heterogeneity of 06-alkylguanine-dna-alkyltransferase …...vol. 4, 475-481, february 1998 clinical...

Vol. 4, 475-481, February 1998 Clinical Cancer Research 475

Heterogeneity of 06-Alkylguanine-DNA-alkyltransferase Measured

by Flow Cytometric Analysis in Blood and Bone Marrow

Mononuclear Cells1

Lili Liu, Keunmyoung Lee, Jane Schupp,

Omer N. Ko#{231},and Stanton L. Gerson2

Division of Hematology and Oncology and the Case Western Reserve

University/University Hospitals Ireland Cancer Center, Case Western

Reserve University School of Medicine, Cleveland, Ohio 44106-4937

ABSTRACT

Alkyltransferase (AGT) repairs alkylation at O6�

guanine in DNA and is a major determinant of susceptibilityto alkylating chemotherapeutic agents and carcinogens.

Using a newly developed flow cytometry assay with the

monoclonal anti-AGT antibody, mT3.1, we compared AGT

expression in single-cell suspensions with standard biochem-

ical and Western blot assays to validate the fluorescence-

activated cell sorting (FACS) method and develop potential

applications. From Chinese hamster ovary cells (CHO)

transfected with human 06-methylguanine-DNA methyl-

transferase cDNA, 6 CHO-06-methylguanine-DNA methyl.

transferase clones were isolated that expressed 0.3 to 64

fmol/p.g DNA (by biochemical assay) of human AGT. FACSyielded a linear relationship between mean fluorescence in-

tensity and both AGT activity by biochemical assay and

AGT protein by Western blot. Using this standard curve,FACS-analyzed AGT protein content in human peripheral

blood mononuclear cells (PBMCs) from normal donorsranged from 6.1 to 12.8 fmol/p.g DNA, similar to thoseobtained by biochemical assay and Western blot. This sug-

gests that the level of immunoreactive protein appears to bean accurate predictor of AGT activity in the steady state.

FACS-AGT in PBMCs from normal donors had a low indexof heterogeneity within the sample. In contrast, by FACS.

AGT analysis of human bone marrow samples and granu-locyte-colony-stimulating factor-mobilized PBMCs, AGTwas lower and had an 8-fold higher index of heterogeneitythan observed in PBMCs from normal donors. After treat-ment with 06-benzylguanine (06-bG), Western and FACS-AGT detected significant levels of AGT protein for up to

Received 8/25/97; revised 10/30/97; accepted 1 1/3/97.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

I This work was supported in part by the Grants R01ES06288,

UO1CA75525, R01CA63193, and P30CA43703 from the USPHS.

2 To whom requests for reprints should be addressed, at Division ofHematology and Oncology. Department of Medicine and the Case

Western Reserve University/University Hospitals Ireland Cancer Cen-

ter, BRB-3, 10900 Euclid Avenue, Cleveland, OH 44106-4937. Phone:

(216) 368-1 176: Fax: (216) 368-1 166; E-mail:[email protected].

24 h, whereas biochemical assay showed AGT activity lessthan 5% of the basal level. Because only the biochemicalassay accurately measures net AGT activity, the AGT-FACSassay will not be useful in clinical trials to assess the efficacy

of 06-bG or other AGT inhibitors. Thus, AGT-FACS canrapidly assess the heterogeneity of steady-state AGT in sin-gle-cell suspensions and may be useful for assay in lympho-cytes, bone marrow cells, leukemic myeloma plasma cells, or

cells transfected with the AGT gene; Western blot analysis is

better for small samples such as tumor biopsies, whereasbiochemical assay is best able to measure enzyme activityand its inactivation by 06-bG or other agents.

INTRODUCTION

Certain environmental chemical agents and several chemo-

therapeutic agents used in patients with cancer are known to

alkylate DNA at the O� position of guanine, resulting in lesions

that are both mutagenic and cytotoxic. It has been observed that

AGT3 is a critical protein for repair of 06-alkylguaninc-DNA

adducts, protecting cells from the cytotoxic, mutagenie, and

carcinogenic effect of alkylating agents (1-3).

