Reciprocal relationship between O6-methylguanine-DNA methyltransferase P140K expression level and chemoprotection of hematopoietic stem cells

Abstract

Retroviral-mediated delivery of the P140K mutant O(6)-methylguanine-DNA methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSC) has been proposed as a means to protect against dose-limiting myelosuppressive toxicity ensuing from chemotherapy combining O(6)-alkylating agents (e.g., temozolomide) with pseudosubstrate inhibitors (such as O(6)-benzylguanine) of endogenous MGMT. Because detoxification of O(6)-alkylguanine adducts by MGMT is stoichiometric, it has been suggested that higher levels of MGMT will afford better protection to gene-modified HSC. However, accomplishing this goal would potentially be in conflict with current efforts in the gene therapy field, which aim to incorporate weaker enhancer elements to avoid insertional mutagenesis. Using a panel of self-inactivating gamma-retroviral vectors that express a range of MGMT(P140K) activity, we show that MGMT(P140K) expression by weaker cellular promoter/enhancers is sufficient for in vivo protection/selection following treatment with O(6)-benzylguanine/temozolomide. Conversely, the highest level of MGMT(P140K) activity did not promote efficient in vivo protection despite mediating detoxification of O(6)-alkylguanine adducts. Moreover, very high expression of MGMT(P140K) was associated with a competitive repopulation defect in HSC. Mechanistically, we show a defect in cellular proliferation associated with elevated expression of MGMT(P140K), but not wild-type MGMT. This proliferation defect correlated with increased localization of MGMT(P140K) to the nucleus/chromatin. These data show that very high expression of MGMT(P140K) has a deleterious effect on cellular proliferation, engraftment, and chemoprotection. These studies have direct translational relevance to ongoing clinical gene therapy studies using MGMT(P140K), whereas the novel mechanistic findings are relevant to the basic understanding of DNA repair by MGMT.

Figure 1

Retrovirus vectors and in vivo selection/protection of transduced bone marrow cells. A, schematic representation of SIN-MGMT retroviral vectors. Vectors were derived by modification of Sin.SF, Sin.EFS, and Sin.PGK (29). Abbreviations: SF, enhancer/promoter from the SFFV LTR; EFS, truncated form of the promoter/enhancer from the human elongation factor 1α 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; ΔU3, deletion in the enhancer/promoter region of the retroviral LTR; Ψ, 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.

Figure 2

Repair of temozolomide-induced DNA damage. A, residual levels of O⁶-methylguanine in the nuclear DNA of treated transduced cells. Mice were sacrificed 2 h posttreatment with O⁶-benzylguanine/temozolomide as described in Materials and Methods. Bone marrow was pooled from three mice within each experimental group and lin^– transduced cells were isolated by flow sorting. Isolated cells were then subjected to immunostaining for O⁶-methylguanine adducts as described in Materials and Methods. The mean arbitrary nuclear fluorescence units (AFU) of ≥100 cells are shown minus background. Representative of two experiments. □, SF-IG; ■, SF-MGMT; , 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).

Figure 3

Competitive transplantation of MGMT vector–transduced cells. A, schematic representation of competitive repopulation assay used to directly compare the engraftment potential of bone marrow populations expressing different MGMT vectors. B, competitive repopulation of transduced bone marrow. Lethally irradiated recipient mice were injected with a mixture of sorted bone marrow transduced with the indicated vectors and freshly isolated bone marrow. At 6 mo posttransplant, the contribution of each gene-modified population to the production of peripheral blood leukocytes was analyzed by flow analysis. A portion of the harvested bone marrow was transplanted into lethally irradiated secondary recipient mice as described in Materials and Methods. At 15 wk posttransplant, the frequency of each gene-marked population in the peripheral blood of secondary recipients was determined by flow analysis. Percentage of SF-MGMT– (■), EFS-MGMT– ( ), or PGK-MGMT–transduced ( ) cells in the peripheral blood. Columns, mean of eight mice per group; bars, SD. **, P < 0.01; *, P < 0.05, versus the SF-MGMT GFP⁺ graft in the same recipients. SF-MGMT Venus⁺ transduced bone marrow is used to control for bias due to type of fluorochrome used.

Figure 4

Very high expression of MGMT^P140K causes a proliferation defect in 32D cells and correlates with altered subcellular localization. A, 32D cells were transduced with the indicated retroviral vectors and the percentage of gene-modified cells in culture was analyzed with time in culture. ■, SF-MGMT; □, SF-IG; , EFS-MGMT; , PGK-MGMT; , 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 ×400 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.

Figure 5

Transduced 32D cells expressing high MGMT^P140K show aberrant cell cycle progression. A, sorted transduced 32D cells were isolated by flow sorting based on fluorescence and were serum starved for 16 h. Cells were then resuspended in IL3/10% serum–containing growth media. At 12 and 24 h postinduction, cells were pulse labeled with BrdUrd for 20 min and then fixed and stained. The proportion of cells in each phase of the cell cycle was determined by analysis of BrdUrd incorporation versus 7-amino-actinomycin D (7-AAD) staining. ■, SF-MGMT; □, SF-IG; , EFS-MGMT; , PGK-MGMT *, P < 0.05; **, P < 0.01, compared with SF-MGMT–transduced group. B, lysate from transduced and sorted 32D cells described in A was prepared at 6 h postinduction. These lysates were then subject to immunoblot and probed with the indicated antibodies. Lysate from starved (S) nontransduced cells is shown as control to illustrate the level of p27 in noninduced cells.