Deficiency of myeloperoxidase and abnormal chromosome 1 occurs in variant (HL60) promyelocytes

Leukemia Research Vol. 10, No. 5, pp. 501-513, 1986. 01~t5-2126/86 $3.00 + .00 Printed in Great Britain. © 1986 Pergamon Press Ltd

DEFICIENCY OF MYELOPEROXIDASE A N D A B N O R M A L CHROMOSOME 1 OCCURS IN VARIANT

(HL60) PROMYELOCYTES*

ARTHUR K. SULLIVAN,5" THOMAS T. AMATRUDA,§ LINDA FITZ-GIBBON,t H. PHILIP KOEFFLER,§ JOHN PEYMAN,~ GEOFFREY ROWDEN,:~ GENE SHEMATEKt and AWATEF

SHIHAB-EL-DEEN+

-~McGill Cancer Centre, McGill University, Montreal, and Division of Hematology, Royal Victoria Hospital, Montreal, Quebec, Canada, :~Department of Pathology, Dalhousie University, Halifax, N.S., Canada and §Department of Medicine, Division of Hematology-Oncology, UCLA

School of Medicine, Los Angeles, CA, U.S.A.

(Received 30 September 1985. Accepted 7 November 1985)

Abstract--Maturation of normal polymorphonuclear neutrophils is characterized by successive periods of granule synthesis, a process which frequently is abnormal in leukemia. Recently, the human leukemic cell line HL60, displaying a promyelocytic phenotype, has been used to study granulocyte maturation.

We describe a variant line of HL60, called HL60-A7, resulting from growth in actinomycin D, which contains atypical large azurophilic granules deficient in myeloperoxidase. The products of in-vitro translation of A7 RNA contained less than 5% of the immunoreactive MPO found in the parent line. Electrophoresis of plasma membrane polypeptides radioiodinated by the lactoperoxidase technique revealed several differences. Karyotypic analysis identified a consistent chromosome lq ÷ abnormality which was not found in any of the parental cells examined.

This constellation of differences between HL60 and HL60-A7, i.e. MPO deficiency, abnormal granule morphology, cell surface changes, and further cytogenetic abnormalities, may point to a common site sensitive to altered regulation in some leukemic promyelocytes.

Key words: Leukemia, peroxidase, promeylocytes, chromosomes, cytoplasmic granules, plasma membrane.

I N T R O D U C T I O N

As EARLY myeloid cells mature into neutrophilic granulocytes, they undergo an ordered sequence of organ- elle synthesis to produce at least two types of granule, each with a characteristic morphology and enzyme content [1, 2]. The appearance of the primary, or azurophilic granules is a major feature of the transition of a myeloblast into a promyelocyte. In acute myeloblastic

*Supported in part by the National Cancer Institute of Canada, National Institutes of Health grants CA 36038, CA 3273, and CA 33936, the Bruce Fowler Memorial Fund, and the Jonsson Comprehensive Cancer Centre. AKS is a recip- ient of a Chercheur Boursier award from the Fonds de la recherche en sant6 du Qu6bec; LF and JP, studentships from the Quebec Cancer Research Society; HPK, a Career Devel- opment Award from NIH; and TTA a fellowship from the Leukemia Society of America.

Abbreviations: BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; MPO, myeloperoxidase; NBT, nitro blue tetrazolium; 6-TG, 6-thioguanine; TCA, trichloroacetic acid.

Correspondence to: Dr A. K. Sullivan, McGill Cancer Centre, Rm. 714, 3655 Drummond St, Montreal, Quebec, H3G 1Y6, Canada.

leukemia several granule-related abnormalities occur within the immature cells such as deficiency of con- stituent enzymes, Auer bodies, and a large variation in quantity [3-5], The factors controlling this early process have received little study beyond ultrastructural descrip- tion, partly because of the difficulty in obtaining sufficient material for biochemical analysis and also because over 90% of the accessible cells of the marrow already have begun the next synthetic phase to produce secondary (specific) granules.

Human myeloid leukemic cell lines provide a model in which to study the process of granule formation. One of these lines, known as HL60, consists predominantly of promyelocytes with abundant azurophilic granules and myeloperoxidase [6]. When cultured in the presence of compounds such as dimethyl sulfoxide or retinoic acid, the cells mature into metamyelocytes and neutrophilic bands which can perform some normal func- tions, but are abnormal in that they lack secondary granules and lactoferrin [7-9]. Variants of HL60, derived by this and other laboratories, lack both primary granules and myeloperoxidase and fail to mature when exposed to known inducers of HL60 [10, 11]. The studies presented here describe the cells of a subline of HL60 which are deficient in myeloperoxidase and

501

502 ARTHUR K. SULLIVAN et al.

possess p r o m i n e n t , bu t a b n o r m a l , azurophi l ic granules . This is a potent ia l ly useful mode l in which to s tudy some of the m e c h a n i s m s of a b n o r m a l granule d e v e l o p m e n t in myelo id leukemia .

