Actu Oncologicu Vol. 35, No. 7, pp.

841-847, 1996

DNA DAMAGE INDUCTION BY '251-ESTROGEN

LINDAS. YASUI,ALUN HUGHESand EUGENER. DESOMBRE

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DNA damage induced by the radioactive decay of 1251-estrogen('"I-VME2) in an estrogen receptor

expressing CHO cell line, CHO-ER, was measured. "'I-VME2 targeted "'I atoms proximal to DNA
estrogen response elements (EREs). ''I decays were accumulated at - 135"C, and thereafter assayed
by alkaline and neutral filter elution techniques to measure DNA single strand break (ssb) and double
strand break (dsb) induction respectively. Increasing DNA damage (both ssbs and dsbs) was detected
after exposure of cells to increasing concentrations of "'I-VME2. DNA ssb and dsb dose-response
curves for "'I-VME2 were multiphasic. The rates of DNA damage induction by the decay of
12'I-VME2 was determined by comparing slopes of all data or by comparing initial slopes. DNA ssb
induction per 12'I-VME2 decay was approximately 2 times greater compared with DNA dsb induction.
1251-VME2decay induced approximately 4-8 times more DNA dsbs than 12'1UdR decay.

Endocrine therapy has been used in the treatment of

breast cancer for nearly a century ( I ) . Breast cancers
expressing estrogen receptors (ER) are responsive to endocrine therapy but may recur after initial remission.
Other ER-containing cancers, such as ovarian carcinoma,
show little response to endocrine therapies. Current endocrine therapies are only effective on hormone-dependent
cancer cells. However, ER-expressing cancers may also
synthesize other growth factors that circumvent the hormone-dependent growth controls, especially under the selective pressure of endocrine therapy (2). We are
investigating the potential for delivering a cytotoxic dose
of radiation (using the decay of "'I emitting Auger electrons) to the cell nucleus of cancers expressing nuclear
ERs. Such therapy would only require ER to deliver an

Received 7 September 1995.

Accepted 29 February 1996.
From the Northern Illinois University, Department of Biological
Sciences, DeKalb (L.S. Yasui) and Ben May Institute. University
of Chicago, Chicago ( A . Hughes, E. R . DeSombre), 11, USA.
Correspondence to: Linda Yasui, Northern Illinois Universiy,
Department of Biological Sciences, DeKalb, Illinois 601 15.
e-mail: lyasui@niu.edu
FAX: (815) 753-0461
Paper presented at the Third International Symposium on Biophysical Aspects of Auger Processes, August 24-25, 1995, Lund,
Sweden.

p Scandinavian University Press 1996. ISSN 0284-1 86X

"51-ER hgdnd in close proximity to DNA for a sufficient

time for the decay of the nuclide.
'251 decay emits low energy Auger electrons. The range
of emitted Auger electrons is short, but the density of
energy deposition is high. Thus. Auger electrons from 'l5I
decay produce an extremely dense and highly localized
pattern of energy depostion, resulting in cytotoxicity (for
example see (3)). ER-bound "51-ligands should effectively
target and kill cells expressing ER (4).
ER-dependent radiotoxicity has been reported previously using 16a-[ '251]iodoestradiol in cultured MCF-7
breast cancer both when cells were frozen to accumulate
decays ( 5 ) and when in vitro conditions were controlled to
maximize retention of ligand in cells (6, 7). In the present
study we investigated DNA ssb and dsb induction to
provide a basis for understanding the cytotoxic effects of
'251 decay.

Material and Methods

Cell lines and mainrenonce

For I2'I-estrogen labeling studies, Chinese hamster

ovary cells transfected with estrogen receptor (CHO-ER)
cDNA prepared from MCF-7 cell mRNA (8) were used.
Molecular characteristics of the CHO-ER cells have been
described (8). The stably transfected CHO-ER cells were

841

842

1 .o

Table 1
Incorporation of '."I-V M E 2 inro CHO-ER cells

I
I1
111
IV

VI
VII

Acra Oncologica 35 ( 1996)

L. S . YASUI ET AL.

'151-VME2
Conc. (nM)

Initial
molecules/
cell

Molecules/
cell after
dilution

Decays/
cell*

5.86
2.76
0.65
1.17
0.22
0.13
0.06

251 163
161 367
52 014
20 892
11 344
4 528
2 363

I20 430
34 140
13 510
12 167
2 042
336
88

28 060.2
8 886.6
3 147.8
2 834.9
475.8
78.3
20.5

0.5

* Decays accumulated after 23 days freezing time when 0.767 '"1

activity remains = ( 0 . 2 3 3molecules/cell
~
after dilution).

