Proc. Natl. Acad. Sci.
USA
Vol. 93, pp. 206-210, January 1996
Genetics
Increasing DNA repair methyltransferase levels via bone marrow
stem cell transduction rescues mice from the toxic effects of
1,3-bis(2-chloroethyl)-1-nitrosourea, a chemotherapeutic
alkylating agent
(chloroethylnitrosourea/retroviral vector/DNA repair/06-methylguanine DNA methyltransferase)
RODNEY MAZE*t, JAMES P. CARNEYt, MARK R. KELLEY*t, BRIAN J. GLASSNER§, DAVID A. WILLIAMS*t1II,
ANDLEONA SAMSON§
*Department of Pediatrics, Herman B. Wells Center for Pediatric Research, James Whitcomb Riley Hospital for Children, Departments of tMedical and
Molecular Genetics and tBiochemistry and Molecular Biology, and IHoward Hughes Medical Institute, Indiana University School of Medicine,
Indianapolis, IN 46202-2552; and §Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, MA 02115
Communicated by Elkan Blout, Harvard Medical School, Boston, MA, September 22, 1995
ABSTRACT The chloroethylnitrosourea (CNU) alkylat-
ing agents are commonly used for cancer chemotherapy, but
their usefulness is limited by severe bone marrow toxicity that
causes the cumulative depletion of all hematopoietic lineages
(pancytopenia). Bone marrow CNU sensitivity is probably due
to the inefficient repair of CNU-induced DNA damage; relative
to other tissues, bone marrow cells express extremely low
levels of the 06-methylguanine DNA methyltransferase
(MGMT) protein that repairs cytotoxic 06-chloroethylgua-
nine DNA lesions. Using a simplified recombinant retroviral
vector expressing the human MGMT gene under control of the
phosphoglycerate kinase promoter (PGK-MGMT) we in-
creased the capacity of murine bone marrow-derived cells to
repair CNU-induced DNA damage. Stable reconstitution of
mouse bone marrow with genetically modified, MGMT-
expressing hematopoietic stem cells conferred considerable
resistance to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-
nitrosourea (BCNU), a CNU commonly used for chemother-
apy. Bone marrow harvested from mice transplanted with
PGK-MGMT-transduced cells showed extensive in vitro
BCNU resistance. Moreover, MGMT expression in mouse
bone marrow conferred in vivo resistance to BCNU-induced
pancytopenia and significantly reduced BCNU-induced mor-
tality due to bone marrow hypoplasia. These data demonstrate
that increased DNA alkylation repair in primitive hematopoi-
etic stem cells confers multilineage protection from the my-
elosuppressive effects of BCNU and suggest a possible ap-
proach to protecting cancer patients from CNU chemothera-
py-related toxicity.
The chloroethylnitrosourea (CNU) chemotherapeutic alkylat-
ing agents have been used to treat certain kinds of cancer for
over two decades (1), and 1,3-bis(2-chloroethyl)-1-nitrosourea
(BCNU, carmoustine) is typical of this class of chemothera-
peutic drug. BCNU is particularly effective in treating child-
hood and adult glial tumors (2, 3) and has been used in
high-dose chemotherapy for lymphomas, breast, lung, and
gastrointestinal cancers (1, 4). Unfortunately, the clinical
success of the CNUs has been limited by their severe bone
marrow and lung toxicities. Myelosuppression is characteris-
tically delayed following CNU treatment and can lead to
severe, prolonged, and cumulative pancytopenia (3). The
extreme sensitivity of bone marrow to CNUs may be explained
by the inability of this tissue to repair CNU-induced cytotoxic
DNA damage efficiently (5).
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
206
CNU alkylation produces numerous DNA base modifica-
tions plus DNA inter- and intrastrand crosslinks. Interstrand
crosslinks are particularly cytotoxic because they grossly in-
terfere with DNA replication; their formation can be initiated
by a chloroethyl group at the 06 position of guanine which
slowly rearranges to form an ethyl bridge between NI of
guanine and N3 of cytosine in the opposite strand (6). The
formation of these cytotoxic DNA interstrand crosslinks can
be prevented by transfer of the 06-chloroethyl group to the
06-methylguanine DNA methyltransferase (MGMT) protein
prior to crosslink formation (7-10). MGMT levels in mam-
malian cells thus correlate with resistance to CNU-induced
cytotoxicity (7, 11, 12).
