Download Differential Effects of Polyamine Homologues on

(CANCER RESEARCH 52. 1840-1847. April 1, 1992]
Differential Effects of Polyamine Homologues on the Prevention of DL-aDifluoromethylornithine-mediated
Inhibition of Malignant Cell Growth
and Normal Immune Response1
Ajay B. Singh, T. J. Thomas, Thresia Thomas, Manjeet Singh, and Richard A. Mann2
Divisions of NepHrology [A. B. S., M. S., R. A. M.] and Rheumatology [T. J. TJ, Department of Medicine, Department of Environmental and Community Medicine
IT. T.J, and the Program in Clinical Pharmacology, Clinical Research Center [T. J. T.], university of Medicine and Dentistry of New Jersey-Roben Wood Johnson
Medical School, New Brunswick, New Jersey 08903
ABSTRACT
Natural polyamines (putrescine, spermidine, and spermine) are ubi
quitous cellular cations that play an important role in cell proliferation
and differentiation. Ornithine decarboxylase is the first and a ratelimiting enzyme in the biosynthesis of polyamines. Polyamine depletion
using DL-a-difluoromethylornithine (DFMO), an inhibitor of ornithine
decarboxylase, has been shown to suppress cell growth in a variety of
settings, including those of tumor and lymphocyte proliferation. The
objective of the present investigation was to examine the inhibitory effects
of DFMO on a variety of murine in vitro immune responses, including
lymphocyte proliferation in response to T-cell mitogen (concanavalin A),
B-cell mitogen (lipopolysaccharide), and alloantigen as well as cytotoxicity. DFMO-mediated inhibition of cell proliferation in these cases
correlated with depletion of intracellular polyamines. The inhibitory
effects of DFMO were reversed by polyamine repletion with putrescine.
Putrescine also reversed the growth-inhibitory effects of DFMO on 4
tumor cell lines that we tested: 28-13-3S, YAC-1, P-815, and K562.
However, putrescine homologues exhibited a differential effect in pre
venting DFMO-mediated inhibition of cell growth in normal lymphocytes
and cancer cell lines. Only putrescine homologues containing a shorter
méthylène
chain were effective in preventing the growth-inhibitory action
of DFMO on normal immune response. In contrast, only the longer chain
homologue 1,5-diaminopentane overcame the effect of DFMO on tumor
cell growth. These findings suggest that supplementation with selected
polyamine homologues may sustain normal immune response in DFMOtreated individuals while effectively suppressing malignant cell growth.
The potential clinical relevance of these observations is discussed.
INTRODUCTION
The low molecular
weight
polyamines
putrescine
[H2N(CH2)4NH2], spermidine [H2N(CH2)4NH(CH2)3NH2], and
spermine [NH2(CH2)3HN(CH2)4NH(CH2)3NH2) are essential
for cell growth and differentiation (1,2). Although polyamines
are detectable in all eukaryotic cells, high levels are found in
those cells which are rapidly proliferating such as cancer cells
or those of embryonic tissue (3-5). Elevated polyamine levels
have also been reported in patients with malignancy, hemolytic
anemia, and systemic lupus erythematosus (6-8).
The biosynthesis of polyamines involves the early conversion
of arginine to ornithine which, in turn, is converted to putres
cine (9). This latter conversion is a rate-limiting step in polyam
ine synthesis and is catalyzed by ODC.1 Subsequently, putres
cine is converted to spermidine
and spermine through the
Received 11/7/90; accepted 1/24/92.
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
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' This work was supported, in part, by the National Institutes of Health grants
DK 40504 (R. A. M.), AR 39020 (T. J. T.), and CA 42439 (T. T.).
2To whom requests for reprints should be addressed.
3The abbreviations used are: ODC, ornithine decarboxylase; B6, C57BL/6;
C2, ethylenediamine; C3, 1,3 diaminopropane; ("4. 1,4 diaminobutane or putres
actions of spermidine synthase and spermine synthase, respec
tively. Under physiologically compatible ionic conditions,
polyamines are positively charged; hence, a target of their action
is the negatively charged phosphate groups on biological macromolecules, including DNA and RNA. Recent evidence dem
onstrates that polyamines stabilize double stranded DNA by
increasing its helix coil transition temperature ('/',„
or melting
temperature) (10) and are capable of condensing DNA into
compact structures such as toroids and spheroids (11). They
also provoke a dramatic alteration in the conformation of
certain polynucleotides (12). Despite these findings, the exact
mechanism by which polyamines support cell growth remains
uncertain. It is also unclear whether the charge, the conforma
tion, or the length of the carbon chain determines the biological
function of a polyamine.
Intracellular polyamine depletion has been accomplished uti
lizing inhibitors of ODC. The most extensively studied ODC
inhibitor is DFMO [H2NCH2CH2CH2C(CHF2)(COOH)NH2]
(13, 14). DFMO competitively inhibits ODC, by binding to its
active site, thereby preventing polyamine synthesis and cell
proliferation. DFMO has been shown to inhibit cell growth in
a variety of tumors, both in vitro and in vivo, and has been
generally well tolerated by animals and humans (15-17).
Previously it has been reported that putrescine reversed the
in vitro inhibitory effects of DFMO on LI210 leukemic cells
(18). However, not all putrescine homologues shared this abil
ity. For example, although 5- and 6-carbon putrescine homo
logues partially restored the proliferative capability of DFMOtreated cells, the 2- and 3-carbon homologues did not do so. In
the present study, we have examined the ability of DFMO to
inhibit in vitro murine immune response. We have also tested
a variety of polyamine homologues for their ability to reverse
DFMO-mediated growth inhibition. We report that DFMO
inhibited normal and malignant cell growth. Putrescine re
versed and prevented this inhibitory effect of DFMO. However,
although 2- or 3-carbon chain putrescine homologues prevented
DFMO-mediated inhibition of normal cells, they did not pre
vent the inhibition of tumor cells. In contrast, a higher homo
logue (5-carbon chain) of putrescine did overcome the growthinhibitory effects of DFMO on tumor cell lines. Thus, selected
polyamine homologue supplementation may sustain normal
immune response in DFMO-treated individuals while main
taining suppression of malignant cell growth.
MATERIALS
AND METHODS
Animals. Male C57BL/6 (B6, H-2") and DBA/2 (H-2d) mice, 8-10
weeks of age, were purchased from The Jackson Laboratory, Bar
Harbor, ME. All animals were housed in approved facilities under the
direction of the Animal Care and Use Committee of UMDNJ-Robert
Wood Johnson Medical School. They were on a 12-h day/12-h dark
cine; C5, 1,5 diaminopentane; C6, 1,6 diaminohexane; Con A, concanavalin A;
light cycle and were fed Purina rodent chow and water ad libitum.
DFMO, DL-a-difluoromethylornithine;
HPLC, high performance liquid chromatography; [3H]dThd, tritiated thymidine.
