retroviral transfer of a bacterial alkyltransferase gene into … · g418 selection of infected nih...

Vol. 1, /359-1368, Not’einher 1995 Clinical Cancer Research 1359

Retroviral Transfer of a Bacterial Alkyltransferase Gene

into Murine Bone Marrow Protects against

Chioroethylnitrosourea Cytotoxicity1

Linda C. Harris,2 Upendra K. Marathi,

Carol C. Edwards, Peter J. Houghton,

Deo Kumar Srivastava, Elio F Vanin,

Brian P. Sorrentino, and Thomas P. Brent

Department of Molecular Pharmacology [L. C. H., U. K. M.,

C. C. E., P. J. H., T. P. B.], Department of Biostatistics [D. K. S.],

Division of Experimental Hematology [E. F. V.], and Department of

Biochemistry [B. P. 5.], St. Jude Children’s Research Hospital,

Memphis, Tennessee 38105; Department of Pharmacology, College

of Medicine, University of Tennessee, Memphis. Tennessee 38163

[T. P. B.1; and Genetic Therapy, Inc., Gaithersburg, Maryland20878 [E. F. V.]

ABSTRACT

The chloroethybnitrosoureas (CENUs) are important

antineoplastic drugs for which clinical utility has been re-

stricted by the development of severe delayed myebosuppres-

sion in most patients. To investigate the potential of DNA

repair proteins to reduce bone marrow sensitivity to the

CENUs, we transferred the Escherichia coli ada gene, which

encodes a Mr 39,000 06-alkylguanine-DNA alkyltransferase

(ATase), into murine bone marrow cells by the use of a

high-titer ecotropic retrovirus. The ada-encoded ATase is

resistant to 06-benzylguanine (06-BG), a potent inhibitor of

the mammalian ATases, thus affording the bone marrow an

additional level of protection against CENUs. In methylcel-

luiose cultures, ada-infected hematopoietic progenitor cells

were twice as resistant as uninfected cells to the toxic effects

of l,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) following

treatment with 06-BG. Although showing no obvious pro-

tective effects against leukopenia, overexpression of the bac-

teriab ATase activity reduced the severity of anemia and

thrombocytopenia in mice treated with 06-BG and BCNU.These effects, which were maximal at a BCNU dose of 12.5

mg/kg, were associated with improved survival when BCNU

was given at this dose. At lower BCNU doses cytotoxicity

was limited in both transduced and control mice, and at

higher doses the protective effect was saturated due to cy-

totoxicity. These results suggest that ada gene therapy may be

a feasible approach to amelioration of delayed myelosuppres-

sion following 06-BG plus CENU combination chemotherapy.

Received 3/29/95: revised 6/21/95; accepted 7/13/95.

I Supported in part by NIH Grants CA23099 and CA14799, Cancer

Center Support (CORE) Grant P30 CA21765, and the American Leba-

nese 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.O.

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

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

INTRODUCTION

O”-alkylguanine-DNA ATases3 are conserved DNA repair

proteins found in all living organisms studied to date (1, 2).

They are responsible for the repair of O”-alkybguanine and

04-alkylthymine, which are toxic, mutagenic DNA adducts gen-

erated by the alkylnitrosoureas MNU and N-methyl-N’ -nitro-N-

nitrosoguanidine (1, 2). ATases also repair O”-chloroethylgua-

nine lesions, the DNA cross-link precursors produced by the

CENUs, such as BCNU and l-(2-chloroethyl)-3-(trans-4-meth-

ylcyclohexyl)- 1 -nitrosourea (3). Because DNA cross-links are

extremely cytotoxic, the CENUs possess low mutagenic poten-

tial (4), and, therefore, these drugs are useful antineoplastic

agents (5). The ATase repair reaction is stoichiometnic and

autoinactivating, so that the degree of cytotoxicity produced by

a CENU depends on the amount of repair protein within the cell

(6). Consequently, human tumor xenografts expressing high

levels of this repair enzyme are resistant to CENU therapy (6),

and tissues expressing very low levels (e.g., bone marrow) are

highly sensitive (7, 8). These observations are especially perti-

nent to BCNU, because of its central role in primary therapy for

brain tumors (9); as for all CENUs, its dose-limiting toxicity is

delayed myebosupression (5, 9).

An attractive strategy to overcome bone marrow sensitivity

to the CENUs would rely on overexpression of an ATase gene

and incorporating a mechanism by which endogenous tumor

ATase activity may be selectively inhibited. Several groups

have introduced drug resistance genes into murine bone marrow

with retroviral vectors [e.g. , the multidrug resistance I gene

(MDRJ; Ref. 10) and mutant dihydrofolate reductase cDNAs

(1 1-13)] demonstrating gene expression by selection of taxob-

resistant marrow in s’is’o or improvement in the survival of

methotrexate-treated mice.

In the study reported here, we tested the feasibility of this

approach using the ada ATase gene of Escherichia coli (14),

which encodes a Mr 39,000 protein consisting of two subunits

that repair either O”-alkylguanine and 04-abkylthymine or alky-

lphosphotniester adducts (14, 15). Previously, its expression in

mammalian cells in vitro has been shown to protect against

nitrosourea cytotoxicity (16, 20). The ada protein is similar to

other ATases in that it is autoinactivated following reaction with

its substrate, although in contrast to mammalian ATases, it has

a very low affinity for O”-BG (21, 22), which has been used to

3 The abbreviations used are: ATase, alkyltransferase; CENU, chloro-ethybnitrosourea; MNU, N-methyl-N-nitrosourea; MNNG, N-methyl-N’-

nitro-N-nitrosoguanidine; BCNU, l,3-bis(2-chloroethyb)- 1 -nitrosourea;

MDRI, mubtidrug resistance 1; O”-BG, O”-benzybguanine; PGK, phos-

phoglycerate kinase; IL, interbeukin; LTR, long terminal repeat; RT,

reverse transcription; PEG, polyethylene glycol.

Research. on February 2, 2020. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

1360 Alkyltransferase Gene Expression in Munine Bone Marrow

sensitize resistant human tumor xenografts to CENU chemo-

therapy by depleting their ATase (23-26).

Theoretically, 06-BG treatment should sensitize tumors to

the cytotoxic effect of the CENUs, whereas overexpression of

the 06-BG-resistant ATase increases the resistance of normal

bone marrow. We describe successful transfer of the bacterial

ada gene into the hematopoietic cells of CB/CaJ mice, and

protection against 06-BG plus BCNU-induced myelosuppres-

sion and death.

