Cytometry 13:653-658 (1992)

0 1992 Wiley-Liss, Inc.

Protooncogene Expression in Subpopulations of Cells From Leukemia Patients' Ben C. Hulette, Shripad D. Banavali, Daniel P. Finke, Venu Gopal, and Harvey D. Preislep University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0508 Received for publication November 11, 1991; accepted March 2, 1992

This report describes a method for preserving the light scatter patterns of cells in which myc and myb expression are being measured. Exposure of cells to 1% paraformaldehyde for 72 h prior to antibody staining for myc and myb proteins preserved the light scatter patterns. Using this method, myc and myb expression was found to be highest in lymphocytes and monocytes and lowest in granulo cytes. The measurement of differences in

INTRODUCTION Protooncogenes are under intense investigation because of their central role in the regulation of proliferation and differentiation. Until recently, identification of protooncogene expression in leukemia cells has been dependent on RNA analysis via Northern blot or dot blot methodologies (2,12-14,161. Recently, methods utilizing monoclonal antibodies and flow cytometry for assessing intracellular levels of myc and myb protein have been described (4,7). These methods provide information on the expression of these genes in the cell population as a whole. Analysis of expression in subpopulations of cells would be possible if light scatter patterns (forward versus orthogonal light scatter) could be maintained. These patterns, however, are destroyed by the use of detergents to permit entry of the antibodies into the cells. In this paper, we describe a technically simple method which permits preservation of the light scatter of cells thereby allowing evaluation of myc and/or myb expression in cell subpopulations obtained from leukemia patients. This method appears to be superior to the permeabilization method described earlier to preserve light scatter for blood cell work (8). MATERIALS AND METHODS Normal peripheral blood (PB) and bone marrow aspirates (BM) from 3 bone marrow transplant donors as well as BM aspirates from 6 patients with acute myelogenous leukemia (AML) were collected in heparinized tubes after informed consent. For this paper, we ran-

the level of expression of these genes in subpopulations of leukemia cells obtained from individual patients is possible as is assessment of the levels of expression amongst normal and leukemia cells present in the same patient. 0 1992 Wiley-Liss, Inc.

Keyterms: myc, myb, flow cytometry, light scatter

domly selected one normal PB, one normal BM, and one BM from a patient with AML from the above mentioned group. The PB and BM were gradient separated over Ficoll-Hypaque (1.077 g/ml) (Pharmacia, Piscataway, NJ). The mononuclear fraction was collected and washed twice in phosphate buffered saline (PBS; Gibco, Grand Island, NY) supplemented with 1% fetal calf serum (FCS; Gibco, Grand Island, NY). A cytospin slide was prepared for morphology. A total of 1 x 10' mononuclear cells were placed into 12 x 75 mm polystyrene round-bottom tubes (Falcon Labware, Lincoln Park, NJ), pelleted, and fixed for 30 min a t 4°Cin 1% reagent grade paraformaldehyde (Fisher Scientific, Fairlawn, NJ) in PBS. According to the standard staining procedures (101,the cells are washed free of paraformaldehyde, permeabilized by resuspending the cells in 0.1% Triton X-100, and stained for myc and myb proteins. This procedure degrades the light scatter pattern so that i t cannot be used to distinguish among different cell types. For the purpose of comparison, a n aliquot of the same cells was kept in 1%paraformaldehyde for 72 h prior to myc and myb staining. As a baseline control, the light scatter pattern of unfixed cells was assessed.

'This work was supported in part by Grant CA41285 from the National Cancer Institute and The Lois MacKay Scripps Drug Development Fund. 'Address reprint requests to Harvey D. F'reisler, M.D., University of Cincinnati Medical Center, K Pavilion, M.L. 508, 231 Bethesda Avenue, Cincinnati, OH 45267-0508.

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FIG.1. Identification of peripheral blood cells. A Forward versus orthogonal (90")light scatter pattern. B: 2-color dot-plot showing reactivity with CD-45 and CD-14 antibodies. M = monocytes; G = granulocytes; L= lymphocytes. 10,000 events are displayed in A and B each. The proportion of events in corresponding cytograms in A and R are equal.

