BRIEF COMMUNICATIONS Novel Retinoic Acid Receptor-a Transcripts in Acute Promyelocytic Leukemia Responsive to All-frans-Retinoic Acid
All-rrans-retinoic acid (RA) induces differentiation of malignant cells and influences normal development (1-3). Actions of RA are thought to be mediated by specific cytoplasmic or nuclear receptors. The RA nuclear receptor alpha (RAR-a) appears to be the predominant RA-binding receptor expressed in various hematopoietic cells (4-8). Retroviral vector-mediated transduction of RAR-a into an RAresistant subclone of the human leukemia cell line HL-60 can restore sensitivity to RA (9), indicating a direct role for RAR-a in this differentiation program. Clinical trials of all-/ra«s-RA have shown that this agent induces complete remission in a high proportion of patients with acute promyelocytic leukemia (70,7/). Since RAR-a maps to chromosome 17 in the region of the characteristic cytogenetic abnormality of acute promyelocytic leukemia, translocation t(15;17)(q22;ql2) (12-14), we studied RAR-a expression in the course of our clinical studies of RA in acute promyelocytic leukemia (75). In this communication, we report the following: 1) leukemic cells from patients with acute promyelocytic leukemia express aberrant messenger RNA (mRNA) transcripts for RAR-a; 2) expression of both normal and aberrant RAR-a transcripts increases after 1 week of RA treatment; and 3) augmentation of the transcript, as well as the clinical response, occurs at low plasma concentrations.
mic cells or the normal cells present in the specimen. The novel bands of lane 2 are not present in cells from the patient with acute promyelocytic leukemia who lacked t(15; 17), as shown in lane 3. In Fig 1,B, lane 1 shows faint expression of normal and abnormal RAR-a in bone marrow mononuclear cells
Received October 16, 1990; revised October 23, 1990; accepted October 23, 1990. Supported in part by grant PDT-381 from the American Cancer Society (ACS) and by grant FDR-000674 from the Food and Drug Administration, Department of Health and Human Services. W. H. Miller, Jr, is the recipient of a Young Investigator Award from the American Society of Clinical Oncology. E. Dmitrovsky is the recipient of ACS Clinical Oncology Career Development Award 89-129. S. R. Frankel was supported by the Mortimer J. Lacher Research Fund and is the recipient of Public Health Service Cancer Chemotherapy Training Grant CA-09207-14 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. W. H. Miller, Jr, E. Dmitrovsky (Laboratory of Molecular MedicineX R. P. WarrelL Jr (Developmental Chemotherapy and Leukemia Services), S. R. Frankel (Department of Medicine), A. Jakubowski, J. L Gabrilove (Laboratory of Leukocyte Biology), J. Muindi (Laboratory of Clinical Pharmacology), Memorial Sloan-Kettering Cancer Center and the Cornell University Medical College, New York, NY. We thank Dr Pierre Chambon, Institut National de la Sante et de la Recherche Medicale (TNSERMX Strasbourg, France, for providing the RAR-a probe. * Correspondence to: Raymond P. Warrell, Jr, MD, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
B «28S
-28S
RAR-a
ll 1
2
RAR-a
^m 1BH1^18S
18S
3 GAPDH
1
2
3
Fig I. Total cellular RNA extracted (17) from the mononuclear cell fraction ofbone marrow aspirates separated by Ficoll-Hypaque sedimentation. Northern Wot analysis was performed on RNA by electrophoresis on a 1% agarose gel and blotted on reinforced nitrocellulose (Schkichcr & SchudL Keene, NH) (18). Blots were hybridized to a 600-base-pair Pstl cut cDNA RAR-a (19) or a 1.2-kilobase ft/1 cut chicken cDNA GAPDH (20\ washed stringently as previously described (27). Fig 1,A shows RAR-a expression from the human teratocarcinoma line NTERA-2 d. D1 (NT2/DIX lane 1; a patient with acute promyelocytic leukemia with the t(15; 17) receiving RA, lane 2; and a patient with phenotypic acute promyelocytic leukemia lacking the t(15; 17X lane 3. Fig 1,B shows RAR-a and GAPDH gene expression from a patient with acute promyelocytic leukemia with the t(15; 17) before treatment, lane 1; the same patient after 1 week on RA treatment, lane 2; and NT2/D1, lane 3. The proportions of malignant cells in the differential cell counts of the two successive bone marrow aspirates (lanes 1 and 2) were comparable.
Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at New York University on July 22, 2015
Wilson H. Miller, Jr, Raymond P. Warrell, Jr* Stanley R. Frankel, Ann Jakubowski, Janice L. Gabrilove, Josephia Muindi, Ethan Dmitrovsky
Four patients with acute promyelocytic leukemia (M3 by French-American-British [FAB] criteria), three with newly diagnosed disease and one after tworelapses,were treated with RA (45 mg/m2 per day). Plasma concentrations of RA were assayed by highpressure liquid chromatography by use of a previously described method (76). Karyotypes were evaluated on unstimulated cultures of bone marrow after 48 hours by use of Giemsa and/or quinacrine banding of chromosome preparations. Three patients had the characteristic t(15;17) translocation, whereas one patient with newly diagnosed disease had no evidence of a t(15;17). Fig 1,A shows Northern blot analysis of RAR-a mRNA expression from a human teratocarcinoma cell line lacking t(15; 17) (22) and expressing RAR-a (23) (lane 1), a leukemic patient with t(15;17) who had received RA for 2 weeks (lane 2), and a patient with morphologic acute promyelocytic leukemia lacking t(15;17) (lane 3). Lane 1 shows two bands, at approximately 3.8 and 2.8 kilobases, that have been reported for human RAR-a from various malignant and normal tissues, including fresh, non-M3 subtype leukemic cells (5-8,24). Lane 2 shows the two normal bands and two additional novel bands. Since this aspirate contained only 59% blasts and promyelocytes, the observed normal bands may have originated from either the leuke-
Vol. 82, No. 24, December 19, 1990
speculate that pharmacologic doses of RA may overcome a maturation block by maximally stimulating a partially active RAR-a, by overcoming a blockade of normal RAR-a protein by an abnormal protein, or by recruiting a related receptor with a lower sensitivity to RA, such as the novel retinoic acid receptor encoded by the RXR gene (27). Our findings suggest that expression of a novel RAR-a molecule from a translocated RAR-a gene may be involved in the pathogenesis of acute promyelocytic leukemia and may in part account for the exquisite clinical responsiveness of this disease to one ligand—all-frcms-retinoic acid.
(12) MATTEI MG, PETKDVICH M, MATTEI JF, ET
AL: Mapping of the human retinoic acid receptor to the q21 band of chromosome 17. Hum Genet 80:186-188, 1988 (73) LEMONS RS, EILENDER D, WALDMANN RA,
ET AL: Cloning and characterization of the t(15;17) translocation breakpoint region in acute promyelocytic leukemia. Genes Chromosomes Cancer 2:79-87, 1990 (14) BORROW J, GODDARD AD, SHEER D, ET AL:
Molecular analysis of acute promyelocytic leukemia breakpoint cluster region on chromosome 17. Science 249:1577-1580, 1990 (75) WARRELL RP J R , FRANKEL SR, SCHEIN-
BERG DA, ET AL: Ali-trans retinoic acid in acute promyelocytic leukemia: Preliminary U.S. clinical experience. Blood. In press (76) VANE FM, STOLTENBORG JK, BUGGE CJL:
Determination of 13-as-retinoic acid and its major metabolite, 4-oxo-13-c«-retinoic acid, in human blood by reverse-phase high-performance liquid chromatography. J Chromatogr 227:471 -484, 1982 (17) CHIROWIN JM, PRZYBYLA
References (7) STRICKLAND S, MAHDAVI V: The induction
of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 15:393-403, 1978 (2) BREITMAN TR, SELONICK SE, COLLINS SJ:
Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc Natl Acad Sri USA 77:2936-2940, 1980 (3) SPORN MB, ROBERTS AB, EDS: Retinoids,