MATERIALS AND METHODS

Cell culture and derivation of the HL60-A7 subline

Cells were maintained under standard conditions of culture in RPMI-1640 medium supplemented with 10% fetal calf serum. Induction of HL60 cells to terminal maturation with DMSO or retinoic acid was performed according to the pro- cedures of Collins et al. [7] or Breitman et al. [8]. The degree of maturation was assessed morphologically and by the capacity to reduce NBT dye upon phorbol stimulation as described by Koeftier et at. [12].

A line with unusual properties was noted after a series of manipulations of HL60 cells, done for other reasons. From an uncloned culture of late-passage HL60 we obtained a clone resistant to 6 ~tg/ml 6-thioguanine (6-TG), and from their progeny subcultured another clone resistant to 20 Ixg/ml 6-TG, as described previously [10]. This line, called 20TG20, was exposed to seven four-day cycles of exposure and recovery from 0.1 nM actinomycin D over a period of six months. At that point the cells showed a high level of spontaneous maturation (15-30% NBT positivity) in the absence of any inducing agent. These cells were difficult to maintain in a proliferative state and over several weeks evolved into the line described here called 2020A7. The A7 lines grew similarly to HL60 except for a tendency to form clumps, in contrast to the parent which grows as single cells in suspension, and showed a much lower degree of spontaneous maturation (<1%).

Cytochemical methods. Staining of cytocentrifuge prep- arations of cells for myeloperoxidase, acid phosphatase and Sudan black were by standard methods [13]. Immuno- peroxidase staining has been described [14]. For electron microscopy cells were washed once in culture medium, fixed in 1.25% buffered paraformaldehyde, pelleted, post-fixed in buffered osmium, dehydrated and embedded in TAAB aral- dite resin (Marivac, Halifax, N.S.) for processing and analysis after staining with uranyl acetate.

Cytogenetic analysis. The standard method described by Sandberg was used in these studies [15]. Briefly, 10 7 cells were incubated with 0.02 Ixg/ml colcemid (Gibco) at 37 ° for 1 h and then in 0.075 M KC1 for 25 min. After pelleting, they were fixed for 30 min in methanol: glacial acetic acid (3:1 vol/vol); this was repeated three times using a 2:1 solution. Slides were prepared by dropping a sample of the cell suspension from 2.5 ft onto chilled, wet, precleaned glass slides. Standard staining was with the Giemsa, and G-banding by the trypsin- Giemsa methods.

Source of antibodies. The antibody to human myeloperoxidase was prepared in rabbits using purified protein (gen- erous gift of R. Lehrer) as the immunogen and has been characterized [16]. Goat antisera to human granulocyte elastase and rabbit antisera to granulocyte chymotrypsin-like enzymes, primary granule products [17], were a gift from Dr L. Moroz (Royal Victoria Hospital, Montreal) [18]. Murine monoclonal antibodies H36/71 and D46 have been described elsewhere [10, 14]. Briefly, H36/71 is reactive with a major granulocyte 3-fucosyl-N-acetyllactosamine antigen called Myl

[19]. The D46 antibody was prepared to a myeloblastic subline of HL60 and is reactive with the surface of these immature cells but not with that of HL60 or most normal myeloid marrow elements. However, it is strongly reactive with internal structures of granulated myeloid cells. The K101 antibody resulted from an immunization of mice with K562 cells from which a clone was selected that reacted with HL60 granules but not with the surface of any normal or cultured cells tested (unpublished results).

Analysis of cell surface proteins. Lactoperoxidase-catalyzed iodination was carried out according to the method of Hubbard and Cohn [29]. Briefly, 107 cells were used for each labeled cell preparation except where noted. Cells were sedimented at 300 × g for 4 rain at room temperature in 50 ml polypropylene tubes. Approximately 98% of the supernatant was aspirated and the cells were gently resuspended and washed with 10 ml of PBS-TE (0.15 M NaCI, 0.01 M phosphate, pH 7.5,100 KIU Trasylol, 0.5 mM EDTA). The cells then were transfered to a fresh 12 ml polystyrene tube to eliminate albumin and other bovine serum proteins adsorbed to the walls, and washed twice more with 10 ml of PBS-TE. To 200 ~1 of cell suspension were added 20 ~tl of 18 mg/ml glucose, 20 ~tl of 1.4 u/ml glucose oxidase, 20 ~tl of 0.2 mg/ml lactoperoxidase, and 10 ~tl of [125I]- NaI containing 0.5 mCi. The mixture was incubated at room temperature for 15 rain, swirled at 0, 5, 10, and 15 min, and the reaction stopped by washing the cells three times with PBS-TE.