Preparation of Cell Suspensions. Spleens and lymph nodes were
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EFFECT OF DFMO ON CELL GROWTH AND IMMUNE RESPONSE
aseptically removed, minced, and passed through a wire screen mesh in
RPMI 1640 supplemented with penicillin G (100 units/ml), strepto
mycin (100 Mg/ml), gentamicin (25 ng/m\), L-glutamine (2 mM), 2mercaptoethanol (5 x IO"2 mM), and 10% heat-inactivated fetal calf
RESULTS
serum (Hyclone lot 115882) preselected for its ability to support
lymphocyte blast transformation and cell-mediated lympholysis. All
cultures were conducted at 37'C in 5% CO;
Cell Lines. The murine tumor lines YAC-1 (a mouse lymphoma line)
(19), P815 (a mouse mastocytoma line) (20), 28-13-3S (a hybridoma
formed by the fusion of the mouse myeloma line SP2/0-Agl4 and cells
derived from a C3H mouse immunized with C3H.SW cells) (21), and
the human chronic myelogenous leukemia cell line K562 (22) were
obtained from American Tissue Culture Collection (Rockville, MD)
and maintained in RPMI 1640 supplemented as outlined above.
Chemicals and Reagents. DFMO was kindly provided by the Merrell
Dow Research Institute (Cincinnati, OH) of Marion Merrell Dow, Inc.
This compound was >99.99% pure, as reported by the manufacturer.
C4, C2, and C3 were obtained in their dihydrochloride form from
Sigma Chemical Co. (St. Louis, MO). C5 and C6 were purchased in
their dihydrochloride form from Aldrich Chemical Co. (Milwaukee,
WI). C5 and C6 were reported to be >99% pure. Compounds C2 to C4
were reported to be >98% pure. We could not detect amine impurities
in these compounds by HPLC assay. Prior to being added to culture,
reagents were reconstituted in RPMI 1640, filter sterilized, and stored
at 4°C.
Lymphocyte Blast Transformation Assay. B6 lymphocytes (5 x IO'1)
were placed in 2-ml culture wells (24 wells/plate) either alone, with an
equal number of irradiated allogeneic lymphocytes (DBA/2), or with
Con A (4 ¿ig/ml)or lipopolysaccharide (10 ^g/ml). After 1-3 days in
culture, the contents of the wells were gently mixed and sampled.
Samples (100 n\) of cell suspensions were placed in microtiter wells in
the presence of 0.25 ^Ci of pH]dThd (New England Nuclear, Boston,
MA). Four h later, the cells were harvested for scintillation counting.
Data are expressed as the mean cpm ±SEM of three replicate wells.
In Vitro Induction and Assay of Cell-mediated Lympholysis. B6
lymphocytes were tested for cytotoxic potential using modifications of
established methods (23). Briefly, 5 x IO6B6 cells were cultured along
with an equal number of irradiated DBA/2 simulators in 2-ml wells.
After 5 days, the remaining cells were harvested and placed in microtiter
wells along with '( r labeled targets in a standard 4-h chromium release
assay. Spontaneous release represented release by targets incubated
with medium alone whereas maximal release was determined by incu
bation of targets with 4% cetrimide. Targets consisted of DBA/2 cells
harvested after 3-4 days in culture containing 4 jig/ml of Con A. These
cells were then chromium labeled by incubating I x IO7cells with 200
ti\ of sodium chromate [5 mCi (185 MBq)/5 ml NEZ-030S; New
England Nuclear] for l h at 37°C.Results are expressed as the per
centage of specific lysis which is calculated as
Experimental release - spontaneous release
Maximal release - spontaneous release
The data presented are the means ±SEM of three replicate wells.
Measurement of Intracellular Polyamines. Cells (IO7) were harvested
from culture, washed with phosphate-buffered saline, and pelletized.
The cell pellet was treated with 2 ml of 8% sulfosalicylic acid and
sonicated for 15 s in ice. The solution was incubated in ice for l h and
centrifuged at 900 x g for 5 min in a Sorvall RC5C centrifuge to
remove the precipitated protein. Intracellular polyamine levels were
determined by HPLC after derivatization to their dansyl derivatives, as
described by Kabra et al. (24). 1,6-Diaminohexane was used as an
internal standard. HPLC was performed on a Perkin-Elmer system
using Binary LC Pump 250 and a LS 40 fluorescence detector. In some
cases, polyamines were determined at the Brain Tumor Research Lab
oratory of the University of California at San Francisco.
Statistical Analysis. Each experiment was carried out in triplicate.
Data have been depicted as the mean ±SEM using data from one
representative experiment. Each experiment was repeated 2-4 times
with similar results obtained. Statistical significance was determined by
a 2-tailed Student's t test.
DFMO Inhibits Both the Proliferation and Cytotoxicity of
Normal Lymphocytes. We investigated the effect of DFMO on
murine lymphocyte response to the T-cell mitogen Con A and
to alloantigen. Lymphocytes harvested from B6 mice were
cultured with mitogen or irradiated allogeneic (H-2d) stimula
tors. DFMO was added to the culture at one of several concen
trations in order to determine the ability of the drug to modulate
the immune response. Cell proliferation was determined by [JH]
dThd incorporation after 3 days in culture. DFMO inhibited,
in a concentration-dependent fashion, lymphocyte proliferation
in response to both of these stimuli (Table 1). Even at low
concentrations of DFMO (0.1 mivi), reductions in cell prolifer
ation were statistically significant. DFMO, at concentrations
above 0.1 mM, also induced a statistically significant reduction
in cell proliferation in response to the B-cell mitogen lipopoly
saccharide (10 Mg/ml) (data not shown).
The inhibition of lymphocyte responsiveness seen in Table 1
was not due to toxicity of DFMO as viability at 48 and 72 h,
as determined by supervital dye exclusion, was roughly com
parable to that seen in cultures which did not contain DFMO
(typically 75-85% of the cells appeared to be viable). Further
more, incubation of cells with 1 mM DFMO for 2 h, followed
by thorough washing prior to being placed in culture, had no
effect on their proliferation in response to Con A or allogeneic
targets (data not shown).
We next examined the ability of DFMO to modulate the
induction of cytotoxic effector cells against allogeneic targets.
B6 lymphocytes were placed in culture along with lethally
irradiated DBA/2 stimulators in a standard mixed lymphocyte
culture. DFMO, ranging in concentration from 0 to 1 mM, was
added on day 0. After 5 days in culture, the remaining cells
were harvested and tested for their ability to lyse 5'Cr-labeled,
Con A-stimulated DBA/2 blasts. Fig. 1 demonstrates that
DFMO, in a concentration-dependent
fashion, inhibited the
generation of B6 anti-DBA/2 cytotoxic lymphocytes. Even at
0.1 mM DFMO, cytotoxicity was reduced although statistically
significant impairment in cytotoxicity was achieved at DFMO
concentrations of 0.25 mM or above only (P < 0.02). Fig. 2
demonstrates that the addition of DFMO to such cytotoxicity
induction cultures after day 1 did not impair effector cell
induction even at 1 mM. Statistically significant reduction in
cytotoxicity (P < 0.008) was seen at each effectontarget ratio
when DFMO was added on day 0 or day 1.
DFMO Inhibits Polyamine Biosynthesis. To confirm that
DFMO was being internalized and was inhibiting intracellular
polyamine synthesis, B6 cells were placed in culture with or
without DFMO along with either Con A or irradiated DBA/2
Table 1 Ability of DFMO to inhibit cell proliferation
to"Con
proliferation in response
Concentration of
DFMO
(mM)0.00
A17.7
±0.73
±0.05
14.2 + 0.11*
1.8±0.02r
0.10
1.3±0.09r
1.9
±0.02'
0.25
1.1 ±0.003r
1.1 ±0.03°
0.50
1.0±0.02fAlloantigen2.9
1.0±0.02r
1.00Cell
" B6 lymphocytes were placed in 2-ml culture wells along with either Con A
(4 ¿ig/ml),or an equal number of irradiated allogeneic (DBA/2, H-2d) targets.