Differentiation, and Disease, CIBA Symposium 10. London: Pitman, 1985 (4) NERVI C, GRIPPO JF, SHERMAN MI, ET AL:
Identification and characterization of nudear retinoic acid-binding activity in human myeloblastic leukemia HL-60 cells. Proc Natl Acad Sci USA 86:5854-5858, 1989 (5) DE THE H, MARCHIO A, TIOLLAIS P, ET AL:
Differential expression and ligand regulation of the retinoic acid receptor alpha and beta genes. EMBO J 8:429-133, 1989 (6) L A R G M A N Q D E T M E R K , CORRAL J Q E T A L :
Expression of retinoic acid receptor alpha mRNA in human leukemia cells. Blood 74:99-102, 1989 (7) GALLAGHER RE, SAID F, PUA I, ET AL:
Expression of retinoic acid receptor-alpha mRNA in human leukemia cells with variable responsiveness to retinoic acid. Leukemia 3:789-795, 1989 (8) KlZAKI M, KOEFFLER HP, LlN CW, ET AL:
Expression ofretinoicacid receptor mRNA in hematopoietic cells. Leuk Res 14:645-655, 1990 (9) COLLINS SJ, ROBERTSON KA, MUELLER L:
AE, M A C -
DONALD RJ, ET AL: Isolation of biologically active ribonudeic acid from sources enriched in ribonudease. J Biochem 18:5294-5299, 1979 (18) SOUTHERN EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503-517, 1975 (79) PETKOVICH M, BRAND NJ, KRUST A, ET AL:
A human retinoic acid receptor which belongs to the family of nudear receptors. Nature 330:444-450, 1987 (20) DUGAICZYK A, HARON JA, STONE EM, ET
AL: Cloning and sequencing of a deoxyribonudeic acid copy of gryceraldehyde-3phosphate dehydrogenase messenger ribonudeic add isolated from chicken musde. Biochemistry 22:1605-1613, 1983 (27) MILLER WH JR, MOY D, LI A, ET AL:
Retinoic acid induces down-regulation of several growth factors and proto-oncogenes in a human embryonal cancer cell line. Oncogene 5:511-517, 1990 (22) DMITROVSKY E, MURTY W V S , MOY D, ET
AL: Isochromosome 12p in non-seminoma cell lines: Karyotogic amplification of c-ki-nu2 without point-mutational activation. Oncogene 5:543-548, 1990 (23) SlMEONE A , ACAMPORA D, ARC1ONI L, ET
AL: Sequential activation of HOX2 homeobox genes by retinoic add in human embryonal carcinoma cells. Nature 346:763-766, 1990 (24) KJUJST A, KASTNER P, PETKOVICH M . E T A L :
A third human retinoic add receptor, hRARgamma. Proc Natl Acad Sci USA 86:5310-5314, 1989
Retinoic acid-induced granulocytic differentiation of HL-60 myeloid leukemia cells is mediated directly through the retinoic acid receptor (RAR-alpha). Mol Cell Biol 10:2154-2163, 1990 (70) HuANoME,YEYC,CHENSR,ETAL:Useof all-fra/u retinoic acid in the treatment of acute promyelocytic leukemia. Blood 72:567-572, 1988
(25) WANG C, CURTIS JE, MINDEN MD, ET AL:
(7/) DEGOS L, CHOMIENNE C, BALLERINI P, ET
(27) MANGELSDORF DJ, ONG ES, DYCK JA, ET
AL: All trans retinoic acid: A novel differentiation therapy for acute promyelocytic leukemia. Proc ASCO 9:207, 1990
AL: Nudear receptor that identifies a novel retinoic acid response pathway. Nature 345:224-229, 1990
Expression of a retinoic add receptor gene in myekiid leukemia cells. Leukemia 3:264-269, 1989 (26) DE THE H, CHOMIENNE C, LANOTTE M, ET
AL: The t(15;17) translocation of acute promyelocytic leukaemia fuses the retinoic arid receptor alpha gene to a novel transcribed locus. Nature 347:558-561, 1990
BRIEF COMMUNICATIONS 1933
Downloaded from http://jnci.oxfordjournals.org/ at New York University on July 22, 2015
