- Email: [email protected]
Formation of a phosphoramide mustard-nucleotide adduct that is not by alkylation at the N7 position of guanine
BIOCHEMICALAND BIOPHYSICALRESEARCH COMMUNICATIONS Pages 843-850
Vo1.163, No. 2,1989 September 15,1989
FORMATION OF A PHOSPHORAMIDE MUSTARD-NUCLEOTIDE ADDUCT THAT IS NOT BY ALKYLATION AT THE N7 POSITION OF GUANINE Alexander E. Maccubbin, L I ~ Caballes, Girish B. Chheda*, Robert F. Struck-- and Hira L. Gurtoo Grace Cancer Drug Center and *Department of Biophysics Roswell Park Memorial Institute 666 Elm Street, Buffalo, NY 14263 **Southern Research Institute, Birmingham, AL 35255 Received July 28, 1989
SUMMARY: The reaction of 2'-deoxyguanosine 3'-monophosphate with phosphoramide mustard resulted in the formation of several adducts. One of these adducts was formed by linking phosphoramide mustard to the phosphate group of 2'-deoxyguanosine 3'-monophosphate rather than by the generally accepted mechanism involving alkylation at the N7 position of guanine. Thls adduct served as an acceptor for the transfer of 32p from [y32p]ATP by polynucleotlde kinase and thus could be detected by the sensitive 32p_ postlabeling assay. ®1989 A c a d e m i c Press, Inc. Metabolism of
the
antlcancer agent cyclophosphamide results
production of the reactive (Figure l)
(1,2).
metabolltes acrolein
in
the
and phosphoramide mustard
Phosphoramide mustard is generally considered to be the
therapeutlcaily active metabolite responsible for the antitumor activity of cyclophosphamlde
(2-5).
A number of
studies have demonstrated that
phosphoramlde mustard alkylates guanine at the 7 position (6-11). been suggested that
this
alkylatlng
activity,
involving
It
has
formation of
monoadducts and cross-links, is responsible for the therapeutic activity of cyclophosphamlde (6,7,11). We have been studying cyclophosphamide:DNA adducts to determine their nature and their role in the biological activity of cyclophosphamide, and to develop ultrasensitlve analytical adducts.
methods for
the quantification
of
such
We report, here, the formation of a novel adduct that does not
involve N7 alkylatlon of guanine but rather Is the result of the formation of phosphoester linkage between phosphoramlde mustard and 2'-deoxyguanosine 3'-monophosphate.
T h l s phosphoester adduct was formed In addition to N7
alkylation products.
MATERIALS AND METHODS Materials. 2'-deoxyguanoslne, 2'-deoxyguanosine 3'-monophosphate, 2'-deoxyguanoslne 5'-monophosphate and 2'-deoxyguanosine 3',5'-bisphosphate were
843
0006-291X/89 $1.50 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 163, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 4-Hydroxycyclophosphomide
Cyclophosphomide ClCHzCH2
H N
MFO, NADPH
\ / / N-P~O
CICHzCH2
H
OH
CH ;CH2
N
\N-P~O
?H 2
O
CICH2CH2
/
CH2
ClCH2CH2
0
CH 2
Acrolein H
CH; C - C
H 0
CICHzCH2
CICHzCH2 \
NH
/N-t° CICHzCH2
NHz
0
2
H C=O \
/c"2
CH2
Aldophosphamide
\N_p/_-O
I
/
\
CICH2CH2
OH
Phosphoromide Mustord
BINDING TO NUCLEOSIDE 3' MONOPHOSPHATE OR DNA
NUCLEOSIDE3'PHOSPHOESTERADDUCT (SUCH AS PEAK A)
ALKYLATION OF GUANINEAT N7 YIELDING FINAL PRODUCTSAFTER ACID HYDROLYSIS
I
ACID HYDROLYSIS i
NUCLEASEP1
1 N-(2-chloroethyl)-N-[2-(7-guanmyl)ethyl]amine(NOR-G) 2 N-(2-hydroxyethyl)-N-[2-(7-guanmyl)ethyl]amine(NOR-G-OH) 3 NN-bis-[2-(7guaninyl)ethyl]amine(G-NOR-G)
i BASE + PHOSPHOESTER ADDUCT
Figure l:
NUCLEOSIDE + PHOSPHOESTER ADDUCT
Metabollsm of cyclophosphamide and binding of phosphoramide mustard to nucleotides.