Human and murine bone marrow cells express extremely
low MGMT levels relative to other tissues (5). Increasing
MGMT expression in bone marrow might therefore decrease
that tissue's sensitivity to CNU-induced cytotoxicity. Indeed,
we recently demonstrated that transduction of murine bone
marrow progenitor cells with a retroviral vector expressing
the human MGMT cDNA provided a moderate level of
protection from the peripheral pancytopenia induced by
repeated BCNU treatments (12). However, protection was
quite modest and we now propose that in order to produce
a more useful level of BCNU protection, it is critical to
express MGMT in hematopoietic stem cells, because the
cumulative and delayed nature of the BCNU-induced bone
marrow toxicity may be related to damage in the stem cell
compartment (13-15). Here we show that expression of the
human MGMT DNA repair protein by a transduction pro-
tocol which targets murine bone marrow stem cells confers
considerable resistance to BCNU-induced toxicity both in
vitro and in vivo.
MATERIALS AND METHODS
Recombinant Retroviral Vector and Packaging Cell Lines.
The human MGMT cDNA was cloned under control of the
phosphoglycerate kinase (PGK) promoter in the N2/Zip retro-
viral construct as described (12) to produce N2/ZipPGK-MGMT
(hereafter called PGK-MGMT). A high-titer producer clone of
PGK-MGMT in GP+E-86 packaging cells (16), OMG-9, was
previously described (12) and was used throughout this study.
Abbreviations: CNU, chloroethylnitrosourea; BCNU, 1,3,-bis(2-chloro-
ethyl)-1-nitrosourea; 06-MeG, 06-methylguanine; MGMT, 06-methyl-
guanine DNA methyltransferase; PGK, phosphoglycerate kinase; CPC,
committed progenitor cell(s); HPP-CFC, high-proliferative-potential
colony-forming cell(s).
IlTo whom reprint requests should be sent at the * address.
 Genetics: Maze et al.
Infection of Bone Marrow Cells, Transplantation, and
BCNU Treatment of Recipient Mice. Bone marrow cells were
harvested from the hind limbs of 8- to 10-week-old female
C57BL/6J mice (The Jackson Laboratory) 48 hr after intra-
peritoneal injection with 5-fluorouracil (5-FU2d, 150 mg/kg of
body weight, SoloPak Laboratories, Franklin Park, IL) (17).
Harvested bone marrow cells were prestimulated (18) and
infected by coculture on confluent, mitomycin C-treated
GP+E-86 cells (mock-infected control) or OMG-9 cells (18).
After cocultivation, nonadherent hematopoietic cells were
harvested and 106 cells were injected into lethally irradiated
(139Cs source, 11 Gy, split dose, with a minimum of 3 hr
between doses; Nordion International, Kanata, ON, Canada)
recipient 8- to 10-week-old female C57BL/6J mice. Three
weeks after transplantation, mice were treated with weekly
intraperitoneal injections of BCNU at 40 mg/kg of body
weight, a regimen previously shown to induce lethal pancyto-
penia with prolonged (10 weeks) treatment (15).
Peripheral Blood Analysis. Total peripheral blood leuko-
cyte and platelet counts and hematocrits were analyzed on tail
vein bleeds (12).
Analysis of BCNU Resistance of Transduced Committed
Progenitor Cells (CPC) and High-Proliferative-Potential Col-
ony-Forming Cells (HPP-CFC). Bone marrow and spleen
hematopoietic cellularity was determined by standard methods
(19), and CNU sensitivity of CPC and HPP-CFC was deter-
mined as described (12, 15, 20).
MGMT DNA Repair Assay. MGMT levels in bone marrow
and spleen cell extracts (50 ,ug of protein) were determined
using a 32P-labeled 18-bp oligonucleotide (a gift of Russell 0.