AGT is the only protein to accept an alkyl group from the

06 position in DNA on its own cysteine residue forming S-

alkylcysteine, resulting in irreversible inactivation. Regenera-

tion of AGT requires protein synthesis and takes 12-24 h (4).

Thus, the number of protein molecules present in cells equals

the number of 06-alkylguanine adducts that can be repaired (5,

6). The cellular level of this protein is a useful biomarker for

predicting cellular response or resistance to DNA damage in-

duced by alkylating agents. A number of different methods for

determination of AGT have been reported (3-8), all of which

measure mean AGT in the sample. The most specific assay is

the HPLC method that separates 06-mcthylguanine and N7-

methylguanine, allowing an internal control for the level of

substrate present in the methylated DNA (9). However, this

assay requires special equipment, is not conveniently estab-

lished in many laboratories, and might not be suitable for rapid

assay of AGT in a large number of samples. In addition, assay

for AGT activity in individual cells has been hampered by the

low amount of the protein. In this report, we describe a sensitive

flow cytometry analysis using the mouse monoclonal anti-

human AGT antibody (mT3.l). Cultured cells, human PBMCs,

and bone marrow cells were analyzed by FACS and compared

3 The abbreviations used are: AGT, alkyltransferase; FACS, fluores-

cenee-aetivated cell sorter: CHO, Chinese hamster ovary cells; MGMT,

06-methylguanine-DNA methyltransferase; PBMC, peripheral blood

mononuclear cell; G-CSF, granuloeyte-eolony-stimulating factor; MFI,

mean fluorescent intensity; 06-bG. 06-benzylguanine; IH, index of

heterogeneity; HPLC, high-performance liquid chromatography.

Research. on August 4, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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BRegion PScatterogram Region R

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476 Heterogeneity of 06-Alkylguanine-DNA-alkyltransferase

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FSC-Height

Scatteroram Isotype

0� 2b0 400 600 800 idooFSC-Height

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FL2-Height

0 �1 i�2 �

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Region IsoR

-rnnui . .��;0 �o’ io2 �

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Fig. 1 Sufficient separation of

AGT� cell population. A, the for-

ward and right angle scatter gates

on K562 cells transfected with

human MGMT and cells were

stained with antibody mT3.l. B,histogram of gate P was pre-

sented AGT� cell population. C,histogram of gate R was predom-

inantly cellular debris and non-

permeabilized cells. D-F, K562

cells stained with mouse IgG1

isotype control. FSC, forward

scatter; SSC, side scatter; FL2,

fluorescence intensity; IsoP, iso-

type antibody, region P; IsoR,

isotype antibody, region R.

with biochemical measurement for AGT activity and Western

blotting of AGT protein. AGT-FACS identified heterogeneity in

single-cell suspensions and could be useful in clinical samples

of normal bone marrow, leukemia, mycloma, circulating tumor

cells, or to monitor MGMT-transduced hematopoictic cells dur-

ing gene therapy.

MATERIALS AND METHODS

Chemicals and Reagents. Mouse monoclonal antibody

(mT3.1) was kindly provided by Dr. Darell D. Bigner (Duke

University Medical Center, Durham, NC) and Dr. Thomas P.

Brent (Department of Pharmacology, St. Jude Hospital for Chil-

dren, Memphis, TN). 06-bG was generously provided by Dr.

Robert Mosehel (Frederick Cancer Research and Development

Center, National Cancer Institute, Frederick, MD). Stock solu-

tions were made in DMSO.

Tnansfection of Human MGMT Vector into CHO Cells.Plasmid pHM expressing human MGMT in CHO cells (CHO/

phMGMT) was constructed by inserting the human MGMT

eDNA coding sequence into the unique NcoI and BamHI re-

striction sites of a retroviral vector plasmid, pHM (10). Six �.ag

of plasmid DNA were transfected into 1.8 X 106 cells with

Lipofectaminc (Life Technologies, Inc., Gaithcrsburg, MD) fol-

lowing the manufacturer’s protocol; meanwhile, 0.6 p.g of

pSV2neo plasmid DNA was cotransfected, because this vector

plasmid lacks a neo cassette for selection. The transfected cells

were selected by addition of G418 (0.1%, w/v) into the media.

Six individual colonies were isolated that expressed variable

levels of human AGT.

