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Cell, Tumor, and Stem Cell Biology |
1 Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; 2 Internal Medicine (Cancer Research) and 3 Institute of Cell Biology, University of Duisberg-Essen, Essen, Germany; 4 Department of Experimental Hematology, Hannover Medical School, Hannover, Germany; 5 Carcinogenesis Group, Cancer Research UK Paterson Institute for Cancer Research, Manchester, United Kingdom; and 6 Division of Hematology/Oncology, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts
Requests for reprints: David A. Williams, Children's Hospital Boston, 300 Longwood Avenue, Karp 07212, Boston, MA 02115. Phone: 617-919-2697; Fax: 617-730-0934; E-mail: DAWilliams{at}childrens.harvard.edu.
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Abstract |
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Introduction |
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Several mutant versions of MGMT are resistant to such pseudosubstrate inhibitors, yet retain alkyltransferase activity. Of these, the P140K mutant (MGMTP140K) seems to be optimal, combining resistance to pseudosubstrate inhibitors with efficient repair of cytotoxic O6-alkylguanine adducts (15–18). Thus, one goal of current studies is to achieve transgenic expression of MGMTP140K in bone marrow HSC/P. Coupled with O6-alkylating agent treatment and O6-benzylguanine depletion, tumor cells will be sensitized while simultaneously protecting the bone marrow compartment to effect a widened therapeutic window. Clinical studies are currently under way using gamma-retrovirus vectors expressing MGMTP140K in patients with poor-prognosis brain tumors (19). In addition, we and others have shown that treatment of mice and dogs that have been transplanted with HSC transduced with O6-benzylguanine–resistant MGMT vectors allows significant in vivo selection of transduced cells (16, 20–23).
The design of safer retroviral vectors has been an area of intense interest in the field of gene therapy since the occurrence of leukemia due to vector insertional activation of the LIM-only protein 2 (LMO2) proto-oncogene in the gene therapy trial for X-linked severe combined immunodeficiency (24). Further observations from clinical trials, as well as numerous in vitro and in vivo assays, have shown that the powerful enhancer elements present in the long terminal repeats (LTR) of gamma-retroviral vectors can readily alter cell fate by insertional up-regulation of genes that confer a growth advantage (reviewed in ref. 25). One potential solution to this side effect of retroviral insertion is to use internal promoter elements in the context of deletions of the 3' LTR viral enhancer, so-called "self-inactivating" or SIN vectors. These modified vectors have recently been shown to have attenuated ability to activate a variety of growth-promoting genes in an in vitro immortalization assay (26). In addition, by coupling the use of a SIN vector backbone with an internal promoter, which has reduced enhancer activity compared with the gamma-retroviral LTR, it is possible to further reduce the risk of transformation via insertional transactivation (27).
The application of this approach to the design of vectors with which to express MGMTP140K creates a conundrum because the detoxification of O6-alkylguanine adducts by MGMT is a stoichiometric process. Thus, whereas using a weaker enhancer/promoter element to drive expression of MGMTP140K may decrease the risk of insertional mutagenesis, the success of such vectors in chemoprotection and selection may be diminished. In addition, theoretically the risk of cell transformation may be increased due to increased frequency of unresolved O6-alkyl adducts related to the lower levels of MGMTP140K protein.
In this study, we used a panel of SIN gamma-retroviral vectors that express a range of MGMTP140K activities to establish the level of MGMT expression that is optimal for selection/protection of HSC/P. Importantly, the promoter/enhancer elements used in these vector constructs have differing potential to promote insertional mutagenesis (27). We show that the lower-level expression of MGMTP140K driven by weaker cellular promoters is sufficient for selection of HSC following O6-benzylguanine/temozolomide treatment and detoxifies O6-methyguanine lesions to the level of untreated controls. Surprisingly, we also found a previously undescribed deleterious effect of very high overexpression of MGMTP140K on HSC reconstitution in vivo and cell proliferation in vitro. These studies may further elucidate the function(s) of MGMT protein and have important implications for the use of MGMTP140K vectors in clinical trials.
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Materials and Methods |
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Mice. All animals were maintained in a specific pathogen-free environment and all experiments were approved by the Institutional Animal Care and Use Committee of Cincinnati Children's Research Foundation. Both C57BL/6J (referred to as C57B6 hereafter) and B6.SJL-Ptprca Pepcb/BoyJ mice were obtained from The Jackson Laboratory. C57BL/6J pUR288(lacZ)-transgenic mice (referred to as lacZTG) have previously been described (28).
