536 Bioconjugate Chem.

1995, 6, 536-540

Quantitative Analysis of 3’-Azido-3‘-deoxythymidine

Incorporation
into DNA in Human Colon Tumor Cells
Minoti Sharma,* Rama Jain, Elizabeth Ionescu, a n d J a m e s W. Darnowski’
Department of Biophysics, Roswell Park Cancer Institute, Buffalo, New York 14263. Received March 14, 1995@

We have previously reported that 3’-azido-3’-deoxythymidine(AZT) can possess significant antineo-

plastic activity in vitro and in vivo when combined with agents which inhibit de novo thymidylate
synthesis. Under these conditions cytotoxicity is closely associated with the degree t o which AZT is
incorporated into DNA. We now report a fluorescence postlabeling technique by which AZT
incorporation into DNA can be quantitated without employing radiolabeled AZT. Cultured human
colon tumor (HCT-8) cells were exposed to various concentrations of AZT alone and in combination
with 5-fluorouracil (FUra). Control cells received the same amount of medium. DNA was isolated
from harvested cell pellets (2 x lo7). Enzymatic digestion of DNA to the mononucleotide level followed
by HPLC analysis of the digest showed that the DNA preparation was free of RNA contamination.
The DNA digest was conjugated with dansyl chloride in situ via the phosphoramidate derivative with
ethylenediamine. HPLC analysis of the postlabeled nucleotides using fluorescence detection detected
105,245, and 479 fmol of 5’-monophosphate of AZT (AZTMP) per pg of DNA from cells exposed to 20,
50, and 100 pM AZT, respectively. FUra (3 pM) doubled the AZT incorporation per pg of DNA in
cells exposed to 50 and 100 pM AZT. These findings generally support our previously reported data
which quantitated (3H)AZTincorporation into cellular DNA and are discussed in light of the potential
clinical utility of this technique in assessing the relationship between AZT incorporation into DNA
and therapeutic action.

