1986 67: 676-681   

 M Andreeff, DE Slater, J Bressler and ME Furth cytometry in hematopoietic cell linesCellular ras oncogene expression and cell cycle measured by flow

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676 Blood, Vol 67, No 3 (March), 1986: pp 676-68 1

Cellular ras Oncogene Expression and Cell Cycle Measured by Flow Cytometry in

Hematopoietic Cell Lines

By Michael Andreeff, Dennis E. Slater, Jan Bressler, and Mark E. Furth

Human hematopoietic malignancies provide an excellent

model for the study of the activity of cellular oncogenes in a

context of known defects in cell proliferation and differen-

tiation. A flow cytometric immunofluorescence assay was

developed to quantitate the expression of the cellular ras

oncogenes in relation to the cell cycle in individual leukemic

cells. Specific binding of a monoclonal antibody to the

21 -kd protein (p21 ras) encoded by the Ha-ras. Ki-ras. and

N-ras genes was measured by flow cytometry and con-

firmed by fluorescence microscopy. P21 ras was detected

in 41 6B. a murine hematopoietic precursor cell character-

ized by a high level of Ki-ras expression, and in the human

D NA ISOLATED from a wide variety of malignant cells

can induce cell transformation when transfected into

cultured NIH 3T3 mouse fibroblasts. In 10% to 20% of

human tumors,’ the activated transforming genes are related

Front Memorial Sloan-Kettering Cancer Center. New York.

Supported in part by grants CA-20194 and CA-29564from the

National Cancer Institute.

Submitted May 31. 1 985; accepted Sept 1 7. 1985.

Address reprint requests to Dr Michael Andreeff, Memorial

Sloan-Kettering Cancer Center. 1275 York Ave. New York, NY

10021.

< 1986 &v Grune & Stratton, lnc.

0006-4971/86/6703--0019$03.00/0

leukemic cell lines P-i 2 and KG-i . The presence of p21 ras

in the cell lines was also shown by immunoprecipitation.

Cellular DNA content was determined simultaneously to

define cell cycle phases. There was an equal distribution of

p2lras in G1. S. and G2M, with considerable heterogeneity

of ras gene expression in the G1 compartment. The assay

allows oncogene expression to be studied in populations of

intact single cells in which cell heterogeneity is maintained.

requires very few cells per sample. and directly correlates

oncogene expression to cell kinetic data.

a 1986 by Grune & Stratton. Inc.

to the oncogenes of the Harvey or Kirsten murine sarcoma

viruses or to the N-ras gene originally described in the

human SK-S-NH neuroblastoma cell line.2 The N-ras gene

is of particular interest since it has been found in human Tand B cell leukemia cell lines, myeloid and Burkitt’s lym-

phoma cell lines, and in primary AML.37 All three cellular

ras genes encode proteins weighing 21,000 daltons

(p2lras)5’9 whose biochemical activity is the binding of

guanine nucleotides.’#{176} H-ras has also been shown to have

GTPase activity.”’4 P2lras is localized on the inner surface

of the cell membrane,’5 Point mutations in the cellular ras

genes involving amino acid substitutions at positions 1 2 or I 3

or 59 through 63 of p2lras can result in oncogenic activa-

tion.’�2’ Cell transformation has also been associated with

Fig 1 . Microphotographs of murine leukemia cell line 41 6B.original magnification. x 545; current magnification x436.(A) phase contrast; (B) immunofluorescence of same cells stainedwith YYG-106 monoclonal antibody (control); (C) immunofluores-

cence of cells stained for p21 ras with Vi 3-259 monoclonal anti-body. The oncogene product is localized on the inner surface of thecell membrane; all cells appear to be positive. Exposure is equal forB and C. Immunoprecipitation of this experiment is shown in Fig 2;flow cytometric analysis is shown in Fig 3.