MATERIALS AND METHODS

The plasmid containing the ada gene p062C (27) was

generously provided by Dr. G. P. Margison (Paterson Labora-

tories, Manchester, England), and the retrovinal plasmid pGlNa

(28) was provided by Genetic Therapy, Inc. (Gaithersburg,

MD). The pUCOO7pgk plasmid contained the PGK promoter

(29), and the N2 control retroviral producer clone (30) expressed

the neo gene. NIH 3T3 cells were obtained from the American

Type Culture Collection (Rockville, MD) and grown in DMEM

(GIBCO-BRL, Gaithensburg, MD) supplemented with 10%

newborn calf serum (Sigma Chemical Co., St. Louis, MO), as

were GP + E86 cells (31). PA317 cells (32) were grown in

DMEM containing 10% FCS (Sigma). CBA/CaJ female mice

were obtained from The Jackson Laboratory (Bar Harbor, ME)

and housed in isolation cages in a negative pressure cubical with

access to food and water ad libitum. Human IL-6 was a gift from

Amgen (Thousand Oaks, CA), whereas munine IL-3 was ob-

tamed from GIBCO-BRL. O”-BG was a gift from Dr. R. C.

Moschel (National Cancer Institute, Frederick, MD), and BCNU

(carmustine) was obtained from Bristol-Myers Squibb Co.

(Princeton, NJ). For in vitro studies, O”-BG was dissolved in

DMSO, and BCNU was dissolved in 100% ethanol. For in vivo

studies, the former compound was dissolved in 40% PEG 400 in

saline, with the latter dissolved in 10% ethanol. All radiolabeled

isotopes were from Amersham (Arlington Heights, IL); other

reagents were from Sigma unless otherwise stated.

Generation of Ecotropic Retroviral Producer Clones byTransinfection. Plasmid DNA was initially transfected into

the amphotropic packaging cell line PA317 by a modified

calcium phosphate coprecipitation method (Stratagene, La Jolla

CA; Ref. 33). Forty-eight h later, the medium-containing virus

was removed and diluted 1:5, 1:10, 1:25, 1:50, and 1:100 with

fresh medium. Polybrene (hexadimethrine bromide) at 6 p.g/ml

was added to each of the dilutions, which then were added to

five plates of the ecotnopic packaging cell line GP+E86. After

another 48 h, each of the GP+E86 plates was divided into five

larger 150 x 20-mm plates at the following dilutions: 1:10,

1:20, 1:40, 1:80, and 1:160. One day later, fresh media contain-

ing 500 p.g/ml active G418 (Geneticin; GIBCO-BRL) were

added to the cells and replaced every 3 or 4 days until discrete

colonies were visible. After 10-14 days, 30 colonies were

isolated using glass cloning rings (Bellco, Vineland, NJ) and

placed in the individual wells of 24-well plates. These producer

clones were expanded and characterized for virus production.

Retroviral Titering by PEG Precipitation of RNA andSlot Blot Hybridization. Viral supernatants, 1 ml each, were

transferred from each producer clone to an Eppendorf tube, to

which 0.5 ml PEG solution (30% PEG 8000, 1.5 M NaC1) was

added. The tubes were held on ice for 30 mm before centnifu-

gation at 14,000 rpm for S mm at 4#{176}C.The pellets were resus-

pended in 0.2 ml VTR [0.5 ml Vanadyl Ribonucleoside Com-

plex (GIBCO-BRL), 10 mg/ml tRNA, and S ml TE (10 m�vt

Tnis-HCI, 1 mM EDTA, pH 8.0)], after which 0.2 ml 2X lysis

buffer (1% SDS, 0.6 M NaC1, 20 m�i EDTA, and 20 msi Tnis, pH

7.4) was added. The mixed solution was extracted once with an

equal volume of U-saturated phenol and then with an equal

volume of chloroform:isoamyl alcohol (24:1). The RNA was

then precipitated by the addition of 0.9 ml ethanol and by

holding on dry ice for 15 mm. After centnifugation at 14,000

rpm for 15 mm at 4#{176}C,the pelleted RNA was dissolved in 250

p.1 15% formaldehyde solution (2.1 ml 37.5% formaldehyde, 2.9

ml H2O); 250 p.1 20X SSC were added; and the samples were

heated at 50#{176}Cfor 15 mm. They were then held on ice before

being applied to the slot blot apparatus (Minifold II; Schleicher

& Schuell, Keene, NH), which contained a GeneScreen plus

membrane (New England Nuclear DuPont, Wilmington, DE)

prewetted in lOX SSC. The membrane was hybridized with a

virus-specific probe using standard procedures (34).

Clones that generated the strongest signals, hence the high-

est levels of viral RNA, were subjected to biological titering by

G418 selection of infected NIH 3T3 cells and counting resistant

colonies.

ATase Assays and Fluorography. The O”-ATase assay

1, used to quantitate the activity in the producer clones, relied on

[3HIMNU-treated calf thymus DNA substrate as previously

described (35). Assay 2, originally described by Wu et a!. (36)

and modified by Marathi et a!. (37), relies on PvuII digestion of

a double-stranded oligonucleotide once the O”-methylguanine

adduct within its cleavage recognition sequence has been re-

paired by ATase. The second assay has greater sensitivity than

the first and therefore allowed qualitative assessment of ATase

activity in WBCs isolated from mouse peripheral blood. Blood

(0.7-1.5 ml) was obtained by cardiac puncture from anesthe-

tized mice, which were subsequently killed by cervical disloca-

tion. RBCs were lysed in 10 ml RBC lysis buffer (150 m�i

NH4C1, 10 mM KHCO3, and 0.1 msi EDTA) and centrifuged for

12 mm at 2000 rpm to pellet the WBCs, from which extracts

were prepared as described for assay 1 (35).

Fluorography was performed by incubating producer cell

extracts with [3HJMNU-treated substrate DNA at 37#{176}Cfor 30

mm before PAGE and electroblotting of proteins to Immobil-

lon-P membranes (Millipore, Bedford MA). Because the ATase

proteins covalently bind [3H]methyl groups following their re-

pair, radiolabeled protein was visualized by the addition of

scintillation fluid (Scint-A XF, Meniden, CT) to the membrane

and autoradiography (14).

Genomic DNA Isolation, Labeling of DNA Probes, and

Southern Blot Analysis. Genomic DNA was isolated from

producer cells using a standard protocol, essentially as described

by Sambrook et al. (34), which involves SDS lysis, incubation

in proteinase K (GIBCO-BRL), phenol-chloroform extraction,

and ethanol precipitation. Southern blot analysis was performed

as described by Sambrook et a!. (34); 32P labeling of DNA

probes was carried out with a Random Prime Labeling kit

(Boehninger Mannheim, Indianapolis, IN).