The fixed mononuclear cells were washed free of paraformaldehyde and permeabilized by resuspending in IFA (10 nM HEPES, 150 nM NaC1, 4% FCS, 0.1% sodium azide in distilled water) containing 0.1% Triton X-100 (IFA-Tx) for 5 min (10,ll). The cells were pelleted and resuspended in 100 pl of IFA-Tx. For myc staining, the cells were incubated with a mouse monoclonal antibody (MAb) against myc peptide (Microbiological Associates, Bethesda, MD) a t a concentration of 1 pg per 1 x lo6 cells for 30 min a t 4°C. As a negative control, the myc antibody was neutralized by pre-incubation with 2 x concentration by weight of myc peptide (Microbiological Associates, Bethesda, MD) for 1 h a t room temperature prior to addition to the cells being tested. For myb staining, cells were incubated with a MAb against myb peptide (Microbiological Associates, Bethesda, MD) a t a concentration of 1 pg per 1 x lo6 cells for 30 min a t 4°C. The negative control consisted of cells treated with a myb antibody which had been neutralized by preincubation with 2.5 x concentration by weight of myb peptide (Microbiological Associates, Bethesda, MD) for 1h r a t room temperature. The cells were then washed twice in IFA-Tx, resuspended in 100 pl of IFA-Tx, and incubated with affinity purified FITC-conjugated goat-anti-mouse Ig (TAG0 Inc., Burlingame, CA) at a concentration of 3.5 pg per l x lo6

FTG.2. Effect of Tx-100 on light scatter pattern. A: Light scatter pattern of fresh unfixed normal peripheral blood cells. B: Effect of Tx-100 on light scatter pattern of fresh cells. C: Effect of Tx-100 on light scatter pattern of cells fixed for 24 h in 1% paraformaldehyde. Note that, as in b, the light scatter pattern is destroyed. D Preservation of light scatter pattern by fixation for 72 h in 1% paraformaldehyde despite treatment with Tx-100. The cytometer gains for a and d are same.

cells for 30 min a t 4°C. The cells were then washed in IFA and resuspended in 0.5 ml of PBS and analyzed by flow cytometry.

Flow Cytometry Flow cytometric analysis was carried out using a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CAI interfaced with a Hewlett Packard Model 310 computer. An excitation source of 488 nm was achieved using a 15 milliwatt air-cooled argon-ion laser. Fluorescence emission was collected through a 530130 bandpass filter for FITC labeled cells and through a 585142 bandpass filter for phycoerythrin (PE) labeled cells. AutoCOMP software, Version 2.0 along with CaliBRITE beads (Becton Dickinson) were used to set-up the instrument. Consort-30 program (Becton Dickinson Immunocytometry Systems, San Jose, CA) was used for acquisition and analysis of data. Routinely, lysed whole PB double stained with FITCconjugated human leukocyte antigen (CD-45) (1,151 and PE-conjugated human monocyte/macrophage antigen (CD-14) (3,9) were used for antigenic confirmation of subpopulations identified by their light scatter characteristics (Fig. 1B). Instrument settings for the baseline unfixed cells were saved and read each time the corresponding light scatter of the preserved specimens were run. Percent positivity was measured by gating the histogram. The gate included < 1% false positive

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FIG. 4. myc expression in subpopulations of normal peripheral blood after Ficoll-Hypaque gradient separation. The histograms on the right side gives the myc data of corresponding gated population of FIG.3. Stability of myc and myb expression during fixation and cells. Dotted histogram is from data of control cells and solid histostorage in 1%paraformaldehyde. The percent positivity of cells for gram from that of antibody-treated cells. The numerical data for this antibody and fluorescence intensity ratios (Ratio of ungated mean figure are also given in Table 1. Note the high expression of myc in channel of histogram from antibody-treated cells to that of the con- lymphocytes and monocytes and the very low expression in polymortrol) are plotted against the number of days the cells were in 1% phonuclear neutrophils (FMN). paraformaldehyde before staining. A: myc expression in HL60 cells. B: myb expression in HL60 cells. C : Percent positivity of myc and myb expression in freshly obtained AML cells. The frequencies of cell populations were: Blast cells (389'01, granulocytes (52%), lymphocytes cells. The mean channels of the ungated histograms (7%), and monocytes (3%). were used to determine the fluorescence intensity ratio -ME

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RESULTS The light scatter pattern of normal peripheral blood is provided in Figure 1A. Three populations are dis-

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HULETTE ET AL.