DFMO was added at the indicated concentration on day 0. After 72 h in culture,
proliferation was determined by ['HjdThd incorporation. Data are expressed as
cpm x 10~4±SEM of three replicate samples.
fP<0.001.
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EFFECT OF DFMO ON CELL GROWTH AND IMMUNE RESPONSE
was added on day 0, 1,2, 3, or 4 and cytotoxicity was measured
on day 5. Controls were not treated with DFMO. Polyamines
were measured from a parallel set of cells, as used in the
cytotoxicity assay. Polyamine concentrations measured in these
cells on day 5 were as follows: putrescine, 1.6, 0, 0, 0.9, 1.0,
and 1.4 nmol/106 cells for control, day 0 DFMO, day 1 DFMO,
LJ
30
day 2 DFMO, and day 4 DFMO, respectively. Spermidine and
spermine concentrations (nmol/106 cells) in these cells were:
U
U
a
H
20
50:1
20:1
5:1
EFFECTOR:TARGET RATIO
Fig. 1. Effect of DFMO on the induction of cytotoxic effector cells. B6 cells
were placed in culture along with irradiated DBA/2 stimulators in a standard
mixed lymphocyte culture. DFMO was added on day 0 at 0 mM (O), 0.1 mM (D),
0.25 mM (•),0.5 mM (A), or 1.0 mM (x). After 5 days, the remaining cells were
harvested and tested for cytotoxicity against s>Cr-labeled DBA/2 blasts. Bars,
SEM.
70
en
60
ÃŽJ 50
O
Ü
O
40
UJ
85
30
*
20
10
50:1
20:1
5:1
EFFECTORiTARGET RATIO
Fig. 2. DFMO effects the afferent phase of the induction of cytotoxic effector
cells. B6 cells were placed in culture along with irradiated DBA/2 stimulators in
a standard mixed lymphocyte culture. The cultures contained either no DFMO
(•),or DFMO (1.0 mM) added on day 0 (O), day 1 (•),day 2 (II), day 3 (A), or
day 4 (A). After 5 days, the remaining cells were harvested and tested for
cytotoxicity against "Cr-labeled DBA/2 blasts. Bars, SEM.
cells. After 72 h, the remaining cells were washed with phos
phate-buffered saline and analyzed for intracellular polyamine
content after prccolumn derivatization procedure, using HPLC
(24). The results of this polyamine assay are presented in Table
2. DFMO (1 HIM)reduced the concentration of putrescine and
spermidine to undetectable levels in Con A-stimulated cultures.
When allogeneic stimulators were used, DFMO again lowered
intracellular putrescine to undetectable levels while spermidine
was reduced to 11% of the level measured in cells harvested
from cultures to which DFMO had not been added. Regardless
of the stimuli present in culture, DFMO failed to markedly
reduce spermine levels (although the reduction seen in the
presence of alloantigen did reach statistical significance). The
relative preservation of spermine levels is consistent with ob
servations in other laboratories and has been attributed to a
spermine salvage pathway (25). DFMO also caused depletion
of polyamines in cells harvested after 48 h of treatment (see
Table 5).
We also determined polyamine levels in our cytotoxicity
assay cultures exposed to DFMO. In these experiments, DFMO
spermidine, 5.7, 1.2, 1.4, 1.8, 3.1, and 4.7; spermine, 4.2, 6.0,
7.4, 5.2, 4.6, and 4.7. These data, in conjunction with results
shown in Fig. 2, clearly show that DFMO addition to cytotox
icity cultures on day 0 or day 1 depletes putrescine to undetect
able levels and spermidine to approximately one-fourth of that
of controls. Polyamine depletion at this stage correlates with
impaired induction of cytotoxic effector cells. Although addi
tion of DFMO on days 2 and 3 substantially reduced putrescine
and spermidine levels, there was no effect on cytotoxicity.
Addition of DFMO to cultures on day 4 had no major effect
on the concentration of any of the polyamines analyzed on day
5. Thus it would appear that polyamines are critical for the
afferent phase of this immune response and that depletion of
polyamines, during later stages of cytotoxic effector cell differ
entiation, has no effect on their lytic potential.
Putrescine Reverses and Prevents the Inhibitory Effects of
DFMO on Cell Proliferation and Cytotoxicity. DFMO is known
to specifically inhibit ODC which in turn catalyzes the conver
sion of ornithine to putrescine. The inhibitory effect of DFMO
is therefore, in large part, due to depletion of intracellular
polyamines. Adding back polyamines to the culture medium
should obviate the need to synthesize them and thereby prevent
the inhibitory effect of DFMO. In order to test this possibility,
we examined the effect of exogenous putrescine on cells treated
with DFMO. Table 3 demonstrates that the addition of putres
cine (100 JIM)to the culture completely prevented the inhibitory
effects of DFMO on cell proliferation in response to Con A
and allogeneic stimulators as well as the inhibition of cytotox
icity by DFMO. Once again DFMO, in the absence of exoge
nous polyamine, markedly inhibited immune response. In the
presence of putrescine, however, even 1 HIM DFMO failed to
diminish proliferation or cytotoxicity. The very small decrease
in proliferation in response to Con A (10.3 ±0.8 versus 11.5 ±
0.07 cpm x IO"4) was not statistically significant.
In Table 3 putrescine is overriding the effects of DFMO,
both agents having been added to the culture on day 0. In order
to prove that putrescine is capable of reversing the effects of
DFMO on polyamine depletion, we treated cells with DFMO
for 1-2 days prior to the addition of putrescine, thus allowing
synthesisConcentration
Table 2 Ability of DFMO to inhibit polyamine
polyamine concen
to"Con
tration in response
of
DFMO
(mM)0
Allotargets0.21
A
±0.03
0.27 ±0.02
Putrescine
Undetectable
Undetectable
1.0
Spermidine
3.33 ±0.53
2.92 ±0.17
0
Undetectable
0.31
±0.02*
Spermidine
1.0
2.65 + 0.26
1.45 ±0.10
Spermine
0
2.36 ±0.14
0.91±0.10r
1.0PolyaminePutrescine
SpermineIntracellular
" B6 lymphocytes were placed in 2-ml culture wells along with either Con A
(4 ¿ig/ml)or an equal number of irradiated allogeneic (DBA/2, H-2'') targets.
Where indicated, DFMO was added on day 0. After 72 h in culture, the remaining
cells were harvested and washed and 10 x Id'1 cells were analyzed for their
intracellular polyamine content using HPLC. Data are expressed in nmol/10'
cells and represent the mean ±SEM of three replicate samples.
"P< 0.001.
CP<0.02.