Genomic DNA was isolated from mouse blood (200 pi) by

lysing RBCs in 10 ml RBC lysis buffer (see above). WBCs were



Sail

Sail pBRO

Clinical Cancer Research 1361

BamHI Salt BamHl

pGlNa

Fig. I Construction of pGlNada, a retroviral vector derived from the pGlNa plasmid, which contains ada under control of the PGK promoter (pgk

Pr.). Ltr, long terminal repeat; pBRO, pBR322 origin of replication; AMP, ampiciblin resistance gene; neo, neomycin resistance gene.

pelleted by centnifugation at 2000 rpm for 12 mm and resus-

pended in 1 ml cell lysis buffer (0.32 M sucrose, 10 mr�t Tnis-

HC1, pH 7.5, 5 mM MgC12, and 1% Triton X-100). After being

held on ice for S mm, the tubes were centrifuged for 10 mm at

3000 rpm to pellet the nuclei. Genomic DNA was then isolated

as described for the producer cells.

Retrovirab Infection of Murine Bone Marrow Cells.

CBAJCaJ mice were thymectomized at 8 weeks of age so that

future studies may use human tumor xenografts, and, at 10

weeks, received i.v. injections of 150 mg/kg 5-fluorouracil

(Sobopak Laboratories, Inc., Elk Grove Village, IL). Forty-eight

h later, they were sacrificed for isolation of bone marrow from

their tibias and femurs (38). Marrow was flushed from the bones

with a needle and syringe containing PBS and 2% FCS (Hy-

clone, Logan, UT) and plated into DMEM containing 15%

Hyclone FCS, 2x glutamine, IL-3 (3 ng/ml), and IL-6 (100

ng/ml) (39) at a concentration of S x 106 cells/100-mm suspen-

sion dish (Corning Glass Works, Corning, NY). After another

48 h, the marrow cells were harvested and cocultured with

retroviral producer cells at the same density and in the same

media as above with the addition of polybrene (6 p.g/ml).

Following a 48-h infection period, marrow cells were harvested

and used for either transplantation of irradiated recipients or in

vitro methylcellulose colony-forming assays.

Assay for Clonogenic Hematopoietic Progenitor Cells.

Methylcellubose (Stem Cell Technologies, Vancouver, British

Columbia, Canada) was divided into 4-ml aliquots for each drug

dose to be tested. Retrovirally infected bone marrow cells were

then added at a concentration of 1 x 105/ml followed by the

addition of 10 �LM O”-BG. After 2 h, BCNU was added, and

1-ml aliquots were plated into 35-mm dishes and incubated for

1 1 days. The number of colonies was counted by light micros-

copy on day 4. Colonies for RT-PCR analysis were isolated on

day 11.

PCR Analysis. DNA isolated from peripheral mouse

blood was subjected to PCR analysis with either ada and �3-glo-

bin or neo- and �3-globin-specific primers. The sequences of

these oligonucleotides were: ada, 5’-GCTGGCCAATCT-

GTCTFAGC-3’ and 5’-CCGATGTAGATGAAATGGAC-3’,

which generated a 3i9-bp fragment; neo, 5’-TCCACTATG-

GCTGATGCAATGCGGC-3’ and 5’-GATAGAAGGCGAT-

GCGCTGCGAATCG-3’, which generated a 404-bp fragment,

and �3-globin, 5’-GAAG1TGGGTGmGGAGAC-3’ and 5’-

GAGACTGCFCCCFAGAATCG-3’, which generated a 401-bp

fragment. Each PCR reaction contained 200 ng DNA, 1 x Pro-

mega PCR buffer (Promega, Madison, WI), 1 m�i MgCl,, 0.2

mM of each of the four nucleotides, 2 �j.Ci [33P]dCTP, 50 ng of

each oligonucleotide primer, and 2.5 units Taq polymerase

(Promega). Amplifications were performed in a minicycler

(M. J. Research, Watertown, MA) for 25 cycles at 94#{176}Cfor 45

s, 56#{176}Cfor 1 mm 30 s, and 72#{176}Cfor 2 mm. Amplified products

were separated by nondenatuning PAGE (6%) and visualized by

autoradiography of the dried gel.

RT-PCR. RNA was prepared from hematopoietic cob-

nies with RNAzol (Tel-Test, Fniendswood, TX), as described by

the manufacturer; RT was performed with a eDNA cycle kit

(Invitrogen, San Diego, CA). The conditions of RT-PCR were

essentially the same as those described for DNA, except that

reactions were extended to 30 cycles.

Hematobogical Analysis. Blood (20 p.1) was diluted with

9980 p.1 hematobogical diluent (0.1 M NaCI, 16 msi boric acid,

0.1 mM sodium-tetraborate, and 0.5 mts.i EDTA) and analyzed

using a Sysmex automated hematology analyzer.

Statistical Analysis. The measurements for the variables

hemoglobin, hematocnit, RBCs, platelets, and WBCs were re-

corded on days 0, 3, 9, 14, 21, 28, 42, 45, 51, 56, 63, and 70 for

the two groups (ada and neo) of mice. Because the observations

for each mouse on various days are correlated, the data were

analyzed using ANOVA for repeated measures (40). For the

analysis, it was assumed that the underlying covaniance structure

had compound symmetry, and the observations were missing at

random; i.e. , information missing because of death or for any

other reason did not provide any information about the process.

The analysis was performed using the program 5V in the BMDP

statistical software package (41). The significance of the param-

eters in the model was tested using a Wald-type x2 statistic. For

the two groups (ada and neo), Kaplan-Meier plots (42) were

obtained and compared using the exact Wilcoxon-Gehan test as

implemented in StatXact statistical software (43).



‘�/ �) �)

49.5 � � .�- ada32.5 -

27.5 �--- � ‘--. .�- human/mouse18.5 -

4 14 18 21 23 29

kb

23.1 -

9.4 -

6.6 -

4.4 -

2.3 -

2.0 -

1.3 -

1.111,1

Cu>

>

(I)0

0

10A A ada-BCNU

.-. neo-BCNU

U #{149}ada-BCNU + O6BG. - . neo-BCNU + 06 BG

1 � � I I I

0 5 10 15 20 25 30

BCNU Dose �M

Fig. 4 Methylcellulose assay for cbonogenic survival of hematopoeitic

progenitor cells among ada- and neo-infected bone marrow cells treated

in vitro with either BCNU alone or BCNU in combination with O”-BG.Points, mean colony number relative to the number in untreated plates;

bars, SDs.


