Retroviral Transfer ofaBacterial Alkyltransferase Gene (ada) … · 2005-09-10 · kDa 112 84 53.2-34.9-28.7-20.5-c’f, ‘ 0 f , q 0 e.4-adalose - ..‘ Clinical Cancer Research

Vol. 3, 301-307, Februars 1997 Clinical Cancer Research 301

Retroviral Transfer of a Bacterial Alkyltransferase Gene (ada) into

Human Bone Marrow Cells Protects against 06-Benzylguanine

plus 1 ,3-Bis(2-chloroethyl)- 1 -nitrosourea Cytotoxicity1

Upendra K. Marathi, Linda C. Harris,

Carol C. Venable, and Thomas P. Brent2

Department of Molecular Pharmacology, St. Jude Children’s Research

Hospital, Memphis. Tennessee 38105 lU K. M.. L. C. H., C. C. V.,

T. P. B.], and Department of Pharmacology. College of Medicine,

University of Tennessee, Memphis, Tennessee 38163 fT. P. B.)

ABSTRACT

The antitumor activity of 1,3-bis(2-chloroethyl)-1-nitro-

sourea (BCNU) is limited by the 06-alkylguanine-DNA al-

kyltransferase (ATase) in tumor cells and by delayed mye-

losuppression. Inactivation of neoplastic ATase by O6�

benzylguanine (BG) improves the therapeutic index for

BCNU. We have demonstrated previously that BG +

BCNU-induced myelosuppression in mice is reduced by ex-

pression of the BG-resistant ATase ada in murine bone

marrow. We have now generated an amphotropic retrovirus

containing the ada gene and tested the effectiveness of ada

expression in preventing BG + BCNU cytotoxicity in human

hematopoietic progenitor cells. A retroviral producer clone

with a biological titer of 6.5 x i04 colony-forming units/ml

and 4.4 pmol ATase/mg protein was used for transduction of

bone marrow. Cocultivation of these ada producer cells with

progenitor cells from six normal individuals resulted in

1.9-3.9-fold protection against BG + BCNU-induced cyto-

toxicity in committed progenitor cell assays. Furthermore,

this cytoprotective effect was associated with a high trans-

duction efficiency (40%) and a 2-fold increase of ATase

activity in the surviving committed progenitor cell colonies.

These data provide a basis for testing the clinical effective-

ness of retroviral ada gene transfer into hematopoietic cells

to increase the therapeutic index of BG + BCNU.

Received 6/24/96; revised 10/15/96: accepted 11/12/96.

The costs of publication of this article were defrayed in part by the

payment 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 by NIH Grants CA23099, CA14799. and

CA09346, Cancer Center Support (CORE) Grant P30 CA2 I 765, and the

American Lebanese and Syrian Associated Charities.

2 To whom requests for reprints should be addressed, at Department of

Molecular Pharmacology, St. Jude Children’s Research Hospital. P. 0.

Box 318, 332 North Lauderdale, Memphis. TN 38105. Phone: (901)

495-3440; Fax: (901) 521-1668.

INTRODUCTION

BCNU3 is a chlonoethylnitrosounea that has been used in

the treatment of Hodgkin’s lymphoma and primary brain tumors

( 1 , 2). However, the clinical utility of BCNU is limited by

cumulative and delayed myelosuppression (3, 4). This hyper-

sensitivity may be related to low levels of MGMT in primitive

hematopoietic cells (5, 6). MGMT is a DNA repair protein that

prevents the major cytotoxic lesion induced by BCNU, the DNA

interstrand cross-link (7). The clinical utility of BCNU may also

be compromised by the expression of MGMT in neoplastic

cells. Because a majority of human tumor specimens tested

contain MGMT and are thought to be BCNU resistant, MGMT-

depleting agents have been developed to reverse BCNU resist-

ance (8-10).