Generation of viral vectors. SIN gamma-retroviral vectors used in this study have previously been described (29). A cassette consisting of the encephalomyocarditis virus internal ribosome entry site, followed by either enhanced green fluorescent protein (eGFP) or Venus, was inserted directly 3' of the MGMTP140K cDNA (Fig. 1A ). Viral supernatants were generated as previously described (30). Vector titers were in the range of 8.0 x 105 to 8.4 x 106 IU/mL.
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Transplant and analysis of chimerism. Bone marrow was injected into the tail vein of lethally irradiated recipient mice [11.75 Gy, 56 cGy/min, 135Cs source Mark I Model 68A Irradiator (J.L. Shepherd and Associates), split dose]. Peripheral blood cell counts were obtained using a Hemavet 850 FS Hematology Analyzer (Drew Scientific). Following red cell lysis (Pharm Lyse, Becton Dickinson), cells were either directly assessed for fluorescence (FACS Canto, Becton Dickinson) or stained with fluorescently labeled monoclonal antibodies [antimouse CD3
(145-2C11), B220 (RA3-6B2), TER-119, GR1 (RB6-8C5), MAC1 (M1/70), CD5 (53-7.3), and CD8a (53-6.7); all from E-Bioscience] following the manufacturer's instructions, before flow analysis. Viable cells were identified by exclusion of 7-amino-actinomycin D.
Drug treatment. O6-Benzylguanine (Sigma) was dissolved at 7.5 mg/mL, using sonication for 1 h at 4°C in polyethylene glycol 400 (Electron Microscopy Sciences). The stock was further diluted to 2.5 mg/mL using ice-cold PBS and then injected i.p. into mice at 30 mg/kg. Temozolomide (Chemodex) was dissolved at 40 mg/mL in DMSO and was diluted to 8 mg/mL in PBS and placed on ice and immediately injected i.p. into mice at 80 mg/kg. Temozolomide was administered 2 h after O6-benzylguanine, and this schedule was repeated on 2 consecutive days.
Analysis of apoptosis and cell cycle. Cell cycle analysis was done using the Becton Dickinson APC bromodeoxyuridine (BrdUrd) Flow Labeling Kit according to the manufacturer's instructions. 32D cells were starved for 16 h in IMDM, 1% FCS and were then incubated in media with 10% FCS, 10 ng/mL mIL3 at 37°C for the time periods indicated. 32D cells were then labeled with BrdUrd for 20 min at 37°C before fixation.
Quantification of active MGMT. Quantification of MGMT activity was done as described by Watson and Margison (31).
Immunoblots. The lysate from 5 x 105 cells was subjected to immunoblot as described by Gu and colleagues (32). The following primary antibodies were incubated with the membranes at the indicated dilutions: rabbit anti-p21 (C-19; Santa Cruz; 1:1,000); rabbit polyclonal anti-p27 (Cell Signaling Technology; 1:1,000); mouse anti–cyclin D1 (DCS6; Cell Signaling Technology; 1:200); mouse anti–cyclin E (E-4; Santa Cruz; 1:500); mouse anti–β-actin (AC-15; Sigma; 1:5,000); mouse anti-MGMT (MT3.1; Millipore; 1:1,000); and rabbit polyclonal anti–phospho-RB (Ser780) (Cell Signalling Technology; 1:1,000). The membranes were then washed thrice and incubated with the appropriate secondary antibody [antirabbit IgG-horseradish peroxidase (HRP) conjugate or antimouse IgG-HRP conjugate, both from Cell Signaling Technologies, diluted 1:2,500]. HRP was visualized using either Lumiglo (Cell Signaling Technologies) or Supersignal West Femto (Pierce). The separation of soluble proteins from chromatin- and nuclear matrix–bound proteins before immunoblot was done as described (33).
Immunocytologic assay for DNA adducts and Comet assay. Lineage-negative (lin–) bone marrow cells were isolated from mice 2 h after treatment with O6-benzylguanine and temozolomide as described above. Immunocytologic assay for DNA alkylation products by adduct-specific antibodies was done as described (34) using either mouse anti–(O6-methylguanine) monoclonal antibody EM2-3 (35) or rabbit anti–imidazole-ring opened (N7-methylguanine) serum (kindly provided by Dr. A. Povey, University of Manchester, Manchester, United Kingdom). The alkaline comet assay was done on sorted lin– bone marrow cells as described (36). Quantification of fluorescence signals relating to DNA adducts and DNA strand breaks was also as described (36).