Isolation Mononuclear Cells. Blood and bone marrow

samples were collected from normal donors who gave informed

consent under an Institutional Review Board approved protocol.

Mononuclear cells were isolated by Ficoll-Hypaquc discontin-

uous gradient separation. Cells at the interface were removed

and washed twice with PBS at 4#{176}C.PBMCs were also collected

from patients undergoing leukopheresis after G-CSF growth

factor treatment to increase the proportion of hematopoictic

progenitor cells in the blood. These patients subsequently un-

derwent high-dose chemotherapy with reinfusion of these pro-

genitor cells.

FACS Analysis of AGT. Cells were prepared for detec-

tion of AGT protein using a method for intracellular staining. To

develop this method, several detergents were tested to determine

which was most efficient in permeabilizing cell membranes.

Twecn-20 permeabilized cells better than either Saponin or

Triton X. Briefly, after cells were stabilized for 30 mm using 1%

paraformaldehyde, the membranes were permeabilized by incu-

bating in 1% Tween 20 for 30 mm at 37#{176}C,followed by

incubation in 2% BSAIPBS for 30 mm. Nonspecific binding

sites were blocked for 30 mm at 22#{176}Cwith 10% normal goat

serum. Human AGT antibody mT3.l (7 p.g/ml) was added, and

cells were incubated at 4#{176}Covernight. Cells were washed twice

with 2% BSAIPBS and incubated with secondary antibody [goat

anti-mouse IgG-1 (gamma) phycoerythrin conjugated] for 1 h at

4#{176}C.After washed as above, cells were resuspended in PBS +

BSA for FACS analysis. Flow cytometry was performed using

FACScan (Becton Dickinson, Mountain View, CA), equipped

with an argon laser. MFI of permeabilized cells was determined

by subtracting the nonspecific background (isotype control)

from the fluorescence intensity of cells stained with specific

AGT antibody (mT3.1). Light scatter was used for gating on

permeabilized cells. To further analyze the heterogeneity of

AGT expression, we used the parameter, IH [width at half peak

(in units of signal intensity)IMFI], based on the histogram of

fluorescent staining from each sample. A larger IH means that

the cell population has more heterogeneous expression of AGT.

Alkyltransferase Assay. The cell pellets were resus-

pended in 0.5 ml cell extract buffer [70 msi HEPES (pH 7.8), 0.1

mM EDTA, 50% glycerol, 1 mr�i Dli.’, and 25 �iM spermidinc]

and sonicated three times for 10 s at 4#{176}Cto complete cell

disruption and centrifuged at 10,000 X g for 2 mm to remove

cellular debris. The protein and DNA content were measured,



A

1 2 3 4 5 6

C

B

Number of CHO-MGMT clones>0

n.L)

AGT �

zea

.�‘E

.�: BQC)

�400.

300�

200�

100’

0 20 40 60 80

Clinical Cancer Research 477

Fig. 2 A, FACS analysis of humanAGT in CHO parental cell line (non-transfeeted: peaks 1 and 2) and

clones (transfeeted with human

MGMT: peaks 3-6). CHO parental

cell lines were stained either with

AGT antibody (mT3.1) or isotype

IgG with similar MFI �peak 1,

stained with isotype IgG). Clones

transfeeted with human MGMT had

a clear AGT staining signal, and

clone 3 (peak 6) expressed the high-

est level of MGMT, whereas other

clones (peaks 3, 4, and 5) had a van-

able amount of AGT. B, immunore-

active protein detected by Western

blot in CHO-hMGMT transfeetants.

The clones expressed different

amounts of human AGT. The highest

level of human AGT was detected in

clone 3. FL2, fluorescence intensity.C. the correlation between AGT ac-tivity and immunoreactive protein by

either FACS (left; r = 0.998) orWestern blot (rig/it: r 0.999).Symbols represent different CHO-

MGMT clones.

