BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 230, 251–253 (1997)ARTICLE NO. RC965937

Possible Involvement of Bcl-2 Pathway in Retinoid XReceptor Alpha-Induced Apoptosis of HL-60 Cells

Neeraj Agarwal1 and Kapil Mehta*Department of Anatomy and Cell Biology, University of North Texas Health Science Center and North TexasEye Research Institute, Fort Worth, Texas 76107; and *Department of Bioimmunotherapy,The University of Texas MD Anderson Cancer Center, Houston, Texas 77030

Received November 17, 1996

vating two distinct types of nuclear receptors: RARsRetinoids induce granulocytic differentiation and and RXRs that function as dimeric, ligand dependent

subsequent apoptosis in human myeloid (HL-60) leuke- transcription factors. Each RAR and RXR family of re-mia cells. Differentiation is induced due to activation ceptors comprise three subtypes: -a-, b, and -g and eachof retinoic acid receptors (RARs) whereas, activation subtype is encoded by a distinct gene (5-6). The RARsof retinoid X receptors (RXRs) seems to be essential binds to both the ATRA and its naturally occuring dou-for driving these cells into apoptosis. In order to un-

ble bond isomer, 9-cis RA whereas RXRs bind only toderstand the mechanism of RXR induced apoptosis, we9-cis RA (7). Like most of the blood cells, HL-60 cellsused a variant HL-60 cell line (HL-60R) with a trans-express both RARs and RXRs (8) that makes it difficultdominant negative mutation. The retroviral vector-to evaluate the involvement of a particular receptor-mediated gene transfer was used to introduce themediated events. With the availability of HL-60R cells,functional RARs or RXRa into HL-60R cells. We stud-it has become feasible to generate HL-60 cells whichied the effect of receptor-selective retinoid treatmentexpress either RARa or RXRa by gene transfection us-on the expression of Bcl-2 and Bax oncogenes by re-ing specific coding sequences (9-10).verse-transcription polymerase chain reaction (RT-

Our earlier results suggested that activation ofPCR) and immunoblot analysis in RARs and RXRatransfected HL-60 cells. Our results show that activa- RARa provides a terminal cell differentiation signal intion of RXRa results in apoptosis via down-modulation HL-60 cells; wheras activation of RXRa induces a sig-of Bcl-2 mRNA as well as its gene product expression nal for direct apoptosis in these cells (4). In this report,with no change in Bax mRNA expression. q 1997 Academic we investigated the expression of Bcl-2 and Bax, thePress two apoptosis-linked gene products during RXRa-in-

duced apoptosis of HL-60R cells. Our results indicatethat RXRa induces apoptosis by lowering Bcl-2 proteinas well as its mRNA expression with no change in Bax

Maturation of normal hemopoietic cells is a result of levels.the progressive differentiation of its precursor cells into

MATERIALS AND METHODSterminally differentiated cells. The terminally differen-tiated cells subsequently undergo apoptosis and thus HL-60R cells, cell culture and treatment with retinoic acid. Thea homeostasis is maintained for stem cells (1). The hu- human myeloid leukemia cell line HL-60, resistant to differentiating

effects of ATRA (designated as HL-60R), was retrovirally transfectedman myelocytic leukemia cells, HL-60 serve as an use-with cDNA coding for full length RARa and RXRa (11). HL60R andful model to study the regulatory mechanisms per-the two tranfectacted sublines thereof were kindly provided by Dr.taining to myeloid cell differentiation and apoptosisSteve Collins (Hutchinson Cancer Center, Seattle, WA). All the three

since all trans-retinoic acid (ATRA) induces terminal cell lines were maintained in RPMI 1640 growth medium supple-differentiation and subsequent apoptosis in these cells mented with 5% fetal calf serum, 2 mM L-glutamine, 100 units/

ml penicillin, and 100 mg/ml streptomycin. The retinoid receptor-(2-4). Retinoids mediate their biological effects by acti-transfected cell lines (HL-60R-RARa and RXRa) were occassionallycultured in the presence of neomycin (1 mg/ml) to prevent selectionagainst retrovirus-negative clones. Treatment with retinoic acid was1 Address for correspondence: Department of Anatomy and Celldone as described earlier (4).Biology, University of North Texas Health Science Center, 3500

Camp Bowie Blvd, Fort Worth, TX 76107-2699. E-mail: nagarwal@ Reverse transcription and polymerase chain reaction (RT-PCR) analy-sis. Total cellular RNA from HL-60R cells was isolated using RNAzolmolly.hsc.unt.edu.

