(CANCER RKSEARCII 50. 38-43. January 1. IOTO]
Enhanced Poly(adenosine diphosphate ribose) Polymerase Activity and Gene
Expression in Ewing's Sarcoma Cells1
Sarada C. Prasad, Peter J. Thraves, Kishor G. Bhatia, Mark E. Smulson, and Anatoly Dritschilo2
Departments of Radiation Medicine ¡S.C. P., P. J. T., A. [>.]and Biochemistry ¡M.E. S.. K. G. B.J, Georgetown I 'niversity Medical Center. Washington. IK' 20007
ABSTRACT
Ewing's sarcoma (ES) is a highly malignant childhood bone tumor and
is considered curable by moderate doses of radiotherapy. The addition of
chemical inhibitors of the activity of the nuclear enzyme poly(adenosine
diphosphate ribose) |poly(ADPR)| polymerase to ES cells in culture
results in increased cell killing, a phenomenon called "inhibitor sensitization." Since poly(ADPR) polymerase is thought to be associated with
DNA repair, it has been suggested that ES cells and other inhibitorsensitized cells may have a reduced capacity for polymer synthesis
resulting in deficient postirradiation recovery. We present here the un
expected observation that in comparison to other cell lines tested, ES
cells exhibit a high enzyme activity, higher constitutive levels of the
protein, and elevated levels of its inKN'A transcript for poly(ADPR)
polymerase. No gross amplifications or rearrangements of the gene were
observed; however, regulation of poly(ADPR) polymerase in these tumor
cells takes place at the level of the gene transcript.
INTRODUCTION
Poly(ADPR)'
polymerase is a chromatin-associated
enzyme
is true that radiation survival parameters measured in vitro
using established cell lines do not always correlate with local
tumor response to fractionated radiotherapy; however, radio
therapy has been proven successful for Ewing's sarcoma (18,
19), in spite of the wide variability in the Da values reported for
various ES cell lines in culture (35-37).
On the basis of these observations, we advanced an initial
hypothesis that ES cells characterized as responsive to radio
therapy (19) and more prone to radiation killing (13) in the
presence of 3AB and BZ would probably show poly(ADPR)
polymerase activity levels to be rate limiting for DNA repair
following radiation. ES cells, therefore, offer a useful in vitro
model system to investigate the biochemical events associated
with tumor cell response to radiation and a possible role of
poly(ADPR) polymerase activity and gene expression.
Here, we report studies on the analysis of this enzyme in five
cell lines of ES in comparison to NHF and cervical carcinoma
(HeLa). We observed that a higher polymerase activity is a
common feature in five cell lines derived from Ewing's sarcoma
in comparison to several other tumor cells tested. We also
demonstrate that such increases in activity of the enzyme are
mediated by constitutively higher amounts of the enzyme pro
tein. The recent development of a cloned complementary DNA
for the polymerase provided an opportunity to test our hypoth
esis at the level of the expression of the poly(ADPR) polymerase
in ES cells.
catalyzing the covalent modification of nuclear proteins by
addition of repeating units of (ADP-ribose) (1). Roles for this
enzyme have been suggested in DNA replication, DNA repair,
RNA synthesis, cell differentiation, cell cycle regulation, DNA
ligase function, chromatin structure, and topoisomerase activi
ties (2-9). The enzyme requires DNA as a cofactor and strand
breaks in DNA act as a stimulus to its activity both in vitro and
in vivo (3, 8). Evidence obtained from two different kinds of
approaches suggests that poly(ADP-ribose) metabolism may be
involved in the expression of the extent of cell killing in re
sponse to ionizing radiation (10-17); (a) studies showing DNA
fragmentation and subsequent rise in poly(ADPR) polymerase
activity as obligatory for DNA repair following treatment of
normal mammalian cells with radiation and alkylating agents;
and (A) use of competitive inhibitors of the polymerase during
exposure of mammalian cells to ionizing radiation resulting in
pronounced radiation sensitization.
Studies from our laboratory indicated that inhibitors of
poly(ADPR) polymerase (3-aminobenzamide) potentiate the
(cytotoxic) damaging effects of ionizing radiation in certain
tumor cells, while others seem unaffected (12, 13). The end
points in our earlier studies were the ratios of the terminal
slopes (A,) of the survival curves in the presence or absence of
inhibitors. Increases in the slopes of the radiation survival
curves, referred to as "sensitization" to radiation, are aug
MATERIALS
AND METHODS
Cell Culture. The cell lines used in the studies included normal
diploid human fibroblasts (NHF7), cervical carcinoma (HeLaS3), and
five lines of human ES cells (A4573, ROES, 5838B, Tel06, Tel77).
