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Experimental Therapeutics |
Howard Hughes Medical Institute, Section of Pediatric Hematology/Oncology [S. R., D. A. W.], Department of Pediatrics, Herman No. Wells Center for Pediatric Research [S. R., J. B., M. D., R. C., S. C., D. A. W.], and Division of Biostatistics, Department of Medicine [R. S.], Indiana University School of Medicine, Indianapolis, Indiana 46202, and Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 [M. X-W., A. E. P.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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8–12% transduced cells; no treatment, 6% transduced cells;
BCNU only, 51% transduced cells; 6-BG/BCNU, 93% transduced
cells). To determine whether this selection occurred at the stem
cell level, bone marrow from each treatment group was infused into
secondary recipients. Whereas animals that received bone marrow from
untreated animals reconstituted with 2% transduced cells, animals
receiving marrow from 6-BG/BCNU-treated animals reconstituted with 94%
transduced cells, demonstrating nearly complete selection for stem
cells in the primary animals. Mice reconstituted with marrow from
animals treated with BCNU only demonstrated 23% transduced cells,
consistent with partial selection of stem cells in the primary mice.
The levels of transduced cells also correlated with survival during a
second round of intensive combination chemotherapy (probability of
survival: 6-BG/BCNU, 1.0; BCNU alone, >0.70; no treatment, <0.1).
These data demonstrate that mutant MGMT expressed in the bone marrow
can protect mice from time- and dose-intensive chemotherapy and that
the combination of 6-BG and BCNU leads to uniform selection of
transduced stem cells in vivo in mice. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One application where a rapid selection of transduced cells would be desirable is the use of hematopoietic cells resistant to certain chemotherapeutic agents. Rapid amplification of drug-resistant cells might allow the use of more intensified chemotherapy regimens to treat tumors resistant to conventional therapy. Among the best-studied mechanisms of tumor resistance is the increased expression of MGMT that mediates resistance to CENUs and other alkylating agents (13, 14, 15) . 6-BG, an effective pharmacological inhibitor of MGMT activity, has been shown to restore tumor cell sensitivity to some alkylators, in particular, CENUs, in vitro (16, 17, 18) and in human xenografts in vivo (19 , 20) . However, pharmacological manipulation of MGMT will likely enhance toxicity to normal tissues simultaneously. In many cases, the use of dose intensification is limited by excessive toxicity of normal cells, including blood cells. Early Phase I human trials appear to show that hematopoietic cells are particularly sensitive to the combination of 6-BG and CENU cytotoxicity (21 , 22) .
Several human mutant MGMT proteins have been shown to confer resistance to 6-BG while retaining the ability to remove alkylator-induced O6 adducts (23, 24, 25) . The identification of such mutants has allowed for the development of new strategies to increase the therapeutic index of alkylating agents by using 6-BG to inactivate tumor MGMT activity and gene transfer of 6-BG-resistant mutant MGMT cDNAs to protect hematopoietic cells. Although current approaches are focused on blood cells, if this approach is successful, it could have implications for use in other sensitive tissues. MGMT mutants P140A and G156A expressed and purified from Escherichia coli have shown a 40- and 240-fold higher resistance to 6-BG depletion compared with WT MGMT (24) . Xu-Welliver et al. (26) have generated several additional MGMT mutant proteins, including the single mutant P140K that has been shown to be over 1000-fold more resistant to 6-BG. All of these mutants showed a reduced rate of repair of methylated DNA substrates in vitro compared with human WT MGMT by a factor of 2.5 (P140A), 10 (P140K), and 25 (G156A). However, each mutant protected Chinese hamster ovary cells from BCNU-induced cytotoxicity to a level comparable with WT MGMT (27) . Expression of either of P140A or G156A has been shown to protect hematopoietic cells from combination 6-BG and BCNU treatment in vitro (28 , 29) . Mice transplanted with MGMT mutant G156A-transduced bone marrow were also protected from 6-BG and BCNU hematopoietic toxicity (10 , 30) . However, the G156A mutant shows severely reduced DNA repair kinetics in addition to being relatively unstable in mammalian cells compared with WT MGMT and P140K (31) . Taken together, all of the in vitro data suggest that the P140K mutant should provide superior protection to primary hematopoietic cells; however, the significance of the reduced kinetic properties of this mutant compared with WT MGMT and P140A is not clear. To date, no studies have directly compared WT MGMT and these MGMT mutants with respect to protection of susceptible tissue from combined 6-BG/BCNU treatment.
