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Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 11557–11562, October 1997
Medical Sciences
The role of polyamine catabolism in polyamine analogue-induced
programmed cell death
(spermidine
y
spermine N
1
-acetyltransferase
y
polyamine oxidase)
HYO CHOL HA*†,PATRICK M. WOSTER‡,JAMES D. YAGER*, AND ROBERT A. CASERO,JR.*†§
*Division of Toxicological Sciences, Department of Environmental Health Sciences, Johns Hopkins School of Hygiene and Public Health, Baltimore, MD 21205;
†The Oncology Center Research Laboratories, Johns Hopkins University School of Medicine, Baltimore, MD 21231; and ‡Wayne State University, Detroit,
MI 48202
Communicated by John W. Littlefield, Johns Hopkins Universit y School of Medicine, Baltimore, MD, August 19, 1997 (received for review
July 9, 1997)
ABSTRACT N
1
-ethyl-N
11
-[(cyclopropyl)methyl]-4,8,-
diazaundecane (CPENSpm) is a polyamine analogue that
represents a new class of antitumor agents that demonstrate
phenotype-specific cytotoxic activity. However, the precise
mechanism of its selective cytotoxic activity is not known.
CPENSpm treatment results in the superinduction of the
polyamine catabolic enzyme spermidineyspermine N
1
-
acetyltransferase (SSAT) in sensitive cell types and has been
demonstrated to induce programmed cell death (PCD). The
catalysis of polyamines by the SSATypolyamine oxidase
(PAO) pathway produces H
2
O
2
as one product, suggesting
that PCD produced by CPENSpm may be, in part, due to
oxidative stress as a result of H
2
O
2
production. In the sensitive
human nonsmall cell line H157, the coaddition of catalase
significantly reduces high molecular weight (HMW) DNA
(>50 kb) and nuclear fragmentation. Important to note,
specific inhibition of PAO by N,N*-bis(2,3-butadienyl)-1,4-
butane-diamine results in a significant reduction of the for-
mation of HMW DNA and nuclear fragmentation. In contrast,
the coaddition of catalase or PAO inhibitor has no effect on
reducing HMW DNA fragmentation induced by N
1
-ethyl-N
11
-
[(cycloheptyl)methyl]-4,8,-diazaundecane, which does not in-
duce SSAT and does not deplete intracellular polyamines.
These results strongly suggest that H
2
O
2
production by PAO
has a role in CPENSpm cytotoxicity in sensitive cells via PCD
and demonstrate a potential basis for differential sensitivity
to this promising new class of antineoplastic agents. Further-
more, the data suggest a general mechanism by which, under
certain stimuli, cells can commit suicide through catabolism
of the ubiquitous intracellular polyamines.
Programmed cell death (PCD) is a fundamental biological
regulatory mechanism involving selective cell deletion. It is an
active and irreversible process in which cells activate the
intrinsic death program for their own demise. PCD is abso-
lutely required for the natural development and homeostasis
of tissues in complex multicellular organisms (1–4). Morpho-
logical characteristics of PCD include cell shrinkage, nuclear
condensation and fragmentation, plasma and nuclear mem-
brane budding, and apoptotic bodies (3). PCD is biochemically
characterized by activation of nucleases that cleave chromo-
somal DNA into high molecular weight (HMW) andyor low
molecular weight oligonucleosomal DNA fragments (5). PCD
can be induced by normal physiological processes and by
multiple nonphysiological stimuli, including oxidative stress
and chemotherapeutic agents (4–7).
The polyamines spermidine and spermine and their diamine
precursor putrescine are intracellular cationic molecules that
are essential for cell proliferation and differentiation (8, 9).
The intracellular concentration of these ubiquitous molecules
is highly regulated by the polyamine metabolic pathway, which
influences the synthesis, degradation, uptake, and excretion of
the cations (9). High ornithine decarboxylase (ODC) activity,
the first rate-limiting step of polyamine biosynthesis, and
increased levels of intracellular polyamines are known to occur
in rapidly proliferating cells or cells undergoing differentiation
and transformation. Depletion of intracellular polyamines by
direct inhibition of polyamine biosynthesis is generally asso-
ciated with a decrease in proliferation and has been the
primary focus in past antiproliferative studies (10).
