1 Division of Pulmonary, Allergy, Critical Care and Occupational Medicine, Department of Internal Medicine, and 2 Department of Pediatrics and Biochemistry and Molecular Biology, Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana 46202
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an important cause of pulmonary toxicity. BCNU alkylates DNA at the O6 position of guanine. O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl groups from the O6 position of guanine. To determine whether overexpression of MGMT in a lung cell reduces BCNU toxicity, the MGMT gene was transfected into A549 cells, a lung epithelial cell line. Transfected A549 cell populations demonstrated high levels of MGMT RNA, MGMT protein, and DNA repair activity. The overexpression of MGMT in lung epithelial cells provided protection from the cytotoxic effects of BCNU. Control A549 cells incubated with 100 µM BCNU had a cell survival rate of 12.5 ± 1.2%; however, A549 cells overexpressing MGMT had a survival rate of 71.8 ± 2.7% (P < 0.001). We also demonstrated successful transfection of MGMT into human pulmonary artery endothelial cells and a primary culture of rat type II alveolar epithelial cells with overexpression of MGMT, resulting in significant protection from BCNU toxicity. These data suggest that overexpression of DNA repair proteins such as MGMT in lung cells may protect the lung cells from cytotoxic effects of cancer chemotherapy drugs such as BCNU.
overexpression; DNA repair protein; interstrand cross-link; alkylation; 1,3-bis(2-chloroethyl)-1-nitrosourea
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1,3-BIS(2-CHLOROETHYL)-1-NITROSOUREA (BCNU) is an important chemotherapeutic agent that can be used in either monotherapy or combination with other therapeutic agents for treatment of various cancers including brain tumors (5, 6). BCNU damages the DNA of mammalian cells by alkylation of guanine at the O6 position, with subsequent formation of interstrand DNA cross-links. The primary limiting complication of BCNU is pulmonary toxicity (1). The mechanisms of pulmonary toxicity are not well understood. Possible explanations include glutathione depletion in cells (10, 38), acceleration of apoptosis (17, 27), or DNA damage to a target cell population of the lung (18). A strategy to inhibit one of these underlying mechanisms of toxicity might not only provide insight into BCNU pulmonary toxicity, it would also suggest a possible therapeutic approach to reduce toxicity in vivo.
Alkylation by nitrosoureas induces various DNA base modifications that result in DNA interstrand and intrastrand cross-links. BCNU alkylates DNA at the O6 position of guanine by addition of a chloroethyl group that rearranges to form an ethyl bridge between N1 of guanine and N3 of cytosine in the opposite strand (23, 26). Interstrand cross-links are particularly cytotoxic because they entirely impede DNA replication. O6-methylguanine-DNA methyltransferase (MGMT) repairs the O6 lesions by removing alkyl groups from the O6-guanine before DNA cross-link formation (4). MGMT accepts both methyl and chloroethyl groups in a stoichiometric fashion that leads to inactivation of the protein (3, 31). The genes encoding alkyltransferases, including human MGMT, have been cloned and characterized (34-36, 42). As a result, it is possible to determine whether overexpression of MGMT in lung cells reduces DNA damage from alkylating drugs such as BCNU.
High MGMT activity and protein levels in tumor cells correlate with resistance to BCNU (15, 16). In normal cells, the amount of MGMT varies widely among tissues. In particular, lung tissue is associated with low levels of MGMT (7). This might suggest one reason for the sensitivity of the lung to BCNU toxicity.