To identify the most promising MGMT mutant for hematopoietic cell protection, we used a murine model to compare the mutant P140K with the previously tested P140A mutant and WT MGMT. Our results demonstrate that the P140K mutant is superior to the P140A mutant and WT MGMT in protecting bone marrow from 6-BG/BCNU toxicity in vivo. The data also suggest that the combined use of 6-BG and BCNU leads to significant selection of chemoresistant primitive hematopoietic stem cells in vivo. To study in vivo selection more carefully, we generated a bicistronic retroviral construct containing the P140K mutant linked to the eGFP marker gene. The expression of GFP from this construct allowed serial determinations of the number of vector-expressing blood cells in individual mice. After several courses of 6-BG/BCNU treatment, transduced stem cells were uniformly selected in vivo. The resistance phenotype was amplified and transmitted to secondary and tertiary transplant recipients following bone marrow transplant confirming modification and resistance at the stem cell level. Animals receiving in vivo selected bone marrow were highly resistant to the cytotoxic effects of 6-BG/BCNU on subsequent exposure. These data further support the potential utility of mutant MGMT gene transfer in effecting chemoresistance and suggest a powerful selection method for hematopoietic cells in cancer patients undergoing intensive chemotherapy.
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MATERIALS AND METHODS |
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Bone Marrow Transduction and Transplantation.
Bone marrow was harvested from femurs of 8–10-week-old C57Bl/6J mice
(Jackson Laboratories, Bar Harbor, ME) 48 h after treatment with
5-fluorouracil (150 mg/kg; SoloPak Laboratories, Franklin Park, IL) as
described previously (35
, 36)
. Harvested bone marrow cells
were prestimulated for 48 h at 37°C in 5%
CO2 with 100 units/ml recombinant human
interleukin 6 (Pepro Tech, Inc. Rock Hill, NJ) and 100 ng/ml
recombinant rat stem cell factor (Amgen, Thousand Oaks, CA) in
-modified Eagle’s medium (Life Technologies, Inc.) supplemented
with 20% FCS (Hyclone, Logan, UT). Retroviral transduction was done on
fibronectin fragment CH296 (RetroNectin-; Takara Shuzo, Biotechnology
Group, Otsu, Japan) at a concentration of 8
µg/cm2 as described previously (36
, 37)
, with a multiplicity of infection of 0.5 in the presence of
cytokines. Over a period of 48 h, the cells were incubated with
viral supernatant twice for 4 h. After infection, hematopoietic
cells were harvested using cell dissociation buffer (Life Technologies,
Inc.), and 1.5 x 106 cells were
injected via tail vein into lethally irradiated mice
(139Cs source, 11 Gy, split dose with a minimum
of 3 h between doses; Nordion International, Kanata, Canada).
Drug Administration and Blood Analysis.
Four weeks after transplantation, the mice were randomly assigned to
the different treatment groups: mice were injected weekly with five
doses of 40 mg/kg BCNU (Drug Synthesis and Chemistry Branch,
Developmental Therapeutics program, Division of Cancer Treatment,
National Cancer Institute, Bethesda, MD) injected i.p. in a solution of
10% (v/v) ethanol and 90% (v/v) normal saline or injected with
30 mg/kg 6-BG (provided by R. Moschel; Frederick Cancer Research
Center, Frederick, MD) in a solution of 40% (v/v) polyethylene
glycol-400 and 60% (v/v) 0.05 M PBS, and BCNU (10 mg/kg)
was injected 1 h after 6-BG injection (both were injected
i.p.). BCNU was used within 10 min of reconstitution.
