1-Nitrosourea, a Chemotherapeutic Alkylating Agent (Chloroethylnitrosourea/Retroviral Vector/DNA Repair/06-Methylguanine DNA Methyltransferase) RODNEY MAZE*T, JAMES P

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, AND LEONA 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- CNU alkylation produces numerous DNA base modifica- ing agents are commonly used for cancer chemotherapy, but tions plus DNA inter- and intrastrand crosslinks. Interstrand their usefulness is limited by severe bone marrow toxicity that crosslinks are particularly cytotoxic because they grossly in- causes the cumulative depletion of all hematopoietic lineages terfere with DNA replication; their formation can be initiated (pancytopenia). Bone marrow CNU sensitivity is probably due by a chloroethyl group at the 06 position of guanine which to the inefficient repair ofCNU-induced DNA damage; relative slowly rearranges to form an ethyl bridge between NI of to other tissues, bone marrow cells express extremely low guanine and N3 of cytosine in the opposite strand (6). The levels of the 06-methylguanine DNA methyltransferase formation of these cytotoxic DNA interstrand crosslinks can (MGMT) protein that repairs cytotoxic 06-chloroethylgua- be prevented by transfer of the 06-chloroethyl group to the nine DNA lesions. Using a simplified recombinant retroviral 06-methylguanine DNA methyltransferase (MGMT) protein vector expressing the human MGMT gene under control of the prior to crosslink formation (7-10). MGMT levels in mam- phosphoglycerate kinase promoter (PGK-MGMT) we in- malian cells thus correlate with resistance to CNU-induced creased the capacity of murine bone marrow-derived cells to cytotoxicity (7, 11, 12). repair CNU-induced DNA damage. Stable reconstitution of Human and murine bone marrow cells express extremely mouse bone marrow with genetically modified, MGMT- low MGMT levels relative to other tissues (5). Increasing expressing hematopoietic stem cells conferred considerable MGMT expression in bone marrow might therefore decrease resistance to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1- that tissue's sensitivity to CNU-induced cytotoxicity. Indeed, nitrosourea (BCNU), a CNU commonly used for chemother- we recently demonstrated that transduction of murine bone apy. Bone marrow harvested from mice transplanted with marrow progenitor cells with a retroviral vector expressing PGK-MGMT-transduced cells showed extensive in vitro the human MGMT cDNA provided a moderate level of BCNU resistance. Moreover, MGMT expression in mouse protection from the peripheral pancytopenia induced by bone marrow conferred in vivo resistance to BCNU-induced repeated BCNU treatments (12). However, protection was pancytopenia and significantly reduced BCNU-induced mor- modest and we now propose that in order to tality due to bone marrow hypoplasia. These data demonstrate quite produce that increased DNA alkylation repair in primitive hematopoi- a more useful level of BCNU protection, it is critical to etic stem cells confers multilineage protection from the my- express MGMT in hematopoietic stem cells, because the elosuppressive effects of BCNU and suggest a possible ap- cumulative and delayed nature of the BCNU-induced bone proach to protecting cancer patients from CNU chemothera- marrow toxicity may be related to damage in the stem cell py-related toxicity. compartment (13-15). Here we show that expression of the human MGMT DNA repair protein by a transduction protocol which murine bone marrow stem cells confers The chloroethylnitrosourea (CNU) chemotherapeutic alkylat- targets ing agents have been used to treat certain kinds of cancer for considerable resistance to BCNU-induced toxicity both in over two decades (1), and 1,3-bis(2-chloroethyl)-1-nitrosourea vitro and in vivo. (BCNU, carmoustine) is typical of this class of chemotherapeutic drug. BCNU is particularly effective in treating child- MATERIALS AND METHODS hood and adult glial tumors (2, 3) and has been used in high-dose chemotherapy for lymphomas, breast, lung, and Recombinant Retroviral Vector and Packaging Cell Lines. gastrointestinal cancers (1, 4). Unfortunately, the clinical The human MGMT cDNA was cloned under control of the success of the CNUs has been limited by their