However, a more recent strategy has been to design poly-
amine analogues that exploit the self-regulating nature of
polyamine metabolism. Porter, Bergeon, and colleagues (11,
12) have led the field in the design and testing of the symmetric
bis(ethyl)polyamines that were designed specifically to down-
regulate polyamine biosynthesis by feedback mechanisms
rather than by direct enzyme inhibition. We and others have
described an additional action of these compounds that, in a
cell type-specific manner, leads to a superinduction of sper-
midineyspermine N
1
-acetyltransferase (SSAT), the first rate-
limiting step in the catabolism of spermine and spermidine
(13). The cell type-specific superinduction of SSAT has been
associated with, but not causally linked to, the cytotoxic
response to several polyamine analogues that have demon-
strated significant antitumor activity against important solid
tumors. We have, therefore, focused our attention on the
design and testing of polyamine analogues that maintain tumor
type-specific SSAT induction and cytotoxicity. Current evi-
dence suggests that the regulation of the intracellular poly-
amine levels plays a pivotal role not only in cell proliferation
and differentiation but also in PCD. The deregulation of the
intracellular polyamine levels and abnormal polyamine met-
abolic enzyme activity have been reported in cells undergoing
PCD (14–20).
We have demonstrated that the cell type-specific cytotox-
icity induced by N
1
-ethyl-N
11
-[(cyclopropyl)methyl]-4,8,-
diazaundecane (CPENSpm) in the human nonsmall cell lung
carcinoma line NCI H157 occurs via a PCD pathway (21).
Similar results were observed in breast (22) and prostate
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
© 1997 by The National Academy of Sciences 0027-8424y97y9411557-6$2.00y0
PNAS is available online at http:yywww.pnas.org.
Abbreviations: CHENSpm, N
1
-ethyl-N
11
-[(cycloheptyl)methyl]-4,8,-
diazaundecane; CM-H
2
DCFDA, 5-(and -6)-chloromethyl-29,79-
dichlorodihydrofluorescein diacetate (mixed isomers); CPENSpm,
N
1
-ethyl-N
11
-[(cyclopropyl)methyl]-4,8,-diazaundecane; CuyZn-SOD,
CuyZn-superoxide dismutase; HMW, high molecular weight; MDL
72,527, N,N9-butadienyl)-1,4-butane-diamine; ODC, ornithine decar-
boxylase; PAO, polyamine oxidase; PCD, programmed cell death;
SSAT, spermidineyspermine N
1
-acetyltransferase.
§To whom reprint requests should be addressed. e-mail: casero@
welchlink.welch.jhu.edu.
11557
cancer cell lines (23). However, the mechanism of analogue-
induced PCD is not known. CPENSpm treatment leads to a
superinduction of SSAT (24), and the resultant N
1
-
acetylspermine and N
1
-acetylspermidine are substrates for the
constitutive, FAD-requiring, intracellular polyamine oxidase
(PAO). This enzyme is present in most cell types and cleaves
N
1
-acetylated polyamines to produce spermidine or pu-
trescine, H
2
O
2
, and 3-acetamidopropanal (25).
The goal of the present study was to determine if the
production H
2
O
2
resultant from analogue induced SSAT is
associated with the observed CPENSpm-induced DNA and
nuclear fragmentation. The results presented here demon-
strate that the inhibition of PAO, thus H
2
O
2
production,
significantly reduced the CPENSpm-induced HMW DNA and
nuclear fragmentation and delayed the onset of PCD in the
NCI H157 cells. To verify that SSAT induction plays a role in
the observed induction of PCD, we compared the effects of
CPENSpm to those of N
1
-ethyl-N
11
-((cycloheptyl)methyl)-
4,8,-diazaundecane (CHENSpm), a related analogue that does
not superinduce SSAT. The data also underscore the possi-
bility that intracellular polyamine catabolism may function in
a general cellular suicide mechanism in response to various
stimuli.
