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Role of glutathione in cellular resistance to alkylating agents
Advan. Enzyme ReguL, Vol. 33, pp. 19-26, 1993
0065-2571/93/$24.00 ~ 1993PergamonPress Lid
Printed in Great Britain.All rights reserved
ROLE OFGLUTATHIONE IN C E L L U L A R R E S I S T A N C E TO ALKYLATING AGENTS O. MICHAEL COLVIN °, HENRY S. FRIEDMANt, MICHAEL P. GAMCSIK$, CATHERINE FENSELAU§ and JOHN HILTON* *Division of Pharmacology and Experimental Therapeutics, The Johns Hopkins Hospital Oncoiogy Center, Baltimore, MD tDepartment of Pediatrics and Preuss Laboratory for Brain Tumor Research, Duke University Medical Center, Durham, NC SDivision of NMR Research, Department of Radiology, The Johns Hopkins University School of Medicine, Baltimore, MD §Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, MD
INTRODUCTION
The tripeptide glutathione (Fig. 1) is known to participate in the detoxification of electrophilic xenobiotics, and a number of investigators have described an association between elevated concentrations of glutathione in cells and resistance of these cells to alkylating agents (1-3). We have explored the mechanism of this association and have demonstrated that the alkylating agents which we have studied react with glutathione to produce conjugates which are stable. The agents for which we have demonstrated conjugates with glutathione are melphalan (4, 5), chlorambucii (6), mechlorethamine (7), cyclophosphamide (8), and phosphoramide mustard (8), the active alkylating agent produced by the metabolism of cyclophosphamide (9). The structure of the monoglutathionyl conjugate of mephalan is shown in Figure 2 (4).
HO-C--CHCHsC~-C-NIt-CH-C-NH'CHj-C-OH Glutothtoe4
FIG. 1. Structure of glutathione.
oh-
thH
CI
FIG. 2. The monoglutathionyl conjugate of melphalan. 19
20
o . M . COLVIN,
et al.
Our studies indicate that these conjugates are stable, even at elevated temperatures, and they do not have alkylating activity as measured by reaction with nitrobenzylpyridine, a standard assay for biological alkylating agents. The melphalan-GSH conjugate is not cytotoxic when incubated with cells, but it is doubtful that this compound or the conjugates of other alkylating agents would enter cells, as glutathione does not. It is likely that most of the GSH-alkylating agent conjugates are formed intracellularly, and our data indicate that these compounds should not be cytotoxic to cells. STUDIES
OF THE MECHANISM AGENT-GLUTATHIONE
OF AN ALKYLATING REACTION
In our studies of the conjugation of melphalan with glutathione we found that the presence of glutathione did not accelerate the rate of disappearance of meiphalan (Fig. 3). This finding is consistent with the hypothesis that the chemical reaction is not a direct displacement of chloride by the thiol group, but rather a reaction of glutathione with the reactive aziridinium ion formed from melphalan (Fig. 4). In relatively low chloride concentrations, such as biological fluids, this reaction is irreversible. Therefore, the rate of removal of the aziridinium will not affect the rate of aziridinium formation, and the presence or absence of glutathione will not affect the rate of disappearance of the parent melphalan. To examine the mechanism of the reaction directly, mechlorethamine was labeled with 13C in the ot carbon of the mustard group and reacted with
1
0.1
i .5
i 0.01 0.001
~oo
~ time
(mln)
~o
~o
FIG. 3. Decomposition of melphalan at physiologic pH in the presence and absence of glutathione.