700�

0’

0 20 40 60 80

AGT activity (fmol/p.tg) DNA

and then fixed amounts of protein from each sample were

incubated with substrate DNA, which was prepared by incubat-

ing calf thymus DNA with [3H]mcthylnitrosourca. The reaction

mixture was incubated for 60 mm at 37#{176}Cand then precipitated

with 14% trichloroacetic acid at 4#{176}C.The determination of AGT

activity in cell extracts was based on the removal of [3H]mcthyl

group from 06-[3H]mcthylguaninc in 06-[methyl-3H]guaninc-

DNA. AGT activity was expressed as fmol removed 06-meth-

ylguanine/p.g DNA or fmol removed 06-methylguanine/mg

protein (9).

Western Blotting. Cell extracts were resolved by SDS-

PAGE (12% polyacrylamide) in a Bio-Rad minigel apparatus at

150 V for 1 h. Proteins were transferred onto polyvinylidene

difluoride membranes, using a Bio-Rad mini Trans-Blot cell for

1 h at 100 V. The blotted membranes were blocked with 5% dry

milk in TBS buffer and then probed for 2 h with mT3.l, which

is specific for human cellular MGMT ( 1 1). After three 5-mm

washes with TBS-Twcen 20 (0.05%), the blots were incubated

with secondary antibody, anti-mouse HRPO-anti-IgG for 1 h.

Antibody binding was visualized by ECL according to the

manufacturer’s instructions. Standard curves of samples with

known activity were included in each blot. To standardize the

Western blotting assay, K562 (chronic myclogenous leukemia)

cell line was transfected with human MGMT eDNA, and 0-50

,_Lg protein of cellular sonicate was subject to SDS-PAGE and

immunoassay. Densitometric analysis of the ECL film was

performed by a SciScan 5000 scanner using BioAnalysis soft-

ware. Regression analysis indicated that antibody-antigen curve

yielded linear plots with correlation coefficients of 0.995, when

band intensity was plotted versus fmol of AGT loaded (range,

0.9-16 fmol).

RESULTS

Identification of AGT Expression in PermeabilizedCells by FACS. After treatment with paraformaldehyde and

Tween 20, most cells were permeabilized and could be stained

with specific AGT antibody, whereas other FACS signals might

consist of platelet and cellular debris. These respective popula-

tions were identified by forward versus right angle scatter. The

scatter gates were established using K562-MGMT cells selected

to be 100% MGMT� by resistance to l,3-bis(2-chlorocthyl)-l-

nitrosourea ( 1 2). An example of the scatter gates used to detect

AGT is shown in Fig. 1 . Cells were stained with antibody mT3. 1



(no

0C)

A

C D

AGT’-#{216}� �- - � �

0

C3C

itFL2-Height

478 Heterogeneity of O�-Alkylguanine-DNA-alkyltransferase

PBMC by G-CSF PBMC-normal donors

Fig. 3 FACS analysis of AGT in PBMCs from normal donors and patients under going cytophenesis. A. PBMCs treated with G-CSF. B, PBMCsfrom normal donors showed a higher fluorescent staining. C, AGT protein detected by Western blot in human PBMCs. Lanes 1-4, PBMCs by G-CSF.PBMCs were collected from four patients receiving G-CSF growth factor treatment. LaneS 5-8, PBMC-normal donors. PBMCs were obtained from

tour normal donors. D, FACS analysis of mononuclear cell AGT of bone marrow. Human bone marrow had a lower MFI than the PBMCs examined

in B. FL2. fluorescence intensity.

(Fig. 1, A-C) and mouse IgG1 isotype control (D-F). Region R

contains predominantly cellular debris and nonpermeabilized

cells, confirmed by antihistone antibody staining (data not

shown). This region contains only a small number of AGT-

staining cells. In contrast, region P contains permeabilized,

AGT-positive cells staining with mT3.1. In Fig. lb. the mT3.l

staining appears uniform. and the background of nonstaining

cells is removed. This allowed us to quantitate AGT expression

in the permeabilized cell fraction.

Correlation of AGT Activity by FACS with WesternBlotting and Biochemical Assay. CHO cells containing very

low levels of AGT (<0. 1 fmol/�i.g DNA) were transfeeted with

human MGMT eDNA as described previously (10). Clones of

CHO-MGMT expressed variable AGT, ranging from 0.3 to 65

fmol/p.g DNA by biochemical assay. As shown in Fig. 14,

FACS analysis, based on MFI. had consistent results with bio-

chemical assay: clone 3 expressed the highest level of human

AGT (peak 6), whereas other clones have lesser amounts. Sim-

ilar results are also obtained by Western blot (Fig. 2B). Immu-

noreactive AGT proteins detected by Western blotting or FACS

are highly correlated with enzyme activity measured by bio-

chemical assay (Fig. 2C; r 0.998 and 0.999, respectively).

indicating that the three methods have equal specificity for

AGT.