Cells were lysed with 1 ml of 1% Triton X-100 in PBS- TENIBAPA (PBS-TE, made to 1 mM each in N-ethyl- maleimide, iodoacetamide, benzamidine, 6-aminocaproic acid, phenylmethylsufonyl fluoride and sodium azide). The cell lysis buffer was prepared immediately before use by the addition of each inhibitor from separate aqueous stock solu- tions, or dry dioxane stock solution (phenylmethylsulfonyl fluoride), directly to the cell suspension in PBS-TE in 10 ml polycarbonate ultracentrifuge tubes (Beckman Instruments) before the addition of the Triton X-100 stock solution. The extraction mixture was vortexed and particulate matter sedimented at 100,000 x g at 4°C for 10rain in a Type 40 or 50 Ti rotor (Beckman Instruments). The clear supernatant was aspirated and immediately (10-20 min after lysis) mixed with 5 ml of absolute ethanol at -20°C, and kept for 18-48 h at -20°C. The precipitated cell proteins were collected by centrifugation at 900 x g for 10 min at -5°C, and the drained pellet was dissolved in 100 ~tl of electrophoresis sample buffer consisting of 2% SDS, 10% glycerol, 0.2% bromphenol blue, 0.1 M dithiothreitol, and 0.01 M tris(hydroxymethyl)amino- methane, pH 6.8, and boiled in a water bath for 2 rain. Aliquots of 5 ~tl of each cooled sample were counted in an NCS 1185 gamma counter (Nuclear Chicago/Searle), or mixed with 5 ml of Aquasol-2 and counted in an LS-7500 beta counter (Beckman Instruments) or in a 1218 Rackbeta counter (LKB Instruments).

For electrophoresis, samples containing equal radioactive counts were applied to discontinuous slab gel sample wells [21]. The separate gel consisted of a 1.5-mm thick, 17-cm long, 7-15% acrylamide gradient; the stacking gel was of 3% acrylamide. Stacking proceeded for 2 h at 50 V, and separation for approx. 15 h at 75 V at ambient temperature. Gels were fixed and stained with 0.025% Coomassie brilliant blue in methanol-water-acetic acid (41:52:7), destained in methanol- water-acetic acid (7:86:7), and then in methanol-water-acetic

Myeloperoxidase-deficient (HL60) promyelocytes 503

acid-glycerol (6:82:6:6), and dried for 125I-autoradiography in a vacuum gel drier (Bio-Rad).

Metabolic labelling of proteins in vivo. Cells were grown to a density of 5 × 105 cell/ml in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum in a 5% CO2 atmosphere, washed and resuspended at 3 × 106cells/ml in methionine-free media (Flow) containing 10% heat-activated fetal calf serum. Metabolic labeling was with 50 ~tCi/ml [35S]- methionine (1400 CI/mmol) for 4 h at 37°C after which the cells were washed three times in media, once in phosphate- buffered saline (PBS: 0.02M sodium phosphate, 0.15M NaCI), and lysed by incubation in 0.05 M Tris-HCl (pH 7.0) buffer containing 0.4% NP-40, 0.4% sodium deoxycholate, 0.15 M NaC1, 5 mM EDTA and 1 mg/ml BSA. Aliquots of 2 ~tl were removed and assayed for TCA-precipitable radioactivity.