GLUTATHIONE AND ALKYLATING AGENTS
21
c¢~" ~ L . ~ c ~ ' ~ ~ID G-S-T
%%% %
c~
~,~
,a-c~-c~o,~
" ~_/'
FIG. 4. Potential reaction mechanisms of melphalan with glutathione.
glutathione (7). In the conjugate formed, the [13C] was found to be equally distributed between the a and 13 carbons of the glutathionyi conjugate, indicating that the reaction had proceeded through the cyclic aziridinium intermediate, with the resulting redistribution of the ethylene carbon atoms. In this study, it was also observed that the aziridinium intermediate was detectable by NMR, with a longer half-life than is usually presumed. Using the same approach, we have also studied aniline mustard, an aromatic nitrogen mustard similar to melphalan, and found that this reaction also proceeds through the aziridinium intermediate. The enzyme glutathione S-transferase (EC2.5.1.18) catalyzes the conjugation of glutathione with electrophiles, and elevated glutathione S-transferase activity in cells has been associated with resistance to alkylating agents (10, 11). Human and murine glutathione S-transferases occur as a number of isozyme families, and the ct,/x, and lr isozymes in particular have been studied to determine their relationship to alkylating agent resistance. Elevated levels of both ~r (12) and a (13, 14) isozymes have been associated with cellular resistance to alkylating agents, and the/~ isozyme has been shown to specifically catalyze the inactivation of BCNU (15). We studied the effect of the et, ~., and "rr on the reaction of melphalan with glutathione and found that only the et isozyme catalyzed this reaction (16). Cacao and colleagues have also reported that the reaction of chlorambucil with glutathione is catalyzed by the a isoenzyme (17), and in very recent studies Hilton found that the reaction of busulfan with glutathione is catalyzed specifically by the a isozyme (unpublished data). In our studies of the enzyme catalyzed conjugation of melphalan, we found that the rate of disappearance of the parent compound is not accelerated by the presence of enzyme, indicating that the enzyme catalyzed reaction also proceeds through the aziridinium intermediate (18).
22
O.M. COLVIN, et al.
Studies of the mechanisms of reaction with glutathione have now been carried out with cyclophosphamide and its aikylating metabolite, phosphoramide mustard. For these studies cyclophosphamide and phosphoramide mustard were synthesized with a deuterium label in the 13position of the chloroethyl group. The reactions were carried out in the absence or presence of glutathione S-transferase (from rabbit liver) and the location of the deuterium labeled carbon determined from the molecular weights of the fragments seen on mass spectrometry. Cyclophosphamide was found to react with glutathione without enzyme at pH 10, and this reaction proceeded through a direct displacement reaction (8) (Fig. 5). At pH 7.4 cyclophosphamide reacted with glutathione in the presence of enzyme, and again this reaction proceeded through a direct displacement reaction. These findings correlate with the fact that cyclophosphamide is not a reactive alkylating agent at physiological pH and does not show evidence of forming an aziridinium in NMR studies. The fact that the direct displacement of chloride in cyciophosphamide can be catalyzed by GST suggests that an analogous reaction can be catalyzed with melphalan and other nitrogen mustards, but is difficult to detect in the presence of a more rapid reaction with the aziridinium moiety. In contrast to cyclophosphamide, phosphoramide mustard reacted at
m/z 213
A.
B.
H 0
r~rl
"!_N
/--N\II /-"u2~
H 0
cN\IpI
N/--C~ cl
H 0
_ /--N\I I + / :
~-.0 / - ~-co~c,
/-
\ll
/
~v
~ _P-N ~-.0 / ~-CD2CI
--CD-Cl
,
~_O/P-N~,c0,
-
~
t
\
.
H 0 /'--N\I I
~
_SG
I /
.F{-CD2
~._ O/P-- N~._COICI FIG. 5. Alternative mechanisms for the conjugation of glutathione with cyclophosphamide. (From Ref. (8).)
23
GLUTATHIONE AND ALKYLATING AGENTS
B.