Inactivation of MGMT with DNA-methylating agents

(e.g., streptozotocin) on the free base BG resulted in an enhance-

ment of BCNU antitumon activity (1 1, 12). However, regimens

targeting the reversal of BCNU resistance by the inactivation of

MGMT also increased BCNU-induced toxicity ( 13-16). Strep-

tozotocin-mediated depletion of MGMT in humans resulted in a

2-3-fold reduction in the maximally tolerated dose of BCNU,

partially due to an exacerbation of BCNU-induced thrombocy-

topenia and leukopenia (14, 15). Similarly. the combination of

BG + BCNU resulted in bone marrow aplasia, splenic lympho-

cyte depletion in rodents (16), suggesting that the potentially

increased therapeutic index of BCNU afforded by BG in hu-

mans may be limited by the hypersensitization of hematopoietic

cells. Therefore, protection of hematopoietie cells with concur-

rent depletion of MGMT in tumor cells by BG should increase

the effectiveness of the combination of BG + BCNU. To this

end, we previously generated a retroviral vector pG 1 Nada eon-

taming a bacterial homologue of MGMT, the BG-resistant

ATase gene ada, under the control of the human phosphoglyc-

erate kinase promoter (17). Expression of ada in munine hema-

topoietic cells resulted in a 2-fold increase in the D37 for BCNU

in combination with BG in CPC assays ( 17). Further, transplan-

tation of ada-transduced bone marrow into CBaJCaJ mice in-

creased the maximally tolerated dose of BG + BCNU and

attenuated anemia and thrombocytopenia produced by the drug

combination (17). In the current study, we have developed an

amphotnopie retrovirus and tested the protective effect of ada

expression against BG + BCNU-induced cytotoxicity in human

hematopoietic progenitor cells. Our data provide a preclinical

3 The abbreviations used are: BCNU. 1 ,3-bis(2-chloroethyl)- I -nitro-

sourea; MGMT. 06-methylguanine-DNA methyltransferase; BG, 0#{176}-

benzylguanine: ATase. 0#{176}-alkylguanine-DNA alkyltransferase: CPC,

committed progenitor cell; CFU, colony-forming unit; RT, reverse tran-

scniption.

Research. on September 29, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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302 Expression of ada Alkyltransferase in Human Bone Marrow

basis for testing in humans the effectiveness of ada gene transfer

in reducing BG + BCNU-indueed myelosuppression.

MATERIALS AND METHODS

Cell Culture. NIH3T3 cells were obtained from the

American Type Culture Collection (Rockville, MD) and grown

in DMEM medium (Life Technologies, Inc., Gaithersburg, MD)

supplemented with 10% newborn calf serum (Sigma Chemical

Co., St. Louis, MO). PA317 cells obtained from the American

Type Culture Collection were grown in DMEM containing 10%

fetal bovine serum.

Drugs. BCNU, purchased from Bristol-Myers Squibb

Co. (Princeton, NJ), was dissolved in 100% ethanol immediately

prior to use. BG, a generous gift from Dr. R. C. Mosehel

(National Cancer Institute, Frederick, MD), was dissolved in

DMSO.

Generation and Characterization of an Amphotropicada Retrovirus. The construction of pGlNada and the gen-

eration and characterization of an ecotropic producer cell line

has been described previously (17). Virus-containing superna-

tant from this ecotropic ada producer cell line was used to infect

the amphotropic producer cell line, PA317, in the presence of 6

p.g/ml polybrene (Sigma Chemical Co.). After 96 h, neo-ex-

pressing cells were selected with 500 p.g/ml G418 (Life Tech-

nologies, Inc.). Selection medium was replaced every 3-4 days

until discrete colonies were visible. Twenty-three G418-resist-

ant colonies were isolated individually using glass cloning rings

(BeIlco, Vineland, NJ). After further expansion, producer cell

clones were characterized in terms of viral production and

expression of ada as described previously (17).

Briefly, viral titer was measured initially by quantitation of

viral RNA by slot blot hybridization using the neo gene as a

probe (17). The biological titer of 10 clones showing the greatest

level of neo gene expression was further determined by G41 8

selection of virally transduced NIH3T3 cells as described pre-

viously (17). Stable integration of the proviral genome into the

various producer cell clones was determined by Southern blot

analysis as described previously (18). The G1Na.40 ampho-

tropic cell line, which produces retrovirus containing neo alone

with biological titer of 1 X 106 CFU/ml, served as a control in

these studies. This cell line was provided by Genetic Therapy,

Inc. (Gaithersburg, MD).

