[CANCER RESEARCH 42, 4546-4552. November 1982]
0008-5472/82/0042-0000$02.00
A Nuclear Matrix Antigen in HeLa and Other Human Malignant Cells
Zbigniew Wojtkowiak,1 David M. Duhl,2 Robert C. Briggs,3 Lubomir S. Hnilica,4 Janet L. Stein,5 and
Gary S. Stein5
Department ol Biochemistry and the A. B Hancock, Jr. Memorial Laboratory of the Vanderbilt University Cancer Center, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232 [Z. W., D. M. D., R. C. B., L. S. H.] and Department of Biochemistry and Molecular Biology, The University of Florida School of
Medicine, Gainesville. Florida 32610¡J. L. S., G. S. S.¡
ABSTRACT
Antisera were obtained in rabbits to preparations of dehistonized chromatin from HeLa cells. By complement fixation
assays, the antisera reacted with HeLa cell chromatin but only
marginally with human placenta chromatin. The complementfixing reactivity of the antisera was inversely related to the
amount of dehistonized chromatin used for immunization.
Immunochemical staining of electrophoretically
separated
chromosomal proteins transferred to nitrocellulose sheets re
vealed numerous antigens in chromatin preparations from sev
eral human tumors, placenta, and normal kidney. While immunoabsorption of the antisera with placenta chromatin removed
some of the immunochemical staining, many of the electropho
retically separated antigens resisted repeated immunoabsorptions. However, further comparisons revealed that only one
major protein antigen (band at an approximate molecular
weight of 81,000) was represented in all the assayed human
tumors while being absent from human placenta or kidney.
Fractionation of HeLa cells into three cytoplasmic and sev
eral nuclear fractions showed that almost all the antigens
recognized by antisera to dehistonized chromatin were nuclear.
The antigenic protein with an approximate molecular weight of
81,000 was found associated with the nuclear matrix fraction.
INTRODUCTION
' Present address: Department of Biochemistry. University of Lodz, Banacha
12-16, 90 237 Lodz, Poland.
2 Supported by Grant CA-09313 from the National Cancer Institute.
3 Supported by Grants CA-18389, CA-27338. and CA-26948 from the Na
of
Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee
37232.
5 Supported by National Science Foundation Grant NSF-PCM 801-8075.
Received April 2, 1982; accepted August 4, 1982
4546
specific distribution.
MATERIALS
AND METHODS
HeLa S3 cells were maintained
in suspension
cultures
in Eagle's
minimum essential medium as modified by Joklik and supplemented
with 7% calf serum. Cells were harvested and kept frozen at —¿70°
until further use. HT-29 human colon adenocarcinoma cells, originally
provided by Dr. Jörgen Fogh, Sloan-Kettering Institute, were grown in
McCoy's Medium 5A supplemented with 15% fetal calf serum. The
GW-39 human adenocarcinoma
Differences in chromosomal nonhistone proteins between
normal and malignant tissues have been demonstrated by
electrophoretic procedures (26, 35) or immunologically (14,
36). Dehistonized chromatin preparations used as immunogens
can elicit cell- or tissue-specific antibodies (3, 9, 14, 28).
Indeed, some of these antibodies can detect immunological
changes in chromosomal proteins during chemical carcinogenesis before the appearance of malignant phenotype (7, 8). We
have shown in our previous studies that the specificity of
antibodies to dehistonized chromatin depends on the presence
in the immunogen of complexes between DNA and chromo
somal nonhistone proteins (11, 15, 28, 29). Recent studies
with HeLa cells showed that irradiation or exposure of chro
matin to alkylating agents stabilized these antigenic complexes,
presumably by cross-linking the interacting macromolecules
(17,18). Since antisera to dehistonized chromatin preparations
tional Cancer Institute.
' To whom requests for reprints should be addressed at the Department
must contain antibodies to a number of various nonhistone
proteins, we have investigated the immunological heterogene
ity of chromosomal nonhistone proteins separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose
for direct detection.
In their comprehensive review of nuclear protein structure
and function during carcinogenesis,
Allfrey and Boffa (1)
pointed out the difficulties of comparing nuclear proteins iso
lated from different organs or tumors. All these tissues contain
mixed cell populations, either of various phenotypes or differing
in their proportions of dividing and nondividing cells. To circum
vent this problem, we have raised antisera to dehistonized
chromatin of cells (HeLa) growing in cultures, i.e., represented
by a singular phenotype and presumed to contain antibodies
to proteins of all the cell cycle phases. Our results show that
many nuclear proteins are antigenic and some may exhibit cell-
University of
transplantable
were obtained
Nuclei from
was supplied by Dr. David Goldenberg,
Kentucky at Lexington, and grown as a solid tumor
in unconditioned Syrian hamsters. Other human tissues
from the Vanderbilt University Hospital.