Immunofluorescence intracellular staining for MGMT. 3T3 cells were grown on glass coverslips in 12-well non–tissue-culture-coated dishes (Becton Dickinson). Fixation and permeabilization were done using the Becton Dickinson Cytofix/Cytoperm Fixation/Permeabilization Solution Kit according to the manufacturer's instructions. The samples were then incubated with mouse anti-MGMT (MT3.1, Millipore) diluted 1:100 in Permeabilization/Wash solution for 1 hour at room temperature. After washing, samples were incubated in Rhodamine-conjugated donkey anti-mouse IgG secondary antibody (Jackson Immunoresearch Laboratories) at 37°C for 45 min. After washing with Permeabilization/Wash solution, samples were mounted using Vectashield containing 4',6-diamidino-2-phenylindole (DAPI). Images were obtained on a Zeiss Axiovert 200M microscope (Carl Zeiss Microimaging, Inc.) and images were collected with a Hamamatsu camera using Openlab software (Improvision).
Mutation frequency analysis. Mutation frequency was determined in lacZTG bone marrow as described (37).
Statistical analysis. All quoted errors are SEs of the mean unless otherwise stated. The Student t test or Wilcoxon rank-sum test was used to determine statistical significance dependent on whether all data conformed to a normal distribution as defined by the Shapiro-Wilk test.
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Results |
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3 orders of magnitude (MGMT activities of 2,091 ± 105, 290 ± 5, 450 ± 53, and <3 fmol/µg DNA for SF-MGMT–, EFS-MGMT–, PGK-MGMT–, and SF-IG–transduced bone marrow, respectively). Inverse correlation between MGMT expression and chemoprotection and HSC selection in vivo. To evaluate the degree of protection against O6-alkylating agents conferred by each SIN gamma-retroviral vector in vivo, we conducted competitive engraftment studies using bone marrow that was separately transduced with the panel of vectors shown in Fig. 1A. Transduced and sorted bone marrow was i.v. injected into lethally irradiated recipient mice (4 x 105 per mouse) along with 5 x 105 freshly isolated nontransduced bone marrow. At 7 weeks posttransplant, mice were treated with 30 mg/kg O6-benzylguanine and, 2 hours later, 80 mg/kg temozolomide for 3 consecutive days. The percentage of gene-marked cells in the peripheral blood and the peripheral blood leukocyte count were subsequently monitored for 4 months following treatment. In spite of several previous reports of successful selection using MGMTP140K vectors, mice transplanted with cells transduced with the SF-MGMT vector showed no evidence of in vivo selection (Fig. 1B; Supplementary data 3). In contrast, robust long-term in vivo selection was shown in mice transplanted with both PGK-MGMT– and EFS-MGMT–transduced bone marrow. Surprisingly, these data show that the highest level of MGMTP140K expression was not associated with chemoselection whereas lower levels of expression were sufficient to elicit a survival advantage over nontransduced bone marrow HSC/P cells. As expected, there was no apparent selection of engrafted cells expressing GFP only (SF-IG) compared with the nontreated group.
These results paralleled the protection from peripheral blood leukopenia affected by these same vectors. By 10 weeks posttreatment with O6-benzylguanine/temozolomide, the peripheral leukocyte counts of animals engrafted with SF-IG– or SF-MGMT–transduced cells had not recovered to the range of nontreated controls (Table 1 ). In contrast, mice transplanted with EFS-MGMT– or PGK-MGMT–transduced cells showed recovery of peripheral blood leukocyte counts to control levels by a maximum of 5 weeks posttreatment, despite the graft consisting of a mixed chimera of protected and nonprotected cells. In addition, the magnitude of early leukopenia observed at 2 weeks posttreatment was less severe in the PGK-MGMT and EFS-MGMT groups compared with the SF-IG or SF-MGMT group (Table 1). These data show that in spite of very high levels of expression of MGMTP140K, animals transplanted with cells transduced with SF-MGMT showed less protection from chemotherapy-induced pancytopenia than animals transplanted with vectors expressing lower levels of MGMTP140K.