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416 B

Control

/�I I I ..‘

H�

B

Y13 -

S

FLOW CYTOMETRY OF ONCOGENE EXPRESSION 677

elevated gene expression, as in the Y I adrenal carcinoma cell

line22 or in 4l6B murine hematopoietic precursor ce11s2324 in

which p21 Ki-ras is overproduced. Similarly, the normal

Ha-ras gene can transform N IH 3T3 cells if it is ligated to a

retroviral strong transcriptional promoter.25’26

A flow cytometric assay of immunofluorescence in which

DNA content can be measured simultaneously would be

useful to determine the range of p2lras levels in human

leukemia and lymphoma, to detect subpopulations of tumor

cells with markedly high levels of p2lras, and to correlate ras

gene expression with cell cycle phases. Hematopoietic malig-

nancies provide an excellent model for the study of the

activity of cellular oncogenes in a context of known defects in

cell proliferation and differentiation.

MATERIALS AND METHODS

Monoclonal antibodies. A panel of rat monoclonal antibodies

that precipitate p2 1ras was developed by Furth et al.27 Y I 3-259 is a

broad spectrum monoclonal antibody that binds p21 encoded by theHa-ras, Ki-ras, and N-ras genes. YYG-106, used as a control

antibody in these experiments, is a rat monoclonal antibody directed

against murine hematopoietic colony-stimulating factor.

Leukemic cell lines. Cells (4 16B) were grown in a I : 1 mixtureof Dulbecco’s modified Eagle’s medium (DMEM) and Ham Fl 2

medium supplemented with antibiotics and 20% horse serum (Flow

Laboratories, Rockville, Md). P-l2and KG-I weregrown in RPMI1640 medium supplemented with 2 mmol/L of glutamine, 1%antibiotics and 10% heated fetal calf serum (FCS). All cells weremaintained at 37 #{176}Cin a 5% CO2 humidified atmosphere.

Simultaneous staining of DNA and intracytoplasmic antigens.

Leukemic cell suspensions were fixed in 100% methanol for ten

minutes at - 20 #{176}Cand were stained by a three-step indirect method.

Fifty microliters of Y13-259 at a concentration of 50 zg/mL were

incubated with 1 to 2 x 106 cells (minimum cell number was 0.5 x

106). This was followed by rabbit anti-rat lgG (Cappel, Cochran-

ville, Pa) at a dilution of 1:200, and fluorescein isothiocyanate

(FITC)-goat anti-rabbit lgG (B&M, Indianapolis) at 1:40. All ofthe reagents were added for 30 minutes at 4 #{176}C.The cells were then

stained with propidium iodide after treatment with RNase as

previously described.28 The fixation and staining conditions were

developed after extensive experiments (data not shown) conducted to

enhance the sensitivity of the assay for the detection of low levels of

p2lras.

A computer-interfaced research cytofluorograph (model FC2OI,

Ortho Instruments, Westwood, Mass) with an argon-ion laser(model 75, Lexel, Palo Alto, Calif) tuned to 488 nm and operated at30 mW was used. The signals were amplified on a linear scale. FITC

green fluorescence (tertiary antibody-anti-p2 I ras), propidium io-

dide red fluorescence (DNA) and red pulse width (nuclear diame-

..�,-- �I1

-F--+--

. �11 �-F---

RAS EXPRESSION

Fig 2. Radioimmunoprecipitation of murine leukemia cell line41 6B with rat monoclonal antibody Vi 3-259 and normal rat lgG(control). The antibody precipitates p21 ras. The same experimentis evaluated by immunofluorescence in Fig 1 and by flow cytometryin Fig 3.

Fig 3. Computer-drawn histograms of correlated measure-ments of cellular DNA content and p21 ras immunofluorescence ofmurine leukemia cell line 416B: y-axis. DNA content (cell cyclestages G011-S-G2M); x-axis. indirect immunofluorescence of con-trol antibody YYG-106 (A) and Y13-259 directed against p2iras(B); z-axis. cell number. Red fluorescence (propidium iodide) pulsewidth was also measured simultaneously to distinguish single cellsfrom cell aggregates (not shown). Data shown here are gated forsingle cells only; ( - ) designates negative and ( + ) designatespositive immunofluorescence. Microphotographs of this experi-

ment are shown in Fig 1 B and C and immunoprecipitation is shownin Fig 2. DNA measurements show progression of cells from G.� toS and G2M. Almost all cells are positive for p21 rca. with no cellcycle-related increase of ras expression in these exponentiallygrowing cells. Considerable heterogeneity is apparent in all cellcycle phases. particularly in G1.

