Cancer Research The Future of Cancer Research: Science and Patient Impact 09 AM Call for Abstracts
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
[Cancer Research 59, 3059-3063, July 1, 1999]
© 1999 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cai, Y.
Right arrow Articles by Dolan, M. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cai, Y.
Right arrow Articles by Dolan, M. E.
[Cancer Research 59, 3059-3063, July 1, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

Role of O6-Alkylguanine-DNA Alkyltransferase in Protecting against Cyclophosphamide-induced Toxicity and Mutagenicity1

Yingna Cai, Michael H. Wu, Susan M. Ludeman, David J. Grdina and M. Eileen Dolan2

Section of Hematology/Oncology, Department of Medicine [Y. C., M. H. W., M. E. D.], Department of Radiation and Cellular Oncology [D. J. G.], and Cancer Research Center [M. E. D.], University of Chicago, Chicago, Illinois 60637, and Duke Comprehensive Cancer Center, Duke University, Durham, North Carolina 27710 [S. M. L.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 REFERENCES
 
Cyclophosphamide is used to treat a wide range of human malignancies. However, it is also a known carcinogen associated with induction of therapy-related leukemia and bladder cancer. The DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), protects cells from the toxic and mutagenic effects of O6-alkylating agents. We report here the contribution of AGT in protecting against the toxic and mutagenic effects of cyclophosphamide. CHO cells transduced with wild-type human AGT (CHOAGT) and pcDNA3 (CHOpcDNA3) were treated with activated cyclophosphamide derivatives, 4-hydroperoxycyclophosphamide (4-HC), 4-hydroperoxydidechlorocyclophosphamide (4-HDC), a progenitor of acrolein, and phosphoramide mustard (PM). The results show that CHOAGT is 7- or 20-fold less sensitive to the toxic effects of 30 µM 4-HC or 300 µM 4-HDC, respectively, than CHOpcDNA3 cells as measured by cell survival using a colony-forming assay. CHOAGT cells treated with 20 µ M 4-HC or 200 µM 4-HDC produced 4- or 7-fold lower mutation frequency as measured at the HPRT locus than CHOpcDNA3 cells treated with the same dose of drugs. At 30 µM acrolein, the cell survival for CHOAGT was 30% compared with 18.7% for CHOpcDNA3. The mutation frequency of acrolein at the same dose was 57 mutants/106 cells in CHOpcDNA3 compared with no mutants in CHOAGT. In contrast, CHOAGT and CHOpcDNA3 cells treated with PM had similar survival curves and exhibited no difference in mutation frequency. The present study demonstrates that AGT plays an important role in protecting against the toxic and mutagenic effect of cyclophosphamide and suggests that acrolein, not PM, is responsible for generating the toxic and mutagenic lesion(s) protected by the AGT protein.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 REFERENCES
 
The DNA repair protein, AGT,3 has been shown to protect cells from the toxic and mutagenic effects of several classes of alkylating agents including alkylnitrosoureas and alkyltriazenes (1) . There is direct removal of alkyl groups from the O6-position of guanine, leaving guanine intact in DNA, thus preventing GC to AT transitions by monofunctional agents or preventing cross-linking by bifunctional agents. The role of AGT in protecting against mutagenic and carcinogenic effects of alkylating agents has been demonstrated in the transgenic models. Transgenic mice carrying the human alkyltransferase gene targeted to T cells had an overall lower incidence of thymic lymphomas (84% in nontransgenic versus 10% in transgenic mice) when treated with a single dose of N-methyl-N-nitrosourea (2) .

Cyclophosphamide belongs to the class of oxazaphosphorines and is an alkylating agent widely used for treating a variety of human malignancies. It is also a known carcinogen associated with therapy-related leukemia and bladder cancer (3 , 4) . Fig. 1Citation shows the metabolism of cyclophosphamide. Bioactivation of cyclophosphamide yields 4-hydroxycyclophosphamide, which then spontaneously interconverts with its tautomer aldophosphamide. Through an elimination mechanism, aldophosphamide fragments to acrolein and PM (5) . Both acrolein and PM are reactive species toward DNA. It has been reported that the covalent DNA adducts formed from PM are intra/interstrand cross-link DNA adducts and mono adducts at the N7 position of guanine (5) . The adducts formed from acrolein are cyclic adducts between the N1 and exocyclic amino nitrogens of deoxyguanosine in DNA (6) . The DNA adducts from PM and acrolein have been found in rodents after administration of cyclophosphamide (7 , 8) . In vivo genotoxic activity including chromosome aberrations, sister chromatid exchange, and gene mutation has been reported in both animals and humans after administration of cyclophosphamide (5) .