INTRODUCTION (St. Louis, MO). Ethylenediamine (EDA), triisopropyl-

We have reported, using a human colon tumor model,
that AZT in combination with FUra or methotrexate
(MTX)exert superior cytotoxic and antineoplastic effects
compared to either drug alone (2-3). As a result, clinical
analysis of AZT-based regimens for the treatment of
cancer have been initiated, and presently phase I1
+
analysis of AZT FULV is underway. Sommadossi et
al. have reported that AZT-induced cytotoxicity in human
bone marrow cells is related to AZT incorporation in DNA
(4). Our group also has observed that AZT cytotoxicity
correlated closely with the size of intracellular pools of
di- and triphosphates of AZT and the amount of AZT
incorporation into cellular DNA (3).
Thus far, all the reports correlating AZT incorporation
in DNA and cytotoxicity have been based on studies
utilizing radiolabeled AZT. As a result, clinical confir-
mation of the therapeutic relevance of AZT incorporation
into DNA is not practical. Therefore, we now report a
technique to analyze AZT incorporation into DNA using
nonisotopic detection and demonstrate its capacity to
quantitate the incorporation into cellular DNA. These
results and the method are discussed in light of their
potential utility in ongoing clinical trials.
MATERIALS AND METHODS
Chemicals and Reagents. The standard deoxy-
nucleotides, 1-methylimidazole, 1-(3,3-bis(methylamino)-
propyl)-3-ethylcarbodiimide hydrochloride (CDI), 5-(di-
methy1amino)naphthalene 1-sulfonylchloride(Dansyl chlo-
ride), protected mononucleotide phosphodiester, the en-
zyme nuclease P1, and FUra were purchased from Sigma
* To whom all correspondence should be addressed. Phone:
(716) 845-8296. Fax: (716) 845-8899.
’ Department of Medicine, Brown University and Roger
Williams Hospital, Providence, Rhode Island 02908.
@ Abstract published in Advance ACS Abstracts, July 15,
1995.
1043-1802/95/2906-0536$09.00/0
benzenesulfonyl chloride (TPSCI), anhydrous pyridine,
and 2-cyanoethyl phosphate (barium salt dihydrate) were
obtained from Aldrich Chemical Co. AZT was the gener-
ous gift of the Burroughs Wellcome Co. (Research Tri-
angle Park, NC), and RPMI 1640 medium and fetal
bovine serum (FBS) were purchased from Gibco (Grand
Island, NY). HPLC grade solvents, chemical, and dispos-
able tissue culture supplies were obtained from Fisher
Scientific (Medford, MA).
Cell Line. Continuous cultures of HCT-8 human colon
adenocarcinoma cells, obtained from the American Type
Culture Collection, were used in these studies. The
biochemical and histological characterization of this cell
line has been reported (5). Cells were cultured in sterile
plastic tissue culture flasks as monolayers in RPMI 1640
medium supplemented with 10% fetal bovine serum
(FBS) and passed twice weekly. Cell cultures were
maintained in a humidified incubator a t 37 “C in an
atmosphere of 5% COZ. Under these conditions their
doubling time was 20 h and cells in logarithmic growth
were used in all studies.
In Vitro Evaluation of Cytotoxicity. HCT-8 cells
(1 x lo5) were added to 10 mL of RPMI 1640 media
containing 10% FBS in 25 mL culture flasks. AZT and
FUra previously dissolved in media were added a t
concentrations of 20,50, and 100 pM AZT and 3 pM FUra
either alone or in various noted combinations. Control
cultures received the same amount of media without
drug. After 5 days, cells were harvested and growth
inhibition was determined as described previously ( 2 , 2 ) .
Each experiment was performed in duplicate and re-
peated a minimum of four times.
For experiments to quantitate AZT incorporation into
DNA and the effect of FUra on this parameter, ap-
proximately 3 x lo6 HCT-8 cells were added to 150 mL
flasks containing 50 mL of RPMI 1640 media, 10%FBS,
and the various noted concentrations of either FUra and/
0 1995 American Chemical Society
AZT Incorporation into DNA
HO{
i
O T O A
OH
HO-/-O{N3 + HO-!;toH-
I1
Figure 1. Scheme for the chemical synthesis of the 5’-monophosphate of AZT and its dinucleotide d(pApAZT).
0
I1
-O-P-OCH~
-b
~
‘coj COI
1 MI0AZOLE
BUFFER, pH6
N3
CD I = 1 -ethyl-3,3-dimethylominopropylcorbodiimide
Figure 2. Scheme for the synthesis of the fluorescence-labeled 5’-monophosphate of AZT
or AZT. After 5 days the cells (approximately 2 x lo7)
were harvested following reported procedure (3).
Preparation of 5‘-Monophosphate of AZT
(AZTMP) and Its Dinucleotide d(pApAZT1. The
scheme for the synthesis of AZTMP and its dinucleotide
d(pApAZT)is shown in Figure 1. AZT was phosphoryl-
ated with 2-cyanoethyl phosphate following a previously
reported procedure (6). The fully protected dinucleoside
monophosphate (111) was synthesized in the solution
phase by a modified phosphotriester approach (7). The
product was purified by column chromatography on silica