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678 ANDREEFF ET AL

ter) were measured in 5,000 cells per sample and stored as correlated

measurements in a Nova 1220 minicomputer (Data General, South-wood, Mass). Red pulse width was used to distinguish single cells

from cell aggregates. Data analysis was performed with a Tektronix

4010-I graphic terminal (Tektronix, Beaverton, Ore) using com-puter programs developed by Sharpless.�

Immunofluorescence. Leukemic cells were stained by the three-

step indirect method described above without counterstaining forDNA. Cell suspensions were mounted under coverslips with Hydro-mount (National Diagnostics, Somerville, NJ) and examined with

appropriate filter combinations for fluorescein.Immunoprecipitation ofp2 I ras proteins. Cells were labeled by

metabolic incorporation of 35S-methionine (0. 1 mCi/mL in methio-nine-free medium supplemented with 5% dialyzed fetal bovine

serum) for five hours at 37 #{176}C.The cells were washed and thenincubated for a further period of one hour in complete medium

containing unlabeled methionine and 10% fetal bovine serum. Cell

lysates were prepared in a buffer containing 1% Triton X-lOO(Sigma Chemical, St Louis) and 0.5% Na deoxycholate. Portions

containing I 0� cpm of incorporated 35S-methionine (assessed byprecipitation with trichloroacetic acid) were diluted to 0.3 mL with

lysis buffer, and Na dodecyl sulfate (SDS) was added to a finalconcentration of 0.25% (wt/vol). One microgram of monoclonal

anti-ras rat lgG or purified normal rat immunoglobulin was added

to each tube, and reactions were incubated at 4 #{176}Cfor I 8 hours.Immune complexes were collected by addition of formalin-fixed Saureus precoated with rabbit antibodies against rat lgG, for one

hour at 4 #{176}C.The bacteria were washed extensively, and labeled

proteins were eluted by boiling in sample buffer containing SDS and2-mercaptoethanol. The samples were analyzed by electrophoresison 15% polyacrylamide gels containing 0.1% SDS, and labeled

proteins were detected by fluorography.

RESULTS

Three different cell lines were used to develop the quanti-

tative flow cytometric assay of intracytoplasmic immuno-

fluorescence: the murine hematopoietic precursor cell line

416B, the human myeloblastic leukemia line KG-l, and the

human T cell lymphoblastic leukemia line P- 1 2. Fig 1 shows

phase contrast, control antibody YYG-106, and specific

staining with anti-p2lras antibody Y13-259 in 4l6B cells.

The photomicrographs were matched for magnification and

for times of exposure and development. Immunoprecipitation

of 35S-methionine-labeled 416B cells is shown in Fig 2:

Yl3-259 precipitates a single band in the 2l-kd region. The

corresponding flow cytometric histograms are shown in Fig

3: the upper histogram shows proliferating cells in the

G,-S-G2M phases of the cell cycle (y-axis = DNA), and

defines the background fluorescence (x-axis = immuno-

fluorescence of YYG-106). The lower histogram shows

positive p2lras immunofluorescence in >90% ofthe cells. G,

cells exhibit considerable heterogeneity, with cells expressing

Fig 4. Microphotographs of human lymphoblastic leukemia cell line P-12. original magnification x 545; current magnification x420. (Aand C) Phase contrast of cells stained with control antibody YVG-106 (B). and anti-p21 ras antibody Y13-259 (D). respectively. Exposure isequal for B and D. Characteristic membrane localization of p21 ras; some cells stain as weakly as in B with control antibody.