MATERIALS AND METHODS
Chemicals and Cell Culture. CPENSpm and CHENSpm
were synthesized as reported (26). The stock solutions of
analogues were prepared at a concentration of 1 mM in 0.1 N
HCl. N,N9-bis(2,3-butadienyl)-1,4-butane-diamine (MDL 72,
527) was a generous gift from Eugene Gerner (University of
Arizona, Tucson, AZ) and Hirak S. Basu (University of
Wisconsin, Madison, WI). Catalase from bovine liver, CuyZn-
superoxide dismutase (CuyZn-SOD) from bovine erythro-
cytes, N-acetyl-L-cysteine, butylated hydroxytoluene, buty-
lated hydroxyanisole, ZnCl
2
, aminoguanidine, and aurintricar-
boxylic acid were purchased from Sigma. Proteinase K and
RNase A were purchased from GIBCOyBRL. 5-(and -6)-
chloromethyl-29,79-dichlorodihydrofluorescein diacetate
(CM-H
2
DCFDA), mixed isomers, was purchased from Mo-
lecular Probes. The nonsmall cell lung carcinoma line NCI
H157 cell was maintained as reported (21). Intracellular
polyamine pools and SSAT and ODC activities were measured
using cellular extracts as reported (21).
Field-Inversion Gel Electrophoresis and Quantitation of
HMW DNA Fragmentation. DNA from treated and control
cells was resolved using field-inversion gel electrophoresis, as
described (21). DNA fragmentation was assayed quantitatively
using a modification of the method described by Hoyt et al.
(27). For qualitative analysis, DNA was stained with ethidium
bromide and photographed using the Eagle Eye system (Strat-
agene). For quantitative analysis, DNA was transferred to a
ZetaProbe nylon membrane (Bio-Rad) and hybridized to a
32
P-labeled human AluI DNA sequence (from Barry Nelkin,
Johns Hopkins University School of Medicine, Baltimore)
using published methods (28). Phosphor image analysis was
performed on a Molecular Dynamics PhosphorImager using
IMAGEQUANT software (Sunnyvale, CA). Five hundred thou-
sand cells, which were shown to be within the linear range of
the quantitative detection without saturation (dat a not shown),
were analyzed for the HMW DNA fragmentation. Percentage
of HMW DNA fragmentation 5DNA migratedytotal DNA
(DNA remaining in the well 1DNA migrated) 3100.
Assessment of Morphology. Exponentially growing NCI
H157 cells were treated with 10
m
M CPENSpm with or without
cotreatment of 500 unitsyml catalase or 250
m
M MDL 72,527
for various times. Adherent cells were harvested with trypsin
and combined with cells f loating in the medium. Cells were
then washed with 1X PBS, fixed with 300
m
l PBS, 20
m
l 10%
Nonidet P-40 in PBS, 36
m
l 37% formaldehyde, and 2
m
lof1
mgyml Hoechst 33342 (Calbiochem–Behring), visualized un-
der UV excitation, and photographed with a Nikon Labophot
microscope.
Flow Cytometric Detection of Peroxides by CM-H
2
DCFDA.
Control cells and cells treated for1hwith10
m
M CPENSpm
with or without cotreatment of 500 unitsyml catalase or 250
m
M MDL 72,527 were analyzed for increase in changes in
fluorescence indicating a change in H
2
O
2
production. Adher-
ent cells were harvested with trypsin and combined with cells
floating in the medium. Cells were then washed with 1X PBS,
and 1 310
6
cells were treated with 10
m
M CM-H
2
DCFDA for
20 min at 37°C. One hundred thousand cells were then
analyzed on a Becton Dickinson FACScan as reported (29).
Flow Cytometry. Flow cytometric analysis of CPENSpm-
and CHENSpm-treated cells was performed using the pro-
pidium iodide (Sigma) staining method as described (30).