In some studies, primary mice transplanted with bone marrow cells transduced with P140K retrovirus that had been left untreated, treated with BCNU only, or treated with a combination of 6-BG and BCNU were randomly chosen as a stem cell source for secondary transplants. After irradiation of recipient mice (as described above), 4 x 106 bone marrow cells harvested from primary mice were transplanted into secondary recipients. Four weeks after infusion of bone marrow cells, all animals were treated with 4 weekly doses of 6-BG and BCNU as described above.
Blood was collected each week before chemotherapy treatment. Blood counts were run on a Cell Dyn 3500 hematology analyzer (Abbott Laboratories, South Pasadena, CA) using veterinary software (Abbott Laboratories).
Analysis GFP Expression by Flow Cytometric Analysis.
For analysis of GFP expression in myeloid and lymphoid lineages,
peripheral blood, bone marrow, and spleen were first depleted of RBCs
using RBC lysis buffer (Gentra Systems, Minneapolis, MN) for 30 min on
ice and blocked with 10% normal rat serum (Caltag Laboratories,
Burlingame, CA) in PBS for 10 min on ice. Cells were then incubated
with one or two antibodies for 30 min on ice, washed once in 0.2% BSA
in PBS, and then analyzed on a FACScan flow cytometer (Becton
Dickinson, San Jose, CA). Antibodies used were: (a)
PE-conjugated GR-1 (RB6-8C5); (b) PE-conjugated B220
(RA3-6B2); (c) PE-conjugated CD3 (145-2C11); (d)
PE-conjugated Rat IgG2a; (e)
PE-conjugated Armenian hamster IgG group 1; and
(f) PE-conjugated Rat
IgG2b [all were purchased from PharMingen (San
Diego, CA) and used at the concentrations recommended by the
manufacturer]. The percentage of GFP-expressing granulocytes (GR-1),
B-cells (B220), and T-cells (CD3, CD4, and CD8) within peripheral
blood, bone marrow, and spleen was determined by dividing the number of
double-positive cells by the number of lineage-positive cells
(corrected for background using isotope-specific control antibody; Ref.
34
).
DNA Isolation and Analysis.
Genomic DNA was isolated using the Puregene DNA Isolation kit (Gentra
Systems) according to the manufacturer’s instructions. Genomic DNA
(10–20 µg) was digested with EcoRI (New England BioLabs,
Beverly, MA) and separated on a 0.8% agarose gel. The DNA was
transferred to a nylon membrane (Magna Graph, Micron Separation INC,
Westboro, MA). The membrane was then hybridized with a
32P-labeled full-length eGFP probe.
Prehybridization and hybridization were completed with ExpressHyb
Hybridization Solution (Clontech) according to the manufacturer’s
instructions. Posthybridization washes were done in 2x SSC/0.05% SCS
at room temperature and then in 0.1x SSC/0.01% SDS at 50°C. The
membrane was then exposed to X-ray film at -70°C.
Statistical Methods.
Sample sizes in this study were sufficient to ensure adequate power.
The normality assumption required for the large sample tests is
frequently not satisfied by these data. Hence, nonparametric tests were
used for all analyses. To compare between two independent samples,
Wilcoxon test was used, and multiple sample comparisons were done using
the Kruskal-Wallis test. When comparing pretreatment values with
posttreatment values (paired data), the sign rank test was used. All
conclusions are made using 0.05 as the level of significance. Survival
rates of the groups (mutant and treatment groups) were described using
Kaplan-Meier curves and compared using the log-rank test. All data are
presented as the mean ± SD unless otherwise indicated.
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RESULTS |
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Comparison of P140A and P140K MGMT Mutant Function in
Vivo.
