Tightly-bound to DNA proteins in rat experimental hepatomas and normal liver cells
Proteins tightly bound to DNA (TBP) comprise a group of proteins that remain bound to DNA even after harsh deproteinization procedures. The amount of these proteins is 20–100 µg for mg of DNA depending on eukaryotic source. This experimental paper examines the possibility to use some TBP for clinica...
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| Zitieren: | Tightly-bound to DNA proteins in rat experimental hepatomas and normal liver cells / D. Labeikyte, V. Borutinskaite, N. Legzdins, N. Sjakste // Experimental Oncology. — 2011. — Т. 33, № 3. — С. 121-125. — Бібліогр.: 30 назв. — англ. |
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| author | Labeikyte, D. Borutinskaite, V. Legzdins, N. Sjakste, N. |
| author_facet | Labeikyte, D. Borutinskaite, V. Legzdins, N. Sjakste, N. |
| citation_txt | Tightly-bound to DNA proteins in rat experimental hepatomas and normal liver cells / D. Labeikyte, V. Borutinskaite, N. Legzdins, N. Sjakste // Experimental Oncology. — 2011. — Т. 33, № 3. — С. 121-125. — Бібліогр.: 30 назв. — англ. |
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| description | Proteins tightly bound to DNA (TBP) comprise a group of proteins that remain bound to DNA even after harsh deproteinization procedures. The amount of these proteins is 20–100 µg for mg of DNA depending on eukaryotic source. This experimental paper examines the possibility to use some TBP for clinical biomarker discovery, e.g. for identification of prognostic and diagnostic cancer markers. The main aim of this study was to designate differences between tightly DNA binding protein patterns extracted from rat liver and rat experimental hepatomas (Zajdela ascites hepatoma and hepatoma G-27) and to evaluate possibility that some of these proteins may be used as biomarkers for cell cancer transformation. Methods: We used proteomics aproach as a tool for comparison of pattern of TBP from rat experimental hepatomas and normal liver cells. Combination of 2DE fractionation with mass spectrometry (MALDI TOF-MS) suitable for parallel profiling of complex TBP mixtures. Results: Intriguingly 2DE protein maps of TBP from rat liver and rat experimental hepatomas (Zajdela acites hepatoma and hepatoma G-27) were quite different. We identified 9 proteins, some of them shared in all TBP patterns. Among identified tightly bound to DNA proteins there were three proteins considered as nuclear matrix proteins (lamin B1, scaffold attachment factor B1, heterogeneous nuclear ribonucleoprotein). Also we identified DNA repair protein RAD50, coiled-coil domain-containing protein 41, structural maintenance of chromosomes protein1A and some ATP –dependent RNA helicases indicating that TBP are of interest with respect to their potential involvement in the topological organization and/or function of genomic DNA. Conclusions: We suppose that proteomic approach for TBP identification may be promising in development of biomarkers, also obtained results may be valuable for further understanding TBP functions in genome.
