(CANCER RESEARCH 51, 4360-4366, August 15. 1991]
Interaction of AyV',/V"-Triethylenethiophosphoramide
Triethylenephosphoramide with Cellular DNA1
and N,N',N"-
Noam A. Cohen, Merrill J. Egorin,2 Stuart W. Snyder, Binial Ashar, Bruce E. Wietharn, Su-shu Pan,
Douglas D. Ross, and John Hilton
Division of Developmental Therapeutics, University of Maryland Cancer Center [N. A.C..M.J.E.,
S. W. S., B. A., B. E, W., S. P., D. D. R.¡,and Division of Medical
Oncology, Department of Medicine [M. J. E., D. D. R.J, University of Maryland School of Medicine, Baltimore, Maryland 21201; and Pharmacology Laboratory
[J. H.I, The Johns Hopkins Oncology Center, Baltimore, Maryland 21205
ABSTRACT
The antineoplastic agents /V,/V',A'"-triethylenethiophosphorainide
(thioTEPA) and jV,/V',jY"-triethylenephosphoramide (TEPA) were stud
ied for their interaction with the DNA of LI 210 cells in the presence and
absence of rat hepatic microsomes and NADPH. Alkaline elution was
used to study 3 types of DNA lesions. When L1210 cells were incubated
with thioTEPA alone, or with thioTEPA in the presence of microsomes
and NADPH, no single-strand breaks were detected. However, incubation
of L1210 cells for 2 h with thioTEPA, at concentrations i-10(1«¿M,
caused
a dose-dependent increase in interstrand cross-linking that reached a
maximum by 2 h after drug exposure. In the presence of rat hepatic
microsomes and NADPH, this cross-linking was eliminated, but a differ
ent DNA lesion, alkali-labile sites, was produced. These alkali-labile
sites were partially reparable with maximum repair achieved by 2 h after
removal of drug. ThioTEPA was >85% consumed by the microsomal
incubation conditions employed, and TEPA was the only product of the
microsomal metabolism of thioTEPA. Alkaline elution studies of L1210
cells that had been incubated with TEPA, alone or in the presence of
microsomes and NADPH, demonstrated an elution pattern identical to
that produced by thioTEPA in the presence of microsomes and NADPH.
Lymphoblastoid cell lines derived from patients with Eanconi's anemia
were far more sensitive to thioTEPA and mechlorethamine hydrochloride
than were lymphoblasts derived from normal humans, but this hypersensitivity was not noted with TEPA or bleomycin. This is consistent with
the known hypersensitivity of cells from patients with Fanconi's anemia
to agents that produce interstrand cross-links and with the alkaline
elution studies described above. In contrast, lymphoblastoid cell lines
derived from patients with ataxia telangiectasia were no more sensitive
to thioTEPA than were lymphoblasts derived from normal humans but
were far more sensitive to bleomycin. One of these cell lines proved
hypersensitive to TEPA, whereas the other was no more sensitive to
TEPA than were lymphoblasts from normal humans. Our data imply
that thioTEPA produces interstrand cross-links but that TEPA, the
primary metabolite of thioTEPA, produces DNA lesions that are alkali
labile.
INTRODUCTION
of this compound by several cell types (28, 29). Those studies
led to the formulation of a scheme wherein thioTEPA liberates
aziridine which, after hydrolysis to ethanolamine, is incorpo
rated via normal cellular metabolic synthetic pathways into
phosphatidylethanolamine
(29, 33). If aziridine, a monofunctional alkylator, is the moiety responsible for the cytotoxicity
of thioTEPA, it is highly unlikely that interstrand cross-linking
would represent the mechanism of action of thioTEPA, and
thus certain mechanisms responsible for resistance to crosslinking agents (39-41) would not pertain to thioTEPA. Similar
considerations were felt likely to pertain to TEPA, the major
in vivo metabolite of thioTEPA. The studies presented in the
current paper were undertaken to test these hypotheses.
MATERIALS AND METHODS
Drugs and Drug Purity. [U-'4C]ThioTEPA (2.3 mCi/mmol) (custom
synthesized by Amersham Corp., Arlington Heights, IL) was produced
by the reaction of chloro-[U-'4C]ethylamine hydrochloride with sodium
hydroxide, followed by reaction of the resulting [U-^Cjaziridine
with thiophosphoryl chloride. The radiochemical purity of the final
[U-HC]thioTEPA preparation was determined to be >98% by TLC as
described previously (36). Nonradioactive thioTEPA and TEPA were
graciously supplied by Lederle Laboratories (Pearl River, NY) and Dr.