Fig. 2 Fluorography of cell extracts showing sizes of active ATaseproteins. Clones 18 and 29 are two ada-expressing retrovirab producer

clones, GP+E86 is the parental munine line from which they were

derived, and CEM is a human T-lymphoblast cell line that served as a

control. Arrows, ada, human, and munine ATase proteins.

RESULTS

Generation of an ada Ecotropic Retrovirus. The

Moloney leukemia virus-based vector pGlNada was generated

from the pGlNa plasmid by inserting the PGK promoter (29)

and the ada gene (Fig. 1; Ref. 27). The resulting plasmid has the

potential to express the ada gene in mammalian cells under

control of the PGK promoter and the neo gene under control of

the viral promoter contained within the LTR. pGlNada was then

used to generate retroviral producer clones, as described in

‘ ‘Materials and Methods.’ ‘ The clone with the highest titer,

clone 18 (approximately 2 x 106 virus particles/ml,) was used

in all subsequent experiments described in this article. It was

shown to be free of the helper virus, because supernatants

collected from infected NIH 3T3 cells failed to infect a second-

any culture as determined by a marker rescue assay (32).

Characterization of Producer Clones. The O”-ATase

activity of clone 18 (1.5 pmol/mg protein) was 5-fold greater

than the endogenous activity of parental GP+E86 cells (0.3

pmol/mg). Moreover, clone 18 ATase was not inhibited by a 1-h

exposure to 10 p.M O”-BG (data not shown). Incubation of

whole-cell extract with [3H]MNU-tneated substrate DNA, fob-

bowed by PAGE and fluorognaphy, enabled us to visualize the

sizes of radiolabeled ATases, namely, the Mr 39,000 ada and Mr

22,000 mouse proteins (Fig. 2). Although clone 18 expresses

both the neo and ada genes, it was designated the ada-express-

ing virus to distinguish it from N2, which expresses only neo.

Genomic DNA isolated from several producer clones was

digested with the NheI restriction endonuclease (which cuts the

viral DNA once within each of its two LTRs) and subjected to

Southern blot analysis. After hybridization with a neo gene

probe, a single 4-kb band was observed in each ofthe lanes (Fig.

3), demonstrating that full-length proviral DNA had been uni-

fonmly inserted into the host cell genome.

ada Gene Transfer to Murine Hematopoietic Commit-

ted Progenitor Cells in Vitro. CB/CaJ bone marrow cells

were infected with either the clone 18 on N2 netrovirus and

exposed to BCNU, added alone on 1 h after treatment with

O”-BG. After 4 days of growth in methylcellulose, the total

1.1 -

0.9 -

Fig. 3 Southern blot analysis of DNA extracted from six retroviral

producer cell lines probed with the izeo gene. The DNA was digested

with N/tel, which cuts once within each viral LTR to generate a 4063-bp

fragment.

number of hematopoeitic progenitor cell colonies was counted.

The survival responses to BCNU alone of cells infected with

either ada- or neo-expressing viruses were similar; however,

with preexposure to O”-BG the neo-transfected cells became

sensitized to BCNU, whereas those beaning the ada gene did not

(Fig. 4). The D37 for neo-tnansfected cells was 10 p.M BCNU

compared with 20 JiM for the the ada-positive cells, representing

a 2-fold increase in sensitivity following preincubation with

O”-BG.

The presence of ada mRNA was demonstrated by RT-PCR

(Fig. 5) within individual colonies, indicative of ada gene ex-

pression. Only two of the eight colonies exposed to O”-BG but

not to BCNU were positive for ada, compared with eight of

eight exposed to both O”-BG and BCNU (30 p.s�; Fig. 5).

Reconstitution of Lethally Irradiated Mice with Bone

Marrow Infected ex Vivo with the ada or neo Gene. Thirty-

seven female CB/CaJ donor mice were sacrificed 48 h after iv.



bp

1 2 3 4 5 6 7 8

+ - + - + - + -+ - + - + - + - RT

0iM BCNU

1 2 3 4 5 6 7 8

+ - + - + -+ - + - #{247}- + -�1- -AT

� � � 30MBCNU310�281- �204-194”

ada_

neo

pglobinlFig. 6 Demonstration of the presence of ada or izeo genes in mouse

genomic DNA. PCR analysis of DNA isolated from the peripheral blood

of eight representative mice is shown. Mice 1-4 (upper panel) werereconstituted with ada virus-infected marrow, and mice 1-4 (lower

panel) were reconstituted with izeo virus-infected marrow. PCRs were

performed with either ada-. iieo-, or 13-gbobin-specific primers.


bp

603 -

310281 -204 -

194 “

603-

Fig. 5 Frequency of bone marrow progenitor cell colonies that expressed ada mRNA. ada RT-PCR analysis of individual colonies isolated from

methylceblubose 1 1 days after treatment with O”-BG and a 2-h exposure to either vehicle only (0 p.M BCNU, top) or 30 �J.M BCNU (hotto,n).

administration of 150 mg/kg 5-fluorouracil (38), and hone mar-

row cells were harvested as described in ‘ ‘ Materials and Meth-

ods.’ ‘ Fifty percent of the cells were infected with the clone 18

virus containing the ada gene, and the remainder were infected

with the N2 virus containing neo. Cells (4 x 10”) from either

manipulation were then injected into tail veins of each of 73

CB/CaJ female recipient mice that had been thymectomized and

lethally irradiated (850 cGy). All mice became reconstituted

with donor marrow cells, as determined by 100% survival

beyond 2 weeks after transplantation. After 3 weeks, DNA was

isolated from 200 pi blood isolated from the retroonbital venous

plexus of each mouse. PCR analysis performed on blood sam-

ples from 28 of 38 ada and 12 of 35 iieo recipient mice revealed

the presence of the respective gene in every case examined (Fig.

6). Thus, all mice were likely positive for the virally inserted

genes. At 12 weeks after transplantation, three ada-positive

mice were sacrificed, and their marrow was used to reconstitute

24 additional mice, all of which became PCR positive for the

gene. This finding, together with the observation that three ada

mice remained PCR positive when tested 8 months after trans-

plantation, indicates that long-lived primitive hematopoietic

progenitors had been infected with the netrovirus ex i’is’o.

