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Overexpression of the MPO gene occurring in a case of APL without unusual genotypic characteristics
Leukemia Research Vol. 14, No. 9, pp. 735-742, 1990. Printed in Great Britain.
0145-2126/90 $3.00 + .00 Pergamon Press plc
O V E R E X P R E S S I O N OF THE MPO G E N E O C C U R R I N G IN A CASE OF APL W I T H O U T U N U S U A L G E N O T Y P I C CHARACTERISTICS* SERGIO FERRARI, ENRICO TAGLIAFICO, PAOLA TEMPERANI, ROSSELLAMANFREDINI, GIOVANNI CECCHERELLI, PATRIZIA ZUCCHINI, ANTONIO TABILIO,t AMEDEA DONELLI, GIUSEPPE TORELLI, GIOVANNI EMILIA and UMBERTO TORELLI Experimental Hematology Center, II Medical Clinic and Hematology Service, University of Modena, Italy and tlnstitute of Medical Clinics, University of Perugia, Italy (Received 17 January 1990. Accepted 4 March 1990)
Abstract--Northern blot analysis of four typical cases of acute promyelocytic leukemia showed that one of the cell population examined was characterized by a very high level of expression of the myeloperoxidase (MPO) gene. Western blot analysis confirms that the protein content of the cells corresponded to the levels of the MPO mRNA. Southern blot studies of the DNA of this cell population ruled out the presence of any genome amplification or rearrangement. Chromosome hybridization studies in situ confirmed that the MPO gene was translocated on the long arm of chromosome 15. The observation that a typical genomic pattern may or may not be associated with the MPO overexpression leads us to believe that so far it is impossible to reach any conclusion about the significance of the translocation in the genesis of MPO overexpression. Key words: Gene expression, t(15;17), acute promyelocytic leukemia, myeloperoxidase, karyotype, in situ hybridization.
INTRODUCTION
17. At variance with the results of these authors Weil et al. [11] have located the MPO gene in the region
IT IS commonly accepted that malignant promyelocytes of acute promyelocytic leukemia (APL) are characterized by an intense myeloperoxidase (MPO) activity which suggests, on cytochemical grounds, a particular abundance of function MPO [1]. In most, if not all cases, the A P L is characterized by a specific reciprocal translocation involving chromosomes 15 and 17 [2, 3], though variant translocations have been also described [4]. The cloning of the MPO gene [5-8] has permitted the localization of the gene in the long arm of chromosome 17 [9] but the precise locus is still debated. Chang et al. [9] and van Tuinen et al. [10] have located the gene on bands q21-q23 and q22-q24 respectively, far from the A P L specific breakpoint, ql 1.2-q12, on chromosome
q12--q21. The high levels of MPO activity seen in A P L have suggested the possibility that the MPO gene expression may be affected by this translocation [9, 11]. Several aspects of the molecular genetics of A P L are still controversial. Weil et al. [11] have found that in two out of four cases examined the MPO gene was rearranged and in all their cases it was translocated on chromosome 15. However this was not confirmed by Miller et al. [12] and by Donti et al. [13], who were unable to find a single case with MPO gene rearrangement, although the t(15;17) was found in several cases. Moreover no data of expression were reported in any of the previously mentioned studies, so that no correlation between gene translocation or rearrangement, and the level of MPO messenger R N A and protein in the malignant cells, has so far been possible. This paper presents the results of our studies concerning the level of expression of MPO gene in cases of classical APL. One of the cases was characterized by a level much higher than the others, although the chromosome analysis gave evidence of a typical t(15;17) reciprocal translocation without any evidence of amplification or rearrangement of the MPO gene.