purchased f r o m Pharmacia. Guanine, nuclease P], K2HPO 4 and KH2PO 4 were obtained from Sigma. 32p-postlabeling materials have been previously described (12,13). Phosphoramide mustard was kindly provided by the Drug Development Branch of the NCI, and N7 alkylated products of guanine were prepared as previously described ( ] l ) . [Chloroethyl-3H] cyclophosphamide (specific activity 460 mCi/mmole) was purchased from Amersham. Incubation of 2'-Deoxyquanosine 3'-MonosDhosDhate with PhQsphoramide Mustard. One mg of 2'-deoxyguanoslne 3'-monophosphate and 6 mg of phosphoramlde mustard were incubated for 6h in 1 ml of O.IM potassium phosphate buffer, pH 7.4. The reaction mixture was then analyzed uslng a Waters HPLC system using a Supelcosll LC-18-S (Supelco, Inc.) reversed phase Cl8 column. The elution was performed with an i n i t i a l 1socratic run of 50 mM KH2PO4 buffer (pH 4.0) containing 2.5% methanol for 3 minutes followed by a linear 844
Vol. 163, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
gradient to 20% methanol in 50 mM KH2PO4 over 12 min at a flow rate of l ml/min. A fraction containing Peak A was collected and dried. The sample was redissolved in 50 mM ammonium formate buffer (pH 4.5) and analyzed on the same HPLC system, using a Vydac Cl8 column (Vydac 20]TPI04, distributed by Alltech), by elution wlth 50mM ammonium formate buffer for 5 minutes followed by a linear gradient incorporating 20% acetonitrile into this buffer over 20 minutes at a flow rate of l ml/mln. Peak A, purified from several runs, was pooled and used for Its characterization. PreParation of Tritium Labeled Peak A. In order to generate 3H-labeled phosphoramlde mustard to produce 3H-labeled Peak A, i t was necessary to metabolize 3H-labeled cyclophosphamide. [3H] Cyclophosphamide, in the presence of 2'-deoxyguanoslne 3'-monophosphate, was metabolized by p a r t i a l l y purified cytochrome P-450 in a reconstituted system as previously described (14). T h i s mixture was incubated overnight at 37°C and the reactlon was stopped by extraction with chloroform to remove protein and unmetabolized cyclophosphamlde. The aqueous layer after extraction was separated by HPLC and a fraction containing 3H-labeled peak A was collected. T h l s fraction was purified as described above to yield 3H-labeled Peak A. Characterization of Peak A. In order to determine i f Peak A could be detected at the low levels likely to be found in DNA, Peak A was tested for its s u i t a b i l i t y for detection by the 32p-postlabeling assay (12). An aliquot of purified Peak A (~l pmole) was dried in a SpeedVac rotary concentrator/drier (Savant Corp.) to remove ammonium formate buffer and was postlabeled wlth 32p essentially as previously described (12,13). Briefly, Peak A was incubated at 37°C for 45 minutes with 50 ~Ci of [y32p]ATP (specific a c t i v i t y >5000 Ci/mmole) and polynucleotide kinase. The postlabeled sample was diluted and I / I 0 of the diluted volume was spotted on a PEl-cellulose TLC plate. Two dimensional chromatography was then carried out using 2.25M ammonium formate, pH 3.5 for the f i r s t dimension and O.3M ammonium sulfate, buffered wlth NaH2PO4 to pH 7.5, for the second dimension. 32p-Labeled Peak A was detected by autoradiography. 32p-Labeled Peak A was eluted from the TLC plate with I.OM NaCl for analysis by HPLC and enzymatic hydrolysis. An aliquot of Peak A (NO.5 nmole) was also postlabeled by incubating i t with unlabeled ATP (600 pmo]e) and polynucleotide kinase. T h l s served as a UV-detectable standard for the bisphosphate of Peak A. Peak A and 32p-labeled peak A were treated with nuclease Pl to remove 3'-phosphate and the reaction products were separated by HPLC using KH2PO4 buffer as described above. Detection was by UV absorbance at 254 nm and fractions were collected at one minute intervals. Peak A and 3Hpeak A were hydrolyzed In O.IN HCI at 80°C for 20 minutes ( l l ) and the reaction products were analyzed by HPLC.
RESULTS Incubation
of
mustard resulted
2'-deoxyguanosine in the formation
3'-monophosphate
of
a number of
with
phosphoramide
products
that
separated by HPLC and detected by absorbance at 254 nm (Figure 2).
could
be
A major
peak, designated Peak A, eluted a f t e r 2'-deoxyguanosine 3'-monophosphate and amounted to
approximately
(Figure 2B). >95%
pure
1.5-2.0%
as
determined
by
deoxynucleoside
the o r i g i n a l and p u r i f i e d
HPLC.
yielded a single spot that migrated, unmodified
of
This peak was collected
amount of resulting
nucleotide
in a product
After 32p-postlabeling,
Peak
in both dimensions, more rapidly
3'-monophosphate 845
standards
that
A
than
had been post-
Vol. 163, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A .010
E
.005
c
to o4
L
0 0 C 0
I
I
0 ¢,1
Pe0k A
.010
.005
I 5
I I0
Retention
labeled
slmllarly
wlth
15
(min)
HPLC separation of 2'-deoxyguanoslne 3'-monophosphate after incubation in O.IM potassium phosphate buffer (pH 7.4) for 6 hr at 37°C either alone (A) or wlth phosphoramlde mustard (B). A major reaction product, Peak A, was isolated and purified for further analysls.
