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Clinical Cancer Research Vol. 5, 163-169, January 1999
© 1999 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Simultaneous Protection of G156A Methylguanine DNA Methyltransferase Gene-transduced Hematopoietic Progenitors and Sensitization of Tumor Cells Using O6-Benzylguanine and Temozolomide1

Jane S. Reese, Brian M. Davis, Lili Liu and Stanton L. Gerson2

Division of Hematology/Oncology, Case Western Reserve University and University Hospitals Ireland Cancer Center, Cleveland, Ohio 44106-4937


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
O6-Benzylguanine (BG) potentiates temozolomide (TMZ) cytotoxicity in tumors by inactivating O6-alkylguanine DNA alkyltransferase but also increases toxicity in hematopoietic cells. To improve the hematopoietic cell tolerance to alkylating agents, we retrovirally transduced the BG-resistant mutant G156A methylguanine DNA methyltransferase gene ({Delta}MGMT) into hematopoietic progenitors and evaluated whether {Delta}MGMT expression in hematopoietic colony-forming units would result in greater drug resistance to TMZ. {Delta}MGMT expression in human and mouse colony-forming units followed by BG treatment resulted in a >7.7-fold increase in the TMZ 90% inhibitory concentration (IC90) and a 5.6-fold increase in the 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) IC90 relative to untransduced cells. This degree of protection enabled {Delta}MGMT-transduced CD34 cells to become much more resistant to BG and TMZ than SW480 cells, which express high O6-alkylguanine DNA alkyltransferase and are normally resistant to TMZ or BCNU alone. {Delta}MGMT-transduced long-term culture initiating cells were also resistant to the BG and TMZ combination, as were untransduced long-term culture initiating cells, suggesting that noncycling early progenitors may be partially protected from TMZ. These data indicate that retroviral transduction of {Delta}MGMT into hematopoietic progenitors followed by BG and TMZ treatment may selectively protect hematopoietic cells more efficiently than BCNU, allowing dose-intensive and repetitive therapy without the risk of cumulative myelosuppression.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The chloroethylating agent BCNU3 and methylating agents such as TMZ, procarbazine, and dacarbazine form adducts at the O6 position of guanine. These agents are associated with cumulative and delayed myelosuppression and secondary leukemia due to early hematopoietic progenitor mutagenic damage. Repair of O6 adducts is mediated by the DNA repair protein alkyltransferase (AGT) via covalent transfer of the alkyl group from the O6 position of guanine to its active site cysteine, which inactivates the protein (1) and either restores the DNA to normal in the case of monoadducts or prevents cross-links in the case of chloroethylating agents. If chloroethyl adducts are unrepaired, the intermediate O6-N1-ethanoguanine will form a highly toxic interstrand DNA cross-link with the complementary cytosine nucleotide residue (2) , resulting in an inhibition of RNA transcription and DNA replication. In contrast, cytotoxicity by methylating agents is due to recognition of the O6-MG:cytosine or O6-MG:thymine base mispair (formed after one round of replication) by the mismatch repair complex and the induction of aberrant repair processes. This process, termed abortive mismatch repair, leads to multiple DNA strand breaks and cell death (3) .

AGT is expressed at high levels in many tumors (4 , 5) and is a primary mechanism of tumor resistance to agents that attack the O6 position of guanine. However, human and murine hematopoietic cells express relatively low levels of AGT (6 , 7) , resulting in dose-limiting myelosuppression after nitrosourea chemotherapy. BG, an AGT inactivator, has been shown to overcome AGT-mediated tumor resistance to chloroethylating agents and methylating agents in vitro (8, 9, 10, 11) and in multiple xenograft studies (12, 13, 14, 15) , but it simultaneously enhances alkylating agent toxicity to hematopoietic progenitors. Several mutant human AGT proteins have been characterized as resistant to BG while retaining the ability to remove drug-induced O6 alkylation. We have studied the mutant G156A human MGMT ({Delta}MGMT), which demonstrated a 240-fold increased resistance to BG inactivation (16) , with the aim of selective protection from BG and BCNU. This resulted in significant protection in vitro of both human CD34-derived hematopoietic progenitors (17) and LTC-ICs (18) as well as murine repopulating progenitors (19) from BG and BCNU cytotoxicity. Furthermore, lethally irradiated mice reconstituted with {Delta}MGMT-transduced hematopoietic progenitors were significantly protected from repeated BG and BCNU exposure compared to mice transplanted with lacZ-transduced progenitors, resulting in reduced myelosuppression and improved survival (19) .