Pieper, Loyola University Medical Center) containing an
06-methylguanine (06-MeG) base within a methylation-
sensitive Pvu II restriction site (21); 06-MeG repair allows Pvu
II digestion, which produces a labeled 8-bp fragment.
Western Blot Analysis of MGMT Expression. Human
MGMT protein was detected with polyclonal antibodies (anti-
MAP1) (22) against human but not murine MGMT (kindly
supplied by Anthony Pegg, Pennsylvania State University
College of Medicine, College Park). Extracts were prepared
from 2-5 x 106 cells (collected on day 35 after bone marrow
transplant), and the proteins were separated by SDS/PAGE,
blotted, probed with 1:500 antiserum, and developed by en-
hanced chemiluminescence (ECL kit, Amersham).
tn
E
._
I-
(9 Control
:3
CD-MGMTBM ,, -BM
I: uw0 1 2 3 4 5 1 2 3
A
CD)cn
E
L-
(D
D co
r-MGMTBM rMGMTSpI
I LU 0 1 2 3 4 5 6 1 2 3 4 5 6
B
*a........
FIG. 1. DNA analysis of recipient mice for integrated PGK-MGMT provirus 10 weeks (A) and 18 weeks (B) after transplantation. Bone marrow
(BM), spleen (Spl), and thymus (Thy) DNA was purified from mice 10 or 18 weeks after bone marrow transplantation with PGK-MGMT-transduced
or mock-infected (control) bone marrow. HuMGMT primers, human MGMT-speclfic primers alone; E86, control producer cells (negative control);
OMG-9, MGMT producer cells (positive control); all other lanes in A and B represent DNA from individual mice. Arrow denotes the predicted
210-bp human MGMT PCR product.
Proc. Natl. Acad. Sci. USA 93 (1996) 207
PCR and Southern Blot Analysis. DNA was prepared as
described (23) for amplification with a PCR kit (Perkin-
Elmer/Cetus) and Taq DNA polymerase. The 5' and 3'
oligonucleotide primers were based on the human MGMT
cDNA sequence (24) and amplify a unit of 210 bp. PCR products
were electrophoresed through a 1.5% agarose gel, blotted, and
hybridized to a 600-bp 32P-labeled BamHI-Sal I fragment of the
human MGMT cDNA (24).
Statistical Analysis. Three plates were scored for each CPC
and HPP-CFC sample. Each mouse was evaluated separately.
Results are expressed as a mean ± SEM derived from the
averages of each individual mouse within a group. The prob-
ability of significant differences between groups was deter-
mined by Student's t test (two-tailed).
RESULTS
We set out to express the human MGMT alkylation repair
protein in primitive hematopoietic stem cells, with the aim
of conferring resistance to the myelosuppressive effects of
BCNU. Murine bone marrow cells were infected with the
PGK-MGMT retrovirus by a protocol previously demon-
strated to transduce reconstituting murine hematopoietic
stem cells at high frequency (18, 25). The transduced bone
marrow cells were transplanted into lethally irradiated re-
cipient mice. Three weeks after transplantation the recipient
mice were administered weekly doses of BCNU at 40 mg/kg,
and treatment was continued for 5 weeks. The transplanted
mice were monitored for integration and expression of the
PGK-MGMT retrovirus, for in vivo and in vitro resistance to
BCNU-induced cytotoxicity among different cell types, and
for BCNU-induced mortality.
PGK-MGMT Retrovirus Is Present in Hematopoietic Tis-
sues up to 18 Weeks After Transplantation. Mice were
analyzed by PCR and Southern blot for the presence of the
human MGMT cDNA at 10 and 18 weeks after bone marrow
transplantation (Fig. 1). Bone marrow, spleen, and thymus
DNA from transplanted mice was subjected to PCR ampli-
fication with human MGMT-specific primers and the prod-
ucts were analyzed on Southern blots probed with a labeled
fragment of the human MGMT cDNA. Ten weeks after
transplantation, bone marrow DNA from four of five mice
transplanted with PGK-MGMT-transduced bone marrow
Control
r MGMT SpI r Spl,ii MGMT Thy-,
1 2 3 4 1 2 3 1 2 3 4 5
208 Genetics: Maze et al.
was positive for the human MGMT sequence (Fig. 1A). At
the same time point, spleen DNA from four of four mice and
thymus DNA from five of five mice were also positive,
indicating that hematopoietic progenitor cells in both the
myeloid and lymphoid lineages were successfully transduced.