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 1. The metabolites of cyclophosphamide, 4-HC, and 4-HDC.

 
The role of AGT in protecting against DNA damage induced by cyclophosphamide is not known. Evidence from a clinical study with 20 ovarian cancer patients demonstrated a positive correlation between high tumor AGT activity and poor initial response to post-operative combination chemotherapy with cyclophosphamide and cisplatin (9) . However, more recent reports have not demonstrated a correlation (10) . Most recently, Friedman et al. (11) demonstrated that the combination of O6-benzylguanine, an AGT inactivator, and 4-HC resulted in a decrease in cell survival compared with 4-HC alone.

The present study is designed to investigate the role of the AGT protein in protecting against the toxic and mutagenic effects of cyclophosphamide. We report cell survival and mutation frequency in the hprt gene in CHO cells transfected with control plasmid and human wild-type agt gene after treatment with 4-HC, 4-HDC, and PM. Our findings confirm the earlier observation (11) that AGT plays a role in 4-HC-induced cytotoxicity and extend this observation to illustrate that acrolein, not PM exposure, results in a toxic and mutagenic lesion protected by AGT. This is the first demonstration that AGT plays an important role in protection against 4-HC-induced mutagenicity Fig. 1Citation .


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 REFERENCES
 
Materials.
Restriction endonucleases, T4 DNA ligase, T4 DNA polymerase, Vent DNA polymerase, deoxynucleotide triphosphates, MgSO4 and Thermopol buffer were purchased from New England Biolabs, Inc. (Beverly, MA). Oligonucleotides used as primers were synthesized by Genosys Biotechnologies, Inc. (The Woodlands, TX). The human wild-type O6-alkylguanine-DNA alkyltransferase cDNA was kindly provided by Dr. Anthony E. Pegg (Pennsylvania State University College of Medicine, Hershey, PA). 4-HC was a gift from O. Michael Colvin (Duke Comprehensive Cancer Center). 4-HC and 4-HDC were synthesized as described previously (12) .4 PM was obtained from the National Cancer Institute Drug Synthesis and Chemistry Branch (Bethesda, MD). All other biochemicals including acrolein were obtained from Sigma Chemical Co. (St. Louis, MO).

Subcloning of Wild-Type agt Genes for Transfection.
PCR was carried out in a 100-µl volume with 250 µM each of the deoxynucleotide triphosphate, 10 µl of Thermopol buffer [10 mM KCl, 20 mM Tris-HCl (pH 8.8), and 10 mM (NH4)2SO4], 2 mM MgSO4, 0.1 µM each of the primers (upper Primer, 44-mer: 5'-ATCACCATAAGCTTGCCGCCACCATGGACAAGGATGTGAAATG-3' and lower Primer, 25-mer: 5'-GGTACCACTCAGTTTCGGCCAGCAG-3'), 1 µl (~50 ng) of DNA template and 2–4 units of Vent DNA Polymerase. Thirty step cycles of amplification were performed in a Perkin-Elmer 480 thermocycler (92°C, 1 min; 61°C, 1 min; 72°C, 45 s), with an initial predenaturation at 94°C for 3 min.

Construction of pcDNA-AGT.
The wild-type agt open reading frame was amplified with primers tagged with appropriate restriction recognition sequence and a consensus Kozak sequence. The proofreading polymerase (Vent DNA polymerase) was used for high-fidelity primer extension. The agt gene amplified was inserted into pcDNA3 under the strong h-CMV immediate-early promoter. The inserts, including sequences at junctures between the insert and the vector, were sequenced on an ABI377 DNA sequencer to ensure that no additional mutations were introduced during the subcloning process. The final constructs (pcDNA-AGT) were propagated in Escherichia coli and purified twice by CsCl-EtBr ultracentrifugation for transfection.