In studies reported herein, we compared the cytotoxicity of BG plus TMZ with that of BG plus BCNU. The use of the BG and TMZ combination is important in this context for the following reasons: (a) TMZ cytotoxicity is mediated solely by O6-MG adducts, which can be repaired by AGT (1) . In contrast, although the primary BCNU toxicity occurs via DNA cross-linking, high AGT expression does not protect against other BCNU-mediated DNA damage and carbamoylation of lysine residues (20) . Whereas only a few cross-links are required for cytotoxicity, over 6000 O6-MG lesions are required for cell death after methylating agent exposure (21) ; and (b) there is evidence in clinical trials that myelosuppression is the predominant toxicity of TMZ treatment, but unlike BCNU, this myelotoxicity is not cumulative (22) . Based on this, it is expected that BG depletion could sensitize cells to TMZ to a greater degree than to BCNU; furthermore, {Delta}MGMT may provide improved selective protection to transduced cells. In a clinical setting, dose escalation of TMZ, when administered with BG, seems much more possible if the marrow can be protected because of the lack of nonhematological toxicity at the current maximum tolerated doses. This is not the case with BCNU, because other organ toxicity (pulmonary and renal) limits dose escalation. In this study, we analyzed the survival of {Delta}MGMT-transduced human and mouse hematopoietic progenitors after treatment with BG and TMZ compared to those treated with BG and BCNU. We found that {Delta}MGMT-transduced CD34 cells are more resistant to BG and TMZ than untransduced or SW480 cells, and BG-treated {Delta}MGMT-transduced cells are more resistant to TMZ than BCNU. Additionally, our results suggest that noncycling early progenitors (LTC-ICs) may be protected from TMZ cytotoxicity.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
CD34 Transduction.
Peripheral blood mononuclear cells were obtained by apheresis from normal donors treated with granulocyte-CSF under an Institutional Review Board-approved protocol. CD34 progenitors were isolated by positive selection using the VarioMACS column (Miltenyi Biotec). For coculture infection, 1 x 105 CD34 cells/ml were cultured with or without MFG-{Delta}MGMT producers as described previously (17) and supplemented with human stem cell factor (100 ng/ml; Amgen Biologicals), IL-3 (100 units/ml) and IL-6 (100 units/ml; both from Systemix), Flt-3 ligand (100 ng/ml; Immunex), and protamine sulfate (4 µg/ml; Sigma). For retroviral supernatant infection, CD34 cells were prepared as described above and cultured in the presence of primary human bone marrow stroma as described previously (17) . Each experiment was performed with cells from a separate donor.

Murine Hematopoietic Progenitor Transduction.
Bone marrow progenitors were obtained from the femur and tibia of 8-week-old male C3H/HeNCrlBR mice (Charles River, Wilmington, MA) 48 h after treatment with 150 mg/kg 5-fluorouracil (Pharmacia, Kalamazoo, MI). Cells (5 x 105 cells/ml) were prestimulated for 24 h in {alpha}-MEM plus 100 units/ml mIL-3 (Genzyme, Cambridge, MA), 100 units/ml mIL-6 (Genzyme), and 100 ng/ml rat SCF (Amgen, Thousand Oaks, CA) and then cocultured for 48 h with Am12 MFG-{Delta}MGMT producer cells that were rendered replication defective by treatment with 5 µg/ml mitomycin C. The cocultures were performed using the cytokines mentioned above plus 6 µg/ml protamine sulfate in {alpha}-MEM supplemented with 20% heat-inactivated FCS, again at a density of 5 x 105 cells/ml.

BG and BCNU or TMZ Treatment of Tumor Cells.
SW480 cells were obtained from American Type Culture Collection and cultured in the appropriate growth medium. For drug treatment, 2000 cells/dish were plated, adhered for 18 h, and exposed to BG for 1 h and exposed to BCNU or TMZ for 2 h as described previously (8) . Colonies containing >50 cells were enumerated after 7 days.