David Poplack of the National Cancer Institute (Bethesda, MD), re
spectively. BLM (Blenoxane; Bristol-Myers, Evansville, IN) and HN2
(Mustargen; Merck Sharp & Dohme, West Point, PA) were used as
their clinical formulations, and methylnitrosourea was a gift from Dr.
Robert Brundrett of the Johns Hopkins Oncology Center (Baltimore,
MD).
Cells and Cell Culture. The LI210 murine lymphoblastic leukemic
cells (42, 43) used for alkaline elution studies were maintained in vitro
by serial culture twice weekly in RPMI 1640 (GIBCO, Grand Island,
NY) containing penicillin (50 units/ml), streptomycin (50 ng/m\), Lglutamine (2 ^mol/ml), and 15% (v/v) heat-inactivated fetal bovine
serum (GIBCO) (medium A). Cells were maintained at 37°Cin an
atmosphere of 5% CO2, 95% air and at 95% humidity. Under these
conditions, the cell population had a doubling time of approximately
14-18 h and achieved a maximum cell density of 1.5-2.0 x 106/ml.
alkylating agents containing pentavalent phosphorus and a/ir
Five human lymphoblastoid cell lines were used in growth inhibition
idine moieties, has been used clinically for over 35 years (1-4).
studies. GM08010 and GM04510B cells were derived from individuals
Recently there has been a resurgence of clinical and pharma
cological interest in thioTEPA, primarily because of its use at with FA and were obtained from the Human Genetic Mutant Cell
Repository (Camden, NJ). These cells cannot repair XL and, conse
high doses in conjunction with autologous bone marrow trans
quently,
have increased sensitivity to bifunctional alkylating agents
plantation
(5-38). Our laboratory has previously used
(44). GM00717A and GM01526B cells were derived from patients with
[14C]thioTEPA to characterize the transport and accumulation
AT and were obtained from the Human Genetic Mutant Cell Reposi
tory. These cells cannot repair SSB and, consequently, are hypersensi
Received 1/14/91; accepted 6/6/91.
tive to agents such as BLM (44). PRC55 cells, obtained from Dr.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
Maimón M. Cohen (University of Maryland School of Medicine,
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Baltimore, MD), were normal with respect to DNA replication and
'Supported in part by American Cancer Society Grant CH-412 and the
repair mechanisms and were used as controls with respect to the FA
Bressler Research Fund.
2 To whom requests for reprints should be addressed, at University of Maryland
and AT cell lines. Lymphoblastoid cell lines were maintained under the
Cancer Center, 655 W. Baltimore St., Baltimore, MD 21201.
same conditions as LI210 cells, except that PRC55 cells were supple
3The abbreviations used are: thioTEPA, /VJV'JV'-triethylenethiophosphormented with 1% nonessential amino acids and 1% L-glutamine. Under
amide; TEPA, N^V'^V'-triethylenephosphoramide;
TLC, thin-layer chromatography; BLM, bleomycin; FA, Fanconi's anemia; AT. ataxia telangiectasia; SSB,
these conditions, the population doubling times and maximum cell
single-strand breaks; XL, DNA/DNA interstrand cross-links.
densities of the lymphoblastoid cell lines are as given in Table 1.
4360
ThioTEPA,3 an antitumor agent that belongs to a family of
Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1991 American Association for Cancer Research.
INTERACTION OF thioTEPA AND TEPA wITH CELLULAR DNA
Table 1 Growth characteristics of human lymphoblastoid cell lines
lineGM08010
Cell
(FA)
GM04510B(FA)
GM00717A(AT)
GM01526B(AT)
PRC55 (normal)Doubling
cell
time
(h)31-55
density4-6
x I0'/ml
5-6 x lO'/ml
130-174
1.5-2.0 x lO'/ml
64-78
2-3 x 10'/ml
45-51
4-6 x 10'/ml
34-59Maximum
Alkaline Elution. All points were run in duplicate, and every experi
ment was repeated a minimum of one time to ensure reproducible
results. The alkaline elution methods used were based on the procedures
of Kohn et al. (45) and Hilton (46). Briefly, L1210 cells were grown in
the presence of 1 MM[HC]thymidine (58.3 mCi/mmol) (New England
Nuclear, Wilmington, DE) for 1 to 2 doubling times. The cells were
then washed once with ice-cold 0.075 M NaCl-0.024 M NajEDTA, pH
7.4, resuspended in fresh medium A, and incubated at 37°Cfor 30 min.