To determine whether overexpression of MGMT in lung cells will reduce BCNU toxicity to these cells, a construct containing the coding region for MGMT was transfected into A549 lung epithelial cells. A549 cells, a malignant cell line thought to be derived from the alveolar epithelium, is a standard model for the study of alveolar epithelial cells in vitro (22, 33, 40). A549 cells have low levels of MGMT activity (8), but the response to BCNU is unknown. Additionally, the MGMT content of normal alveolar epithelial cells or pulmonary endothelial cells is unknown. The data demonstrate that MGMT can be overexpressed in lung cells in vitro as measured by MGMT RNA and protein levels as well as by MGMT DNA repair activity assays. BCNU toxicity in both control cells and MGMT- expressing cells was determined by several different methods. Regardless of the method used to assay toxicity, elevation of MGMT levels in lung cells resulted in significant protection from BCNU toxicity. In addition, we have performed transfection of MGMT of normal human pulmonary artery endothelial (HPAE) cells and a primary culture of rat type II alveolar epithelial cells. In all lung cell types tested, overexpression of MGMT resulted in significant protection from BCNU toxicity.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture
A549 cells are a malignant cell line derived from alveolar epithelial cells (American Type Culture Collection, Manassas, VA). A549 cells were cultured in DMEM medium (BioWhittaker, Walkersville, MD) with 10% fetal bovine serum (FBS; HyClone, Logan, UT), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Biofluids, Rockville, MD). HPAE cells were obtained from Clonetics (Walkersville, MD) and grown in endothelial cell growth medium-2 with 2% FBS (Clonetics). Type II alveolar epithelial cells were isolated from the lungs of 180- to 200-g, pathogen-free female rats (Harlan, Indianapolis, IN) as previously described (37). Type II cells were plated at a density of 1.7 × 105 cells/cm2 and grown in primary culture in DMEM medium with 10% FBS.Preparation of MGMT Vector
The pcDNA 3.0 consists of a neo gene and cytomegalovirus promoter driving high-level constitutive gene expression in many mammalian cells. The coding region for human MGMT (624 bp) was inserted into the pcDNA 3.0 vector (Invitrogen, Carlsbad, CA) at BamHI-EcoRI. Expressed MGMT protein had a molecular mass of ~21 kDa. After the pcDNA-MGMT was transformed into HB101 cells, the plasmid DNA was extracted using a QIAGEN Plasmid Midi kit (QIAGEN, Santa Clarita, CA). The MGMT DNA was quantified with a Gene Quant spectrophotometer. Yields were 500-700 µg/ml.In Vitro Transfection
Transfection of A549, type II, or HPAE cells was performed using the liposome reagent GenePORTER (Gene Therapy System, San Diego, CA) following the manufacturer's instructions. Conditions for liposome-mediated transfection were optimized for gene transfection as previously described (11, 39, 45). Cells (2 × 105) were incubated with the transfection mixture (30 µl of GenePORTER and 4-12 µg of MGMT plasmid) in 1 ml of serum-free medium for 6 h at 37°C, then 1 ml of DMEM containing 20% FBS was added to the culture dish, and the cells were incubated for 18 h. Following incubation, the transfection mixture was removed and cells were cultured with DMEM and 10% FBS. Three days later, the cells were incubated in 800 µg/ml G418 (GIBCO BRL, Grand Island, NY) for 10 days to select the transfected cells. The cells were then assessed for overexpression of MGMT. To determine the reproducibility of transfection results, each transfection experiment was performed using four 6-cm dishes of cells under the same conditions including degree of cell confluency, liposome concentration, and DNA concentration. 3T3 cells that were previously transfected with MGMT were used as positive controls.MGMT RNA Transcripts
Northern blot analysis. Total RNA was isolated from control cells and from cells following MGMT transfection using TRIzol reagent (GIBCO BRL) or RNA Stat-60 (Tel-Test, Friendswood, TX). The RNA was quantified by Gene Quant and analyzed by agarose gel (0.8% wt/vol). RNA (25 µg) was denatured by heating to 65°C in 50% (vol/vol) formamide and 4.4 mol/l formaldehyde, electrophoresed through a 1.4% low electroendosmosis agarose gel (Fisher Scientific, Fair Lawn, NJ) containing 2.2 mol/l formaldehyde, and transferred by capillary blotting to a nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The RNA was cross-linked to the membrane by ultraviolet irradiation at the dose of 12,000 µJ/cm2 for 2 min.