Bone marrow DNA and spleen DNA from mice transplanted
with mock-infected bone marrow were negative for the
human MGMT sequence (Fig. 1A). At 18 weeks after
transplantation, bone marrow DNA from four of six mice
and spleen DNA from three of six mice were positive for the
human MGMT sequence (Fig. 1B). Thymic DNA was not
examined at this time point. Bone marrow reconstitution is
considered stable in mice if donor hematopoietic cells are
present 16 weeks after transplantation (26); the results in
Fig. 1B thus confirm that some mice were transplanted with
PGK-MGMT-transduced cells capable of long-term, stable
hematopoietic reconstitution.
Human MGMT Protein and Increased 06-MeG Repair
Activity in Hematopoietic Tissues of Recipient Mice. Using
antiserum that specifically binds the human (but not mouse)
MGMT protein (22), we demonstrated that human MGMT
was expressed in bone marrow in three of three PGK-MGMT-
transplanted mice and also in spleen in two of these mice; both
bone marrow and spleen from three of three control mice were
negative for the human MGMT protein (Fig. 2). Further,
MGMT activity was higher in hematopoietic cells from mice
transplanted with PGK-MGMT-transduced bone marrow
compared with the same tissues from control mice (Fig. 3). The
methyltransferase assay is based upon the ability of 06-MeG
located in a Pvu II site to inhibit Pvu II digestion; digestion thus
reflects 06-MeG repair (21). Methyltransferase activity was
assayed 3 days after a second BCNU treatment (day 35 after
bone marrow transplantation) and was higher in bone marrow
and spleen cell extracts of mice transplanted with PGK-
MGMT-transduced bone marrow (Fig. 3, lanes 9-14) com-
pared with extracts from control mice (lanes 3-8). The spleen
cell extract from one control mouse (lane 4) had a higher
MGMT activity compared with the other control animals.
However, the average methyltransferase activity was lower in
control tissues than in tissues derived from mice transplanted
with PGK-MGMT-transduced bone marrow (Table 1). No
effect on the endogenous level of MGMT DNA repair activity
was detected after two weekly doses of BCNU (Fig. 3, lanes 15
and 16).
Bone Marrow Cells Removed from PGK-MGMT-Trans-
planted Mice Display BCNU Resistance in Vitro. Bone marrow
cells were harvested from eight mice transplanted with mock-
infected bone marrow and eight mice transplanted with PGK-
MGMT-transduced bone marrow; for both groups, cells were
harvested after the fifth BCNU dose (56 days after transplan-
tation). The harvested bone marrow cells were challenged with
Bone Marrow Spleen
MOCkMGMT MMc MG
MOCk MGMT
;;~~_*-r ;4;- -MGMT
-21.5kDa
1 2 3 45 6 1 2 3 4 56
FIG. 2. Western analysis of =20 ,ug of total protein from bone
marrow and spleen cell extracts of mice transplanted with mock- and
PGK-MGMT-transduced bone marrow, probed with human MGMT-
specific antibodies. Lanes 1-3, mock-infected; lanes 4-6, PGK-
MGMT transduced. Negative control lane (-) contained 100 ,ug of
total protein from GP+E-86 mouse fibroblast packaging cell extract.
Positive (+) control lane contained 100 jig of total protein from
human Mer+ HeLa S3 cell extract. The control lanes have higher
background and hence a shorter exposure is shown for these samples.
All other lanes were developed and exposed simultaneously. The
positions of the MGMT protein and molecular size standards are
indicated at right.