To study SSB and alkali-labile lesion formation, [14C]thymidinelabeled cells were treated with specified concentrations of drug for
specified times, in the presence or absence of aroclor 1254-induced rat
hepatic microsomes (Microbiological Associates, Rockville, MD) and
2 mM NADPH (Sigma Chemical Co., St. Louis, MO). At the end of
the drug exposure, cells were centrifuged at 700 x g for 10 min and
resuspended in ice-cold 0.075 M NaCl-0.024 M NaÃ-EDTA,pH 7.4. The
cells were then loaded onto l-^m polycarbonate filters (25-mm diame
ter; Nuclepore Corp., Pleasanton, CA) and lysed with a solution of
0.2% Sarkosyl (CIBA-GEIGY Corp., Greensboro, NC) in 2 M NaCl0.02 M N34EDTA, pH 10, containing 0.5 mg/ml proteinase K (Boehringer Mannheim, Indianapolis, IN). The lysis solution was pumped
(Gilson Minipuls III; Gilson Medical Electronics, Inc., Middleton, WI)
through the filter for 5 min at a rate of 100 ¿il/min.The pump was
then stopped, and the lysis solution was allowed to incubate in the filter
at room temperature for 50 min, after which the solution was again
pumped through the filter at 100 ¿il/minfor 5 min. Proteinase Kdigested material was then removed from the filter by pumping 0.02 M
NaÃ-EDTA,pH 10, through the filter for 30 min at 100 ^l/min. Cellular
DNA was then eluted from the filter witn 0.02 M EDTA (free acid
form) adjusted to pH 12.1 with tetrapropylammonium hydroxide (RSA
Corp., Ardsley, NY). This elution buffer was pumped for 7.5 h at 40
Ml/min, after which the excess elution buffer was poured off, and the
tubing connecting the filter to the collecting vessel was pumped dry.
The filter was removed, treated with 0.5 ml of 0.5 M HC1 at 65°Cfor
30 min, and then mixed with 5 ml of Ready Safe scintillation fluid
(Beckman Instruments, Fullerton, CA). Radioactivity was determined
with a Beckman LS5801 liquid scintillation counter. The filter support
and tubing were washed with three 1-ml aliquots of l M NaOH. These
washes were pooled, mixed with 100 /<!of glacial acetic acid and 8 ml
of Ready Safe, and the radioactivity was determined. A portion of the
IX nil elution volume was removed and treated like the wash fraction
with regard to determination of radioactivity. All experiments studying
SSB included a standard curve, created by irradiating untreated cells at
4 different doses in the same manner as described below for XL
experiments, and a positive drug treatment control, which consisted of
cells incubated for l h with 300 Mg/ml of BLM.
For detection of alkali-labile sites, the DNA eluate was collected in
twenty 1-h fractions, which were then treated and counted as described
above for the DNA eluate and wash fractions. All experiments studying
alkali-labile site formation included a positive control of cells incubated
for l h with 100 ¿iM
A'-methyl-Ar-nitrosourea.
For detection of XL, cells were labeled and prepared for drug
exposure as described above. Cells were then incubated at 37°C,with
specified concentrations of drug for 2 h, in the presence or absence of
aroclor 1254-induced rat hepatic microsomes and 2 mM NADPH.
Following drug exposure, the cells were centrifuged at room tempera
ture (700 x g) for 10 min, resuspended in fresh medium A, and
incubated at 37°Cfor an additional 1, 2, or 4 h. After this additional
min. To induce SSB in the sedimented cells, radiation was delivered
with a Westinghouse Coronado X-ray unit which was operated at 240
kVp, 15 mA, with 0.5 mm Cu plus 1.0 mm Al filtration yielding a halfvalue of 1.5 mm of Cu and a dose rate of approximately 3.0 Gy/min.
Each of the four equal cell pellets received one of the following radiation
doses: 0, 150, 300, or 450 rads at room temperature. After being
radiated, cell pellets were placed back on ice. All XL studies included
a positive control of cells incubated for 1 h with 5 ^M HN2 followed by
a 1-h posttreatment incubation in drug-free medium A.
For data analysis, percentage DNA retained on the filter was calcu
lated as
Filter dpm + wash dpm
Filter dpm + wash dpm + eluate dpm
Growth Inhibition Studies. GM08010, GM04510B, GM00717A,
GM01526B, and PRC55 lymphoblasts were resuspended to 3-4 x 10*
cells/ml in fresh medium A that contained the designated concentra
tions of thioTEPA, TEPA, HN2, or BLM. Control cultures, lacking
antineoplastic agents, were prepared concomitantly. Cultures, in trip
licate and with final volumes of 1 ml, were incubated in sterile, capped,
16 x 150 mm glass test tubes at 37°Cin an atmosphere of 95% air, 5%
CO2, at 95% humidity. After 96 h (GM08010, GM01526B, and
PRC55) or 168 h (GM04510B and GM00717A) of incubation, cell
concentrations were determined with a ZB, Coulter Counter (Coulter
Electronics, Hialeah Park, FL). In several experiments, designed to
determine the radiosensitivity of LI210 cells, cell cultures, with an
initial cell concentration of 105/ml and containing no drug, were
exposed to specified doses of X-irradiation and then cultured for 72 h
before cell growth was assayed as just described. The concentration of
drug or radiation which produced a 50% inhibition of cell growth with
respect to untreated controls was calculated with the median-effect
equation (47) using a program written by Chou and Chou (48).