Cell Transfection.
CHO cells were transfected with pcDNA3 or with pcDNA-AGT by electroporation using Electro Cell Manipulator (ECM 600; BTX Electronic Genetics, San Diego, CA) as described previously (13) and cultured to produce cloning stable cell lines.

Assay for AGT Activity.
Extracts were prepared from CHO cells with stable expression of wild-type AGT cDNA or plasmid by homogenization in 50 mM Tris (pH 7.5), 0.1 mM EDTA, 5 mM DTT, and 50 µg of DNA. AGT activity was determined as described previously (14 , 15) . Briefly, cell extracts were incubated with [3H]-methylating DNA substrate (5.77 Ci/mmol). The DNA was precipitated by adding ice-cold perchloric acid at a final concentration of 0.25 N and hydrolyzed in 0.1 N HCl at 70°C for 30 min. The modified bases were eluted on a C18 reverse phase column. Protein concentration was determined by the method of Bradford (16) . The results were expressed as fmol of O6-methylguanine released from DNA per mg of protein.

Assay for Cell Survival.
The cytotoxicity induced by 4-HC, 4-HDC, PM, or acrolein was determined by loss of colony-forming ability as described previously (17) . Briefly, CHO cells transduced with pcDNA3 and pcDNA-AGT were plated at a density of 1.4 x 106 cells/T75 flask. On the following day, cells were treated with increasing concentrations of 4-HC, 4-HDC, or PM in serum-free DMEM medium for 1 h and then replaced with fresh DMEM with serum. Solutions of 4-HC, 4-HDC, and PM were made fresh as needed and were used immediately upon preparation. Cells were replated at a density of 200 or 400 cells/100-mm dish 16 h after drug treatment. Cell colonies (>50 cells) were counted 10–12 days later after staining with methylene blue. Survival of colony-forming efficiency was expressed as a percentage of the appropriate set of control cells exposed to vehicle alone.

Assay for Mutation Frequency in Transduced CHO Cells.
Cells were plated, treated as described above, and maintained in exponential growth for an additional 7-day expression period before 1 x 105 cells were plated into 100-mm dish with 5 µg/ml 6-TG. Cells were incubated for 10 days, stained as described above, and counted. Mutation frequency was determined by counting 6-TG-resistant colonies and expressed as number of 6-TG-resistant colonies per 106 cells.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
REFERENCES
 
AGT Activity in Transfected CHO Cells.
CHO cells were transfected using electroporation with pcDNA3 (CHOpcDNA3) or wild-type AGT (CHOAGT). AGT activity in stably expressed cell lines were below the limit of detection and 1913 fmol/mg of protein in CHOpcDNA3 and CHOAGT cells, respectively (Fig. 2)Citation .



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 2. AGT activity in CHOpcDNA3 and CHOAGT cells. AGT activity was determined as described in "Materials and Methods.".

 
Role of AGT in Protecting against Cyclophosphamide-induced Cytotoxicity.
4-HC is an activated form of cyclophosphamide that spontaneously decomposes to acrolein and PM. As shown in Fig. 3ACitation , the percentage of cell survival in CHOpcDNA3 cells treated with 4-HC decreased in a dose-dependent manner, with 10% cell survival observed at 30 µM 4-HC. In contrast, CHOAGT cells were resistant to the toxic effects of 4-HC, with 90% survival at 30 µM 4-HC (Fig. 3A)Citation . This is consistent with the results of Friedman et al. (11) . To identify the metabolite of cyclophosphamide that produced the toxic lesion protected by AGT, CHOAGT cells and CHOpcDNA3 cells were treated with 4-HDC, which generates acrolein but not PM because it contains no chlorine atoms (Fig. 1)Citation . The ED50 of 4-HDC against CHOpcDNA3 was 110 µ M compared with 207 µM for CHOAGT cells, demonstrating that AGT protects against the products of 4-HDC (Fig. 3B)Citation . In contrast, there was no difference in cell survival after treatment with PM between CHOAGT and CHOpcDNA3 cells (Fig. 3C)Citation .