0.1

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maintained in monolayer culture, subcultured by trypsinization, suspended in medium, and cell number determined using a Coulter counter as previously described (9).

10

Fraction Number

Svnthesis of E - 1 7 ~ -"'I]iodovinyl[
1 I~-methos~estradiol,
12'1- V M E 2
Synthesis of I2'I-VME2 by iododestannylation of a tributyltin precursor and determination of specific activity by
sedimentation analysis were performed as previously described (9). The specific activity of the Iz5I-VME2 was
78.26 kBq/pmol (2.1 15 pCi/pmol).
Cell labeling and freezing for dose accumulation
Cellular DNA was radiolabeled with I4C-TdR for detection by liquid scintillation counting used in the alkaline
and neutral filter elution assays. I4C-TdR (740 Bq/ml or
0.02 pCi/ml) was added to the culture medium for 24 h
followed by a 2-h incubation in normal medium lacking
'"C-TdR. Cells were then collected by trypsinization. diluted to a concentration of 6 x lo6 cells/ml and incubated
with various concentrations of "'I-VME2 with mixing at
37C for 1 h. Following Iz5I-VME2incubation, unincorporated label was removed by washing in medium without
added estrogen or iodoestrogen by 2 cycles of centrifugation ( 5 min at 500 x g) and resuspension in I0 ml medium
to allow dissociation of low affinity ligand. The final cell
pellet was resuspended in 5 ml freezing medium, containing 10 % glycerol and frozen to - 135C using a constant
freezing rate. Similar incubations were also carried out
with the same concentrations of unlabeled estradiol alone.
or medium alone.
Incorporations of "'I-VME2 was assayed using an
aliquot of 2.5 x 10' cells. Cell pellets were counted for
radioactivity.
After accumulation of the desired number of decays, the
cells were quickly thawed by immersion of the vial in a
37C water bath. The cell samples were immediately assayed for D N A damage.

10

Fraction Number
Fig I a ) DNA alkaline and b) neutral filter elution profiles for
CHO-ER cells damaged by "'I-VME2 decay Cells analyzed by a)
alkaline elution after 23 days of dose accumulation, accumulated
28 060 2 dpc ( 0 ) ,8 886 62 dpc (U), 3 I47 8 dpc ( A), 2 834 9 dpc
(-), 475 8 dpc (V).78 3 dpc ( 0 ) .20 5 dpc (8)
and 0 dpc ( B )
Cells analyzed the next day by b) neutral elution accumulated
3 269 42 ( A), 2 944 41 dpc
29 144 06 dpc ( 0). 9 220 88 dpc (a).
(+). 49416dpc ( O ) , 8 1 3 l d p c ( 0 ) .2 1 3 d p c (0)and Odpc

(a)
D N A damage detection

The alkaline (10) and neutral (1 I ) filter elution assays

were performed to measure D N A single strand breaks
(ssbs) and DNA double strand breaks (dsbs) respectively,
as previously described (12, 13). CHO-ER cells are tetraploid ( J . Schwartz, personal communication), and
therefore only 2.5 x lo5cells were loaded on each polycarbonate filter ( I p m pore size) for both assays. No assumptions were made about the shapes of elution profiles (both
alkaline and neutral elution data) from '*'I decay irradiated cells; however, each elution profile had different y-in-

Acta Oncologica 35 (1996)

843

DNA D A M A G E BY '"I-ESTROGEN

tercepts, and so one cannot use points on the elution

profiles t o compare the lines, i.e., I2'I elution data does not
lend itself to analysis by a strand scission factor. Instead,
all points on the elution profile curves were averaged
together to obtain the mean value for each column as
previously described (1 3).
Replicate samples were stored at -90C for additional
time to permit additional decay accumulation. The freezing and thawing process did not introduce more DNA
damage.

Results

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1251VME2 incorporation into CHO-ER cells

Increasing I2'I-VME2 concentrations in the medium,
increased amounts of ligand incorporated into the cells.
The amounts of I2'I-VME2 found in the initially washed
cells (initial molecules/cell) and in cells diluted to dissociate ligand from low-affinity sites (molecules/cell after dilution), and the calculated number of accumulated 1251
decays per cells are presented in Table 1.

a.

10

Fraction Number

DNA ssh detection

With exposure to 7 different increasing '251-VME2 concentrations, the D N A eluted at faster rates through the
filters, indicating more D N A ssb damage, was induced
(Figs. la) and 2a) as measured by alkaline elution. The
alkaline elution profile of each "'I-VME2 concentration
was approximately linear which indicates that DNAprotein cross-links did not interfere with sample elution
through the filters, in agreement with previous studies
using I2'IUdR (13).