0006-291X/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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B RNA isolation kit (Biotecx, Houston). First strand complementaryDNA was synthesized from total RNA and stored at 0207C until used.The RT-PCR analysis was performed following the procedure as de-scribed by Wilson et al., 1993 (12). The PCR primers were obtainedfrom Continental Laboratory Products, Inc., San Diego, CA and were,for Bcl-2: TGC ACC TGA CGC CCT TCA C (S) and AGA CAG CCAGGA GAA ATC AAA CAG (A); for Bax: ACC AAG AAG CTG AGCGAG TGT C (S) and ACA AAG ATG GTC ACG GTC TGC C (A); andfor b-actin: TGT GAT GGT GGG AAT GGG TCA G (S) and TTT GATGTC ACG CAC GAT TTC C (A). The (S) is forward primer whereas(A) denotes backward primer. All test samples were amplified simulta-neously with a particular primer pair using a master mix containingall of the components in the PCR reaction, except the target cDNA orin the case of the control, water. PCR reactions were run using theClontech hot start method which utilizes a monoclonal antibody toTaq polymerase. Programmable temperature cycling (Perkin Elmer,Norwalk, CT) was performed as per the following: one initial denatur-ation cycle for 5 minutes at 947C, 5 minutes at 607C, followed by 40amplification cycles for 2 minutes at 727C, 1 minute at 947C and 1minute at 607C with a final extension phase consisting of one cycle of FIG. 1. Reverse transcription polymerase chain reaction (RT-10 minutes at 727C. Control reactions without template were included PCR) analysis of Bcl-2, Bax and b-actin in RARa and RXRa treatedwith each amplification for each pair of primers. b-actin primers served HL-60 cells. Total RNA was extracted from the cells after treatmentas internal control and to confirm cDNA synthesis. Southern blot hy- (0.1 mM, 16 h) with ATRA or 9-cis retinoic acid. The RT-PCR analysisbridization of the PCR products with either 32P-labeled antisense oligo- was performed after synthesizing the first strand cDNA as describednucleotides which hybridize to a region within the amplified sequence in the methods and using commercially available sense and antisenseor 32P-labeled cDNAs were used to confirm that the amplified sequences primers for Bcl-2, Bax, and b-actin. The numbers above each lanewere specific (data not shown). are the treatments: lanes 1 and 4 are untreated controls; lanes 2

Immunoblot analysis. After treatment (0.1 mM, 16 h) with ATRA and 5 are ATRA treated (0.1 mM); and lanes 3 and 6 are 9-cis RAor 9-cis retinoic acid, the cells were lysed in a lysis buffer, 100 mM (0.1 mM) treated. RARa-(lanes 1-3) or RXRa-transfected (lanes 4-6)Tris-HCL buffer, pH 7.4 containing 150 mM NaCl, 3% CHAPS, 1 mM HL-60R cells.EDTA and 1 mM PMSF. The proteins (50 mg) were electrophoresed ina 12% sodium dodecyl sulphate (SDS)-polyacrylamide gel electropho-resis (PAGE) using Lammelie gels. After the electrophoresis, the of Bcl-2 mRNA following their treatment with 9-cis RA.proteins were transferred to a nitrocellulose membrane. The mem-

Treatment of HL60 cells with either ATRA or 9-cis RA,branes were probed with anti-Bcl-2 (NeoMarkers, Fremont, CA) anti-did not result in any major alteration of Bax mRNAbody and the antigen-antibody reaction was detected by using IgG-

conjugated horseraddish peroxidase secondary antibody (1:5,000 di- expression (Fig. 1). b-actin mRNA expression servedluted) and enhanced chemiluminescence (ECL) reagent (Amersham as an internal control to show the extent of synthesisCorp) as per the manufacturer’s recommendation. In order to detect of cDNA for each treatment (Fig. 1).the Bax protein expression, the membrane was stripped and re-