Table 1 myc and myb Expression in Subpopulations of Normal Peripheral Blood and Bone Marrow Normal peripheral blood MYB

Normal bone marrow MYC MYB %( + ) %( + ) F . I ratio %( + 1 F . I ratio %(+) F . I ratio 34.8 1.93 32.3 1.18 56.3 1.72 34.4 1.16 100.0 11.01 84.3 2.31 97.4 10.25 59.3 1.93 93.1 5.15 100.0 1.93 96.5 6.5 84.7 1.75 6.2 1.5 1.1 1.15 6.5 0 1.43 1.0 ratio of ungated mean channel of histogram from antibody treated cells to that of the control. MYC F . I ratio"

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"F . I ratio = 'PMN = polymorphonuclear neutrophils.

cernable, one which is separated from the others (G) and two which are contiguous (L,M). Figure l B , using antibodies against CD-45 and CD-14, also identifies 3 different cell populations. Figure 2 demonstrates the effects of Tx-100 on the light scatter pattern of peripheral blood. Note that fixation in 1% paraformaldehyde for 24 h does not prevent destruction of the light scatter pattern by Tx-100 but that fixing for 72 h does. To determine the effects of prolonged fixation on myc and myb expression, HL60 cells were placed in 1% paraformaldehyde and aliquots removed a t various time intervals for myc and myb analyses. Figure 3A demonstrates that the percent cells containing myc and the fluorescence intensity are stable for at least 75 days. The fluorescence intensity (F.1.) is defined by the mean fluorescence intensity of antibody treated cells divided by the mean fluorescence intensity of cells treated with antibody which was preincubated with the corresponding peptide. Figure 3B demonstrates that myb protein levels are stable only for 15 days. Figure 3C demonstrates that myc and myb stability in freshly obtained human AML cells is similar to that of HL60 cells. Figure 4 presents a n analysis of myc expression in subpopulations of cells in normal peripheral blood. In the analysis of the populations a s a whole, note the complex patterns of the nonspecific binding of the antibody and that the binding of the antibody is only marginally different than the highest level of nonspecific binding. When myc expression in specific subpopulations of cells are assessed using the light scatter profile to select these cells, the level of gene expression is easily discernable. Similar analysis was carried out for myb expression in the same sample and for myb and myc expression in normal bone marrow. The data is summarized in Table 1. Myc and myb expression are very low in granulocytes while these two genes are highly expressed in the lymphoid and monocytic populations. If one had relied upon the analysis of the peripheral blood or marrow cell population as a whole, the high level of expression of these genes in these two normal cell populations would have been unappreciated.

Table 2 myc and myb Expression in Subpopulations of Cells in A M L Bone Marrow

%(+I All data

Lymphocytes Monocytes PMN~ Blasts

69.6 98.8 100.0 35.3 100.0

mYc F . I ratio" 3.07 17.1 13.78 1.88 7.78

%(+)

81.8 91.9 91.6

83.4 86.0

mYb F . I ratio 4.84 9.1 8.35 3.79 7.97

"F . I ratio = Ratio of ungated mean channel of histogram from antibody treated cells to that of the control.

bPMN = Polymorphonuclear neutrophils.

Figure 5 provides a n analysis of myc expression in the bone marrow of a patient in whom leukemic blast cells represent a minority of the cells present. Analysis of the marrow cell population as a whole is similar to that of normal marrow as are the levels of myc and myb expression in the various normal cell populations identifiable by light scatter (Table 2). The levels of myc and myb expression are easily measured in the blast cell population and are similar to that present in the monocyte and the lymphocyte populations.

DISCUSSION Assessment of gene expression before and during therapy is of increasing clinical importance. Protooncogene expression in AML has prognostic significance (6) and bioactive agents, such as rh-GMCSF, can alter protooncogene expression in leukemia cells in patients (5). While flow cytometric assessment of gene expression is perhaps superior to RNA analyses, conventional flow cytometric methods cannot readily assess gene expression in subpopulations of cells. This limitation is not a trivial one since, in many clinical situations, the leukemia cells may not be the predominant cell population. For these reasons, preservation of the light scatter profile of cells during assessment of myc and myb ex pression is of major significance. The method described

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here presents the light scatter profile while maintain ing myc and myb protein levels for at least 2 weeks. One added advantage of this method is that the specimens in a single study, which may extend over several days, can be accumulated and analyzed on the same day thereby minimizing differences in conditions which can affect the measurement of gene expression on different days. Additionally, the stability of myc protein levels when the cells are kept in 1%paraformaldehyde provides a standard which can be run whenever analysis of myc expression is being carried out.