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3. Effects of human AGT on cytotoxicity induced by 4-HC (A), 4-HDC (B), and PM (C). CHOpcDNA3 (•) and CHOAGT ({blacksquare}) were treated with 4-HC, 4-HDC, and PM in serum-free medium for 1 h. The data represent an average from two to three separate experiments, and each points represents 10–15 replicate dishes; bars, SD.

 
Effect of AGT on Protecting against Cyclophosphamide-induced Mutagenicity.
There was a dramatic difference in mutation frequency induced by 4-HC between CHOpcDNA3 and CHOAGT cells. As shown in Fig. 4ACitation , the mutation frequency (TG-resistant colonies/106 cells) at the HPRT locus was decreased by 8- and 4-fold after treatment with 10 or 20 µM 4-HC, respectively, in the presence of AGT (Fig. 4A)Citation . Treatment of CHOpcDNA3 cells with 100 and 200 µM 4-HDC resulted in dose-dependent increase in mutation frequency at the HPRT locus from 25 ± 5.9 mutants/106 cells to 80 ± 8.1 mutants/106 cells; yet CHOAGT cells treated with the same doses of 4-HDC result in only 4 ± 6.3 and 12 ± 6.1 mutants/106 cells (Fig. 4B)Citation . Interestingly, both CHOpcDNA3 cells and CHOAGT cells were equally sensitive to the mutagenic effects of PM (Fig. 4C)Citation . Treatment with PM generated 144 ± 10 mutants/106 cells compared with 150 ± 9 mutants/106 cells for CHOpcDNA3 cells and CHOAGT cells at 300 µM PM.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 4. Effects of human AGT on mutagenicity induced by 4-HC (A), 4-HDC (B), and PM (C). CHOpcDNA3 (•) and CHOAGT ({blacksquare}) were treated with 4-HC, 4-HDC, and PM in serum-free medium for 1 h. The data represent an average of two separate experiments, and each point represents 40–60 replicate dishes; bars, SD.

 
Effect of AGT on Protecting against Acrolein-induced Cytotoxicity and Mutagenicity.
There was a difference in both the cytotoxic and mutagenic effects induced by acrolein in CHOpcDNA3 and CHOAGT cells. At a dose of 30 µM acrolein, the percentage of survival was 18.7% compared with 30% for CHOpcDNA3 and CHOAGT cells, respectively. More dramatic was the difference in mutation frequency at this same dose of drug. There were no mutations found in CHOAGT cells. However, 57 mutants/106 cells were scored in CHOpcDNA3 cells (Fig. 5)Citation .



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 5. Effects of human AGT on cytotoxicity and mutagenicity induced by acrolein. CHOpcDNA3 and CHOAGT were treated with acrolein in serum-free medium for 1 h, and survival (A) or mutation frequency (B) was determined. The data represent an average from two separate experiments, and each column represents 10–20 replicate dishes; bars, SD.

 
Discussion
This report describes the role of AGT in protecting against the toxic and mutagenic effects of cyclophosphamide. The results demonstrate that CHO cells overexpressing the human wild-type AGT protein are resistant to the cytotoxic and mutagenic effects of 4-HC, an activated form of cyclophosphamide, to 4-HDC, an agent that produces acrolein and a nontoxic PM analogue and to acrolein itself. The difference in cytotoxicity and mutagenicity in the presence and absence of AGT is not observed after exposure to PM, suggesting that a DNA adduct common to 4-HC, 4-HDC, and acrolein is recognized and repaired by the AGT protein.

There are conflicting reports in the literature regarding the correlation between AGT activity and antitumor response to cyclophosphamide. Mattern et al. (18) reported that human lung tumor xenografts expressing high AGT activity were less sensitive to the effects of cyclophosphamide. In contrast, Preuss et al. (19) reported that high AGT activity failed to protect against cell killing or sister chromatid exchange induced by cyclophosphamide. Furthermore, D’ Incalci (20) reported no correlation between AGT activity in tumor xenografts and response to cyclophosphamide. PM is thought to be responsible for generating DNA cross-links, which contribute to its antitumor effect. Acrolein has been implicated primarily in the toxic and carcinogenic side effects of cyclophosphamide (11) . Our results show that the biological effects associated with PM are independent of AGT activity, indicating that AGT is not a mechanism of resistance to the effects of PM. Therefore, the contribution of AGT activity to resistance to the cytotoxic effects of cyclophosphamide are due solely to the effects by acrolein, which may be minor in terms of antitumor activity. Cellular factors, such as high levels of glutathione or aldehyde dehydrogenase in tumor cells, may contribute to a greater extent to the resistance of cells to the antitumor effects of cyclophosphamide (21) .