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EFFECT OF DFMO ON CELL GROWTH AND IMMUNE RESPONSE
Table 3 Ability of putriscine (Pu) to prevent DFMO-mediated inhibition of cell proliferation and the induction ofcytotoxic effector cells
cpm x 10"* in response to"
% of specific lysis*
Concentration of
DFMO
(misi)0
ANo
Pu8.4
Pu13.2
Pu67.1
5 ±0.07
±0.09
±0.8
±3.8
±1.9
1.4±0.09J
12.7± 1.4'
11.1 ±0.1
8.2 ±0.2
66.6
3.273.5
±
0.51.0Con
1.0
±0.05''Puc7.2
8.4
±1.2'Puc61.8
±4.8
10.3 ±0.8AllotargetsNo
8.5 ±0.12No
" B6 lymphocytes (5 x 10') were placed in 2-ml culture wells along with either Con A (4 /ig/ml) or an equal number of irradiated allogeneic (DBA/2, H-2') targets.
In some instances DFMO and/or putrescine (Pu) were added on day 0. After 48 h in culture, cell proliferation was determined by ['HJdThd incorporation. Data are
expressed as cpm x 10 4 ±SEM of three replicate samples.
*B6 lymphocytes (5 x 10*) were placed in 2-ml culture wells along with an equal number of irradiated DBA/2 stimulators. In some instances DFMO and/or Pu
were added on day 0. After 5 days in culture, the remaining cells were tested for cytotoxicity against "Cr-labeled DBA-2 blasts in a 4-h chromium release assay. Data
are expressed as percentage of specific lysis at effectortarget ratio of 50:1 and is the mean ±SEM of three replicate samples. Similar data were obtained at
effectortarget ratios of 20:1 and 5:1.
' The final concentration of putrescine (Pu) was 100 ¡¡M.
"P< 0.001.
±0.08
1.5 ±0.01''
1.3 ±0.04''Puf11.
Table 4 Ability of putrescine homologues to prevent DFMO-mediated inhibition of cell proliferation and the induction ofcytotoxic effector cells
cpm x 10 ' in response to"
. of specific lysis*
Polyamine
addedNone
±0.2"
±0.02'
±1.5'
±0.4
±0.2
±2.4
4.8 ±0.08''
4.2 ±0.04'
C2
7.7 ±0.4
9.0 ±0.2
59.7 ±1.4
60.3 ±0.2
7.2 ±0.1'
5.7 ±0.4
5.7 ±0.4
12.2 ±0.07
68.3 ±3.4
C3
65.1 ±3.7
6.0 ±0.4
5.7 ±0.3
10.3 ±0.2
11.7 ±0.6
60.2 ±1.3
61.5 ±2.9
C4
1.1
±0.06'
2.1
±0.027
11.0 ±0.7
6.8 ±1.2
55.4 ±3.7
0.0 ±OV
C5
0.7
±
0.04'DFMO(-)11.2
0.4
±
0.01'DFMO(-)59.6
0.0 ±0.7'
C6DFMO(-)5.3
6.3 ±0.3DFMO(+)f2.7
3.9 ±0.1DFMO(+)f1.7
61.1 ±0.7DFMO(+)r8.4
" B6 lymphocytes (5 x 10") were placed in 2-ml culture wells along with either Con A (4 «ig/ml)or an equal number of irradiated allogeneic (DBA/2, 112'') targets.
In some instances DFMO and/or putrescine (C4) or a 2-, 3-, 5-, or 6-carbon putrescine homologue were added, at a final concentration of 100 «M.
on day 0. After 48
h in culture proliferation was determined by | 'H|d I lid incorporation. Data are expressed as cpm x IO"4 ±SEM of three replicate samples.
* B6 lymphocytes (5 x 10') were placed in 2-ml culture wells along with an equal number of irradiated DBA/2 targets. In some instances DFMO and/or one of
the above mentioned polyamines were added, at a final concentration of 100 JIM. on day 0. After 5 days in culture, the remaining cells were tested for cytotoxicity
against "Cr-labeled DBA/2 blasts in a standard 4-h chromium release assay. Data are expressed as percentage of specific lysis an effectortarget ratio of 50:1 and are
the means ±SEM of three replicate samples. Similar data were obtained at effectortarget ratios of 20:1 and 5:1.
c The final concentration of DFMO was 1 mM.
" P< 0.005.
..
fP<0.02.
DFMO to become internalized and to inhibit ODC prior to the
addition of exogenous polyamine. For these experiments, B6
cells were again placed in culture with either Con A or irradiated
DBA/2 cells. DFMO (1 HIM) was added on day 0. In some
cases putrescine (100 MM) was added on day 1 or day 2.
Proliferation was determined by [3H]dThd incorporation on
day 3. Cytotoxicity was determined on day 5. The addition of
putrescine during the last 24 or 48 h of 72-h proliferation assays
completely reversed the inhibition of proliferation by DFMO
in response to Con A (proliferation was reduced to 10-40% of
control values in wells to which DFMO was added on day 0
whereas the addition of DFMO on day 0, and subsequent
addition of putrescine on day 1 or 2, resulted in 101-104% of
control proliferation) and substantially reversed inhibition of
proliferation in response to DBA/2 stimulators (restoring pro
liferation to 65-70% of control values). Putrescine was also
capable of reversing DFMO-mediated inhibition of cytotoxic
effector cell induction. DFMO was added to such cultures on
day 0. The addition of putrescine on day 2 completely restored
cytotoxicity (percentage of specific lysis at effectontarget ratios
of 50:1, 20:1, 5:1 = 59.6%, 55.8%, 40.6% versus 8.4%, 4.1%,
0% versus 60.4%, 59.2%, 44.2% for control cultures, those
containing DFMO from day 0, and those to which DFMO was
added on day 0 and putrescine added on day 2, respectively).
C2 and C3 Putrescine Homologues Prevent DFMO-mediated
Inhibition of Normal Immune Response. Having demonstrated
that putrescine could reverse and prevent DFMO-mediated
inhibition of normal immune response, we next determined
whether putrescine homologues would also overcome the effects
of DFMO. Specifically, we examined the ability of putrescine
homologues to prevent DFMO-mediated inhibition of lympho
cyte proliferation in response to Con A and allogeneic targets.
Additional studies were designed to determine their ability to
prevent inhibition of induction of cytotoxic effector cells with
specificity for allogeneic targets. B6 lymphocytes were cultured
with putrescine, or a putrescine homologue, and either Con A
or irradiated DBA/2 stimulators with and without DFMO. The
compounds tested included both the shorter and longer carbon
chain homologues of putrescine, with the méthylène
bridging
region ranging from 2- to 6-carbon molecules long. Table 4
demonstrates that the 5- and 6-carbon homologues could not
substantially overcome inhibition of normal immune response
by DFMO. However, the 3-carbon homologue (and to a lesser
degree the 2-carbon homologue) supported normal immune
response in the presence of DFMO.
Results presented in Table 4 also show that certain homo
logues, especially C5 and C6, have a significant effect on the
proliferation of B6 lymphocytes stimulated with Con A and
alloantigen. C5 enhanced proliferation in response to the non
specific T-cell mitogen Con A whereas both C5 and C6 inhibited
alloantigen specific proliferation. Despite the decreased prolif
eration in response to allotargets seen in the presence of C5
and C6 there was no demonstrable decrement in cytotoxicity.
These finding suggest that T-cell subsets may be differentially
affected by polyamine homologues with certain subsets being
more susceptible to the inhibitory effects of selected polyamine
homologues.