severe bone phosphoglycerate kinase (PGK) promoter in the N2/Zip retro- marrow and lung toxicities. Myelosuppression is characteris- viral construct as described (12) to produce N2/ZipPGK-MGMT tically delayed following CNU treatment and can lead to (hereafter called PGK-MGMT). A high-titer producer clone of severe, prolonged, and cumulative pancytopenia (3). The PGK-MGMT in GP+E-86 packaging cells (16), OMG-9, was extreme sensitivity of bone marrow to CNUs may be explained previously described (12) and was used throughout this study. by the inability of this tissue to repair CNU-induced cytotoxic DNA damage efficiently (5). Abbreviations: CNU, chloroethylnitrosourea; BCNU, 1,3,-bis(2-chloroethyl)-1-nitrosourea; 06-MeG, 06-methylguanine; MGMT, 06-methylguanine DNA methyltransferase; PGK, phosphoglycerate kinase; CPC, The publication costs of this article were defrayed in part by page charge committed progenitor cell(s); HPP-CFC, high-proliferative-potential payment. This article must therefore be hereby marked "advertisement" in colony-forming cell(s). accordance with 18 U.S.C. §1734 solely to indicate this fact. IlTo whom reprint requests should be sent at the * address. 206 Downloaded by guest on October 2, 2021 Genetics: Maze et al. Proc. Natl. Acad. Sci. USA 93 (1996) 207

Infection of Bone Marrow Cells, Transplantation, and PCR and Southern Blot Analysis. DNA was prepared as BCNU Treatment of Recipient Mice. Bone marrow cells were described (23) for amplification with a PCR kit (Perkin- harvested from the hind limbs of 8- to 10-week-old female Elmer/Cetus) and Taq DNA polymerase. The 5' and 3' C57BL/6J mice (The Jackson Laboratory) 48 hr after intra- oligonucleotide primers were based on the human MGMT peritoneal injection with 5-fluorouracil (5-FU2d, 150 mg/kg of cDNA sequence (24) and amplify a unit of 210 bp. PCR products body weight, SoloPak Laboratories, Franklin Park, IL) (17). were electrophoresed through a 1.5% agarose gel, blotted, and Harvested bone marrow cells were prestimulated (18) and hybridized to a 600-bp 32P-labeled BamHI-Sal I fragment of the infected by coculture on confluent, mitomycin C-treated human MGMT cDNA (24). GP+E-86 cells (mock-infected control) or OMG-9 cells (18). Statistical Analysis. Three plates were scored for each CPC After cocultivation, nonadherent hematopoietic cells were and HPP-CFC sample. Each mouse was evaluated separately. harvested and 106 cells were injected into lethally irradiated Results are expressed as a mean ± SEM derived from the (139Cs source, 11 Gy, split dose, with a minimum of 3 hr averages of each individual mouse within a group. The prob- between doses; Nordion International, Kanata, ON, Canada) ability of significant differences between groups was deter- recipient 8- to 10-week-old female C57BL/6J mice. Three mined by Student's t test (two-tailed). 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- RESULTS penia with prolonged (10 weeks) treatment (15). We set out to express the human MGMT alkylation repair Peripheral Blood Analysis. Total peripheral blood leuko- protein in primitive hematopoietic stem cells, with the aim cyte and platelet counts and hematocrits were analyzed on tail of conferring resistance to the myelosuppressive effects of vein bleeds (12). BCNU. Murine bone marrow cells were infected with the Analysis of BCNU Resistance of Transduced Committed PGK-MGMT retrovirus by a protocol previously demon- Progenitor Cells (CPC) and High-Proliferative-Potential Col- strated to transduce reconstituting murine hematopoietic ony-Forming Cells (HPP-CFC). Bone marrow and spleen stem cells at high frequency (18, 25). The transduced bone hematopoietic cellularity was determined by standard methods marrow cells were transplanted into lethally irradiated re- (19), and CNU sensitivity of CPC and HPP-CFC was deter- cipient mice. Three weeks after transplantation the recipient mined as described (12, 15, 20). mice were administered weekly doses of BCNU at 40 mg/kg, MGMT DNA Repair Assay. MGMT levels in bone marrow and treatment was continued for 5 weeks. The transplanted and spleen cell extracts (50 ,ug of protein) were determined mice were monitored for integration and expression of the using a 32P-labeled 18-bp oligonucleotide (a gift of Russell 