Microsomal Metabolism Studies. Initial studies, designed to measure
the consumption of thioTEPA, utilized a 2-ml incubation mixture
containing 20 mM NADPH, 200 mM potassium phosphate, pH 7.4,
0.5 mM thioTEPA, and 200 p\ of aroclor 1254-induced rat liver
microsomes. Incubations were performed at 37°Cwith shaking. After
0, 10, 20, 30, 60, and 90 min, 200 M' of incubation mixture were
removed and extracted with 800 n\ of ethyl acetate containing 12.5 Mg/
ml diphenylamine internal standard, and thioTEPA concentrations
were determined with the gas chromatography method of Grochow et
al. (9, 10). Subsequent experiments, examining the metabolic fate
of thioTEPA incubated with microsomes, used [MC]thioTEPA
in lieu of unlabeled compound. After 0, 15, 30, and 60 min
of incubation, 100 ^1 of incubation mixture were removed, mixed with
0.2 g of NaCl, and extracted with 400 //I of chloroform. The resulting
organic extracts were then spotted on 250-nm silica gel 60 plates (E.
Merck, Darmstadt, Germany) that were developed to 15 cm in an
ascending fashion with chloroform:acetone (4:1, v/v). Nonradioactive
thioTEPA and TEPA standards were included on each plate. Thio
TEPA and TEPA were visualized by reaction with 4-(p-nitrobenzyl)pyridine (Sigma Chemical Co.) (28). The blue 4-(/>-nitrobenzyl)pyridine-reactive spots corresponding to thioTEPA and TEPA were
scraped from the plate, placed into scintillation vials, mixed with 0.1
ml glacial acetic acid and 5 ml scintillation fluid, and the radioactivity
was determined. The remainder of each lane was divided into 1-cm
segments that were scraped from the plate and handled in a manner
similar to that for thioTEPA and TEPA spots.
RESULTS
Alkaline Elution
Single-strand Breaks. The mean coefficient of variation for
duplicate determinations was 2.5%. If the limit of detection is
defined as a signal-to-noise ratio of 2, this implies a limit of
detection of 5% change in DNA retention. Therefore, the
alkaline elution procedure employed in these studies was able
incubation, the cell suspensions were divided into 4 equal portions,
sedimented by centrifugation at room temperature (700 x g) for 10
min in 12 x 75 mm polypropylene tubes, and then placed on ice for 10
4361
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INTERACTION
OF thioTEPA AND TEPA WITH CELLULAR DNA
to detect SSB induced by as little as 50 rads of X-irradiation
(Fig. 1). Growth inhibition studies showed that doses up to 300
rads produced no reduction in LI210 cell growth and that 1000
rads were required to reduce growth by approximately 50%. As
expected, progressively increasing doses of irradiation resulted
in a dose-dependent increase in DNA strand breakage which
was manifested as a correspondingly decreased retention of
DNA on the filter (Fig. 1). Similarly, incubation of LI 210 cells
for l h with 300 ng/ml BLM reduced the percentage of DNA
retained on the filter to 50%. There was no alkaline elution
evidence of SSB in LI 210 cells that had been incubated for 2 h
with thioTEPA concentrations ranging from 0 to 3000 ¿IM.
Similarly, less than 50 rad-equivalents of SSB were detected in
LI210 cells that had been incubated with 30 pM thioTEPA for
up to 6 h. Likewise, less than 50 rad-equivalents of SSB were
present in L1210 cells that had been incubated with thioTEPA
in the presence of microsomes and 2 niM NADPH.