R O D I . C I ~ - - / N -- P - N H 2 1 D
Ao D |
D
R O I . . N -- P - NH 2
Ct D
O-
R
D
O - - p - - NH 2
I
I
O-
OGSH transferase
GSH tronsferase
323 GS
N I R
P - NH 2 I O-
GS
°
N --
t ,. :,
!_
NH 2
o-
FIG. 6. Alternative mechanisms for the conjugation of glutathione with phosphoramide mustard. (From Ref. (8).)
pH 10 in the absence of enzyme to form a glutathione conjugate via an aziridinium intermediate (Fig. 6). At pH 7.0 the spontaneous reaction was undetectable by mass spectrometry, but in the presence of enzyme the conjugate was formed, again through the aziridinium intermediate (8). The lack of the observed spontaneous reaction at pH 7.0 with phosphoramide mustard indicates that the aziridinium intermediate of phosphoramide mustard forms more slowly than those of mechlorethamine or melphalan. Fenselau and colleagues also showed that glutathione can react with one of the activated forms of cyclophosphamide, iminocyclophosphamide (or possibly 4-hydroxycyclophosphamide), to form 4-glutathionylcyclophosphamide (18) (Fig. 7). Lee et al. (19) have provided evidence that this reaction interferes with the antitumor effect of activated cyclophosphamide. We are currently conducting further studies of the spontaneous and enzyme catalyzed reactions of phosphoramide mustard and the other activated metabolites to establish the rates of these reactions and the isozyme(s) responsible for the catalysis of the reactions.
24
O . M . C O L V I N , et al.
o,E_
,:---,7
FIG. 7. Conjugation of glutathione at the 4 position of cyclophosphamide.
STUDIES
OF HUMAN MEDULLOBLASTOMA TOCYCLOPHOSPHAMIDE
CELLS RESISTANT
In a recent study the roles of glutathione and other mechanisms of cellular resistance were investigated in medulloblastoma cell lines resistant to cyclophosphamide in vivo and 4-hydroperoxycyclophosphamide in vitro (3). In three out of four resistant cell lines, glutathione was responsible for all or a significant part of the resistance, as demonstrated by increased glutathione levels in the resistant cells and sensitization of the cells by depletion of glutathione by buthionine sulfoximine. A linear relationship between the cellular concentration of glutathione and the sensitivity of the cells to 4-hydroperoxycyclophosphamide could be demonstrated (Fig. 8). The two other mechanisms of resistance detected were increased aldehyde dehydrogenase and an undetermined mechanism, which we believe may be the cdc2 kinase alteration recently described by O'Connor et al. (20).
20
5
J
•
•
y - 6.67 + 0.324x
5 R z - 0.647
0
0
5
10
15
i
J
20
25
GSH (nmol/mg protein)
30
FIG. 8. Relation between intracelluiar GSH concentration and concentration of 4-hydroperoxycyclophosphamide (4-HC) required for 1 log cell kill in clonogenic assay. O, Cells without BSO treatment; II, cells after BSO treatment. (From Ref. (3).)
GLUTATHIONE AND ALKYLATING AGENTS
25
SUMMARY