Stained nuclei were analyzed on a Becton Dickinson FACScan
with an argon ion laser at an excitation wavelength of 488 nm.
RESULTS
The Formation of HMW DNA Fragments Induced by
CPENSpm Treatment in NCI H157 Cells Occurs Concur-
rently with SSAT Superinduction. CPENSpm was found to
induce formation of HMW DNA fragments at a concentration
as low as 8
m
M in 24 h, and fragments could be detected as
early as 18 h with 10
m
M CPENSpm treatment (data not
shown). Based on these results, the treatment of 10
m
M
CPENSpm for 24 h was chosen for further experimentation.
Treatment with 10
m
M CPENSpm produced 37 65% frag-
Table 1. Comparison of effects of 10
m
M CPENSpm and CHENSpm with or without catalase or MDL 72,527 on HMW DNA fragmentation
in NCI H157 cells
Treatment*
Polyamines, nmolymg protein†SSAT activity,
pmolymg
proteinymin§
HMW DNA
fragmentation,
%¶
Relative inhibition
of DNA
fragmentation, %Put Spd Spm Analogue
None 3.1 12.9 24.0 146 63161—
CPENSpm ND‡ND 1.2 53.8 28,561 6286 37 65—
CHENSpm 8.5 9.3 19.7 35.5 468 624367
CPENSpmycatalase 3.4 0.5 2.1 45.0 29,718 6892 8 6377
CHENSpmycatalase 18.5 7.0 10.9 36.3 532 6239699
CPENSpmyMDL 72,527 ND ND 2.4 47.7 24,073 6722 12 6867
CHENSpmyMDL 72,527 5.5 6.7 18.2 43.0 580 63386912
*Treatment of NCI H157 cells, where indicated, was performed with 10
m
M CPENSpm for 24 h in the presence or absence of 500 unitsyml catalase
or 250
m
M MDL 72,527.
†Values represent the means of duplicate determinations.
‡ND, ,0.05 nmolymg protein.
§Values for SSAT enzyme activities represent the means of triplicate determinations 6SD.
¶Values represent the means of triplicate determination SD, quantitation based on phosphoimage analysis as described in Materials and Methods.
11558 Medical Sciences: Ha et al. Proc. Natl. Acad. Sci. USA 94 (1997)
mented DNA after 24 h (Table 1). The formation of apoptotic
nuclei (condensed or fragmented) was observed with 10
m
M
CPENSpm treatment at 24 h (Fig. 1B). During this period, cells
became detached from the flask and aggregated. During the
same treatment time, CPENSpm-induced SSAT activity from
146 pmolymgymin to .28,000 pmolymgymin (Table 1) and
reduced ODC activity (.95%) from 8980 670 pmolymgyhto
290 63 pmolymgyh. The increased SSAT activity and reduced
ODC activity were accompanied by a significant decrease in
intracellular polyamine pools and accumulation of the ana-
logue (Table 1). These results are in contrast to those observed
with CHENSpm, which does not induce SSAT to nearly the
same extent as CPENSpm and does not deplete intracellular
polyamine to the same levels (Table 1). However, 10
m
M
CHENSpm does produce significant HMW DNA fragmenta-
tion (Fig. 2 Aand B).