To compare resistance of MGMT mutants to 6-BG depletion in
vivo, retroviral vectors expressing a mutant containing an amino
acid substitution of alanine for proline (P140A) and a mutant
containing a substitution of lysine for proline (P140K) at position 140
were constructed. The full-length cDNA of each mutant was cloned into
the MSCV 2.1, allowing cDNA expression to be directed from the viral
long terminal repeat. Each plasmid DNA was transfected into
GP+envAmma12 packaging cells, and transient virus supernatant harvested
from the transfected populations was used to infect GP+E86 producer
cells. Subsequently, producer populations were used to harvest virus
supernatant for each vector. Titers of recombinant virus used for
infection of bone marrow cells were as follows: P140A vector,
4 x 105 infectious units/ml; and
P140K vector, 5 x 105 infectious
units/ml assayed on NIH/3T3. Cells infected with either virus
demonstrated MGMT repair activity using a standard oligonucleotide
repair assay and were resistant to BCNU in standard survival assay
in vitro (data not shown).
To determine the relative resistance of hematopoietic cells transduced
with each vector in vivo, murine bone marrow cells were
infected with supernatant virus on FN-CH296. To simulate the low number
of transduced cells characteristically seen in human clinical trials,
the multiplicity of infection was kept close to 0.5 (virus:bone marrow
cells). Lethally irradiated mice were reconstituted with 1.5 x 106 transduced bone marrow cells
expressing either no transgene-encoded MGMT (mock-infected)-, P140A
mutant-, or P140K mutant-transduced bone marrow cells. Four weeks after
initial transplantation of transduced bone marrow cells, mice were
randomly assigned to receive five weekly injections of the combination
treatment of 6-BG (30 mg/kg) and BCNU (10 mg/kg) or BCNU (40 mg/kg)
alone. The third group was left untreated. We used mice treated with
BCNU at the same dose used in the pilot study as a control arm. Before
treatment, both MGMT mutant groups were statistically identical with
regard to ANCs and platelet count (Table 1)
. Mice transplanted with bone marrow cells transduced with P140K or
P140A vectors uniformly survived treatment with BCNU alone. There was
no difference in the ANC or platelet counts among these groups after
treatment (Table 1)
. Thus, the resistance of each mutant to BCNU alone
appeared similar both in vitro and in vivo. This
is in agreement with the findings of Loktionova et al.
(27)
and Maze et al. (28)
, who
have shown in cell culture experiments that no difference in cell
survival after transduction with these mutants is observed, even at
high BCNU doses, despite the reduced rate of DNA repair of the mutants
P140A and P140K compared with WT MGMT.
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. None of the animals transplanted with mock-infected control cells
survived this treatment at 120 days (P < 0.001). Peripheral blood counts were measured each week before
chemotherapy. Before initiation of treatment, both groups were
statistically identical with regard to ANC (P = 0.4) and platelet counts (P = 0.43;
Table 2
). Between the third and fourth treatment, the animals transplanted with
cells transduced with P140K demonstrated significantly higher ANCs
(P140K, 496 ± 266 cells/µl; P140A, 149 ± 75 cells/µl; P < 0.001) and
platelet counts (P140K, 1027 ± 671 x 103
cells/µl; P140A, 239 ± 127 x 103
cells/µl;
P < 0.001) compared with mice transplanted
with cells transduced with P140A. In mice transplanted with P140K,
peripheral blood counts reached pretreatment values for ANC and
platelets after the last weekly treatment dose (Table 2
and Fig. 2
). In contrast, mice transplanted with cells transduced with
P140A-expressing retrovirus demonstrated significantly lower ANC and
platelet counts at the end of the treatment period (Table 2
and Fig. 2
).
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. Although mice randomly assigned to BCNU-only treatment showed a
slightly higher number of neutrophils expressing GFP than the two other
groups (Table 3
; P = 0.044), this difference,
which is probably due to differences in the kinetics of recovery from
the transplant, does not alter the conclusions derived from the
subsequent treatment. As seen in Fig. 3
, after only three treatments, the mean percentage of GFP+ peripheral
blood neutrophils increased markedly in mice treated with the
combination treatment (from 8% to 80%; P < 0.001) but only increased from 12% to 24% (P = 0.008) in mice treated with BCNU alone (6-BG/BCNU group
versus BCNU only, P = 0.008) and
remained unchanged in the untreated group (8% versus 8%).