BG and BCNU or TMZ Treatment of Hematopoietic Cells.
Transduced human CD34 cells were pretreated for 1 h with 10 µM BG in serum-free media followed by treatment with either BCNU or TMZ for 2 h. Cells were plated in triplicate in methylcellulose containing SCF, IL-3, hemin, erythropoietin, granulocyte macrophage-CSF, and 5 µM BG, and colonies of >50 cells were enumerated on day 10. Mouse bone marrow cells were separated from producer cells after transduction and pretreated with 0, 10, or 25 µM BG in serum-free media supplemented with pokeweed spleen cell-conditioned medium for 1 h as described previously (19) . Cells were then treated with BCNU or TMZ for 2 h and plated in triplicate in methylcellulose plus rat SCF, mIL-3, erythropoietin, pokeweed mitogen spleen cell-conditioned medium, hemin, and 20 µM BG. Total counts of CFU colonies of >50 cells were enumerated on day 7. Transduction efficiencies of both human and mouse cells were determined by PCR using proviral-specific primers on genomic DNA isolated from individual CFUs.

LTC-IC Assay.
Cells were cultured in 6-well plates (1–10 x 105 cells/well in duplicate for each dose of TMZ) over a confluent layer of allogeneic human bone marrow stroma prepared and irradiated as described previously (17) . Cultures were grown at 33°C and 5% CO2 with half of the culture media [MyeloCult H5100 (Stem Cell Technologies, Vancouver, Canada) supplemented with 10-6M hydrocortisone] replaced weekly. After 5 weeks, cultures were visually assessed for cobblestone formation (cobblestone-forming units are a marker of LTC-ICs). Both nonadherent and adherent cells were harvested and plated in methylcellulose (Stem Cell Technologies) containing SCF, IL-3, hemin, erythropoietin, and granulocyte macrophage-CSF in duplicate to measure secondary CFUs derived from LTC-ICs. Progenitor colonies containing 50 or more cells were enumerated after 12–14 days.

Alkyltransferase Assay.
The measurement of AGT activity in cell extracts was performed as described previously (23) . Briefly, AGT activity was measured in sonicated cell extracts by the removal of the [3H]methyl group from the O6-[3H]MG present in substrate DNA that was prepared by incubating calf thymus DNA with N-[3H]methylnitrosourea for 1 h at 37°C in a HEPES buffer. The alkylated [3H]methyl O6-MG and N7-methylguanine bases were separated by high-performance liquid chromatography and quantified by liquid scintillation. AGT activity was expressed as fmol of O6-MG removed/mg protein. To measure the depletion of AGT after TMZ treatment, bone marrow and CD34+ enriched peripheral blood mononuclear cells from patients pretreated with cyclophosphamide and granulocyte-CSF (24) and a {Delta}MGMT-transduced hematopoietic cell line (K562) were incubated with or without BG for 1 h in serum-free media and exposed to 0–400 µM TMZ for 4 h. Cells were harvested, washed twice with PBS, and frozen for AGT assay.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
{Delta}MGMT Expression and Drug Resistance in Retrovirally Transduced Human CD34 Cells.
We have previously shown that {Delta}MGMT retrovirally transduced human CD34 progenitor cells were 3-fold resistant to BG and BCNU at the IC50 when compared to untransduced cells. In these experiments, we evaluated the relative resistance to TMZ conferred by {Delta}MGMT. After a transduction period of 72–96 h, progenitors were pretreated for 1 h with 10 µM BG followed by exposure to increasing concentrations of TMZ for 2 h and then plated in methylcellulose supplemented with growth factors and 5 µM BG to maintain depletion of endogenous AGT. CFU-granulocyte, macrophage, BFU-E (burst-forming unit erythroid), and CFU-granulocyte, erythrocyte megakaryocyte, macrophage (collectively referred to as CFU) were enumerated. Individual CFUs were collected from 10 independent experiments, each from a different donor (5 coculture and 5 supernatant plus stroma transductions), and demonstrated transduction efficiencies of 67 ± 5% for coculture and 50 ± 11% for exposure to retroviral supernatant plus human stroma infection methods. Clonogenic {Delta}MGMT-transduced cells were 2.5-fold more resistant to BG and TMZ compared to untransduced cells at the TMZ IC50 and >7.7-fold more resistant at the TMZ IC90 (Table 1Citation ; Fig. 1ACitation ), indicating that 10–35% of cells from the transduced culture were highly resistant to TMZ.