Prevention of the Formation of HMW DNA Fragmentation
by Antioxidants. To determine whether CPENSpm-induced
DNA fragmentation was mediated by the production of H
2
O
2
,
we examined the effect of catalase on CPENSpm-induced
DNA fragmentation. Catalase inhibited the formation of
HMW DNA fragments by 77% compared with the treatment
of CPENSpm alone (Fig. 2 Aand B; Table 1). The prevention
of the formation of HMW DNA fragments was seen at $250
unitsyml of catalase. Heat-inactivated catalase and CuyZn-
SOD did not effect the formation of HMW DNA fragments
(Fig. 2A). However, the protective effect of catalase was
observed to diminish with time. Few CPENSpm-induced
apoptotic nuclei were observed with cotreatment of catalase at
24 h (Fig. 1C), but an increasing number of fragmented nuclei
were observed by 48 h (data not shown). N-acetyl-L-cysteine
(10
m
M) also was found to reduce CPENSpm-induced forma-
tion of HMW DNA fragmentation by '42% during 24-h
exposure. To a lesser extent, butylated hydroxytoluene (400
m
M) and butylated hydroxyanisole (400
m
M) reduced
CPENSpm-induced formation of HMW DNA fragmentation
(data not shown). Catalase was most effective in preventing the
CPENSpm-induced formation of HMW DNA fragmentation
among the antioxidants examined. In contrast, neither catalase
nor CuyZn-SOD had a significant effect on CHENSpm-
induced DNA fragmentation (Fig. 2 Aand B).
Source of Reactive Oxygen Species. The above results
demonstrated that NCI H157 cells treated with CPENSpm
were under oxidative stress. Therefore, the possibility that the
breakdown of the natural polyamines was the source of the
reactive oxygen species that induces HMW DNA fragmenta-
tion was examined. Both the copper requiring serum amine
oxidase and the FAD-dependent intracellular PAO produce
H
2
O
2
as a by-product of polyamine catabolism. Therefore, to
verify which enzyme was responsible for producing H
2
O
2
, the
effects of inhibitors of these enzymes on the CPENSpm-
induced formation of HMW DNA fragmentation was deter-
mined. Increasing concentrations of aminoguanidine (0.1–2
mM), an inhibitor of serum amine oxidase, were used alone
and in combination with 10
m
M CPENSpm. Aminoguanidine
produced no damage on its own and was unable to inhibit
HMW DNA fragmentation produced by 24-h CPENSpm
treatment (data not shown). MDL 72,527, a specific inhibitor
of PAO, was similarly tested alone and in combination with
CPENSpm. Based on the result of a dose-response analysis,
250
m
M MDL 72,527 was chosen for further testing. At 250
m
M, MDL 72,527 was found to significantly reduce the gen-
eration of HMW DNA fragmentation in CPENSpm-treated
cells by 67% (Fig. 2C; Table 1). Similar to results with catalase,
there was little CPENSpm-induced nuclear fragmentation
with cotreatment of MDL 72,527 at 24 h (Fig. 1D), but the
nuclear fragmentation was again observable at later time
points (48 h). As stated above, CHENSpm is not a potent
inducer of SSAT and has a relatively minor effect on intra-
cellular polyamine concentrations (Table 1). Therefore, it is
highly significant that neither aminoguanidine nor the specific
PAO inhibitor had a significant effect on CHENSpm-induced
FIG. 1. The effects of catalase and MDL 72,527 on CPENSpm-induced apoptotic nuclei. Cells were (A) untreated, (B) treated with 10
m
M
CPENSpm, (C) treated with 10
m
M CPENSpm plus 500 unitsyml catalase, or (D) treated with 10
m
M CPENSpm plus 250
m
M MDL 72,527. All
treatment times were 24 h. Cells were fixed with Hoechst dye, visualized under U V excitation, and photographed with a Nikon Labophot microsc ope.
Medical Sciences: Ha et al. Proc. Natl. Acad. Sci. USA 94 (1997) 11559
DNA fragmentation (Fig. 2 Aand B), suggesting that
CHENSpm-induced damaged is not mediated through the
same oxidative stress pathway. These results strongly suggest
that PAO activity is a source of reactive oxygen species in
CPENSpm-treated NCI H157 cells.
The results with catalase and MDL 72,527 indicate that the
generated reactive oxygen species is H
2
O
2
. In an attempt to
substantiate this hypothesis, CM-H
2
DCFDA, an oxidation-
sensitive fluorescent probe, was used. CM-H
2
DCFDA is oxi-
dized to the fluorescent compound dichlorodihydrofluorscein,
which is retained in the cell. The induction of SSAT by
CPENSpm and similar compounds occurs very rapidly (13).