O”-BG and BCNU Treatment of Mice Transplantedwith Retrovirally Infected Bone Marrow Cells. Five weeks

after transplantation, 35 ada- and 35 iieo-beaning mice, pro-

duced as described above, were divided into five treatment

groups each comprising 7 ada and 7 neo mice. They were

treated as follows: (group 1) vehicle alone; (group 2) 30 mg/kg

O”-BG and 7.5 mg/kg BCNU; (group 3) 30 mg/kg O”-BG and

10 mg/kg BCNU; (group 4) 30 mg/kg O”-BG and 12.5 mg/kg

BCNU; and (group 5) 30 mg/kg O”-BG and 15 mg/kg BCNU.

The drugs were injected i.p., with O”-BG administered 2 h

before BCNU. The mice in group 1 received both vehicles, 40%

PEG in saline and 10% ethanol.

Blood samples (20 �i.l each) were taken from the tail vein

of each mouse on the day of drug treatment (day 0) and subse-

quently on days 3, 9, 14, 21, and 28. On day 42, surviving mice

p globin

1 2 3 4I I I 1 1 1 I I

1 2 3 4I ii 1 1 1 1

received a second dose of O”-BG and BCNU that was identical

to the first treatment, with additional blood collected on days 42,

45, 51, 56, 63, and 70. Measurements of hemoglobin, hemato-

cnit, erythrocytes, WBCs, and platelets were obtained for all

doses at each time point and subjected to statistical analysis.

The two phases of the experiment from day 0 through 28

and from day 42 through 70 were analyzed either separately and

also after combining them together. The most significant differ-

ences in the blood parameters for the two groups were observed

in group 4 following treatment with O”-BG and 12.5 mg/kg

BCNU. Fig. 7 shows the hematopoietic data for this dose. For

group 4, the combined statistical analysis showed there was a

significant difference (at a level of significance a 0.01)

between the two groups (ada and ,zeo) for the variables hemo-

gbobin (P < 0.0001), hematocrit (P 0.0001), and RBCs (P



Hemoglobin70

Hematocrit

60

6

50

4

40

2

30

16

14

12

10

-... 8

bO

0

10

8

2

0

20

10

0

-u--ad�id��D�.neoI

E

1200

1000

800

600

400

200

0

p = 0.0002

0

Days Days

1364 Alkyltransfenase Gene Expression in Murine Bone Marrow

Red Blood Cells

I I I I

10 20 30 40 50 60 70 80

Fig. 7 Comparison of findings in mouse blood sampled at various times before and after O”-BG and 12.5 mg/kg BCNU treatment. Panels, groupof seven ada mice and seven neo control mice each. Drugs were administered on days 0 and 42. Data points, mean measurements for surviving mice;

bars, SE. Because only one neo mouse survived at day 70, a SE could not be calculated for this point. The statistical significance (a = 0.01) for thedifferences between the two groups of mice analyzed using ANOVA for repeated measures over the entire 70-day time course is shown as the P value.

0.0002). The P value corresponding to the platelet counts for the

two groups was 0.014, which indicates that the two groups differ

significantly at the 0.05 level of significance but not at the 0.01

level of significance. However, there was no difference in the

mean of WBC levels in the two groups (P = 0.136). A similar

trend was also noted when the data in the two phases were

analyzed separately, with the only exception that the difference

between ada and neo in platelet counts was significant at a =

0.01 in the second phase (P = 0.004). There was no difference

in any of the variables in the two groups (ada and neo) when

treated with vehicle alone.

The vast majority of mice survived the first drug treatment;

however, death rates increased substantially after the second

dose (Fig. 8). A clear survival advantage was conferred by the

ada protein in mice treated with BCNU at 12.5 mg/kg (P =

0.021) consistent with the protection of hematobogical parame-

tens shown in Fig. 7. There was no significant difference in the

survival of the two groups for all other dose levels. Necropsies

performed on four mice within 1 h of death revealed severe

myeboid hypoplasia and anemia due to BCNU-induced deficits

in blood elements.

ATase Activity Assay of WBCs Isolated from Trans-

duced Mouse Blood. ATase assay 2 was performed on WBC

extracts isolated from 12 ada mice and 2 neo mice 21 weeks

after reconstitution with transduced marrow (Fig. 9). Extracts

(50 gig) were pretreated for 30 mm at 37#{176}Cwith 50 p.M O”-BG

to inhibit endogenous munine ATase prior to incubation with

0.05 pmol oligonucleotide substrate. A positive control (Fig. 9,

C3) consisted of 10 �i.g ada producer cell extract expressing 3

pmol/mg ada ATase. As indicated in Fig. 9, O”-BG-resistant

activity was evident in at least three of the mice analyzed,

demonstrating that ada ATase was being expressed, albeit at



7.5 mg/kg BCNU

30 mg/kg 06 BG

100

10 mg/kg BCNU

30 mg/kg 06 BG

L1�

>

>

(1)

50

0-0 1020304050607080

1’ 1’

12.5 mg/kg BCNU30 mg/kg 06 BG

100

50ada

� neo

0 � � � �

0 1020304050607080

t t15 mg/kg BCNU

30 mg/kg 06 BG

�100

50

‘.

t

1.� I I I I,J �,

0 1020304050607080 0

1’

10 20 30 40

1’

50 60 70 80


Time (Days)

Fig. 8 Kaplin-Meien survival curves for mice exposed to 30 mg/kg 06-BG and various doses of BCNU. Arrows, days at which drugs were

administered, days 0 and 42. Each group contained seven mice. Curves for two groups (ada and neo) were compared using the exact Wilcoxon-Gehantest. A clean survival advantage was conferred by ada in the mice treated with 12.5 mg/kg BCNU (P 0.021), but there was no significant differencein the survival of the two groups at all other doses (7.5 mg/kg, P 0.44; 10 mg/kg, P = 0.93; 15 mg/kg, P = 0.92).

low levels, in WBCs after transduction and transplantation of

virally infected marrow cells. ATase cannot be measured in any

other hematopoietic cell compartment.

DISCUSSION

We have generated a high-titer netnoviral producer clone

that expresses high levels of the bacterial ATase gene, desig-

nated ada. This replication-defective retnovirus infects long-

lived munine hematopoetic progenitor cells, as demonstrated by

the persistence of ada gene sequences in peripheral blood and

by the generation of ada-positive secondary bone marrow re-

cipient mice. The functionally active, full-length Mr 39,000 ada

protein expressed by producer clone 18 was easily distinguished

from the smaller Mr 22,000 endogenous munine ATase ex-

pressed by GP+E86 cells (Fig. 2). The identity of the ada

protein was confirmed by its resistance to the mammalian

ATase inhibitor 06-BG (21). Southern blot analysis of genomic

DNA isolated from clone 18 and other producer clones (Fig. 3)

showed that the correct full-length proviral DNA had been

inserted into all clones analyzed, without gross rearrangements

on deletions.

