Volume 7
Number I
Spring I �7'1
May 191)3
Medi.::-.I Joumll of Ih.·
Isl:tmic: Republic uf Ir.m
STU DIES ON THE BINDING OF THE ALKYLATING
A GENT SULFUR MUSTARD T O C ALF THYMUS
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CHROMATIN
A. RABBANI Ph.D., AND M. KHODABANDEH, M.Sc.
From the Instilllte oj Biochemistry amI Biophysics, Tehran University oj Medical Sciences, Tf'hrlllJ,
Islamic R"pllblic or Iran.
ABSTRACT
In this study the effect of the alkylating agent, sulfur mustard, on calf thymus
chromatin was investigated using UVfVis spectroscopy, gel electrophoresis and
thermal denaturation techniql,cs. The results show that treatment of isolated
chromatin with sulfur mustard releases histones from the core particles but does
not affect histone H I and nonhistone chromosomal proteins. The content of
released proteins for the amounts lower than 50Ilg/mL are both dose and time
dependent. It is suggested that they have cross linked reaction to other chromo­
somal proteins or to DNA. Thermal melting analysis also indicate that the
interaction of sulfur mustard with DNA increases thermal melt(Tm) of DNA
revealing condensation of DNA molecule by sulfur mustard. The results in relation
to the functional states of chromatin are discussed.
M.lIRI, 1101. 7, No.1, 43-46,1993.
INTRODUCTION
MATERIALS AND METHODS
Bifunctional alkylating agents are the most important
Calf thymus was obtained from Ziaran slaughter-house
and heterogeneous class of antineoplastic drugs in clinic,�
and transferred to the lab in ice. After removing the
use.'" DNA is genemlly thought to be the princip,� target
membranes, tissues were immediately frozen in liquid
for these drugs. They alkylate DNA molecule preferen­
nitrogen before use. DNA was prep'tred from thymus
tially at guanine-N7 position.'" It has been reported that
according to Kayet al. method." It was dissolved in 50 mM
'�kylating agents produce DNA inter-and intrastrand cross­
tris-HCI buffer by stirring overnight at 4°C. The amount of
links in a number of cell lines.'·7 In addition, seve,,�
DNA was determined optically using A26 0=0.6 . Sulfur
workers have suggested UJat these drugs, especially nitro­
mustard was obtained from Dr. Riasi in Defense Research
gen mustard, cause DNA-protein complexes in treated
cells which arc resistant to deproteination procedurcs,H.9
Establishment. Stock solutions of I OO)lg/mL were made in
2 5 111M trieUumolamine-HCI buffer pH 7.5 containing I
At the molecular level, in the cell nucleus, DNA is
mM EDTA and diluted to desired concentrations with the
complexed with special sets of proteins called histones ,md
same huffer.
nonhistone proteins. To date five main fractions of his­
Isolation of nuclei: Nuclei from frozen c,M thymus
tones, namely H I , H2 A, H2 B, H3 and H4 have been
(10 g) lVere prepared essenti'�ly according to the proce­
isolated and charactcrized,lO Interaction of the proteins
dure described by Hewish and Burgoyne."
with DNA molecule in the chromatin produces a be:ld on
a string structure defined.L'i nucleosomes.lO·n
huffer and the sediment suspended in 1 0 mL of the same
In this study we have attempted to exmnine the scl!!c­
buffer. 100)lL of the suspension was diluted into 3 mL of
Nuclei peliet lVas washed three times with TMKC
tive release and or cross-linking of chromatin components
N NaOH and the absorb'mce at 2 6 0 nm was measured.
by treatment of calf thymus nuclei with the aIkylating
Binding assay: Nucleus suspension in TMKC buffer
agent sulfur mustard.
pH 7.4 diluted I: 1 with triethanolmnine-EDTA buffer and
43
Sulfur Mustard Binding to Chromatin
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Fig. 1. Changes in the absorbance (260 om) of
the supernatants
obtained from the treatment of calf thymus chromatin with
differentconccnlralions of sulfurmuslard.lncubation times were
30. 60. and 120 min. at A. 25°C; and S, 37°C. Rcsuhs arc
expressed
as
o
difference to controls and each point is the average
30
60
120
Time (min)
of five determinations.
Fig. 2. Time course of sulfur mustard uptake by thymus chroma­
tin. Measurements were performed at 260 nm. a: 25°C, b: 37°C.
the nuclei were lysed by a brief homogenization. Suspen­
sion was divided into several equal portions each contain­
was diluted into desired concenlnltion and then mixed Wi01
ing I mg DNA permL and sulfur mustard at0 .5,10,20 and
different amounls of sulfur muslard. The mixtures were
50 Jlg/mL were added. 111e samples were incubated 30,60,
incubaled for 30 min m25°C. Trealed and conlrols were­
and 120 min. at either 25°C or 3rC while occasiomdly
subjected to an increase in lempemture at a mle of I 'C/
mixing. Treated smnplcs and the controls at time intervals
min. 111e absorb'mce was read at 260 nm on a single beam
were taken and centrifuged for 5 min m6000g, 4°C. The
Gilford240 spectrophotometer using tris bufferas a blank.