HeLa cells were purchased from the American Type Culture Collection,
Rockville, MD: Ewing's sarcoma cells were kindly provided by Dr.
Timothy Kinsella of the National Cancer Institute, Bethesda, MD. The
normal diploid human fibroblasts were established as primary cultures
from newborn foreskin tissue. All cell lines were maintained in Eagle's
minimal essential medium with nonessential amino acids, 10% fetal
bovine serum, 1% L-glutamine, 110 mg/liter sodium pyruvate, and 500
lU/ml penicillin-500 Mg/"1' streptomycin at 37°Cin a humidified
atmosphere of 95% air and 5% CO2. Stock cultures were routinely
tested for Mycoplasma contamination at 3-6-month intervals.
Radiation Survival Curves. Stock cultures in exponential growth were
detached by trypsinization and appropriate cell numbers were plated in
25-cm2 flasks and incubated to permit cell attachment for 6 h. Following
irradiation, refeeding was performed every 3 days and after 10-14 days,
cells were fixed with Bouin's solution and colonies were stained with
mented by poly(ADPR) polymerase inhibitory agents in some
tumor cells. Specifically ES cells are sensitized to radiation
killing while lung carcinoma and osteosarcoma are not (13). It
Giemsa. Only colonies of 50 cells or more were scored as survivors.
Survival fractions were determined from colony count data. Survival
curves were constructed using the single-hit multitarget mode
Received 12/15/88; revised 9/27/89; accepted 10/3/89.
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.
1This work was supported in part by grants from the American Cancer Society
and the NIH.
2 To whom requests for reprints should be addressed.
1The abbreviations used are: poly(ADPR). poly(adenosine diphosphate ribose);
ES, Ewing's sarcoma; 3AB. 3-aminobenzamide: BZ, benzamide; NHF, normal
All experiments were performed in triplicate and the survival data are
expressed as the mean ±SE of three separate survival experiments.
Radiations were performed using a clinical "'Co radiation unit
human fibroblasts. DTT. dithiothreitol: SDS, sodium dodecyl sulfate; PBS.
phosphate-buffered saline; cDNA. complementary DNA.
(Atomic Energy Commission Limited of Canada). Cells were irradiated
at room temperature in air at a distance of 80 cm from the source at a
S= \ -(1 -exp-D|00)"
38
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l'OI.V(ADl'R)
PO1.YMERASE AND KWING'S SARCOMA
dose rate of 1.80 Gy/min. Dose rate measurements were made using a
standardized Victoreen chamber.
Specific Activity of Poly(ADPR) Polymerase in Human Tumor Cells.
Cells in log phase were harvested by trypsinization and washed in a
balanced salt solution prior to use. Activity of poly(ADPR) polymcrase
was determined using [I2P]NAD at 100 MMfinal substrate concentra
Northern Analysis of Human I mum mRNA. Total RNA from the
tumor cells was isolated by guanidine thiocyanate and CsC'l gradient
centrifugation (30). Approximately 20 Mgof RNA from each tumor cell
line were size fractionated by electrophoresis on formaldehyde (2.2 M)agarose (1.0%) gels (31). Following electrophoresis, the resolved RNA
was capillary blotted, without pretreating the gel, onto nylon and
subsequently probed with poly(ADPR) polymerase cDNA (3.7 kilobases) or ¿i-actin(2.0 kilobases). Equal quantities of DNA and RNA
were loaded as determined by absorbance and ethidium bromide stain
ing. The conditions of hybridization and washing in high stringency
buffers were the same for both Southern and Northern blots and as
described (32).
tion. Initial velocity assay (45 s) was performed in sonicated extracts as
described by Cherney el al. (20), while activity in permeabilized cells
(10 min) was carried out according to the method of Berger et al. (2).
NAD levels were determined by the procedure of Bernofsky and Swan
(21).