MGMT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plasmids (pBSKT-GAPDH) were extracted with a QIAGEN midi preparation kit and digested with endonucleases (BamHI-EcoRI for MGMT and PstI-XhoI for GAPDH). The digested samples were electrophoresed on a 0.8% agarose gel, and fragments were purified on a Supelco spin column (Sigma-Aldrich, St Louis, MO). cDNA probes were labeled with 1-3 ×109 counts · minMGMT Protein Expression and Activity
Western blot. Cell lysates in SDS-PAGE (10%) were electroblotted to presoaked Immobilon-P membrane (Millipore, Bedford, MA) for 18 h at 40-mA constant current in transfer buffer (192 mM glycine, 20 mM Tris · HCl, and 15% methanol). The membrane was blocked with 1.5% BSA in TBS-T (50 mM Tris · HCl pH 7.5, 150 mM NaCl, and 0.05% Tween-20) for 2 h at room temperature. Each blot was incubated with murine monoclonal antibodies to human MGMT (Lab Vision, Fremont, CA) at a dilution of 1:330 at room temperature for 1-1.5 h. The membrane was washed five times with TBS-T (50 mM Tris · HCl pH 7.5, 150 mM NaCl, and 0.5% Tween 20) for 10 min followed by a quick rinse with deionized water (46). Secondary anti-mouse peroxidase-conjugated antibodies (Sigma-Aldrich) at 1:500 were incubated with the membrane at room temperature for 45 min. The membrane was extensively washed and exposed on Kodak OMAT film using a chemiluminescence kit (Pierce, Rockford, IL).
MGMT activity assay.
MGMT activity was assayed by detecting removal of
O6-methylguanine using a restriction
endonuclease inhibition assay (14). Cell lysates from
control cells and cells transfected with MGMT (50-µg protein samples)
were reacted separately with 0.1 pmol of [-32P]dTTP
end-labeled 18-bp O6-methylguanine-containing
DNA substrate at 37°C for 2 h in 150 µl of analysis buffer (50 mM Tris · HCl, pH 8.0, 0.1 mM EDTA, 5 mM
dithiothreitol, and 5% glycerol). The 18-bp oligonucleotide substrate, containing a single methyl lesion at the
O6 position of guanine within a PvuII
site was custom synthesized using an automated DNA synthesizer and
purified (14, 29). The DNA substrate was precipitated by
centrifugation and air-dried. The samples were reacted with
PvuII for 1 h at 37°C, and the reaction was
terminated by addition of 90% formamide loading buffer. The samples
were electrophoresed on a 20% denaturing gel. The gel was dried, and
DNA oligonucleotides with or without cleavage were visualized by X-ray
film. Densitometry analysis was done using Kodak analysis software.
MGMT activity was expressed as a cleavage index indicating the ratio of
cleaved oligonucleotide to uncleaved oligonucleotide.
Assays of BCNU Toxicity
To determine the toxicity of BCNU to cells and to determine whether overexpression of MGMT was protective, three different assays of cellular toxicity were used: 1) 3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay, 2) [3H]thymidine incorporation assay, and 3) colony formation assay.MTS assay. Cells (2×104) were plated on a flat-bottomed 96-well plate (Falcon) in the presence of 1, 10, 100, 200, and 300 µM BCNU. After incubation for up to 48 h at 37°C, the percentage of cell survival was determined with the MTS/phenazine methosulfate reagents (Promega, Madison, WI) (13, 46). The MTS assay (Promega CellTiter 96 Aqueous Assay) is a nonradioactive, colorimetric method for determining the number of viable cells in the proliferation assay.
[3H]thymidine incorporation assay.
Cells were cultured in 96-well plates at 37°C for 24 h in the
presence or absence of BCNU, as described above. The cells were pulsed
with 1 µCi of [3H]thymidine (Amersham) for 8 h.
Incorporation of [3H]thymidine in the cell lysates was
enumerated with a scintillation counter.
Colony formation assay. MGMT-transfected cells were plated onto 10-cm culture dishes with 400 cells/dish in 5 ml of DMEM in the presence or absence of BCNU for 2 h. The cells were then cultured in fresh medium for 10-15 days (19). When the colonies became visible, they were stained with 2% methyl blue solution to assess the survival rate. Single colonies were selected and grown separately.