ATase Assays and Fluorography. ATase activity was

determined in various producer cell clones by measuring the

transfer of {3H]06-methylguanine from [3H]1-methyl-l-nitro-

sourea-treated calf thymus DNA (19), and the component of

activity due to ada was confirmed by SDS-PAGE and fluorog-

raphy as described previously (20). The latter assay distin-

guishes by molecular weight the relative levels of endogenous

Mr 23,000 murine MGMT protein from the transduced Mr

39,000 ada protein (17). ATase activity in CPC colonies was

measured by modification of a method originally described by

Wu et a!. (21) that has greater sensitivity than the first assay,

allowing measurement of considerably lower levels of ATase

activity. In this assay, ATase-mediated repair of 06-methylgua-

nine in a PvuII restriction site in an 18-men allows its digestion

to an 8-men. The percentage of PvuII digestion (percentage of

probe cleavage) of the lesion-containing oligonueleotide is di-

reetly proportional to the level of ATase. Briefly, total cellular

extracts were prepared by lysing 60-100 CPC colonies in 100

jil of 50 mM Tris-HC1 (pH 7.5), 1 ms� EDTA, 1 msi DTF, and

0.1 M NaC1 via three cycles of freezing and thawing. Approxi-

mately 1-2 �ig of total protein were incubated with 2 fmol of the

06-methylguanine-eontaining oligonueleotide for 2 h. Under

these conditions, the basal level of detection was approximately

3 fmollmg protein in crude extracts. After gel eleetrophoresis,

ATase activity was quantitated by measuring pixel intensity of

the radiolabeled fragments using a Phosphorlmager 445 SI

(Molecular Dynamics, Sunnyvale, CA):

Pixels of 8-mer% probe cleavage

Pixels of 18-mer + pixels of 8-merx 100

fmol ATase/mg protein is calculated as the percentage of probe

cleavage X 2 fmol of 06-methylguanine-eontaining oligonu-

eleotide/mg protein.

Preparation of Mononuclear Cells. Hematopoietie pro-

genitor cells were derived either from bone marrow of five

healthy individuals who had donated marrow for allogeneic

bone marrow transplantation or from umbilical cord blood of

two normal neonates. Each source of progenitor cells will be

referred to by donor numbers 1-Vil. Bone marrow aspirates

from the anterior iliac crest and umbilical cord blood samples

were transferred to Media-l99 (Life Technologies, Inc.) supple-

mented with 1000 units of hepanin (Fujasawa, Inc., Deerfield,

IL). Mononuclear cells were isolated using a Histopaque (Sigma

Chemical Co.) cushion gradient within 2-3 h of harvest. Mono-

nuclear cells from sample VI were enriched for CD34-positive

cells using Ceprate LC CD34 cell selection kit (CellPro, Inc.

Bothell, WA) prior to further analysis. Samples III, VI, and VII

were cryogenically preserved prior to retroviral gene transdue-

tion, whereas all other samples were transduced immediately

after harvest.

Retroviral Gene Transduction. Mononuclear cells

were incubated at 37#{176}Cfor 48 h in stimulation medium consist-

ing of DMEM supplemented with 15% fetal bovine serum

(Hyclone Laboratories, Logan, UT), 2 mrsi glutamine, 20 ng/ml

interleukin 3 (Amgen, Inc., Thousand Oaks, CA), and 50 ng/ml

interleukin 6 (Amgen, Inc.). One-half of these cells were trans-

duced with ada-containing virus, and one-half of these were

transduced with neo-eontaining control virus. Transduction was

carried out by coculture of the mononuclear cells with neo or

ada producer cell lines for 48 h in stimulation medium supple-

mented with 6 p.g/ml polybrene. The stimulation medium for

samples Ill-Vu was supplemented with 100 ng/ml stem cell

factor (Genzyme, Inc. Cambridge, MA). Mononuclear cells

from the first three individuals (I-Ill) were cocultured with

log-phase producer cells, whereas those from the next four

samples (IV-VII) were cocultured with producer cells that had

been sterilized with 0.01 mg/ml mitomycin C to eliminate

producer cell contamination in the subsequent assays for pro-

genitor cell survival and ada RNA expression. Additionally,

because polybrene has been shown to be cytotoxie to CD34-

positive cells, the CD34-enriehed cells from sample VI were

incubated with producer cells in the presence of 4 pg/ml prot-

amine sulfate (Elkins-Sinn, Cherry Hill, NJ; Ref. 22).