Proc. Natl. Acad. Sci. USA 93 (1996)
Mock- Control MGMT-
F Recipients 17 Recipients 71
* ..#.. . * *. ,*.~~~~~Q .4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
FIG. 3. MGMT DNA repair activity in recipient mice after trans-
plantation with PGK-MGMT-transduced or mock-infected bone mar-
row. Bone marrow (BM) and spleen cell (Spl) extracts were analyzed
for the ability to demethylate an 06-methylated guanine base located
in a methylation-sensitive Pvu II restriction enzyme site contained
within an 18-bp oligonucleotide. The oligonucleotide (0.2 pmol, added
in excess) was exposed to cell extracts containing 50 Ag of total protein.
Lane 1, oligonucleotide; lane 2, OMG-9 producer cells; lanes 3-8,
bone marrow and spleen from three mock-infected recipient mice;
lanes 9-14, bone marrow and spleen from three PGK-MGMT-
transduced recipient mice; lane 15, bone marrow from nontrans-
planted C57BL/6J mouse treated with BCNU; lane 16, bone marrow
from untreated C57BL/6J mouse.
BCNU in vitro and the survival of both HPP-CFC and CPC was
measured. Bone marrow cells from the PGK-MGMT trans-
planted mice were considerably more BCNU-resistant than
those from control mice, and the resistance was seen in CPC
as well as the more primitive HPP-CFC (Fig. 4).
PGK-MGMT-Transduced Bone Marrow Cells and Their
Progeny Are Resistant to BCNU Exposure in Vivo. Repeated
BCNU treatment leads to severe and cumulative pancytopenia
in both mice and humans (3, 15). Hematocrits and total
peripheral leukocyte and platelet counts were used as a
measure of bone marrow cytotoxicity induced during 5 weeks
of repeated BCNU treatments. Peripheral blood leukocytes
and hematocrits were similar in the control and PGK-MGMT
mice prior to BCNU treatment, but platelet counts were
significantly higher in control mice than in PGK-MGMT mice;
the reason for this discrepancy is unclear. BCNU-induced
pancytopenia was less severe in mice expressing the human
MGMT protein in their hematopoietic tissues, compared with
control mice (Fig. 5). Although platelet counts were initially
slightly lower in the PGK-MGMT mice than in the control
mice, by 1 week after the first BCNU treatment the platelet
counts were significantly higher in the PGK-MGMT mice than
in the control mice. The difference between PGK-MGMT and
control mice for total leukocyte counts and hematocrits
reached significance by 3 weeks of BCNU treatment and
remained significant at 5 weeks. Indeed, after 5 weeks of
BCNU treatment, when control mice were clearly pancytope-
nic (Fig. 5), the PGK-MGMT mice displayed platelet counts
and hematocrits that were not significantly different from
those of nontransplanted, untreated mice (P > 0.05) and
leukocyte counts that were only reduced by 50% compared
with the same mice (data not shown). The cellularity of the
bone marrow and spleen provides another in vivo measure of
BCNU-induced cytotoxicity. Bone marrow and spleen cellu-
larities after five weekly BCNU doses (8 weeks after trans-
plantation), and the cellularities in untreated, nontransplanted
Table 1. Analysis of MGMT DNA repair activity in recipient mice
Activity, % conversion of
18-bp band to 8-bp band
Tissue Control MGMT
Bone marrow 20.0 + 3.1 51.0 + 9.9
Spleen 19.3 ± 12.6 60.3 ± 5.8
Values are mean ± SEM (n = 3).
 Genetics: Maze et al. Proc. Natl. Acad. Sci. USA 93 (1996) 209

HPP-CFC Table 2. Analysis of hematopoietic cellularity in mice after

BCNU treatment
Cell no. x 10-6
BCNU-treated
Untreated Control PGK-MGMT
Bone marrow 22.7 ± 1.2 2.47 ± 0.3 9.66 ± 2.2**
Spleen 63.3 ± 3.5 26.7 ± 11.1 102.6 + 25.3*
Values are mean ± SEM [n = 8 femurs (bone marrow) or spleens
at week 5 of BCNU treatment).