ACKNOWLEDGMENTS The authors wish to thank Mr. Joseph Evola for the preparation of excellent illustrations and Ms. Sandra Buchman for excellent secretarial assistance. Forward Scatter

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FIG.5. myc expression in subpopulations of AML bone marrow cells after Ficoll-Hypaque gradient separation. The marrow contained 29% blasts; 9% promyelocytes; 52% myelocytes, metamyelocytes, and granulocytes; 7% lymphocytes; and 3% monocytes. The histogram on the right provides the myc data for corresponding population of cells. The dotted histogram represents data from control cells, whereas solid histograms, the data from antibody-treated cells. Note the high expression of myc in blasts, lymphocytes, and monocytes and the low expression in polymorphonuclear neutrophils (PMN). Numerical data for this figure are given in Table 2.

LITERATURE CITED 1. Beverley PCL: Production and use of monoclonal antibodies in transplantation immunology. In: Transplantation and Clinical Immunology XI, Touraine J L , Trager J, Beteul H, Brochier J , Dubernard JM, Revillard JP, Triau R (eds). Excerpta Medica, Amsterdam, 1980, pp 87-94. 2. Blick M, Westin E, Gutterman J, Wong-Staal F, Gallo RC, McCredie K, Keating M, Murphy E: Oncogene expression in human leukemia. Blood 64(6):1234-1239, 1984. 3. Dimitriu-Bona A, Burmester GR, Waters SJ,Winchester RJ: Human mononuclear phagocyte differentiation antigens I. Patterns of antigenic expression on the surface of human monocytes and macrophages defined by monoclonal antibodies. J Immunol 130: 145-152, 1983. 4. Evan GI, Lewis GK, Ramsay G, Bishop JM: Isolation of monoclonal antibodies specific for human c-myc protooncogene product. Mol Cell Biol 5:3610-3616, 1985. 5. Freeman J, Gopal V, Hulette B, Flessa H, Bhaskaran J , Hun R, Raza A, Preisler HD: rh-GMCSF in AML: Clinical and biological effects. Blood 78(10):452a, 1991. 6. Gopal V, Hulette B, Li YQ, Kuvelkar R, Raza A, Larson R, Goldberg J, Tricot G, Bennett J, Preisler HD: Myc and myb expression in acute myelogenous leukemia. In press, Leukemia Research, 1991. 7. Gowda SO, Koler RD, Bagby, Jr., GC: Regulation of c-myc expression during growth and differentiation of normal and leukemic human myeloid progenitor cells. J Clin Invest 77:271-278, 1986. 8. Hallden G, Anderson U, HEd J , Johanson SGO: A new membrane permeabilization method for the detection of intracellular antigens by flow cytometry. J Immunol Methods 124:103-109, 1989. 9. Herrmann F, Komischke B, Odenwald E, Ludwig WD: Use of monoclonal antibodies as a diagnostic tool in human leukemia I. Acute myeloid leukemia and acute phase of chronic myeloid leukemia. Blut 47:157-163, 1983. 10. Kasten MB, Salmon DJ, Civin CI: Expression of protooncogene c-myb in normal hematopoietic cells. Blood 73:1444-1451, 1989. 11. Loke SL, Neckers LM, Schwab G, Jaffe ES: C-myc protein in normal tissue: Effects of fixation on its apparent subcellular distribution. AJP 131:29-37, 1988. 12. McClain K L Expression of oncogenes in human leukemias. Cancer Research 445382-5389, 1984. 13. Preisler HD and Raza A: Protooncogene transcript levels and acute nonlymphocytic leukemia. Semin Oncol 14(2):207-216, 1987. 14. Preisler HD, Raza A, Larson R, LeBeau M, Browman G, Goldberg J, Grunwald H, Vogler R, Verkh L, Singh P, Block AM, Sandberg A: Protooncogene expression and the clinical characteristics of

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acute nonlymphocytic leukemia. A Leukemia Intergroup Pilot Study. Blood 73(1):255-262, 1989. 15. Rowe DJ, Beverley PCL: Characterisation of breast cancer infiltrates using monoclonal antibodies to human leucocyte antigens. Br J Cancer 49:149-159, 1984.

16. Westin EH, Wong-Staal F, Gelmann EP, Favera RD, Papas TS, Lautenberger J A , Eva A, Reddy EP, Tronick SR, Aaronson SA, Gallo KC: Expression of cellular homologues of retroviral onc genes in human hematopoietic cells. Proc Natl Acad Sci USA 79:2490-2494, 1982.

Protooncogene expression in subpopulations of cells from leukemia patients.

This report describes a method for preserving the light scatter patterns of cells in which myc and myb expression are being measured. Exposure of cell...
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