DNA/DNA Interstrand Cross-links. A 1-h incubation of
LI210 cells with specified concentrations of HN2 and a subse
quent incubation for l h in drug-free medium produced a dosedependent retardation in the elution of irradiated DNA, i.e., a
dose-dependent increase in cross-linking (Fig. 1). Incubation of
LI210 cells with thioTEPA at concentrations <100 /¿M
had no
detectable impact on the DNA elution profile, but thioTEPA
i
I
150
300
«50
Radiation Dose (Radi
Fig. 3. Effect of aroclor 1254-induced rat liver microsomes and NADPH on
the cross-linking activity of thioTEPA. As described in "Materials and Methods,"
L1210 cells were labeled overnight with [MC]thymidine and incubated for 2 h
with 0 (•)or 300 MMthioTEPA in the presence (»)or absence (•)of NADPH
and aroclor 1254-induced rat liver microsomes. After a 2-h incubation in drugfree medium, cells were exposed to specified doses of X-irradiation, and alkaline
elution was performed as described in "Materials and Methods." A, 5 /IM HN2,
the concomitant positive control included in each experiment. Points, individual
determinations from a representative experiment performed in duplicate.
100
20
200
4OO
Radiation Date (Rod)
ISO
300
Radiation Oase (Rads)
450
Fig. 1. Ability of HN2 to produce DNA/DNA XL. L12IO cells were labeled
overnight with ['4C]thymidine and incubated for l h with 0 (O), 0.5 (•),1.0 (A).
or 5.0 (A) nM HNj. as described in "Materials and Methods." After a 1-h
incubation in drug-free medium, cells were exposed to specified doses of Xirradiation. and alkaline elution was performed as described in "Materials and
Methods." Points, individual determinations from a representative experiment
performed in duplicate.
Fig. 4. Ability of TEPA, in the presence or absence of aroclor 1254-induced
rat liver microsomes and NADPH. to produce DNA/DNA XL in LI210 cells.
As described in "Materials and Methods," L1210 cells were labeled overnight
with ['"C]thymidine and incubated for 2 h with 0 (•)or 500 ¿IM
TEPA in the
presence (•)or absence (A) of NADPH and aroclor 1254-induced rat liver
microsomes. After a 2-h incubation in drug-free medium, cells were exposed to
specified doses of X-irradiation. and alkaline elution was performed as described
in "Materials and Methods." •,5 JIM HN2, the concomitant positive control
included in each experiment. Points, individual determinations from a represent
ative experiment performed in duplicate.
concentrations >100 MMproduced a dose-dependent retarda
tion in the elution of DNA which was similar to that seen with
HN2 (Fig. 2). Increasing the post-thioTEPA exposure incuba
tion in drug-free medium from 2 to 4 h resulted in no detectable
HN2 increase in cross-linking, implying that cross-linking had
60-reached a maximum by 2 h after thioTEPA exposure.
In contrast to the XL observed in L1210 cells that had been
40-500 fj.M TT
or
incubated with thioTEPA, LI210 cells incubated with thio
<
TEPA in the presence of aroclor 1254-induced rat liver micro
20lOO^MTT
somes and NADPH showed a reduction in XL (Fig. 3). Their
alkaline elution pattern approached that of cells irradiated after
No Drug
incubation with no drug. In these experiments, concomitantly
450
300
150
analyzed cells that had been incubated with 300 ^M thioTEPA
Radiolio n Dose (Rod)
alone or 5 n\i HN2 produced XL elution patterns consistent
Fig. 2. Ability of thioTEPA to produce DNA/DNA XL. L1210 cells were
with
those described above (Figs. 1 and 2). As expected, LI210
labeled overnight with [MC]thymidine and incubated for 2 h with 0 (•).100 (A),
500 (•),or 1000 (T) ^M thioTEPA, as described in "Materials and Methods."
cells that had been incubated with 500 UM TEPA in lieu of
After a 2-h incubation in drug-free medium, cells were exposed to specified doses
thioTEPA showed no alkaline elution evidence of XL (Fig. 4).
of X-irradiation. and alkaline elution was performed as described in "Materials
and Methods." #, 5 i¡\tHN2, the concomitant positive control included in each
Rather, the alkaline elution pattern of LI210 cells incubated
with TEPA resembled that of LI210 cells incubated with
experiment. Points, mean of a representative experiment performed in duplicate.
100
4362
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INTERACTION
" ••è A•
thioTEPA•
•••
m*
ts
8°-Remaining
•« _ lO^iM
*
*
A
•
*
OF thioTEPA AND TEPA wITH CELLULAR DNA
as a positive control for SSB. As expected the elution profile of
cells that had been incubated with TEPA alone resembled that
of cells that had been incubated with thioTEPA in the presence
of microsomes and NADPH (Fig. 6).
If, after exposure to thioTEPA with microsomes and
NADPH or to TEPA alone, LI210 cells were allowed to
incubate in drug-free medium, they were able to repair some of
the alkali-labile DNA lesions induced by these drugs. This
repair was time dependent during the first 2 h of incubation in
drug-free medium but did not increase after that time (Fig. 7).