from a patient with acute promyelocytic leukemia who had t(15; 17) before treatment. Lane 2 shows enhanced expression of both the normal and aberrant mRNA transcripts in cells from the same patient after 1 week of RA treatment in vivo. Hybridization to a probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cellular housekeeping enzyme (20), conFirms that comparable amounts of intact RNA were added to both lanes. Another patient with t(15;17) expressed novel RAR-a bands migrating identically to those in Fig 1 (data not shown) and achieved a complete remission with RA treatment. Leukemic cells from two patients with other FAB subtypes of acute myelocytic leukemia revealed only the two normal bands during Northern blot analysis (data not shown), consistent with prior reports (6,7,25). Thus, we found new RAR-a mRNA transcripts in leukemic cells from three patients with acute promyelocytic leukemia, each of whom exhibited the characteristic t(15; 17) and achieved complete remission with RA treatment. The transcript was not found in the single patient who lacked t(15;17) and who failed to respond clinically to RA. Clinical failure in this patient was not due to inadequate serum RA levels, since this individual achieved plasma RA concentrations somewhat higher, 21 to 82 ng/mL (7 to 27 X lO"7 M), than those of responding patients. While correlation of the molecular genetic abnormality and clinical response clearly requires further study, the presence of the described RAR-a mRNA species (and characterization of its translated protein) may ultimately prove useful in the prediction of clinical response to RA therapy. The translocation and altered RAR-a transcripts, however, are not prerequisites for response in vitro in various cell lines (1,2). The finding of two new transcripts reported here and the independent account of a new transcript in one other patient with acute promyelocytic leukemia (26) extend the discovery of a genomic rearrangement of RAR-a in acute promyelocytic leukemia (14,26) to the level of gene expression. We
All-rrans-retinoic acid (RA) induces differentiation of malignant cells and influences normal development (1-3). Actions of RA are thought to be mediated by specific cytoplasmic or nuclear receptors. The RA nuclear receptor alpha (RAR-a) appears to be the predominant RA-binding receptor expressed in various hematopoietic cells (4-8). Retroviral vector-mediated transduction of RAR-a into an RAresistant subclone of the human leukemia cell line HL-60 can restore sensitivity to RA (9), indicating a direct role for RAR-a in this differentiation program. Clinical trials of all-/ra«s-RA have shown that this agent induces complete remission in a high proportion of patients with acute promyelocytic leukemia (70,7/). Since RAR-a maps to chromosome 17 in the region of the characteristic cytogenetic abnormality of acute promyelocytic leukemia, translocation t(15;17)(q22;ql2) (12-14), we studied RAR-a expression in the course of our clinical studies of RA in acute promyelocytic leukemia (75). In this communication, we report the following: 1) leukemic cells from patients with acute promyelocytic leukemia express aberrant messenger RNA (mRNA) transcripts for RAR-a; 2) expression of both normal and aberrant RAR-a transcripts increases after 1 week of RA treatment; and 3) augmentation of the transcript, as well as the clinical response, occurs at low plasma concentrations.