AGT in human PBMCs and bone marrow exhibit a range

of enzyme activity. In PBMCs (ti = 6) from normal donors,

AGT activity measured by biochemical assay was 9.3 ± 2.7

fmoL/p.g DNA (range, 6.1-12.8 fmol/p.g DNA). In contrast,

PBMCs (pi 7) from leukopheresis collections obtained from

cancer patients receiving G-CSF had a much higher concentra-

tion of hematopoietic progenitors than normal blood (CD34+

cells ranged from 0.4-5%, compared with <0.1% CD34+ cells

in routine blood collections) and contained lower AGT activity

(4.6 ± 1.2 fmol!)ig DNA; range, 2.9-5.9, P < 0.002). In Fig. 3,

A and B, histograms of fluorescent staining of AGT by FACS

confirmed these differences. PBMCs from normal donors had

higher MFI (107 ± 14) than that in G-CSF mobilized-PBMCs

(38 ± 27; P = 0.015). The distribution of activity in individual

cells indicated that there is a normal distribution in most in-

stances, but that in some histograms, there appears to be a

shoulder of either high- or low-expressing cells. AGT protein

detected by FACS correlates with AGT biochemical activity

measured by the HPLC method and by Western analysis (see

also Fig. 3C), with correlation coefficients of 0.873 and 0.787,



100

80 #{149} #{149}

60

40

20 #{149}

Iv

0 5 10 15

AGT activity (fmoI/�tg DNA)

by HPLC

z>,

I’

AGT protein (fmol4tg DNA)

by Western blot assay

AGT activity (fmol1�1g DNA)by HPLC


Fig. 4 The correlation of hu-

man AGT protein analyzed by

FACS, Western blot, and AGT

activity assay in human PB-

MCs. #{149},PBMCs from normaldonors; U, PBMCs from do-nors receiving G-CSF.

Tabl e 1 Comparison of heterogeneity of AGT in PBMC s from normal don ors or patientsundergoingcytophenesis

PBMCs from normal donorsPBMCs from donors receiving G-CSF

No. of samplesNo. of samples Width” at half peak MFI” IH Width” at half peak MFId IH

1 194 22 8.81 1 60 87 0.68

2 137 26 5.27 2 100 104 0.963 114 20 5.70 3 46 119 0.38

4 120 41 2.92 4 114 127 0.89

S 128 40 3.20 5 88 99 0.88

6 288 20 14.40 6 86 104 0.837 136 97 1.40

Mean 160 ± 36 38 ± 28 6.0 ± 4.4 82 ± 25” 107 ± 14’ 0.7 � 0.2”

a In units of signal intensity.

“p = 0.016.‘� P = 0.015.

“P = 0.0002.

respectively (P < 0.001 ; Fig. 4). These results demonstrate that

FACS analysis accurately measures AGT in clinic blood cell

samples and provides valuable information on the distribution of

activity in various subpopulations of cells.

With the FACS method, it is possible to assay the hctcrogc-

neity within each sample visually and mathematically. As one

measure of heterogeneity, we defined the parameter IH [width at

half peak (in units of signal intensity)IMFI]. This value is deter-

mined by two factors: the width at half peak, a measure of the

distribution of cells with positive staining; and MH, a measure of

mean level ofAGT in the cell population. Although the IH does not

directly account for subpopulations, the overlap in histograms for

these populations will tend to broaden the width at half peak. IH

values listed in Table 1 reveal that there was a much greater degree

of heterogeneity seen in the expression of AGT in the G-CSF

mobilized PBMC samples than that in the unstimulated PBMC

samples from normal donors (6.0 ± 4.4 versus 0.7 ± 0.2; P =

0.0002). As shown in Fig. 3D, analysis of the samples from bone

marrow (n = 3) indicates that their MR is even lower than that of

the G-CSF mobilized PBMC cell samples (MR. 16.8 ± 8.6) and

that they had a similar high degree of heterogeneity in the samples

(IH, 9. 1 ± 1.9).