Cells that had been incubated for 2 h with 1, 10, 100, or 300
/UMthioTEPA, but without microsomes or NADPH, showed
no evidence of alkali-labile lesions.
•
100/lM
•thioTEPA•
AJOOM-g/ml
§a
A'"
40-ûô
•
'*•*,.A
20-•s0-•
°
I 2 3 4 5 6
7 8 9 10 II 12 13 14 15 16
Elution Time (h)
Metabolic Studies
Fig. 5. Elution patterns of DNA from LI210 cells incubated with thioTEPA
in the presence of aroclor 1254-induced rat liver microsomes and NADPH or
with BLM. L1210 cells were labeled overnight with |'"C]thymidine and incubated
for 2 h with either 300 ^M BLM (•)or with 10 P) or 1000 (A) ^M thioTEPA in
the presence of NADPH and aroclor 1254-induced rat liver microsomes. After
this 2-h incubation, cells were analyzed for alkali-labile sites with the alkaline
elution methodology described in "Materials and Methods." Points, cumulative
There was no decrease in thioTEPA concentration when the
drug was incubated at 37°Cfor 90 min with NADPH but
without microsomes. In contrast, addition of microsomes
caused a rapid decrease in thioTEPA concentration, with ap
proximately 50% gone by 10 min and >85% gone by 60 min
(Fig. 8). Although the gas chromatography analysis employed
in initial studies demonstrated the disappearance of thioTEPA
under these conditions, it was unable to characterize the
products of microsomal metabolism of thioTEPA. Use of
[14C]thioTEPA and the TLC system described in "Materials
and Methods" showed TEPA to be the only radioactive product
elution patterns from a representative experiment performed in duplicate.
100
•I
80
CONTROL
• •_
resulting from microsomal metabolism of thioTEPA. In the
TLC system used, thioTEPA and TEPA were well resolved
with R( of 0.45 and 0.07, respectively (28), and there were no
radioactive areas on the TLC plate other than those correspond
ing to authentic thioTEPA and TEPA standards.
TEPA
o
"5
40
£
o 20
S?
Growth Inhibition Studies
GM08010 and GM04150B lymphoblastoid cell lines, derived
from patients with FA, were each far more sensitive to the
growth-inhibitory effects of thioTEPA and the known XLproducing agent HN2 than was the PRC55 lymphoblastoid line
that was derived from a human with normal DNA replication
8 9 IO I 112 13 14 15 16 17 18 19 20
ElutionTime (h)
Fig. 6. Elution patterns of DNA from LI210 cells incubated with TEPA.
L1210 cells were labeled overnight with ['"C]thymidine and incubated for 2 h
with either 0 (•)or 500 (•)/JM TEPA. After this 2-h incubation, cells were
analyzed for alkali-labile sites with the alkaline elution methodology described in
"Materials and Methods." Points, cumulative elution patterns from a represent
I
2 3 4 5 67
ww80-60-40-2O-o.f***lls--...*^A
ative experiment performed in duplicate.
""*•••*»•
thioTEPA in the presence of microsomes and NADPH (Fig.
3). Inclusion of microsomes and NADPH in the incubation
mixture with TEPA and L1210 cells had no effect on the
alkaline elution pattern, i.e., it resembled that of cells incubated
with TEPA alone (Fig. 4).
Alkali-labile Sites. In order to detect alkali-labile DNA le
sions, the alkaline elution protocol was modified slightly so
that the eluate was collected as twenty 1-h fractions. Treatment
of cells with varying concentrations of thioTEPA in the pres
ence of microsomes and NADPH produced elution profiles
consistent with the dose-dependent production of alkali-labile
lesions (Fig. 5) (49). This is indicated by the 6-h delay preceding
a sudden increase in the rate of DNA eluting from the filter
(Fig. 5). This pattern is identical to that produced by methylnitrosourea, a monofunctional alkylating agent that is known
to produce alkali-labile DNA lesions. Alkali-labile lesion for
mation was detectable in cells after incubation of microsomes
and NADPH with thioTEPA concentrations as low as 10 pM
(Fig. 5). This elution pattern can be contrasted with that pro
duced by 300 Mg/ml BLM, which was included in experiments
IE
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E
•
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es•*:.^r
2/0.5
A2/1A2/2O2/6
CL
O
^*£**A*
*** *
•?Control•2/0*
I 234567
Elution Time (h)
Fig. 7. Repair of alkali-labile sites induced in the DNA of LI 210 cells incubated
with thioTEPA in the presence of aroclor 1254-induced rat liver microsomes and
NADPH. L1210 cells were labeled overnight with [MC]thymidine and incubated
for 2 h with either 0 (•)or 300 I¡M
thioTEPA in the presence of aroclor 1254induced rat liver microsomes and NADPH. After this 2-h incubation, cells were
incubated for an additional 0 (•),0.5 (+), 1 (A), 2 (A), or 6 (O) h in drug-free
medium before being analyzed for alkali-labile sites with the alkaline elution
methodology described in "Materials and Methods." Points, cumulative elution
patterns from a representative experiment performed in duplicate.