kDa

112

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34.9-

28.7-

20.5-

c’f, �‘ �0

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� - ..‘ �

Clinical Cancer Research 303

CPC Assay. Transduced mononuclear cells were nesus-

pended at a density of 1-2.5 X l05/ml in complete methyleel-

lulose Methocult containing 30% fetal bovine serum, 2 mtvi

glutamine, 1 % BSA, 0. 1 msi 2-mereaptoethanol, 5% serum-free

phytohemagglutinin leukocyte conditioned medium, 3 units/ml

erythropoietin (Stem Cell Technologies, Inc., Vancouver, Bnit-

ish Columbia, Canada). Thereafter, cells were sequentially

treated at an interval of I h with BG (10 or 25 p.M) and BCNU

(30-100 fLM). Colony formation was measured 4-9 days after

drug treatment. Survival values for the combination of BG +

BCNU were normalized to the values obtained with BG alone.

Because plating efficiency was relatively low, the methylcellu-

lose used for samples Ill-Vu was further supplemented with 50

ng/ml stem cell factor, 20 ng/ml granulocyte/macnophage cob-

ny-stimulating factor, 20 ng/mI interleukin 3, 20 ng/ml interleu-

kin 6, and 20 ng/ml granulocyte colony-stimulating factor (Stem

Cell Technologies, Inc), which increased plating efficiency 2-3-

fold. The additional growth factors facilitate differentiation and

promote survival of all hematopoietie cell lineages and are

thought not to affect transduction efficiency.

RT-PCR Analysis of CPC Colonies. Seven to 14 days

after drug treatment, colonies were plucked from methyleellu-

lose, RNA was isolated, and transduction efficiency was deter-

mined by RT-PCR analysis as previously described ( I 7) with

the following modifications. The absence of contaminating pro-

ducer cells was confirmed by microscopic examination of the

colonies and by testing for the producer cell-specific ampho-

tropic ENV mRNA using PCR primers as described previously

(23). Additionally, the RNA was precipitated in the presence of

20 jig of glycogen (Boehringer Mannheim, Indianapolis, IN) as

a carrier to increase the extent of recovery.

RESULTS

Generation and Characterization of Producer Cell

Clones. We have previously constructed a Moboney leukemia

virus-based retrovinal vector pGlNada containing the ada gene

driven by the human phosphoglycerate kinase promoter to gen-

erate an ecotropie producer cell line (clone 18) capable of

conferring functional ada expression to munine hematopoietie

cells ( 17). Virus-containing supernatant from this producer cell

line was used to transduce the amphotropie packaging cell line

PA3 17, as described in “Materials and Methods.” A clone

designated “clone 5” with a biological titer of 6.4 X I 0’� CFU/ml

was used in all the experiments described in this study. This

producer line was shown to be free of helper virus by the lack

of acquisition of 0418 resistance after two serial supernatant

infections of NIH3T3 cells using a marker rescue assay (24).

The ATase activity in clone 5 was 4.4 pmol/mg protein,

which represents a 25-fold increase over the parental PA3 17

cells. This activity was completely resistant to inactivation by 10

p.M BG (data not shown). To further demonstrate that this ATase

activity was due to ada, we radiolabeled the ATases in clone 5

using [3H]l-methyl-l-nitrosourea-treated calf thymus DNA as a

substrate. As seen in Fig. I , the vast majority of ATase activity

in this clone was associated with BG-resistant ada protein.