In order to examine whether the addition of homologues, in
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EFFECT OF DFMO ON CELL GROWTH AND IMMUNE RESPONSE
Table 5 Intracellular polyamine levels in B6 cells after treatment with Con A,
DFMO, and/or putrescine homologues
these compounds had very little effect on suppression of tumor
cells by DFMO. However, the C5 homologue, which was largely
Polyamine concentration
(nmol/10'cells)"
ineffective in preventing the effects of DFMO on normal lym
phocytes, completely abrogated the inhibitory effect of DFMO
TreatmentCon
on the 28-13-3S line and substantially prevented inhibition of
ACon
all tumor lines tested.
DFMOCon
A+
We also determined polyamine levels in all cell lines treated
C2Con
A+
DFMOCon
A + C2 +
with putrescine homologues in the absence and presence of
C3Con
A+
DFMO. Representative data from P815 cell line are presented
DFMOCon
A + C3 -1C4Con
A+
in Table 7. As in the case of normal lymphocytes, there was a
DFMOCon
A + C4 +
substantial decrease in putrescine and spermidine levels in cells
C5Con
A+
treated with DFMO in combination with putrescine homo
DFMOCon
A + C5 +
C6Con
A+
logues, except in the case of putrescine. C6 was the only
A + C6 + DFMOPutrescine0.200.080.140.050.220.080.290.250.240.130.150.10Spermidine0.540.130.620.140.520.120.640.600.090.050.160.05Spermine0.480.490.430.420.380.410.390.
homologue that substantially reduced polyamine levels in the
" B6 lymphocytes were placed in 2-ml culture wells along with Con A (4 Mg/
absence
of DFMO. Transport of homologues into the cell
ml). Putrescine homologues C2-C6 (100 MM)and DFMO (1 HIM)were added to
occurred in the presence and absence of DFMO, as seen in Fig.
culture on day 0, as indicated. After 48 h in culture, the remaining cells were
harvested and washed, and 10 x 10" cells were analyzed for their intracellular
4 for C5. The presence of DFMO, however, substantially in
polyamine content using HPLC. Results reported represent the average of dupli
creased
(5 transport and its metabolism to an aminopropyl
cate assays.
derivative (see the peak at 19.1 min). Analysis of the chromatograms of other homologues showed similar derivatization of
the absence or presence of DFMO, had an effect on intracellular
these compounds to a spermidine homologue within the cell.
polyamine levels, we quantified polyamine pools in these cells Comparable data was obtained for other tumor cell lines: 28by HPLC. Table 5 presents our data on polyamine levels in 13-3S, YAC-1, and K562 (data not shown).
cells harvested after 48 h. The addition of DFMO decreased
putrescine and spermidine levels in control cells as well as those
treated with all homologues except putrescine. There was a DISCUSSION
rapid uptake of putrescine in cells treated with or without
In this report, we present data to demonstrate that depletion
DFMO. A similarly high level of C5 transport occurred in both
of polyamines by DFMO results in significant immunosupprescases, with a turnover of C5 to a spermidine homologue (Fig.
sion of normal murine lymphocytes. Depletion of putrescine
3) that was identified as NH2(CH2)5NH(CH2),NH2 using a and spermidine in DFMO-treated cells and the ability of ex
purified sample of this compound (10). We found similar con
ogenous putrescine to both prevent and reverse the effects of
version of other putrescine homologues to their aminopropyl
DFMO provide evidence that the target of action of DFMO is
derivatives within the cell. Our results also show that C6
polyamine biosynthesis. Several investigators have shown that
substantially reduced putrescine and spermidine even in the
absence of DFMO. C6, in the absence of DFMO, reduced
5-1
putrescine and spermidine levels to 75 and 30%, respectively,
of control sample level.
)
t
We also observed that DFMO inhibited putrescine and sper
z
midine synthesis in lymphocytes stimulated with alloantigen in
the presence and absence of putrescine homologues (data not
IT
shown).
2H
The C5 Homologue of Putrescine Supports Tumor Cell
Growth. We next examined whether DFMO would inhibit the
growth of a variety of tumor cell lines and, if so, whether this
inhibition would be prevented by the addition of either putres
cine or its homologues. We tested the murine tumor lines 2810.0
15.0
20.0
13-3S, YAC-1, and P815 and the human leukemia line K562
C/)
5
with (1 HIM)and without DFMO. In some instances the culture
medium was supplemented with one of several putrescine hom
4ologues in an attempt to abrogate the inhibitory effects of
O
DFMO. The agents tested again included the C2 to C6 homo
3logues described above. After 48 h in culture, proliferation in
UJ
U
each well was determined by ['H]dThd incorporation. As shown
V)
than that seen in the absence of DFMO (19.8 ±0.7 with C4
and DFMO versus 22.1 ±0.4 with C4 alone, cpm x 10~4,
P < 0.05).
In contrast to the ability of C2 and C3 homologues to
overcome the effects of DFMO on normal immune response,
2-
UJ
in Table 6, DFMO inhibited the proliferation of all tumor lines
tested. In all instances putrescine was able to overcome this
inhibition, the one possible exception being the K562 line.
Although C4 allowed for much greater cell growth in the
presence of DFMO (19.8 ±0.7 with C4 versus 3.2 ±0.3 without
C4, cpm x 10~4), that cell growth was still significantly less
ce
O
0-
10.0
15.0
20.0
ELUTIONTIME
(min)
Fig. 3. Chromatogram showing internalization of C5 in normal lymphocytes
and its conversion to an aminopropyl derivative. HPLC analysis of precolumn
derivatized polyamines was conducted as described in Ref. 24. B6 cells were
cultured for 48 h with either Con A + C5 (A) or Con A + C5 + DFMO (B).
a, putrescine; b, C5; c, C6; d, spermidine; e, AP5 (aminopropyl derivative of C5);
/ spermine.
1844
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EFFECT OF DFMO ON CELL GROWTH AND IMMUNE RESPONSE
Table 6 Ability ofputrescine analogues to prevent DFMO-mediated inhibition of tumor cell growth
10""28-1
cpm x
3-3Sadded
DFMO(-)None
±0.07'
±0.3"
±0.01'
±1.7
±0.8
±2.8
±0.05
7.0 ±0.2'
2.7 ±0.3'
0.6 ±0.01'
3.1 ±0.3'
26.3 ±1.6
12.9 ±0.7
23.0 ±0.5
C2
.7 ±0.05
8.9 ±0.5'
0.8 ±0.02'
4.9 ±0.2'
3.5 ±0.4'
.6 ±0.2
25.7 ±0.5
15.3 ±0.9
23.2 ±0.3
C3
19.8±0.7/
C4
.6 ±0.1
1.3 ±0.2
19.5 ±2.4
20.4 ±1.5
13.7 ±2.5
10.7 ±0.7
22.1 ±0.4
9.4 ±0.3'
15.6 ±0.01''
18.1 ±0.4
.4 ±0.1
1.4 ±0.3
25.5 ±1.7
20.8 ±0.6
21.1 ±0.8
C5
0.6 ±0.06'YAC-1DFMO(-)24.5
6.9 ±0.08'P-815DFMO(-)11.1
2.0 ±0.4'K562DFMO(-)21.0
3.2 ±0.07'
15.4 ±0.2DFMO(+)*3.3
22.4 ±0.2DFMO(+)*3.2
.2 ±0.01DFMO(+)*0.4
6.6 ±0.2DFMCH+)*1.8±0.08C
C6.7
" Tumor cells (5 x 10') were placed in 2-ml culture wells. In some instances DFMO and/or putrescine (C4) or a 2-, 3-, 5-, or 6-carbon putrescine homologues were
added, at a final concentration of 100 UM,on day 0. After 48 h in culture, cell proliferation was determined by | 'I l|d Ilui incorporation. Data are expressed as cpm x
IO"4 ±SEM of three replicate samples.