mic cells or the normal cells present in the specimen. The novel bands of lane 2 are not present in cells from the patient with acute promyelocytic leukemia who lacked t(15; 17), as shown in lane 3. In Fig 1,B, lane 1 shows faint expression of normal and abnormal RAR-a in bone marrow mononuclear cells
Received October 16, 1990; revised October 23, 1990; accepted October 23, 1990. Supported in part by grant PDT-381 from the American Cancer Society (ACS) and by grant FDR-000674 from the Food and Drug Administration, Department of Health and Human Services. W. H. Miller, Jr, is the recipient of a Young Investigator Award from the American Society of Clinical Oncology. E. Dmitrovsky is the recipient of ACS Clinical Oncology Career Development Award 89-129. S. R. Frankel was supported by the Mortimer J. Lacher Research Fund and is the recipient of Public Health Service Cancer Chemotherapy Training Grant CA-09207-14 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. W. H. Miller, Jr, E. Dmitrovsky (Laboratory of Molecular MedicineX R. P. WarrelL Jr (Developmental Chemotherapy and Leukemia Services), S. R. Frankel (Department of Medicine), A. Jakubowski, J. L Gabrilove (Laboratory of Leukocyte Biology), J. Muindi (Laboratory of Clinical Pharmacology), Memorial Sloan-Kettering Cancer Center and the Cornell University Medical College, New York, NY. We thank Dr Pierre Chambon, Institut National de la Sante et de la Recherche Medicale (TNSERMX Strasbourg, France, for providing the RAR-a probe. * Correspondence to: Raymond P. Warrell, Jr, MD, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
B «28S
-28S
RAR-a
ll 1
2
RAR-a
^m 1BH1^18S
18S
3 GAPDH
1
2
3
Fig I. Total cellular RNA extracted (17) from the mononuclear cell fraction ofbone marrow aspirates separated by Ficoll-Hypaque sedimentation. Northern Wot analysis was performed on RNA by electrophoresis on a 1% agarose gel and blotted on reinforced nitrocellulose (Schkichcr & SchudL Keene, NH) (18). Blots were hybridized to a 600-base-pair Pstl cut cDNA RAR-a (19) or a 1.2-kilobase ft/1 cut chicken cDNA GAPDH (20\ washed stringently as previously described (27). Fig 1,A shows RAR-a expression from the human teratocarcinoma line NTERA-2 d. D1 (NT2/DIX lane 1; a patient with acute promyelocytic leukemia with the t(15; 17) receiving RA, lane 2; and a patient with phenotypic acute promyelocytic leukemia lacking the t(15; 17X lane 3. Fig 1,B shows RAR-a and GAPDH gene expression from a patient with acute promyelocytic leukemia with the t(15; 17) before treatment, lane 1; the same patient after 1 week on RA treatment, lane 2; and NT2/D1, lane 3. The proportions of malignant cells in the differential cell counts of the two successive bone marrow aspirates (lanes 1 and 2) were comparable.
Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at New York University on July 22, 2015
Wilson H. Miller, Jr, Raymond P. Warrell, Jr* Stanley R. Frankel, Ann Jakubowski, Janice L. Gabrilove, Josephia Muindi, Ethan Dmitrovsky
Four patients with acute promyelocytic leukemia (M3 by French-American-British [FAB] criteria), three with newly diagnosed disease and one after tworelapses,were treated with RA (45 mg/m2 per day). Plasma concentrations of RA were assayed by highpressure liquid chromatography by use of a previously described method (76). Karyotypes were evaluated on unstimulated cultures of bone marrow after 48 hours by use of Giemsa and/or quinacrine banding of chromosome preparations. Three patients had the characteristic t(15;17) translocation, whereas one patient with newly diagnosed disease had no evidence of a t(15;17). Fig 1,A shows Northern blot analysis of RAR-a mRNA expression from a human teratocarcinoma cell line lacking t(15; 17) (22) and expressing RAR-a (23) (lane 1), a leukemic patient with t(15;17) who had received RA for 2 weeks (lane 2), and a patient with morphologic acute promyelocytic leukemia lacking t(15;17) (lane 3). Lane 1 shows two bands, at approximately 3.8 and 2.8 kilobases, that have been reported for human RAR-a from various malignant and normal tissues, including fresh, non-M3 subtype leukemic cells (5-8,24). Lane 2 shows the two normal bands and two additional novel bands. Since this aspirate contained only 59% blasts and promyelocytes, the observed normal bands may have originated from either the leuke-
Vol. 82, No. 24, December 19, 1990
speculate that pharmacologic doses of RA may overcome a maturation block by maximally stimulating a partially active RAR-a, by overcoming a blockade of normal RAR-a protein by an abnormal protein, or by recruiting a related receptor with a lower sensitivity to RA, such as the novel retinoic acid receptor encoded by the RXR gene (27). Our findings suggest that expression of a novel RAR-a molecule from a translocated RAR-a gene may be involved in the pathogenesis of acute promyelocytic leukemia and may in part account for the exquisite clinical responsiveness of this disease to one ligand—all-frcms-retinoic acid.