*Supported by a grant from AIRC (Associazione Italiana per la Ricerca sul Cancro). Abbreviations: A P L , acute promyelocytic leukemia; MPO, myeloperoxidase; PHA, phytohemagglutinin; BrdU, Bromodeoxyuridin; GTG, G banding-trypsinGiemsa; FPG, fluorochrome-protolysis-Giemsa. Correspondence to: Dr Sergio Ferrari, Experimental Haematology Center, II Medical Clinic, Policlinico, Via del Pozzo 71-41100 Modena, Italy. 735
736
S. FERRARIet al. MATERIALS AND METHODS
Cell characterization The blast cell populations were obtained by leukapheresis. Morphological evaluation was performed on May-Grunwald-Giemsa stained smears. The following cytochemical reactions were performed: PAS, Sudan Black B, diaminobenzidine MPO and alpha-naphthol acetate esterase. Surface marker analysis was performed by indirect immunofluorescence using a panel of monoclonal antibodies. D N A extraction and Southern blot analysis Leukocyte enriched plasma was diluted in RPMI and centrifuged to obtain pellets from which DNA was extracted according to the technique of Gross-Bellard et al. [14], with minor modifications. Control DNA was extracted from leukocytes of a normal blood donor. The DNA was digested with different restriction endonucleases: Bam HI, Bgl II, Pst I, Xba I, Kpn I and Sma I, size fractionated on 0.8% agarose gel, and transferred to Gene Screen membrane (NEN Research Products, Boston). The analysis was performed as described by Southern [15]. After prehybridization, the filters were hydridized with the different 32P-labeled probes, as already described by Selleri et al. [16]. After washing in 0.2 × SSC and 0.5% SDS at 65°C for several hours, the filters were exposed at -80°C with intensifying screens. Cell culture and cytogenetics Phytohemagglutinin-stimulated peripheral blood lymphocytes from a normal male donor were cultured at 37°C for 72 h in RPMI-1640 medium supplemented with 15% fetal calf serum and 2mM L-glutamine. Bone marrow and/or peripheral blood cells of APL patients were cultured without mitogen in RPMI-1640 medium, 15% FCS, 2 mM L-glutamine at 37°C for 24-72 h. To obtain early metaphase chromosomes BrdU-Thymidine synchronization was used [17]. The chromosome preparations were harvested by means of standard procedures and banded with trypsin (GTG) and with a fluorochrome-protolysis-Giemsa (FPG) method [18]. A total of 20-36 metaphases were analyzed for each sample. Karyotypes were described according to the ISCN 1985 [19]. Chromosomal hybridization in situ The MPO probe was radiolabeled using the multiprime labeling procedure [20] with 3H-dCTP and 3H-dTTP to a specific activity of 1 × 10-8 cpm/lxg DNA. Hybridization in situ was performed using a minor modification of the protocol described by Zabel et al. [21]. Briefy, slides were treated with ribonuclease A and chromosomal DNA was denatured at 70°C for 2 min in a 70% formamide. Probe DNA was denatured at 100°C, then added to a solution of 50% formamide. The final concentration of the probe used on each slide was 40-60 ng DNA/ml. Chromosome hybridization was carried out at 42°C for 16-18 h. Autoradiography was performed by dipping the slides in a nuclear-track emulsion and, at different intervals of exposure, 8-10 days, slides were developed and fixed. Chromosome banding was obtained using the fluorochrome-protolysis-Giemsa (FPG) method of Perry and Wolf [18]. Only methaphases containing a complete chromosome spread with precise grain locations were analyzed and scored.
Molecular probes The DNA fragments obtained from the plasmids carrying the gene probes used in this study were: a 2.2kb Eco RI-Hind III fragment from the human MPO cDNA as described by Johnson et al. [7], and kindly provided by G. Rovera; a 0.55 kb Pst I fragment of the human-/32microglobulin cDNA [22]; and a 1.9 kb Xba I fragment of the human p53 coding region, isolated from the recombinant plasmid pLSVH p53-62 described by Zakut-Houry et al. [23]. DNA labeling All the purified DNA fragments were labeled using the random prime labeling procedure described by Feinberg and Vogelstein [20]. The specific activities obtained ranged from 2-3 × 10 9 cpm/IxgDNA. Northern blot analysis The total cellular RNA was extracted from normal and leukemic cells using a modification of the guanidinium/ isothiocyanate/phenol procedure as described by Chomczynski and Sacchi [24]. The Northern blot analysis was performed as described by Ferrari et al. [25] with minor modifications. Western blot analysis Western blot analysis was performed as described by Burnette [26] with minor modifications, as described by Ferrari et al. [27].
RESULTS Characteristics o f the cell populations studied All the myeloid populations studied had the morphological, cytochemical, immunological and cytogenetic characteristics of the acute promyelocytic leukemia. Southern blot analysis Figure 1 shows the restriction pattern of MPO in the D N A extracted from the four A P L populations. Other control D N A s , including a normal donor, are reported. The samples in the figure were digested with Bam H I and Bgl II, but four other restriction enzymes were used, without obtaining any evidence of rearrangements or amplification. To have some indication of the behaviour of a gene located on the short arm of chromosome 17, restriction patterns were obtained with a probe of the p53 oncogene. A rearrangement was found only in HL-60 D N A (Fig.
2). C h r o m o s o m a l hybridization in situ with the M P O probe Figure 3 reports the distribution of labeled sites on normal metaphase chromosomes obtained from normal P H A stimulated lymphocytes, after hybridization of the M P O specific probe. 118 metaphase cells from this hybridization were examined. The
FIG. 1. Restriction pattern of the MPO gene in high molecular weight DNA isolated from peripheral blood cells of four classical APL (M3) patients (RC, GOM, GM, BS), from HL-60 cells, from an ALL patient (PG) and from a normal donor (BA). After endonuclease digestion with several restriction enzymes (see Materials and Methods) the DNA fragments were separated, blotted and hybridized to the MPO probe as described. The size of the marker bands are specified in the figure. The restriction patterns of the DNAs studied are in keeping with the genomic organization of the MPO gene as described by Morishita et al. [29].