using 0-5% methanol in dichloromethane. The pure
product was phosphorylated with 2-cyanoethyl phosphate
as in the case of AZT. After being deblocked with
ammonia and pyridine (9:l v/v), the product (IV) was
isolated by C18 reversed phase chromatography (5 ym,
10 mm x 25 cm) using a 30 min linear gradient of 0-20%
acetonitrile in 0.1 M ammonium acetate buffer and
desalted on the same system using a linear gradient of
0-100% methanol in water. AZTMP (11) was also
isolated by reversed phase HPLC under similar condi-
tions. The chemical shifts in ppm with reference to TSP
a t 1.93 (d, 3H, CHd, 2.49-2.52 (m, 2H, CH-2’ and CH-
2’7, 4.06-4.23 (m, 3H, CH-5’, C H - 5 , CH-4’1, 4.50-4.53
(m, l H , CH-3’), 6.26-6.30 (t, l H , CH-l’), and 7.7 (s, l H ,
CH-6) were in agreement with the structure of AZTMP.
Bioconjugafe Chem., Vol. 6,No. 5, 1995 537
I
.“-Of OR N,
I11
1 * O T
HO-[;tO-[;$N,
v IV
N3
4
The downfield chemical shifts of the dinucleotide shown
at 8.52 (s, l H , AH-8), 8.27 (s, l H , AH-2), 7.5 (s, l H , AZT
H-6), 6.46-6.49 (t, lH, AH-1’1, and 6.13-6.16 (t, lH, AZT
H-1’) with respect to TSP supported the structure of
d(pApAZT). Nuclease P1 digestion of IV afforded U P
(V) and AZTMP (11) as shown in Figure 1. ‘H NMR
measurements were done on a Bruker WP 200 spectrom-
eter.
Figure 2 shows the scheme used for the synthesis
of dansylated nucleotide. The phosphate group of
AZTMP (1)reacts with water-soluble l-(3,3-bis(methyl-
amino)propyl)-3-ethylcarbodiimide (CDI) in l-methyl-
imidazole buffer a t pH 6 to generate the phosphor-
imidazolidate 2 which when exposed to ethylenediamine
(EDA) results in the formation of a stable 5’-phosphor-
amidate derivative 3. The free amino group of the
phosphoramidate reacts readily with dansyl chloride in
50 mM borate buffer, pH 9.5, to yield the fluorescent-
labeled nucleotide 4.
Fluorescence Postlabeling Assay of d(pApAZT1.
The dinucleotide (1 OD) was digested with nuclease P1
following reported procedure (8). The digest was filtered
on an ultrafree microunit with 10 000 NMWL polysulfone
membrane and labeled with dansyl chloride as described
below. The filtrate was adjusted to pH 6 with 0.1 M
NaOH and lyophilized. A cocktail (50 yL) prepared by
538 Bioconjugate Chem., Vol. 6,No. 5, 1995
mixing 40 mg of CDI, 15 pL of EDA, and 8 pL of
1-methylimidazole in 1mL of water, pH 6, was added to
the lyophilized material. The reaction mixture was left
a t room temperature overnight to prepare the phos-
phoramidate derivative of the digested nucleotides. The
pH of the reaction mixture was then adjusted to 9.5 with
0.2 M NaZC03, and 50 p L of dansyl chloride (1g/10 mL
acetone) was added. After being stirred a t room tem-
perature in the dark for 1h, the dansyl-labeled reaction
mixture was filtered on a microcentrifuge to remove any
particulate material, and the filtrate was frozen until
ready for HPLC analysis. A sample of AZTMP (1 OD)
was also dansylated following the same procedure.
Postlabeling and HPLC Analysis of Postlabeled
DNA Digests. Typically, DNA was isolated from a pellet
containing 2.5 x lo7 cells. The pellet was lysed by a
guanidinethiocyanate procedure (9). The protocol was
modified by adding 0.75 vol of ethanol to the lysate. After
2 h a t -22 "C, the mixture was centrifuged a t l O O O O g
for 20 min a t 4 "C, and DNA was isolated from the
supernatant by ethanol precipitation, proteinase K treat-
ment of the precipitate, and organic extraction following
the standard procedure. DNA was digested enzymati-
cally to the nucleotide level, and the nucleotide profile
of the digest was routinely monitored by HPLC to
determine the purity of the preparation. Typically, 100
pg of DNA (2 OD) was digested with 2 pL of DNAse-1
(2000 KU/lOO pL) and 2 pL of nuclease P l ( 1 mg/mL) in
40 pL of Tris (10 mM), EDTA (0.1 mm), MgClz (4 mM)
buffer, pH 7.5 containing 2 pL of ZnS04 (10 mM) and 4
p L of NaOAc (38 mM, pH 5.0) at 37 "C overnight. The
digest was derivatized with EDA cocktail following the
procedure described for the DNA model study. However,
in order to enrich the modified nucleotide from the
normal nucleotides in the DNA digest, the phosphor-
amidate reaction mixture was fractionated by HPLC. A
fraction corresponding to the retention time of phosphor-
amidate derivative of AZTMP was collected, lyophilized,
and labeled by adding 25 pL of dansyl chloride in 100 pL
of 0.1 M carbonate bicarbonate buffer, pH 9.5. An aliquot
of the labeled, enriched fraction was analyzed by HPLC
using fluorescence detection as shown in Figure 5.
Alternatively, the EDA reaction mixture was reacted
with dansyl chloride following the procedure described
in the model study, and the postlabeled digest was
fractionated by HPLC using fluorescence detection. A