Figure 2:
plate
Time
(Table
I).
32p-Labeled
Peak A,
eluted
from
1.0M NaCl and analyzed by HPLC, cochromtographed
the
with
TLC
a UV-
detectable standard of peak A that had been prepared by postlabeling Peak A with non-radioactive ATP (Figure 3B).
Table 1:
Two dimensional TLC Rf values for 32p-postlabeled compounds
Dimension ID 2D
a dA3'P dC3'P dG3'P dT3'P
= = = =
dA3'P
dC3'P
0.61 0.29
0.53 0.34
Compounda dG3'P 0.20 0.14
2'-deoxyadenosine 3'-monophosphate. 2'-deoxycytldlne 3'-monophosphate. 2'-deoxyguanoslne 3'-monophosphate. 2'-deoxythymldlne 3'-monophosphate. 846
dT3'P 0.39 0.49
Peak A .74 .63
Vol. 163, No. 2, 1989
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
.05
L_
A
.04 .03
I .02 E e• 1" In OJ
.01
25
r- I
eO
~
~i i
.020
15~
i
m ~t .010 <~ .005
Io
I L.]
I
'
.025 .020
5
j"
C -15
,,,~ x
"3
l ] I
.015 .010 .005
- 20
I
.015
0
B
~-t_~
d
4
8
6
3
,_ 12
Retention Time (rain) HPLC proflles of postlabeled 2'-deoxyguanoslne 3'-monophosphate (A) and peak A (B) and postlabeled peak A after treatment with nuclease Pl (C).
Figure 3:
Inltlal
characterization of
nuclease Pl that wlll 3'-mononucleotides, respectively. was treated
Peak A was by enzymatic hydrolysis
remove the 3'-phosphate group of
with
bisphosphates or
resulting in 5'-mononucleotide and nucleoside products,
32p-Labeled Peak A blsphosphate, eluted from TLC plates, wlth
nuclease Pl
at
demonstrated that essentlally all
37°C for
30 minutes.
authentic
2'-deoxyguanoslne 5'-monophosphate (Figure
digestion
of
unlabeled
2'-deoxyguanosine
Peak A,
HPLC analysis
the radioactiv|ty cochromatographed with i.e.,
3'-monophosphate,
3C).
Nuclease Pl
phosphoramlde mustard modified
resulted
in
a
product
that
cochromatographed wlth an authentic 2'-deoxyguanosine standard (Table 2). Peak A was also characterized for 20 minutes.
by acid hydrolysls in O.IN HCI at 80°C
HPLC analysls of acld hydrolyzed Peak A demonstrated the
presence of a product that cochromatographed wlth guanine but not NOR-G or NOR-G-OH (Table 2).
Moreover, when 3H-Peak A was acid
hydrolyzed,
the
radioactivity was found in the void volume and not with the N7 alkylated 847
Vol. 163, No. 2, 1 9 8 9 Table 2:
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
HPLC retention time of chemical standards and Peak A a f t e r enzymatic and chemical treatment
Compounda
Treatmentb
Retention Time (minutes)
dG3'P Peak A Guanine NOR-G NOR-G-OH dG3'P Peak A dG dG3'P Peak A Peak A dG3'5'P dG3'P Peak A dG5'P dG3'P Peak A
..... ..... ..... ..... ..... O.IN HCI O.IN HCI ..... NPI NPI NPI then O.IN HCI ..... PNK + ATP PNK + ATP ..... PNK + ATP then NPI PNK ÷ ATP then NPI
6.5 II.9 4.9 5.7 3.8 4.9, 6.5 4.9 14.4 14.4 14.4 4.9 2.4 2.4 3.7 7.0 7.0 7.0
adG3'P = 2'-deoxyguanoslne 3'-monophosphate; NOR-G = N-(2-chloroethyl)-N[2-(7-guanlnyl)ethyl]amlne; NOR-G-OH = N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amlne; dG = 2'deoxyguanosine; dG3'5'P = 2'-deoxyguanoslne 3',5'-blsphosphate; dG5'P = 2'-deoxyguanoslne 5'-monophosphate. bO.IN HCI = O.IN HCI 80°C, 20 minutes; NPI = Nuclease Pi 37°C, 30 minutes; PNK + ATP = polynucleotide kinase plus ATP 37°C, 45 minutes. All retention times are from samples run on Supelcosil LC-18-S reversed-phase column using KH2P04 buffer as described in Materials and Methods.
products ( d a t a not
shown).
Finally,
acid hydrolysis of
nuclease P l - t r e a t e d
Peak A a l s o y i e l d e d a product t h a t cochromatographed w i t h guanine (Table 2).