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Experimental Oncology 33, 121–125, 2011 (September) 121
TIGHTLY-BOUND TO DNA PROTEINS IN RAT EXPERIMENTAL
HEPATOMAS AND NORMAL LIVER CELLS
D. Labeikytė1,*, V. Borutinskaitė2, N. Legzdiņš3, N. Sjakste3,4
1Department of Biochemistry and Biophysics, Vilnius University, Vilnius LT-03101, Lithuania
2Department of Developmental Biology, Institute of Biochemistry, Vilnius university, Vilnius LT-08662, Lithuania
3Latvian Institute of Organic Synthesis, Riga LV1006, Latvia
4Faculty of Medicine, University of Latvia, Riga LV1001, Latvia
Proteins tightly bound to DNA (TBP) comprise a group of proteins that remain bound to DNA even after harsh deproteinization
procedures. The amount of these proteins is 20–100 μg for mg of DNA depending on eukaryotic source. This experimental paper
examines the possibility to use some TBP for clinical biomarker discovery, e.g. for identification of prognostic and diagnostic cancer
markers. The main aim of this study was to designate differences between tightly DNA binding protein patterns extracted from rat liver
and rat experimental hepatomas (Zajdela ascites hepatoma and hepatoma G-27) and to evaluate possibility that some of these proteins
may be used as biomarkers for cell cancer transformation. Methods: We used proteomics aproach as a tool for comparison of pattern
of TBP from rat experimental hepatomas and normal liver cells. Combination of 2DE fractionation with mass spectrometry (MALDI
TOF-MS) suitable for parallel profiling of complex TBP mixtures. Results: Intriguingly 2DE protein maps of TBP from rat liver and
rat experimental hepatomas (Zajdela acites hepatoma and hepatoma G-27) were quite different. We identified 9 proteins, some of them
shared in all TBP patterns. Among identified tightly bound to DNA proteins there were three proteins considered as nuclear matrix
proteins (lamin B1, scaffold attachment factor B1, heterogeneous nuclear ribonucleoprotein). Also we identified DNA repair protein
RAD50, coiled-coil domain-containing protein 41, structural maintenance of chromosomes protein1A and some ATP –dependent
RNA helicases indicating that TBP are of interest with respect to their potential involvement in the topological organization and/
or function of genomic DNA. Conclusions: We suppose that proteomic approach for TBP identification may be promising in develop-
ment of biomarkers, also obtained results may be valuable for further understanding TBP functions in genome.
Key Words: tightly to DNA bound proteins, nuclear matrix proteins, biological markers, proteomic analysis.
INTRODUCTION
Search for novel tumour markers is an important
research branch in modern oncology. Several tumour
markers have been found among the nuclear matrix
proteins [1]. The nuclear matrix, operationally defined
as nuclear structure resistant to high salt and detergent
extraction, contains proteins that contribute to the
preservation of nuclear shape and its organization.
Nuclear matrix enables spatial organization of DNA
replication, transcription and repair processes; it har-
bours numerous enzymes and transcription factors
[2–4]. One of the bladder cancer-specific nuclear
matrix proteins (NMP-22) was proposed for use in di-
agnostics commercial kit for its detection in urine
is available from Matritech, Cambridge, Massachusetts.
Utility of the marker and restrictions for its application
are discussed in more than hundred publications [5].
Development of other nuclear-matrix-derived tumour
markers is on line [6]. Nuclear matrix proteins are
tightly bound to DNA as DNA-protein bonds in this
structure are resistant to high salt and mild detergent
treatments. Some researchers doubt in existence
of nuclear matrix in living cells. Isolation procedure
is prone to many artefacts and isolated nuclear matrix
structures are supposed to form due to “molecular
crowding” [7]. Another group of proteins forming tight
complexes with DNA cannot be suspected in artefac-
tual nature due to above considerations. These are the
so-called tightly-bound proteins (TBP). These proteins
remain attached to DNA with covalent or non-covalent
bonds after harsh deproteinization procedures like
treatment with phenol, chloroform, ionic detergents,
proteases, etc. Enrichment of the TBP in specific DNA
sequences was a special interest with respect to spec-
ulation on the potential function of such sequences
in higher order structures of the genome of different
organisms including human, mouse, and chicken [8,
9 and references therein]. Unlike nuclear matrix isolation
procedure that is prone to many artefacts [7], TBP are
easily isolated, their spectrum is well-reproducible.
To our opinion the TBP are prospective for search
of novel tumour markers. Finding of a tissue-specific
spectrum of TBP in plants [9, 10] and animals [11] has
encouraged us to compare spectrum of TBP in rat
normal tissue (liver) and experimental hepatomas with
a goal to find prospective proteins for tumor marker
development.
MATERIALS AND METHODS
Animals. Animals were obtained from the Labora-
tory of Experimental Animals, Riga Stradins University,
Riga, Latvia. All experimental procedures were carried
out in accordance with guidelines of the Directive
86/609/EEC ”European Convention for the Protection
of Vertebrate Animals Used for Experimental and other
Scientific Purposes” (1986) and were approved by the
Received: June 14, 2011.