Acceptor Gel Analysis. Cells, after trypsinization and washing, were
sonicated in 50 mM Tris-HCI (pH 8.0), 0.25 M sucrose. 2 imi MgCI:,
1 mM DTT, and 0.1 mM phenylmethylsulfonyl fluoride. Protein (100
Mgfrom each extract) was incubated with 0.1 ^Ci [12P]NAD (1000 Ci/
mmol) for 5 min at 25°Cfollowed by precipitation with cold ethanol.
The pellets were washed, dried, and electrophoresed on 7.5% SDSpolyacrylamide gels (22). Subsequently, the gels were dried and exposed
to X-ray film.
Polymer Gel Analysis. For analysis of "P-labcled poly(ADP-ribosc)
RESULTS
We have analyzed the steady state levels of poly(ADPR)
polymerase in five ES cell lines and have compared these with
a cervical carcinoma and normal human fibroblasts (Table 1).
Contrary to expectations based upon their in vitro sensitization
by 3AB. the specific activities of the polymerase in either
from human tumor cells, 500 Mgof protein from each extract (prepared
as above) were incubated with 10 ¿iCiof [l:P]NAD (100 MM.final) for
permeabilized ES cells or sonicated ES extracts were approxi
30 min at 25°Cfollowed by precipitation with 10% trichloroacetic acid
mately 10-fold higher than normal human fibroblasts (NHFat 0°Cfor 1 h. The precipitates were washed with cold trichloroacetic
7). A similar comparison of polymerase activity of ES and
acid, suspended in 100 M'of 1.0 M KOH, and heated at 60°Cfor 1 h.
HeLa cells showed a 2-fold increase. We determined the enzyme
After neutralizing, 2 M!of each sample were mixed with 8 n\ of sample
activities by two methods that complement each other. The
buffer (8.0 M urea 1% SDS) and subjected to electrophoresis on 20%
quantitative differences in poly(ADPR) polymerase activity
SDS-urea-polyacrylamide gel (23) with xylene cyanol and bromophenol
measured in the sonicated extracts of cells may be interpreted
blue as markers.
indirectly as an estimation of the number of polymerase mole
Activity Gel Analysis of Poly(ADPR) Polymerase. Exponentially
cules per cell. This increased incorporation of |':P]NAD was
growing human tumor cells were trypsinized, washed in balanced salt
completely inhibited by the presence of 3-aminobenzamide in
solution, and sonicated on ice in 1.5 M NaCl-50 m\i Tris-HCI (pH
all cell lines (data not shown).
7.5), 0.5 mM DTT, I mM EDTA, and 1 MNIpepstatin. Following
There is compelling evidence to suggest that reduction in the
centrifugation at 10,000 x g for 10 min, the supernatant protein was
cellular level of NAD accompanies the increases in poly(ADPR)
assayed by the method of Lowry et al. (24). In situ activity gel analysis
was performed by separating 40 Mgof protein from each cell extract on
polymerase activity (5). Our estimates of the baseline NAD
7.5% SDS-polyacrylamide gels (containing sonicated salmon sperm
levels were inversely related to the polymerase activities (Table
DNAat lOUMg/ml).
1) in ES-1, HeLa, and NHF-7, respectively.
The gels were subsequently icnatural and assayed directly with 10
In order to demonstrate the relationship between the
ml of 100 mM Tris-HCI (pH 8.0), 10 IHMMgCI2, l IHM DTT, l ¡IM poly(ADPR) polymerase activity analysis with that of radioNAD, and 10 ^Ci/ml of [a«/«ii/ie-2,8-"P]NAD(1000 Ci/mmol) at
biological parameters in Fig. 1, we present radiation dose37°Cfor 16 h (25). The washed gels were exposed to X-ray film at
response curves of the various cell lines used in the present
-70°C.
study. ES-1 and ES-5 show greater sensitivity than ES-3 and
Western Blot Analysis of Human Tumor Cell Kxtracts. Whole cell
ES-4. Cells of ES-2 grew diffusely and were not amenable to
extracts were made by sonicating cell pellets in 0.3 M KC1, 50 mM TrisHCI (pH 7.5), 1 mM EDTA, 1 mM DTT, 2 mM MgCl:, 0.1 mM the clonogenic assay. These survival curves tend to have larger
"shoulder" regions and larger /),>values than early passage ES
phenylmethylsulfonyl fluoride, and 1 MMpepstatin for 6 s (3 times) on
ice (9). Following sonication, the extracts were left on ice for 1 h and
then centrifuged at 10,000 x g for 5 min at 4°C.The protein content
cells reported by Weichselbaum et al. (36), a characteristic we
have seen with prolonged passage in culture. It is important to
note that at lower doses of radiation (<3.0 Gy) all of the four
of each extract was determined by the assay of Lowry et al. (24).