Southern blot analysis of clone 5 DNA revealed that the proviral

genome was integrated into the host genome without gross

rearrangements or deletions (data not shown).

Fig. I Fluorographic analysis of Alase protein in producer cell clones.Clones 2, 4, and S are retroviral producer cell clones derived from

parental PA317 cells. CEM is a human T-lymphoblast cell line thatserved as a positive control. Arrows, Mr 39,000 tidti and M, 22,000

MGMT proteins.

Cytoprotective Effects of ada against BG + BCNU-

induced Cytotoxicity. Mononuclear cells isolated from the

bone marrow of five normal individuals and one umbilical cord

blood specimens were transduced with either ada or tteo retro-

viruses, and the cytotoxic effects of the combination of BG +

BCNU were determined. Cells were sequentially treated with

BG and BCNU, and the total number of CPC colonies was

determined 4-9 days later. Fig. 2 shows the survival data for

day 4 when the predominant colony type was erythroid (CFU-

E). At later times (days 7-9) when other lineages (CFU-GM.

BFU-E, and CFU-GEMM) matured, we observed essentially the

same survival patterns. Moreover, when the lineage-specific

survival scores were analyzed separately, no obvious differences

in their intrinsic sensitivity to BG + BCNU or selective cyto-

protection by ada was detected; hence in this report, we present

only the combined colony survival values. BG alone up to a

dose of 25 f.LM was nontoxie. Donor characteristics for samples

1-VI are shown in Table 1, and a representative CPC assay

illustrating the cytoprotective effective ofada is depicted in Fig.

2A. In this experiment, bone marrow mononuclear cells (sample

I) were cocultured with log phase ada or control two producer

cells and treated with the combination of BG (10 tiM) + BCNU

(30-100 �i.M). Bone marrow cells cocultured with the neo pro-

ducer line were extremely sensitive the cytotoxie effects of the

combination with a one log cell kill dose of approximately 35

�LM BCNU. In contrast, bone marrow cells cocultured with the

ada producer line became essentially refractory to combination-

induced cytotoxicity up to as much as 100 p.M BCNU, exem-

plifled by a 3.9-fold increased survival advantage at a 30 p.M

dose of BCNU (Fig. 2A and Table 1). Using the identical

protocol, the cytoprotective effect of ada was determined in two

additional bone marrow samples (II and III). Samples II and III

were intrinsically 2- and 4-fold less sensitive than any other

sample tested after BG (10 p.M) + BCNU (30 p.M), as indicated

by the control neo cells in Fig. 2C. The extent of ada-mediated

cytoproteetion after this drug treatment in samples II and HI was

I . 1 - and 2.9-fold, respectively.



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ada

I II III IV V VI


BCNU (pM)

Fig. 2 Comparison of the cytotoxic effects of BG + BCNU in ada- or

neo-transduced hematopoietic progenitor cells. The cytotoxic effects of

BG + BCNU were determined by counting the total number of eryth-roid, myeloid, and mixed progenitor cell colonies 4 days after drug

treatment. A, representative CPC assay showing ada- or control tieo-

transduced mononuclear cells treated with 10 p.M BG followed by30-100 p.M BCNU. B, average (mean ± SD) survival values from ,ieo-

or ada-transduced cells from the six samples tested at the 30 p.M dose of

BCNU. *, paired t test analysis of the survival data indicated that ada-

modified bone marrow is more resistant than neo-transduced bone

marrow (P < 0.015). C. CPC survival values for transduced mononu-

clear cells from bone marrow from five normal individuals (I-V) and

CD34-enriched cells from an umbilical cord blood sample (VI) aftertreatment with BG followed by a 30-p.M dose of BCNU.