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Bone marrow cells transduced with SF-MGMT detoxify O6-alkyl adducts in vivo. Given the inferior in vivo selection and chemoprotection conferred by the SF-MGMT vector, we sought to verify that this construct was able to mediate the detoxification of cytotoxic O6-alkylguanine adducts. Bone marrow was harvested 2 hours posttreatment with O6-benzylguanine/temozolomide from transplanted mice as described above. Lin– GFP+/Venus+ bone marrow cells were isolated and O6-alkylguanine adducts were quantified by immunocytologic assay. Unexpectedly, the SF-MGMT–expressing cells showed equivalent repair of O6-alkylguanine adducts comparable to cells expressing lower levels of MGMTP140K via PGK-MGMT and EFS-MGMT vectors. Nonprotected SF-IG–transduced cells harbored 10-fold higher levels of residual lesions (Fig. 2A
). As expected, due to the specificity of MGMT for O6-adducted guanines, the frequency of N7-alkylguanine lesions and of excision repair–induced DNA single-strand breaks as assessed by alkaline comet assay were equivalent across all groups (data not shown). These data show that all groups received a comparable biological dose of temozolomide and suggest that the defect in chemoprotection and selection of SF-MGMT–transduced cells is not due to defective O6-alkylguanine adduct repair or to an altered processing of other DNA alkylation products.
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SF-MGMT–transduced bone marrow shows a competitive engraftment defect in the absence of chemoselection. We next sought to analyze whether very high levels expression of MGMTP140K had a detrimental effect on engraftment of gene-modified HSC in the absence of treatment with temozolomide. To do this, we directly compared engraftment of bone marrow transduced with different vectors expressing MGMTP140K from different promoters (Fig. 1A) within the same recipient mouse. Bone marrow was separately transduced with vectors encoding MGMTP140K from different promoters and coexpressing either eGFP or Venus. Transduced bone marrow cells were isolated by flow sorting and equivalent numbers of eGFP+ or Venus+ cells were coinjected into lethally irradiated recipient mice along with 5 x 105 freshly isolated nontransduced competitor cells (Fig. 3A ). The percentage contribution of each transduced population to the peripheral blood of recipient mice was analyzed at 6 months posttransplant. At the time of sacrifice and analysis, 25% of a femur equivalent of bone marrow from these mice was also transplanted into lethally irradiated secondary recipients to definitively assess HSC activity in each transduced population. The percentage of transduced cells in the peripheral blood of secondary recipients was evaluated at 15 weeks posttransplant.
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32D cells transduced with SF-MGMT show a growth defect. To better identify the potential mechanism(s) by which very high levels of MGMTP140K have a detrimental effect on HSC engraftment, we sought to determine if MGMTP140K expression was detrimental to the growth of the 32D myeloid progenitor cell line. 32D cells were separately transduced with the SF-MGMT, EFS-MGMT, and PGK-MGMT vectors and the percentage of gene-marked cells was followed with time in culture. As seen in primary bone marrow, expression of MGMT was highest in 32D cells transduced with SF-MGMT and considerably lower in cells transduced with PGK-MGMT and EFS-MGMT (MGMT activities of 1,532 ± 147, 371 ± 14, 185 ± 7, and <3 fmol/µg DNA for SF-MGMT–, EFS-MGMT–, PGK-MGMT–, and SF-IG–transduced 32D cells, respectively). In the absence of drug treatment, SF-MGMT–transduced 32D cells showed a pronounced growth defect resulting in an 50% reduction in the frequency of transduced cells over 3 weeks of culture (Fig. 4A
). In contrast, there was no significant change in the percentage of gene-marked cells in cultures containing SF-IG–, EFS-MGMT–, or PGK-MGMT–transduced cells, indicating that low levels of MGMTP140K expression did not alter cell growth in culture relative to the nontransduced 32D cells. The growth defect exhibited by SF-MGMT–transduced 32D cells was confirmed at the clonal level, as there was a 40% reduction in colony formation of these cells compared with 32D cells expressing only eGFP (Supplementary data 4). Finally, there was a >30% reduction in tritiated thymidine uptake by SF-MGMT–transduced 32D cells compared with control groups (Supplementary data 4).