fraction corresponding to the retention time of dansylated
AZTMP was collected isocratically with 23% acetonitrile
in 0.1 M ammonium acetate. Reanalysis of an aliquot
of the enriched fraction using the high-sensitive detector
accessory under the same elution conditions detected the
peak of interest.
HPLC Separation of Nucleotides of Normal and
Modified Bases. Prior to labeling, the nucleotides were
separated on a Radial-Pak LC cartridge 8MBC18 (10 pm,
8 mm x 10 cm) using a 30 min linear gradient of 0-20%
acetonitrile in 0.1 M ammonium acetate, pH 7. The
profiles were monitored a t 254 nm using a Beckman
variable wavelength detector with an Altex spectropho-
tometer cell. The postlabeled nucleotides were analyzed
on a microsorb C18 column (5 pm, 4.6 mm x 25 cm) using
a McPherson detector model 750B equipped with a highly
sensitive detection accessory to monitor fluorescence. The
elution conditions are described in the figure legends.
RESULTS AND DISCUSSION
In the present study the ICE,, of AZT in HCT-8 cells
after a 5-d exposure was approximately 67.5 pM. Under
these conditions the ICsoof FUra was 2.3 pM. Analysis
of combined effect of AZT and FUra revealed that these
Sharma et al.
Table 1. Analysis of the Combined Effect of FUra and
AZT on IC50 of FUra or AZT in HCT-8 Cellsa
incubation condition of FUra of AZT
control 2.3 i 0.2 67.5 i 4.8
0.5 pM FUra 49.4 i 6.2
1.5p M FUra 32.0 i 4.6
20 pM AZT 1.8 6 0.3
50 pM AZT 1.4 f 0.4
Twenty-five mL tissue culture flasks containing 10 mL of
RPMI 1640 media + 10% FBS, 1 x lo5 cells and various
concentrations of AZT alone, FUra alone, or their noted combina-
tions, were incubated at 37 "C. After 5 days, the cells were
harvested and the cell number was determined. Percent growth
inhibition was quantitated using cells incubated without AZT or
FUra as control. Each value represents the mean f SE from four
determinations.
agents exerted additive cytotoxic effects (Table 1). Upon
closer examination, however, it was apparent that while
FUra was able to increase the cytotoxic activity of AZT,
AZT exerted less of a n effect on the cytotoxicity of FUra.
Previous results using radiolabeled AZT have suggested
that, in this model, AZT-induced cytotoxicity was closely
associated with the degree to which AZT was incorpo-
rated into DNA (1-3). Reflecting both the expense of
these isotopic studies and their lack of utility in the
clinical setting we have therefore attempted to develop
a method to assess AZT incorporation into DNA by
directly quantitating the AZT nucleotide content of DNA
in cells exposed to this thymidine analogue.
To this end, the 5'-monophosphate of AZT and its
dinucleotide derivative were synthesized and labeled as
described in the methods (Figures 1and 2). The labeling
reactions, though shown in three steps in Figure 2, were
carried out in a single pot. The overall yield was 90%.
Thereafter, chromatographic conditions were devised to
resolve the nucleotides of normal and modified bases both
before and after labeling. Figure 3 shows the reversed
phase HPLC profiles of (a) the dinucleotide d(pApAZT1,
(b) nuclease P1 digest of the dinucleotide, and (c) the
normal nucleotides and AZTMP. It has been reported
that nuclease P1 releases the normal nucleotides from
modified DNA as 5'-monophosphate (pN) whereas the
modified nucleotides, depending on the nature of modi-
fication, are excised as dinucleotide (pNpX, X = modified
base) (10). A DNA model study shows that nuclease P1
excised AZT as 5'-monophosphate (pX). The retention
times of the two peaks in profile b matched the retention
times of authentic dAMP and AZTMP shown in profile
c. This was further confirmed by cochromatography
(results not shown). HPLC analysis of the postlabeled
digest also supported such an observation (Figure 4b).
Cochromatography (profile 4c) of the postlabeled digest
of d(pApAZT) with the postlabeled dinucleotide demon-
strated clearly that the peak a t 21 min in the profile 4b
is from labeled dAMP and not from the labeled dinucleo-
tide. The labeled dinucleotide is eluted a t 22.5 min as
shown in the profile 4a. HPLC analysis of the post-
labeled nucleotides of known concentrations also revealed
that the labeling yields of AZTMP and its dinucleotide
derivative were quantitative.
Figure 5 shows fluorescence postlabeling assay from
control (profile c) and AZT exposed (profile b) HCT-8 cells.
Unlike the DNA model study, enrichment of modified
nucleotide is essential in order to detect the peak of
interest in modified, cellular DNA by postlabeling tech-
nique from the huge background of normal nucleotides
(10).As described in the methodology, the enrichment
of the modified nucleotides from the normal nucleotides
AZT Incorporation into DNA Bioconjugate Chem., Vol. 6,No. 5, 1995 539