Cyclophosphamide has been reported as a mutagen and carcinogen to humans. Evidence suggests that its mutagenic effects are associated with DNA adducts generated from both metabolites, acrolein and PM (11) . Acrolein has been shown to initiate bladder carcinogenesis in rats (22) . Our data are consistent with mutations in the HPRT locus in CHO cells after treatment with either acrolein or PM. High AGT activity in normal tissue may act to protect cells against the toxic and mutagenic side effects of acrolein. AGT activity is known to be low in hematopoietic progenitors (23) and decreased in the peripheral blood lymphocytes of patients with therapy-related acute nonlymphocytic leukemia compared with non-therapy-related leukemia patients (24) . In addition, cyclophosphamide has been shown to decrease alkyltransferase activity in peripheral lymphocytes of patients undergoing bone marrow transplantation, and this decrease is thought to be mediated through the production of acrolein (25) . Together with our data, it is likely that low or decreased AGT activity in stem cells may be responsible for unrepaired mutagenic DNA lesions formed from acrolein that could eventually lead to therapy-related leukemias.

The fact that the AGT protein removes alkylating groups from the O6 position of guanine residue at DNA has been well established (2) . To date, there is no evidence in the literature to suggest that the mammalian AGT recognizes lesions other than O6-alkylguanine and O4-alkylthymine adducts. In addition, there is no clear evidence that O6-alkylguanine or O4-alkylthymine lesions are formed after exposure of cells or animals to cyclophosphamide or acrolein. The DNA adducts formed from acrolein have been found to be N1 and exocyclic amino nitrogen of deoxyguanosine (6) . Several reasonable possibilities exist to explain the protection of acrolein-induced toxicity and mutagenicity in the presence of AGT: (a) acrolein could undergo a Michael addition with the O6 position of guanine producing an O6-propyl aldehyde moiety (O-CH2CH2CHO), and it is also possible that the O6 position of guanine could react with the aldehyde group of acrolein, yielding a hemiacetal [O-CH(OH)CH= CH2], as suggested by Friedman et al. (11) . By close analogy with O6-2-chloroethyl- and O6-2-hydroxyethyl-guanines, both acrolein adducts would be expected to be typically structured substrates of AGT. (b) it is possible, although unlikely, that the previously identified N-alkylation products formed from acrolein and guanine could be substrates for the AGT protein. Repair of such adducts has not been reported; and (c) the thiol group in the active site of AGT could simply be scavenging acrolein. Consistent with this are reports that cyclophosphamide, acrolein, and other aldehydes can inhibit AGT (25, 26, 27) .

Cyclophosphamide has been linked to the onset of therapy-related cancer, including bladder cancer and leukemia (3 , 4) . Because AGT protects against toxicity and mutagenicity induced by acrolein, then a therapeutic strategy to limit the toxicity and mutagenicity of this chemotherapeutic agent is by overexpression of the AGT protein in hematopoietic stem cells.


   ACKNOWLEDGMENTS
 
We thank Honglin Hao for valuable advice on mutation analysis.


   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 NIH Grants CA71627 (to M. E. D.), CA57725 (to M. E. D.), CA37323 (to S. M. L.), CA68438 (to S. M. L.), and CA37435 (to D. J. G.). Back

2 To whom requests for reprints should be addressed, at Section of Hematology/Oncology, University of Chicago, 5841 South Maryland Avenue, Box MC2115, Chicago, IL 60637. Phone: (773) 702-4441; Fax: (773) 702-0963; E-mail: medolan{at}mcis.bsd.uchicago.edu Back

3 The abbreviations used are: AGT, O6-alkylguanine-DNA alkyltransferase; CHO, Chinese hamster ovary; CHOAGT, wild-type human AGT-expressed CHO cells; CHOpcDNA3, pcDNA3-transfected CHO cells; 6-TG, 6-thioguanine; 4-HC, 4-hydroperoxycyclophosphamide; 4-HDC, 4-hydroperoxydidechlorocyclophosphamide; PM, phosphoramide mustard, HPRT, hypoxanthine phosphoribosyl transferase. Back