Western blot reactivity of tumor cell extracts was carried out accord
ing to the procedure of Towbin et al. (26). Briefly, 100 Mgof protein
were electrophoresed on denaturing polyacrylamide (7.5%) gels and
electrophoretically transferred onto nylon paper. Following blocking of
the unoccupied sites on the membrane overnight at 25°Cwith PBS
Table 1 Specific activities ofpolyf.-iDI'R) polymcrase and cellular NAD levels in
human tumor cells in culture
Human tumor cells in logphase were harvested by trypsinization and washed
prior to use. Activity assays for poly(ADPR) polymerase were performed in
sonicated extracts (20) and permeabili/ed cells (2). NAD determinations were
done according to the method of Bernofsky and Swan (21 ). Data points are means
±SD of three separate determinations. The experimental codes assigned to the
cell lines «ereadhered to throughout the paper.
containing 5% casein, the membrane was incubated with polyclonal
antiserum to poly(ADPR) polymerase (1:200 diluted in PBS with 1%
casein) for 3 h at 25°C.The filter was then washed in PBS containing
0.5% Nonidet P-40 followed by incubation with 125l-protein A at 0.1
MCi/ml (68 MCi/Mg)for 1.5 h at 25°C.After extensive washing, the
nylon membrane was exposed to .X-ray film.
Restriction Enzyme Analysis and Southern Blot Hybridization. Hu
man tumor DNA was isolated by the method of Blin and Stafford (27).
Tumor DNAs (10-20 Mg)were digested with EcoRI (5 units/Mg DNA)
for 3 h followed by electrophoresis in an 0.8% agarose gel in Ix TAE
(0.04 M Tris-acétate,0.001 M EDTA) buffer. The digests were then
blotted according to the method of Southern (28) and probed with a
cDNA for poly(ADP-ribose) polymerase labeled and with ["P]dCTP
Foly(ADPR) polymerase ac
tivity (pmol ADP-ribose)/
min/mg protein
CellsNormal
eel
codeNHF7HeLa-S3ES-1ES-2ES-3ES-4ES-5Permeabilized
cells249
extracts149 protein)3984260013172637126
fibroblastsCervical
carcinomaEwing's
sarcomaA4573RD-ES5838BTCI
77TCI
06Experimenta
by the random primer method of Feinberg and Vogelstein (29). Human
placenta! DNA was used as control DNA.
89944
±
1674801
±
13749
±
401408
±
9242098
±
14311±
1441957
±
±59I688±
15
±6741901
761308
2522395
±
611557
±
±201Sonicated ±54(pmol/mg±69
39
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POLY(ADPR) POLYMERASE AND EWING'S SARCOMA
A
B
,
-I LU T I I I I
LU I CO CO CO CO CO
X Z LU LU LU LU LU
2345678
Radiation Dose (Gy)
-xc
234567
Radiation Dose (Gy)
Fig. 1. Effect of radiation dose on the cell survival. A, normal human fibroblasts (NHF-7) and cervical carcinoma (HeLa S-3); B, ES cells. Points, means;
bars, SE
*-BPB
ES cell lines studied appear to be more resistant than either
NHF or HeLa. In spite of such apparent heterogeneity in the
radiosensitivities among ES cell lines, our data (13) using the
same cell lines showed that the ES and NHF are inhibitor
sensitized to an equal extent by 3AB and BZ.