Statistical Methods
All experiments were conducted in triplicate, and data are expressed as means ± SE. Data were compared using Student's t-test or one-way ANOVA with paired comparisons, with significance at P < 0.05 (20). ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Overexpression of MGMT
Hybridization bands were identified by Northern blot analysis at the expected transcript site for human MGMT (Fig. 1). Control A549 cells also demonstrated a band at the position of the MGMT transcript; however, the amount of transcript in control cells was much less than that in the MGMT-transfected cells (P < 0.001). The blot was reprobed with human GAPDH to monitor sample loading and to ensure that the RNA loading was consistent.
|
Western blot of MGMT-transfected A549 cell populations revealed MGMT as
a 21-kDa protein (Fig. 2). The control
A549 cells did not reveal a detectable amount of MGMT protein even
though subsequent studies (see below) demonstrated a small amount of baseline MGMT activity in control A549 cells. This is consistent with
the results of others (8). In contrast, the cells
transfected with the MGMT gene had an easily detectable amount of MGMT
protein.
|
MGMT activity in the cells was assessed by a radiolabeled
oligonucleotide cleavage assay. As a representation of the MGMT activity assays, two sets of transfected A549 cells are shown that were
positive for MGMT activity (Fig. 3).
Activity of MGMT-transfected A549 cells cleaved 59.6 ± 3.1% of
the methyl-containing oligonucleotide, whereas untransfected A549 cells
exhibited 8.8 ± 0.6% cleavage (P = 0.004).
|
Effect of MGMT Overexpression on BCNU Toxicity
MGMT-transfected A549 cells were protected from BCNU toxicity (Fig. 4). The MTS assay demonstrated that overexpression of MGMT in A549 cells provided significant protection against BCNU toxicity compared with that in the nontransfected control cells (Fig. 4A). As an example, in the presence of 150 µM BCNU, only 46.8 ± 6.4% of the A549 control cells survived compared with 108.0 ± 9.1% of the A549 cells overexpressing MGMT (P < 0.001).
|
A [3H]thymidine incorporation proliferation assay also showed that overexpression of MGMT provided protection from BCNU toxicity (Fig. 4B). In the presence of 150 µM BCNU, [3H]thymidine incorporation in A549 cells without MGMT was reduced to 34.2 ± 2.8% of nontreated cells, whereas [3H]thymidine incorporation in A549 cells overexpressing MGMT was 75.5 ± 2.8% of that in nontreated cells (P < 0.05).
A colony formation assay also confirmed that overexpression of MGMT was protective against BCNU toxicity. In the presence of 100 µM BCNU, only 12.5 ± 1.2% of the A549 control cells survived compared with 71.8 ± 2.7% of the transfected A549 cells (P < 0.001; Fig. 4C).
HPAE cells and rat type II alveolar epithelial cells were also
successfully transfected with MGMT, and expression was verified by
Western blot (Fig. 5,
A-C). At baseline, neither HPAE cells nor rat type II
cells had detectable MGMT. We demonstrated that HPAE cells
overexpressing MGMT were significantly protected from BCNU toxicity as
measured by MTS assay (Fig. 5B). In the presence of 150 µM
BCNU, 49.5 ± 8.3% of the control cells survived, whereas 95.1 ± 7.5% of the HPAE cells expressing MGMT survived
(P < 0.05). Similarly, MGMT was successfully
transfected into a primary culture of rat type II alveolar epithelial
cells. The overexpression of MGMT in type II cells was confirmed by
Western blot (Fig. 5C), and again overexpression of MGMT was
associated with significant protection from BCNU toxicity (Fig.
5D).
|
To assess the relationship between MGMT activity and BCNU resistance,
we assessed MGMT activity assays and MTS cytotoxicity assays in
different populations of MGMT-transfected A549 cells. The results
showed that the levels of MGMT DNA repair activity in lung cells were
positively correlated with the resistance to BCNU toxicity in A549
cells (r = 0.92) (Fig.
6).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A plasmid construct containing the coding region for human MGMT was successfully transfected into A549 alveolar epithelial cells using liposome delivery. High-level expression of MGMT in lung cells was confirmed by increased mRNA transcripts, protein expression, and MGMT DNA repair activity. The overexpression of MGMT in A549 cells was associated with a significant reduction in the toxicity of BCNU. Protection of lung epithelial cells from the cytotoxic effects of BCNU was verified using various methods including a MTS assay, a [3H]thymidine incorporation assay, and a colony formation assay. Our study suggests that MGMT is a critically important DNA repair protein capable of reducing BCNU toxicity to lung cells. We also transfected MGMT into HPAE cells and a primary culture of type II alveolar epithelial cells and obtained similar protection of the target cells from BCNU toxicity.