4 J. L. Flowers, S. M. Ludeman, M. P. Gamcsik, O. M. Colvin, K-L. Shao, J. H. Boal, J. B. Springer, and D. J. Adams. Airborne cytotoxicity in 4-hydroperoxycyclophosphamide multi-well assays: diffusion of chloroethylaziridine in vitro, submitted for publication. Back

Received 3/15/99. Accepted 5/13/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 REFERENCES
 

  1. Pegg A. E., Dolan M. E., Moschel R. C. Structure, function, and inhibition of O6-alkylguanine-DNA alkyltransferase. Progress Nucleic Acid Res. Mol. Biol., 51: 167-223, 1995.[Medline]
  2. Dumenco L. L., Allay E., Norton K., Gerson S. L. The prevention of thymic lymphomas in transgenic mice by human O6-alkylguanine-DNA alkyltransferase. Science (Washington DC), 259: 219-222, 1993.[Abstract/Free Full Text]
  3. Gibbons R. B., Westerman E. Acute non-lymphocytic leukemia following short-term, intermittent, intravenous cyclophosphamide treatment of lupus nephritis. Arthritis Rheum., 31: 1552-1554, 1988.[Medline]
  4. Ortiz A., Gonzalez-Parra E., Aloarez-Costa G., Egido J. Bladder cancer after cyclophosphamide therapy for lupus nephritis. Nephron, 60: 378-379, 1992.[Medline]
  5. Anderson D., Bishop J. B., Garner R. C., Ostrosky-Wegman P., Selby P. B. Cyclophosphamide. Review of its mutagenicity for an assessment of potential germ cell risks. Mutat. Res., 330: 115-181, 1995.[Medline]
  6. Chung F., Young R., Hecht S. S. Formation of cyclic 1, N2-propanodeoxyguanosine adducts in DNA upon reaction with acrolein or crotonaldehyde. Cancer Res., 44: 990-995, 1984.[Abstract/Free Full Text]
  7. Hemminki K. Binding of metabolites of cyclophosphamide to DNA in a rat liver microsomal system and in vivo in mice. Cancer Res., 45: 4237-4243, 1985.[Abstract/Free Full Text]
  8. Jacobson-Kram D., Albertini R. J., Branda R. F., Falta M. T., Iype P. T., Kolodner K., Liou S., McDiarmid M. A., Morris M., Nicklas J. A., O’Neill J. P., Poirier M. C., Putman D., Strickland P. T., William J. R., Xiao S. Measurement of chromosomal aberration sister chromatid exchange, hprt mutation and DNA adducts in peripheral lymphocytes of human populations at increased risk for cancer. Environ. Health Perspect. Suppl., 101: 121-125, 1993.
  9. Chen S. S., Citron M., Spiegel G., Yarosh D. O6-Methylguanine-DNA methyltransferase in ovarian malignancy and its correlation with postoperative response to chemotherapy. Gynecol. Oncol., 52: 172-174, 1994.[Medline]
  10. Codegoni A. M., Broggini M., Pitelli M. R., Pantapotto M., Torri V., Mangioni C., Incalci M. D. Expression of genes of potential importance in the response to chemotherapy and DNA repair in patients with ovarian cancer. Gynecol. Oncol., 65: 130-137, 1997.[Medline]
  11. Friedman H. S., Pegg A. E., Johnson S., Loktionova N. A., Dolan M. E., Modrich P., Moschel R. C., Struck R., Brent T. P., Ludeman S., Bullock N., Kilborn C., Keir S., Dong Q., Bigner D. D., Colvin O. M. Modulation of cyclophosphamide activity by O6-alkylguanine-DNA alkyltransferase. Cancer Chemother. Pharmacol., 43: 80-85, 1998.
  12. Zon G., Ludeman S. M., Brandt J. A., Boyd V. L., Ozkan G., Egan W., Shao K. L. NMR spectroscopic studies of intermediary metabolites of cyclophosphamide. A comprehensive kinetic analysis of the interconversion of cis- and trans-4-hydroxycyclophosphamide with aldophosphamide and the concomitant partitioning of aldophosphamide between irreversible fragmentation and reversible conjugation path ways. J. Med. Chem., 27: 466-485, 1984.[Medline]