Detection of 06-bG-inactivated AGT by FACS andWestern Blot Analysis Compared with Biochemical Assay.06-bG, a specific inhibitor of AGT, potentiatcs the cytotoxicity

of chemotherapeutic alkylating agents and has entered clinical

trials (13). Monitoring depletion of AGT by 06-bG would help

define the efficiency of drug effect in vivo. We tested whether

FACS analysis could differentiate between inactive and active

AGT. After human MGMT-CHO cells were treated with 06-bG

(25 pM) for 2, 6, and 24 h, >98% of human AGT activity was

depleted at 2 h, and little recovery was found up to 24 h,

measured by biochemical assay. However, immunorcactive

AGT protein detected by Western blot assay was unchanged at

2 h and gradually decreased (Fig. SA). FACS analysis also

revealed a gradual time-dependent decrease in fluorescent stain-

ing of cellular AGT protein after 06-bG (Fig. SB). The rate of

degradation of inactivated AGT protein measured by FACS and

Western blot assay was similar; 24 h after 06-bG, 25-35% of

baseline AGT protein was detected, which was inactive, as

proven by biochemical assay (Fig. SC). Thus, only the biochem-

ical assay accurately reflects the level of AGT activity. Western

blot and FACS analysis remain efficient methods to trace the

degradation of inactivated AGT, and this can help define con-

ditions (drug treatment or perhaps other cell stress) in which a

proportion of AGT is inactive.

DISCUSSION

FACS analysis of cellular AGT content is shown here to be

an accurate, rapid assay for single-cell suspensions, highly cor-

related with the traditional assay methods of biochemical assay



U

100

0 5 10 15 20 25

480 Heterogeneity of 06-Alkylguanine-DNA-alkyltransferase

Time (h) after 06b-G

0 2 6 24

AGT -*� �

.�)

0

I-

F-

C4��

0

0

ci

ci

Time afterO6-bG (hr)

A

Fig. 5 Comparison of human AGT measurement after inactivation of

AGT by 06-bG in CHO-MGMT cells. A, Western blot of inactivated

human AGT protein in CHO-MGMT transfeetants after O�-bG treat-

ment. Lrnze 1, untreated CHO-MGMT cell extract: Lanes 2-4, 2, 6, and

24 h after 06-bG treatment, respectively. B, FACS analysis of human

AGT in CHO-MGMT cells after 06-bG treatment. The high human

AGT detected by FACS decayed slowly from baseline �peak 6), 2 h

�peak 5). 6 h �peak 4). and 24 h (peak 3) after treatment but still

remained above that of nontransfeeted cells (peak 2). Peak 1. cells were

stained with isotype IgG as background. FL2. fluorescence intensity. C.comparison between AGT activity assay, Western blot, and FACS for

the rate of inactivation and degradation of human AGT after 06-bG

treatment. #{149}.FACS analysis; #{149},Western blot assay: A, HPLC meas-

urement.

and Western blot. Furthermore, as has been shown previously,

the amount of immunoreactive protein, measured previously by

Western blot, is an accurate measure of AGT activity in unper-

turbed cells (1 1). This has a number of important implications;

it suggests that the AGT-FACS assay could be used as a rapid

assay of AGT activity in a variety of samples, both laboratory

cells and clinical samples. In the clinic, samples could include

blood and marrow samples, as well as samples of malignant

cells including leukemia, lymphomas, myclomas, and disaggre-

gated tumor samples. Because only a small number of cells need

to be analyzed by FACS, it is also possible to analyze AGT in

samples too small to be measured by the conventional activity

assay. A second application is in the area of gene therapy, where

we have used FACS to detect the degree of transduced cells

(14), and in which it could be used to screen and select viral

producer cell clones and transduced, retrovirally infected cell

populations.

The AGT-FACS assay can also reveal subpopulations of

cells with different levels of AGT and the overall heterogeneity.