Extraction of RNA. HL60 and A7 cells were harvested in log phase of growth and frozen in liquid nitrogen. Total cellular RNA was extracted by the method of Chirgwin et al. [22] using guanidine thiocyanate and cesium chloride gradient centrifugation. Poly A+ RNA was selected by chromatography over an oligo dT-column [23]. One gram of frozen cells was thawed in 16 ml 4 M guanidine thiocyanate containing 0.5% N-lauroyl sarcosine and 0.i M 2-mercaptoethanol, suspended with 5 passes with a hard Dounce homogenizer, layered on 3 ml of CsCI (5.7 M) and centrifuged in a Beckman type SW41 ultracentrifuge rotor for 16 h at 36,000 rpm at 20°C. The RNA pellet was washed in ethanol, resuspended in H20 and guanidine HC1 7.5 M (2.6 ml), and precipitated by addition of cold ethanol, 0.5vol. This pellet was resuspended in H20 and precipitated by addition of 0.1 volume of potassium acetate (2 M) and 2.2 vol of cold absolute ethanol. Poly A+ RNA, prepared by a modification of the method of Aviv and Leder [24] was heated to 80 ° for 1 min, cooled rapidly, suspended at 150 ~tg/ml in oligo dT binding buffer (0.5 M LiCI, 0.1% SDS, 10 mM Tris-Cl pH 7.5, 1 mM EDTA), and passed twice over a column of oligo dT cellulose. The column was washed with 15 ml of oligo dT binding buffer, and the RNA eluted in 7 ml of elution buffer (0.1% SDS, 10 mM Tris-HCl pH 7.5, 1 mM EDTA). Samples were precipitated by addition of 2.2 vol of cold absolute ethanol and 0.1 vol of 3 M sodium acetate.

Translation of RNA in vitro. Poly A+ RNA was suspended in H20. Two microlitres of Poly A+ RNA from HL60 cells (1.7 mg/ml) and A7 cells (1.2 mg/ml) were incubated with 2 ~tl of 5 mM mercuric hydroxide for 5 min. In-vitro translation [25] was performed by addition of 6 ~tl rabbit reticulocyte lysate (nuclease treated), 40~tCi of [35S]-methionine and 4 units RNAsin to 1 ~tl of the RNA: mercuric hydroxide solution; K + was adjusted to 100 raM, and samples were incubated at 30°C for 90 min. Reaction was terminated by placing samples in ice. Aliquots of 2~tl were removed and assayed for TCA precipitable radioactivity.

Immunoprecipitation. Cell lysate (2 × 105-106 counts/rain of TCA-precipitable radioactivity) or in-vitro translation mix (5 x 104-106counts/rain of TCA-precipitable radioactivity) were diluted in immunoprecipitation buffer (PBS containing BSA 5mg/ml, methionine l mg/ml, N-lauroyl sarcosine 0.05%). Samples were cleared by mixing with 20 p.l of a 1:1 suspension of protein A agarose: immunoprecipitation buffer, incubated for 15 min at 4°C and centrifuged at 12,000 x g. The supernatant was treated with 10 ~tg of myeloperoxidase antibody and incubated for 3 h at 4°C. In selected experiments, 20 ~tg of unlabeled MPO was added to the translation mix to

compete with binding of antibody to radiolabeled MPO. Pro- tein A agarose (35 ~tl of a 1:1 suspension) was added to the supernatant, and samples incubated for an additional 15 min at 4°C, with occasional agitation. The protein A agarose:antibody complex was sedimented by centrifugation at 12,000 x g for 2min and washed 5 x with PBS containing 0.1% sodium lauroyl sarcosine. Immune complexes were dissociated by boil- ing for 6min in SDS sample buffer (1% SDS, 1% fl-mercaptoethanol) and analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE was performed according to Laemmli [21]. All gels contained 10% polyacrylamide. Gels were prepared for fluorography with ENHANCE (New England Nuclear) according to the manu- facturer's instructions.

Enzyme assays

Myeloperoxidase activity was determined by a microtiter plate adaptation of an assay by Babior and Cohen [26] using o-dianisidine, hydrogen peroxide, Triton X-100, and pH 5.5 citrate buffer. One unit of peroxidase activity is defined here as the initial rate of change in A450 given by 0.1 ng/ml horseradish peroxidase (Sigma, Type X) or 30ng/ml lactoperoxidase (Calbiochem).

R E S U L T S

Morphology

The light microscopic appearance of the HL60-A7 cells is shown in Fig. 1B, compared to the parental line (Fig. 1A). In A7 cultures there was a greater percentage of granulated cells, and the granules were more promi- nent than in HL60. Al though the percentage of HL60 cells with visible granules (30-90%) varied widely under different condit ions of culture (unpublished observations), that of A7 was uniformly greater than 90%. Large vacuoles also were seen consistently in A7, even under optimal culture condit ions where the viability, assessed by trypan blue exclusion, was over 95%. On thin sections of embedded cell pellets, these vacuoles clearly were distinguishable from granules (not shown).

Electron-microscopic examinat ion of HL60 revealed several discrete granular structures with a fine, lacy staining pat tern (Fig. 2A). In contrast, the granules of the A7 subline (Figs 2B and C) often showed partially disrupted limiting membranes , many of which appeared to be fused together, and contained a dense "bull 's eye" precipitate.