FIG. 2. Restriction pattern of the p53 gene in high molecular weight DNA isolated from the same populations as described in Fig. 1. After endonuclease digestion with several restriction enzymes (see Materials and Methods) the DNA fragments were separated, blotted and hybridized to the p53 probe as described. The size of the marker bands are specified in the figure. 737
FIG. 5. Composite panel of the autoradiographs from Northern blot analysis of total cellular RNA isolated from a patient with ALL (lane 1); different APL patients (lanes 2 to 5); HL-60 cells (lane 6); Daudi cell !ine (lane 7); peripheral lymphocytes obtained from a normal donor (lane 8). The genes hybridized and the sizes of the mRNA are labeled on the figure. The levels of expression of the fl2-microglobulin gene show that the amount of RNA for each lane is almost the same.
FIG. 7. Western blot analysis of the four APL populations expressing the MPO mRNA. Lane 1: ALL patient; lanes 2 to 5: APL patients; lane 6 : H L 6 0 cells; lane 7: a CML patient; lane 8: peripheral blood lymphocytes obtained from a normal donor. 100 ~tg of protein lysate were loaded in each lane. Protein concentration was determined by the Folin technique. After S D S - P A G E the amount of protein was controlled by silver staining (Bio-Rad) and, following electroblotting to nitrocellulose membrane, by Red Ponceau staining.
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FIG. 3. Results of hybridization in situ. Idiogram displays examples of G-banded chromosomesfrom different normal cells and shows the grain distribution. A total of 186 chromosomally located grains on 118 metaphases was analyzed. The predominant site of hybridization in each experiment was the long arm of chromosome 17 with a total of 31 grains localized on bands 17q22-q24. The number of grains in this region was at least four times the number found in any other chromosomal segment of similar length.
distribution of 186 labeled sites indicates a significant labeling on the long arm of chromosome 17. About 14% of labeling is observed on band 17q22-24. Figure 4 shows the distribution of MPO hybridizing sites on chromosomes of patient G.O. carrying the t(15;17). 26 metaphase cells from this hybridization were examined. The frequency of labeled sites indicates that MPO is present on a chromosome 15 and on a chromosome 17. Study of the MPO gene expression Figure 5 shows the results of Northern blot analysis of the RNA extracted from the APL cells. The RNA extracted from cells of a MPO expressing ALL, of the HL60 line and of two lymphoid populations without MPO expression were also analyzed. All the APL cell populations were expressing MPO at elevated, although variable, levels (lanes 2, 3, 4 and 5). However, the abundance of the MPO mRNA in the cells from patient G.O. (lane 3),was at least ten times higher than that observed in other cell populations. A quite different pattern of expression was observed studying the p53 gene. In one of the APL patients, the gene was not expressed. In the other three cases, including the case of G.O. overexpressing the MPO, quite similar levels of p53 mRNA were observed. In the HL-60 cell, a truncated p53 mRNA was observed.
Figure 6 shows the densitometric scanning of the autoradiographs of the Northern blot of Fig. 5. Western blot analysis Figure 7 shows the results of Western blot analysis in the different populations studied. The figure indicates that the concentration of the MPO protein was fairly in keeping with that of the corresponding mRNA, with the exception of the lymphoid cells of patient P.G. on which no MPO protein was detectable, as already reported [27]. The amount of MPO protein present in cells from patient G.O. was again at least ten times higher than that of other APL patients. This holds not only for the mature peptide (55 kD), but also for the precursor peptide (80 kD). Figure 8 shows a densitometric comparison between the levels of MPO mRNA and the levels of the corresponding protein. DISCUSSION Our results are in agreement with those reported by Miller et al. [12] and by Chang et al. [5], who have concluded that no rearrangement of the MPO gene may be observed in the APL cells by Southern blotting analysis. Our data are also in keeping with those of the authors who reported the mapping of the MPO
740
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FIG. 4. Distribution of labeled sites on normal chromosomes 15 and 17 and on the translocation derivatives 15q+ and 1 7 q - , after hybridization with the MPO-specific probe to metaphase cells from a bone marrow aspirate of an APL patient (G. O.). A total of 52 chromosomally located grains on 26 metaphase cells from this hybridization were examined. The results of this hybridizaton indicate that the MPO is translocated to the rearranged chromosome 15.
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FIG. 6. Densitometric scanning of the autoradiographs of the Northern blot analysis reported in Fig. 5 showing the levels of expression of the MPO m R N A and p53 mRNA.