After five treatments, 93% of the neutrophils in the peripheral blood
expressed GFP in mice treated with 6-BG/BCNU compared with 51% of the
neutrophils in the group of mice treated with BCNU alone
(P < 0.001; Table 3
and Fig. 3
).
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. This
may suggest that treatment with BCNU alone selects less efficiently
than the combination therapy for long-lived stem cells compared with
short-lived progenitor cells. The persistence of GFP+ cells at high
levels in mice treated with combination therapy after completion of
treatment suggested that selection of a more primitive hematopoietic
progenitor cell was occurring in vivo. To examine mice for evidence of stem and progenitor cell selection after BCNU treatment alone or with the combination of 6-BG/BCNU, serial transplants were performed. Theoretically, combination treatment may provide greater selective pressure because endogenous MGMT in some hematopoietic cells may protect against treatment with BCNU alone. Furthermore, if transduced stem cells were enriched with either treatment, then the drug resistance phenotype seen in peripheral blood of treated mice should be transplantable to secondary recipients. Moreover, effective elimination of nonresistant stem cells in the primary donor animals should increase the resistance of secondary recipient mice to hematopoietic toxicity during subsequent chemotherapy treatment. Primary mice transplanted with bone marrow cells transduced with the P140K vector that were left untreated, treated with BCNU, or treated with combination 6-BG and BCNU were used as bone marrow donors in secondary transplants. Seven weeks after the fifth treatment of these primary mice, 4 x 106 bone marrow cells were harvested and transplanted into secondary irradiated recipients. Four weeks after the infusion of cells, the secondary recipients were treated with 4 weekly doses of 6-BG and BCNU.
All secondary transplant recipients of donor stem cells derived from
the 6-BG/BCNU-treated mice survived an additional four weekly doses of
6-BG/BCNU, whereas only 75% of recipients of stem cells derived from
BCNU-treated donors survived this treatment (Fig. 4)
. None of the animals transplanted with cells derived from untreated
donors survived (Fig. 4
; P < 0.001). Before
treatment, the three groups were similar with regard to the ANC
(P = 0.31) and the platelet count
(P = 0.11; data not shown). The effects of
combination therapy on peripheral blood counts varied significantly
among the three groups. Mice transplanted with cells harvested from
previously untreated mice or mice treated with BCNU alone demonstrated
significant declines in ANC (Fig. 5A)
. In contrast, mice transplanted with bone marrow cells
harvested from primary mice treated previously with combination
6-BG/BCNU showed no significant neutropenia in the face of retreatment
with combination drugs (Fig. 5A)
. There was no significant
drop in the platelet counts in secondary transplant recipients derived
from BCNU-pretreated or 6-BG/BCNU-pretreated primary animals (Fig. 5B)
. However, secondary transplant recipients derived from
untreated mice had significantly lower platelet counts after the second
treatment (Fig. 5B)
. These data suggest that selection of
stem cells is occurring in primary mice treated with 6-BG/BCNU to a
greater extent than in mice treated with BCNU alone. In addition, the
lack of significant hematopoietic toxicity in these secondary
transplant recipients suggests that few nontransduced cells
survive treatment in the primary animals.
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. The number of GFP+ peripheral blood cells remained low (4–5%) and
constant in the representative mouse from the untreated group during
the entire experimental observation period and in secondary transplant
recipients (Fig. 6
, A-C). In the representative
mouse treated only with BCNU, the number of GFP+ cells increased from
12% (pretreatment) to 63% after BCNU treatment and then fell to 28%
after transplantation into a secondary animal (Fig. 6
,
D—F). However, subsequent treatment of this
secondary animal with combination 6-BG/BCNU led to GFP expression in
99% of peripheral blood cells of this secondary animal (Fig. 6
G). In contrast, in the representative mouse treated with
6-BG/BCNU, the number of GFP+ cells increased from 8% to 98% after
combined treatment in primary mice. This high level of GFP-expressing
cells was maintained in the secondary animal before and after treatment
(Fig. 6
, H—K). The high level of GFP+ cells
persisted in tertiary transplant recipients of bone marrow derived from
this animal (Fig. 6
L). Of interest, the level of GFP
expression (analyzed by GFP intensity) in neutrophils was identical in
cohorts of mice transplanted from the same initial donor.