*Correspondence: Fax: 370-2398231
E-mail: danute.labeikyte@gf.vu.lt
Abbreviations used: TBP — the tightly bound to DNA proteins;
2DE – two dimensional gel electrophoresis.
Exp Oncol 2011
33, 3, 121–125
122 Experimental Oncology 33, 121–125, 2011 (September)
Animal Ethics Committee of the Food and Veterinary
Service (Riga, Latvia). Wistar male rats, each weighing
215.00±5.63 g at the beginning of the experiments,
were used in all the work. The environment was main-
tained at a temperature of 22±0.5 °C with a 12-h light/
dark cycle. The animals were fed a standard laboratory
diet. Strains of Zajdela ascites hepatoma (ZAH) and
G-27 hepatoma were obtained from the Cancer Re-
search Centre (Moscow). 0.5 ml of ZAH ascetic liquid
was inoculated to rats with five day interval. To passage
the G-27 hepatoma 1 ml of tumor suspension was in-
oculated, tumor tissue was collected 14 days later [12].
DNA isolation. Chopped liver or hepatoma tissue
was placed in a Dounce homogenizer, 10-fold extent
of homogenization buffer (w/v) was added (0.25M su-
crose, 0.05M Tris-HCl, pH 7.4; 0.002M CaCl2), 10 fric-
tions were produced. Homogenate was centrifuged
at 1000 g for 10 min. The pellet was again homogenized
by 15 frictions in a buffer containing 50 mM Tris-HCl
pH 7.5; 0.25 M sucrose, 2 mM CaCl2, 1% Triton X-100.
Crude nuclei were pelleted at 1000 g for 10 min. Ex-
traction was repeated. The nuclear pellet was mixed
with appropriate amount of buffer (100 mM Tris HCl
pH7.5; 500 mM NaCl, 50 mM Na2EDTA, 1.25% SDS;
3.8 g/l of sodium bisulfite, 4ml/l of 2-mercaptoetha-
nol). Two latter components were added to the buffer
just before the extraction to obtain a slightly viscous
suspension. The lysate was incubated for 45 minutes
at 65 °C. DNA was extracted with the same volume
of chloroform/isoamylic alcohol mixture (24:1), the
suspension was centrifuged and the water phase
was separated. RNA was separated by precipitation
in 4M LiCl at 4 °C for at least one hour, 12M LiCl solu-
tion was added to the water phase to reach the neces-
sary salt concentration. RNA was pelleted for 10 min
at 10000 g. The chloroform extraction procedure of the
supernatant was repeated again. DNA was precipitated
from the water phase with two volumes of ethanol and
washed in 70% ethanol.
Isolation of TBP complexes with DNA by means
of exhaustive nuclease digestion.
The DNA was digested with DNase I (Fermentas)
(0.01 U/g, room temperature, overnight) in 10 mM Tris-
HCl, pH 7.6; 5 mM MgCl2. Digestion was performed
in a dialysis sack with constant dialysis against digestion
buffer. Completeness of digestion was monitored by gel
electrophoresis. Protein concentration was determined
with the BCA according [13], using crystalline bovine
serum albumine as standard [13]. Proteins were pre-
cipitated with ice-cold 10% (v/v) TCA for 1 h on ice, and
protein pellet was washed twice with ice-cold acetone.
Resulting pellet were dissolved in rehydration buffer
(7M urea, 2M thiourea, 2% CHAPS, 0.5% IPG buffer,
40 mM DTT, 0.002% bromphenol).
2D electrophoresis (IEF/SDS-PAGE).
An 11 cm Immobiline DryStrips with linear pH gra-
dient 5.3–6.5, and Excel Gel SDS, gradient 8–18%
(GE Healthcare, Piscataway, NJ,USA) were used for
2DE. The TBP corresponding 1 mg of DNA dissolved
in 200 l of rehydration buffer and were loaded onto
IPG gel strip; then were reswollen overnight. The iso-
electrofocusing was carried out up to a total of 70 kVh.