  13. Wahl G. M., Lewis K. A., Ruiz J. C., Rothenberg B., Zhao J., Evan G. A. Cosmid vectors for rapid genomic walking, restriction mapping, and gene transfer. Proc. Natl. Acad. Sci. USA, 84: 2160-2164, 1987.[Abstract/Free Full Text]
  14. Dolan M. E., Moschel R. C., Pegg A. E. Depletion of mammalian O6-alkylguanine-DNA alkyltransferase activity by O6-benzylguanine provides a means to evaluate the role of this protein in protection against carcinogenic and therapeutic alkylating agents. Proc. Natl. Acad. Sci. USA, 87: 5368-5372, 1990.[Abstract/Free Full Text]
  15. Dolan M. E., Mitchell R. B., Mummert C., Moschel R. C., Pegg A. E. Effect of O6-benzylguanine analogues on sensitivity of human tumor cells to the cytotoxic effects of alkylating agents. Cancer Res., 51: 3367-3372, 1991.[Abstract/Free Full Text]
  16. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254, 1976.[Medline]
  17. Domoradzki J., Pegg A. E., Dolan M. E., Maher V. M., McCormick J. J. Correlation between O6-methylguanine DNA-methyltransferase activity and resistance of human cells to the cytotoxic and mutagenic effect of N-methyl-N'-nitro-N-nitrosoguanidine. Carcinogenesis (Lond.), 5: 1641-1647, 1984.[Abstract/Free Full Text]
  18. Mattern J., Eichhorn U., Kaina B., Volm M. O6-Methylguanine-DNA methyltransferase activity and sensitivity to cyclophosphamide and cisplatin in human lung tumor xenografts. Int. J. Cancer, 77: 919-922, 1998.[Medline]
  19. Preuss I., Thust R., Kaina B. Protective effect of O6-methylguanine-DNA methyltransferase (MGMT) on the cytotoxic and recombinogenic activity of different antineoplastic drugs. Int. J. Cancer, 65: 506-512, 1996.[Medline]
  20. D’Incalci M., Bonfanti M., Pifferi A., Mascellani E., Tagleabue G., Berger D., Fiebig H. H. The antitumor activity of alkylating agents is not correlated with the levels of glutathione, glutathione transferase and O6-alkylguanine-DNA alkyltransferase of human tumor xenografts. Eur. J. Cancer, 34: 1749-1755, 1998.
  21. Crook T. R., Souhami R. L., Whyman G. D., Mclean A. E. M. Glutathione depletion as a determinant of sensitivity of human leukemia cells to cyclophosphamide. Cancer Res., 46: 5035-5038, 1986.[Abstract/Free Full Text]
  22. Cohen S. M., Garland E. M., John M., Okamura T., Smith R. Acrolein initiates rat urinary bladder carcinogenesis. Cancer Res., 52: 3577-3581, 1992.[Abstract/Free Full Text]
  23. Gerson S. L., Phillips W., Kastan M., Dumenco L. L., Donovan C. Human CD34+ hematopoietic progenitors have low, cytokine-unresponsive O6-alkylguanine-DNA alkyltransferase and are sensitive to O6-benzylguanine plus BCNU. Blood, 88: 1649-1655, 1996.[Abstract/Free Full Text]
  24. Sagher D., Karrison T., Schwartz J. L., Larson R., Meier P., Strauss B. Low O6-alkylguanine DNA alkyltransferase activity in the peripheral blood lymphocytes of patients with therapy-related acute nonlymphocytic leukemia. Cancer Res., 48: 3084-3089, 1988.[Abstract/Free Full Text]
  25. Lee S. M., Crowther D., Scarffe J. H., Dougal M., Elder R. H., Rafferty J. A., Margison G. P. Cyclophosphamide decreases O6-alkylguanine-DNA alkyltransferase activity in peripheral lymphocytes of patients undergoing bone marrow transplantation. Br. J. Cancer, 66: 331-336, 1992.[Medline]
  26. Krokan H., Grafstrom R. C., Sundqvist K., Esterbauer H., Harris C. C. Cytotoxicity, thiol depletion and inhibition of O6-methylguanine-DNA methyltransferase by various aldehydes in cultured human bronchial fibroblasts. Carcinogenesis (Lond.), 6: 1755-1759, 1985.[Abstract/Free Full Text]
  27. Wlodek L The reaction of sulfhydryl groups with carbonyl compounds. Acta Biochim. Pol., 35: 307-317, 1988.[Medline]