The in vitro methylcellubose assay demonstrated that the

clone 18 retrovirus was able to infect hematopoietic progenitor

cells (Fig. 4). Functional expression of the ATase was indicated

by the 2-fold greaten resistance of clone 18 versus N2-infected

cells to BCNU-induced cytotoxicity. Because these cells were

only partially transduced (-25%), it is likely that the resistance

of the ada transduced component was greaten than 2-fold. It was

surprising that after exposure to BCNU alone, the ada ATase

provided no additional protection to cells beyond that provided

by endogenous ATase levels (7, 8). It is possible that the levels

of ada activity were so low that the total increase in DNA repair

capacity was too small to be significant. However, once the

munine enzyme was inhibited with 10 p.M 06-BG, the neo-

expressing cells became sensitized to BCNU, whereas the ada-

expressing cells retained their resistance, indicating that the

bacterial protein was functionally active in munine hematopoi-

etic cells. The 2-fold increase in resistance generated by clone

18 may have been greater if a higher proportion of cells ex-

pressed the gene. ada mRNA was detected in only two of eight

colonies exposed to the virus in vitro, suggesting that a low

proportion (-25%) of cells expressed the ada gene (Fig. 5).

However, this proportion increased to 100% following treatment

with 10 �LM O”-BG and 30 p.M BCNU, presumably due to

selection for ada-expressing cells containing functionally active

ATase encoded by the bacterial gene.



ada neo

12.5mg/kg

BCNUI 10mg/kg

Cl C2 C3 Vehicle BCNU

18 mer 4 -�--� . - - �-

8 mer -�

Vehicle


Fig. 9 ATase activity assay of peripheral WBCs isolated from mice 21 weeks after retroviral transduction. Lane Cl, unreacted double-stranded

O”-methybguanine-containing oligonucleotide 18-men substrate; Lane C2, the same oligonucleotide following incubation with P11411, demonstrating

that no cleavage occurs prior to repair of the O”-guanine adduct; Lane C3, incubation with a control producer cell extract-expressing ada. Remaining

lanes, extracts of peripheral blood leukocytes from surviving mice that were transduced with ada or neo and treated with vehicle or different doses

of BCNU as indicated in the legends to Figs. 7 and 8. All of the cell extracts were incubated with 50 p.M O”-BG for I h at 37#{176}Cto inactivate

endogenous mouse ATase prior to addition of the oligonucleotide substrate.

The ability of the ada ATase protein to protect hematopoi-

etic progenitor cells against BCNU in vitro prompted us to use

clone 18 to infect CB/CaJ mouse bone marrow. The modified

marrow was then injected into marrow-depleted recipients for

the study of ada protective effects in vivo. We chose thymec-

tomized CB/CaJ mice for our experiments because they have

been successfully used at this center in therapeutic studies with

human tumor xenografts (6). Our in vivo experiments revealed

that transduction of the ada gene into hematopoietic cells can

protect against nitnosounea-induced myebosupression. After the

first treatment of O”-BG and BCNU, all measured blood ele-

ments (platelets, enythnocytes, hematocnit, hemoglobin, and

WBCs) were significantly suppressed (Fig. 7), but there were

few deaths (Fig. 8). The rationale for giving a second treatment

after the blood values had returned to normal was that ada-

expressing cells might be selected during the first treatment, so

that marrow would become enriched with such cells and be

more resistant when challenged with a second dose. However,

validity of this approach could not be determined because of the

large number of animal deaths that occurred shortly after the

second treatment. Although some blood components were pro-

tected after both 12.5-mg/kg doses of BCNU in ada-positive

compared with neo-positive mice, only one of the seven animals

given the latter gene was alive on day 70, precluding a mean-

ingful statistical test of differences in blood profiles. Nonethe-

less, the fact that six times more ada-positive than neo-positive

mice survived this treatment cleanly indicates a protective effect

from the bacterial enzyme. The low frequency of deaths in both

treatment groups at doses below 12.5 mg/kg and the equally

high frequency at 15 mg/kg suggest a very narrow BCNU dose

range, over which the therapeutic benefits of ATase gene trans-

fen could be realized. This is consistent with the known steep

dose response for BCNU (44).

One might be able to enhance the protective effects of ada

gene transfer by increasing the level of gene expression. Assay

of ATase activity in transduced mouse peripheral blood leuko-

cytes demonstrated a relatively low level ofada expression (Fig.

9). The pattern of ada expression in these cells is probably

different from that in the rapidly proliferating stem cells from

which they were derived and that are the presumed target for

BCNU. The present data suggest that the erythnoid progenitor

cells from which RBCs and platelets are derived express a

higher level of ada ATase, because it was in these lineages that

protection was evident. Because the blood was taken for ATase

assay 21 weeks after transplantation, it is also possible that the

measured levels of ada expressed undernepresent the level of

expression at the time of drug treatment at weeks S and 1 1 after

reconstitution.

The question also arises as to whether the bacterial ada

ATase, with no nuclear localization signal, is able to accumulate

in the cell nucleus (19). Its ability to protect mammalian cells in

vitro against DNA damage (16-20) suggests that at least some

of the protein reaches the nucleus, but it is unknown how much

ATase activity measured in these cells is available for DNA

repair in the nucleus. The human ATase also lacks a recogniz-

able nuclear localization signal, yet it is localized there (45-47),

possibly as a result of its DNA binding properties. To address

these issues in future studies, we plan to use a mutant human

ATase that resists inhibition by O”-BG (48).

We have demonstrated that ada gene transfer to munine

hematopoietic cells confers protection against the toxic effects

of O”-BG plus BCNU, both in vitro and in viva. Such selective

protection of the bone marrow while retaining the capacity for

sensitizing tumor cells to BCNU with O”-BG provides a stnat-

egy for increasing the therapeutic index for BCNU.




ACKNOWLEDGMENTS

We thank Deepthi Jayawardene and the St. Jude Biostatistics

Department for help with statistical analysis. We also thank Pam

Cheshire and Joanna Remack for their excellent technical assistance,

John Gilbert for editing the manuscript, and Dr. Arthur W. Nienhuis for

his helpful comments and discussions.

REFERENCES

1. Pegg, A. E. Mammalian 06-alkylguanine-DNA alkyltransferase: reg-ulation and importance in response to alkylating carcinogenic and ther-

apeutic agents. Cancer Res., 50: 6119-6129, 1990.

2. Pegg, A. E., and Byers, T. L. Repair of DNA containing O”-abkyl-guanine. FASEB J., 6: 2302-2310, 1992.

3. Brent, T. P. Suppression of cross-link formation in chloroethylnitro-

sourea-treated DNA by an activity in extracts of human leukemic

bymphoblasts. Cancer Res., 44: 1887-1892, 1984.