DL#CUSSION It
Is commonly accepted that phosphoramide mustard derived from cyclo-
phosphamide reacts wlth DNA vla the a l k y l a t i o n of the N7 position of guanine (Figure
l).
We expected to
isolate
deoxynucleoslde 3'-monophosphates after
N7 alkylated treatment of
products the
as modifled
nucleotide
with
phosphoramlde mustard to test whether these adducts could serve as substrates for polynucleotlde klnase-catalyzed transfer of 32p from [y32p]ATP. However, among the reaction products obtained by the incubation of phosphoramlde mustard and 2-deoxyguanosine 3'-monophosphate was a major product that had characteristics d i f f e r e n t than the expected N7 a l k y l a t l o n products (Figure l ) . 32p-Postlabeling analysis demonstrated that t h l s product, Peak A, is a modified 2'-deoxyguanoslne 3'-monophosphate derivative that has retained the 3'-phosphate
group.
Polynucleotide
klnase 848
transferred
32p from
[y32p]_
Vol. 163, No. 2, 1 9 8 9
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ATP to Peak A resulting in a modified 2'-deoxyguanoslne 3',5'-bisphosphate that could be separated from unmodified 2'-deoxyguanoslne 3',5'-bisphosphate and other 2'-deoxynucleoslde 3',5'-bisphosphates
by two dlmenslonal TLC and
HPLC. Enzymatic and acid hydrolysis of peak A yielded unmodified 2'-deoxyguanosine and guanine, respectively, 2'-deoxyguanoslne and guanine.
rather
than N7 alkylated
modified
These data suggest that 2'-deoxyguanosine
3'-monophosphate was modified by phosphoramide mustard to form a phosphodiester linkage with the 3'-phosphate group. Preliminary NMR data of Peak A are consistent with the identification of Peak A as a phosphodiester adduct. The signlficance of Peak A in the antitumor activity of cyclophosphamide has yet to be established.
We have detected this adduct in DNA treated wlth
phosphoramide mustard or
cytochrome P-450-metabollzed cyclophosphamide (a
detailed
report
therefore,
of
these studies is
in preparation).
It
would appear,
that Peak A is not an artifact of incubating the monomer, 2'-de-
oxyguanoslne
3'-monophosphate, with
phosphoramide mustard.
To
our
knowledge, only one other group has speculated on the possibllity of phosphoester adduct formation by cyclophosphamide (15,16). ing given the fact
that
most studies
This is not surpris-
examining DNA adducts formed by
phosphoramide mustard derived from cyclophosphamide have used 3H-labeled drug and have included acid hydrolysis of DNA to release adducts. data have shown, under these conditions,
Peak A is
unmodified guanine that would be devoid of radlolabel,
As our
destroyed to yield thus rendering i t
undetectable by radiometric methods. The formatlon of DNA adducts involving phosphate groups in phosphoester linkages has been reported for other antlcancer drugs (17).
The adduction
of nucleic acids by phosphoramide mustard may occur in an analogous manner. The phosphate ester adduct of phosphoramide mustard characterized in this report is relatively stable, except under very acidic conditions,
and may
represent a relatively persistent adduct. Moreover, phosphoester adducts can theoretically form with all nucleotldes and thus could potentially outnumber adducts formed by N7 alkylation of guanine. potential
for
stability, effects
of
wlde distribution
wlthin
nucleic
these adducts may contribute cyclophosphamide.
chemotherapeutic effects
At
Thus, given their
acids and their
signiflcantly
to
present, however, their
and toxicologic
effects
of
relative
the blologlcal role
in
the
cyclophosphamide is
unknown and will require further study.
ACKNOWLEDGMENTS We wish to thank Miss Karen Marie Schrader f o r preparation
of
this
manuscript.