Although the subsequent experiments were primarily de-

signed to determine the functional expression of ada, we also

consistently observed a 2.1-2.6-fold ada-mediated cytoprotee-

tive effect. Treatment of sample IV with BG (10 p.M) followed

by BCNU (30 p.M) resulted in a 2.6-fold reduction in ada-

transduced cells. When the dose of BG in samples V and VI was

increased to 25 p.M. a comparable 2.4- and 2. 1-fold decrease in

BCNU-induced cytotoxicity was observed in ada-transduced

cells. A 2-fold enrichment for CD34-positive mononuclear cells

derived from umbilical cord blood (sample VI) did not provide

greater cytoproteetion than non-enriched bone marrow. The

mean survival after BG + BCNU of neo- or ada-infected cells

from all six trans-infection experiments is illustrated in Fig. 2B.

ada significantly (P < 0.015) conferred a 2.1-fold protection

against BG + BCNU-indueed cytotoxicity.

Functional Expression of ada in CPC Colonies. Trans-

duetion efficiency was determined 7-14 days after drug treat-

ment by measuring the frequency of expression of ada mRNA

in individual CPC colonies. The transduction efficiency and

extent of in vitro selection is summarized in Table 1 . The

determination of ada transduction efficiency in the CPC cob-

nies from samples I-Ill was precluded because of contaminating

producer cells, identified by RT-PCR analysis of the producer-

specific amphotropie ENV mRNA. Despite harvesting CPC

colonies that appeared microscopically free of producer cells,

we were unable to determine whether ada mRNA signal was

derived from producer or transduced bone marrow cells, be-

cause a majority of ada-expressing colonies also appeared to

express the amphotropie ENV. To overcome this difficulty in

subsequent experiments, producer cells were sterilized with

mitomycin C prior to coculture with samples IV-VII. The

transduction efficiency in samples IV-VI was 40% in CPCs

treated with only BG. In sample IV treated with BG (10 p.M) +

BCNU, there was no evidence for in vitro selection of ada-

expressing cells (Table 1). In samples V and VI treated with a

higher dose of BG (25 p.M), it appeared that in vitro selection did

occur. The combined data for samples V and VI (Table 1 and

Fig. 3), 8 of 20 ada-expressing colonies without BCNU treat-

ment versus 1 3 of 1 7 after BCNU treatment, indicate an approx-

imately 2-fold enrichment (P = 0.045 by Fisher’s exact test;

Ref. 25). All of the tested colonies from samples IV-VI were

ENV negative (data not shown), indicating that the amplified

ada mRNA was indeed from transduced hematopoietie CPCs.

The level of ATase activity in CPC colonies pooled from a

non-enriched umbilical cord blood sample VII is illustrated in

Fig. 4. Extracts prepared from control ,ieo-transdueed, un-

treated, or BCNU-treated CPC colonies respectively elicited

0.163 and 0.192% probe cleavage/jig protein, representing 4-5-

fold above background cleavage. Although no greaten ATase

activity was observed in untreated, ada-transduced cells, the

level of ATase activity in BCNU-treated ada-cells ( 1 .25% probe

eleavage/p.g protein) was approximately 7-fold greater than in

BCNU-treated neo cells, suggesting that BCNU treatment re-

sulted in selection of ada-expressing cells. This level of ATase

activity in ada-transduced cells was estimated to be 25.0

fmollmg protein.

DISCUSSION

We have generated an amphotropic, retroviral producer cell

line that expresses high levels of the bacterial ATase ada under

control of the human phosphoglycerate kinase promoter. The

majority of ATase activity in this producer cell line was due to

the Mr 39,000 ada protein (Fig. 1), which was highly refractory

to BG-mediated depletion. This cell line produces replication-

incompetent retrovirus that is capable of delivering functional

proviral genome to human hematopoietie CPCs, as indicated by

a 2-fold attenuation of BG + BCNU-indueed cytotoxicity, and



BG BG+BCNU


TahI e 1 Effects of ada transduction on the response of hematopoietic progenitor cells to BG + BCNU

Sample no.” Donor traits Fold protectionb

Frequency o f ada expression

BG only’ BG + BCNU”

III111IVVVI

Male, age 27

Male, age 14Female. age 12Female, age 34Female, age 18Neonate

3.9

1.1

2.92.62.42.1

ND

ND

ND2/54/104/10

ND

ND

ND1/3’7/7’6/10’

“ Samples I-V are unenniched mononuclear cells from bone marrow, whereas sample VI represents CD34-enniched mononuclear cells from

umbilical cord blood.