4. Erickson, L. C., Bradley, M. 0., and Kohn, K. W. Measurements of

DNA damage in Chinese hamster cells treated with equitoxic andequimutagenic doses of nitrosoureas. Cancer Res., 38: 3379-3384,

1978.

5. Carter, S. K., Schabel, F. M., Bnoder, L. E., and Johnston, T. P.1,3-Bis(2-chboroethyl)-l-nitrosourea (BCNU) and other nitrosoureas incancer treatment: a review. Adv. Cancer Res., /6: 273-332, 1972.

6. Brent, T. P., Houghton, P. J., and Houghton, J. A. 06-alkylguanine-

DNA alkyltransferase activity correlates with the therapeutic response

of human rhabdomyosarcoma xenografts to 1-(2-chboroethyl)-3-(trans-4-methybcyclohexyl)-1 -nitrosourea. Proc. NatI. Acad. Sci. USA, 82:

2985-2989, 1985.

7. Gerson, S. L., Trey, J. E., Miller, K., and Bergen, N. B. Comparisonof 06-alkybguanine-DNA alkyltransferase activity based on cellularDNA content in human, rat and mouse tissues. Carcinogenesis, 7:

745-749, 1986.

8. Gerson, S. L., Trey, J. E., Miller, K., and Benjamin, E. Repair of06-abkylguanine during DNA synthesis in murine bone marrow hema-topoietic precursors. Cancer Res., 47: 89-95, 1987.

9. Philips, G. L., Wolff, S. N., Fay, J. W., Herzig, R. H., Lazarus, H. M.,

Schobd, C., and Herzig, G. P. Intensive 1,3-bis-(2-chloroethyl)-1-nitro-sourea (BCNU) monotherapy and autobogous bone marrow tnansplan-tation for malignant glioma. J. Clin. Oncob., 4: 639-645, 1986.

10. Sorrentino, B. P., Brandt, S. T., Bodine, D., Gottesman, M., Pastan,

I., Cline, A., and Nienhuis, A. W. Selection of drug-resistant bone

marrow cells in vito after retroviral transfer of human MDRI. Science

(Washington DC), 257: 99-103, 1992.

11. Williams, D. A., Hsieh, K., DeSilva, A., and Mulligan, R. C.Protection of bone marrow transplant recipients from lethal doses of

methotrexate by the generation of methotrexate-resistant bone marrow.J. Exp. Med., 166: 210-218, 1987.

12. Corey, C. A., DeSilva, A., Holland, C., and Williams, D. A. Serial

transplantation of methotrexate resistant bone marrow: protection of

murine recipients from drug toxicity by progeny of transduced stem

cells. Blood, 75: 337-343, 1990.

13. Li, M-X., Banerjee, D., Zhao, S-C., Schweitzer, B. I., Mineishi, S.,

Gilboa, E., and Bertino, J. R. Development of a retroviral construct

containing a human mutated dihydrofobate reductase eDNA for hema-

topoietic stem cell transduction. Blood, 83: 3403-3408, 1994.

14. Margison, G. P., Cooper, D. P., and Brennand, J. Cloning of the E.coli 06-methylguanine and methylphosphotniesten gene using a func-

tionab DNA repair assay. Nucleic Acids Res., /3: 1939-1952, 1985.

15. Olsson, M., and Lindahl, T. Repair of alkybated DNA in Esche-

richia coli: methyl group transfer from Ob�methybguanine to a protein

cysteine residue. J. Blob. Chem., 255: 1069-1071, 1980.

16. Brennand, J., and Margison, G. P. Reduction in the toxicity and

mutagenicity of alkylating agents in mammalian cells harbouning the E.coli alkyltransferase. Proc. NatI. Acad. Sci. USA, 83: 6292-6296, 1986.

17. Samson, L., Derfier, B., and Waldstein, E. A. Suppression of human

DNA alkybation repair defects by E. coli DNA repair genes. Proc. Natl.Acad. Sd. USA, 83: 5607-5610, 1986.

18. Kataoka, H., Hall, J., and Karran, P. Complementation of sensitivity

to alkylating agents in E. coli and Chinese hamster ovary cells byexpression of a cloned bacterial DNA repair gene. EMBO J., 5: 3195-

3200, 1986.

19. Dumenco, L. L., Warman, B., Hatzogbou, M., Lim, I. K., Abboud,S. L., and Gerson, S. L. Increase in nitrosourea resistance in mammalian

cells by retroviralby mediated gene transfer of bacterial 06-alkybgua-nine-DNA alkyltransferase. Cancer Res., 49: 6044-6051, 1989.

20. Jelinek, J., Kleibl, K., Dexter, T. M., and Mangison, G. P. Trans-

fection of munine multi-potent haemopoietic stem cells with an E. coliDNA alkyltransferase gene confers resistance to the toxic effects ofalkylating agents. Cancinogenesis, 9: 81-87, 1988.

21. Doban, M. E., Pegg, A. E., Dumenco, L. L., Moschel, R. C., and

Gerson, S. L. Comparison of the inactivation of mammalian and bac-terial Ob�alkylguanine�DNA alkyltransferase by 06-benzybguanine and

06-methylguanine. Carcinogenesis, 12: 2305-2309, 1991.

22. Crone, T. M., and Pegg, A. E. A single amino acid change in human

06-alkybguanine-DNA-alkyltransfenase decreases sensitivity to inactiva-tion by 06-benzylguanine. Cancer Res., 53: 4750-4753, 1993.

23. Dolan, M. E., Stine, L., Mitchell, R. B., Moschel, R. C., and Pegg,

A. E. Modulation of mammalian 06-alkylguanine-DNA alkyltransferasein vivo by 06-benzybguanine and its effects on the sensitivity of a human

glioma tumor to 1-(2-chloroethyl)-3-(4-methylcycbohexyl)-1-nitno-

sourea. Cancer Commun., 2: 371-377, 1990.

24. Mitchell, R. B., Moscheb, R. C., and Dolan, M. E. Effect of

O�’-benzylguanine on the sensitivity of human tumor xenografts to1,3-bis(2-chboroethyl)-1-nitrosourea and on DNA interstrand cross-linkformation. Cancer Res., 52: 1171-1175, 1992.

25. Doban, M. E., Pegg, A. E., Biser, N. D., Moschel, R. C., and

English, H. F. Effect of 06-benzybguanine on the response to 1,3-bis(2-

chboroethyl)-l-nitrosourea in the Dunning R3327G model of prostaticcancer. Cancer Chemothen. Pharmacol., 32: 221-225, 1993.