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and Table 4
; P < 0.001). After combination treatment,
there was no significant difference in the number of GFP+ cells between
secondary animals derived from mice treated initially with BCNU only or
with combination therapy (Table 4
and Fig. 7
). The increase in GFP+
neutrophils in the peripheral blood was also reflected in the bone
marrow after treatment (Table 4)
.
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Analysis of Lymphoid Recovery.
Our laboratory has demonstrated previously that BCNU treatment of mice
posttransplant is toxic to immune recovery (9)
. To
evaluate whether lymphocytes were protected from combination therapy in
mice transplanted with bone marrow cells transduced with the P140K
retrovirus, the percentage of GFP+ B and T cells in peripheral blood
and spleen were evaluated before and after treatment in primary and
secondary animals. Thymic cell numbers could not be analyzed due to the
lack of identifiable thymic tissue in mice treated with 6-BG/BCNU.
Primary transplant recipients showed a marked reduction in peripheral
blood lymphocyte count after treatment (before treatment,
4405 ± 1435 cells/µl; after treatment, 1611 ± 1014 cells/µl; P = 0.002), whereas
secondary transplant recipients showed lower but stable peripheral
blood lymphocyte counts throughout the treatment period (before
treatment, 1614 ± 841 cells/µl; after treatment,
1572 ± 427 cells/µl; P > 0.99). In primary mice, the percentage of GFP+ B220+/CD3+ cells in the
peripheral blood increased to 32% (P = 0.008) in BCNU only-treated mice and 53% (P < 0.001) in 6-BG/BCNU-treated mice compared with 5%
(P = 0.375) in untreated mice (Table 3)
. In
addition, all secondary animals showed a significantly higher
percentage of GFP+ B220+/CD3+ cells in the peripheral blood after
treatment (Table 4
; 6-BG/BCNU, P = 0.002;
BCNU, P = 0.004). There was no significant
difference in the number of GFP+ B220+/CD3+ cells between the
peripheral blood and spleen of primary and secondary transplant
recipients (data not shown). Flow cytometric analysis of splenocytes
showed heterogeneous GFP expression in lymphocytes of primary
transplant recipients (Fig. 8A and B
, bottom panel). These data
suggest that although protection and selection of transduced
lymphocytes occurs in vivo, the clonal nature of the
selected lymphocyte population appears to be more complex than that
seen in the myeloid compartment in the time course analyzed in these
studies.
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and 8)
, with a discrete, narrowly confined
pattern of expression suggestive of oligoclonality. To confirm
identical integration patterns in primary and secondary mice at the
molecular level, which would provide further evidence of selection of
the stem cell compartment, DNA was digested with EcoRI,
which cuts only once in the proviral genome, and probed with the
full-length eGFP probe to determine the integration fragments of
transduced cells. As seen in Fig. 8, A and B
,
identical hybridizing bands were seen in the DNA of bone marrow and
spleen cells of the primary and all corresponding secondary and
tertiary (data not shown) animals. These data confirm at the molecular
level repopulation of multiple secondary animals with transduced stem
cells. The limited number of integration sites seen in each animal also
suggests oligoclonality of hematopoiesis. Thus, these data are
consistent with engraftment and effective protection of mice by a
limited number of transduced stem cells. |
DISCUSSION |
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Genetic strategies to decrease sensitivity of bone marrow cells to CENUs have been explored by a number of investigators using retrovirus-mediated gene transfer of MGMT (9 , 10 , 12 , 29 , 45, 46, 47, 48) . In addition, many MGMT mutants have been generated using random in vitro mutagenesis or based on the primary sequence and structure of the bacterial gene product, ada, that is naturally resistant to 6-BG (24, 25, 26) . The stability and resistance of these mutant proteins to 6-BG depletion and BCNU treatment varies over several logs (27 , 31) . The use of gene transfer to express these mutants in bone marrow cells thus provides a unique approach to increase resistance of normal blood forming cells to the combined effects of 6-BG and CENUs, while allowing sensitization of tumor cells in vivo. To date, limited data are available that analyze MGMT mutants in this context, particularly with respect to in vivo resistance to the combination of both classes of agents.