Expression of Bcl-2 and Bax proteins following reti-probed with anti-Bax (kindly provided by Dr. Tim McDonald, UTM. D. Anderson Cancer Center) monoclonal antibody as described. noic acid treatments. Since the results of RXRa acti-

vation showed dramatic effect on Bcl-2 mRNA levelsin HL-60 cells, we next determined its effect on expres-RESULTSsion of gene products for both the Bcl-2 and Bax by

Expression of Bcl-2 and Bax mRNAs following reti- immunoblot analysis using monoclonal antibodiesnoic acid treatments. We and others have shown ear- raised against Bcl-2 and Bax. Since these proteins arelier that ligand activation of retinoic acid receptor expressed at low levels, the immunoblot analysis for(RARs) results in differentiation but the activation of Bcl-2 and Bax was performed using enhanced chemilu-RXRa receptor can directly drive HL-60 cells into minescence (ECL) reagent (Amersham Corp). The re-apoptosis without their differentiation to granulocytes sults of immunoblot analysis showed a single band of(3-4). One can observe all the typical characteristics of expected molecular weight of 26-kDa for Bcl-2 and aapoptosis such as laddering of the genomic DNA as well 21-kDa band for Bax proteins (Fig. 2). In contrast toas in situ labeling of fragmented DNA with biotinylated the effect of retinoids treatment on Bcl-2 mRNA expres-dUTP (TUNEL) following retinoic-induced activation sion, the protein product was reduced in both the RARaof RXRa in HL60 cells (4). Since Bcl-2/Bax have been as well as RXRa-transfected cells following treatmentlinked to apoptosis, we studied the expression of Bcl-2 with ATRA or 9-cis RA. However, as per the results ofand Bax mRNA using RT-PCR analysis. Our results expression of Bcl-2 mRNA expression, the Bcl-2 proteinshow that the treatment of RARa transfected HL-60R expression was also reduced to almost negligible levelscells with nuclear receptor panagonist, 9-cis RA, or following specific activation of RXRa receptors (Fig. 2).even with RAR-selective retinoid, ATRA, did not alter To our surprise, a small but consistent reduction inBcl-2 mRNA expression (Fig. 1). However, HL-60 cells expression of Bax was also observed following activa-

tion of RARa with HL60 cells (Fig. 2).transfected with RXRa, showed a complete suppression

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vation of retinoid receptors. Particularly, ligand-medi-ated activation of RXRa, induced strong suppression ofBcl-2 expression in HL-60 cells, both at protein and itsmRNA level. Levels of Bax were not affected in responseto ATRA treatment of HL-60 cells. Recently, it was re-ported that activation of RARs in HL-60 cells down-mod-ulates Bcl-2 expression, whereas, activation of RXRs byreceptor-selective retinoids did not alter Bcl-2 expression(15). While we don’t know the reason for this discrepancy,the observation that Bcl-2 is completely downregulatedby RXRa activation, complements our observation thatFIG. 2. Immunoblot analysis of Bcl-2, and Bax in RARa andligand-induced activation of RXRa is sufficient signal forRXRa treated HL-60 cells. After appropriate treatment with eitherinduction of apoptosis in HL-60 cells (4).ATRA or 9-cis RA, the cells were lysed and subjected to SDS–PAGE

and immunoblotted using antibodies against Bcl-2 and Bax, as de-scribed in the methods. As expected the antibodies against Bcl-2 ACKNOWLEDGMENTSrecognized a protein at 26 kDa and Bax, a 21 kDa protein. The resultsshow the levels of Bcl-2 and Bax expression in RARa-(lanes 1-3) and The authors thank the expert technical help of Teresa McQueen,RXRa-(lanes 4-6) transfected HL-60R cells following 16 h culture in Michael Rooney and Rajnee Agarwal. We also thank the generouspresence of medium alone (lanes 1 and 4) or medium containing 0.1 supply of Bax antibodies from Dr. Tim McDonald of UT MD CancermM either ATRA (lanes 2 and 5) or 9-cis RA (lanes 3 and 6). Center and HL60R and the two transfected sublines by Dr. Steve

Collins of Hutchinson Cancer Center, Seattle, WA.