Nitrosoureas induce many different types of DNA damage, but the O6-guanine lesions are known precursors of interstrand DNA cross-links that can result in cell death (9). DNA methyltransferases such as MGMT are the only class of DNA repair proteins that directly remove alkyl groups from the O6 position of guanine (24). Because repair of the O6-guanine lesions by MGMT is a direct reversal of the DNA damage, MGMT is the most efficient mechanism to repair the DNA lesion (21). Repair of the O6-guanine lesion is considered to be the rate-limiting step in the repair of the DNA damage from nitrosoureas (4, 28). Previous studies demonstrated a direct correlation between MGMT levels in cancer cells and resistance to nitrosourea derivatives (4, 41).
Our study demonstrates that enhanced MGMT activity in lung cells is proportional to the resistance of these cells to BCNU toxicity. This suggests that an increase in specific DNA repair activity may rapidly correct drug-induced DNA damage in susceptible lung cells before DNA cross-linking and cell death occurs. There is in vivo evidence that further suggests a protective role for MGMT. Mice that received repeated infusions of MGMT-transfected hematopoietic progenitors have significantly increased peripheral blood cell counts during multiple rounds of BCNU administration (12, 30). In addition, transgenic mice overexpressing MGMT have a significant reduction in carcinogenic mutations due to nitrosoureas (25, 47).
In our study, however, overexpression of MGMT did not result in complete protection of lung cells from BCNU toxicity. This may be because nitrosoureas induce lesions at several sites on DNA, such as the N7 position of guanine, a site of DNA damage not affected by MGMT (26). It is also possible that the O6-chloroethylguanine lesion escapes repair by MGMT and rearranges into a 1-O6-ethanoguanine lesion, the immediate precursor to the interstrand cross-link. There is also evidence to suggest that BCNU induces toxicity by reducing levels of glutathione (38), which is unlikely to be affected by MGMT. In our model, however, overexpression of MGMT was associated with significant protection from BCNU toxicity; thus DNA damage, and specifically O6-guanine lesions, are likely key intermediate steps in cell death.
We modified lung cells to overexpress MGMT to test the hypothesis that lung cells would become more resistant to BCNU toxicity. High-level expression of MGMT in vitro was achieved using liposome gene delivery. However, use of liposomes has some perceived limitations. Studies in vitro reveal that expression of liposome-delivered genes may last only a few days (32). Similarly, the use of liposomes in vivo results in loss of expression as a function of time (43, 44). However, if MGMT gene therapy were considered a possible intervention to reduce BCNU pulmonary toxicity in vivo, pretreatment of the lung to achieve transient high-level expression would be a distinct advantage. Thus these initial studies in vitro suggest that liposome delivery of the MGMT gene may prove valuable as a method to deliver the MGMT gene to reduce BCNU pulmonary toxicity in vivo.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. David A. Williams for helpful advice on this manuscript and Dr. Yi Xu for providing help with the technical aspects of the experiment.
![]() |
FOOTNOTES |
---|
These studies were supported by National Cancer Institute Grant P01-CA-75426 (to W. J. Martin II and M. R. Kelley), and National Institutes of Health Grants CA-76643, ES-07815, and NS-38506 (to M. R. Kelley).
Address for reprint requests and other correspondence: W. J. Martin II, Division of Pulmonary, Allergy, Critical Care and Occupational Medicine, 1001 W. 10th St., OPW 425, Indianapolis, IN 46202-2879.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 July 2000; accepted in final form 7 November 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bhalla, KS,
Wilczynski SW,
Abushamaa AM,
Petros WP,
McDonald CS,
Loftis JS,
Chao NJ,
Vredenburgh JJ,
and
Folz RJ.
Pulmonary toxicity of induction chemotherapy prior to standard or high-dose chemotherapy with autologous hematopoietic support.
Am J Respir Crit Care Med
161:
17-25,
2000
2.
Biswas, T,
Ramana CV,
Srinivasan G,
Boldogh I,
Hazra TK,
Chen Z,
Tano K,
Thompson EB,
and
Mitra S.
Activation of human O6-methylguanine-DNA methyltransferase gene by glucocorticoid hormone.