F, Ratio of the survival fraction of ada- and neo-transduced bone marrow after treatment with BG + BCNU.

‘. Frequency of CPC colonies expressing ada mRNA after BG treatment.d Frequency of CPC colonies expressing ada mRNA after BG + BCNU treatment.,. The difference between BG versus BG + BCNU treatments is insignificant by Fisher’s exact test (P 1.0).

1The difference between BG versus BG + BCNU treatments (combined for samples V and VI) is significant by Fisher’s exact test (P = 0.043).

1 2 3 4 5 6 7 8 9 10 11 12 13141516171819

319 bp

Fig. 3 Frequency of CPC colonies expressing ada mRNA. ada RT-PCR analysis of individual colonies isolated from methylcellulose 9 days after

treatment with BG alone (Lanes 3-12) or BG followed by BCNU (30 riM; Lanes 13-19). Lanes 1 and 2, respectively. represent a positive control

for PCR using ada eDNA and a positive control for the reverse transcription reaction using RNA from clone 5.

a BCNU-induced enrichment of cells expressing ada mRNA

and protein. These data demonstrate that efficient retroviral

transfer of the ada gene into human bone marrow cells is

feasible and can confer functional expression of the ada protein

which, at least in vitro, can decrease the cytotoxic effects of the

combination of BG + BCNU. We have made similar observa-

tions previously with murine bone marrow cells both in vitro

and in t’ivo using an analogous ecotropie ada retrovirus that was

capable of infecting long-lived hematopoietic progenitor cells,

preventing lethal myebotoxicity induced by the combination of

BG + BCNU ( I 7). A 2-fold cytoprotective effect of ada in vitro

was associated with a modest reduction in the anemic and

thrombocytopenie effects of the drug combination in mice (17).

Others have reported recently that overexpression of hu-

man MGMT in primitive murine progenitor cells not only

attenuates the paneytopenic effect of BCNU in vitro and in vito

but also increases the maximally tolerated dose of BCNU in the

munine paradigm (26-28). It was proposed that MGMT trans-

duetion of human bone marrow in an autologous bone marrow

transplant setting could facilitate aggressive dose escalation of

BCNU to eradicate refractory tumors. However, the dose esca-

lation afforded by ovenexpression of MGMT in bone marrow

using currently available retroviral constructs may be insuffi-

cient to kill tumors with high levels of MGMT. Therefore,

clinical use of MGMT in myebopnoteetive strategies may be

limited to patients with tumors devoid of on having very low

levels of MGMT expression. The use of BG to sensitize resistant

tumor cells with high levels of MGMT would be confounded by

the fact that MGMT in the transduced hematopoietic cells and

tumor cells would be equally depleted. Transduction with BG-

resistant ATases such as ada to protect bone marrow has a

marked advantage oven MGMT in that it would allow more

selective BG-mediated inactivation of MGMT in tumor cells,

sensitizing them to BCNU-indueed cytotoxicity while simulta-

neously sparing the ada-transduced bone marrow. We suggest

that with this 2-fold approach of sparing bone marrow and

targeting tumor cell drug resistance will achieve a greater in-

crease in the therapeutic index of BCNU than either modality

alone.

Although we have been able to establish the feasibility of

ada-mediated cytoprotection in both murine and human progen-

itor cells, the level of ATase expression and the extent of

protection against BG + BCNU-induced cytotoxicity is modest.

With a transduction efficiency of 40%, the levels of ATase

activity remained below 30 fmol/mg protein, and survival was

increased only 2-fold. Whether such levels of in vitro expression

and protection will enable sufficient dose escalation of BCNU in

vit,o in humans is unknown. Levels of ada ranging from ap-



s,) ��(i neo ada

�, <- 18 mer

.�. 1#{248}.� <-8mer


0 30 0 3OuMBCNUFig. 4 Alase activity in CPC colonies. ATase activity in pooled CPCcolonies was measured based on the repair of 0#{176}-methylguanine in aPvuII restriction site of an I 8-mer end-labeled oligonucleotide. Levelsof ATase activity are directly proportional to the extent of Pt’i�Il endo-

nuclease-mediated cleavage of the 18-mer to an 8-mer. Lane 1, unre-

acted 06-methylguanine-containing 18-mer substrate; Lzine 2, the same

oligomer incubated with P11411 alone; Lane 3. oligomer incubated with1 p.g of CEM extract prior to Ps’i,II digestion; Lanes 4-7, oligomer

incubated with 1-2 p.g of extracts prepared from CPC colonies trans-

duced with ada or neo prior to P11111 digestion.