Prior to second dimension gel stripes were reduced
and acylated according to the manufacturer’s rec-
ommendations. IEF/ SDS PAGE was performed with
Multiphor II device (GE Healthcare, Piscataway, NJ,
USA). Gels were silver stained according to Shevc-
henko A et al. [14].
Preparation of samples for MALDI-TOF. After
staining, the spots of interest were excised from gel,
crushed to 1 mm2 sized slices and dehydrated with
50% acetonitrile. The gel slices were then dried un-
der vacuum and rehydrated with 30 μl 25 mM NH4H-
CO3 (pH 8.3). Proteins in the gel slices were digested
overnight with 100 ng of modified trypsin (Promega,
Madison, WI, USA) at 37 °C. The peptides were then
washed twice from the gel with 50 μl 5% TFA in 50%
acetonitrile. The wash-outs were collected and dried
under vacuum. For MALDI-TOF, peptides were diluted
in 3 μl of 0.01% trifluoracetic acid (TFA) in 30% aceto-
nitrile. 0.8 μl of each sample with matrix (α-Cyano-4-
hydroxycinnamic acid) were loaded on MALDI plate.
MALDI-TOF (Matrix-Assisted Laser Desorp-
tion/Ionization Time-Of-Flight) analysis. Samples
were analyzed with a MALDI–TOF MS using a Voyager-
DE™ Pro (Applied Biosystems, Framingham, MA).
Positive ionization, acceleration voltage 20 kV, grid
voltage 75%, guide wire 0.02 and the extraction delay
time 200 ns were used to collect spectra in the mass
range of 700−4000 Da. Reflector mass spectra were
acquired and calibrated either externally or inter-
nally, using trypsin autolysis peptides (m/z 842.5200,
1045.5642, 2211.1046).
Data processing of the spectra was performed with
Data Explorer™ Version 4.0 (Applied Biosystems).
Protein identification was performed by searching
in protein sequence database (SwisPro) using Pep-
tIdent and MS-Fit programs available on the ExPaSY
server (http://us.expasy.org/). The following param-
eters were used for databases searches: monoiso-
topic mass accuracy 50–100 ppm, missed cleavages
0–1 and complete carbomethylation of cysteines.
RESULTS
2D electrophoresis. We screened three sepa-
rate sets of TBP for changes of the proteome. The
separation by pI was limited to the range of 5.3–6.5 for
a better resolution of TBP proteins. Proteins separated
by 2D electrophoresis were visualized by staining with
AgNO3. As seen in Figure, the resulting 2DE maps
of tightly bound protein patterns from rat liver and rat
experimental hepatomas (Zajdela ascites hepatoma
and G-27 hepatoma) appeared quite different. 2DE
TBP patterns of Zajdela ascites hepatoma (Figure,
b) and hepatoma G-27 (Figure, c) appeared dif-
ferent also. It was expected that these proteomas
might share similar spot patterns. We suggest that
protein spot pattern alterations hardly occur due
to different protein abundance in TBP patterns. For all
2-DE fractionations we subjected TBP amount which
Experimental Oncology 33, 121–125, 2011 (September) 123
corresponded to 1 mg of DNA. As seen in Figure,
the least amount of TBP is characteristic for hepa-
toma G-27 cells. TBP is represented by numerous
peptides in all TBP samples. The images of 2DE gels
could not be readily overlapped to determine dif-
ferences. Protein spot profiles were analyzed using
image analysis software — Melanie 7 (Swiss Institute
of Bioinformatics). Due to the different spot pattern
of the three sets of TBP, software matching did not
generate useful identification of protein differences.
Using the same spot detection parameters different
number of spots was detected in all preparations. For
example, comparative analysis of corresponding gel
sections as denoted in Figure by frames (pH range
of 5.8–6.4 and molecular mass within the range
of 20–80 kDa) revealed 35 spots in Zajdela acites
hepatoma TBP pattern, 24 spots in liver TBP patern
and 18 in hepatoma G-27 TBP pattern. Only 7 spots
were shared among the all TBP sets. In general, the
TBP pattern appeared to be different in normal and
malignant tissues. Zajdela ascites hepatoma TBP pat-
tern appear to be more numerous and heterogenous.