Both elevated glutathione levels and increased activity of the enzyme glutathione S-transferase have been associated with the resistance of cells to alkylating agents. We have demonstrated that one mechanism of this resistance is the inactivation of the alkylating agents by conjugation with glutathione. This conjugation can be catalyzed by glutathione S-transferase. For the nitrogen mustard agents we have studied, both the spontaneous and enzyme catalyzed reactions proceed through the aziridinium intermediates of the alkylating agents, and the cx isoenzymes of GST are involved. In a study of cyclophosphamide resistant medulloblastoma cell lines elevated cellular concentrations of glutathione correlated well with the resistance of the cell lines. REFERENCES I. G. CALCUTI" and T. A. CONNERS, Tumor sulfhydryl levels and sensitivity to the nitrogen mustard merophan, Biochem. Pharmacol. 12, 839--845(1963). 2. D. SUZUKAKA, B. J. PETRO and D. T. VISTICA, Reduction in glutathione content of L-PAM resistant L1210 cell confers drug sensitivity, Biochem. Pharmacol. 31, 121-124 (1982). 3. H. S. FRIEDMAN, O. M. COLVIN, S. H. KAUFMANN, S. M. LUDEMAN, N. BULLOCK, D. D. BIGNER and O. W. GRIFFITH, Cyclophosphamide resistance in medulloblastoma, Cancer Res. 32, 5373-5378 (1992). 4. D. M. DULIK, C. FENSELAU and J. HILTON, Characterization of melphalanglutathione adducts whose formation is catalysed by glutathione S-transferases, Biochem. Pharmacol. 35, 3405--3409(1986). 5. D . M . DULIK and C. FENSELAU, Conversion of meiphalan to 4-(glutathionyl)phenylalanine. A novel mechanism for conjugation by glutathione-S-transferases, Drug Metab. Dispos. 15, 195-199 (1987). 6. D. M. DULIK, O. M. COLVIN and C. FENSELAU, Characterization of glutathione conjugates of chlorambucil by fast atom bombardment and thermospray liquid chromatography/mass spectrometry, Biomed. Environ. Mass Spectrom. 19, 248-252 (1990). 7. M.P. GAMCSIK, T. G. HAMILL and O. M. COLVIN, NMR studies of conjugation of mechlorethamine with glutathione, J. Med. Chem. 33, 1009-1014 (1990). 8. Z.M. YUAN, P. B. SMITH, R. B. BRUNDRETI', O. M. COLVIN and C. FENSELAU, Glutathione conjugation with phosphoramide mustard and cyclophosphamide. A mechanistic study using tandem mass spectrometry, Drug Metab. Dispos. 19, 625--629 (1991). 9. O. M. COLVIN and B. A. CHABNER, Alkylating agents, pp. 276-313 in Cancer Chemotherapy: Principles and Practice (B. A. CHABNER and J. M. COLLINS, eds.) J. L. Lippincott, Philadelphia, Pennsylvania (1990). 10. C. N. ROBSON, A. D. LEWIS, C. R. WOLF, J. D. HAYES, A. HALL, S. J. PROCTOR, A. L. HARRIS and I. D. HICKSON, Reduced levels of drug-induced DNA cross-linking in nitrogen mustard-resistant Chinese hamster ovary cells expressing elevated glutath/one S-transferase activity, Cancer Res. 47, c~z2-6027 (1987). 11. A. L. BUTLER, M. L. CLAPPER and K. D. "FEW, Glutathione S-transferase in nitrogen mnstard-resistant and -sensitive cell lines, Mol. Pharmacol. 31, 575-578 (1987). 12. K. NAKAGAWA, N. SAIJO, S. TSUCHIDA, M. SAKAL, Y. TSUNOKAWA, J. YOKOTA, M. MURAMATSU, K. SATO, M. TERADA and K. D. "FEW,
26
13. 14.
15. 16. 17. 18. 19. 20.