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2-fold increase in levels of MGMT relative to MGMTP140K-transduced cells (MGMT activities of 1,230 and 2,330 fmol/µg DNA for P140K- and WT MGMT–transduced cells, respectively). To determine if the MGMTP140K mutant protein expression mimicked WT protein with respect to intracellular distribution, we next examined the localization of protein in transduced cells. To provide better visualization of protein localization, we used NIH 3T3 fibroblasts and examined fluorescence distribution. As shown in Fig. 4B, WT MGMT was expressed in both the nucleus and the cytoplasm. As a control, the non–DNA-binding mutant MGMTY114E (39) was examined and seemed to be predominantly cytoplasmic, suggesting that DNA binding is required for nuclear retention. In marked contrast to WT protein, MGMTP140K was almost exclusively localized to the nucleus (Fig. 4B; Supplementary data 5). MGMTP140K expressed at lower levels via the PGK-MGMT and EFS-MGMT vectors was also restricted predominantly to the nucleus, suggesting that the restricted localization was independent of expression level. Additionally, the largely nuclear localization of MGMTP140K was not observed when a range of other MGMT mutants were used (Supplementary data 5).
The lack of cytoplasmic MGMTP140K expression compared with other MGMT mutants was also observed in transduced 32D cell lines, but localization in these cells was more difficult to discern due to the low ratio of cytoplasmic/nuclear volume (data not shown). Therefore, to examine these cells, transduced 32D cells were fractionated by differential lysis of nuclear and cytoplasmic components and analyzed by immunoblot. MGMTP140K expression was largely restricted to the chromatin-bound fraction (Fig. 4C). In contrast, WT MGMT was abundant in both the chromatin-bound fraction and the cytoplasmic fraction, whereas the MGMTY114E mutant was expressed primarily in the cytoplasmic fraction. Overall, these data show that the defect in growth of 32D cells overexpressing MGMT is specific to MGMTP140K, and in multiple cell types MGMTP140K shows a more restricted localization to the nucleus, correlating with elevated association with chromatin. This restriction may be associated with DNA binding as the Y114E mutant is predominantly cytoplasmic and does not associate with chromatin.
SF-MGMT expression in 32D cells interferes with cell cycle progression. To determine the basis of the impaired growth of 32D cells transduced with SF-MGMT, cell cycle analysis was done 12 and 24 hours following release from serum starvation. SF-MGMT–transduced 32D cells showed a pronounced delay in the G0/G1 to S phase transition compared with SF-IG–, PGK-MGMT–, and EFS-MGMT–transduced cells (Fig. 5A ). This delay in cell cycle was associated with a 1.4- to 1.6-fold reduction in SF-MGMT–transduced cells in S phase at 12 hours postinduction, compared with SF-IG–, PGK-MGMT–, and EFS-MGMT–transduced cells, and a 1.2-fold reduction at 24 hours postinduction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...References |
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In this study, we show that weaker human cellular promoters located internal to the viral LTRs are sufficient to drive the expression of MGMTP140K in HSC/P at levels that efficiently repair O6-alkylguanine DNA adducts. This repair leads to protection and in vivo selection of gene-modified HSC after combined pharmacologic inhibition of WT MGMT and temozolomide treatment. Although the EFS and PGK promoters mediate low-level MGMTP140K expression relative to vectors that incorporate the viral SF promoter, the level of exogenous MGMT activity encoded by these vectors is still 2 orders of magnitude higher than endogenous MGMT activity in hematopoietic cells. Because vectors that incorporate weaker enhancer elements are thought to reduce the risk of insertional mutagenesis, these data suggest that the combination of the EFS or PGK promoter with the MGMTP140K cDNA in a SIN retroviral backbone makes up a superior vector configuration to develop and test in the next generation of clinical trials.
Very high levels of MGMTP140K precluded efficient selection and protection of gene-modified HSC/P, an unexpected finding given the many previous reports of successful use of this cDNA. This finding seems to be unrelated to the efficiency of repair of the O6-methylguanine lesion. In support of this finding, we define an engraftment defect resulting from very high levels of MGMTP140K expression. The observed defect in cellular proliferation seems to be specific to the overexpression of the P140K mutant of MGMT. We surmise that very high levels of MGMTP140K may result in defective in vivo HSC/P protection and selection due to a proliferation defect analogous to the one we define in 32D cells.