Oonsyi A Z T mD
Sulfonic Acid

AZTmp 10 20 30 40

c, c

10
4
AZTmp

20

Time (min)
1, 10
d
I
20

Figure 3. HPLC profiles of (a) d(pApAZT), (b) nuclease P1

digest of d(pApAZT), and (c) 5’-monophosphates of C, T, G, A,
and AZT eluted with a 30 min linear gradient of 0-20%
(0

10
20

20
30

30
40

40

acetonitrile in 0.1 M ammonium acetate buffer, pH 7. Time (min)

Figure 4. HPLC profiles of dansylated (a) d(pApAZT), (b)
can be achieved by HPLC fractionation of the DNA digest nuclease P1 digest of d(pApAZT), and (c) cochromatography of
d(pApAZT) and its nuclease P1 digest eluted with a 30 min
both before and after labeling. The latter approach offers linear gradient of 18-30% acetonitrile in 0.1 M ammonium
the advantage of enriching the modified nucleotide not acetate buffer, pH 7.
only from the normal nucleotides but also from the excess
labeling reagent. However, the successful application of A Z T Monoplimphate
this procedure depends on the chemical nature of the -
modified nucleotide. We observed that AZTMP can be E (a1 AZT Expored DNA Digest
resolved from the normal nucleotides both before and ON
ln t
E Authentic AZT Monophosphate
after labeling. The peak a t 19 min in profile b of Figure W
5 was identified as AZTMP by cochromatography of the 0
I

postlabeled DNA digest with authentic, dansylated

AZTMP (profile a). The percent digestion of DNA was X
_.
.o
Is,
N

calculated by HPLC, prior to labeling, from the integrated w

area of excised dAMP peak and the response factor of
standard dAMP. The efficiency of digestion of modified cz
v)
( I d AZT Exposed DNA Digest
DNA was 90% of control. The profiles in Figure 5
represent analysis of approximately 1pg of DNA.
i!!
z I I