supernatants and pellets were studied as follows:
The derivative of hyperchromicity al lempcmture T was
The absorbance of the supernatants were monitored at
.
dhT
c,dculated by the equauon
dT
260 and230 nm. The supernatanls were made10% trichlo­
roacetic acid (TCA) by respect to 70% TCA. The precipi­
hCT+I)-hCT-IJ
2
tates were then collected by centrifugation al6000 g for5
min, dissolved in SDS sample solvenumd loaded onto the
RESULTS AND DISCUSSION
gels. SDS-polyacrylamide gel electrophoresis was essen­
tially carried oul as described by Lammeli." Destained
Fig. I shows the effect of various concentrations of the
gels were sc,mned by densitometric gel scanner Beckm,m
alkylaling agent sulfur mustard on the possible release of
Model (R-112) ,md the amount of each protein was deter­
DNA from c,df thymus chromatin inlo the supernatanls as
mined by calculating the area under the peaks.
detennined by measuring the absorbances at 260 nm. The
rcsulis are expressed as difference to controls. Incubation
Thermal analysis: DNA was dissolved in50 mM tris­
was carried out at two different temperatures, 25°C and
HCI buffer pH 7 5
. at a final concentration of I mg/mL. II
37°C, and three time intervals 30, 60 , and 120 minutes
44
A. Rabbani, Ph.D, and M. Khodabancleh, M.Sc.
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Fig. 3. SDS-polyacrylamide gel electrophoresis paltcrn of mus­
tard treated and controls aI25°C. a.cof each c;\pcrimenl arc zero
to 50 ug./mL concentrations of sulfur mustard. respectively. A;
standard calf thymus histone.'>.
B; standard nonhistone proteins
(HMG).
were used. 111e resuHs indicate thal
010 release of DNA is
proceeded in a dose dependent manner. In bOOl conditions.
the maximum release is obtained at 10-20 Jlg/mL of
Sulfur mustard (J..Lg/llll)
Fig. 4. Absorbance detcnnination of the supernatants at 230 nm.
mustard level in absorb:U1ces. As is also seen in Fig. 2 Olere
is a considemble difference between two incubation con­
Details are
ditions as a function of time. At 30 min and 120 min of
as
indicated in Fig. 1.
incubations at 25°C. the amount of released DNA is
increased remarkably in comparsion 10 60 min incubation.
Sediments from lfeated and conlfols were subjected to
But when the experiment is perfonned at 37°C and 30 min
DNA and protein isolation procedures 100. In Ole presence
incubation, 010 lowest absorbances are observed.
of aIkylating agent DNA resists against eXlfaction by
The proteins from treated and control samples were
phenol-chloroform procedure. Perchloric acid (5%) ex­
isolated and analyzed for identification of Ole materials
traction and fractional acetone precipitation method was
remained in the supernatants. The proteins were precipi­
used to isolate HI and HMG proteins. Analysis of the
tated with 10% TCA and the contents were eleclfophore­
proteins on the SDS polyacrylamide gel eleclfophoresis
sed on 15% SDS-polyacryhunide slab gels. The gel pallem
revealed a complex pauem-indicating thal both HMG and
is given in Fig. 3. It is shown that Ole highest release is at
Hl are present in the sediment (data nO! shown).
20Jlg/mL of sulfur mustard, however, below this value the
In order to determine whether the binding of sulfur
panems are similar to controls. The resuHs suggest an
mustard alters isolated DNA slfUcture, we examined the
insufficient amount of mustard for acting on chromatin.
effect of different concenlfations of mustard on free DNA
On Ole oOler hand, at higher concenrrations of mustard (50
in solution. Thermal denaturation analysis is a technique to
Jlg/mL) although the gel panem is identical to Ole controls.
demonstrate any SlfUCtural changes occurred in DNA.
the interpretation is completely different from what has
Therefore DNA and sulfur mustard were mixed under
been given above. since in the laller presumably cross
experimental conditions mentioned in the methods section
linking of proteins 10 proteins or 10 DNA has occurred by
and incubated. Treated and conlfols were Olen thennally
sulfur mustard action. 111erefore after centrifugation the
analyzed. Derivative melting profiles of the samples WiOl
cross linked products remain auached to Ole insoluble part
10 :U1d 50 Jlg/mL sulfur mustard is shown in Fig. 5 in
of the reaction mixlfUe. According to the gel pauem. it is
comparison 10 unlfeated DNA. It is seell Omtthe binding of
also apparent that Ole released proteins are only core
sulfur mustard protects DNA against thermal denaturation
histones H2A. H2B. H3 and H4. No hislOne HI or nonhi­
and shifts its Tm (thermal mells) to higher temperatures.
SlOne prOleins especially high mobility group (HMG) is
This condensation pattern of DNA induced by mustard
observed on Ole gel. To conflfIn the above resuHs, data
action can be explained by inter-or intraslfand cross link­
obtained from the 230 nm absorbance measurements is
ing of DNA as a function of aIkylating agent.
also given in Fig.4. The results are in aggreement wiOl thc
Data mentioned so
gel pauem.
far indicate O,at when calf Olymus
chromatin is lfeated with sulfur mustard, DNA and pro-
45
Sulfur Mustard Binding to Chromatin
effects of these agents on the purified proteins and to look
at the possible structural changes of individual chromatin
components.