0. PGK-MGMT retrovirus, for in vivo and in vitro resistance to Pieper, Loyola University Medical Center) containing an BCNU-induced cytotoxicity among different cell types, and 06-methylguanine (06-MeG) base within a methylation- for BCNU-induced mortality. sensitive Pvu II restriction site (21); 06-MeG repair allows Pvu PGK-MGMT Retrovirus Is Present in Hematopoietic Tis- II digestion, which produces a labeled 8-bp fragment. sues up to 18 Weeks After Transplantation. Mice were Western Blot Analysis of MGMT Expression. Human analyzed by PCR and Southern blot for the presence of the MGMT protein was detected with polyclonal antibodies (anti- human MGMT cDNA at 10 and 18 weeks after bone marrow MAP1) (22) against human but not murine MGMT (kindly transplantation (Fig. 1). Bone marrow, spleen, and thymus supplied by Anthony Pegg, Pennsylvania State University DNA from transplanted mice was subjected to PCR ampli- College of Medicine, College Park). Extracts were prepared fication with human MGMT-specific primers and the prod- from 2-5 x 106 cells (collected on day 35 after bone marrow ucts were analyzed on Southern blots probed with a labeled transplant), and the proteins were separated by SDS/PAGE, fragment of the human MGMT cDNA. Ten weeks after blotted, probed with 1:500 antiserum, and developed by en- transplantation, bone marrow DNA from four of five mice hanced chemiluminescence (ECL kit, Amersham). transplanted with PGK-MGMT-transduced bone marrow

tn E ._ I- (9 Control Control -BM MGMT r MGMT :3 CD-MGMTBM ,, r SpI Spl ,ii Thy-, I: uw0 1 2 3 4 5 1 2 3 1 2 3 4 1 2 3 1 2 3 4 5 A

CD)cn E

L- (D r-MGMTBM rMGMTSpI D co 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. Downloaded by guest on October 2, 2021 208 Genetics: Maze et al. Proc. Natl. Acad. Sci. USA 93 (1996)

was positive for the human MGMT sequence (Fig. 1A). At Mock- Control MGMT- the same time point, spleen DNA from four of four mice and F Recipients 17 Recipients 71 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 * ..#.. .* *. ,*.~~~~~Q .4 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 human MGMT sequence (Fig. 1B). Thymic DNA was not examined at this time point. Bone marrow reconstitution is FIG. 3. MGMT DNA repair activity in recipient mice after trans- or mar- considered stable in mice if donor cells are plantation with PGK-MGMT-transduced mock-infected bone hematopoietic row. Bone marrow (BM) and spleen cell (Spl) extracts were analyzed present 16 weeks after transplantation (26); the results in for the ability to demethylate an 06-methylated guanine base located Fig. 1B thus confirm that some mice were transplanted with in a methylation-sensitive Pvu II restriction enzyme site contained PGK-MGMT-transduced cells capable of long-term, stable within an 18-bp oligonucleotide. The oligonucleotide (0.2 pmol, added hematopoietic reconstitution. in excess) was exposed to cell extracts containing 50 Ag of total protein. Human MGMT Protein and Increased 06-MeG Repair Lane 1, oligonucleotide; lane 2, OMG-9 producer cells; lanes 3-8, Activity in Hematopoietic Tissues of Recipient Mice. Using bone marrow and spleen from three mock-infected recipient mice; lanes 9-14, bone marrow and spleen from three antiserum that specifically binds the human (but not mouse) PGK-MGMT- transduced recipient mice; lane 15, bone marrow from nontrans- MGMT protein (22), we demonstrated that human MGMT planted C57BL/6J mouse treated with BCNU; lane 16, bone marrow was expressed in bone marrow in three of three PGK-MGMT- from untreated C57BL/6J mouse. transplanted mice and also in spleen in two of these mice; both bone marrow and spleen from three of three control mice were BCNU in vitro and the survival of both HPP-CFC and CPC was negative for the human MGMT protein (Fig. 2). Further, measured. Bone marrow cells from the PGK-MGMT trans- MGMT activity was higher in hematopoietic cells from mice planted mice were considerably more BCNU-resistant than transplanted with PGK-MGMT-transduced bone marrow those from control mice, and the resistance was seen in CPC compared with the same tissues from control mice (Fig. 3). The as well as the more primitive HPP-CFC (Fig. 4). methyltransferase assay is based upon the ability of 06-MeG PGK-MGMT-Transduced Bone Marrow Cells