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FIG. 8. The graphic shows the results of the densitometric scanning of the Western blots compared with the densitometric scanning of the MPO m R N A levels. Different densitometric units were used for R N A and protein.
Very different levels of the MPO gone expression in classical APLs gone to h u m a n c h r o m o s o m e 17(q22-q24). Since the A P L specific breakpoint on c h r o m o s o m e 17 is mapped at a location m u c h m o r e proximal at the centromere, it is difficult to believe that the M P O gone may be rearranged as a consequence of the translocation. A control Southern blot experiment performed with a p r o b e of the p53 gene, located on the short arm of c h r o m o s o m e 17, confirmed the absence of rearrangements of both the p53 and M P O genes. The high levels of M P O activity in A P L have been so far related to the differentiation arrest at the promyelocytic stage [9, 28], or to the t(15;17) chrom o s o m e translocation [9]. So far it is impossible to reach any conclusion about the significance of the translocation in the genesis of M P O overexpression. Presently, there is no indication that the two events are necessarily associated, and a much m o r e detailed molecular analysis is required, in order to clarify the changes in the genetic control mechanisms involved in the disorder of M P O gone expression. Acknowledgement--The authors are grateful to Dr G. Rovera, Wistar Institute, Philadelphia, for generously supplying the myeloperoxidase cDNA probe.
REFERENCES 1. Hayhoe E. G. S. & Quaglino D. (1980) Haematological Cytochemistry, p. 275. Churchill Livingstone, Edinburgh. 2. Rowley J. D., Golomb H. M., Vardiman J., Fukuhara S., Dougherty C. & Potter D. (1977) Further evidence for a non-random chromosomal abnormality in acute promyelocytic leukemia. Int. J. Cancer 20, 869. 3. Fourth International Workshop on Chromosomes in Leukemia, 1982 (1984) Chromosomes in acute promyelocytic leukemia. Cancer Genet. Cytogenet. 11, 288. 4. Heim S., Kristoffersson U., Mandhal N., Maim C. & Mitelman F. (1988) Variant translocation t(3;15) (q21;q22) in a patient with acute promyelocytic leukemia. Leukemia 2, 65. 5. Chang K. S., Trujillo J. M., Cook R. G. & Stass S. A. (1986) Human myeloperoxidase gone: molecular cloning and expression in leukemic cells. Blood 68, 1411. 6. Johnson K. R., Nauseef W. M., Care A., Wheelock M. J., Shane S., Hudson S., Koeffler H. P., Selsted M., Miller G. & Rovera G. (1987) Characterization of cDNA clones for human myeloperoxidase: predicted amino acid sequence and evidence for multiple RNA species. Nucleic Acids Res. 5, 2013. 7. Weil S. C., Rosner G. L., Reid M. S., Chisholm R. L., Farber N. M., Spetznagel J. K. & Swanson M. S. (1987) cDNA cloning of human myeloperoxidase: Decrease in myeloperoxidase mRNA upon induction of HL60 cells. Proc. natn. Acad. Sci. U.S.A. 84, 2057. 8. Morishita K., Kubota N., Asano S., Kaziro Y. & Nagata S. (1987) Molecular cloning and characterization of cDNA for human myeloperoxidase. J. biol. Chem. 262, 3844.
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9. Chang K. S., Schroeder W., Siciliano M. J., Thompson L. H., McCredie K., Beran M., Freireich E. J., Liang J. C., Trujillo J. M. & Stass S. A. (1987) The localization of the human myeloperoxidase gone is in close proximity to the translocation breakpoint in acute promyelocytic leukemia. Leukemia 1, 458. 10. van Tuinen P., Johnson K. R., Ledbetter S. A., Nussbaum R. L., Rovera G. & Ledbetter D. H. (1987) Localization of myeloperoxidase to the long arm of human chromosome 17: relationship to the 15 ;17 translocation of acute promyelocytic leukemia. Oncogene 1, 319 11. Weil S. C., Rosner G. L., Reid M. S., Chisholm R. L., Lemons R. S., Swanson M. S., Carrino J. J., Diaz M. O. & Le Beau M. M. (1988) Translocation and rearrangement of myeloperoxidase gene in acute promyelocytic leukemia. Science 240, 790. 12. Miller C. W., Rovera G., Venturelli D., Huebner K. F., van Tuinen P., Ledbetter D. H., Kitchingam G., Mirro J. & Koeffler H. P. (1989) The myeloperoxidase gene in acute promyelocytic leukemia. Science 244, 823. 13. Donti E., Montanucci M., Longo L., Mencarelli A., Pandolfi P., Tabilio A., Nanni M., Alimena G., Avanzi G., Pegoraro L., Grignani E. & Pellicci P. G. (1989) The myeloperoxidase gene in acute promyelocytic leukemia. Science 224, 824. 14. Gross-Bellard M., Oudet P. & Chambon P. (1973) Isolation of high molecular weight DNA from mammalian cells. Eur. J. Biochem. 36, 32. 