Cytogenetic analysis Cells in metaphase or mitosis were prepared from the

parental and A7 lines and compared (Fig. 3). Of 10 HL60 cells banded and examined in detail, 5 contained 44 chromosomes, 3 contained 45, 1 had 46, and 1 had 43. All contained double minutes. Other abnormalit ies found most consistently included: - X , - 5 , - 8 , - 1 6 , - 1 7 , +18; as well as C, acrocentric D and E group markers. The findings are consistent with those reported previously for HL60 by Gallagher et al. [6]. Analysis of 15 cells of the A7 line showed 9 with 46 chromosomes, 4 with 45, and 2 with 44. Abnormali t ies included: - X , - 5 , - 8 , - 1 6 , - 1 7 , +18 and A ,C ,D, and E group


markers; double minutes were not found in any of the metaphases. A new marker, lq ÷, was observed in all cells analysed. Banding studies suggested that the abnormality was a duplication of the q21---~qter segment. This structure was not seen in any HL60 metaphases, among cells of the 6TG-resistant line (clone 6TG20) preceding A7, or at the stage of high-spontaneous maturation after actinomycin D treatment while the predominant population still expressed myeloperoxidase.

Granule content A striking feature of the A7 cells was the lack of

myeloperoxidase (MPO) activity and sudanophilia assessed by histochemistry (Figs 1E/F and I/J) . The deficiency of MPO was quantitative and not due to an inactive enzyme, since a rabbit antibody to purified human MPO failed to visualize more than 2% of cells by immunoperoxidase staining (Fig. 1 M/N). This was confirmed by direct assay for enzymatic activity of cell extracts (Fig. 4). The possibility of low MPO content

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0.1" 2"

• - _ _ , - - ~ . . . . . . . . . . . . . . . . . . i !

4 8 12 16 20 24 TIME (mini

FIG. 4. Colorometric assay for mye|operoxidase in cell extracts. (A) HL60. (11) HL60-A7.

on the basis of synthesis and premature secretion was eliminated by the measurement of less than 0.5% of the activity in the A7 culture supernatant than in that of HL60 (not shown). Failure to detect MPO did not reflect a general defect in synthesis or localization of granule enzymes since acid phosphatase activity and' immunoreactive granulocyte elastase and chymotrypsin-like enzymes were present in at least as great a quantity in A7 as in HL60 (Figs 1 C/D, S/T and U/V). The pattern of acid phosphatase staining as well as that of three monoclonal antibodies to granule- related structures (D46, K101 and H36/71; Figs 1 G/H, K/L and O/P) further visualized the coarse nature of the A7 granules, in contrast to the fine, discrete structures observed in the parental HL60 cells.

Cell surface properties Electrophoretic analysis of the total cell Triton-

extractable proteins indicated that the major peptides of HL60 and A7 were of an identical molecular weight (Fig. 5). In contrast, the surface structures radioiodinated by the lactoperoxidase method were quite

different, indicating either changes in the overall peptide composition of the plasma membrane or differences in their relative accessibility to the enzyme. Other experiments have shown that these differences were not merely due to endogenous myeloperoxidase in HL60, which potentially can catalyse radioiodination (Peyman et al., manuscript submitted).

Metabolic labeling and mRNA translation in vitro To further define the defect in MPO production,

biosynthetic experiments were performed with whole cell lysates and purified RNA from parental HL60 and A7.

Metabolic labeling of proteins Electrophoresis of cell lysate from A7 revealed a

pattern of proteins similar to that of HL60 cells. Immu- noprecipitation of HL60 cell lysate (2 x 105 counts/min) with antibodies to MPO showed bands of radioactivity with approx, mol. wt of 85, 60 and 45 kilodaltons (kd) (Fig. 6; lane 3). These bands are specific for MPO, as has been demonstrated previously by cold-competition studies [16]. In contrast, immunoprecipitation of either A7 or Raji (a B-lymphoblastoid cell line) cell lysate

5 (2 x 10 counts/min of TCA precipitable radioactivity) with MPO-specific antibody did not reveal any bands of radioactivity corresponding to myeloperoxidase proteins (Fig. 6; lanes 1 and 2).

In-vitro translation of mRNA Translation in vitro of intact poly A + RNA from

A7 and HL60 cells produced a pattern of radioactive proteins ranging in size from 14 to 100 kd. Immuno- precipitation of the translation mix from mRNA of HL60 (1 × 105 and 2 × 105 counts/min) revealed protein bands of approx, mol. wt 74, 45, 46 and 15 kd (Fig. 7; lanes a and b). Addition of unlabeled MPO to the translation mix (cold competition experiment) inhibited immunoprecipitation of labeled MPO, causing the protein bands of 74, 45 and 15 kd to disappear (Fig. 8; lanes d and e). The protein band of 46kd was not MPO specific and may be actin [27].