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  		Table 1 Drug resistance of hematopoietic progenitors compared to SW480 cells after treatment with or without BG followed by TMZ

 


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  		Fig. 1. Clonogenic survival of transduced CD34+ cells compared to SW480 cells after treatment with BG and TMZ or BCNU. Cells were treated with 10 µM BG before the administration of 0–400 µM TMZ (A) or 0–40 µM BCNU (B). Data points are the means ± SE. The curve of CD34+ cells represents experiments from 15 separate donors. {Delta}MGMT-transduced CD34+ cells, {blacksquare}; SW480 cells, •; untransduced CD34+ cells, {blacktriangleup}.

 
Selective Protection of {Delta}MGMT Hematopoietic Cells versus Tumor Cells.
SW480 is a colon cancer cell line that expresses high AGT and is resistant to both BCNU and TMZ (8) . Our recent studies demonstrated that BG potentiates TMZ and BCNU toxicity in SW480 cells. In these experiments, we asked whether hematopoietic progenitors, rather than being as sensitive as SW480 to BCNU alone, could become more resistant than SW480 to BG and BCNU after {Delta}MGMT transduction. In the presence of BG, {Delta}MGMT-transduced CD34 cultures yielded CFUs that were more resistant to both BCNU and TMZ than SW480 cells. Using survival curves of {Delta}MGMT-transduced, CD34-derived CFUs that had not been preselected for {Delta}MGMT-expressing cells, the ratio of drug resistance of {Delta}MGMT+CD34 versus SW480 was 3.1 at the BCNU IC90 but was >3.6 at the TMZ IC90 (survival of 11% at the highest dose). Thus, even without selection for transduced cells expressing AGT (which is expected to increase the survival observed; Ref. 19 ), pretreatment of CD34 cells with BG to deplete endogenous AGT allows selective protection of hematopoietic progenitors transduced with {Delta}MGMT while sensitizing AGT-overexpressing tumor cells (Tables 1Citation and 2Citation ; Fig. 1, A and BCitation ).


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  		Table 2 Drug resistance of hematopoietic progenitors compared to SW480 cells after treatment with or without BG followed by BCNU

 
Comparison of BCNU and TMZ Cytotoxicity in Hematopoietic Cells.
To evaluate BG potentiation of TMZ compared to BCNU, the relative resistance patterns observed in {Delta}MGMT-transduced cells were compared to untransduced cells. We demonstrate that {Delta}MGMT-transduced cells have greater resistance to the BG and TMZ combination than to BG and BCNU. The ratio of TMZ resistance in {Delta}MGMT-CD34 versus untransduced cells was >7.7 at the IC90 compared to a ratio of 5.6 at the IC90 for BCNU (Fig. 1, A and B)Citation . Additionally, at doses that resulted in a 1% survival of untransduced cells, a mean of 40% of {Delta}MGMT cells survived TMZ treatment, as compared to a mean of 27% of BCNU-treated cells. Of interest, however, the mean proportion of highly resistant cells was similar (approximately 11% for TMZ and 8% for BCNU).

TMZ Resistance in Primary Murine Hematopoietic Progenitors.
We also evaluated BG and TMZ resistance in primary murine bone marrow transduced with wt MGMT, {Delta}MGMT, or lacZ. In these studies, AGT levels were much higher in murine bone marrow cells transduced with wt MGMT than in {Delta}MGMT-transduced cells due to reduced stability of the mutant protein (25) and to lacZ-transduced cells that only express endogenous murine AGT. After transduction, the marrow cells were treated with 0, 10, or 25 µM BG for 1 h followed by a 2-h treatment in 0–1600 µM TMZ; then they were plated in methylcellulose supplemented with 20 µM BG. The resulting survival curves are shown in Fig. 2Citation . Cells transduced with wt MGMT had TMZ IC50 values similar to those of lacZ-transduced marrow, although they demonstrated improved survival at the TMZ IC90, possibly due to incomplete BG inactivation of the provirally expressed wt MGMT in cells expressing high levels. The ratio of resistance of {Delta}MGMT cells versus untransduced cells was 8.6 and 4.7 at the TMZ IC50 and IC90, respectively, compared to 4.6 and >2.2 at the BCNU IC50 and IC90 (Ref. 19 ; Fig. 2Citation ; Tables 1Citation and 2Citation ). Thus, after methylating agent exposure, there is greater relative protection from TMZ than BCNU in {Delta}MGMT-transduced cells, although the levels of AGT in {Delta}MGMT-transduced cells are lower than in wt MGMT transduced cells. These studies lay the foundation for murine xenograft models and other studies to compare the ability to select for MGMT+ long-term repopulating cells in mice after the administration of BG and BCNU or BG and TMZ.