In the study reported here, we compared in vivo the effects of expression of the MGMT mutants P140A and P140K with WT MGMT in bone marrow cells. Our results demonstrate that the P140K mutant is superior to both the P140A mtuant and human WT MGMT in protecting bone marrow from 6-BG/BCNU toxicities in vivo. Mice expressing P140K showed significantly higher resistance to the combination therapy of 6-BG/BCNU, resulting in increased survival and protection of myeloid, lymphoid, and megakaryocytic lineages. In contrast, the dose-intensive chemotherapy leads to severe bone marrow aplasia in mice reconstituted with mock-infected bone marrow cells or with bone marrow cells transduced with WT MGMT or the P140A mutant. These data confirm and extend the results of Loktionova et al. (27) showing that the reduction in repair kinetics of the P140K mutant compared with WT MGMT and the P140A mutant is not as critical for determining cellular resistance to combined 6-BG/BCNU as the increase in resistance to 6-BG. This may be due to the overall rapid kinetics of this protein.
Studies examining the effect of expression of other mutants in vivo on the sensitivity to drug therapy have used different treatment protocols, making comparison of data derived from different mutants difficult. For example, Davis et al. (49) reported increased survival of mice expressing G156A in bone marrow after exposure of mice to two doses of 30 mg/kg 6-BG and 10 mg/kg BCNU given 3 weeks apart. An additional study in nonablated mice examined infusion of G156A-expressing cells during treatment with higher doses of 6-BG and BCNU. Mice expressing G156A in bone marrow demonstrated significant survival compared with mice infused with cells not expressing a MGMT transgene. Thus, in each of these studies, resistance to BCNU and moderate resistance to 6-BG depletion were noted along with protection from myelosuppression. However, Davis et al. (31) also reported reduced stability of the G156A mutant in mammalian cells compared with the P140K mutant, making this mutant a less optimal candidate to confer protection to hematopoietic stem cells in a dose-intensified chemotherapy trial.
The P140A/G156A double mutant has been shown to be highly resistant to 6-BG depletion (24) . However, there are conflicting results with respect to the degree of protection of hematopoietic and other cells after gene transfer of this mutant. Hickson et al. (50) have previously demonstrated protection of fibroblasts cells using P140A/G156A expressed off a cytomegalovirus promoter, despite a 10-fold decrease in repair activity, whereas Maze et al. (28) demonstrated no protection in murine L1210 cells after retroviral transduction. The P140A/G156A mutant protein appears unstable and is therefore unlikely to be superior to P140K in hematopoietic cell protection. Christians et al. (51) and Encell et al. (52) have isolated several additional mutants generated by random mutagenesis that show a high degree of resistance to 6-BG as well as protection from alkylating agent cytotoxicity in bacteria. One potential problem with all MGMT mutants when used in human studies is the risk of immunogenicity, which may be increased by multiple mutations. Direct comparison of these mutants in vivo with P140K is warranted to determine the optimal mutant to be used in future studies.