This
CA-43567 and CA-24538. 849
work
her assistance in the
was supported
by NIH grants
Vol. 163, No. 2, 1 9 8 9
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
REFERENCES I. Montgomery, J.A. and Struck, R.F. (1971). Prog. Drug. Res. 17, 320-409. 2. Friedman, O.M., Myles, A. and Colvin, M. (1979). Ln: Advances in Cancer Chemotherapy, A. Rosowsky (ed.), Marcel Dekker Inc., New York, pp. 143-204. 3. Connors, T.A., Cox, P.J., Farmer, P.B., Foster, A.B. and Jarman, M. (1974). B1ochem. Pharmacol. 23, 115-124. 4. Struck, R.F., Kirk, M.C., Witt, M.H. and Laster, W.R. Jr. (1975). Biomed. Mass Spectrom. 2, 46-42. 5. Colvin, M., Brundrett, R.B., Kan, M.N., Jardine, I. and Fenselau, C. (1976). Cancer Res. 36, 1121-1126. 6. Mehta, J.R., Przybylsk~, M. and Ludlum, D.B. (1980). Cancer Res. 40, 4183-4186. 7. Vu, V.T., Fenselau, C.C. and Colvln, O.M. (1981). 3. Am. Chem. Soc. 103, 7362-7364. 8. Mehta, 3.R. and Ludlum, D.B. (1982). Cancer Res. 42, 2996-2999. 9. Chetsanga, C.3., Polldori, G. and Mainwaring, M. (1982). Cancer Res. 42, 2616-2621. I0. Kallama, S. and Hemm~nkl, K. (1984). Acta Pharmacol. Toxlcol. 54, 214-220. 11. HemmlnkI, K. (1985). Cancer Res. 45, 4237-4243. 12. Reddy, M.V., Gupta, R.C., Randerath, E. and Randerath, K. (1984). Carclnogenesls 5, 231-243. 13. Dunn, B.P., Black, 3.3. and Maccubbin, A. (1987). Cancer Res. 47, 6543-6548. 14. Struck, R.F., Kari, P., Kalln, 3., Montgomery, 3.A., Marlnello, A.J., Love, 3., Bansal, S.K. and Gurtoo, H.L. (1984). B1ochem. B1ophys. Res. Comm. 120, 390-396. 15. Lindemann, H. and Harbers, E. (1980). Arzneim-Forsch/Drug Res. 30, 2075-2080. 16. Lindemann, H. (1984). Antlcancer Res. 4, 53-58. 17. Carter, C.A., Kirk, M.C. and Ludlum, D.B. (1988). Nucl. Acids Res. 16, 5661-5672.
850
Vo1.163, No. 2,1989 September 15,1989
FORMATION OF A PHOSPHORAMIDE MUSTARD-NUCLEOTIDE ADDUCT THAT IS NOT BY ALKYLATION AT THE N7 POSITION OF GUANINE Alexander E. Maccubbin, L I ~ Caballes, Girish B. Chheda*, Robert F. Struck-- and Hira L. Gurtoo Grace Cancer Drug Center and *Department of Biophysics Roswell Park Memorial Institute 666 Elm Street, Buffalo, NY 14263 **Southern Research Institute, Birmingham, AL 35255 Received July 28, 1989
SUMMARY: The reaction of 2'-deoxyguanosine 3'-monophosphate with phosphoramide mustard resulted in the formation of several adducts. One of these adducts was formed by linking phosphoramide mustard to the phosphate group of 2'-deoxyguanosine 3'-monophosphate rather than by the generally accepted mechanism involving alkylation at the N7 position of guanine. Thls adduct served as an acceptor for the transfer of 32p from [y32p]ATP by polynucleotlde kinase and thus could be detected by the sensitive 32p_ postlabeling assay. ®1989 A c a d e m i c Press, Inc. Metabolism of
the
antlcancer agent cyclophosphamide results
production of the reactive (Figure l)
(1,2).
metabolltes acrolein
in
the
and phosphoramide mustard
Phosphoramide mustard is generally considered to be the
therapeutlcaily active metabolite responsible for the antitumor activity of cyclophosphamlde
(2-5).
A number of
studies have demonstrated that
phosphoramlde mustard alkylates guanine at the 7 position (6-11). been suggested that
this
alkylatlng
activity,
involving
It
has
formation of
monoadducts and cross-links, is responsible for the therapeutic activity of cyclophosphamlde (6,7,11). We have been studying cyclophosphamide:DNA adducts to determine their nature and their role in the biological activity of cyclophosphamide, and to develop ultrasensitlve analytical adducts.
methods for
the quantification
of
such
We report, here, the formation of a novel adduct that does not
involve N7 alkylatlon of guanine but rather Is the result of the formation of phosphoester linkage between phosphoramlde mustard and 2'-deoxyguanosine 3'-monophosphate.
T h l s phosphoester adduct was formed In addition to N7
alkylation products.
MATERIALS AND METHODS Materials. 2'-deoxyguanoslne, 2'-deoxyguanosine 3'-monophosphate, 2'-deoxyguanoslne 5'-monophosphate and 2'-deoxyguanosine 3',5'-bisphosphate were
843
0006-291X/89 $1.50 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 163, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 4-Hydroxycyclophosphomide
Cyclophosphomide ClCHzCH2
H N
MFO, NADPH
\ / / N-P~O
CICHzCH2
H
OH
CH ;CH2
N
\N-P~O
?H 2
O
CICH2CH2
/
CH2
ClCH2CH2
0
CH 2
Acrolein H
CH; C - C
H 0
CICHzCH2
CICHzCH2 \
NH
/N-t° CICHzCH2
NHz
0
2
H C=O \
/c"2
CH2
Aldophosphamide
\N_p/_-O
I
/
\
CICH2CH2
OH
Phosphoromide Mustord
BINDING TO NUCLEOSIDE 3' MONOPHOSPHATE OR DNA
NUCLEOSIDE3'PHOSPHOESTERADDUCT (SUCH AS PEAK A)
ALKYLATION OF GUANINEAT N7 YIELDING FINAL PRODUCTSAFTER ACID HYDROLYSIS
I
ACID HYDROLYSIS i
NUCLEASEP1
1 N-(2-chloroethyl)-N-[2-(7-guanmyl)ethyl]amine(NOR-G) 2 N-(2-hydroxyethyl)-N-[2-(7-guanmyl)ethyl]amine(NOR-G-OH) 3 NN-bis-[2-(7guaninyl)ethyl]amine(G-NOR-G)
i BASE + PHOSPHOESTER ADDUCT
Figure l:
NUCLEOSIDE + PHOSPHOESTER ADDUCT
Metabollsm of cyclophosphamide and binding of phosphoramide mustard to nucleotides.