Oncogene
18:
525-532,
1999[ISI][Medline].
3.
Bobola, MS,
Tseng SH,
Blank A,
Berger MS,
and
Silber JR.
Role of O6 methylguanine DNA methyltransferase in resistance of human brain tumor cell lines to the clinically relevant methylating agents temozolomide and streptozotocin.
Clin Cancer Res
2:
735-741,
1996[Abstract].
4.
Brent, TP,
Houghton PJ,
and
Houghton JA.
O6-alkylguanine-DNA alkyl-transferase activity correlates with the therapeutic response of human rhabdomyosarcoma xenografts to CENU.
Proc Natl Acad Sci USA
82:
2985-2989,
1985[Abstract].
5.
Cagnoni, PJ,
Nieto Y,
Shpall EJ,
Bearman SI,
Baron AE,
Ross M,
Matthes S,
Dunbar SE,
and
Jones RB.
High-dose chemotherapy with autologous hematopoietic progenitor-cell support as part of combined modality therapy in patients with inflammatory breast cancer.
J Clin Oncol
16:
1661-1668,
1998[Abstract].
6.
Chen, ZP,
Yarosh D,
Garcia Y,
Tampieri D,
Mohr G,
Malapetsa A,
Langleben A,
and
Panasci LC.
Relationship between O6-methylguanine-DNA methyltransferase levels and clinical response induced by chloroethylnitrosourea therapy in glioma patients.
Can J Neurol Sci
26:
104-109,
1999[ISI][Medline].
7.
Citron, M,
Graver M,
Schoenhaus M,
Chen S,
Decker R,
and
Kleynerman L.
Detection of messenger RNA from O6-methylguanine-DNA methyltransferase gene MGMT in human normal and tumor tissues.
J Natl Cancer Inst
84:
337-340,
1992[Abstract].
8.
Day, RS, III,
Ziolkowski CH,
Scudiero DA,
Meyer SA,
Lubiniecki AS,
Girardi AJ,
Galloway SM,
and
Bynum GD.
Defective repair of alkylated DNA by human tumour and SV40-transformed human cell strains.
Nature
288:
724-727,
1980[ISI][Medline].
9.
Eadie, JS,
Conrad M,
Toorchen D,
and
Topal MD.
Mechanism of mutagenesis by O6-methylguanine.
Nature
308:
201-203,
1984[ISI][Medline].
10.
Egyhazi, S,
Edgren MR,
Hansson J,
Krockel D,
Mannervik B,
and
Ringborg U.
Role of O6-methylguanine-DNA methyltransferase, glutathione transferase M3-3 and glutathione in resistance to carmustine in a human non-small cell lung cancer cell line.
Eur J Cancer
33:
447-452,
1997[ISI][Medline].
11.
Felgner, PL,
Gadek TR,
Holm M,
Roman R,
Chan HW,
Wenz M,
Northrop JP,
Ringold GM,
and
Danielsen M.
Lipofectin: a highly efficient, lipid-mediated DNA-transfection procedure.
Proc Natl Acad Sci USA
84:
7413-7417,
1987[Abstract].
12.
Gerson, SL,
Phillips W,
Kastan M,
Dumenco LL,
and
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
13.
Gillies, RJ,
and
Denton M.
Determination of cell number in monolayer cultures.
Anal Biochem
159:
109-113,
1986[ISI][Medline].
14.
Hansen, WK,
Deutsch WA,
Yacoub A,
Williams DA,
and
Kelley MR.
Creation of a fully functional human chimeric DNA repair protein.
J Biol Chem
273:
756-765,
1998
15.
Hongeng, S,
Brent TP,
Sandford RA,
Li H,
Kun LE,
and
Heideman RL.
O6 methylguanine DNA methyltransferase protein levels in pediatric brain tumors.
Clin Cancer Res
3:
2459-2463,
1997[Abstract].
16.
Jaeckle, KA,
Eyre HJ,
Townsend JJ,
Schulman S,
Knudson HM,
Belanich M,
Yarosh DB,
Bearman SI,
Giroux DJ,
and
Schold SC.
Correlation of tumor O6 methylguanine-DNA methyltransferase levels with survival of malignant astrocytoma patients treated with bis-chloroethylnitrosourea: a Southwest Oncology Group study.