15. Southern E. M. (1975) Detection of specific sequences among the DNA fragments separated by gel electrophoresis. J. Molec. Biol. 98, 503. 16. Selleri L., Narni F., Emilia G., Colo' A., Zucchini P., Venturelli D., Donelli A., Torelli U. & Torelli G. (1987) Philadelphia-positive chronic myeloid leukemia with a chromosome 22 breakpoint outside the breakpoint cluster region. Blood 70, 1659. 17. Dutrillaux B. & Viegas-Pequignot E. (1981) High resolution R- and G-banding on the same preparation. Human Genet. 57, 93. 18. Perry P. & Wolff S. New Giemsa method for the differential staining of sister chromatids. Nature 251, 156. 19. ISCN (1981) An international system for human cytogenetic nomenclature: high resolution banding. Cytogenet. Cell Genet. 31, 1. 20. Feinberg A. P. & Vogelstein B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Analyt. Biochem. 132, 6. 21. Zabei B. U., Naylor S. L., Sakaguchi A. Y., Bell G. I. & Shows T. B. (1983) High resolution chromosomal localization of human genes for amylase, propriomelanocortin, somatostatin, and a DNA fragment (D3S1) by in situ hybridization. Proc. hath. Acad. Sci. U.S.A. 80, 6932. 22. Suggs S. V., Wallace R. B., Hirose T., Kawashima L. H. & Itakura K. (1981) Use of synthetic oligonucleotides as hybridization probe: isolation of cloned cDNA sequences for human fl2-microglobulin. Proc. hath. Acad. Sci. U.S.A. 78, 6613. 23. Zakut-Houry R., Beinz-Tadmor B., Givol D. & Oren M. (1985) Human p53 cellular tumor antigen: cDNA sequence and expression in COS cells. E M B O J. 4, 1251. 24. Chomczynski P. & Sacchi N. (1987) Single-step method
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of RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction. Anal. Biochem. 162, 156. 25. Ferrari S., Torelli U., Selleri L., Donelli A., Venturelli D., Narni F., Moretti L. & Torelli G. (1985) Study of the levels of expression of two oncogenes, c-myc and c-myb, in acute and chronic leukemias of both lymphoid and myeloid lineage. Leukemia Res 9, 833. 26. Burnette W. N. (1981) 'Western blotting': electrophoretic transfer of proteins from sodium dodecyl sulphate-polyacrylamide gel to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analyt. Biochem. 112, 194. 27. Ferrari S., Mariano M. T., Tagliafico E., Sarti M., Ceccherelli G., Selleri L., Merli F., Narni F., Donelli
A., Torelli G. & Torelli U. (1988) Myeloperoxidase gene expression in blast cells with a lymphoid phenotype in cases of acute lymphoblastic leukemia. Blood 72, 873. 28. Ferrari S., Tagliafico E., CecchereUi G., Selleri L., Calabretta B., Donelli A., Temperani P., Sarti M., Sacchi M., Emilia G., Torelli G. & Torelli U. (1989) Expression of the myeloperoxidase gene in acute and chronic myeloid leukemias: relationship to the expression of cell cycle-related genes. Leukemia 3, 423. 29. Morishita K., Tsuchiya M., Asano S., Kaziro Y. & Nagata S. (1987) Chromosomal gene structure of human myeloperoxidase and regulation of its expression by granulocyte colony-stimulating factor. J. biol. Chem. 262, 15208.
0145-2126/90 $3.00 + .00 Pergamon Press plc
O V E R E X P R E S S I O N OF THE MPO G E N E O C C U R R I N G IN A CASE OF APL W I T H O U T U N U S U A L G E N O T Y P I C CHARACTERISTICS* SERGIO FERRARI, ENRICO TAGLIAFICO, PAOLA TEMPERANI, ROSSELLAMANFREDINI, GIOVANNI CECCHERELLI, PATRIZIA ZUCCHINI, ANTONIO TABILIO,t AMEDEA DONELLI, GIUSEPPE TORELLI, GIOVANNI EMILIA and UMBERTO TORELLI Experimental Hematology Center, II Medical Clinic and Hematology Service, University of Modena, Italy and tlnstitute of Medical Clinics, University of Perugia, Italy (Received 17 January 1990. Accepted 4 March 1990)
Abstract--Northern blot analysis of four typical cases of acute promyelocytic leukemia showed that one of the cell population examined was characterized by a very high level of expression of the myeloperoxidase (MPO) gene. Western blot analysis confirms that the protein content of the cells corresponded to the levels of the MPO mRNA. Southern blot studies of the DNA of this cell population ruled out the presence of any genome amplification or rearrangement. Chromosome hybridization studies in situ confirmed that the MPO gene was translocated on the long arm of chromosome 15. The observation that a typical genomic pattern may or may not be associated with the MPO overexpression leads us to believe that so far it is impossible to reach any conclusion about the significance of the translocation in the genesis of MPO overexpression. Key words: Gene expression, t(15;17), acute promyelocytic leukemia, myeloperoxidase, karyotype, in situ hybridization.
