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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.]
Direct reversal of O6 adducts caused by
chemotherapy agents is accomplished in mammalian cells by the protein
O6-methylguanine DNA methyltransferase
(MGMT). Some tumors overexpress MGMT and are resistant to alkylator
therapy. One future approach to treatment of these tumors may rely on
concurrent pharmacological depletion of tumor MGMT with
O6-benzylguanine (6-BG) and protection of
sensitive tissues, such as hematopoietic stem and progenitor cells,
using genetic modification with 6-BG-resistant MGMT mutants. We have
used retroviral-mediated gene transfer to transduce murine
hematopoietic bone marrow cells with MGMT point mutants showing
resistance to 6-BG depletion in vitro. These mutants
include proline to alanine and proline to lysine substitutions at the
140 position (P140A and P140K, respectively), which show 40- and
1000-fold resistance to 6-BG compared with wild-type (WT) MGMT.
Lethally irradiated mice were reconstituted with murine stem cells
transduced with murine stem cell virus retrovirus expressing each
mutant, WT MGMT, or mock-infected cells and then treated with a
combination of 30 mg/kg 6-BG and 10 mg/kg
1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or with 40 mg/kg BCNU
alone. Compared with mice treated with BCNU alone, significant myeloid
toxicity and death occurred in mice reconstituted with mock-infected or
WT MGMT (<0.1 probability of survival) or the P140A mutant (0.13
probability of survival) MGMT cDNAs. In contrast, after an
initial period of mild cytopenia, mice reconstituted with the P140K
mutant (0.83 probability of survival) recovered nearly normal blood
counts, even during continued treatment. Comparison of peripheral blood
neutrophils after completion of 5 weekly treatments in these animals
showed a direct correlation between the treatment and in
vivo selection for progeny of transduced cells (pretreatment,
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.
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