The enhancement of ADP ribosylation of proteins in ES cells
may be directed to all cellular acceptors in general or to an
increased modification of one of the several major acceptors
[e.g., histones, high mobility group proteins, topoisomerases,
or poly(ADPR) polymerase itself]. Accordingly, we compared
the acceptor proteins for ADP-ribose in the cell extracts by
electrophoresis and found that most of the incorporated ["P]
Fig. 2. Poly(ADP-ribosylation) in human tumor cells. Tumor cell extracts
were prelabeled with ("P|NAD in vitro and subjected to (A) acceptor gel and (B)
polymer gel analysis. Procedures for the preparation of cellular extracts, labeling
of acceptor proteins, and subsequent gel electrophoresis were as described in
"Materials and Methods." I. autoradiographic resolution of cellular acceptor
proteins on a 7.5% polyacrylamidc gel (22). 113 Marker, position of poly(ADPR)
polymerase as a major acceptor of poly(ADP-ribose). kDa, molecular weight in
thousands. B, autoradiogram of a 20% SDS-urea-polyacrylamide gel (2.1) with
"P-labeled polymers of varying lengths in the cell lines. Xylene cyanol (AT) and
bromophenol blue (BPB) were used as markers.
NAD is associated with a M, 113,000 protein representing the
automodified polymerase (Fig. 2A) with no additional acceptors
with a molecular size of >113,000. The levels of [12P]NAD
incorporated into the automodified polymerase (Fig. 2A) reflect
the enzyme activity differences represented in Table 1 with the
basal activity in NHF-7 being very low in comparison to HeLa
and ES-1. The other acceptors were observed to form only a
minor component of the activity measured. Furthermore, an
examination by SDS-urea-polyacrylamide gel electrophoresis
of the relative capacities of the cells to incorporate [32P]NAD
into ADP-ribose polymer in ES-1, NHF-7, and HeLa cells,
indicated only quantitative differences (Fig. 2B). Consequently,
the various species of polymer found on protein were the same,
indicating that each cell type had the capacity to synthesize the
same types of polymer. Several lines of evidence indirectly
suggest a role for poly(ADPR) polymerase in DNA repair (24, 14-16), in particular, excision repair. The activity of the
polymerase is stimulated by DNA damage resulting from treat
ment of cells with ionizing radiation as well as with other DNAdamaging agents (14). However, we failed to detect any further
change in polymer chain size or new acceptor proteins following
a test dose of radiation (up to 10.0 Gy) in ES cells as compared
to its steady state levels (data not shown).
In ES-1 cells, the significantly elevated levels of polymerase
activity noted in Table 1 might be a result of: (a) altered kinetic
parameters of the enzyme; and/or (b) an increase in the number
of enzyme molecules per se in the cell. Determination of kinetic
constants in crude cellular extracts indicated an apparent Kmof
54 UMfor HeLa cells and one of 21 p\\ for ES-1 cells. In view
of the large number of nuclear proteins which act as acceptors
of the ADP-ribose moiety from NAD, the significance of an
enzyme with an apparent low Km as in ES cells is not clear. It
possibly reflects on the regulation of the poly(ADP ribosylation)
process itself, namely the choice of acceptor proteins and/or
the extent of modification. In order to determine if ES cells
have constitutively higher amounts of polymerase, we carried
out the enzyme activity analysis //; situ by its localization in
polyacrylamide gel (Fig. 3/1) and compared it with the meas
urement of immunoreactive polymerase protein present by
Western analysis (Fig. 3Ä).In all of the cell lines tested, the
amount of the automodified polymerase observed in the "activ
ity gel" (Fig. 3/4) corresponded to the antibody-positive enzyme
detected in the Western blot (Fig. 3B). Also, these analyses are
in agreement with enzyme activity data presented in Table 1
indicating that there are higher numbers of polymerase mole
cules per cell in ES as compared to other cells.
These observations suggest either that ES cells express the
gene at a higher level or that the mRNA is more stable. The
recent cloning and sequencing of the cDN A for the poly(ADPR)
polymerase (33, 34) permitted us to study the genomic events
associated with poly(ADP-ribose) metabolism in these cells.
Based on the molecular weight of the complete protein the
expected size of the cDNA for the polymerase was 3.7 kilobases.
Using RNA gel blot analysis and subsequent immunoprecipitation of the translated product one of us (33, 34) has confirmed
that the 3.7-kilobase cDNA represents the complete coding
sequence for the enzyme. Restriction enzyme digestions of the
genomic DNA from the four ES lines, HeLa, NHF-7, and
human placenta with £coRI (//««/IIIand ///'/;</!11, data not
shown) and subsequent Southern-blotting and hybridization
with the "P-labeled cDNA, revealed no gross rearrangements
or amplifications of the gene (Fig. 4A). The slot-blot analysis
of sequentially diluted DNA samples from the same cell lines
40
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POLV(ADPR) POLYMERASE AND EWING'S SARCOMA
I
Z
*-
CM
CO
LU
CO
111
co
I
CO
HI
•¿Â«t
io
I
I
co
co
LU
LU
kDa
-
113
T-
U.