J Clin Oncol
16:
3310-3315,
1998[Abstract].
17.
Kaina, B,
Ziouta A,
Ochs K,
and
Coquerelle T.
Chromosomal instability, reproductive cell death and apoptosis induced by O6-methylguanine in Mex, Mex+ and methylation-tolerant mismatch repair compromised cells: facts and models.
Mutat Res
381:
227-241,
1997[ISI][Medline].
18.
Kehrer, J,
and
Klein-Szanto A.
Enhanced acute lung damage in mice following administration of 1,3-bis(2-chloroethyl)-1-nitrosourea.
Cancer Res
45:
5707-5713,
1985[Abstract].
19.
Koc, ON,
Reese JS,
Davis BM,
Liu L,
Majczenko KJ,
and
Gerson SL.
MGMT-transduced bone marrow infusion increases tolerance to O6-benzylquanine and 1,3-bis(2-chloroethyl)-1-nitrosourea and allows intensive therapy of 1,3-bis(2-chloroethyl)-1-nitrosourea-resistant human colon cancer xenografts.
Hum Gene Ther
10:
1021-1030,
1999[ISI][Medline].
20.
Kuebler, RR,
and
Smith H.
Statistics. New York: Wiley, 1976.
21.
Lawley, PD,
and
Philips DH.
DNA adducts from chemotherapeutic agents.
Mutat Res
355:
13-40,
1996[ISI][Medline].
22.
Lazrak, A,
Samanta A,
and
Matalon S.
Biophysical properties and molecular characterization of amiloride-sensitive sodium channels in A549 cells.
Am J Physiol Lung Cell Mol Physiol
278:
L848-L857,
2000
23.
Limp-Foster, M,
and
Kelley MR.
DNA repair and gene therapy: implications for translational uses.
Environ Mol Mutagen
35:
71-81,
2000[ISI][Medline].
24.
Lindahl, T.
DNA repair enzymes.
Annu Rev Biochem
51:
61-87,
1982[ISI][Medline].
25.
Liu, L,
Qin X,
and
Gerson SL.
Reduced lung tumorigenesis in human methylguanine DNA-methyltransferase transgenic mice achieved by expression of transgene within the target cell.
Carcinogenesis
20:
279-284,
1999
26.
Ludlum, DB.
DNA alklation by the haloethylnitrosoureas: nature of modifications produced and their enzymatic repair or removal.
Mutat Res
233:
117-126,
1990[ISI][Medline].
27.
Meikrantz, W,
Bergom MA,
Memisoglu A,
and
Samson L.
O6alkylguanine DNA lesions trigger apoptosis.
Carcinogenesis
19:
369-372,
1998[Abstract].
28.
Mitra, S,
and
Kaina B.
Regulation of repair of alkylation damage in mammalian genomes.
Prog Nucleic Acid Res Mol Biol
44:
109-142,
1993[ISI][Medline].
29.
Morgan, SE,
Kelley MR,
and
Pieper RO.
The role of the carboxyl-terminal tail in human O6-methylguanine DNA methyltransferase substrate specificity and temperature sensitivity.
J Biol Chem
268:
19802-19809,
1993
30.
Moritz, T,
Mackay BJ,
Glassner BJ,
Williams DA,
and
Samson L.
Retrovirus-mediated expression of a DNA repair protein in bone marrow protects hematopoietic cells from nitrosourea-induced toxicity in vitro and in vivo.
Cancer Res
55:
2608-2614,
1995[Abstract].
31.
Pieper, RO.
Understanding and manipulating O6-methylguanine-DNA methyltransferase expression.
Pharmacol Ther
74:
285-297,
1997[ISI][Medline].
32.
Piva, R,
Lambertini E,
Penolazzi L,
Facciolo MC,
Lodi A,
Aguiari G,
Nastruzzi C,
and
del Senno L.
In vitro stability of polymerase chain reaction-generated DNA fragments in serum and cell extracts.
Biochem Pharmacol
56:
703-708,
1998[ISI][Medline].
33.
Pottratz, ST,
Paulsrud J,
Smith JS,
and
Martin WJ II.
Pneumocystis carinii attachment to cultured lung cells by Pneumocystis gp120, a fibronectin binding protein.