and Their located in a Pvu II site to inhibit Pvu II digestion; digestion thus Progeny Are Resistant to BCNU Exposure in Vivo. Repeated reflects 06-MeG repair (21). Methyltransferase activity was BCNU treatment leads to severe and cumulative pancytopenia assayed 3 days after a second BCNU treatment (day 35 after in both mice and humans (3, 15). Hematocrits and total bone marrow transplantation) and was higher in bone marrow peripheral leukocyte and platelet counts were used as a and spleen cell extracts of mice transplanted with PGK- measure of bone marrow cytotoxicity induced during 5 weeks MGMT-transduced bone marrow (Fig. 3, lanes 9-14) com- of repeated BCNU treatments. Peripheral blood leukocytes pared with extracts from control mice (lanes 3-8). The spleen and hematocrits were similar in the control and PGK-MGMT cell extract from one control mouse (lane 4) had a higher mice prior to BCNU treatment, but platelet counts were MGMT activity compared with the other control animals. significantly higher in control mice than in PGK-MGMT mice; However, the average methyltransferase activity was lower in the reason for this discrepancy is unclear. BCNU-induced control tissues than in tissues derived from mice transplanted pancytopenia was less severe in mice expressing the human with PGK-MGMT-transduced bone marrow (Table 1). No MGMT protein in their hematopoietic tissues, compared with effect on the endogenous level of MGMT DNA repair activity control mice (Fig. 5). Although platelet counts were initially was detected after two weekly doses of BCNU (Fig. 3, lanes 15 slightly lower in the PGK-MGMT mice than in the control and 16). mice, by 1 week after the first BCNU treatment the platelet Bone Marrow Cells Removed from PGK-MGMT-Trans- counts were significantly higher in the PGK-MGMT mice than planted Mice Display BCNU Resistance in Vitro. Bone marrow in the control mice. The difference between PGK-MGMT and cells were harvested from mice transplanted with mock- eight control mice for total leukocyte counts and hematocrits infected bone marrow and eight mice transplanted with PGK- reached significance by 3 weeks of BCNU treatment and MGMT-transduced bone marrow; for both groups, cells were harvested after the fifth BCNU dose (56 days after transplan- remained significant at 5 weeks. Indeed, after 5 weeks of BCNU treatment, when control mice were clearly pancytope- tation). The harvested bone marrow cells were challenged with nic (Fig. 5), the PGK-MGMT mice displayed platelet counts Bone Marrow Spleen and hematocrits that were not significantly different from those of nontransplanted, untreated mice (P > 0.05) and MOCkMGMT MMcMOCk MGMTMG leukocyte counts that were only reduced by 50% compared ;;~~_*-r ;4;- -MGMT with the same mice (data not shown). The cellularity of the -21.5kDa bone marrow and spleen provides another in vivo measure of 1 2 3 45 6 1 2 3 4 56 BCNU-induced cytotoxicity. Bone marrow and spleen cellularities after five weekly BCNU doses (8 weeks after trans- FIG. 2. Western analysis of =20 ,ug of total protein from bone plantation), and the cellularities in untreated, nontransplanted marrow and spleen cell extracts of mice transplanted with mock- and PGK-MGMT-transduced bone marrow, probed with human MGMT- Table 1. Analysis of MGMT DNA repair activity in recipient mice specific antibodies. Lanes 1-3, mock-infected; lanes 4-6, PGK- MGMT transduced. Negative control lane (-) contained 100 ,ug of Activity, % conversion of total protein from GP+E-86 mouse fibroblast packaging cell extract. 18-bp band to 8-bp band Positive (+) control lane contained 100 jig of total protein from human Mer+ HeLa S3 cell extract. The control lanes have higher Tissue Control MGMT background and hence a shorter exposure is shown for these samples. Bone marrow 20.0 + 3.1 51.0 + 9.9 All other lanes were developed and exposed simultaneously. The Spleen 19.3 ± 12.6 60.3 ± 5.8 positions of the MGMT protein and molecular size standards are indicated at right. Values are mean ± SEM (n = 3). Downloaded by guest on October 2, 2021 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 0.01 patients against the effects of cancer chemotherapies that are 0.01-0 20 . 