W
The results shown in Table 2 reveal the relationship u .3 n
between the media concentration of AZT and the degree z c &I
w
of AZT incorporation into cellular DNA detected by xw
fluorescence postlabeling technique. Each number in this a
0 (c) Coritrol DNA Digest
table is a n average of three independent measurements. 3
Having demonstrated that the fluorescence postlabeling U
w
technique could be used to quantitate AZT incorporation L
into cellular DNA, we next assessed the effect of exposing t-
cells to both FUra and AZT on the degree to which AZT 4
W
was incorporated into cellular DNA in this model using a
nonisotopic detection. The results of these studies indi-
cated that FUra (3 pM) coexposure doubled the incorpo-
TIME (inin)
ration of AZT into DNA from cells exposed to 50 pM ( I C d
and 100 pM (Table 2). Biochemical analysis of acid Figure 5. HPLC profiles of postlabeled DNA digests of HCT-8
(PCA)-insoluble material from HCT-8 cells exposed to 5 cells (c) control, (b) AZT exposed, and (a) AZT exposed cochro-
pM FUra increased the incorporation of radiolabeled AZT matographed with dansylated AZTMP, eluted with 25% aceto-
nitrile in 0.1 M ammonium acetate buffer, pH 7.
into the nucleic acid fraction by 52% and also decreased
the IC50 of U T considerably. Furthermore, there ap- effect, perhaps reflecting more thymidylate synthatase
peared to be a FUra-related dose dependency to this inhibition at higher FUra concentration (1).
540 Bioconjugate Chem., Vol. 6,No. 5, 1995
Table 2. Effect of Various Concentrations of AZT Alone
and AZT plus FUra on AZT Incorporation into DNA of
Exposed HCT-8Cells
drug and concn AZTMP (fmoyun DNA)
none
AZT, 20 pM 109 f 5
AZT, 50 ,uM 245 f 7
AZT, 100 pM 479 f 11
AZT, 20 pM + FUra, 3 pM 114 & 5
AZT, 50 pM + FUra, 3 pM 476 i 9
+
AZT, 100 ,uM FUra, 3 pM 980 i 10
An AZT concentration-dependent relationship between
AZT incorporation into cellular DNA and cytotoxicity has
been reported by quantitating the tritium content of PCA
insoluble material obtained from HCT-8 cells after
exposure to (3H)AZT( I 1. Fluorescence postlabeling assay
of DNA in this model also shows a similar trend (Table
2) although the absolute amount of AZT incorporated into
DNA as determined using radiolabeled AZT is 1order of
magnitude higher than those observed by nonisotopic
detection. The labeling efficiency of AZTMP, as moni-
tored by HPLC analysis of each reaction step shown in
Figure 2, was 90-95%. It is unlikely that the procedural
loss can account for the observed result reported by
radiolabeling study ( I ) . This difference may reflect,
therefore, several differences between these methods. In
the fluorescence postlabeling assay, prior to labeling, the
nucleotide profile of DNA digest is routinely assessed by
HPLC analysis to check the purity of DNA preparation.
No such information was available for PCA insoluble
material used to quantitate the radiolabeled AZT. In
addition, it is difficult to make PCA insoluble material
completely free of unwanted radiolabel, especially when
material of relatively high specific activity is used.
AZT is presently used extensively in the treatment of
AIDS and ARC, and significant research is directed
toward identifying the mechanism(s) responsible for both
its therapeutic activity and toxicity. In addition, several
groups have reported that AZT can possess antineoplastic
activity in combination with drugs which disrupt DNA
synthesis and chemical evaluation of this potential is
underway. Our present findings utilizing the fluores-
cence postlabeling technique to quantitate AZT incorpo-
ration into DNA suggest the important potential of this
technique in studies to clinically monitor the fate of AZT
without using radiolabeled drug. Clearly, further in vitro
studies are essential in order to evaluate the therapeutic
Sharma et al.
relevance of the two-drug regimen in cancer chemo-
therapy under clinical settings.
ACKNOWLEDGMENT
Supported in part by National Cancer Institute grants
CA46896 and CA55358.
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