Therefore, a 1-h exposure to CPENSpm was chosen to exam-
ine the early effects on H
2
O
2
production. During the 1-h
treatment, there is a large increase in enzyme activity, and high
concentrations of the natural polyamines are available as
substrates. CPENSpm treatment alone produced a significant
increase in detected fluorescence (Fig. 3). However, cotreat-
ment with either catalase or the PAO inhibitor resulted in no
increased fluorescence over untreated cells. It should be noted
that Fig. 3 represents one of four experiments that demon-
strated identical trends. However, the baseline fluorescence of
controls varied between experiments. This shift in background
fluorescence was possibly caused by the necessity of trypsiniza-
tion of the monolayer cells.
CHENSpm-, but not CPENSpm-Induced PCD, is Associ-
ated with G
2
yM Arrest. Cell cycle analysis of H157 cells after
treatment with CPENSpm and CHENSpm demonstrated dis-
tinctly different profiles. The cell cycle profile of cells treated
with 10
m
M CPENSpm for 24 h was essentially unchanged
compared with control although cells treated with 10
m
M
CPENSpm were clearly undergoing PCD by this time (Fig. 4).
By contrast, cells treated with 10
m
M CHENSpm for 24 h
demonstrated a profound G
2
yM block ('61% cells in G
2
yM),
which was observable as early as 16 h (Fig. 4).
DISCUSSION
The unsymmetrically substituted polyamine analogue
CPENSpm has been shown to exhibit a cell type-specific
cytotoxic activity against human nonsmall cell lung carcinoma
lines in culture (24). Although the treatment of analogue-
sensitive cells is accompanied by a superinduction of SSAT
activity, reduction of ODC activity, depletion of intracellular
polyamines, and accumulation of analogue, the precise mech-
anisms underlying the cytotoxic response have not been elu-
cidated. It recently has been demonstrated that CPENSpm-
treated NCI H157 cells undergo PCD (21). The results of the
current study demonstrate that the PCD is associated with
HMW DNA and nuclear fragmentation in CPENSpm-treated
NCI H157 cells and suggest a mechanism, demonstrating that
the observed damage is at least partially a result of oxidative
FIG. 2. HMW DNA fragmentation induced by polyamine ana-
logues. (A) The effects of catalase, heat-inactivated catalase (p), and
CuyZn-SOD on HMW ($50 kb) DNA fragmentation induced by 10
m
M CPENSpm and CHENSpm in NCI H157 cells. Cells were exposed
to 10
m
M CPENSpm for 24 h with or without 500 unitsyml catalase,
500 unitsyml heat-inactivated catalase, or 1000 unitsyml CuyZn-SOD
and analyzed by field-inversion gel electrophoresis to assess HMW
($50 kb) DNA fragmentation. Quantitation of HMW DNA fragmen-
tation was determined by hybridizing transferred DNA to a
32
P-labeled
human AluI DNA sequence as described in Materials and Methods.(B)
The effects of catalase and MDL 72,527 on HMW ($50 kb) DNA
fragmentation induced by 10
m
M CPENSpm or CHENSpm in NCI
H157 cells. Cells were exposed to 10
m
M CPENSpm or CHENSpm for
24 h with or without 500 unitsyml catalase or 250
m
M MDL 72,527.
FIG. 3. The effects of catalase and MDL 72,527 on CPENSpm-
induced fluorescence detected by CM-H
2
DCFDA. Cells were (A)
treated with 10
m
M CPENSpm, (B) treated with 10
m
M CPENSpm
plus 500 unitsyml catalase, or (C) treated with 10
m
M CPENSpm plus
250
m
M MDL 72,527. After a 1-h treatment, cells were harvested and
treated with 10
m
M CM-H
2
DCFDA for 20 min, and 1 310
5
cells were
analyzed by flow cytometry.
11560 Medical Sciences: Ha et al. Proc. Natl. Acad. Sci. USA 94 (1997)
stress from the production of H
2
O
2
by the SSATyPAO path-
way.