purchased f r o m Pharmacia. Guanine, nuclease P], K2HPO 4 and KH2PO 4 were obtained from Sigma. 32p-postlabeling materials have been previously described (12,13). Phosphoramide mustard was kindly provided by the Drug Development Branch of the NCI, and N7 alkylated products of guanine were prepared as previously described ( ] l ) . [Chloroethyl-3H] cyclophosphamide (specific activity 460 mCi/mmole) was purchased from Amersham. Incubation of 2'-Deoxyquanosine 3'-MonosDhosDhate with PhQsphoramide Mustard. One mg of 2'-deoxyguanoslne 3'-monophosphate and 6 mg of phosphoramlde mustard were incubated for 6h in 1 ml of O.IM potassium phosphate buffer, pH 7.4. The reaction mixture was then analyzed uslng a Waters HPLC system using a Supelcosll LC-18-S (Supelco, Inc.) reversed phase Cl8 column. The elution was performed with an i n i t i a l 1socratic run of 50 mM KH2PO4 buffer (pH 4.0) containing 2.5% methanol for 3 minutes followed by a linear 844
Vol. 163, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
gradient to 20% methanol in 50 mM KH2PO4 over 12 min at a flow rate of l ml/min. A fraction containing Peak A was collected and dried. The sample was redissolved in 50 mM ammonium formate buffer (pH 4.5) and analyzed on the same HPLC system, using a Vydac Cl8 column (Vydac 20]TPI04, distributed by Alltech), by elution wlth 50mM ammonium formate buffer for 5 minutes followed by a linear gradient incorporating 20% acetonitrile into this buffer over 20 minutes at a flow rate of l ml/mln. Peak A, purified from several runs, was pooled and used for Its characterization. PreParation of Tritium Labeled Peak A. In order to generate 3H-labeled phosphoramlde mustard to produce 3H-labeled Peak A, i t was necessary to metabolize 3H-labeled cyclophosphamide. [3H] Cyclophosphamide, in the presence of 2'-deoxyguanoslne 3'-monophosphate, was metabolized by p a r t i a l l y purified cytochrome P-450 in a reconstituted system as previously described (14). T h i s mixture was incubated overnight at 37°C and the reactlon was stopped by extraction with chloroform to remove protein and unmetabolized cyclophosphamlde. The aqueous layer after extraction was separated by HPLC and a fraction containing 3H-labeled peak A was collected. T h l s fraction was purified as described above to yield 3H-labeled Peak A. Characterization of Peak A. In order to determine i f Peak A could be detected at the low levels likely to be found in DNA, Peak A was tested for its s u i t a b i l i t y for detection by the 32p-postlabeling assay (12). An aliquot of purified Peak A (~l pmole) was dried in a SpeedVac rotary concentrator/drier (Savant Corp.) to remove ammonium formate buffer and was postlabeled wlth 32p essentially as previously described (12,13). Briefly, Peak A was incubated at 37°C for 45 minutes with 50 ~Ci of [y32p]ATP (specific a c t i v i t y >5000 Ci/mmole) and polynucleotide kinase. The postlabeled sample was diluted and I / I 0 of the diluted volume was spotted on a PEl-cellulose TLC plate. Two dimensional chromatography was then carried out using 2.25M ammonium formate, pH 3.5 for the f i r s t dimension and O.3M ammonium sulfate, buffered wlth NaH2PO4 to pH 7.5, for the second dimension. 32p-Labeled Peak A was detected by autoradiography. 32p-Labeled Peak A was eluted from the TLC plate with I.OM NaCl for analysis by HPLC and enzymatic hydrolysis. An aliquot of Peak A (NO.5 nmole) was also postlabeled by incubating i t with unlabeled ATP (600 pmo]e) and polynucleotide kinase. T h l s served as a UV-detectable standard for the bisphosphate of Peak A. Peak A and 32p-labeled peak A were treated with nuclease Pl to remove 3'-phosphate and the reaction products were separated by HPLC using KH2PO4 buffer as described above. Detection was by UV absorbance at 254 nm and fractions were collected at one minute intervals. Peak A and 3Hpeak A were hydrolyzed In O.IN HCI at 80°C for 20 minutes ( l l ) and the reaction products were analyzed by HPLC.