* The final concentration of DFMO was 1 mM.
'/>< 0.001.
"P< 0.005.
..
fP<0.05.
of DFMO on MRL-lpr/lpr mice, a strain of mice which spon
taneously develops an autoimmune disorder which closely re
Polyamine concentration
cells)"TreatmentNoneDFMOC2C2 (nmol/106
sembles the human disorder systemic lupus erythematosus
(SLE or lupus). The observed effects of DFMO included dimin
ished autoantibody production, prolongation of life span, and
a significant reduction of renal injury. Comparable effects of
another polyamine inhibitor, (2Ä,5Ä)-6-heptyne-2,5-diamine,
DFMOC3C3
+
were also reported by Claverie et al. (34) for MRL-lpr/lpr mice.
In view of the effect of DFMO on nonmalignant cells reported
DFMOC4C4
+
above, these observations could reflect DFMO-mediated
DFMOC5C5
+
suppression of either helper/inducer or effector cells, or both,
in the MRL-lpr/lpr mouse.
DFMOC6C6
+
We also present data to suggest that putrescine homologues
+ DFMOPutrescine0.110.050.160.050.19ND0.580.60.090.050.070.05Spermidine1.490.161.250.261.550.421.191.160.800.070.17NDSpermine0.500.730.420.700.540.690.370.180.510.50
substantially
differ with regard to their effect on normal lym
" P815 cells were placed in culture and putrescine homologues C2-C6 (100
phocytes as compared to their effect on tumor cells. To our
MM)and DFMO (1 mM) were added on day 0, as indicated. After 48 h in culture,
knowledge this is the first time that such a structure-dependent
the remaining cells were harvested and washed, and 10 x 10' cells were analyzed
disparity has been reported. Putrescine and its 2- and 3-carbon
for their intracellular polyamine content using HPLC. Results reported represent
Table 7 Inlracellular polyamine levels in PS 15 cells after treatment with DFMO
and/or putrescine homologues
the average of duplicate assays.
5i
DFMO is a specific inhibitor of the polyamine biosynthetic
enzyme ODC and that this drug has no other cellular target
(13-15,26).
Recent studies have suggested that polyamines play a signif
icant role in normal lymphocyte response (27-30). Pasquali et
al. (27) showed that DFMO caused a reduction in pokeweed
mitogen-induced cell proliferation and immunoglobulin synthe
sis of cultured normal human lymphocytes. Ehrke et al. (28)
demonstrated that the in vivo administration of DFMO to mice
significantly reduced the cytotoxic potential and antibody pro
duction of cells harvested from these animals. Bowlin et al. (29)
reported that diacetylputrescine potentiated tumor immunity.
Furthermore, 3 ODC inhibitors were shown to potentiate spe
cific tumoricidal effector cell generation in vivo (32). The find
ings in this paper confirm and extend these initial observations
on the immunosuppressive properties of DFMO and related
compounds.
Although one might argue that the impaired immune re
sponse following incubation with DFMO reflects the elimina
tion of a critical subpopulation of lymphoid cells (viability after
exposure to DFMO was 78-85%), we think this unlikely inas
much as the population incubated with medium alone exhibited
similar viability. Furthermore, the inhibitory effects were re
versed by the addition of putrescine.
Recent studies have also suggested that polyamines play a
significant role in the lymphocyte response of autoimmunity
(31, 32). Thomas and Messner (33) reported a beneficial effect
(0
4-
oc
2-1
ir
m 1oc
—¿o10.0
15.0
20.0
5-1
LU
O
4H
3-
O
2Vi
LU
OC
O
010.0
15.0
20.0
ELUTION TIME (min)
Fig. 4. Chromatogram showing internalization of C5 in P815 tumor cell line
and its conversion to an aminopropyl derivative. HPLC analysis of precolumn
derivatimi polyamines was conducted as described in Ref. 24. P815 cells were
cultured for 48 h with either C5 (A) or C5 + DFMO (B). a, putrescine; *, C5; c,
C6; d, spermidine; e, APS (aminopropyl derivative of C5);/ spermine.
1845
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EFFECT OF DFMO ON CELL GROWTH AND IMMUNE RESPONSE
homologues are effective in preventing the growth-inhibitory
effects of DFMO on normal lymphocytes. These homologues
are, however, ineffective in preventing the effects of DFMO on
tumor cell lines. In contrast, putrescine and its 5-carbon hom
ologue are efficacious in preventing the effects of DFMO on
tumor cells, but the 5-carbon homologue cannot significantly
prevent the effect of DFMO on normal lymphocytes. This
differential effect may have important therapeutic implications.
HPLC analysis shows that putrescine homologues are rapidly
internalized in both normal lymphocytes and tumor cell lines.
These homologues are converted to their aminopropyl deriva
tives, as reported earlier by Porter and Bergeron ( 18). Polyamine data presented in Tables 5 and 7 suggest that putrescine
homologues and/or their aminopropyl derivatives, rather than
putrescine or spermidine, are responsible for reversing the
growth-inhibitory effects of DFMO as putrescine and spermi
dine levels are very low in cells treated with DFMO and
putrescine homologues (C3, C5, or C6).
The use of a chemotherapeutic or immunosuppressive agent
is often limited by the adverse effect of the drug on normal
cells. DFMO has been studied as an anticancer agent in tissue
culture, animals, and humans (15-17). It has been reported that
DFMO inhibited the growth of a human leukemia line, 1.1210
(18). Interestingly, the DFMO-mediated
inhibition of cell
growth in this cell line was reversed by the addition of putrescine
and was partially reversed by the addition of 5- and 6-carbon
homologues of putrescine and spermidine. This observation,
along with those reported above, suggests that malignant cells
and normal lymphocytes may differ with regard to their ability
to utilize polyamine homologues. Therefore, the administration
of selective putrescine homologues would allow for the preven
tion of the inhibitory effects of DFMO on normal cells, while
allowing DFMO to continue to suppress malignant cell growth.
Despite extensive research on polyamines during the past
several years, the specific role of these compounds in cell growth
is not precisely known. It is clear that they bind to and stabilize
DNA, RNA, and proteins such as estrogen receptors (10, 3538). Under physiological ionic conditions, polyamines are pos
itively charged; hence electrostatic interaction between poly
amines and the negatively charged regions of macromolecules,
including the DNA phosphate backbone, is a possible mode of
their action. Thus, Manning's counterion condensation theory
(39), based on small cation-polyelectrolyte anión interactions,
was utilized to explain the condensation and aggregation of
DNA in the presence of polyamines (40). However, electrostatic
interactions could not adequately explain all DNA-polyamine
interactions. When a series of spermidine and spermine hom
ologues were evaluated with regard to their ability to convert
right-handed B-DNA to left-handed Z-DNA, it became appar
ent that chemical structure of polyamines significantly influ
enced their ability to provoke conformational alterations of
DNA (41). A comparison of the results of physical chemical
(41 ) and tissue culture experiments (18) showed that the efficacy
of a given spermidine homologue in modulating DNA confor
mational transition correlated well with the ability of the com
pound to reverse the growth-inhibitory effects of DFMO on
LI210 leukemic cells. Further studies by Basu et al. (42, 43)
showed a correlation of structural and functional properties of
spermine homologues and derivatives. In light of these reports,
it is tempting to speculate that polyamine-DNA interactions
might be involved in the effects of putrescine- and diaminopentane-induced reversal of the growth of DFMO-treated cancer
cells. Since the short carbon chain homologues, including eth-
ylenediamine (C2), reversed the effects of DFMO on normal
lymphocytes, it may be that electrostatic effects predominate in
this setting.