Thus, within each PBMC sample from normal donors, there was

a relatively uniform level of AGT protein. reflecting the rela-

tively homogeneous population of cells (about 15% monocytes

and 85% lymphocytes) and the similar levels of AGT in these

cells, as we have reported previously (15). In contrast, in PB-

MCs from G-CSF mobilized samples. as in bone marrow sam-

ples, there was much more heterogeneity in AGT levels than

that in the unstimulated PBMC samples. This difference is not

surprising, given the markedly heterogeneous cell population of

both samples, which in the case of the G-CSF mobilized cells

include activated monocytes. cosinophils. lymphocytes, CD34+

cells and other hematopoictic progenitors, and late maturing

myclocytes; and in the case of bone marrow, includes the entire

differentiation lineage of hcmatopoiesis. These results confirm

our previous observations with marrow subpopulations and

CD34+ cells, indicating that hematopoietic progenitors have

lower AGT activity than lymphocytes and monocytes (16). The

use of double or triple antibody staining techniques, while

challenging when one of the epitopes is nuclear and the others

react with surface molecules, is an appealing way to identify

specific subpopulations with either high or low AGT-FACS

levels.

We did not observe samples with an incongruity between

mean AGT activity and the level of immunoreactive protein on

Western blot, nor did we find samples in which a subpopulation

of cells lacked AGT activity but had evidence of AGT protein

by AGT-FACS or Western blot. Thus, in the steady state, the

level of immunoreactive protein appears to be an accurate

predictor of AGT activity. Furthermore, this suggests that the

normal breakdown of AGT, which appears to involve ubiquiti-

nation and proteosome digestion ( 17), does not result in excess

immunorcactive protein relative to active protein. Given the

long half-life of this protein, in excess of 24 h, this result may

not be surprising but reiterates the suggestion that the level of

MGMT mRNA is the major determinant of AGT activity in

cells (18).

Finally, our data show that after treatment with 06-bG,

only the biochemical assay accurately measures active AGT.

Although the AGT is degraded more rapidly after inactivation,

the monoclonal antibody we used. mT3. 1 , and most if not all




other antibodies react with the inactive as well as active protein

(19). Accordingly, both AGT-FACS and Western blot assays

failed to distinguish active protein from inactive protein. We

have found that the time course of loss of immunoreactive

protein after 06-bG exposure varies between cells and that it

matches the AGT-FACS measurement of AGT content, al-

though new AGT is being synthesized by the cells at a slow but

constant rate (20). This indicates that the AGT-FACS assay will

not be useful in clinical trials to assess the efficacy of 06-bG or

other AGT inhibitors, and that the biochemical assay will need

to be used unless an antibody is developed that reliably distin-

guishes between the active and inactive forms of AGT.

In summary, AGT-FACS is a highly sensitive technique to

rapidly and accurately analyze AGT content in single-cell sus-

pensions and to assess the degree of heterogeneity that exists

within that population. Potential applications include: detection

of tumor cells with high AGT amenable to AGT inactivation

therapy; gene therapy detection of transduced cells; and analysis

of subpopulations by multiparameter surface or internal marker

analysis.

ACKNOWLEDGMENTS

We thank Deborah A. Corey for assistance with the establishment

of this method.

REFERENCES

1. Dumenco, L. L., Allay, E., Norton, K., and Gerson, S. L. The

prevention of thymic lymphomas in transgenic mice by human O6�

alkylguanine-DNA AGT. Science (Washington DC), 259: 219-222,

1993.

2. Dolan, M. E., Mosehel, R. C., and Pegg, A. E. Depletion of mam-malian 06-alkylguanine-DNA AGT by 06-benzylguanine provides a

means to evaluate the role of this protein in protection against carcino-genie and therapeutic alkylating agents. Proc. Natl. Acad. Sci. USA, 87:

5368-5372, 1990.

3. Liu, L., Allay, E., Dumenco, L. L., and Gerson, S. L. Rapid repair of

O�-methylguanine-DNA adducts protects transgenic mice from N-

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1998;4:475-481. Clin Cancer Res L Liu, K Lee, J Schupp, et al. marrow mononuclear cells.measured by flow cytometric analysis in blood and bone Heterogeneity of O6-alkylguanine-DNA-alkyltransferase

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