RESULTS Incubation
of
mustard resulted
2'-deoxyguanosine in the formation
3'-monophosphate
of
a number of
with
phosphoramide
products
that
separated by HPLC and detected by absorbance at 254 nm (Figure 2).
could
be
A major
peak, designated Peak A, eluted a f t e r 2'-deoxyguanosine 3'-monophosphate and amounted to
approximately
(Figure 2B). >95%
pure
1.5-2.0%
as
determined
by
deoxynucleoside
the o r i g i n a l and p u r i f i e d
HPLC.
yielded a single spot that migrated, unmodified
of
This peak was collected
amount of resulting
nucleotide
in a product
After 32p-postlabeling,
Peak
in both dimensions, more rapidly
3'-monophosphate 845
standards
that
A
than
had been post-
Vol. 163, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A .010
E
.005
c
to o4
L
0 0 C 0
I
I
0 ¢,1
Pe0k A
.010
.005
I 5
I I0
Retention
labeled
slmllarly
wlth
15
(min)
HPLC separation of 2'-deoxyguanoslne 3'-monophosphate after incubation in O.IM potassium phosphate buffer (pH 7.4) for 6 hr at 37°C either alone (A) or wlth phosphoramlde mustard (B). A major reaction product, Peak A, was isolated and purified for further analysls.
Figure 2:
plate
Time
(Table
I).
32p-Labeled
Peak A,
eluted
from
1.0M NaCl and analyzed by HPLC, cochromtographed
the
with
TLC
a UV-
detectable standard of peak A that had been prepared by postlabeling Peak A with non-radioactive ATP (Figure 3B).
Table 1:
Two dimensional TLC Rf values for 32p-postlabeled compounds
Dimension ID 2D
a dA3'P dC3'P dG3'P dT3'P
= = = =
dA3'P
dC3'P
0.61 0.29
0.53 0.34
Compounda dG3'P 0.20 0.14
2'-deoxyadenosine 3'-monophosphate. 2'-deoxycytldlne 3'-monophosphate. 2'-deoxyguanoslne 3'-monophosphate. 2'-deoxythymldlne 3'-monophosphate. 846
dT3'P 0.39 0.49
Peak A .74 .63
Vol. 163, No. 2, 1989
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
.05
L_
A
.04 .03
I .02 E e• 1" In OJ
.01
25
r- I
eO
~
~i i
.020
15~
i
m ~t .010 <~ .005
Io
I L.]
I
'
.025 .020
5
j"
C -15
,,,~ x
"3
l ] I
.015 .010 .005
- 20
I
.015
0
B
~-t_~
d
4
8
6
3
,_ 12
Retention Time (rain) HPLC proflles of postlabeled 2'-deoxyguanoslne 3'-monophosphate (A) and peak A (B) and postlabeled peak A after treatment with nuclease Pl (C).
Figure 3:
Inltlal
characterization of
nuclease Pl that wlll 3'-mononucleotides, respectively. was treated
Peak A was by enzymatic hydrolysis
remove the 3'-phosphate group of
with
bisphosphates or
resulting in 5'-mononucleotide and nucleoside products,
32p-Labeled Peak A blsphosphate, eluted from TLC plates, wlth
nuclease Pl
at
demonstrated that essentlally all
37°C for
30 minutes.
authentic
2'-deoxyguanoslne 5'-monophosphate (Figure
digestion
of
unlabeled
2'-deoxyguanosine
Peak A,
HPLC analysis
the radioactiv|ty cochromatographed with i.e.,
3'-monophosphate,
3C).
Nuclease Pl
phosphoramlde mustard modified
resulted
in
a
product
that
cochromatographed wlth an authentic 2'-deoxyguanosine standard (Table 2). Peak A was also characterized for 20 minutes.
by acid hydrolysls in O.IN HCI at 80°C
HPLC analysls of acld hydrolyzed Peak A demonstrated the
presence of a product that cochromatographed wlth guanine but not NOR-G or NOR-G-OH (Table 2).
Moreover, when 3H-Peak A was acid
hydrolyzed,
the
radioactivity was found in the void volume and not with the N7 alkylated 847
Vol. 163, No. 2, 1 9 8 9 Table 2:
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
HPLC retention time of chemical standards and Peak A a f t e r enzymatic and chemical treatment
Compounda
Treatmentb
Retention Time (minutes)
dG3'P Peak A Guanine NOR-G NOR-G-OH dG3'P Peak A dG dG3'P Peak A Peak A dG3'5'P dG3'P Peak A dG5'P dG3'P Peak A
..... ..... ..... ..... ..... O.IN HCI O.IN HCI ..... NPI NPI NPI then O.IN HCI ..... PNK + ATP PNK + ATP ..... PNK + ATP then NPI PNK ÷ ATP then NPI
6.5 II.9 4.9 5.7 3.8 4.9, 6.5 4.9 14.4 14.4 14.4 4.9 2.4 2.4 3.7 7.0 7.0 7.0
adG3'P = 2'-deoxyguanoslne 3'-monophosphate; NOR-G = N-(2-chloroethyl)-N[2-(7-guanlnyl)ethyl]amlne; NOR-G-OH = N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amlne; dG = 2'deoxyguanosine; dG3'5'P = 2'-deoxyguanoslne 3',5'-blsphosphate; dG5'P = 2'-deoxyguanoslne 5'-monophosphate. bO.IN HCI = O.IN HCI 80°C, 20 minutes; NPI = Nuclease Pi 37°C, 30 minutes; PNK + ATP = polynucleotide kinase plus ATP 37°C, 45 minutes. All retention times are from samples run on Supelcosil LC-18-S reversed-phase column using KH2P04 buffer as described in Materials and Methods.