Fraction of proteins with lower pI is more pronounced
in G-27 TBP pattern.
170
130
100
70
55
40
35
5.3 5.7 6.1 6.5
170
130
100
70
55
40
35
5.3 5.7 6.1 6.5
170
130
100
70
55
40
35
5.3 5.7 6.1 6.5
a b
c
Figure. 2D electrophoresis patterns of TBP isolated from rat liver
cells (a), experimental Zajdela ascites hepatoma (b) and experi-
mental G-27 hepatoma (c). First dimension — Immobiline Dry
Strips 5.3–6.5 pH; second dimension — Excel Gel SDS, gradient
8–18%. Gels stained with AgNO3. Positions of molecular weight
markers are indicated on the left, pH — below. Numbers denote
spots which were identified by MALDI-TOF. Frame denoted the
gel section comparatively analyzed with Melanie 7
Mass spectrometric analysis. Some of protein
spots were subjected to mass spectrometry. Our
criteria for accepting spots for mass spectrometry
analysis were as follows: i) due to the limited sensitivity
of the mass spectrometer only protein spots contain-
ing no less than 1 pmol of protein were subjected
to analysis; we selected similar spots (pI, Mw) shared
among all TBP sets, and some spots which were pres-
ent only in separate TBP pattern; iii) spots for analysis
were chosen following methodological approaches for
proteomic analysis of nuclear matrix proteins [15, 16];
Protein spots were cut out from the gels and sub-
jected to in gel tryptic digestion. Mass fingerprints of the
peptide mixtures were obtained by MALDI TOF mass
spectrometry. Protein identification was performed
by searching in protein sequence database (SwisPro)
using PeptIdent and MS-Fit programs. The following
parameters were used for databases searches: monoiso-
topic mass accuracy 50–100 ppm, missed cleavages
0–1 and complete carbomethylation of cysteines. The list
of proteins identified by PeptIdent program is presented
in Table. The Table presents only proteins with a MASCOT
total protein score > 50 so were considered as real and
as high confidence proteins and covered by peptides
at least 15% of the entire protein sequence.
Surprisingly, we found that the resulting list of iden-
tified proteins from different TBP patterns appeared
quite similar. We consider that such result to some
extent is determined by our criteria of accepting spots
for mass spectrometry and the limited sensitivity of the
mass spectrometer.
Spots (e.g., 3, 4, 7, 8, 10) were detected as frag-
ments of identified proteins. Intriguingly similar spots
(the same pI, Mw) from different gels contained the
same TBP as identified by mass spectrometry. Pep-
tides from spots 3 (liver), 7 (Zajdela acites hepatoma)
and 10 (hepatoma G-27) are identified as DNA repair
protein RAD50. Spots 5 (Zajdela acites hepatoma)
and 11 (hepatoma G27) are homologous to lamin B1.
Spots 1 (liver) and 12 (hepatoma G27) contain pep-
tides homologous to coiled-coil domain-containing
protein 41. In all above cases the spots occupy
similar positions in all the three gels. However spots
2 (liver; Mw≈35 kDa; pI 6.2) and 8 (hepatoma-G27;
Mw ≈60kDa; pI 6.4) are corresponding to peptides
of different size. It was revealed that these peptides
are homologous to “Structural maintenance of chro-
mosomes protein 1A” in both cases. Spot 4 solely
contained peptides homologous to ATP-dependent
RNA helicase. Spot 9 was the only to be homologous
to scaffold attachment factor B1.
Spots 1, 2 and 6 were found to contain two proteins
as identified by MALDI-TOF MS.
DISCUSSION
The identified proteins in all TBP samples are nucle-
ar proteins (Table). This excludes possibly artefactual
binding of the proteins in vitro observed in some cases
[11, 17]. Moreover, ability to form very tight complexes
with DNA was reported to several of these identified
proteins.