Previously, no studies using gene transfer of MGMT have critically examined the effect of CENUs or a combination of CENUs with 6-BG on hematopoietic stem cell populations. Here we have examined the effect of a 6-BG/BCNU combination on the stem cell compartment because hematopoietic toxicity has been seen in early human safety trials. The recovery of peripheral blood counts during ongoing treatment with combination therapy along with the emergence of uniformly GFP+ cells in the peripheral blood of primary animals strongly suggested that P140K expression and combination drug treatment lead to selection of transduced hematopoietic cells in vivo. Data from serial transplants demonstrated that this selection is likely occurring at the level of the stem cell. Secondary transplanted mice reconstituted with bone marrow cells derived from primary animals expressing P140K and treated with combination therapy demonstrated no hematopoietic toxicity on subsequent exposure to the dose of intensive chemotherapy regimen. In contrast, mice derived from primary animals not treated previously developed severe and fatal cytopenias. The level of blood cells derived from transduced stem/progenitor cells after secondary and even tertiary transplants remained high and unchanged without any further treatment. In addition, the pattern of GFP expression in peripheral blood was nearly identical in secondary and tertiary transplant recipients compared with the initial donor. Finally, DNA analysis demonstrated common retrovirus integration bands in primary and multiple secondary mice, confirming survival and expansion of transduced and selected stem cells in these mice in vivo. Interestingly, selection in the lymphoid compartment appeared less uniform, suggesting that some T-cell precursors are more resistant to the combination therapy. Delayed maturation of T cells after high-dose chemotherapy is seen in humans and may also contribute to the recovery kinetics seen in the model described here. In addition, the heterogeneity in GFP expression in the lymphoid compartment may relate to peripheral expansion of transduced mature T lymphocytes as seen in human studies (53 , 54) .
Data derived from mice treated with BCNU alone show a decrease in the number of GFP+ cells in the peripheral blood after cessation of selective pressure. Studies by Allay et al. (6) also demonstrated incomplete selection of stem cells after gene transfer of mutant dihydrofolate reductase and treatment with trimetrexate and nitrobenzylmercaptopurine riboside 5' monophosphate. In both cases, the decline in transduced cells is likely due to higher selective pressure on more differentiated progenitors compared with transduced stem cells. The contribution of these more mature progenitors to the peripheral blood declines over time due to the limited proliferative capacity of cells in this compartment. Taken together, these data suggest that stringent selection of MGMT-expressing stem cells occurs in the setting of combined 6-BG/BCNU treatment in vivo and that such a selection requires expression of a mutant MGMT highly resistant to 6-BG depletion in the setting of retrovirus-encoded expression. Such a selection may not be possible with other drug resistance markers, such as MDR or dihydrofolate reductase.
The clinical utility of this approach remains to be defined, and issues related to potential toxicities will need further clarification in mice, in human xenograft models, and in nonhuman primates. The data presented here and recent improvements in gene transfer technology suggest that the combined use of pharmacological depletion of tumor MGMT activity and genetic manipulation of hematopoietic cells may ultimately be useful in human chemotherapy protocols.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grant P01 CA75426-02, a William
Kennedy Research Fellowship of the National Childhood Cancer
Foundation, and a Fellow Scholar Award of the American Society of
Hematology. 
2 To whom requests for reprints should be
addressed, at Herman B. Wells Center for Pediatric Research, Indiana
University, Cancer Research Institute, 1044 West Walnut Street, #414,
Indianapolis, IN 46202.
3 The abbreviations used are: MGMT,
O6-methylguanine DNA methyltransferase;
6-BG, O6-benzylguanine; CENU,
chloroethylnitrosourea; WT, wild-type; MSCV, murine stem cell virus;
BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; GFP, green fluorescence
protein; eGFP, enhanced GFP; PE, phycoerythrin; ANC, absolute
neutrophil count; IRES, internal ribosome entry site.
Received 3/17/00. Accepted 7/20/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MGMT-transduced bone marrow infusion increases tolerance to O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea and allows intensive therapy of 1,3-bis(2-chloroethyl)-1-nitrosourea-resistant human colon cancer xenografts. Hum. Gene Ther., 10: 1021-1030, 1999.[Medline]
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; n = 13), P140A (•; n = 16), or
mock-transduced (control, 
; n = 11)
cells were treated weekly with 6-BG/BCNU (arrows).
P < 0.001, log-rank test.