26. Felker, G. M., Friedman, H. S., Dolan, M. E., Moschel, R. C., and

Schold, C. Treatment of subcutaneous and intracranial brain tumorxenografts with 06-benzylguanine and l,3-bis(2-chloroethyb)-1-nitro-sourea. Cancer Chemothen. Pharmacob., 32: 471-476, 1993.

27. Brennand, J., and Margison, G. P. Expression of the E. coli O�-methybguanine-methylphosphotniester methyltransfenase gene in mam-malian cells. Carcinogenesis, 7: 185-188, 1986.

28. McLachbin, J. R., Mittereder, N., Daucher, M. B., Kadan, M., andEglitis, M. A. Factors affecting retroviral function and structural integ-

nity. Virology, 195: 1-5, 1993.

29. Bodine, D. M., Moritz, T., Donahue, R. E., Luskey, B. D., Kessler,

S. W., Martin, D. I. K., Onkin, S. H., Nienhuis, A. W., and Williams,D. A. Long-term in vivo expression of a munine adenosine deaminasegene in Rhesus monkey hematopoietic cells of multiple lineages afterretroviral mediated gene transfer into CD34� bone marrow cells. Blood,

82: 1975-1980, 1993.

30. Armentano, D., Yu, S-F., Kantoff, P. W., von Ruden, T., Anderson,

W. F., and Gibboa, E. Effect of internal viral sequences on the utility of

retroviral vectors. J. Vinol., 61: 1647-1650, 1987.

31. Markowitz, D, Goff, S., and Bank, A. A safe packaging line forgene transfer separating viral genes on two different plasmids. J. Virob.,

62: 1120-1124, 1988.

32. Miller, A. D., and Buttimore, C. Redesign of retrovirus packaging

cell lines to avoid recombination leading to helper virus production.Mol. Cell. Biol., 6: 2895-2902, 1986.

33. Chen, C., Okayama, H. High efficiency transformation of mamma-

han cells by plasmid DNA. Mob. Cell. Biob., 7: 2745-2752, 1987.

34. Sambrook, J., Fnitsch, E. F., and Maniatis, T. Molecular Cloning: A

Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor

Laboratory, 1989.

35. Brent, T. P. Isolation and purification of 06-alkylguanine-DNAalkyltransferase from human leukemic cells. Prevention of chboroeth-



1368 Alkyltransfenase Gene Expression in Munine Bone Marrow

ylnitrosoures-induced cross-links by purified enzyme. Phanmacol. &

Ther., 31: 121-140, 1985.

36. Wu, R. S., Hust-Calderone, S., and Kohn, K. W. Measuement of06-alkylguanine-DNA alkyltransferase activity in human cells and tu-

mon tissue by restriction endonuclease inhibition. Cancer Res., 47:

6229-6235, 1987.

37. Marathi, U. K., Dolan, M. E., and Erickson, L. C. Extended deple-tion of O�-methylguanine-DNA methyltransferase activity following06-benzyl-2’-deoxyguanosine or 06-benzylguanine combined with

streptozotocin treatment enhances l,3-bis(2-chloroethyl)-1-nitrosouneacytotoxicity. Cancer Res., 54: 4371-4375, 1994.

38. Bodine, D. M., McDonagh, K. T., Seidel, N. E., and Nienhuis,A. W. Survival and retrovirus infection of murine hematopoietic stemcells in vitro: effects of 5-FU and method of infection. Exp. Hematol.,19: 206-212, 1991.

39. Bodine, D. M., Karlsson, S., and Nienhuis, A. W. Combination ofinterleukins 3 and 6 preserves stem cell function in culture and enhances

netrovirus-mediated gene transfer into hematopoietic stem cells. Proc.

NatI. Acad. Sci. USA, 86: 8897-8901, 1989.

40. Crowder, M. J., and Hand, D. J. Analysis of Repeated Measures.New York: Chapman and Hall, 1991.

41. W. J. Dixon (ed.). BMDP Statistical Software Manual. Los Ange-les: University of California Press, 1992.

42. Kaplan, E. L., and Meien, P. Nonparametnic estimation from incom-plete observations. J. Am. Statist. Assoc., 53: 457-481, 1958.

43. StatXact Statistical Software for Exact Nonparametnic Inference.Cambridge, MA: Cytel Software Corp., 1992.

44. Mitchell, R. B., Moschel, R. C., and Dolan, M. E. Effect of06-benzylguanine on the sensitivity of human tumor xenognafts to

1,3-bis(2-chloroethyl)-1-nitrosourea and on intenstrand cross-link for-mation. Cancer Res., 52: 1171-1175, 1992.

45. Lee, S. M., Rafferty, J. A., Elder, R. H., Fan, C-Y., Bromley, M.,Harris, M., Thatcher, N., Potter, P. M., Altermatt, H. J., Peninat-Frey, T.,Cemy, T., O’Connor, P. J., and Mangison, 0. P. Immunohistologicalexamination of the inter- and intracellular distribution of 06-alkylgua-nine-DNA alkyltnansferase in human liver and melanoma. Br. J. Cancer,66: 355-360, 1992.

46. Aya, T. C., Loh, K. C., Ali, R. B., and Li, B. F. L. Intracellularlocalization of human DNA repair enzyme methylguanine-DNA meth-yltransferase by antibodies and its importance. Cancer Res., 52: 6423-

6430, 1992.

47. Brent, T. P., von Wnonski, M. A., Edwards, C. C., Bromley, M.,Margison, 0. P., Rafferty, J. A., Pegnam, C. N., and Bigner, D. D.Identification of nitrosourea-nesistant human rhabdomyosarcomas by in

situ immunostaining of O�-methylguanine-DNA methyltransfenase. On-cob. Res., 5: 83-86, 1993.

48. Crone, T. M., Goodtzova, K., Edara, S., and Pegg, A. E. Mutationsin human 06-alkylguanine-DNA alkyltransfenase imparting resistance to

O�-benzylguanine. Cancer Res., 54: 6221-6227, 1994.



1995;1:1359-1368. Clin Cancer Res L C Harris, U K Marathi, C C Edwards, et al. cytotoxicity.murine bone marrow protects against chloroethylnitrosourea Retroviral transfer of a bacterial alkyltransferase gene into

Updated version

http://clincancerres.aacrjournals.org/content/1/11/1359

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/1/11/1359To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



retroviral transfer of a bacterial alkyltransferase gene into … · g418 selection of infected nih...

Documents