As seen in Table some of identified proteins belong
to nuclear matrix. Lamins, heterogeneous nuclear
ribonucleoproteins and scaffold attachment factor
B1 are considered as nuclear matrix proteins. The
nuclear matrix is considered a proteinaceous structure
spatially organizing the interphase nucleus. Neverthe-
les it was demonstrated that TBP-DNA and nuclear
matrix-DNA complexes are different structures [9],
most likely some of nuclear matrix proteins shared
in TBP pattern.
124 Experimental Oncology 33, 121–125, 2011 (September)
Table. Description of TBP proteins identified after 2D electrophoresis and
MALDI-TOF MS1. Proteins are listed with their respective SWISS-PROT pri-
mary accession numbers
Spot2 Identified Protein pI
Swiss-
Prot Ac-
cess Nr
Mass, Da
Nu
m
be
r o
f p
ep
tid
es
3
%
o
f s
eq
ue
nc
e4
Ex
pe
c t
ed
M
ea
su
re
d
1(A) Coiled-coil domain-containing
protein 41
6,2 Q66H89 82 ≈60 18 18
Heterogeneous nuclear ribonu-
cleoprotein M
Q62826 56 19 18
2(A) Structural maintenance of chro-
mosomes protein 1A
6,2 Q9Z1M9 56 35 54 30
Nucleophosmin P13084 33 18 46
3(A) DNA repair protein RAD50 5,7 Q9JIL8 154 ≈35 96 47
4(B) Putative ATP-depen-dent RNA
helicase DHX39
5,7 Q5BJS0 134 ≈38 27 20
5(B) Lamin-B1 5,4 P70615 66 66 43 48
6(B) Heterogeneous nuclear ribonu-
cleoprotein M
6,4 Q62826 74 ≈70 71 40
Structural maintenance of chro-
mosomes pr.1A
Q9Z1M9 143 28 34
7(B) DNA repair protein RAD50 5,7 Q9JIL8 154 ≈35 65 33
8(C) Structural maintenance of chro-
mosomes pr.1A
6,4 Q9Z1M9 143 60 37 18
9(C) Scaffold attachment factor B1 5,4 O88453 105 ≈105 20 15
10(C) DNA repair protein RAD50 5,7 Q9JIL8 154 ≈35 84 42
ATP-dependent RNA helicase
DHX39
Q5U216 49 ≈35 23 41
11(C) Lamin-B1 5,4 P70615 66 ≈70 18 22
12(C) Coiled-coil domain-containing
protein 41
6,0 Q66H89 69 ≈70 27 25
Notes: 1proteins characterized with a MASCOT total protein score > 50 were
considered as real and as high confidence proteins; 2Spots corresponds num-
bers in Fig. TBP isolated from rat liver cells (A); rat experimental Zajdela as-
cites hepatoma B); rat experimental G-27 hepatoma (C); 3number of pep-
tides — number of peptides of the protein identified by mass spectrometry
as peptides belonging to the sequence of identified protein; 4% of the the se-
quence — part of the sequence of identified protein covered by the above —
mentioned peptides
Some TBP proteins are identified as RAD50 are
components of a single protein complex, Mre11-
Rad50-Nbs1 (MRN). The MRN complex consists
of dimers of each subunit and this heterohexamer
controls key sensing, signaling, regulation, and effec-
tor responses to DNA double-strand breaks including
ATM activation, homologous recombinational repair,
microhomology-mediated end joining and, in some
organisms, non-homologous end joining. To organize
the MRN complex, the Mre11 exonuclease directly
binds Nbs1, DNA, and Rad50. Rad50, a structural
maintenance of chromosome (SMC) related protein,
employs its ATP-binding cassette (ABC) ATPase,
Zn hook, and coiled coils to bridge DSBs and fa-
cilitate DNA end processing by Mre11 [18, 19]. Thus
RAD50 keeps attached to DNA a huge multiprotein
complex.