C\J
I
I
confirmed that there was no gross amplification of gene. We
also have demonstrated that the transcriptionally
active
poly(ADPR) polymerase gene is located on chromosome 1 and
its pseudogene counterparts on chromosomes 13 and 14 (33,
34). In the present study karyotypic analysis suggested chro
mosome 1 trisomy as a common feature of the four ES lines
(data not shown) among other chromosomal abnormalities.
To determine if the observed enhancement in enzyme activi
ties and protein levels corresponded to an elevated expression
of the coding gene, total cellular RNA from NHF-7, HeLa, and
ES-1 cells was subjected to Northern analysis (Fig. 40). Densitometric scanning of the autoradiograms revealed a greater
than 5- and 2-fold increase in the levels of the polymerase
mRNA in the representative ES cell line (Fig. 4B) in compari
son to NHF-7 and HeLa, respectively. When the same Northern
blot was hybridized with the cDNA probe for actin, the 2kilobase transcript is comparable in HeLa and ES-1 while it
appears to be overexpressed in NHF-7. In addition, experiments
which assessed the effects of actinomycin D on the inhibition
of transcription of poly(ADPR) polymerase gene suggested a
significant increase in the stability of the mRNA in Ewing cells
compared to controls.4
I
CO
CO
ni
uj
co
i
co
UJ
•¿^-in
co
tu
co
m
kDa
-113
DISCUSSION
Fig. 3. Activity gel (/<) and Western blot (A) analysis of human tumor cell
extracts. kDa. molecular weight in thousands. In A, 40 <*gof protein from
sonicated cellular extracts were electrophorescd on a 7.5% polyacrylamidc gel
and assayed for activity in situ as described by Scovassi et al. (25). In B, 100 >jg
of protein from 0.3 M KO extracts of the cell lines were subjected to electrophoresis and Western blotting (26). Conditions applied for electrophoretic separation
transfer to nylon membrane and subsequent reaction with poly(ADPR) polymerase specific antibody were described previously (9). The cell lines used are
represented by the same experimental code numbers assigned in Table I. In the
case of Fig. 3, both A and B, ¡13markers indicate mobility of homogeneous
poly(ADPR) polymerase.
<
*- CM Tf U5
Q U. _i l l I I
0. I UJ CO CO CO CO
X
Kb
25-
2
X
LU LU LU LU
B
u.
I UJ CO
Z
I
LU
Kb
-3.7
8.27/7.35.3-2.0
2.3Fig. 4. Southern (/4) and Northern (B) blot hybridization analysis of
poly(ADPR) polymerase in various cell types. A, Southern blot hybridization of
human tumor DNAs digested with EcoRl and probed with "P-labeled
poly(ADPR) polymerase cDNA. DNA (10-20 Mgfrom each cell line) was used
for restriction digestion and subsequent agarose gel clcctrophoresis. Ethidium
bromide-stained gel indicated that the amounts of DNA loaded on the gel were
comparable. B, comparison of the expression of poly(ADPR) polymerase gene by
Northern hybridization of 20 jig of total cellular RNA from the various cell lines
as indicated (29-32). The probes used were 12P-labeled poly(ADPR) polymerase
cDNA [3.7 kilobases (kb)] or rf-actin (2.0 kilobases|. The conditions of hybridi
zation and washing in high stringency buffers are the same for both the DNA and
RNA blots. Relative molecular sizes of the bands are indicated in the margins (in
kilobases).
Evidence obtained from in vitro cell survival studies suggests
that ES cells are more sensitive to ionizing radiation than are
other tumor cells using several radiobiological parameters for
comparison (35-37). Furthermore, the effects of inhibitors of
poly(ADPR) polymerase in enhancing the cytotoxic effects of
radiation in ES cells have pointed to an interplay of events
associated with their DNA damage accumulation and repair
capacities. Additionally, the success of radiotherapeutic man
agement for local control of this sarcoma in vivo suggests a
relationship to the radiosensitivity of the tumor cells. If the
degree of radiosensitization caused by agents like 3AB and BZ
reflects the capacity of human tumors for ADP ribosylation in
response to ionizing radiation, one may advance an initial
hypothesis that the more radiosensitive cell lines may have a
lower polymerase activity which is rate limiting for DNA repair
and may in turn be sensitized in the presence of inhibitors.