*P < 0.01 vs. BCNU-treated control.
0.1 4-W1 0.1 t **P < 0.05 vs. BCNU-treated control.

A B gous bone marrow transplantation is commonly used to protect

patients against the effects of cancer chemotherapies that are
0.01-0 20 40 60 .

BCNU, ,uM
80 0.01
0 20 40 60 80
BCNU,
FIG. 4. BCNU survival curves of bone marrow harvested from
mice transplanted with PGK-MGMT-transduced (0) or mock-infected
control (a) bone marrow. Curves show HPP-CFC (A) and CPC (B)
survival after BCNU exposure. BCNU resistance was evaluated as
described in Materials and Methods. Each point represents assays done
in triplicate for HPP-CFC and CPC for eight mice per group from two
separate experiments; each animal was evaluated separately and the
error bars represent SEM.
mice, are shown in Table 2. Bone marrow and spleen cells
clearly survived repeated BCNU treatments better in mice
expressing the human MGMT protein than in control mice.
Moreover, the bone marrow cellularity in BCNU-treated
PGK-MGMT mice was only moderately reduced (by 50%)
versus untreated mice, and the spleen cellularity was not
reduced at all (increased spleen size is frequently seen in mice
responding to hematopoietic stress).
PGK-MGMT-Transduced Bone Marrow Rescues Recipient
Mice from BCNU-Associated Mortality. Weekly BCNU treat-
ment in mice produces significant mortality, presumably due to
BCNU-related pancytopenia (15). However, increasing
MGMT activity in murine hematopoietic cells (by expressing
the human MGMT protein) confers significant protection
against BCNU-induced mortality. Fig. 6 represents data from
three separate experiments; mortality after 5 weeks was sig-
nificantly reduced in the PGK-MGMT mice versus control
mice; 23 of 25 (92%) PGK-MGMT mice survived the repeated
BCNU treatments, whereas only 19 of 36 (53%) control mice
survived the same treatment (P < 0.001).
DISCUSSION
Hematopoietic cells present a particularly amenable target for
somatic gene therapy because these cells are accessible, he-
matopoiesis is well understood, and procedures for bone
marrow transplantation are well established (27-29). Autolo-
7 12-
6-
5.
x 4-
0 6-
3.
0z
,LM
particularly toxic to the bone marrow (30, 31) and it seems
reasonable to expect that the in vitro transfer of appropriate
genes into harvested bone marrow cells (prior to transplanta-
tion) could generate bone marrow that is resistant to further
cancer chemotherapy (after transplantation) (28). This ap-
proach has been attempted previously with dihydrofolate
reductase and multidrug-resistance cDNAs (32, 33). Using a
murine model, we have shown that transfer of a DNA alkyla-
tion repair gene into bone marrow stem cells provides con-
siderable resistance to subsequent treatments with the com-
monly used chemotherapeutic alkylating agent BCNU.
BCNU produces 06-chloroethylguanine DNA adducts that,
if left unrepaired by the MGMT repair protein, produce highly
cytotoxic DNA interstrand crosslinks (6, 34). Expression of the
MGMT repair protein varies by as much as 100-fold between
different tissues, being highest in liver and lowest in bone
marrow (5, 12, 35). However, the reasons underlying this tissue
variation are not clear. We demonstrate here that the extreme
sensitivity of hematopoietic tissues to alkylating agents is due,
at least in part, to their low methyltransferase levels, because
increasing the methyltransferase levels in these cells conferred
considerable BCNU resistance. MGMT expression provided
sustained protection against severe peripheral pancytopenia, a
clinically important side effect normally associated with
BCNU chemotherapy. While it is quite clear that increasing
the MGMT levels in hematopoietic tissues confers alkylation
resistance, it is not yet clear whether very long-term high-level
MGMT expression in a tissue that is normally relatively
MGMT-deficient will have any unexpected detrimental ef-
fects.