INTRODUCTION
17. At variance with the results of these authors Weil et al. [11] have located the MPO gene in the region
IT IS commonly accepted that malignant promyelocytes of acute promyelocytic leukemia (APL) are characterized by an intense myeloperoxidase (MPO) activity which suggests, on cytochemical grounds, a particular abundance of function MPO [1]. In most, if not all cases, the A P L is characterized by a specific reciprocal translocation involving chromosomes 15 and 17 [2, 3], though variant translocations have been also described [4]. The cloning of the MPO gene [5-8] has permitted the localization of the gene in the long arm of chromosome 17 [9] but the precise locus is still debated. Chang et al. [9] and van Tuinen et al. [10] have located the gene on bands q21-q23 and q22-q24 respectively, far from the A P L specific breakpoint, ql 1.2-q12, on chromosome
q12--q21. The high levels of MPO activity seen in A P L have suggested the possibility that the MPO gene expression may be affected by this translocation [9, 11]. Several aspects of the molecular genetics of A P L are still controversial. Weil et al. [11] have found that in two out of four cases examined the MPO gene was rearranged and in all their cases it was translocated on chromosome 15. However this was not confirmed by Miller et al. [12] and by Donti et al. [13], who were unable to find a single case with MPO gene rearrangement, although the t(15;17) was found in several cases. Moreover no data of expression were reported in any of the previously mentioned studies, so that no correlation between gene translocation or rearrangement, and the level of MPO messenger R N A and protein in the malignant cells, has so far been possible. This paper presents the results of our studies concerning the level of expression of MPO gene in cases of classical APL. One of the cases was characterized by a level much higher than the others, although the chromosome analysis gave evidence of a typical t(15;17) reciprocal translocation without any evidence of amplification or rearrangement of the MPO gene.
*Supported by a grant from AIRC (Associazione Italiana per la Ricerca sul Cancro). Abbreviations: A P L , acute promyelocytic leukemia; MPO, myeloperoxidase; PHA, phytohemagglutinin; BrdU, Bromodeoxyuridin; GTG, G banding-trypsinGiemsa; FPG, fluorochrome-protolysis-Giemsa. Correspondence to: Dr Sergio Ferrari, Experimental Haematology Center, II Medical Clinic, Policlinico, Via del Pozzo 71-41100 Modena, Italy. 735
736
S. FERRARIet al. MATERIALS AND METHODS
Cell characterization The blast cell populations were obtained by leukapheresis. Morphological evaluation was performed on May-Grunwald-Giemsa stained smears. The following cytochemical reactions were performed: PAS, Sudan Black B, diaminobenzidine MPO and alpha-naphthol acetate esterase. Surface marker analysis was performed by indirect immunofluorescence using a panel of monoclonal antibodies. D N A extraction and Southern blot analysis Leukocyte enriched plasma was diluted in RPMI and centrifuged to obtain pellets from which DNA was extracted according to the technique of Gross-Bellard et al. [14], with minor modifications. Control DNA was extracted from leukocytes of a normal blood donor. The DNA was digested with different restriction endonucleases: Bam HI, Bgl II, Pst I, Xba I, Kpn I and Sma I, size fractionated on 0.8% agarose gel, and transferred to Gene Screen membrane (NEN Research Products, Boston). The analysis was performed as described by Southern [15]. After prehybridization, the filters were hydridized with the different 32P-labeled probes, as already described by Selleri et al. [16]. After washing in 0.2 × SSC and 0.5% SDS at 65°C for several hours, the filters were exposed at -80°C with intensifying screens. Cell culture and cytogenetics Phytohemagglutinin-stimulated peripheral blood lymphocytes from a normal male donor were cultured at 37°C for 72 h in RPMI-1640 medium supplemented with 15% fetal calf serum and 2mM L-glutamine. Bone marrow and/or peripheral blood cells of APL patients were cultured without mitogen in RPMI-1640 medium, 15% FCS, 2 mM L-glutamine at 37°C for 24-72 h. To obtain early metaphase chromosomes BrdU-Thymidine synchronization was used [17]. The chromosome preparations were harvested by means of standard procedures and banded with trypsin (GTG) and with a fluorochrome-protolysis-Giemsa (FPG) method [18]. A total of 20-36 metaphases were analyzed for each sample. Karyotypes were described according to the ISCN 1985 [19]. Chromosomal hybridization in situ The MPO probe was radiolabeled using the multiprime labeling procedure [20] with 3H-dCTP and 3H-dTTP to a specific activity of 1 × 10-8 cpm/lxg DNA. Hybridization in situ was performed using a minor modification of the protocol described by Zabel et al. [21]. Briefy, slides were treated with ribonuclease A and chromosomal DNA was denatured at 70°C for 2 min in a 70% formamide. Probe DNA was denatured at 100°C, then added to a solution of 50% formamide. The final concentration of the probe used on each slide was 40-60 ng DNA/ml. Chromosome hybridization was carried out at 42°C for 16-18 h. Autoradiography was performed by dipping the slides in a nuclear-track emulsion and, at different intervals of exposure, 8-10 days, slides were developed and fixed. Chromosome banding was obtained using the fluorochrome-protolysis-Giemsa (FPG) method of Perry and Wolf [18]. Only methaphases containing a complete chromosome spread with precise grain locations were analyzed and scored.