REFERENCESDISCUSSION

1. Steller, H. (1995) Science 267, 1445–1449.ATRA treatment induces morphological changes in2. Brietman, T. R., Selonick, S. E., and Collins, S. J. (1980) Proc.

HL-60 cells that are characteristic of mature granulo- Natl. Acad. Sci. USA 77, 2936–2940.cytes. Like in vivo matured granulocytes, in vitro differ- 3. Nagy, L., Thomazy, V. A., Shipley, G. L., Fesus, L., Lamph, W.,entiated HL-60 cells are destined to die by a genetically Heyman, R. A., Chandraratna, R. A. S., and Davies, P. J. A.

(1995) Mol. Cell. Biol. 15, 3540–3551.regulated process called, apoptosis or programmed cell4. Mehta, K., McQueen, T., Neamati, N., Collins, S., and Andreef,death. Recently, it was shown that in mature granulo-

M. (1996) Cell. Growth Differ. 7, 1–8.cytes ATRA can provide a signal for apoptosis (3). Suscep-5. Pfahl, M., Aplet, R., Bendlk, I., Fanjul, A., Graupner, G., Lee,tibility of cells to undergo apoptosis is regulated by sev-

M., La-Vista, N., Lu, X., Piedrafita, J., Ortiz, M. A., Salbert, G.,eral genes, including the Bcl-2 gene encoding a 26-kDa and Zhang, X. (1994) Vit. Horm. 49, 327–381.mitochondrial-associated protein, which prolongs the cell 6. Mangefsdorf, D. J., Umesono, K., and Evans, R. M. (1994) Thesurvival by delaying apoptosis. Previous studies have Retinoids (Sporm, M. B., Roberts, A. B., and Goodman, D. S.,suggested that ATRA treatment of HL-60 cells results in Eds.), pp. 319–346, Raven Press, New York.a decreased expression of Bcl-2 (13). However, it is not 7. Heyman, R. A., Mangefsdorf, D. J., Dyck, J. A., Stein, R. B.,

Eichele, G., Evans, R. M., and Thaller, C. (1992) Cell 68, 397–clear whether reduced levels of Bcl-2 are necessary for406.differentiation or apoptosis to occur. Overexpression of

8. Hashimoto, Y., Kagechika, H., and Shudo, K. (1990) Biochem.Bcl-2 in retrovirally transfected HL-60 cells did blockBiophys. Res. Comm. 166, 1300–1307.apoptosis in differentiated cells but did not affect their

9. Robertson, K. A., Emami, B., and Collins, S. J. (1992) Blood 80,ability to differentiate (14). Both, the differentiation as 1885–1889.well as apoptosis events induced by ATRA in HL-60 cells, 10. Robertson, K. A., Emami, B., Lueller, L., and Collins, S. J. (1992)are regulated by RARs and RXRs (4). Since the HL-60 Mol. Cell. Biol. 12, 3743–3749.cells contain both types of receptors, we determined the 11. Collins, S., Robertson, K. A., and Mueller, L. (1990) Mol. Cell.

Biol. 10, 2154–2163.role of these receptors in the expression of Bcl-2 and Bax,12. Wilson, S. E., Walker, J. W., Chwang, E. L., and He, Y-G. (1993)the proteins that are known to play a role in apoptosis.

Invest. Ophthalmol. Vis. Sci. 34, 2544–2561.Retrovirally transfected HL-60R cells overexpressing13. Delia, D., Aiello, A., Soligo, D., Fontanella, E., Melani, C., Pez-RARa or RXRa were used for this purpose. These trans-

zella, F., Pierotti, M., and Della Porta, G. (1992) Blood 79, 1291–fected HL-60 cell lines can be induced to differentiate into 1298.granulocytic pathway or to undergo apoptosis by ligand- 14. Naumovski, L., and Clearly, M. L. (1994) Blood 83, 2261–2267.induced activation of RARa and RXRa respectively (4). 15. Nagy, L., Thomazy, V. A., Roshantha, A., Chandraratna, S., Hey-The results presented in this report suggested a global man, R. A., and Davies, P. J. A. (1996) Leukemia Res. 20, 499–

505.suppression of Bcl-2 protein expression following the acti-

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