This article has been cited by other articles:


Home page
 MutagenesisHome page
R. Nagasubramanian, R. J. Hansen, S. M. Delaney, M. M. Cherian, L. D. Samson, S. C. Kogan, and M. E. Dolan
Survival and tumorigenesis in O6-methylguanine DNA methyltransferase-deficient mice following cyclophosphamide exposure
Mutagenesis, September 1, 2008; 23(5): 341 - 346.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
B. Yamini, X. Yu, M. E. Dolan, M. H. Wu, D. W. Kufe, and R. R. Weichselbaum
Inhibition of Nuclear Factor-{kappa}B Activity by Temozolomide Involves O6-Methylguanine Induced Inhibition of p65 DNA Binding
Cancer Res., July 15, 2007; 67(14): 6889 - 6898.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
R. J. Hansen, R. Nagasubramanian, S. M. Delaney, L. D. Samson, and M.E. Dolan
Role of O6-methylguanine-DNA methyltransferase in protecting from alkylating agent-induced toxicity and mutations in mice
Carcinogenesis, May 1, 2007; 28(5): 1111 - 1116.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
M. F. Paz, R. Yaya-Tur, I. Rojas-Marcos, G. Reynes, M. Pollan, L. Aguirre-Cruz, J. L. Garcia-Lopez, J. Piquer, M.-J. Safont, C. Balana, et al.
CpG Island Hypermethylation of the DNA Repair Enzyme Methyltransferase Predicts Response to Temozolomide in Primary Gliomas
Clin. Cancer Res., August 1, 2004; 10(15): 4933 - 4938.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
P. Depeille, P. Cuq, S. Mary, I. Passagne, A. Evrard, D. Cupissol, and L. Vian
Glutathione S-Transferase M1 and Multidrug Resistance Protein 1 Act in Synergy to Protect Melanoma Cells from Vincristine Effects
Mol. Pharmacol., April 1, 2004; 65(4): 897 - 905.
[Abstract] [Full Text]

Home page
 Molecular Cancer TherapeuticsHome page
H. S. Friedman, S. Keir, A. E. Pegg, P. J. Houghton, O. M. Colvin, R. C. Moschel, D. D. Bigner, and M. E. Dolan
O6-Benzylguanine-mediated Enhancement of Chemotherapy
Mol. Cancer Ther., September 1, 2002; 1(11): 943 - 948.
[Abstract] [Full Text] [PDF]

Home page
 JNCI J Natl Cancer InstHome page
M. E. Dolan and R. L. Schilsky
Silence Is Golden: Gene Hypermethylation and Survival in Large-Cell Lymphoma
J Natl Cancer Inst, January 2, 2002; 94(1): 6 - 7.
[Full Text] [PDF]

Home page
 JNCI J Natl Cancer InstHome page
M. Esteller, G. Gaidano, S. N. Goodman, V. Zagonel, D. Capello, B. Botto, D. Rossi, A. Gloghini, U. Vitolo, A. Carbone, et al.
Hypermethylation of the DNA Repair Gene O6-Methylguanine DNA Methyltransferase and Survival of Patients With Diffuse Large B-Cell Lymphoma
J Natl Cancer Inst, January 2, 2002; 94(1): 26 - 32.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Y. Cai, M. H. Wu, M. Xu-Welliver, A. E. Pegg, S. M. Ludeman, and M. E. Dolan
Effect of O6-Benzylguanine on Alkylating Agent-induced Toxicity and Mutagenicity in Chinese Hamster Ovary Cells Expressing Wild-Type and Mutant O6-Alkylguanine-DNA Alkyltransferases
Cancer Res., October 1, 2000; 60(19): 5464 - 5469.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cai, Y.
Right arrow Articles by Dolan, M. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cai, Y.
Right arrow Articles by Dolan, M. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online