40 60 80 0 20 40 60 80 particularly toxic to the bone marrow (30, 31) and it seems BCNU, ,uM BCNU, ,LM reasonable to expect that the in vitro transfer of appropriate FIG. 4. BCNU survival curves of bone marrow harvested from genes into harvested bone marrow cells (prior to transplanta- mice transplanted with PGK-MGMT-transduced (0) or mock-infected tion) could generate bone marrow that is resistant to further control (a) bone marrow. Curves show HPP-CFC (A) and CPC (B) cancer chemotherapy (after transplantation) (28). This ap- survival after BCNU exposure. BCNU resistance was evaluated as proach has been attempted previously with dihydrofolate described in Materials and Methods. Each point represents assays done reductase and multidrug-resistance cDNAs (32, 33). Using a in triplicate for HPP-CFC and CPC for eight mice per group from two murine model, we have shown that transfer of a DNA alkyla- separate experiments; each animal was evaluated separately and the tion repair gene into bone marrow stem cells provides con- error bars represent SEM. siderable resistance to subsequent treatments with the com- mice, are shown in Table 2. Bone marrow and spleen cells monly used chemotherapeutic alkylating agent BCNU. clearly survived repeated BCNU treatments better in mice BCNU produces 06-chloroethylguanine DNA adducts that, expressing the human MGMT protein than in control mice. if left unrepaired by the MGMT repair protein, produce highly Moreover, the bone marrow cellularity in BCNU-treated cytotoxic DNA interstrand crosslinks (6, 34). Expression of the PGK-MGMT mice was only moderately reduced (by 50%) MGMT repair protein varies by as much as 100-fold between versus untreated mice, and the spleen cellularity was not different tissues, being highest in liver and lowest in bone reduced at all (increased spleen size is frequently seen in mice marrow (5, 12, 35). However, the reasons underlying this tissue responding to hematopoietic stress). variation are not clear. We demonstrate here that the extreme PGK-MGMT-Transduced Bone Marrow Rescues Recipient sensitivity of hematopoietic tissues to alkylating agents is due, Mice from BCNU-Associated Mortality. Weekly BCNU treat- at least in part, to their low methyltransferase levels, because ment in mice produces significant mortality, presumably due to increasing the methyltransferase levels in these cells conferred BCNU-related pancytopenia (15). However, increasing considerable BCNU resistance. MGMT expression provided MGMT activity in murine hematopoietic cells (by expressing sustained protection against severe peripheral pancytopenia, a the human MGMT protein) confers significant protection clinically important side effect normally associated with against BCNU-induced mortality. Fig. 6 represents data from BCNU chemotherapy. While it is quite clear that increasing three separate experiments; mortality after 5 weeks was sig- the MGMT levels in hematopoietic tissues confers alkylation nificantly reduced in the PGK-MGMT mice versus control resistance, it is not yet clear whether very long-term high-level mice; 23 of 25 (92%) PGK-MGMT mice survived the repeated MGMT expression in a tissue that is normally relatively BCNU treatments, whereas only 19 of 36 (53%) control mice MGMT-deficient will have any unexpected detrimental ef- survived the same treatment (P < 0.001). fects. We previously demonstrated that MGMT expression in hematopoietic progenitor cells conferred a somewhat modest DISCUSSION level of protection against BCNU-induced peripheral pancy- Hematopoietic cells present a particularly amenable target for topenia (12). It is important to note that in this study we somatic gene therapy because these cells are accessible, he- expressed the methyltransferase in more primitive and longer- matopoiesis is well understood, and procedures for bone lived hematopoietic cells and that expression in these cells (and marrow transplantation are well established (27-29). Autolo- their progeny) conferred a more substantial level of protection

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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. Downloaded by guest on October 2, 2021 210 Genetics: Maze et al. Proc. Natl. Acad. Sci. USA 93 (1996)

100 - Toxicology Scholar. B.J.G. was supported by National Institutes of Health Training Grant 5T32 CA09078-19.