J Clin Invest
88:
403-407,
1991[ISI][Medline].
34.
Rydberg, B,
Spurr N,
and
Karran P.
cDNA cloning and chromosomal assignment of the human O6-methylguanine DNA methyltransferase: cDNA expression in Escherichia coli and gene expression in human cells.
J Biol Chem
265:
9563-9569,
1990
35.
Shiraishi, A,
Sakumi K,
Nakatsu Y,
Hayakawa H,
and
Sekiguchi M.
Isolation and characterization of cDNA and genomic sequences for mouse O6-methylguanine-DNA methyltransferase.
Carcinogenesis
13:
289-296,
1992[Abstract].
36.
Sihota, S,
von Wronski MA,
Tano K,
Bigner DD,
Brent TP,
and
Mitra S.
Characterization of cDNA encoding mouse DNA repair protein O6-methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant protein in Escherichia coli.
Biochemistry
31:
1897-1903,
1992[ISI][Medline].
37.
Skillrud, DM,
and
Martin WJ II.
Paraquat-induced injury of type II alveolar cells.
Am Rev Respir Dis
129:
995-999,
1984[ISI][Medline].
38.
Smith, AC,
and
Boyd MR.
Preferential effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on pulmonary glutathione reductase and glutathione/glutathione disulfide ratios: possible implications for lung toxicity.
J Pharmacol Exp Ther
229:
658-663,
1984[Abstract].
39.
Spragg, DD,
Alford DR,
Greferath R,
Larsen CE,
Lee DK,
Gurtner GC,
Cybulsky MI,
Nicolau C,
and
Gimbrone MAJ
Immunotargeting of liposomes to activated vascular endothelial cells: a strategy for site-selective delivery in the cardiovascular system.
Proc Natl Acad Sci USA
94:
8795-8800,
1997
40.
Spragg, RG,
and
Li J.
Effect of phosphocholine cytidylyltransferase overexpression on phosphatidylcholine synthesis in alveolar type II cells and related cell lines.
Am J Respir Cell Mol Biol
22:
116-124,
2000
41.
Tano, K,
Dunn WC,
Darroudi F,
Shiota S,
Preston RJ,
Natarajan AT,
and
Mitra S.
Amplification of the DNA repair gene O6 methylguanine DNA methyltransferase associated with resistance to alkylating drugs in a mammalian cell line.
J Biol Chem
272:
13250-13254,
1997
42.
Tano, K,
Shiota S,
Collier J,
Foote RS,
and
Mitra S.
Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6-akylguanine.
Proc Natl Acad Sci USA
87:
686-690,
1990[Abstract].
43.
Vacik, J,
Dean BS,
Zimmer WE,
and
Dean DA.
Cell-specific nuclear import of plasmid DNA.
Gene Ther
6:
1006-1014,
1999[ISI][Medline].
44.
Veit, K,
Boissel JP,
Buerke M,
Grosser T,
Meyer J,
and
Darius H.
Highly efficient liposome-mediated gene transfer of inducible nitric oxide synthase in vivo and in vitro in vascular smooth muscle cells.
Cardiovasc Res
43:
808-822,
1999[ISI][Medline].
45.
Wheeler, CJ,
Felgner PL,
Tsai YJ,
Marshali J,
Sukhu L,
Doh SG,
Hartikka J,
Nietupski J,
Manthorpe M,
Nichols M,
Plewe M,
Liang X,
Norman J,
Smith A,
and
Cheng SH.
A novel cationic lipid greatly enhances plasmid DNA delivery and expression in mouse lung.
Proc Natl Acad Sci USA
93:
11454-11459,
1996
46.
Wu, M,
Brown WL,
and
Stockley PG.
Cell-specific delivery of bacteriophage-encapsidated ricin A chain.
Bioconjug Chem
6:
587-595,
1995[ISI][Medline].
47.
Zaidi, NH,
Pretlow TP,
O'Riordan MA,
Dumenco LL,
Allay E,
and
Gerson SL.
Transgenic expression of human MGMT protects against azoxymethane-induced aberrant crypt foci and G to A mutations in the K-ras oncogene of mouse colon.
Carcinogenesis
16:
451-456,
1995[Abstract].