 Bcr-Abl is also often found in adult Placid, NY, USA), for Bcr using monoclonal anti-human Bcr acute lymphocytic leukemia (ALL).3 The Bcr-Abl gene enco- antibodies N 2 (Oncogene Science, Calbiochem, San Diego, des for character chimeric p210 and p185 proteins.4 These CA, USA). are constitutively active tyrosine kinases which may induce resistance to apoptotic cell death.5–12 A number of protein kinases, including Raf-1, Src, and ErbB, depend on association with the chaperone protein, heat-shock MTT assay protein 90 (Hsp90), for proper function and stability. The benzoquinone ansamycin geldanamycin (GA) binds to Hsp90 Fifteen thousand cells were plated in 0.1 ml in 96-well flat and specifically inhibits this chaperone’s function, resulting in bottom plates and then exposed to tested agents (final volume ␮ degradation of HSP90-associated proteins.13,14 The chimeric 0.2 ml per well). At the indicated time, 20 l of 5 mg/ml MTT BCR-ABL exists in a complex with Hsp90, and GA causes solution in PBS was added to each well for 4 h. After removal ␮ degradation of BCR-ABL after 3–5 h of treatment.15 Since Bcr- of the medium, 170 l of DMSO was added to each well to Abl expression is associated with resistance to cytotoxic dissolve the formazan crystals. The absorbance at 540 nm was agents, we hypothesized that treatment of Bcr-Abl-expressing determined using a Biokinetics plate reader (Bio-Tek Instru- cells with GA would restore their sensitivity to chemothera- ments, Inc, Winooski, VT, USA). Triplicate wells were assayed peutic agents. Indeed our results confirm this prediction. for each condition and standard deviations were determined. Importantly, while sensitizing Bcr-Abl-expressing cells, GA protected parental cells from cytotoxic effects of doxo- rubicin (DOX). Number of dead and live cells Cells were plated in 24-well plates in 1 ml of medium, or in 96-well plates in 0.2 ml, and were treated with drugs. After the indicated time, cells were counted in triplicate on a Coul- ter Z1 cell counter (Hialeah, FL, USA). In addition, cells were Correspondence: MV Blagosklonny, Medicine Branch, Bldg 10, R incubated with trypan blue and the numbers of blue (dead) 12N226, NIH, Bethesda, MD 20892, USA cells and transparent (live) cells were counted in a hemocyto- Received 30 January 2001; accepted 26 June 2001 meter. Sensitization of Bcr-Abl-expressing cells MV Blagosklonny et al 1538 Figure 2 Reversal of resistance/sensitivity ratios in HL60 and K562 cells by GA. Effects of increasing concentrations of GA on the cytotox- icity caused by 500 nm DOX in either K562 (closed triangles) or HL60 (open triangles) cells. MTT assay was performed after 3 days as described in Materials and methods. At GA = 0 nM axis X, the values represent the cytotoxicity of 500 ng/ml alone. Results were calculated as the percent of values obtained with untreated cells and represent mean ± s.d. Figure 1 Bcr-Abl downregulation and cytotoxic effects of GA in K562 cells. (a) K562 cells were incubated with indicated concen- trations of GA and then immunoblot for Bcr-Abl was performed as Results described in Materials and methods. Multiple lower molecular bands may represent products of BCR-ABL degradation in GA-treated cells. (b) K562 cells and HL60 cells (shown for comparison) were incubated Pharmacological depletion of Bcr-Abl and cytotoxicity with GA. MTT assay was performed after 3 days, as described in Materials and methods. Results were calculated as the percent of In agreement with previous report,15 treatment of K562 cells values obtained with untreated cells and represent mean ± s.d. with 30–810 nm GA completely depleted Bcr-Abl protein (Figure 1a). Similarly, 30 nm of GA caused a near-maximal growth inhibition of K562 cells (Figure 1b). Previous studies have shown that K562 leukemia cells expressing Bcr-Abl are Cell cycle analysis resistant to numerous therapeutic agents including anti-CD95 mAb, high-dose AraC, etoposide, paclitaxel, actinomycin D, 5,8,9,16,18,19 Cells were washed with PBS and resuspended in 75% ethanol staurosporine, and proteasome inhibitors. In con- in PBS and kept at 4°C for at least 30 min. Prior to analysis, trast, GA was more cytotoxic in K562 cells than in HL60 cells cells were washed again with PBS and resuspended and incu- (Figure 1b). bated for 30 min in propidium iodide staining solution con- taining 0.05 mg/ml propidium iodide (Sigma), 1 mm EDTA, 0.1% Triton-X-100 and 1 mg/ml RNAse A in PBS. The suspen- GA sensitized K562 cells but protected HL60 cells sion was then passed through a nylon mesh filter and ana- lyzed on a Becton Dickinson FACScan (Franklin Lakes, NJ, K562 cells were much more resistant to doxorubicin (DOX) USA). than were HL60 cells. (60% and 4% survival, respectively; see Figure 2, X-axis: GA = 0). Addition of 10–100 nm GA decreased survival of DOX-treated K562 cells. If sensitization by GA was due to depletion of Bcr-Abl, this effect should not Nuclear fragmentation assay be observed in HL60 cells. In fact, GA did not sensitize HL60 cells (Figure 2). Unexpectedly, GA rendered these cells more Cells were incubated with drugs for the indicated time. Cells resistant to DOX. Sensitization of K562 coupled with desensit- were washed with PBS, pelleted on to glass slides in a cyto- ization of HL60 resulted in the reversal of their relative sensi- centrifuge, fixed with 90% ethanol with 10% glacial acid and tivities to DOX in the presence of GA (Figure 2). stained with DAPI as described previously.18 Nuclei were When added alone as well as with either PTX or DOX, GA visualized under UV microscopy. depleted p210BCR-ABL in K562 cells (Figure 3a). Also, PTX and Leukemia Sensitization of Bcr-Abl-expressing cells MV Blagosklonny et al 1539 Figure 3 Combinations of drugs with GA. K562 cells (a) and HL60 cells (b) were treated with 90 nm GA, 500 ng/ml DOX, 100 nm PTX or their combinations, as indicated. After 16 h, cells were lysed and immunoblots for PARP and BCR were performed as described in Materials and methods. K562 cells (c) and HL60 cells (d) were treated as described in (a) and (b). MTT assay was performed after 48 h as described in Materials and methods. Results were calculated as the percent of values obtained with untreated cells and represent mean ± s.d. DOX slightly downregulated Bcr-Abl. This downregulation of gated combinations of GA with either PS-341, an inhibitor of Bcr-Abl occurred before apoptosis, since PARP was still intact the proteasome, or DOX (Figure 6). As expected, HL60-Bcr- 16 h after the treatment (Figure 3a). In HL60 cells, Bcr levels Abl were more resistant to PS-341 than were the parental cells were not affected by GA (Figure 3b).