: | ,__, MGMT 90 - ,- (23/25) 1. Carter, S. K., Schabel, F. M., Jr., Broder, L. E. & Johnston, T. P. (1972) Adv. Cancer Res. 16, 273-332. 2. Walker, M. D., Alexander, E., Hung, W. E., MacCarty, C. S., Maha- 804 _ ,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ley, M. S., Jr., Mealey, J., Jr., Norrell, H. A., Owens, G., Ransohof, F., Wilson, C. B., Gehan, E. A. & Strike, T. A. (1978) J. Neurosurg. 49, 333-343. 3. Schabel, F. M., Jr. (1976) Cancer Treat. Rep. 60, 665-698. 70t 4. Colvin, M. (1993) in CancerMedicine, eds. Holland, J. F., Frei, E., III, Bast, R. C., Jr., Kufe, D. W., Morton, D. L. & Weichselbaum, R. R. 60± (Lea & Febiger, Philadelphia), pp. 733-754. ' Mock 5. Gerson, S. L., Trey, J. E., Miller, K. & Berger, N. A. (1986) Carcino- - Infected genesis 7, 745-749. 50- Control 6. Ludlum, D. B. (1990) Mutat. Res. 233, 117-126. (19/36) 7. Erickson, L. C., Laurent, G., Sharkey, N. A. & Kohn, K. W. (1980) Nature (London) 288, 727-729. 4 T 7 8. Robins, P., Haris, A. L., Goldsmith, I. & Lindahl, T. (1983) Nucleic 0 7 14 28 35 42 Acids Res. 11, 7743-7758. BCNU treatment, days 9. Samson, L., Derfler, B. & Waldstein, E. A. (1986) Proc. Natl. Acad. Sci. USA 83, 5607-5610. FIG. 6. curves of mice treated with weekly doses of BCNU. 10. Brent, T. P. & Remack, J. S. (1988) Nucleic Acids Res. 16, 6779-6788. Survival 11. Lindahl, T., Sedgwick, B., Sekiguchi, M. & Nakabeppu, Y. (1988) Data represent three separate experiments. Numbers in parentheses give Annu. Rev. Biochem. 57, 133-157. the number of animals surviving/total animals per group. 12. Moritz, T., Mackay, W., Glassner, B. J., Williams, D. A. & Samson, L. (1995) Cancer Res. 55, 2608-2614. against BCNU toxicity. The phenotypic characterization and 13. Botnick, L. E., Hannon, E. C., Vigneulle, R. & Hellman, S. (1981) Cancer Res. 41, 2338-2341. purification of stem cells from normal human bone marrow by 14. Neben, S., Hemman, S., Montegomery, M., Ferrera, J. & Mauch, P. use of immunologic and functional characteristics have been (1993) Exp. Hematol. 21, 156-162. reported (ref. 36; reviewed in ref. 37), presenting the possibility 15. Maze, R., Moritz, T. & Williams, D. A. (1994) Cancer Res. 54, 4947-4951. that, ultimately, we could express the MGMT repair protein 16. Markowitz, D., Goff, S. & Bank, A. (1988) J. Virol. 62, 1120-1124. (or other repair proteins) in these cells, thus ensuring expres- 17. Dick, J. E., Magli, M. C., Huszar, D., Phillips, R. A. & Bernstein, A. sion in all the human hematopoietic cell lineages. Although (1985) Cell 42, 71-79. 18. Luskey, B. D., Rosenblatt, M., Zsebo, K. & Williams, D. A. (1992) gene transfer protocols in human cells are still inefficient, it has Blood 80, 396-402. been shown that bone marrow cells harvested from cancer 19. Du, X. X., Keller, D. C., Maze, R. & Williams, D. A. (1993) Blood 82, patients for autologous bone marrow transplantation (and 1016-1022. 20. Yoder, M. C., Du, X. X. & Williams, D. A. (1993) Blood 82, 385-391. marked with the neomycin phosphotransferase gene) consis- 21. Wu, R. S., Hurst-Calderone, S. & Kohn, K. W. (1987) Cancer Res. 47, tently contribute to long-term multilineage recovery of hema- 6229-6235. topoiesis (38). It therefore seems likely that one could suc- 22. Pegg, A. E., Wiest, L., Mummert, C., Stine, L., Moschel, R. C. & Dolan, M. E. (1991) Carcinogenesis 12, 1679-1683. cessfully express MGMT in human bone marrow cells being 23. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: used for autologous transplantation and subsequently protect A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, patients against chemotherapy-induced hematotoxicity. In- NY), 2nd Ed. 24. Tano, K., Shiota, S., Collier, J., Foote, R. S. & Mitra, S. (1990) Proc. deed, modification of the retroviral transduction protocol has Natl. Acad. Sci. USA 87, 686-690. led to efficient transduction of human primitive long-term- 25. Apperley, J. F., Luskey, B. D. & Williams, D. A. (1991) Blood 78, culture-initiating cells by a clinically applicable protocol (39). 310-317. 26. Lemischka, I. R., Raulet, D. H. & Mulligan, R. C. (1986) Cell 45, The increased expression of DNA repair methyltransferase 917-927. activity in bone marrow cells may be expected to provide 27. Moritz, T. & Williams, D. A. (1994) Curr. Opin. Hematol. 1, 423-428. resistance to a wide variety of chromotherapeutic alkylating 28. Moritz, T. & Williams, D. A. (1994) Mol. Biol. Can., in press. 29. Nienhuis, A. W., Walsh, C. E. & Liu, J. (1993) in Viruses and Bone agents in addition to BCNU; e.g., cyclophosphamide, melpha- Marrow, ed. Young, N. S. (Dekker, New York), pp. 353-414. lan, chlorambucil, streptozotocin, procarbazine, dacarbazine, 30. Gale, R. P. & Butturini, A. (1989) Lancet ii, 315-317. and busulphan are all expected to produce cytotoxic 06-alkyl- 31. Advisory Committee of the International Autologous Bone Marrow Transplant Registry (1989) Lancet ii, 317-318. guanine DNA adducts that could be repaired by the MGMT 32. Williams, D. A., Hsieh, K., DeSilva, A. & Mulligan, R. C. (1987) J. protein (4). Furthermore, all of these agents are expected to Exp. Med. 166, 210-218. produce 3-alkyladenine DNA adducts that may be subject to 33. Sorrentino, B. P., Brandt, S. J., Bodine, D., Gottesman, M., Pastan, R., Cline, A. & Nienhuis, A. W. (1992) Science 257, 99-103. repair by 3-methyladenine DNA glycosylases (11, 34, 40, 41) 34. Mitra, S. & Kaina, B. (1993) Prog. Nucleic Acid Res. Mol. Biol. 44, and we recently found that 3-methyladenine levels are ex- 109-142. tremely low in mouse bone marrow (B.J.G. and L.S., unpub- 35. Gerson, S. L., Miller, K. & Berger, N. A. (1985) J. Clin. Invest. 76, of 3-methyladenine 2106-2114. lished). Thus, the increased expression 36. Berardi, A. C., Wang, A., Levine, J. D., Lopez, P. & Scadden, D. T. DNA glycosylase in bone marrow stem cells may also provide (1995) Science 267, 104-108. resistance to the toxic effects of these chemotherapeutic 37. Morrison, S. J., Uchida, N. & Weissman, I. L. (1995) Annu. Rev. Cell of retroviral Dev. Biol. 11, 35-71. alkylating agents. Indeed, with the development 38. Brenner, M. K., Rill, D. R., Holladay, M. S., Heslop, H. E., Moen, polycistronic gene expression vectors (42), it may be possible R. C., Buschle, M., Krance, R. A., Santana, V. M., Anderson, W. F. & to simultaneously express the MGMT and 3-methyladenine Ihle, J. N. (1993) Lancet 342, 1134-1137. 39. Moritz, T., Patel, V. P. & Williams, D. A. (1994) J. Clin. Invest. 93, DNA glycosylase repair proteins in alkylation repair-deficient 1451-1457. bone marrow cells, thus providing an even higher level of 40. Matijasevic, Z., Boosalis, M., Mackay, W., Samson, L. & Ludlum, protection against alkylation-induced cytotoxicity. D. B. (1993) Proc. Natl. Acad. Sci. USA 90, 11855-11859. 41. Engelward, E. P., Boosalis, M. S., Chen, B. J., Beng, A., Siciliano, M. J. & Samson, L. D. (1993) Carcinogenesis 14, 175-181. R.M., D.A.W., and L.S. are supported by National Cancer Institute 42. Zitvogel, L., Tahara, H., Cai, 0., Storkus, W. J., Muller, G., Wolf, Program Project Grant SPOl CA59348. L.S. is supported by National S. F., Gately, M., Robbins, P. D. & Lotze, M. T. (1994) Hum. Gene Cancer Institute Grant CA55042 and is a Burroughs Wellcome Ther. 5, 1493-1506. Downloaded by guest on October 2, 2021