REFERENCES
Downloaded from mjiri.iums.ac.ir at 6:31 IRDT on Sunday June 18th 2017
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CRC Handbook of Chemotherapeutic Agents. Vol.2, pp.
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Macromolecular targets and metabolic properties of al1.:ylat­
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and Targets for Cancer Chemo-therapeutic Agents. pp. 4583, New York: Academic press,1981.
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antineoplastic agents. J Nat! Cancer Inst 73:1021-8 1984.
4. Mnttes WB, Hartley JA and Kohn KW: DNA sequence
selectivity of guanine N7 alkylation by nitrogen mustard.
Nucl Acids Res. 14:2971-87, 1986,
5. Hannson J, Lewensohn R, Ringborg U, Nilsson B: Fonnation
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6. Carrano A V,Thompson LH,Stetka DO,Minkler JL,Mazrirnas
JA, Fang S: DNA cross linking, sister chromatid exchange,
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7. Murray D, Meyn RE: Enhancement of the DNA cross linking
activity of nitro gen mustard by misonidazo I and diethylmalate
in a mouse lihrosiln.:oma tumor in vivo. Cancer Res 44: 916, 1984.
8. Ewig RAG, Kohn KW: Protein dependent and protein inde­
pendent DNA cross links in mammalian cells treated with
a1kylating agent'. Feu Proc 36: 336-9, 1977.
9. Orunicke H, Bock KW, Becher H, Oang V, Schnierda J,
Puschendorf B: Effect on alk.y
- laling
binding of DNA to protein. Cancer Res 33: 1048-1053,1973.
10. Hnilica LS: The Structure and Biological Functions of
Hislones. CRC press, 1972.
I I. Kornberg RD, Klug A: The nucleosomes. Sci Arner Feb: 4860,1981.
12. Bradbury EM, Maclean N, Mathews HR: DNA,Chromatin
and Chromosomes. London: Blackwell, 1981.
13. Kay ERM, Simmons NS, Dounce AL: Improved preparation
of DNA. J Am Chelll Soc,74: 1724-6, 1952.
14. Burgonyne LA, Wagar MA and Atkinson MR: Calcium
dependent priming of DNA synthesis in isolated rat liver
nuclei. Biochem Biophys Res Comm. 39: 254-9,1970.
15. Lanuneli UK: Cleavage of structural proteins during the
assembly of the head of bacteriophagcs. Nature 227: 680-5,
1970.
16. Jeney A, Dzuvillay E, Lapis K and Valko E: Chromatin
proteins as a possible target for antitumor agents. Chern Bioi
Interact 26: 349-61, 1979.
17. Riches PG, Selwood SM, Harrap KR: Some effects of
chlorambucil on nUcleoprotein phosphorylation in Yoshida
ascites sarcoma. Chem BioI Interact 18: 11-22, 1977.
18. WolfH, Raydt 0, Puschendorf B, Grunicke H: Effects of the
alkylating agent triethyleneiminobenzoquinone and the syn­
thesis of DNA. FEBS Lett 35: 336-40,1973.
l)()
Tempratufl! (CO)
Fig. S. Thermal denaturation profile of the interaction of DNA
with sulfur mustard at 10 and 50�g/mL concentrations. Deriva­
tive of the points obtained from four separate experiments is
shown.
DNA; - - - -, 20, �g/mL; ....... , 50�g/IllL mustards.
_,
teins in the core particles are more susceptible to alkylation
than linker DNA, Attachment of mustard to DNA in these
regions render core histones to be released from the chro­
matin. On the other hand, when high concentrations of
mustard is used, condensation of chromatin components
occurs producing insoluble DNA-protein or protein-pro­
tein cross links. In vivo treatment of Yoshida sarcoma
chromatin components Witll dibromodulcitol (DBD, 16)
suggest that certain chromosomal proteins are degraded
and subsequently newly synthesized resulting in an ex­
change of chromatin components. The fonnation of a
nucleohistone complex between HI and DNA is inhibited
by pretreaunent of HI with DBD, The effect of other
antitumor alkylating agents such as nitrogen mustard and
trenimon on the nuclear proteins of chromatin has been
reported by Riches and Harrapl7 and Wolf et ai," However,
the type of alterations observed were quite different from
the effects reported here. In spite of these findings our
results do not show any reduced ability of histone HI to
interact with DNA but in contrast the interaction is quite
stronger which tends for HI to remain attached to DNA and
cosediment with it.
From the results it is concluded tlJat alkylating agents
react chemicalIy with chromosomal proteins some being
more susceptible to alkylation that others. In view of the
considerable imponance of chromatin proteins in tlle cell
functions such as replication and tnmscription they to­
gether with DNA are considered as important targets for
alkylating agents. In studies on the mechanism of alkylat­
ing agents action it would seem desirable to measure the
46