O.M. COLVIN, et al. Glutathione-S-transferase "n as a determinant of drug resistance in transfectant cell lines, J. Biol. Chem. 265, 4296--4301 (1990). R. B. PUCHALSKI and W. E. FAHL, Expression of recombinant glutathione-Stransferase ~, Ya or Ybl confers resistance to alkylating agents, Proc. Natl. Acad. Sci. USA 87,2443-2447 (1990). M.T. SMITH, C. G. EVANS, P. DOANE-SETZER, V. M. CASTRO, M. K. TAHIR and B. MANNERVIK, Denitrosation of 1,3~bis(2-chlorethyl)-l-nitrosourea by class ~t glutathione transferases and its role in cellular resistance in rat brain tumor cells, Cancer Res. 49, 2621-2625 (1989). M . G . BOLTON, O. M. COLVIN and J. HILTON, Specificity of isozymes of routine hepatic glutathione S-transferase for the conjugation of glutathione with L-phenylalanine mustard, Cancer Res. $1, 2410--2415 (1991). P.J. CIACCIO, K. D. TEW and F. P. LACRETA, The spontaneous and glutathione S-transferase mediated reaction of chlorambucil with glutathione, Cancer Commun. 2, 279-285 (1990). M. G. BOLTON, D. A. NOE, O. M. COLVIN and J. HILTON, Kinetic analysis of glutathione and glutathione S-transferase in the detoxification of phenylalanine mustard, Proc. Am. Assoc. Cancer Res. 31,374 (1990). S. L. PALLANTE, C. A. L1SEK, D. M. DULIK and C. FENSELAU, Glutathione conjugates. Immobilized enzyme synthesis and characterization by fast atom bombardment mass spectrometry, Drug Metab. Dispos. 14, 313-318 (1986). F.Y. LEE, Glutathione diminishes the anti-tumor activity of 4-hydroperoxycyciophosphamide by stabilizing its spontaneous breakdown to alkylating metabolites, Br/t. J. Cancer 63, 45-50 (1991). P.M. O'CONNOR, D. K. FERRIS, G. A. WHITE, J. PINES, T. HUNTER, D. L. LONGO and K. W. KOHN, Relationship between cdc2 kinase, DNA cross-linking, and cell cycle perturbations induced by nitrogen mustard, Cell Growth Diff. 3, 43-52 (1992).
0065-2571/93/$24.00 ~ 1993PergamonPress Lid
Printed in Great Britain.All rights reserved
ROLE OFGLUTATHIONE IN C E L L U L A R R E S I S T A N C E TO ALKYLATING AGENTS O. MICHAEL COLVIN °, HENRY S. FRIEDMANt, MICHAEL P. GAMCSIK$, CATHERINE FENSELAU§ and JOHN HILTON* *Division of Pharmacology and Experimental Therapeutics, The Johns Hopkins Hospital Oncoiogy Center, Baltimore, MD tDepartment of Pediatrics and Preuss Laboratory for Brain Tumor Research, Duke University Medical Center, Durham, NC SDivision of NMR Research, Department of Radiology, The Johns Hopkins University School of Medicine, Baltimore, MD §Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, MD
INTRODUCTION
The tripeptide glutathione (Fig. 1) is known to participate in the detoxification of electrophilic xenobiotics, and a number of investigators have described an association between elevated concentrations of glutathione in cells and resistance of these cells to alkylating agents (1-3). We have explored the mechanism of this association and have demonstrated that the alkylating agents which we have studied react with glutathione to produce conjugates which are stable. The agents for which we have demonstrated conjugates with glutathione are melphalan (4, 5), chlorambucii (6), mechlorethamine (7), cyclophosphamide (8), and phosphoramide mustard (8), the active alkylating agent produced by the metabolism of cyclophosphamide (9). The structure of the monoglutathionyl conjugate of mephalan is shown in Figure 2 (4).
HO-C--CHCHsC~-C-NIt-CH-C-NH'CHj-C-OH Glutothtoe4
FIG. 1. Structure of glutathione.
oh-
thH
CI
FIG. 2. The monoglutathionyl conjugate of melphalan. 19
20
o . M . COLVIN,
et al.
Our studies indicate that these conjugates are stable, even at elevated temperatures, and they do not have alkylating activity as measured by reaction with nitrobenzylpyridine, a standard assay for biological alkylating agents. The melphalan-GSH conjugate is not cytotoxic when incubated with cells, but it is doubtful that this compound or the conjugates of other alkylating agents would enter cells, as glutathione does not. It is likely that most of the GSH-alkylating agent conjugates are formed intracellularly, and our data indicate that these compounds should not be cytotoxic to cells. STUDIES
OF THE MECHANISM AGENT-GLUTATHIONE
OF AN ALKYLATING REACTION
In our studies of the conjugation of melphalan with glutathione we found that the presence of glutathione did not accelerate the rate of disappearance of meiphalan (Fig. 3). This finding is consistent with the hypothesis that the chemical reaction is not a direct displacement of chloride by the thiol group, but rather a reaction of glutathione with the reactive aziridinium ion formed from melphalan (Fig. 4). In relatively low chloride concentrations, such as biological fluids, this reaction is irreversible. Therefore, the rate of removal of the aziridinium will not affect the rate of aziridinium formation, and the presence or absence of glutathione will not affect the rate of disappearance of the parent melphalan. To examine the mechanism of the reaction directly, mechlorethamine was labeled with 13C in the ot carbon of the mustard group and reacted with
1
0.1
i .5
i 0.01 0.001
~oo
~ time
(mln)
~o
~o
FIG. 3. Decomposition of melphalan at physiologic pH in the presence and absence of glutathione.