proximately 10-170 fmol/mg protein in leukocytes from mice

transplanted with ada-transduced bone marrow failed to prevent

BG + BCNU-induced leukopenia (17). Moreover, this modified

marrow afforded a very marginal therapeutic benefit because

only a 1.25 dose escalation of BCNU could be tolerated. The

low levels of ada and the small dose modification of BCNU in

mice would suggest that these levels of expression and protee-

tion of human bone marrow may not be sufficient to achieve an

appreciable increase in the therapeutic index of BCNU. There-

fore, before this strategy is tested in the clinic, it may be

necessary to generate improved retroviral vectors that yield

higher levels of ada expression in human bone marrow cells.

Approaches to improve the level of expression of func-

tional ATase in transduced cells include the use of stronger

promoters and mammalian ATases. Mutant human MGMT that

is insensitive to BG-mediated inactivation (29) may be more

effective than ada because of its ability to localize into the

nucleus; it is also less likely to elicit an immunogenic response

in vivo. Recently, Reese et a!. (30) have observed that the human

mutant MGMT (G 156A) effectively confers resistance to BG +

BCNU-indueed cytotoxicity in human bone marrow.

In addition to the low levels of expression of ada, our

approach may be limited by low in vito transduction efficiency.

Although we have been able to achieve 40% transduction effi-

ciency in vitro, in vivo gene marking studies suggest that with

Moboney-based retroviral vectors, the extent of drug-resistant

hematopoiesis following autobogous bone marrow transplanta-

(ion may be limited due to low transduction efficiency (3 1 , 32).

Expression of dominant selectable markers, such as the mu!ti-

drug resistance-I (33) and dihydrofo!ate reductase (34) genes,

have been suggested as a means to allow in vitro enrichment of

transduced cells. Our observations that in vitro enrichment of

transduced human as well as murine progenitor cells to nearly

100% after BG + BCNU administration support the potential

use of ada as such a dominant selectable marker for ex vivo

expansion of transduced progenitor cells. Therefore, selection of

ada-expressing cells with BG + BCNU prior to transplant may

improve the it? 1110 transduction efficiency, the extent of drug-

resistant hematopoiesis, and the therapeutic index of BCNU.

In summary, we have established that retroviral transfer of

the ado gene can protect human hematopoietic committed pro-

genitor cells against BG + BCNU cytotoxidity. The pnedomi-

nant limitation of this approach using the pGlNada retrovinal

construct is the low level of ATase expression. With develop-

ment of retroviral constructs that confer greater levels of ATase,

the transplantation of bone marrow transduced with BG-resis-

tant ATases such as ada may not only achieve attenuation of

BCNU-indueed myelotoxicity but also increase the tumor se-

leetivity of BG-mediated modulation of BCNU resistance.

ACKNOWLEDGMENTS

We are grateful to Dr. Malcom Brenner and Barbara Alexander for

providing human bone marrow samples and to Amgen Corp. for pro-

viding interleukins 3 and 6. We are also grateful to Dr. Xiaolong Luo for

providing statistical consultation.

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1997;3:301-307. Clin Cancer Res U K Marathi, L C Harris, C C Venable, et al. plus 1, 3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity.human bone marrow cells protects against O6-benzylguanine Retroviral transfer of a bacterial alkyltransferase gene (ada) into

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Retroviral Transfer ofaBacterial Alkyltransferase Gene (ada) … · 2005-09-10 · kDa 112 84 53.2-34.9-28.7-20.5-c’f, ‘ 0 f , q 0 e.4-adalose - ..‘ Clinical Cancer Research