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  		Fig. 2. BG and TMZ resistance in murine hematopoietic progenitors transduced with {Delta}MGMT. Cells were treated with 0 (A), 10 (B), or 25 µM (C) BG for 1 h and 0–1600 µM TMZ for 2 h. Data are the means ± SD from five independent marrow preparations. {Delta}MGMT-transduced progenitors, {blacksquare}; wt, •; lacZ-transduced cells, {blacktriangleup}.

 
AGT Activity in {Delta}MGMT-transduced Cells.
Although cells expressing {Delta}AGT demonstrated an overall greater resistance to low-dose TMZ than BCNU in the presence of BG, the cells were equally sensitive at higher doses. This suggests that an abundance of O6-MG lesions deplete AGT, reducing the protective effect. To confirm this, we measured the kinetics of AGT depletion in human hematopoietic cells and {Delta}MGMT-transduced K562 cells after treatment with TMZ. Bone marrow and peripheral blood progenitors that exhibit AGT levels comparable to CD34+ cells (6 , 26) were studied because it is technically impractical to measure AGT depletion in CD34+ cells, due to the number of cells required for biochemical assay at each drug concentration. Fifty percent of AGT activity remained after 45 and 65 µM TMZ in bone marrow and peripheral blood, respectively (Fig. 3A)Citation . In both samples, AGT activity was reduced to <10% after 100 µM TMZ. The inactivation curve was shifted to the right in K562 cells transduced with {Delta}MGMT and selected for drug resistance. BG and TMZ treatment of these K562-{Delta}MGMT cells resulted in a TMZ EC50 for AGT depletion of 18 µM(Fig. 3B)Citation . At 200 µM TMZ, 37% of {Delta}AGT activity remained, and at 400 µM, 20% of {Delta}AGT was active. These results demonstrate that high concentrations of TMZ depleted wt or mutant AGT in human hematopoietic cells due to the repair of O6-MG adducts and may have diminished the protective effect of the {Delta}AGT over that of endogenous AGT.



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  		Fig. 3. Inactivation of wt or {Delta}AGT after TMZ. Bone marrow (•) and peripheral blood progenitors ({blacksquare}) were exposed to 0–250 µM TMZ for 4 h (A), and {Delta}MGMT-transduced K562 cells were incubated with ({diamondsuit}) or without ({blacktriangleup}) BG for 1 h followed by 0–400 µM TMZ for 4 h (B). Cells were washed free of the drugs and harvested immediately, and AGT activity in protein extracts was determined by the biochemical assay. Each data point represents the percentage of AGT activity relative to that of untreated control.

 
TMZ Resistance of LTC-ICs.
To determine the effect of TMZ on quiescent early hematopoietic progenitors and the degree of protection provided by {Delta}MGMT, transduced and untransduced human CD34 cells were subject to long-term survival for LTC-ICs. Some cultures were established after treatment with BG and 10, 100, 250, and 400 µM TMZ. After 5 weeks of culture, individual wells were scored for the presence of stromal-associated hematopoietic clones (cobblestone-forming units), and both adherent and nonadherent cells were plated in methylcellulose for secondary CFU analysis. We evaluated the drug resistance of LTC-ICs, as determined by either cobblestone formation or secondary CFU growth. In four experiments, 26% of untransduced LTC-ICs survived 10 µM BG and 100 µM TMZ, a dose that killed >99% of primary CFUs. This suggests that quiescent LTC-ICs are more resistant to BG and TMZ than proliferating committed progenitor cells. Analysis of LTC-ICs derived from cultures of {Delta}MGMT-transduced CD34 cells indicated that 19% (12 of 64 secondary CFUs) were transduced. This is lower than the transduction rate of 50% in primary CFUs. {Delta}MGMT-transduced LTC-ICs showed a BG and TMZ resistance similar to that of untransduced LTC-ICs, with a 21% survival at up to 400 µM TMZ. Thus, TMZ treatment did not appear to enrich for transduced secondary CFUs. This is in contrast to our previous data showing BG and BCNU enrichment for transduced LTC-ICs (18) .