Immunoprecipitation of this experiment is shown in Fig 5. lane 1 ; flow cytometric analysis is shown in Fig 6.

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P12

Control

A

JI/L��0

�T;�j

� �,

B

FLOW CYTOMETRY OF ONCOGENE EXPRESSION 679

low, intermediate, and high amounts of the cellular ras gene

protein. There is no apparent increase in p2lras when the

cells progress to S and G2M if the increase in unspecific

background fluorescence due to increased cell size is taken

into consideration.

The human myeloid leukemic cell line KG-I was also

stained with Y I 3-259 and YYG- I 06. Approximately 60% of

the cells in all cell cycle phases had fluorescence that was

indistinguishable from background level. Forty percent of

the cells were positive, but there was much less heterogeneity

in G, than was found in 4l6B cells. Again, no increase in

p2lras was seen during cell cycle progression (data not

shown).

N-ras-transforming gene sequences were identified in

P-l2 human lymphoblastic leukemic cells in a transfection

assay.3 Fig 4 shows the typical ring pattern of p2lras

immunofluorescence. Fig 5 depicts a one-dimensional gel in

which P-12 cells were immunoprecipitated using two dif-

ferent monoclonal antibodies directed against p2lras. YI3-

238 immunoprecipitates human p21 Ha-ras and p21 Ki-ras

but does not immunoprecipitate p21 N-ras (Furth et al,

unpublished observations). The band precipitated by Y I 3-

238, corresponding primarily to p21 Ki-ras, was weak, but

the flow cytometric assay using this antibody was positive

(data not shown). A double band of p2lras appeared when

P-12 cells were immunoprecipitatcd with Y13-259, since

P-I 2 cells produce both a normal and an altered form of p21

N-ras with slightly different electrophoretic mobilities.3’4 Fig

6 shows the corresponding DNA/p2lras histograms: -25%

of the cells in all cell cycle phases were negative, whereas the

remaining cells were positive in the flow cytometric immuno-

fluorescence assay.

The staining procedure used in these experiments yielded

minimal background fluorescence, and permitted the low

levels of p2lras found in the human leukemic cells to be

detected. The assay has a greater sensitivity than the method

previously described by Andreeff et al for the measurement

of p21 ras in leukemic cells.3#{176}

DISCUSSION

Specific binding ofa monoclonal antibody directed against

the protein product of the three cellular ras oncogenes

(p2lras) can be measured in a quantitative flow cytometric

assay of immunofluorescence. The presence of p2 I ras was

confirmed by direct microscopy in three hematopoietic cell

lines and by immunoprecipitation experiments using the

same antibody. Oncogene expression can be directly corre-

lated to the cell cycle since total DNA content can be

determined simultaneously.

The role of p2lras in cell transformation and its possible

relationship to the cell cycle are not completely understood.

GoiiS

Y13- 259

RAS EXPRESSION

Fig 5. Radioimmunoprecipitation of human lymphoblastic leu-kemia cell line P-i 2 with antibodies against p21 ras and control ratlgG (lanes 1 through 3). Lane 4 shows results of anti-p2lrasantibody Y13-259 with ras-transfected NIH 3T3 cells.

Fig 6. Computer-drawn histograms of correlated measure-ments of cellular DNA content and p21 ras immunofluorescence ofhuman lymphoblastic leukemia cell line P-i 2: y-axis. DNA content(cell cycle stages G011-S-G2M); x-axis. indirect immunofluores-cence of control antibody YYG-106 (A) and Y13-259 directedagainst p2i ras (B); z-axis. cell number; ( - ) negative and ( +)positive immunofluorescence. Microphotographs of the immuno-fluorescence of this experiment are shown in Fig 4(B) and (D).respectively. DNA distribution shows high proliferation. Approxi-mately 75% of cells are positive for p21 ras. with no cell cyclerelated increase of ras expression. Heterogeneity of rca expres-sion with non-Gaussian distribution is apparent in all cell cyclephases.

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680 ANDREEFF ET AL

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amounts of p2lras. It is also possible that a subpopulation

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