Molecular probes The DNA fragments obtained from the plasmids carrying the gene probes used in this study were: a 2.2kb Eco RI-Hind III fragment from the human MPO cDNA as described by Johnson et al. [7], and kindly provided by G. Rovera; a 0.55 kb Pst I fragment of the human-/32microglobulin cDNA [22]; and a 1.9 kb Xba I fragment of the human p53 coding region, isolated from the recombinant plasmid pLSVH p53-62 described by Zakut-Houry et al. [23]. DNA labeling All the purified DNA fragments were labeled using the random prime labeling procedure described by Feinberg and Vogelstein [20]. The specific activities obtained ranged from 2-3 × 10 9 cpm/IxgDNA. Northern blot analysis The total cellular RNA was extracted from normal and leukemic cells using a modification of the guanidinium/ isothiocyanate/phenol procedure as described by Chomczynski and Sacchi [24]. The Northern blot analysis was performed as described by Ferrari et al. [25] with minor modifications. Western blot analysis Western blot analysis was performed as described by Burnette [26] with minor modifications, as described by Ferrari et al. [27].
RESULTS Characteristics o f the cell populations studied All the myeloid populations studied had the morphological, cytochemical, immunological and cytogenetic characteristics of the acute promyelocytic leukemia. Southern blot analysis Figure 1 shows the restriction pattern of MPO in the D N A extracted from the four A P L populations. Other control D N A s , including a normal donor, are reported. The samples in the figure were digested with Bam H I and Bgl II, but four other restriction enzymes were used, without obtaining any evidence of rearrangements or amplification. To have some indication of the behaviour of a gene located on the short arm of chromosome 17, restriction patterns were obtained with a probe of the p53 oncogene. A rearrangement was found only in HL-60 D N A (Fig.
2). C h r o m o s o m a l hybridization in situ with the M P O probe Figure 3 reports the distribution of labeled sites on normal metaphase chromosomes obtained from normal P H A stimulated lymphocytes, after hybridization of the M P O specific probe. 118 metaphase cells from this hybridization were examined. The
FIG. 1. Restriction pattern of the MPO gene in high molecular weight DNA isolated from peripheral blood cells of four classical APL (M3) patients (RC, GOM, GM, BS), from HL-60 cells, from an ALL patient (PG) and from a normal donor (BA). After endonuclease digestion with several restriction enzymes (see Materials and Methods) the DNA fragments were separated, blotted and hybridized to the MPO probe as described. The size of the marker bands are specified in the figure. The restriction patterns of the DNAs studied are in keeping with the genomic organization of the MPO gene as described by Morishita et al. [29].
FIG. 2. Restriction pattern of the p53 gene in high molecular weight DNA isolated from the same populations as described in Fig. 1. After endonuclease digestion with several restriction enzymes (see Materials and Methods) the DNA fragments were separated, blotted and hybridized to the p53 probe as described. The size of the marker bands are specified in the figure. 737
FIG. 5. Composite panel of the autoradiographs from Northern blot analysis of total cellular RNA isolated from a patient with ALL (lane 1); different APL patients (lanes 2 to 5); HL-60 cells (lane 6); Daudi cell !ine (lane 7); peripheral lymphocytes obtained from a normal donor (lane 8). The genes hybridized and the sizes of the mRNA are labeled on the figure. The levels of expression of the fl2-microglobulin gene show that the amount of RNA for each lane is almost the same.