GLUTATHIONE AND ALKYLATING AGENTS
21
c¢~" ~ L . ~ c ~ ' ~ ~ID G-S-T
%%% %
c~
~,~
,a-c~-c~o,~
" ~_/'
FIG. 4. Potential reaction mechanisms of melphalan with glutathione.
glutathione (7). In the conjugate formed, the [13C] was found to be equally distributed between the a and 13 carbons of the glutathionyi conjugate, indicating that the reaction had proceeded through the cyclic aziridinium intermediate, with the resulting redistribution of the ethylene carbon atoms. In this study, it was also observed that the aziridinium intermediate was detectable by NMR, with a longer half-life than is usually presumed. Using the same approach, we have also studied aniline mustard, an aromatic nitrogen mustard similar to melphalan, and found that this reaction also proceeds through the aziridinium intermediate. The enzyme glutathione S-transferase (EC2.5.1.18) catalyzes the conjugation of glutathione with electrophiles, and elevated glutathione S-transferase activity in cells has been associated with resistance to alkylating agents (10, 11). Human and murine glutathione S-transferases occur as a number of isozyme families, and the ct,/x, and lr isozymes in particular have been studied to determine their relationship to alkylating agent resistance. Elevated levels of both ~r (12) and a (13, 14) isozymes have been associated with cellular resistance to alkylating agents, and the/~ isozyme has been shown to specifically catalyze the inactivation of BCNU (15). We studied the effect of the et, ~., and "rr on the reaction of melphalan with glutathione and found that only the et isozyme catalyzed this reaction (16). Cacao and colleagues have also reported that the reaction of chlorambucil with glutathione is catalyzed by the a isoenzyme (17), and in very recent studies Hilton found that the reaction of busulfan with glutathione is catalyzed specifically by the a isozyme (unpublished data). In our studies of the enzyme catalyzed conjugation of melphalan, we found that the rate of disappearance of the parent compound is not accelerated by the presence of enzyme, indicating that the enzyme catalyzed reaction also proceeds through the aziridinium intermediate (18).
22
O.M. COLVIN, et al.
Studies of the mechanisms of reaction with glutathione have now been carried out with cyclophosphamide and its aikylating metabolite, phosphoramide mustard. For these studies cyclophosphamide and phosphoramide mustard were synthesized with a deuterium label in the 13position of the chloroethyl group. The reactions were carried out in the absence or presence of glutathione S-transferase (from rabbit liver) and the location of the deuterium labeled carbon determined from the molecular weights of the fragments seen on mass spectrometry. Cyclophosphamide was found to react with glutathione without enzyme at pH 10, and this reaction proceeded through a direct displacement reaction (8) (Fig. 5). At pH 7.4 cyclophosphamide reacted with glutathione in the presence of enzyme, and again this reaction proceeded through a direct displacement reaction. These findings correlate with the fact that cyclophosphamide is not a reactive alkylating agent at physiological pH and does not show evidence of forming an aziridinium in NMR studies. The fact that the direct displacement of chloride in cyciophosphamide can be catalyzed by GST suggests that an analogous reaction can be catalyzed with melphalan and other nitrogen mustards, but is difficult to detect in the presence of a more rapid reaction with the aziridinium moiety. In contrast to cyclophosphamide, phosphoramide mustard reacted at
m/z 213
A.