However, our present studies on the intrinsic polymerase activ
ity show that several lines of ES cells have a higher polymerase
activity than do normal fibroblasts. Yet, both cell lines are
sensitized by inhibitors of the polymerase enzyme. These data
are not consistent with the hypothesis that poly(ADP-ribose)
metabolism is rate limiting for the repair of DNA damage in
these tumor cell lines. In addition, our observations with the in
situ activity gel and Western analysis demonstrate that there
are higher levels of polymerase enzyme in the A4573, as com
pared to HeLa-S3 and NHF-7 cell, complementing the data
obtained by activity analysis. These enzyme levels are at least
in part, if not totally, responsible for higher polymerase activi
ties observed. Restriction enzyme analysis of the DNA of these
tumor cells showed no gross amplifications or rearrangement
of the poly(ADPR) polymerase gene in any of the cell lines
irrespective of either their intrinsic radiosensitivity or response
to the inhibitors of polymerase following irradiation.
The analysis of the RNA species demonstrates a higher level
of the polymerase mRNA in the Ewing's sarcoma (A4573), and
cervical carcinoma (HeLa-S3), than in human fibroblasts
(NHF-7). Although these observations do not correlate either
4 Unpublished data.
41
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POLY(ADPR) POLYMERASE AND EWING'S SARCOMA
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with the radiosensitivity or the response to inhibitors of
poly(ADPR) polymerase in these cells, they do indicate that
the polymerase activity of a cell in the absence of DNA damage
is regulated at the transcriptional level. Studies are presently in
progress to determine whether these higher steady state titers
of poly(ADP-ribose) polymerase mRNA are due to an increased
expression or an increased stability of the gene. In addition, an
understanding of the modulation of poly(ADPR) polymerase
activity and expression in conjunction with postradiation events
that lead to DNA repair and cell survival may be needed.
Analysis of poly(ADPR) polymerase activity in various or
gans and tissues by Gill (38) has shown that the highest activi
ties are to be seen in bone marrow, thymus, and spleen. It is of
note that the origin of Ewing's sarcoma, a highly malignant,
pediatrie bone tumor, has been recently placed in the most
undifferentiated position of a family spectrum of neural tumors
(39). Using biochemical and immunohistochemical markers,
Lipinski et al. (40) have shown the presence of neuroectodermal
markers in certain Ewing's tumor cells. Four of the five ES cell
lines used in the present study have demonstrated the potential
to undergo marked neural differentiation in vitro (40). Hence,
it may be of significance that studies of Ikai et al. (41) using
skin cells and granulocytes (42) have suggested that terminal
differentiation appears to be associated with a loss of poly(ADPribose)-synthesizing capacity. In this context, it may be noted
that agents such as phytohemagglutinin are reported to give
rise to a similar enhancement in expression of poIy(ADPR)
polymerase gene in T-lymphocytes with regulatory events as
sociated with the translational process (43). Our findings are
compatible with the postulated primitive and undifferentiated
nature of ES cells. Future studies will follow the course of the
poly(ADP ribosylation) process in the presence of specific
agents that may cause differentiation in ES.
In evaluating the initial hypothesis, it appears that the radi
osensitivity of ES cells may be indirectly related to an excess,
rather than limitation of poly(ADPR) polymerase activity. We
also recognize the potential clinical relevance of the high
poly(ADPR) polymerase activity, both in the histological diag
nosis and treatment of ES. In addition, there are several chem
ical inhibitors of the polymerase activity which may be tested
to enhance the clinical therapeutic ratio (44)
ACKNOWLEDGMENTS
We thank Dr. Preston Hensley for assistance in the determination
of kinetic constants of the enzyme and Sandra Hawkins for help in
preparation of the manuscript. The expert technical assistance of Jane
Boyle is also gratefully acknowledged.
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43
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Enhanced Poly(adenosine diphosphate ribose) Polymerase
Activity and Gene Expression in Ewing's Sarcoma Cells
Sarada C. Prasad, Peter J. Thraves, Kishor G. Bhatia, et al.
Cancer Res 1990;50:38-43.
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