Myeloperoxidase immunoprecipitation of the translation of poly A RNA (1.5 × 105 counts/min) from A7 cells did not reveal any radioactive protein bands (Fig. 7; lane d). Electrophoresis of the whole translation mix from A7 and HL60 (Fig. 7; lanes e and f) (2 x 104counts/min) revealed a protein of approx. 75 kd which was present in the HL60 translation but absent from A7 translation.

In a subsequent experiment, immunoprecipitation of the translation mix from A7 mRNA labeled to 5 × 105 and 10 6 counts/min also failed to reveal MPO-specific radioactivity (Fig. 8; lanes a and b). Numerous faint bands of radioactivity of 15-90 kd were noted after immunoprecipitation of 10 6 counts/min. These did not change with cold competition and therefore represent non-specific immunoprecipitations (Fig. 8; lane c).

Response to inducers of maturation When cultured in the presence of DMSO or retinoic

acid, A7 cells acquired the capacity to reduce NBT dye

FIG 1. Photomicrographs comparing HL60 parent and HL60- I/J, myeloperoxidase activity; K/L, anti-granule antibody A7 cells. HL60: first and third vertical rows (A, E, I, M, Q K101; M/N, rabbit anti-MPO; O/P, antibody H36/71 (Myl and C, G, K, O, S, U). AT: second and fourth vertical rows antigen); Q/R, Jenner-Giemsa of cells incubated three days (B, F, J, N, R and D, H, L, P, T, V). Staining reactions are with retinoic acid; S/T, goat anti-neutrophil elastase; U/V, as follows: A/B, Jenner-Giemsa; C/D, acid phosphatase; rabbit anti-chymotrypsin like proteinase. E/F, Sudan black; G/H, immune staining with antibody D46;

505

FIG. 2. Electron micrograph of HL60 and HL60-A7 granules. (A) HL60 (30,600×). (B) A7 (30,600×). (C) Detail of A7

fused granules (48,700×).

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14 15 MA iC ME MD 16 17

20 21 22 FIG. 3. Representative karyotypes of HL60 and HL60-A7 cells. MA, MC, MD, ME are abnormal markers from the lettered group. DM: double minute. HL60 karyotype: 44X (-X, -5~ -8, -16, -16, -17, +18, +MC, +MD, +ME, +DM); HL60-A7 karyotype: 46X (-X, lq ÷, -5, -8 , -16, -16, -17, +18, +18, +MA, +MC, +MD, +ME). Some cells showed three or four chromosome 18 in an inconsistent

fashion.

I x

1 8

Y

507

A B C D 1 2 3 4

92

69

46

30

14

FIG. 5. Polyacrylamide gel electrophoresis of total cell Triton extract and radioiodinated cell surface polypeptides. Lanes A and B: Stained for total protein with Coomassie brilliant blue. Middle Lane, mol. wt standards noted on the corresponding lane between C and D. Lanes C and D: Autoradiogram of gel electrophoresis shown in A and B of cells radioiodinated by the lactoperoxidase method to visualize cell surface peptides.

Lanes A and C: HL60. Lanes B and D: HL60-A7.

FIG. 6. Metabolic labeling of proteins in vitro. Cells were grown for 4 hr in the presence of [35S]-methionine and lysed; the lysate was immunoprecipitated with MPO-specific antibody. Lane 1:A7 cell lysate containing 2 x 105 counts/rain of TCA-precipitable radioactivity. Lane 2: Raji cell lysate, containing 2 x 105 counts/rain of TCA precipitable radioactivity. Lane 3:HL60 cells containing 2 x l0 s counts/rain of TCA precipitable radioactivity. Arrows indicate MPO specific radioactivity. Lane 4: mol. wt markers (C ~4 methylated

proteins: 92, 69, 46, 30, 14.3 kd).