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
AGT is the primary repair mechanism of alkylating agent damage at the O6 position of guanine. The sensitivity of bone marrow and tumor resistance to these agents can be explained by low and high AGT levels in these cells, respectively. Therefore, the gene transfer approach described here was devised to provide a degree of drug protection sufficient to allow bone marrow tolerance to therapy that is cytotoxic to tumors. These studies form the platform on which to develop clinical trials of drug resistance gene transfer into marrow cells. Expression of drug resistance genes in hematopoietic progenitors has been shown to decrease the myelosuppressive effects of chemotherapeutic drugs in animal models. In these studies, transplantation of multiple drug resistance-, dihydrofolate reductase-, or MGMT-transduced bone marrow into mice resulted in higher peripheral blood counts and increased survival after paclitaxel (27 , 28) , methotrexate (29) , or BCNU (30 , 31) , respectively.

In this study, we report that {Delta}MGMT-transduced hematopoietic progenitors are protected from the combination of BG and TMZ. There is accumulating evidence that the methylating agent TMZ is effective as an antitumor drug, and its cytotoxicity can be further enhanced by the use of the AGT inhibitor BG (10 , 11 , 15) . However, in vivo murine and in vitro human studies as well as early Phase I data have indicated that hematological toxicity is dose limiting (32) .

We demonstrated that {Delta}MGMT-transduced hematopoietic progenitors expressing BG-resistant AGT were protected from BG and TMZ, whereas SW480 tumor cells expressing wt AGT became sensitive. The TMZ IC90 for {Delta}MGMT-transduced human CFUs was >3.6-fold higher than that for SW480 cells. Furthermore, in the presence of BG, TMZ sensitized SW480 cells to a greater degree than BCNU (8) . Exposure to 10 µM BG resulted in a 14-fold potentiation of the TMZ effect (defined as the ratio of the IC50 with BG to the IC50 without BG) compared to only a 4-fold potentiation effect with BCNU. Based on this, we predicted that an overexpression of {Delta}MGMT in hematopoietic progenitors would also result in a greater resistance to TMZ than to BCNU. In the current study, {Delta}MGMT expression in human and mouse CFUs resulted in a >7.7-fold increase in the TMZ IC90 after BG treatment when compared to that of untransduced cells. This is greater than the resistance observed with BG and BCNU. Analysis of human CFU survival curves indicated that {Delta}MGMT-transduced cells were more resistant to BG and concentrations up to 300 µM TMZ. In this dose range, which is up to eight times the IC50, the survival advantage was greater with TMZ than with BCNU. At 400 µM TMZ plus BG, resistance decreased because {Delta}AGT had been consumed by the repair of O6-MG adducts formed by TMZ. If AGT expression were higher, the degree of protection at higher TMZ doses would be greater as well. The dose-response toxicity curve for CFUs is steeper after BCNU exposure, suggesting that BCNU toxicity is a result of DNA cross-links and other lesions that cannot be repaired by AGT and that may be more cytotoxic than non-O6-MG adducts. This implies that higher {Delta}AGT expression might generate a higher resistance to TMZ than to BCNU.

We achieved significant potentiation of TMZ cytotoxicity in SW480 tumor cells by maintaining AGT depletion with continuous BG exposure. Prolonged AGT depletion had a greater effect on TMZ cytotoxicity than BCNU cytotoxicity because unrepaired O6-MG adducts become cytotoxic after DNA replication, which may require 12–36 h. The regeneration of AGT activity has a lesser effect on BCNU toxicity because cross-link formation occurs rapidly, with a half-life of 7–8 h (33) , after which AGT is not protective. If BG is given only before BCNU or TMZ, the recovery of AGT in hematopoietic cells begins approximately 12 h after treatment (34) . Therefore, pre-cross-link repair is reduced during this critical period, resulting in BCNU toxicity. However, methyl adducts are not cytotoxic until mismatch repair recognition of the O6-MG:thymine base mispair, which occurs after one cycle of DNA replication (3) . If the O6-MG is repaired at any time, the resulting G:T mismatch is resolved by mismatch repair without cytotoxicity. Otherwise, it is persistently a target for mismatch repair-induced cytotoxicity. Therefore, as soon as BG is removed, newly synthesized AGT will repair O6-MG lesions, reducing the cytotoxic effect of TMZ. This concept was elucidated by Wedge et al. (11) , who demonstrated that TMZ cytotoxicity exceeded that of BCNU only after a prolonged depletion of AGT with 1 µM BG, and to an even greater degree after multiple doses of TMZ, which is highly cytotoxic due to the accumulation of additional O6-MG adducts (11) . On the other hand, if BG is only given before the cytotoxic drug, BCNU is more potent than TMZ, particularly in hematopoietic cells (34 , 35) .