products ( d a t a not
shown).
Finally,
acid hydrolysis of
nuclease P l - t r e a t e d
Peak A a l s o y i e l d e d a product t h a t cochromatographed w i t h guanine (Table 2).
DL#CUSSION It
Is commonly accepted that phosphoramide mustard derived from cyclo-
phosphamide reacts wlth DNA vla the a l k y l a t i o n of the N7 position of guanine (Figure
l).
We expected to
isolate
deoxynucleoslde 3'-monophosphates after
N7 alkylated treatment of
products the
as modifled
nucleotide
with
phosphoramlde mustard to test whether these adducts could serve as substrates for polynucleotlde klnase-catalyzed transfer of 32p from [y32p]ATP. However, among the reaction products obtained by the incubation of phosphoramlde mustard and 2-deoxyguanosine 3'-monophosphate was a major product that had characteristics d i f f e r e n t than the expected N7 a l k y l a t l o n products (Figure l ) . 32p-Postlabeling analysis demonstrated that t h l s product, Peak A, is a modified 2'-deoxyguanoslne 3'-monophosphate derivative that has retained the 3'-phosphate
group.
Polynucleotide
klnase 848
transferred
32p from
[y32p]_
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ATP to Peak A resulting in a modified 2'-deoxyguanoslne 3',5'-bisphosphate that could be separated from unmodified 2'-deoxyguanoslne 3',5'-bisphosphate and other 2'-deoxynucleoslde 3',5'-bisphosphates
by two dlmenslonal TLC and
HPLC. Enzymatic and acid hydrolysis of peak A yielded unmodified 2'-deoxyguanosine and guanine, respectively, 2'-deoxyguanoslne and guanine.
rather
than N7 alkylated
modified
These data suggest that 2'-deoxyguanosine
3'-monophosphate was modified by phosphoramide mustard to form a phosphodiester linkage with the 3'-phosphate group. Preliminary NMR data of Peak A are consistent with the identification of Peak A as a phosphodiester adduct. The signlficance of Peak A in the antitumor activity of cyclophosphamide has yet to be established.
We have detected this adduct in DNA treated wlth
phosphoramide mustard or
cytochrome P-450-metabollzed cyclophosphamide (a
detailed
report
therefore,
of
these studies is
in preparation).
It
would appear,
that Peak A is not an artifact of incubating the monomer, 2'-de-
oxyguanoslne
3'-monophosphate, with
phosphoramide mustard.
To
our
knowledge, only one other group has speculated on the possibllity of phosphoester adduct formation by cyclophosphamide (15,16). ing given the fact
that
most studies
This is not surpris-
examining DNA adducts formed by
phosphoramide mustard derived from cyclophosphamide have used 3H-labeled drug and have included acid hydrolysis of DNA to release adducts. data have shown, under these conditions,
Peak A is
unmodified guanine that would be devoid of radlolabel,
As our
destroyed to yield thus rendering i t
undetectable by radiometric methods. The formatlon of DNA adducts involving phosphate groups in phosphoester linkages has been reported for other antlcancer drugs (17).
The adduction
of nucleic acids by phosphoramide mustard may occur in an analogous manner. The phosphate ester adduct of phosphoramide mustard characterized in this report is relatively stable, except under very acidic conditions,
and may
represent a relatively persistent adduct. Moreover, phosphoester adducts can theoretically form with all nucleotldes and thus could potentially outnumber adducts formed by N7 alkylation of guanine. potential
for
stability, effects
of
wlde distribution
wlthin
nucleic
these adducts may contribute cyclophosphamide.
chemotherapeutic effects
At
Thus, given their
acids and their
signiflcantly
to
present, however, their
and toxicologic
effects
of
relative
the blologlcal role
in
the
cyclophosphamide is
unknown and will require further study.
ACKNOWLEDGMENTS We wish to thank Miss Karen Marie Schrader f o r preparation
of
this
manuscript.
This
CA-43567 and CA-24538. 849
work
her assistance in the
was supported
by NIH grants
Vol. 163, No. 2, 1 9 8 9
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