Treatment with exogenous catalase significantly reduced
CPENSpm-induced HMW DNA fragmentation, demonstrat-
ing that H
2
O
2
contributes to the DNA fragmentation produced
in CPENSpm-treated cells. It should be noted that exogenous
catalase is thought to act on endogenously produced H
2
O
2
in
at least two ways. First, H
2
O
2
is a long-lived, readily diffused,
reactive oxygen species, and once outside the cell it can be
detoxified by the exogenous enzyme (30). Alternatively, a cell
type-specific accumulation of exogenous catalase through an
apparent receptor-mediated, energy-dependent system has
been observed (31). It is currently not known which mecha-
nism, or combination of mechanisms, is operative in the NCI
H157 cells. Also consistent with the hypothesis that H
2
O
2
is
contributing to the induction of PCD is the trend observed in
the CM-H
2
DCFDA experiments. These experiments indicated
that there was an increase in H
2
O
2
production leading to an
increase in fluorescence in response to CPENSpm treatment.
However, treatment with exogenous catalase has no significant
effect on PCD produced by CHENSpm. The extracellular
serum amine oxidase was excluded as a possible source of
H
2
O
2
through the lack of effect of the serum amine oxidase
inhibitor, aminoguanidine. By contrast, the specific inhibitor of
the intracellular PAO, MDL 72,527, significantly decreased
the amount of CPENSpm-induced HMW DNA fragmenta-
tion. The effect of MDL 72,527 was not related to competition
for uptake of CPENSpm by MDL 72,527; the treatment with
the PAO inhibitor had no effect on the ability of cells to
accumulate CPENSpm. The results with the f luorescent probe
in the cotreatment experiments suggest that both catalase and
MDL 72,527 reduced the amount of H
2
O
2
produced by
CPENSpm treatment, completely consistent with the results of
the HMW DNA fragmentation experiments. However, similar
to results with catalase, the PAO inhibitor had no effect on
CHENSpm-induced PCD as measured by HMW DNA frag-
mentation, suggesting that cells undergoing PCD induced by
CHENSpm are not under oxidative stress similar to those cells
treated with CPENSpm. Basu et al. (32) have demonstrated
that small changes in the structure of polyamine analogues can
have significant effects on the interaction with DNA. Their
data suggest the potential of different direct effects on DNA
by CPENSpm and CHENSpm. It is also interesting to note
that, in the 24-h exposure experiments, CHENSpm appeared
to accumulate to a greater extent in those cells treated with the
PAO inhibitor compared with cells treated only with
CHENSpm. These results suggest the possibility that
CHENSpm may be a substrate of PAO. However, no break-
down products attributable to CHENSpm were observed in the
HPLC analysis of these samples.
That CPENSpm and CHENSpm initially kill cells by dif-
ferent mechanisms is underscored by the completely different
cell cycle profiles observed after treatment with the individual
agents. The cytotoxic activity of CPENSpm does not appear to
have a profound effect on the cell cycle, killing cells without
an apparent block at any stage of the cell cycle progression. In
contrast, CHENSpm treatment produces a significant G
2
yM
block concurrent with its cytotoxic activity. This effect by
CHENSpm is somewhat unusual in that interference with
polyamines metabolism and polyamine depletion generally
produce a G
1
yS block if they have any measurable effect on the
cell cycle at all (33, 34).
It should be noted that protection against CPENSpm tox-
icity provided by treatment with exogenous catalase, other
antioxidants, or the PAO inhibitor in these experiments was
neither complete nor permanent. Therefore, it must be
stressed that these results indicate that oxidative stress is only
one component of CPENSpm-induced PCD. Inhibition of only
one component in a multi-component system may be expected
to only influence the kinetics of the PCD process. These data
are consistent with the possibility that CPENSpm and
CHENSpm may share a common reactive oxygen species-
independent mechanism that cannot be altered by antioxi-
dants.