508

92 a b c m d e f

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4 6

30

14

FIG. 7. RNA translation in vitro and immunoprecipitation. Poly A+ RNA from A7 and HL60 cells was translated in oitro and immunoprecipitated with MPO specific antibody. Lanes a, b, c and d represent MPO immunoprecipitations. (a) Immunoprecipitation of HL60 mRNA translation products containing 2 x 105 counts/min of TCA precipitable radioactivity. (b) HL60 mRNA translation products containing 1 x 105 counts/min of TCA precipitable radioactivity. (c) HL60 mRNA translation products containing 5 x 104 counts/rain of TCA precipitable radioactivity. (d) A7 mRNA translation products containing 1.5 x 105 counts/rain of TCA precipitable radioactivity. Lanes e and f represent translation without immunoprecipitation. (e) A7 mRNA translation containing 2 x 104 counts/min of TCA precipitable radioactivity. (f) HL60 mRNA translation containing 2 x 104 counts/min of TCA precipitable radioactivity. Arrows in lane (a) indicate MPO specific radioactivity. Arrow in lane (f) indicates a protein band of approximately 75 kd which is present in HL60 translation products but absent in A7. (m) mol.

wt markers (C14-methylated proteins: 92, 69, 46, 30, 14.3 kd).

509

a b e

FIG. 8. RNA translation in vitro and immunoprecipitation in the presence and absence of unlabeled MPO. Poly A + RNA from A7 and HL60 cells was translated in vitro and immunoprecipitated with MPO-specific antibody. Immunoprecipitations in lanes (c) and (e) were performed after the addition of 40 Ixg of unlabeled MPO to the translation mix (cold competition). (a) Immunoprecipitation of A7 mRNA translation products containing 106counts/min of TCA precipitable radioactivity. (b) Immunoprecipitation from A7 mRNA translation containing 5 × 105 counts/min of TCA precipitable radioactivity. (c) Immunoprecipitation of A7 mRNA translation containing 5 × 105 counts/min of TCA precipitable radioactivity, in the presence of 20 ~tg of unlabeled MPO. (d) Immu- noprecipitation of HL60 mRNA translation containing 4 x 105 counts/min of TCA precipitable radioactivity. (e) Immunoprecipitation of HL60 mRNA translation containing 4 × 105 counts/min of TCA precipitable radioactivity, in the presence of 20 Ixg of unlabeled MPO. Arrows in lane (d) indicate MPO specific radioactivity. (m) mol. wt. markers (C 14-

methylated proteins: 92, 69, 46, 30 kd).

510


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NBT / , /

/ 20

0 1 2 3 T IME(days)

FIG. 9. Course of development of the capacity to reduce NBT dye during exposure to retinoic acid. (0) HL60; (&)

HL60-A7.

(Fig. 9) and decreased their capacity to form colonies in soft agar in a manner similar to the parent HL60 cells (not shown). However, unlike the parental line, after 72 h they rapidly lost viability and did not show mor- phological maturation past the myelocyte stage (Fig. 1 Q/R).

D I S C U S S I O N

The A7 subline of human HL60 promyelocytes contains a constellation of abnormalities in primary granule structure and content, and differences in plasma membrane polypeptides that may or may not originate from a defect caused by the altered chromosome 1. Overall, the karyotype of the parental HL60 cells in our laboratory is similar to that reported by Gallagher et al. [6] and Nowell et al. [28] and to that of the A7. A striking dissimilarity in this HL60 cell pair is the loss of double minutes from the parent and the appearance of an elongated, possibly duplicated, lq ÷ in the MPO- deficient A7 subline. Rowley and others [29-32] have shown that a lq segment often is duplicated in some of the myeloproliferative disorders. Bernard has reported three cases of refractory anemia with excess blasts show- ing a chromosome l trisomy [33]. Also, fragile sites have been identified at the q25 band and terminus of this chromosome [34,35]. Studies on the gene rearrangements in Burkitt's lymphoma suggest that cytogenetic alterations tend to occur at sites most active in expressing differentiated features of the neoplastic cells, and that some oncogenes, e.g. c-myc, may interact with stage-specific differentiation factors to cause pathological changes [36]. Although inspection of the gene map of chromosome 1 [37] does not reveal the presence of any known markers of myeloid differentiation, a possible defect in the MPO gene, in association with this lq + anomaly, may be a fruitful direction for further search.

The role that actinomycin D may have played in generating chromosomal instability at such sites remains

unknown. This complex binds to double stranded DNA most avidly at G-C pairs at several chromosomal locations throughout the genome [38, 39]. Studies on its capacity to protect certain sites from DNAase I digest- ion in vitro, suggest that the binding of actinomycin D is not random [40]. Similarly, it inhibits RNA elongation in the vicinity of 5' XGCY 3' sequences [41]. Should this occur also within living cells, it would be reasonable to suspect that the drug might have potentiated a mutation or rearrangement in a gene related to the expression of MPO, a major component of promyelocytes, comprising over 1% of their total protein content [42].