Our finding of differential effects of putrescine homologues
on normal and malignant cells suggests different pathways for
the utilization of polyamines in different cell types, and perhaps
in different subpopulations of the same cell type. The situation
is further complicated by differential uptake and metabolism of
polyamines by normal and malignant cells. Membrane perme
ability of polyamines is higher in tumor cells compared to
normal cells (44, 45). In addition, DFMO treatment is known
to facilitate putrescine uptake in tumor cells (46, 47). Thus
differences in the uptake of putrescine and its homologues by
normal and tumor cells may contribute to the differential effects
observed in this study although it may be that several discreet
mechanisms are involved. Certainly, a direct effect of DFMO
is apparent on tumor cells grown in vitro. However, ODC
inhibition by a-(fluoromethyl)dehydroornithine
methyl ester
has resulted in enhanced macrophage-mediated tumoricidal
activity (29). Natural killer cell activity is also affected by
polyamines or their homologues (30).
In summary, the data presented demonstrate that DFMO is
effective in suppressing both normal immune response and
tumor cell growth in vitro. In all instances, this suppression is
markedly reversed by the addition of exogenous putrescine.
However, normal cells and tumor cells exhibit striking differ
ences with regard to their response to putrescine homologues.
The inhibition by DFMO of normal lymphocyte response is
overcome only by short carbon chain homologues whereas the
only homologue capable of preventing the effect of DFMO on
tumor cells is C5. This observation may have clinical relevance
in that the administration of short chain homologues may allow
DFMO to effectively combat tumor cell growth, leaving normal
immune response intact. Use of this therapeutic strategy may
limit immunosuppression, and the incidence of opportunistic
infection, in recipients of DFMO.
ACKNOWLEDGMENTS
We are indebted to Laurence J. Marlon, M.D., Professor and Chair
man, Department of Laboratory Medicine, University of California at
San Francisco; Warren Lubic of the Brain Tumor Research Center,
University of California at San Francisco; and Ratna Seethala and Urna
B. Gunnia of the Robert Wood Johnson Medical School for their help
in polyamine assay. We also thank Dr. Peter P. McCann of the Merrell
Dow Research Institute for the generous supply of DFMO for our
studies.
REFERENCES
1. Tabor, C. W., and Tabor, H. Polyamines. Annu. Rev. Biochem., S3: 749790, 1984.
2. Pegg, A. E. Recent advances in the biochemistry of polyamines in eukaryotes.
Biochem. J., 234: 249-262, 1986.
3. Janne. J., Pösö,
H., and Raina. A. Polyamines in rapid growth and cancer.
Biochim. Biophys. Acta, 473: 241-293, 1978.
4. Takami, H., Romsdahl, M. M., and Nishioka, K. Polyamines in blood cells
as a cancer marker. Letter. Lancet, 2: 912-914, 1979.
5. Caldarera, C. M.. Barbiroli, H . and Moruzzi, G. Polyamines and nucleic
acids during development of the chick embryo. Biochem. J., 97:84-88, 1965.
6. Russell, D. H., and Durie, B. G. M. Polyamines as biochemical markers of
normal and malignant growth. Prog. Cancer Res. Ther., 8: 157-165, 1978.
7. Tierney, D. F., Marlon, L. J., Hacker, A. D., and Lowe, N. Polyamines in
clinical disorders. West. J. Med., 142: 63-73, 1985.
8. Puri. H., Campbell. R. A., Puri, A., Harner, V. M. H., Talwalker, Y. B.,
Musgrave, J. E., Bartos. M., Bartos, F. D., and Loggan, B. Serum-free
polyamines in children with systemic lupus erythematosus. Adv. Polyamine
Res.. 2: 359-367. 1978.
9. Pegg, A. E., and Williams-Ashman, H. G. Biosynthesis of putrescine. In: D.
1846
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1992 American Association for Cancer Research.
EFFECT OF DFMO ON CELL GROWTH AND IMMUNE RESPONSE
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
R. Morris and I . J. Marlon (als.). Poly-amines in Biology and Medicine, pp.
3-42. New York: Marcel Dekker, Inc., 1979.
Thomas, T. J., and Bloomfield, V. A. Ionic and structural effects on the
thermal helix-coil transition of DNA complexed with natural and synthetic
polyamines. Biopolymers, 23: 1295-1306, 1984.
Gosule, L. ('.. and Si-hellnum. J. A. DNA condensation with polyamines. I.
Spectroscopic studies. J. Mol. Biol., ¡21:311-327, 1978.
Behe, M., and Felsenfeld, G. Effects of methylation on a synthetic polynucleotide: the B-Z transition in poly(dG-m!dC).poly(dG-m!dC). Proc. Nati.
Acad. Sci. USA, 78: 1619-1623, 1981.
Metcalf, B. W., Bey, P., Danxin, C, Jung, M. J., Casara, P., and Vevert, J.
P. Catalytic irreversible inhibition of mammalian ornithine decarboxylase
(E. C. 4.1.1.17) by substrate and product analogues. J. Am. Chem. Soc., 100:
2551-2553, 1978.
McCann, P. P., Pegg, A. E., and Sjoersdma, A. (eds.). Inhibition of Polyamine
Metabolism. Biological Significance and Basis for New Therapies. New York:
Academic Press, 1987.
Prakash, N. J., Schechter, P. J., Grove, J., and Koch-Weser, J. Effect of adifluoromethylornithine, an enzyme activated irreversible inhibitor of orni
thine decarboxylase, on L1210 leukemia in mice. Cancer Res., 38: 30593062, 1978.
Mamont, P. S., Duchesne, M. C., Grove, J., and Bey, P. Antiproliferative
properties of OL-a-difluoromethylornithine in cultured cells. A consequence
of irreversible inhibition of ornithine decarboxylase. Biochem. Biophys. Res.
Commun., 81: 58-66, 1978.
Levin, V. A., Chamberlain, M. C., Prados, M. D., Choucair, A. K., Berger,
M. S., Silver, P., Seager, M., Gutin, P. H., Davis, R. L., and Wilson, C. B.
Phase III study of eflornithine and methylglyoxal-bis(guanylhydrazone)
combined in the treatment of recurrent primary brain tumors. Cancer Treat.
Rep., 71: 459-464, 1987.
Porter, C. W., and Bergeron, R. J. Spermidine requirement for cell prolifer
ation in eukaryotic cells: structural specificity and quantitation. Science
(Washington DC), 219: 1083-1085, 1983.
Kiessling, R., Klein, E., and Wigzell, H. "Natural" killer cells in the mouse.