(12) MATTEI MG, PETKDVICH M, MATTEI JF, ET
AL: Mapping of the human retinoic acid receptor to the q21 band of chromosome 17. Hum Genet 80:186-188, 1988 (73) LEMONS RS, EILENDER D, WALDMANN RA,
ET AL: Cloning and characterization of the t(15;17) translocation breakpoint region in acute promyelocytic leukemia. Genes Chromosomes Cancer 2:79-87, 1990 (14) BORROW J, GODDARD AD, SHEER D, ET AL:
Molecular analysis of acute promyelocytic leukemia breakpoint cluster region on chromosome 17. Science 249:1577-1580, 1990 (75) WARRELL RP J R , FRANKEL SR, SCHEIN-
BERG DA, ET AL: Ali-trans retinoic acid in acute promyelocytic leukemia: Preliminary U.S. clinical experience. Blood. In press (76) VANE FM, STOLTENBORG JK, BUGGE CJL:
Determination of 13-as-retinoic acid and its major metabolite, 4-oxo-13-c«-retinoic acid, in human blood by reverse-phase high-performance liquid chromatography. J Chromatogr 227:471 -484, 1982 (17) CHIROWIN JM, PRZYBYLA
References (7) STRICKLAND S, MAHDAVI V: The induction
of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 15:393-403, 1978 (2) BREITMAN TR, SELONICK SE, COLLINS SJ:
Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc Natl Acad Sri USA 77:2936-2940, 1980 (3) SPORN MB, ROBERTS AB, EDS: Retinoids,
Differentiation, and Disease, CIBA Symposium 10. London: Pitman, 1985 (4) NERVI C, GRIPPO JF, SHERMAN MI, ET AL:
Identification and characterization of nudear retinoic acid-binding activity in human myeloblastic leukemia HL-60 cells. Proc Natl Acad Sci USA 86:5854-5858, 1989 (5) DE THE H, MARCHIO A, TIOLLAIS P, ET AL:
Differential expression and ligand regulation of the retinoic acid receptor alpha and beta genes. EMBO J 8:429-133, 1989 (6) L A R G M A N Q D E T M E R K , CORRAL J Q E T A L :
Expression of retinoic acid receptor alpha mRNA in human leukemia cells. Blood 74:99-102, 1989 (7) GALLAGHER RE, SAID F, PUA I, ET AL:
Expression of retinoic acid receptor-alpha mRNA in human leukemia cells with variable responsiveness to retinoic acid. Leukemia 3:789-795, 1989 (8) KlZAKI M, KOEFFLER HP, LlN CW, ET AL:
Expression ofretinoicacid receptor mRNA in hematopoietic cells. Leuk Res 14:645-655, 1990 (9) COLLINS SJ, ROBERTSON KA, MUELLER L:
AE, M A C -
DONALD RJ, ET AL: Isolation of biologically active ribonudeic acid from sources enriched in ribonudease. J Biochem 18:5294-5299, 1979 (18) SOUTHERN EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503-517, 1975 (79) PETKOVICH M, BRAND NJ, KRUST A, ET AL:
A human retinoic acid receptor which belongs to the family of nudear receptors. Nature 330:444-450, 1987 (20) DUGAICZYK A, HARON JA, STONE EM, ET
AL: Cloning and sequencing of a deoxyribonudeic acid copy of gryceraldehyde-3phosphate dehydrogenase messenger ribonudeic add isolated from chicken musde. Biochemistry 22:1605-1613, 1983 (27) MILLER WH JR, MOY D, LI A, ET AL:
Retinoic acid induces down-regulation of several growth factors and proto-oncogenes in a human embryonal cancer cell line. Oncogene 5:511-517, 1990 (22) DMITROVSKY E, MURTY W V S , MOY D, ET
AL: Isochromosome 12p in non-seminoma cell lines: Karyotogic amplification of c-ki-nu2 without point-mutational activation. Oncogene 5:543-548, 1990 (23) SlMEONE A , ACAMPORA D, ARC1ONI L, ET