4363
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INTERACTION
OF thioTEPA AND TEPA WITH CELLULAR DNA
The alkaline elution studies presented are consistent with an
earlier report in which Teicher et al. (21) indicated that thio
TEPA can produce XL. At the same time, these results must
be reconciled with previous reports from our laboratory that
provided strong evidence for thioTEPA liberating aziridine and
showed little evidence of [MC]thioTEPA-derived radiolabel
being associated with L1210 cell nuclei or trichloroacetic acidprecipitable material. It is unclear what chemical species pro
duces the XL observed. One candidate would be thioTEPA
itself; however, this material is quite stable under physiological
conditions and alkylates 4-(p-nitrobenzyl)pyridine very slowly
at 37°Cand pH 7.4. Alternatively, XL could be due to the
A^V'-diethylenethiophosphoramide
produced after one aziri
IOO
9080706050
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g
30
»eio
IO
20
30
40
50
60
Time (min)
Fig. 8. Metabolism of thioTEPA by aroclor 1254-induced rat liver microsomes.
ThioTEPA (500 >iM)was incubated with NADPH and aroclor 1254-induced rat
liver microsomes, as described in "Materials and Methods." At specified times,
aliquots of the incubation mixture were analyzed by gas chromatography for
thioTEPA content. Points, mean of duplicate determinations from a representa
tive experiment.
and repair mechanisms (Table 2). In contrast, these two FA
lymphoblastoid cell lines did not give evidence of increased
susceptibility to TEPA or the known SSB-inducing agent BLM
(Table 2).
GM00717A and GM01526B, lymphoblastoid cell lines de
rived from patients with AT, gave no evidence of markedly
increased sensitivity to HN2 or thioTEPA (Table 2). In contrast,
each cell line demonstrated the expected increased sensitivity
to BLM (Table 2). The increased sensitivity of GM00717A to
TEPA was comparable to its increased sensitivity to BLM. The
increased sensitivity of GM01526B to TEPA was less clear;
however, this cell line was also less sensitive to BLM than was
GM00717A.
dine is lost from thioTEPA, because that compound is analo
gous to isophosphoramide mustard, a known cross-linking
agent and cytotoxic metabolite of ifosfamide. Regardless of
which of these moieties is responsible for the XL observed,
there are certain constraints required to reconcile the current
studies with the previously mentioned studies, which showed
80-85% of cellular [14C]thioTEPA-derived radiolabel to reside
in [14C]phosphatidylethanolamine and detected no measurable
increase in nuclear I4C (29). If thioTEPA is the agent producing
the XL observed, XL formation must be very slow or quanti
tatively trivial in comparison to the liberation of aziridine from
thioTEPA. In addition, any alkylation of DNA by aziridine
must occur much more slowly than the hydrolysis of that
material to ethanolamine. If yV,A"-diethylenethiophosphoram-
DISCUSSION
ide is the agent producing the XL observed, XL formation must
occur much more slowly than either hydrolysis of the diaziridinyl compound or further loss of aziridine moieties from this
material. Again, any aziridine liberated in this scheme must be
hydrolyzed to ethanolamine much more facilely than it alkylates
DNA.
Irrespective of which chemical species is responsible for the
XL observed, this process is obviated by including rat liver
microsomes and NADPH in the incubation system. ThioTEPA
is known to be metabolized to TEPA by such a microsomal
system (22, 30). The current data support this observation and
fail to detect any other radiolabeled metabolite of thioTEPA.