We previously demonstrated that MGMT expression in
hematopoietic progenitor cells conferred a somewhat modest
level of protection against BCNU-induced peripheral pancy-
topenia (12). It is important to note that in this study we
expressed the methyltransferase in more primitive and longer-
lived hematopoietic cells and that expression in these cells (and
their progeny) conferred a more substantial level of protection
50T
I
-E 10-
In
404-
6 8-
0. 30.
x
6 CZ
20
41
-'

co 2. r_
coe
4- 1)
0 , B C
0- // II 0- h#
-13 0 1 2 3 4 5 -3 0 1 2 3 4 5 -13 0 1 2 3 4 5
BMT BCNU, weeks BMT BCNU, weeks BMT BCNU, weeks
FIG. 5. Analysis of peripheral blood leukocyte counts (A), platelet counts (B), and hematocrits (C) in mice that received PGK-MGMT-
transduced bone marrow. Each point represents the mean + SEM from three separate experiments with 10-15 mice per group. BMT, time of bone
marrow transplant. *, P < 0.001; **, P < 0.05 for MGMT vs. vehicle control.
210 Genetics: Maze et al.
100 - Toxicology Scholar. B.J.G. was supported by National Institutes of
90 - : | ,__, MGMT
,- (23/25)
804 _ ,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
70t
60±
' Mock
- Infected
50 - Control
(19/36)
4 T 7
0 7 14 28 35
BCNU treatment, days
FIG. 6. Survival curves of mice treated with weekly doses of BCNU.
Data represent three separate experiments. Numbers in parentheses give
the number of animals surviving/total animals per group.
against BCNU toxicity. The phenotypic characterization and
purification of stem cells from normal human bone marrow by
use of immunologic and functional characteristics have been
reported (ref. 36; reviewed in ref. 37), presenting the possibility
that, ultimately, we could express the MGMT repair protein
(or other repair proteins) in these cells, thus ensuring expres-
sion in all the human hematopoietic cell lineages. Although
gene transfer protocols in human cells are still inefficient, it has
been shown that bone marrow cells harvested from cancer
patients for autologous bone marrow transplantation (and
marked with the neomycin phosphotransferase gene) consis-
tently contribute to long-term multilineage recovery of hema-
topoiesis (38). It therefore seems likely that one could suc-
cessfully express MGMT in human bone marrow cells being
used for autologous transplantation and subsequently protect
patients against chemotherapy-induced hematotoxicity. In-
deed, modification of the retroviral transduction protocol has
led to efficient transduction of human primitive long-term-
culture-initiating cells by a clinically applicable protocol (39).
The increased expression of DNA repair methyltransferase
activity in bone marrow cells may be expected to provide
resistance to a wide variety of chromotherapeutic alkylating
agents in addition to BCNU; e.g., cyclophosphamide, melpha-
lan, chlorambucil, streptozotocin, procarbazine, dacarbazine,
and busulphan are all expected to produce cytotoxic 06-alkyl-
guanine DNA adducts that could be repaired by the MGMT
protein (4). Furthermore, all of these agents are expected to
produce 3-alkyladenine DNA adducts that may be subject to
repair by 3-methyladenine DNA glycosylases (11, 34, 40, 41)
and we recently found that 3-methyladenine levels are ex-
tremely low in mouse bone marrow (B.J.G. and L.S., unpub-
lished). Thus, the increased expression of 3-methyladenine
DNA glycosylase in bone marrow stem cells may also provide
resistance to the toxic effects of these chemotherapeutic
alkylating agents. Indeed, with the development of retroviral
polycistronic gene expression vectors (42), it may be possible
to simultaneously express the MGMT and 3-methyladenine
DNA glycosylase repair proteins in alkylation repair-deficient
bone marrow cells, thus providing an even higher level of
protection against alkylation-induced cytotoxicity.
R.M., D.A.W., and L.S. are supported by National Cancer Institute
Program Project Grant SPOl CA59348. L.S. is supported by National
Cancer Institute Grant CA55042 and is a Burroughs Wellcome
Proc. Natl. Acad. Sci. USA 93 (1996)
Health Training Grant 5T32 CA09078-19.
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