AL: Sequential activation of HOX2 homeobox genes by retinoic add in human embryonal carcinoma cells. Nature 346:763-766, 1990 (24) KJUJST A, KASTNER P, PETKOVICH M . E T A L :
A third human retinoic add receptor, hRARgamma. Proc Natl Acad Sci USA 86:5310-5314, 1989
Retinoic acid-induced granulocytic differentiation of HL-60 myeloid leukemia cells is mediated directly through the retinoic acid receptor (RAR-alpha). Mol Cell Biol 10:2154-2163, 1990 (70) HuANoME,YEYC,CHENSR,ETAL:Useof all-fra/u retinoic acid in the treatment of acute promyelocytic leukemia. Blood 72:567-572, 1988
(25) WANG C, CURTIS JE, MINDEN MD, ET AL:
(7/) DEGOS L, CHOMIENNE C, BALLERINI P, ET
(27) MANGELSDORF DJ, ONG ES, DYCK JA, ET
AL: All trans retinoic acid: A novel differentiation therapy for acute promyelocytic leukemia. Proc ASCO 9:207, 1990
AL: Nudear receptor that identifies a novel retinoic acid response pathway. Nature 345:224-229, 1990
Expression of a retinoic add receptor gene in myekiid leukemia cells. Leukemia 3:264-269, 1989 (26) DE THE H, CHOMIENNE C, LANOTTE M, ET
AL: The t(15;17) translocation of acute promyelocytic leukaemia fuses the retinoic arid receptor alpha gene to a novel transcribed locus. Nature 347:558-561, 1990
BRIEF COMMUNICATIONS 1933
Downloaded from http://jnci.oxfordjournals.org/ at New York University on July 22, 2015
from a patient with acute promyelocytic leukemia who had t(15; 17) before treatment. Lane 2 shows enhanced expression of both the normal and aberrant mRNA transcripts in cells from the same patient after 1 week of RA treatment in vivo. Hybridization to a probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cellular housekeeping enzyme (20), conFirms that comparable amounts of intact RNA were added to both lanes. Another patient with t(15;17) expressed novel RAR-a bands migrating identically to those in Fig 1 (data not shown) and achieved a complete remission with RA treatment. Leukemic cells from two patients with other FAB subtypes of acute myelocytic leukemia revealed only the two normal bands during Northern blot analysis (data not shown), consistent with prior reports (6,7,25). Thus, we found new RAR-a mRNA transcripts in leukemic cells from three patients with acute promyelocytic leukemia, each of whom exhibited the characteristic t(15; 17) and achieved complete remission with RA treatment. The transcript was not found in the single patient who lacked t(15;17) and who failed to respond clinically to RA. Clinical failure in this patient was not due to inadequate serum RA levels, since this individual achieved plasma RA concentrations somewhat higher, 21 to 82 ng/mL (7 to 27 X lO"7 M), than those of responding patients. While correlation of the molecular genetic abnormality and clinical response clearly requires further study, the presence of the described RAR-a mRNA species (and characterization of its translated protein) may ultimately prove useful in the prediction of clinical response to RA therapy. The translocation and altered RAR-a transcripts, however, are not prerequisites for response in vitro in various cell lines (1,2). The finding of two new transcripts reported here and the independent account of a new transcript in one other patient with acute promyelocytic leukemia (26) extend the discovery of a genomic rearrangement of RAR-a in acute promyelocytic leukemia (14,26) to the level of gene expression. We