DNA dsh detection

The D N A dsb induction data (Figs. Ib and 2b) indicates
that there was increasing elution of the DNA through the
filter with exposure to increasing I2'I-VME2 concentrations, i.e., increased DNA damage corresponds to increasing numbers of accumulated ''I decays. These profiles
(Figs. Ib and 2b) were also approximately linear indicating
that DNA-protein cross-links did not affect elution.
Alkaline and neutral elution experiments were performed on replicate samples of cells that were stored
frozen for an additional 10- I 1 days and then assayed (Fig.
2). Increased D N A ssb and dsb damage (measured as
increased rate of elution) was observed for these samples
which had accumulated more decays during the additional
10-11 days (Fig. 2).

Dose response analysis

The amount of "'I-VME2 for each of the groups (1VII) is listed in Table 1 relative to the concentration of

1.o

0.5

0
'57

-s
3

&
C

0
-5
E
L4

0.1 -

0.05 -

b.

Fraction Number

Fig. 2. a) DNA alkaline and b) neutral filter elution profiles for

CHO-ER cells damaged by '251-VME2 decay. CHO-ER cells
analyzed by alkaline elution ( a ) after 34 days of dose accumulation, accumulated 39 019.32dpc ( CJ). 12 357.32 dpc (17).
6 587.37 dpc ( A ). 5 932.53 dpc ( 0 ) .661.61 dpc ( V), 108.86 dpc
(
28.51 dpc ( 0 )and 0 dpc ( ), Cells analyzed the next day
by neutral filter elution ( b ) accumulated 39 982.76 dpc ( ' 3 ) ,
12 662.48 dpc (El). 4 485.32 dpc ( L ) .4.039.44 dpc ( 0 ) .677.94 dpc
( O), 1 1 1.55 dpc ( 0 ), 29.22 dpc ( 0 )and 0 dpc ( H).

a),

844

Acta

L S. YASUl ET AL

Oncologica 35 ( 1996)

Table 2
Regression analysis .of dose response curves

F6 data

Mean value data

_

y-intercept
Slope
r

'

AE

NE

____
~
AE

64.63'%)
-3.33 x 10-5
-0.9121

58.71o/u
-0.0001
-0.8932

60.48%
-4.703 x
- 0.9529

_________

NE
56.82?40
0.0001
-9.171

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' All data included in regression analysis except for the highest concentration of '''I-VME2.
'251-VME2 in the incubation medium, as the initial number
of molecules per cell in the washed, concentrated cell
suspension, as the number of molecules per cell found in
dilute cell suspensions (allowed dissociation of at least
some of the non-specifically bound ligand), or as the
number of accumulated decays per cell, based on the
molecules per diluted cell and the time at which the cells
were stored frozen to accumulate decays. D N A damage
induction was estimated by calculating the mean value of
the fractions (13) or determination of the fraction of
radioactivity retained at fraction 6 (F6) for each elution
profile.
More D N A ssbs were induced by I2'I-VME2 decay
compared with DNA dsb induction (approximately 1.6 to
3 times more depending on the method of elution profile
analysis). Alkaline and neutral filter elution profiles (assays
D N A ssbs and dsbs respectively) were analyzed by mean
values, F6 data, and initial slopes. Comparions of ssb
versus dsb induction data yielded 3, 2 or 1.6 times more
ssbs when comparing mean value data, F6 data, or initial
slope data respectively.
The rate of damage induction was determined from the
slopes of dose-response curves. The slopes of the curves
were calculated by regression analysis for all data except
the highest concentration of '"I-VME2 (almost 6 nM)
(Table 2). The biological rationale for not including the
highest "51-VME2 concentration point in the regression
analysis follows. When the concentration of estrogen is
appreciably higher than the affinity constant, the receptor
should be saturated and any additive effects on D N A
breaks should be minimal, assuming that most of the
non-specific binding is distant from the DNA. In addition,
the calculated regression line was a much better fit of the
data ( r values 2 -0.9) when the nearly 6 nM "'I-VME2
data were not included in the regression analysis.
The slopes of DNA ssb induction data (both F 6 data in
Fig. 3 and mean value in Fig. 4) were compared with the
slopes from the DNA dsb induction data (both F6 data in
Fig. 3 and mean value data in Fig. 4).
Initial slopes
Further examination of all the dose-response curves
suggested a multiphasic response (Figs. 3 and 4). This

probably reflects the nature of the binding of '251-VME2

with the estrogen receptor. At lower concentrations of
added '251-VME2, a higher proportion of the "51-VME2
molecules are expected to be receptor bound. At higher

0
B

0.1 I
10.000
0

a.