B.
H 0
r~rl
"!_N
/--N\II /-"u2~
H 0
cN\IpI
N/--C~ cl
H 0
_ /--N\I I + / :
~-.0 / - ~-co~c,
/-
\ll
/
~v
~ _P-N ~-.0 / ~-CD2CI
--CD-Cl
,
~_O/P-N~,c0,
-
~
t
\
.
H 0 /'--N\I I
~
_SG
I /
.F{-CD2
~._ O/P-- N~._COICI FIG. 5. Alternative mechanisms for the conjugation of glutathione with cyclophosphamide. (From Ref. (8).)
23
GLUTATHIONE AND ALKYLATING AGENTS
B.
R O D I . C I ~ - - / N -- P - N H 2 1 D
Ao D |
D
R O I . . N -- P - NH 2
Ct D
O-
R
D
O - - p - - NH 2
I
I
O-
OGSH transferase
GSH tronsferase
323 GS
N I R
P - NH 2 I O-
GS
°
N --
t ,. :,
!_
NH 2
o-
FIG. 6. Alternative mechanisms for the conjugation of glutathione with phosphoramide mustard. (From Ref. (8).)
pH 10 in the absence of enzyme to form a glutathione conjugate via an aziridinium intermediate (Fig. 6). At pH 7.0 the spontaneous reaction was undetectable by mass spectrometry, but in the presence of enzyme the conjugate was formed, again through the aziridinium intermediate (8). The lack of the observed spontaneous reaction at pH 7.0 with phosphoramide mustard indicates that the aziridinium intermediate of phosphoramide mustard forms more slowly than those of mechlorethamine or melphalan. Fenselau and colleagues also showed that glutathione can react with one of the activated forms of cyclophosphamide, iminocyclophosphamide (or possibly 4-hydroxycyclophosphamide), to form 4-glutathionylcyclophosphamide (18) (Fig. 7). Lee et al. (19) have provided evidence that this reaction interferes with the antitumor effect of activated cyclophosphamide. We are currently conducting further studies of the spontaneous and enzyme catalyzed reactions of phosphoramide mustard and the other activated metabolites to establish the rates of these reactions and the isozyme(s) responsible for the catalysis of the reactions.
24
O . M . C O L V I N , et al.
o,E_
,:---,7
FIG. 7. Conjugation of glutathione at the 4 position of cyclophosphamide.
STUDIES
OF HUMAN MEDULLOBLASTOMA TOCYCLOPHOSPHAMIDE
CELLS RESISTANT
In a recent study the roles of glutathione and other mechanisms of cellular resistance were investigated in medulloblastoma cell lines resistant to cyclophosphamide in vivo and 4-hydroperoxycyclophosphamide in vitro (3). In three out of four resistant cell lines, glutathione was responsible for all or a significant part of the resistance, as demonstrated by increased glutathione levels in the resistant cells and sensitization of the cells by depletion of glutathione by buthionine sulfoximine. A linear relationship between the cellular concentration of glutathione and the sensitivity of the cells to 4-hydroperoxycyclophosphamide could be demonstrated (Fig. 8). The two other mechanisms of resistance detected were increased aldehyde dehydrogenase and an undetermined mechanism, which we believe may be the cdc2 kinase alteration recently described by O'Connor et al. (20).
20
5
J
•
•
y - 6.67 + 0.324x
5 R z - 0.647
0
0
5
10
15
i
J
20
25
GSH (nmol/mg protein)
30
FIG. 8. Relation between intracelluiar GSH concentration and concentration of 4-hydroperoxycyclophosphamide (4-HC) required for 1 log cell kill in clonogenic assay. O, Cells without BSO treatment; II, cells after BSO treatment. (From Ref. (3).)