I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Speci
ficity and distribution according to serotype. Eur. J. Immunol., 5: 112-117,
1975.
Dunn, T. B., and Potter, M. A transplantable mast-cell neoplasm in the
mouse. J. Nat. Cancer Inst., 18: 587-601, 1957.
Ozato, K., and Sachs, D. H. Monoclonal antibodies to mouse MHC antigens.
III. Hybridoma antibodies reacting to the H-2b haplotype reveal genetic
control of isotype expression. J. Immunol., 126: 317-321, 1981.
Lozzio, C. B., and Lozzio, B. B. Human chronic myelogenous leukemia cellline with positive Philadelphia chromosome. Blood, 45: 321-334, 1975.
Levy, R. B., and Shearer, G. M. Regulation of T-cell-mediated lympholysis
by the murine major histocompatibility complex. J. Exp. Med., 149: 13791392, 1979.
Kabra, P. M., Lee, H. K., Lubich, W. P., and Marton, L. J. Solid phase
extraction and determination of dansyl derivatives of unconjugated and
acetylated polyamines by reversed phase liquid chromatography: improved
separation systems for polyamines in cerebrospinal fluid, urine, and tissue.
J. Chromatog. Biomed. Appi., 380: 19-32, 1986.
Bowlin, T. L., McKown, B. J., Davis, G. F., and Sunkara, P. S. Effect of
polyamine depletion in vivo by DL-a-difluoromethylornithine on functionally
distinct populations of tumoricidal effector cells in normal and tumor bearing
mice. Cancer Res., 46: 5494-5498, 1986.
Pegg, A. E. Polyamine metabolism and its importance in neoplastic growth
and as a target for chemotherapy. Cancer Res., 48: 759-774, 1988.
Pasquali, J. L., Urlacher, A., Weryha, A., Storck, D., and Mamont, P. S.
Inhibition by Di.-a-difluoromethylornithine of pokeweed-mitogen-induced
immunoglobulin production in cultured human lymphocytes. Immunopharmacology, 7: 145-149, 1984.
Ehrke, M. J., Porter, C. W., Eppolito, C., and Mihich, E. Selective modula
tion by a-difluoromethylornithine of T-lymphocyte and antibody mediated
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
cytotoxic responses to mouse tumor allografts. Cancer Res., 46: 2798-2803,
1986.
Bowlin, T. L., Rosenberger, A., Stemerick, D., and Edwards, M. L. Potentiation of natural killer cell activity and tumor immunity by diacetylputrescine.
Cancer Res., SO: 5460-5463, 1990.
Bowlin, T. L., Hoeper, B. J., Rosenberger, A. L., Davis, G. F., and Sunkara,
P. S. Effects of three irreversible inhibitors of ornithine decarboxylase on
macrophage-mediated tumoricidal activity and antitumor activity in B16F,
tumor-bearing mice. Cancer Res.. 50: 4510-4514, 1990.
Proctor, M. S., Fletcher, H. V., Jr., , Shukla, J. B., and Rennert, O. M.
Elevated spermidine and spermine levels in the blood of psoriasis patients.
J. Invest. Dermatol., 65: 409-411, 1975.
Flescher, E., Bowlin, T. L., Ballester, A., Houk, R., and Talal, N. Increased
polyamines may downregulate interleukin 2 production in rheumatoid ar
thritis. J. Clin. Invest., 83: 1356-1362, 1989.
Thomas, T. J., and Messner, R. P. Beneficial effects of a polyamine biosyn
thesis inhibitor on lupus in MRL-lpr/lpr mice. Clin. Exp. Immunol., 78:
239-244,1989.
Claverie, N., Pasquali, J. L., Mamont, P. S., Danzin, C., Weil-Bousson, M.,
and Siat, M. Immunosuppressive effects of (2Ä,5Ä)-6-heptyne-2,5-diamine,
an inhibitor of polyamine synthesis. II. Beneficial effects on the development
of lupus-like disease in MRL-lpr/lpr mice. Clin. Exp. Immunol., 72: 293298, 1988.
Feuerstein, B. G., and Marton, L. J. Specificity and binding of polyamine/
nucleic acid interactions. In: U. Bachrach and Y. Heimer (eds.). The Physi
ology of Polyamines, Vol. 1, pp. 109-124. Boca Raton, FL: CRC Press,
1989.
Sakai, T. T., and Cohen, S. S. Effects of polyamines on the structure and
reactivity of tRNA. Prog. Nucleic Acid Res. Mol. Biol., 17: 15-42, 1976.
Morgan, J. E., Calkins, C. C., and Matthews, H. R. Discovery and mapping
of discrete binding sites on nucleosome core particles for a photoaffmity
derivative of spermine. Biochemistry', 28: 5095-5106, 1989.
Thomas, T., and Kiang, D. T. Structural alterations and stabilization of
rabbit uterine estrogen receptor by natural polyamines. Cancer Res., 47:
1799-1804, 1987.
Manning, G. S. The molecular theory of polyelectrolyte solutions with
applications to the electrostatic properties of polynucleotides. Q. Rev. Bio
phys., //: 179-246, 1978.
Wilson, R. W., Rau, D. C., and Bloomfield, V. A. Comparison of polyelec
trolyte theories of the binding of cations to DNA. Biophys. J., 30: 317-325,
1980.
Thomas, T. J., and Messner, R. P. Structural specificity of polyamines in
left-handed Z-DNA formation. Immunological and spectroscopic studies. J.
Mol. Biol., 201: 463-467, 1988.
Basu, H. S., Feuerstein, B. G., Deen, D. F., Lubich, W. P., Bergeron, R. J.,
Samejima, K., and Marton, L. J. Correlation between the effects of polyamine
analogs on DNA conformation and cell growth. Cancer Res., 49:5591-5597,
1989.
Basu, H. S., Pellarin, M., Feuerstein, B. G., Deen, D. F.. Bergeron, R. J.,
and Marton, L. J. Effect of Wv/V-bisfethyHhomospermine on the growth of
U-87 MG and SF-126 human brain tumor cells. Cancer Res., 50: 31373140, 1990.
Pakala, R., Kreisel, M., and Bachrach, U. Polyamine metabolism and interconversion in NIH 3T3 and ros-transfected NIH 3T3 cells. Cancer Res., 48:
3336-3340,1988.
Fasulo, L., Fulgosi, B., Colombatto, S., and Grillo, M. A. Uptake of poly
amines by human lymphocytes and their effect on láclateformation from
glucose. Adv. Exp. Med. Biol., 250: 509-516, 1988.
Heston, W. D., and Kadmon, D. n-Difluoromethylornithine enhancement of
"C-putrescine uptake by an androgen-dependent prostatic tumor. J. Urol..
136:944-948, 1986.
Maddox, A. M., Orengo, I., and Haddox, M. K. Difluoromethylornithine
enhances the uptake of methylglyoxal-bis(guanylhydrazone) prior to inhibit
ing leukemic cell proliferation. Chemotherapy, 33: 110-122, 1987.
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Differential Effects of Polyamine Homologues on the Prevention
of dl- α-Difluoromethylornithine-mediated Inhibition of
Malignant Cell Growth and Normal Immune Response
Ajay B. Singh, T. J. Thomas, Thresia Thomas, et al.
Cancer Res 1992;52:1840-1847.
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