FIG. 7. Western blot analysis of the four APL populations expressing the MPO mRNA. Lane 1: ALL patient; lanes 2 to 5: APL patients; lane 6 : H L 6 0 cells; lane 7: a CML patient; lane 8: peripheral blood lymphocytes obtained from a normal donor. 100 ~tg of protein lysate were loaded in each lane. Protein concentration was determined by the Folin technique. After S D S - P A G E the amount of protein was controlled by silver staining (Bio-Rad) and, following electroblotting to nitrocellulose membrane, by Red Ponceau staining.
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FIG. 3. Results of hybridization in situ. Idiogram displays examples of G-banded chromosomesfrom different normal cells and shows the grain distribution. A total of 186 chromosomally located grains on 118 metaphases was analyzed. The predominant site of hybridization in each experiment was the long arm of chromosome 17 with a total of 31 grains localized on bands 17q22-q24. The number of grains in this region was at least four times the number found in any other chromosomal segment of similar length.
distribution of 186 labeled sites indicates a significant labeling on the long arm of chromosome 17. About 14% of labeling is observed on band 17q22-24. Figure 4 shows the distribution of MPO hybridizing sites on chromosomes of patient G.O. carrying the t(15;17). 26 metaphase cells from this hybridization were examined. The frequency of labeled sites indicates that MPO is present on a chromosome 15 and on a chromosome 17. Study of the MPO gene expression Figure 5 shows the results of Northern blot analysis of the RNA extracted from the APL cells. The RNA extracted from cells of a MPO expressing ALL, of the HL60 line and of two lymphoid populations without MPO expression were also analyzed. All the APL cell populations were expressing MPO at elevated, although variable, levels (lanes 2, 3, 4 and 5). However, the abundance of the MPO mRNA in the cells from patient G.O. (lane 3),was at least ten times higher than that observed in other cell populations. A quite different pattern of expression was observed studying the p53 gene. In one of the APL patients, the gene was not expressed. In the other three cases, including the case of G.O. overexpressing the MPO, quite similar levels of p53 mRNA were observed. In the HL-60 cell, a truncated p53 mRNA was observed.
Figure 6 shows the densitometric scanning of the autoradiographs of the Northern blot of Fig. 5. Western blot analysis Figure 7 shows the results of Western blot analysis in the different populations studied. The figure indicates that the concentration of the MPO protein was fairly in keeping with that of the corresponding mRNA, with the exception of the lymphoid cells of patient P.G. on which no MPO protein was detectable, as already reported [27]. The amount of MPO protein present in cells from patient G.O. was again at least ten times higher than that of other APL patients. This holds not only for the mature peptide (55 kD), but also for the precursor peptide (80 kD). Figure 8 shows a densitometric comparison between the levels of MPO mRNA and the levels of the corresponding protein. DISCUSSION Our results are in agreement with those reported by Miller et al. [12] and by Chang et al. [5], who have concluded that no rearrangement of the MPO gene may be observed in the APL cells by Southern blotting analysis. Our data are also in keeping with those of the authors who reported the mapping of the MPO
740
S. FERRAR1et al.
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FIG. 4. Distribution of labeled sites on normal chromosomes 15 and 17 and on the translocation derivatives 15q+ and 1 7 q - , after hybridization with the MPO-specific probe to metaphase cells from a bone marrow aspirate of an APL patient (G. O.). A total of 52 chromosomally located grains on 26 metaphase cells from this hybridization were examined. The results of this hybridizaton indicate that the MPO is translocated to the rearranged chromosome 15.
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FIG. 6. Densitometric scanning of the autoradiographs of the Northern blot analysis reported in Fig. 5 showing the levels of expression of the MPO m R N A and p53 mRNA.
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FIG. 8. The graphic shows the results of the densitometric scanning of the Western blots compared with the densitometric scanning of the MPO m R N A levels. Different densitometric units were used for R N A and protein.
Very different levels of the MPO gone expression in classical APLs gone to h u m a n c h r o m o s o m e 17(q22-q24). Since the A P L specific breakpoint on c h r o m o s o m e 17 is mapped at a location m u c h m o r e proximal at the centromere, it is difficult to believe that the M P O gone may be rearranged as a consequence of the translocation. A control Southern blot experiment performed with a p r o b e of the p53 gene, located on the short arm of c h r o m o s o m e 17, confirmed the absence of rearrangements of both the p53 and M P O genes. The high levels of M P O activity in A P L have been so far related to the differentiation arrest at the promyelocytic stage [9, 28], or to the t(15;17) chrom o s o m e translocation [9]. So far it is impossible to reach any conclusion about the significance of the translocation in the genesis of M P O overexpression. Presently, there is no indication that the two events are necessarily associated, and a much m o r e detailed molecular analysis is required, in order to clarify the changes in the genetic control mechanisms involved in the disorder of M P O gone expression. Acknowledgement--The authors are grateful to Dr G. Rovera, Wistar Institute, Philadelphia, for generously supplying the myeloperoxidase cDNA probe.
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