GLUTATHIONE AND ALKYLATING AGENTS
25
SUMMARY
Both elevated glutathione levels and increased activity of the enzyme glutathione S-transferase have been associated with the resistance of cells to alkylating agents. We have demonstrated that one mechanism of this resistance is the inactivation of the alkylating agents by conjugation with glutathione. This conjugation can be catalyzed by glutathione S-transferase. For the nitrogen mustard agents we have studied, both the spontaneous and enzyme catalyzed reactions proceed through the aziridinium intermediates of the alkylating agents, and the cx isoenzymes of GST are involved. In a study of cyclophosphamide resistant medulloblastoma cell lines elevated cellular concentrations of glutathione correlated well with the resistance of the cell lines. REFERENCES I. G. CALCUTI" and T. A. CONNERS, Tumor sulfhydryl levels and sensitivity to the nitrogen mustard merophan, Biochem. Pharmacol. 12, 839--845(1963). 2. D. SUZUKAKA, B. J. PETRO and D. T. VISTICA, Reduction in glutathione content of L-PAM resistant L1210 cell confers drug sensitivity, Biochem. Pharmacol. 31, 121-124 (1982). 3. H. S. FRIEDMAN, O. M. COLVIN, S. H. KAUFMANN, S. M. LUDEMAN, N. BULLOCK, D. D. BIGNER and O. W. GRIFFITH, Cyclophosphamide resistance in medulloblastoma, Cancer Res. 32, 5373-5378 (1992). 4. D. M. DULIK, C. FENSELAU and J. HILTON, Characterization of melphalanglutathione adducts whose formation is catalysed by glutathione S-transferases, Biochem. Pharmacol. 35, 3405--3409(1986). 5. D . M . DULIK and C. FENSELAU, Conversion of meiphalan to 4-(glutathionyl)phenylalanine. A novel mechanism for conjugation by glutathione-S-transferases, Drug Metab. Dispos. 15, 195-199 (1987). 6. D. M. DULIK, O. M. COLVIN and C. FENSELAU, Characterization of glutathione conjugates of chlorambucil by fast atom bombardment and thermospray liquid chromatography/mass spectrometry, Biomed. Environ. Mass Spectrom. 19, 248-252 (1990). 7. M.P. GAMCSIK, T. G. HAMILL and O. M. COLVIN, NMR studies of conjugation of mechlorethamine with glutathione, J. Med. Chem. 33, 1009-1014 (1990). 8. Z.M. YUAN, P. B. SMITH, R. B. BRUNDRETI', O. M. COLVIN and C. FENSELAU, Glutathione conjugation with phosphoramide mustard and cyclophosphamide. A mechanistic study using tandem mass spectrometry, Drug Metab. Dispos. 19, 625--629 (1991). 9. O. M. COLVIN and B. A. CHABNER, Alkylating agents, pp. 276-313 in Cancer Chemotherapy: Principles and Practice (B. A. CHABNER and J. M. COLLINS, eds.) J. L. Lippincott, Philadelphia, Pennsylvania (1990). 10. C. N. ROBSON, A. D. LEWIS, C. R. WOLF, J. D. HAYES, A. HALL, S. J. PROCTOR, A. L. HARRIS and I. D. HICKSON, Reduced levels of drug-induced DNA cross-linking in nitrogen mustard-resistant Chinese hamster ovary cells expressing elevated glutath/one S-transferase activity, Cancer Res. 47, c~z2-6027 (1987). 11. A. L. BUTLER, M. L. CLAPPER and K. D. "FEW, Glutathione S-transferase in nitrogen mnstard-resistant and -sensitive cell lines, Mol. Pharmacol. 31, 575-578 (1987). 12. K. NAKAGAWA, N. SAIJO, S. TSUCHIDA, M. SAKAL, Y. TSUNOKAWA, J. YOKOTA, M. MURAMATSU, K. SATO, M. TERADA and K. D. "FEW,
26
13. 14.
15. 16. 17. 18. 19. 20.
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