+-

30,000

Decays/cell

x
.->
.*
c1

.-0

d
c

0
.-c

Lr,
0I

b.

d
30,000

-2.
0

10.000

Decayskell

Fig. 3. a) Alkaline filter elution dose response curve and b)

neutral filter elution dose-response curve for fraction of radioactivity retained at fraction 6 . Decays per cell were calculated as:
(0.233) ( N o . molecules/cells after dilution). The data were included in the regression analysis except for the highest concentration of IZ5I-VME2. The calculated regression line is drawn
through the data.

Acru

Oncologica 35 ( 1996)

be by membranes). Thus, a multiphasic nature of the

dose-response curve may reflect saturation of the receptor. Initial concentrations of "51-VME2 higher than 1 n M
may lead to a greater proportion of the radioactivity
non-specifically bound ( DeSombre, own data). A I n M
threshold for binding is also observed by Lippman et al.
(14) in human breast cancer cell lines, MCF-7 and ZR-751. Therefore, the effects observed at lower doses (less than
or equal to 1 nM "'I-VME2 added to culture medium) or
the initial slopes of the dose-response curves should contain biologically relevant information about DNA damage
induction by receptor-bound iodoestrogen.

0.1

i
0

2-L-

10.000

30.000

Decay sicell

a.

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845

D N A I)AM,AGE UY ''SI-FSTROGEN

al

5
0

0.I
0

b.

10,000

Discussion

30.000

Decayskell

Fig. 4. a ) Alkaline and b) neutral filter elution dose response

curve. The mean value of the fractions is plotted relative to the
number of decays accumulated per cell. Decays per cell were
calculated as indicated in Fig. 3. The data were analyzed by
regression analysis. The regression line calculated for all the data
(except for the highest concentration of '251-VME2)is drawn as a
solid line through the data. The regression line calculated for all
"doses" of IZsI when < 1 nM "51-VME2 was added to the culture
medium is drawn as a dotted line. The dotted line is the initial
slope of the dose-response curves.

concentrations of "'I-VME2 added to the medium, lower

proportions are likely to even be associated with the
nucleus (as much of the non-specific binding is believed to

The idea of utilizing Auger emitting radionuclides as a

part of cancer therapy is not new. In fact, clinical trials
with "'IUdR in brain cancer are ongoing (15). 12'1iodoestrogens have been shown to be effective in killing
cells in vitro (6. 7). Furthermore, recent advances in estrogen-ER binding to EREs provide a detailed molecular map
of such interactions (16) which facilitates prediction of
energy deposition patterns and prediction of DNA damage
and cytotoxicity. An earlier report by Sundell-Bergman &
Johanson ( 1 7) utilized another steroid receptor system to
target DNA. They (17) indicated that the efficiency of
inducing DNA dsbs with the thyroid hormone [ "'I]-triiodothyronine, mediated by the T3 receptor (also a mem-

Table 3
Compurison between "-'IlJdR trntl 1251-P " E 2 DNA dumage induction using rtiiriul slopes

Number
retained
Number
retained
ssb:dsb

decays to reduce DNA

to 50% mean value by ssbs
decays to reduce DNA
to 50% mean values by dsbs

* No. decays

f251-VME2*
566 dpc

I?sIUdR**
300 dpc

"51-VME2/"51UdR

358 dpc

3000 dpc

0.1

0.6

10

calculated from regression analysis.