Between a gene and a final polypeptide product are many possible sites for a defect that could result in failure to detect enzyme activity. The MPO in A7 cells was analysed both cytochemically and for activity in a solubilized cell extract. In these cells there was less than 0.5% of the MPO found in HL60. Metabolic labelling studies demonstrated that HL60 synthesized known precursor forms of MPO [16, 43, 44], but A7 cells did not. Likewise, in-vitro translation studies suggested that A7 cells did not contain mRNA which could be translated to a polypeptide that bound to a polyclonal MPO anti- serum. No MPO protein could be detected even when using 106 counts/min of [35S]-methionine in the immunoprecipitation mix after translation of A7 mRNA. Conversely, MPO proteins could be detected in a preparation of HL60 products labeled to only 5 × 104 counts/ min. These results indicate that synthesis of MPO- specific mRNA was decreased by more than 95% in A7 cells in comparison with HL60. The weak immuno- staining with a rare strongly-staining cell in A7, in contrast to the complete lack of staining of Raji (not shown), suggests that the gene is not totally deleted but may be partially transcribed or give rise to an unstable or inefficient RNA as is noted in some thalassemic syndromes [45].

A direct relationship between the MPO defect and the abnormality of the primary granules might exist, as suggested by their simultaneous appearance. The associated finding of decreased staining by the lipophilic dye Sudan black may reflect an underlying abnormality of the granule lipids. On the other hand, it has been suggested that the stable, chloroform-insoluble sudanophilia of granulocytes is dependent upon a prior inter- action or activation by MPO [46]. Our observations are consistent with this hypothesis. Other structural abnormalities of primary granules, with and without MPO deficiency, have been noted in neoplasms of the myeloid lineage [47]. Loss of MPO and failure to form normal secondary granules occurs in the morphologically-mature neutrophils of some patients with dysmyelopoietic syndromes and acute leukemia [48]. Indeed, Bessis has commented that the primary granules of leukemic myeloblasts can be abnormally large and have a tendency to fuse, and has suggested that these fused forms are the precursors of Auer rods [49]. To our knowledge, Auer rods have not been reported in HL60 promyelocytes. Schmalzl et al. [50], in a study of the ultrastructure of leukemic cell granules, showed that blasts from one patient contained large, lobulated


structures somewhat similar to those that we have noted in HL60-A7. More recently, Brederoo et al. [2] have made a detailed electron micrographic study of devel- oping granules of promyelocytes and myelocytes from normal human marrow and have identified three types, nucleated, azurophilic and specific, each of which changes in appearance during cell maturation. In their photographs both the homogeneous (HL60 type) and the condensed-centre (A7 type) form can be seen. At present we are not able to state whether the forms of granule observed in these HL60 cell lines reflect stages in normal or diseased promyelocytic granule maturation, or if the abnormal structure of A7 granules (Fig. 2) is related to the differences in the plasma membrane peptide composition (Fig. 5). From the study of lectin-resistant CHO mutants, Stanley has suggested that abnormalities in surface glycoproteins might result from defects in the internal membrane-sorting apparatus [51]. Whether the pathology of A7 granules reflect a pathway of progression similar to that followed by leukemic cells in vioo awaits further study.

Another relevant aspect of the HL60 phenotype is the capability to mature into neutrophil or macrophage- like cells when stimulated by a variety of compounds. Both DMSO and retinoic acid stimulate A7 cells to activate the apparatus necessary to reduce NBT dye. However, the high rate of cell death after the third day of induction precludes assessment of whether A7 cells can mature to the same extent as the parent over a five- day period. Nauseef et al. [52] reported that some MPO- deficient granulocytes have exaggerated production and release of superoxide and peroxide, as well as enhanced HMP shunt and phagocytic activities. Furthermore, Standahl et al. [53] have shown that the MPO-H202 system may retard neutrophil activity by altering Fc and C3b receptors. In HL60, MPO may have a similar protective effect during activation of the oxygen radicals following maturation induction, whereas in A7 there may not be sufficient protection from the effects of these toxic metabolites. Conceivably, in such a situation, granule damage, lysosomal instability and fusion events might occur.

All of these findings illustrate in a culture model some of the pathological changes that have been observed in neoplastic myeloid cells. By studying the biochemical basis for these defects and their possible relationship to the concurrent cytogenic abnormalities, one may further understand the cellular events giving rise to the leukemic phenotype.

Acknowledgements--the authors thank Mrs E. Jenkins for typing the manuscript, Ms L. Morishita for assistance in preparation of figures, and Dr J. Prchal of the Hematology Division of the Royal Victoria Hospital for supervision of cytogenetic analyses.

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Deficiency of myeloperoxidase and abnormal chromosome 1 occurs in variant (HL60) promyelocytes