In addition, MGMTP140K protein shows an unusual localization to the nucleus/chromatin relative to WT MGMT. This mislocalization of protein is apparently related to DNA binding. The increased propensity of MGMTP140K protein to bind to chromatin could be responsible for the observed proliferation defect. However, if this is the case, there is a threshold for this effect because vectors using weaker cellular promoters to drive expression of MGMTP140K result in intracellular levels of protein that do not elicit a perceptible proliferation defect but show similar protein distribution abnormalities. An excess of mislocalized protein could be deleterious to several nuclear processes (e.g., by inhibiting the synthesis of DNA and/or RNA or interfering with gene transcription or replication functions). This hypothesis would correlate with the observation that MGMT localizes to the sites of active transcription in a cell cycle–specific manner in the absence of treatment with an O6-alkylating agent (40). These data also imply that retroviral vectors containing weak internal promoters in the SIN configuration to express MGMTP140K may be superior in the context of a clinical trial of chemoprotection. This conclusion is in contrast to conventional predications, which would maintain that the achievement of optimal chemoprotection of HSC/P would require higher levels of MGMT expression.
In the setting of numerous previous studies that have documented effective in vivo protection and selection of HSC using retroviral overexpression of MGMTP140K (16, 18, 21–23, 41), the data presented here showing that very high levels of this protein result in a deleterious effect on HSC engraftment seem to be unique. The experimental parameters leading to this observation likely include the level of expression of MGMTP140K, which was achieved using the combination of the spleen focus-forming virus (SFFV) promoter and the Pre element within this retroviral construct. Using 32D cells, we observed that the SF-MGMT vector mediated a 10-fold higher level of MGMT activity compared with a monocistronic MSCV vector encoding MGMTP140K coupled with a Pre (data not shown), which is currently being used in clinical trials (19). Indeed, a 28-fold differential of transgene expression level has previously been documented between a gamma-retroviral vector incorporating the SFFV enhancer coupled with the Pre, versus the weaker MSCV-based backbone (42). Thus, the level of expression of MGMTP140K mediated by the SF-MGMT vector used in this study likely exceeds that achieved by other vector configurations that have previously been reported.
With respect to the chemotherapeutic regimen used, we have attempted to model a high-dose myelosuppressive schedule administered soon after bone marrow transplant. This model may mimic schedules used in some clinical protocols designed for HSC chemoprotection for the purpose of increasing dose intensity for antitumor therapy. A similar treatment schedule could in the future be used to select for transduced HSC/P in autologous reinfusion and treatment of genetic diseases. This approach places a significant proliferative stress on reconstituting HSC/P, increasing the likelihood that a modest proliferation defect would be noted. In contrast, other published studies have used less severe chemotherapeutic regimens. Examples of these schedules include using a gradual dose escalation of temozolomide, delaying the administration of drug until after bone marrow reconstitution is complete, and/or using an extended schedule that allows more time for hematopoietic recovery between rounds of treatment (21–23, 41). Thus, these less dose-intense or time-compressed treatment schedules may mask any reconstitution defect of gene-modified HSC by reducing the proliferative stress on these cells.
Aside from the direct translational application of our findings to clinical trials using gene transfer of MGMT for HSC chemoprotection, these studies may also have importance for further understanding the basic biology of MGMT. For example, these studies show that MGMTP140K may have a higher binding affinity for nonalkylated DNA. One might interpret this finding as a possible explanation for the slower kinetics of O6-alkylguanine detoxification by MGMTP140K relative to WT protein (29) in that increased DNA binding affinity may correlate with a reduced rate of processive scanning of DNA. Furthermore, understanding the precise mechanism(s) through which very high levels of MGMTP140K elicit an effect on cell cycle regulatory proteins may allow further insight into the coordination of DNA repair and cellular proliferation in HSC, where a large fraction of cells remain in a quiescent state for prolonged periods.
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Disclosure of Potential Conflicts of Interest |
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Elke Grassman, Ina Rattmann, Todd Schuesler, Anthony Pegg, Victoria Summey, Shelli Homan, Paul Andreasson, and Christina Sexton for technical assistance and helpful discussions, and Eva Meunier for administrative support.
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Footnotes |
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Received 1/25/08. Revised 3/22/08. Accepted 4/11/08.