**No. decays estimated from dose response curves from Yasui ( 13)

I .9

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846

L S. YASUI ET AL

ber of the steroid receptor superfamily) ( I 8) was as high as

that due to '251UdR incorporated into DNA. This suggests
that the radiolabeled thyroid hormone must be held in
close proximity to the D N A by its association with the T 3
receptor.
Comparison of damage induction (ssb, dsb and ssb:dsb
ratios) by '251-VME2 compared with "'IUdR decay (1 3)
turns up some interesting differences. Although DNA ssb
induction by I2'IUdR compared with "'I-VME2 did not
differ greatly from each other (approximately 1-2 times
greater than D N A ssb induction by I2'I-VME2 comparing
initial slopes or percentages of retention values at mean
values of 50% respectively), DNA dsb induction by "'IVME2 was approximately 4-8 times greater compared with
"'IUdR. In other words, it took fewer "'I decays to produce
DNA dsbs using lZ51-VME2compared with "51UdR. The
4-8-fold increase in dsbs produced by IZ5I-VME2 over
"'IUdR was determined by comparison of initial slopes for
"'I-VME2 with the slopes for I2'IUdR or comparion of the
number of decays required to reduce the mean value of the
fractions to 50% for D N A dsb induction respectively.
The ratio of ssb to dsb induction by "'I-VME2 was also
different from the value obtained for I2'IUdR. Using the
number of decays required to reduce the mean values to
50% shown in Table 3, "'I-VME2 induced per decay, 1.6
DNA ssb and 1 D N A dsb. l'2I decay, when incorporated
into DNA when using I2'IUdR, induced about 10 ssbs and
1 dsb per decay (13).
The surprising increase in apparent DNA dsb damage
induction by '"I-VME2 over '251UdR could have several
explanations. Indeed, there are a number of differences
between the VME2 and UdR vectors used to transport the
radioactive iodine molecule to its targeted site. Three
obvious differences are 1) the placement of the I2'I atom
relative to DNA ( IZ5IUdRplaces theI2'I atom in a central
location between the 2 strands of DNA while the 1251VMEZ is tightly associated with a protein, which is bound
to DNA and may also bind to the palindromic response
element as a dimer which places two "'I-VME2s in the
same region of DNA, increasing the radiation density), 2)
the possible insulation of the I2'I by receptor or other
proteins, and 3 ) the distance which the '"I is from the
sensitive bonds in the DNA. For the I2'I-VME2 to be
more effective than the "'IUdR, one's model would have
to predict that radiation from the iodoestrogen should be
more ideally placed relative to the sensitive DNA bonds
than would be the nucleoside. One critical site of the D N A
polymer is the phosphate linkage which is the key to the
integrity of the polymer. Thus, given our results suggesting
the greater effectiveness of the '"I-VME2 over "'1UdR
decay, one might expect that the I2'I might be more
strategically located to irradiate the phosphate bond. Alternatively, chromatin organization in the proximity of the
ERE may also affect the DNA damage produced by the
(13).
decay of L251

Acta Oncologico 35 ( 1996)

Related to item No. I above and an additional relevant

factor in the analysis of the comparative results of DNA
filter elution studies of VMEZ and UdR is the likely
topological distribution of the breaks. While it is likely to
a first approximation the "'IUdR will be randomly incorporated into DNA, the number of EREs o r their distribution in DNA is completely unknown. One might expect,
however, that the EREs are less likely to be randomly
distributed and therefore the size distribution of the DNA
fragments from the decay of 12'I-VME2 are likely to be
different from those produced from the decay of '251UdR
suggesting differences may exist in the absolute number of
strand breaks assayed after "'I-VME2 compared to
"'IUdR. Studies to calibrate lZ51-VME2damage induction
are currently ongoing.
Recent studies suggest that the placement of the 1251atom
adjacent to the sugar-phosphate backbone is at least as
efficient at producing DNA dsbs as when the "'I atom is
incorporated centrally between the complementary strands
of DNA. When lZs1is placed in association with DNA, using
the "'I labeled DNA minor groove binder, '2'I-Hoechst
33258, DNA dsbs are efficiently produced but no additional
DNA ssbs are dectected (19, 20). Additionally, Panyutin &
Neumann (21) found that approximately 1 DNA dsb was
formed opposite from the site of "'I incorporation and
within 10 base pairs of the site of incorporation when they
used lZ5l labeled triplex forming oligonucleotide which
specifically binds at particular nucleotide sequence in the
major groove of double stranded DNA. These recent studies
may support our findings, i.e., increased number of DNA
dsbs per "'I-VME2 decay over I2'IUdR and approximately
the same number of DNA ssbs induced per decay of
"'I-VME2 compared with "'IUdR. We propose a dimer of
the "'I-VME2-ER complex places two 12'1 atoms in the
proximity of the sensitive sugar-phosphate backbone of the
DNA and produces more DNA dsbs that I2'IUdR decay.
These results may offer avenues t o improved endocrine
therapy for breast cancer.
ACKNOWLEDGEMENTS
The authors would like to thank Dr. David Lotshaw for critical
review of the manuscript and Dr. Jeffrey Schwartz for helpful
discussion. This work was supported by DOE grant DE-FGO293ER61655. grant CAI4599 from the National Cancer Institute
and the Boothroyd Foundation to ERD and also in part by the
PMBC at Northern Illinois University to LY.
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Acta Oncologica 35 (1996)

D N A [ ) A M I C E BY '-"I-ESTROGEN

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