It is unlikely that polyamine depletion alone is responsible
for the observed apoptosis because, in NCI H157 cells, deple-
tion of natural polyamines by 2-difluoromethylornithine does
not result in cell death (36), and CHENSpm only has a minor
effect on polyamine levels. These results are consistent with
the results of Albanese et al., which demonstrated in ODC
overproducing cells that the accumulation of N
1
,N
12
-
bis(ethyl)spermine, not polyamine depletion, corresponded
best with the cytotoxic effect of the analogue (37).
Extra- and intracellular polyamines have been shown to be
involved in the cell death process. Pierce, Parchment, and
colleagues first postulated that H
2
O
2
from extracellular serum
amine oxidase-dependent catabolism of polyamines was a
mediator of PCD in the murine embryo, limb buds, and
blastocysts (38–41). Other recent studies have implicated the
intracellular polyamines to be involved in the cell death
process. Induction of ODC (both mRNA and activity), deple-
tion of intracellular polyamines, and induction of SSAT activ-
ity were shown in dexamethasone-induced PCD in rat thymo-
cytes (14). An imbalance of polyamine metabolism was pro-
posed to be a trigger of PCD in heat shock treatment- and
g
-irradiation-induced apoptosis, in which induction of ODC
mRNA and activity was observed without subsequent increase
FIG. 4. Flow cytometric analysis of NCI H157 nonsmall lung carcinoma cells. Cells were untreated, treated with 10
m
M CPENSpm, and treated
with 10
m
M CHENSpm for 24 h. Cell number and DNA content are represented by the ordinate and abscissa, respectively. The G
1
,S,andG
2
yM
fractions were shaded and quantitated by using the MULTICYCLE software package.
Medical Sciences: Ha et al. Proc. Natl. Acad. Sci. USA 94 (1997) 11561
intracellular spermidine and spermine levels (20). Packham
and Cleveland (18) suggested the possibility that the produc-
tion of H
2
O
2
by the SSATyPAO pathway might mediate PCD
in interleukin 3-dependent murine myeloid cells. In their
study, PCD was induced by enforced expression of ODC
concurrent with overexpression of c-myc, and inhibition of
ODC reduced this apoptotic process. They proposed that
excessive intracellular polyamines produced by overexpression
of ODC might be catalyzed by the SSATyPAO pathway, thus
producing H
2
O
2,
which mediates PCD. However, in a more
recent study, Packham et al. (42) have demonstrated that, in a
myeloid system, cytokine withdrawal or c-myc-enforced death
can occur without increases in reactive oxygen species.
In summary, CPENSpm treatment of NCI H157 cells su-
perinduces SSAT activity concurrently with the production of
HMW DNA fragmentation. The early generation of HMW
DNA fragments can be inhibited by various antioxidant com-
pounds, suggesting that the molecular insult is a reactive
oxygen species. The prevention of DNA fragmentation by
catalase, but not by CuyZn-SOD, suggests that the reactive
oxygen species is H
2
O
2
. Most importantly, the specific inhibi-
tion of PAO by MDL 72,527 significantly reduces the amount
of HMW DNA fragmentation, suggesting that the source of
H
2
O
2
is from the two-step polyamine catabolic pathway. These
results provide the first evidence that the superinduction of
SSAT may have a direct role in DNA damage and cell death
in specific cell types. The mechanism of CPENSpm-induced
cytotoxicity described here has important implications for the
development of new antitumor agents. Specifically, it provides
a unique pathway that may be exploited because the superin-
duction of SSAT is a relatively rare, tumor-specific response to
certain agents (43, 44). Furthermore, the possibility that the
production of H
2
O
2
by polyamine catabolism may be a general
mechanism for cellular suicide must be considered. Additional
studies will be necessary to determine how widespread this
phenomenon is and what other mechanisms are responsible for
the apparent H
2
O
2
-independent DNA damage observed.
We thank Drs. John T. Isaac and Christophe Lengauer for their
advice concerning the morphologic studies. This research was sup-
ported in part by National Institutes of Health Grants ES07141,
CA57545, CA63552, and CA51085.
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