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References |
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retroviral and lentiviral SIN vectors for expression of O6-methylguanine-DNA methyltransferase in hematopoietic cells. Mol Ther 2006;13:391–400.[CrossRef][Medline]This article has been cited by other articles:
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  H. Zhao, T. I. Pestina, M. Nasimuzzaman, P. Mehta, P. W. Hargrove, and D. A. Persons Amelioration of murine {beta}-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both {gamma}-globin and the MGMT drug-resistance gene Blood, June 4, 2009; 113(23): 5747 - 5756. [Abstract] [Full Text] [PDF]
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M. D. Milsom, B. Schiedlmeier, J. Bailey, M.-O. Kim, D. Li, M. Jansen, A. M. Ali, M. Kirby, C. Baum, L. J. Fairbairn, et al. Ectopic HOXB4 overcomes the inhibitory effect of tumor necrosis factor-{alpha} on Fanconi anemia hematopoietic stem and progenitor cells Blood, May 21, 2009; 113(21): 5111 - 5120. [Abstract] [Full Text] [PDF]
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B. C. Beard, R. Sud, K. A. Keyser, C. Ironside, T. Neff, S. Gerull, G. D. Trobridge, and H.-P. Kiem Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients Blood, May 21, 2009; 113(21): 5094 - 5103. [Abstract] [Full Text] [PDF]
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gene; PGK, promoter/enhancer from the human phosphoglycerate kinase gene; MGMT, the human O6-methylguanine-DNA methyltransferase cDNA (P140K mutant unless otherwise stated); IRES, the internal ribosome entry site from encephalomyocarditis virus; eGFP, enhanced green fluorescent protein; Venus, modified version of yellow fluorescent protein; Pre, truncated version of the woodchuck hepatitis virus posttranscriptional regulatory element that lacks any X protein coding sequence; 
U3, deletion in the enhancer/promoter region of the retroviral LTR; 
, packaging signal. B, change in the percentage chimerism of gene-marked cells following treatment with O6-benzylguanine/temozolomide. Lethally irradiated recipient mice were injected as described in Materials and Methods. Mice from each cohort were then either treated or not with a O6-benzylguanine/temozolomide regimen at 7 wk posttransplant. The percentage of GFP+ cells in the peripheral blood at 15 wk posttreatment versus 1 wk pretreatment was used to determine the change in frequency of gene-marked cells with chemoselection. 
, nontreated age-matched control mice; 
, O6-benzylguanine/temozolomide treatment. **, P < 0.01 (Wilcoxon's rank sum test); ns, not significant. Data represent the sum of three independent experiments with 11 to 23 mice per experimental group. C, Kaplan-Meier plot of survival of O6-benzylguanine/temozolomide–treated mice from each experimental group with time posttreatment. Vertical arrows, timing of O6-benzylguanine/temozolomide (6BG/TMZ) treatment. **, P < 0.01, versus other groups (log-rank test). Data represent the sum of three independent experiments with 13 to 23 mice per experimental group.
100 cells are shown minus background. Representative of two experiments. 
, EFS-MGMT; 
, PGK-MGMT. **, P < 0.01, versus all other groups (Student's t test). B, absolute mutation frequency in transduced bone marrow. At 6 mo posttreatment, the surviving mice were sacrificed. Bone marrow was harvested and LacZ reporter plasmids were subsequently rescued from the resulting genomic DNA as described in Materials and Methods. The mutation frequency in the rescued reporter plasmids was then determined. Each cross represents the mutation frequency determined for bone marrow from a single mouse. Dashed line, the upper range of mutation frequency in nontreated controls (CON).
, SF-MGMT vector containing WT cDNA. Columns, mean of three to nine independent experiments; bars, SD. **, P < 0.01, compared with other groups (Student's t test). B, NIH 3T3 fibroblast cells were transduced with the indicated retroviral vectors. Cells were fixed and stained with DAPI and anti-MGMT antibody. Photomicrographs of fixed and stained cells were at x400 magnification. Bar, 9.75 µm. C, 32D cells were transduced with SF-MGMT vectors containing the WT MGMT sequence, the P140K mutant, or the Y114E mutant as a non–DNA-binding control. Transduced cells were isolated by flow sorting and cells were lysed and cellular constituents separated as described in Materials and Methods before immunoblot with the indicated antibody. SC, cytoplasmic and soluble cytoplasmic fraction; SN, soluble nuclear fraction; C, chromatin/nuclear matrix–bound fraction. Lanes 1 to 3, P140K; lanes 4 to 6, WT; lanes 7 to 9, Y114E.