Structural maintenance of chromosomes protein
1A (SMC1A) is structural component of cohesin. Co-
hesin regulates sister chromatid cohesion during the
mitosis and meiosis. In addition, cohesin has been
demonstrated to play a critical role in the regulation
of gene expression. Furthermore, multiple proteins
in the cohesin pathway are also involved in addi-
tional fundamental biological events such as double
strand DNA break repair, chromatin remodeling and
maintaining genomic stability. Composed of several
essential subunits, cohesin forms a ring-like complex
that is thought to embrace sister chromatids, thereby
physically linking them until their timely segregation
during cell division [20].
Several identified polypeptides were homolo-
gous to RNA helicases (Spots No. 4 and 10). These
proteins are now of major interest because they are
known to play important roles in virtually all aspects
of RNA synthesis and function, including nuclear
transcription, pre mRNA splicing, ribosome biogen-
esis, nucleocytoplasmic transport, translation, RNA
decay and organellar gene expression, processes that
involve multi-step association/ dissociation of large
RNP complexes as well as the modulation of complex
RNA structure [21]. Helicases were resently identified
among barely TBP [10].
Lamin B1 is identified exclusively in preparations
of tumour TBP (Spots No.5 and 11). Traditionally lamins
were not considered to form very tight complexes with
DNA. Actually besides being structural components
of the nuclear envelope lamins turn out to be involved
in multiple functions and complex interactions with
other nuclear proteins and DNA. Lamin B1 is involved
in sequestration of the transcription factors [22]. Lamin
B1 maintains the functional plasticity of nucleoli inter-
acting with nucleophosmin, intriguingly nucleophos-
min also revealed in one of the spots [23]. In whole
the nuclear envelope is considered to be a signalling
node in development and disease [24, 25]. Some data
indicate possible involvement of lamins and lamin
receptors in tumour phenotype development [26].
Lamin B interacts with DNA via lamin B receptor [27].
The interaction occurs via linker DNA and is enhanced
by DNA curvatures [28]. Certain genomic elements are
attached to the nuclear lamina, this contributes to the
spatial organization of chromosomes inside the nucle-
us. Sequences in the human genome that interact with
the nuclear lamina in vivo have been already identified.
A map of the interaction sites of the entire genome
with the nuclear lamina shows that genome-lamina
interactions occur through more than 1,300 sharply
defined large domains 0.1–10 megabases in size.
These lamina-associated domains (LADs) are typi-
fied by low gene-expression levels, indicating that
LADs represent a repressive chromatin environment.
The borders of LADs are demarcated by the insulator
protein CTCF, by promoters that are oriented away
from LADs, or by CpG islands, suggesting possible
mechanisms of LAD confinement [29]. Interaction
of silenced genes with the nuclear lamina is mediated
by lamins [30]. Probably presence of Lamin B in the
TBP preparations of rat hepatomas indicates tumour-
progression associated modifications of the above
DNA-lamin interactions.
The nucleus contains many potential cancer
markers [1, 3]. The present study was designed
to determine changes in TBP patterns from normal
and malignant cells. We have analyzed TBP from rat
liver and rat experimental hepatomas (Zajdela ascites
Experimental Oncology 33, 121–125, 2011 (September) 125
hepatoma and hepatoma G-27) to search for candi-
dates of malignant transformation markers. Altogether
we have identified some TBP by mass spectrometry.
TBP pattern proteome alterations of normal and malig-
nant cells evidenced by comparative 2DE-gel analysis
proved that proteomic approach may be promising
in development of biomarkers. At present the nature
and function of many TBP have not been established,
awaiting further investigation. Function of the tightly
bound to DNA proteins in vivo remains an open ques-
tion. Lamins were identified as prospective markers,
however furher research is nessesery to test their utility
for practical applications. Although the further elucida-
tion of the TBP potential for biomarker trawling is nec-
essary due to the limited number of identified TBP,
the proteomic approach has proven to be promising.
ACKNOWLEDGEMENTS
Costs of the work were covered in part from the
Latvian National Research program “A multidisciplinary
study of the main pathologies threatening the quality
of life and longevity of the Latvian population” project
“Creation of diagnostics methods for determination
of cancer risk factors, early diagnostics of tumors and
predisposing diseases, optimization of cancer treat-
ment”, task “To create markers of malignant tumors
on the basis of tightly bound to DNA proteins”.