The successful clinical application of {Delta}MGMT gene therapy requires gene transfer into early hematopoietic progenitors that can contribute to long-term hematopoiesis. Because early hematopoietic progenitors are usually resting cells, we expected that they might have different sensitivities to BCNU and TMZ. The difference in the BCNU and TMZ sensitivity of quiescent cells may be explained by the time involved in formation and the persistence of the cytotoxic AGT reparable DNA lesions as noted above. Thus, quiescent cells, in which AGT is depleted by BG but recovers 12 h later, have a greater opportunity to repair O6-MG adducts until they are removed from quiescence, enter S-phase, and begin DNA synthesis. Indeed, we found that a portion of human early progenitors (LTC-ICs), presumably those resting at the time of drug exposure, were less sensitive to TMZ than committed progenitors. Treatment of CD34 cells with 100 µM TMZ, a dose that killed >99% of primary CFUs, gave rise to hematopoietic clones during a 5-week culture period. Because we observed a similar proportion of {Delta}MGMT-transduced LTC-ICs before and after TMZ treatment and also observed a 21% survival at 400 µM TMZ, we conclude that the degree of quiescence is a significant factor in the resistance of LTC-ICs to TMZ. These results are in contrast to the LTC-IC response to BCNU that resulted in the survival of <1% of secondary CFUs at doses that were cytotoxic to 99% of the primary CFUs. Furthermore, treatment with 10 µM BCNU resulted in significant enrichment for transduced LTC-ICs (18) . Enrichment for long-term repopulating cells was confirmed in mouse transplant experiments in which mice transplanted with {Delta}MGMT-transduced bone marrow maintained normal bone marrow and peripheral blood cellularity after BG and BCNU treatment and survived doses that were lethal to mice transplanted with lacZ-transduced bone marrow (26 , 36) . Whether TMZ, like BCNU, mediates enrichment for relatively quiescent cells in vivo needs to be determined through either murine or clinical studies.

Based on our data, we conclude that BG and TMZ would be effective agents to selectively enrich for {Delta}MGMT-transduced CFUs in vivo, and that {Delta}MGMT should protect the bone marrow from drug-induced myelosuppression. Whether or not BG and TMZ may be used to enrich for {Delta}MGMT-transduced quiescent progenitors needs to be evaluated in a murine model similar to that which has indicated that BG and BCNU significantly enrich for {Delta}MGMT-transduced cells (36) . Importantly, TMZ may become a useful agent for chemotherapy selection because its only dose-limiting toxicity is noncumulative myelosuppression. Unlike selection with BG and BCNU, the lack of other organ toxicities may allow a repetitive and dose-intensive therapy that might amplify the selective pressure in favor of the {Delta}MGMT+ cells. This may improve the marrow tolerance to high doses of TMZ in cancer patients and allow the selection of genetically altered hematopoietic cells in other gene therapy applications.


   ACKNOWLEDGMENTS
 
We thank Dr. Hillard Lazarus for providing the peripheral blood mononuclear cells and Dr. Omer Koç for helpful discussions.


   FOOTNOTES
 
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.

1 Supported in part by USPHS Grants RO1CA73062, RO1ES06288, UO1CA75525, and P30CA43703. Back

2 To whom requests for reprints should be addressed, at Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106. Phone: (216) 368-1176; Fax: (216) 368-1166; E-mail: slg5{at}po.CWRU.edu Back

3 The abbreviations used are: BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; BG, O6-benzylguanine; AGT, O6-alkylguanine DNA alkyltransferase; MGMT, methylguanine DNA methyltransferase gene; {Delta}MGMT, mutant G156A MGMT; TMZ, temozolomide; LTC-IC, long-term culture initiating cell; CFU, colony-forming unit; O6-MG, O6-methylguanine; CSF, colony-stimulating factor; IL, interleukin; mIL, mouse interleukin; wt, wild-type; SCF, stem cell factor. Back

Received 4/27/98; revised 10/28/98; accepted 10/29/98.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pegg A. E. Mammalian O6-alkylguanine-DNA alkyltransferase: regulation and importance in response to alkylating carcinogenic and therapeutic agents. Cancer Res., 50: 6119-6129, 1990.[Free Full Text]
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