The failure to detect [14C]aziridine or [14C]ethanolamine can be
ThioTEPA has been in clinical use for more than 35 years
(1) and has achieved an accepted role in the treatment of certain
tumors (2-4). However, the current clinical application of
thioTEPA is largely empirically derived. Work is still ongoing
to elucidate the mechanism of action of the drug so that it can
be used alone or in combination with other agents in the most
rational fashion. The studies presented in the current paper are
an extension of earlier mechanistic work (20, 23, 28, 29, 31,
33) and complement information already available (21, 22, 24,
25, 30, 37).
explained by the relatively slow rate at which aziridine is
liberated from thioTEPA (28,29) and the relatively low specific
activity of the radiolabeled thioTEPA used in our studies. That
the loss of thioTEPA-derived XL activity caused by rat liver
microsomes and NADPH is due to metabolism of thioTEPA
to TEPA is further substantiated by our observation that TEPA
does not produce XL detectable by alkaline elution. Therefore,
although thioTEPA has been described as a potential prodrug
for TEPA, its conversion to TEPA results in a metabolite that
interacts with cellular DNA in a manner different from that of
Table 2 Sensitivity of lymphoblastoid cell lines derived from patients with Fanconi's anemia or patients with ataxia telangiectasia
to growth inhibition by various cytotoxic agents
AgentHN2
±59(6-1 21)'
BLMthioTEPA
1.2 +0.5(0.6-2.0)5.4.
±5(5-15)
0.04 ±0.01
(0.02-0.05)29.5,
(AT)3
±2 (0.3-5.5)
n=6
6 ±
(3-9)0.8.4
2
1(1-3)
3.5 ±(2.9-3.9)1.0,4.1
0.4
164"
111
0.4, 0.9GM00717A
8, 10GM01526B(AT)2±
1.4,3.6
TEPAGMOSOIO(FA)56°'* 0.3, 1GM04510B(FA)10
°Ratio of IC50 for normal PRC55 cells:IC50 for mutant cell line. A ratio >1 indicates increased susceptibility and a ratio <1 indicates reduced susceptibility. IC50,
concentration producing 50% inhibition.
* Mean ±SD.
' Range if n > 2.
* Values from 2 separate experiments each performed in triplicate.
4364
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INTERACTION
OF thioTEPA AND TEPA wITH CELLULAR DNA
the parent compound. The alkali-labile DNA lesions produced
by TEPA are quite consistent with monofunctional adducts.
Whether these are caused by TEPA itself or by aziridine is
unknown. As with thioTEPA, loss of one aziridine from TEPA
should produce a compound with known cross-linking activity,
i.e, isophosphoramide mustard. If isophosphoramide mustard
is produced, it is unclear why XL are not observed in cells
incubated with TEPA. In any case, the striking difference
between the XL observed in cells incubated with thioTEPA and
the alkali-labile lesions observed in cells incubated with TEPA
is consistent with a previous publication by Teicher et al. (30)
in which differences in requirements for thioTEPA- and TEPAinduced cytotoxicity were noted.
Finally, the growth inhibition studies reported in the current
paper are consistent with, and highly supportive of, the results
of the alkaline elution studies. Not only did thioTEPA and
TEPA differ in their cytotoxic effects on the cell lines tested,
but the differences were those expected based on the known
defects in DNA repair mechanisms present in the mutant
lymphoblastoid cell lines used (44). More specifically, the in
creased sensitivity to thioTEPA of lymphoblasts derived from
patients with FA is consistent with the inability of those cells
to repair XL (44) and the alkaline elution evidence that thio
TEPA, but not TEPA, produces such lesions. Conversely, the
increased sensitivity to TEPA of lymphoblasts derived from
patients with AT is consistent with the defective repair of SSB
by such cells (44) and the alkaline elution evidence that TEPA,
but not thioTEPA, produces alkali-labile lesions.
In conclusion, the current set of experiments provide more
information regarding the mechanism of action of thioTEPA,
but they also provide even more evidence of the complexity
involved in the interaction of thioTEPA with cellular DNA.
This complexity becomes even greater when considered in light
of the clinical pharmacological and metabolic data documenting
the extensive hepatic metabolism of thioTEPA to TEPA (11,
13-15, 17, 22, 30). Ongoing efforts are being directed at clari
fying a number of these issues such as the actual chemical
species that interact with DNA and the potential sequence
specificities of such materials.
20.
ACKNOWLEDGMENTS
21.
We thank B. Knickman and L. Mueller for excellent secretarial
assistance in preparation of this manuscript; Dr. Hugh Eddy and Jin
Ro for irradiation of the cell cultures; Drs. Maimón Cohen, Roger
Schultz, and Jeffrey Chinsky for helpful discussions; Bill and Ted for
an excellent adventure; and Little Feat for musical inspiration. We
dedicate this manuscript to the memory of Stuart Snyder, a valued
colleague and friend, whose untimely death has greatly diminished our
laboratory spirit and productivity.
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Interaction of N,N′,N″-Triethylenethiophosphoramide and N,N′,N
″-Triethylenephosphoramide with Cellular DNA
Noam A. Cohen, Merrill J. Egorin, Stuart W. Snyder, et al.
Cancer Res 1991;51:4360-4366.
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