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Copyright © Experimental Oncology, 2011
|
| id | nasplib_isofts_kiev_ua-123456789-138657 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 1812-9269 |
| language | English |
| last_indexed | 2025-12-07T15:13:29Z |
| publishDate | 2011 |
| publisher | Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| record_format | dspace |
| spelling | Labeikyte, D. Borutinskaite, V. Legzdins, N. Sjakste, N. 2018-06-19T10:59:24Z 2018-06-19T10:59:24Z 2011 Tightly-bound to DNA proteins in rat experimental hepatomas and normal liver cells / D. Labeikyte, V. Borutinskaite, N. Legzdins, N. Sjakste // Experimental Oncology. — 2011. — Т. 33, № 3. — С. 121-125. — Бібліогр.: 30 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/138657 Proteins tightly bound to DNA (TBP) comprise a group of proteins that remain bound to DNA even after harsh deproteinization procedures. The amount of these proteins is 20–100 µg for mg of DNA depending on eukaryotic source. This experimental paper examines the possibility to use some TBP for clinical biomarker discovery, e.g. for identification of prognostic and diagnostic cancer markers. The main aim of this study was to designate differences between tightly DNA binding protein patterns extracted from rat liver and rat experimental hepatomas (Zajdela ascites hepatoma and hepatoma G-27) and to evaluate possibility that some of these proteins may be used as biomarkers for cell cancer transformation. Methods: We used proteomics aproach as a tool for comparison of pattern of TBP from rat experimental hepatomas and normal liver cells. Combination of 2DE fractionation with mass spectrometry (MALDI TOF-MS) suitable for parallel profiling of complex TBP mixtures. Results: Intriguingly 2DE protein maps of TBP from rat liver and rat experimental hepatomas (Zajdela acites hepatoma and hepatoma G-27) were quite different. We identified 9 proteins, some of them shared in all TBP patterns. Among identified tightly bound to DNA proteins there were three proteins considered as nuclear matrix proteins (lamin B1, scaffold attachment factor B1, heterogeneous nuclear ribonucleoprotein). Also we identified DNA repair protein RAD50, coiled-coil domain-containing protein 41, structural maintenance of chromosomes protein1A and some ATP –dependent RNA helicases indicating that TBP are of interest with respect to their potential involvement in the topological organization and/or function of genomic DNA. Conclusions: We suppose that proteomic approach for TBP identification may be promising in development of biomarkers, also obtained results may be valuable for further understanding TBP functions in genome. Costs of the work were covered in part from the Latvian National Research program “A multidisciplinary study of the main pathologies threatening the quality of life and longevity of the Latvian population” project “Creation of diagnostics methods for determination of cancer risk factors, early diagnostics of tumors and predisposing diseases, optimization of cancer treatment”, task “To create markers of malignant tumors on the basis of tightly bound to DNA proteins”. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Tightly-bound to DNA proteins in rat experimental hepatomas and normal liver cells Article published earlier |
| spellingShingle | Tightly-bound to DNA proteins in rat experimental hepatomas and normal liver cells Labeikyte, D. Borutinskaite, V. Legzdins, N. Sjakste, N. Original contributions |
| title | Tightly-bound to DNA proteins in rat experimental hepatomas and normal liver cells |
| title_full | Tightly-bound to DNA proteins in rat experimental hepatomas and normal liver cells |
| title_fullStr | Tightly-bound to DNA proteins in rat experimental hepatomas and normal liver cells |
| title_full_unstemmed | Tightly-bound to DNA proteins in rat experimental hepatomas and normal liver cells |
| title_short | Tightly-bound to DNA proteins in rat experimental hepatomas and normal liver cells |
| title_sort | tightly-bound to dna proteins in rat experimental hepatomas and normal liver cells |
| topic | Original contributions |
| topic_facet | Original contributions |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/138657 |
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