HeLa, normal and leukemic blood cells, and HT-29 cells
were isolated as described by Wilhelm ef al. (34). Human placentas or
the GW-39 tumors were freed of connective tissues, minced, and
suspended in 0.25 M sucrose:! 0 mw Tris, pH 7.5. The minced tissue
was blended in a Waring blendor until all cells were broken as observed
under light microscope. The suspension was filtered through 2 layers,
4 layers, and finally 6 layers of cheesecloth. Crude nuclei were col
lected by centrifugation of the filtrate at 660 x g and washed twice
with 0.25 M sucrose: 10 mw Tris-HCI, pH 7.5. Washed crude nuclei
were then homogenized in 2.2 M sucrose:5 mM MgCI? and centrifuged
at 100,000 x gma. for 1 hr. The transparent nuclear pellet was sus
pended by gentle homogenization in 0.25 M sucrose: 10 mM Tris-HCI,
pH 7.5, containing 0.5% Triton X-100, and the nuclei were collected
by centrifugation at 900 x g for 15 min. Finally, the isolated nuclei
were washed (by gentle homogenization) with 2 mw Tris-HCI, pH 7.5,
and used for the isolation of chromatin (23).
The cytoplasmic protein fractions were prepared from the individual
cell types by extensive homogenization in 0.25 M sucrose: 10 mM Tris,
pH 7.5, (until more than 90% nuclei were released) followed by
centrifugation of the homogenate at 1000 x g for 10 min. The centrif
ugation was repeated once more, and the supernatant was centrifuged
at 100,000 x g for 1 hr. The final supernatant was used as cytoplasmic
protein extract.
The isolation of nucleoli followed the procedure of Busch (4). HeLa
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VOL. 42
Nuclear Antigens in HeLa Cells
cell nuclei were sonicated in 0.34 M sucrose containing divalent cations
until most nuclei were broken. Nucleoli were purified by centrifugation
through 0.88 M sucrose as recommended by the author. Microscopic
examination of the final pellet indicated that nucleoli of considerable
purity were obtained by this procedure. Sonication was also used to
release RNP6 particles from isolated nuclei (20). The sonicate was
centrifuged at 16,300 x g for 15 min to collect the RNP particles (30).
Monomeric nucleosomes were obtained by digestion of isolated
nuclei with micrococcal nuclease followed by extraction of the digested
nuclei with 0.2 mw EDTA and differential centrifugation (32).
Nuclear matrix was isolated according to the method of Berezney
and Coffey (2) by extracting the nuclei with low-magnesium buffer, 2 M
NaCI and Triton X-100. The extracted nuclei were digested with DNase
and RNase before final washing with buffered 0.25 M sucrose contain
ing 5 mM MgCI2. The method of Berezney and Coffey (2) was also
followed to obtain nuclear membranes, with the addition of heparin
treatment as described by Widmer and Parish (33).
Chromatin was dehistonized by treatment with 2.0 M NaCI:5 M
urea: 10 mM sodium phosphate buffer, pH 6.0, as described by Spelsberg ef al. (24) and used for the immunization of New Zealand White
rabbits (28). After the final booster injection, blood was collected by
cardiac puncture, and sera were heat inactivated at 58°and stored at
—¿
20°. It was important to freeze the antisera in small aliquots since
frequent freezing and thawing destroyed their complement-fixing
activ
ity.
The quantitative microcomplement fixation of Wasserman and Levine
(31) was used to test the immunological activity of each antiserum.
Increasing amounts of chromatin were incubated at 4°for 18 hr in the
presence of titrated complement and 0.15 ml of antiserum diluted
1:200. Activated sheep erythrocytes were added and, after incubation
at 37°for 30 min, the extent of hemolysis was determined spectrophotometrically at 413 nm.
For immunoabsorption,
chromatin was washed by suspending it in
10% calf serum:3% bovine serum albumin in 10 mw sodium phosphate:0.14M NaCI, pH 7.2, and centrifuging 30,000 x gmaxfor 15 min.
The chromatin pellet was resuspended in the above solution by homogenization, and antiserum was added in proportion of 1 /il of antiserum to 4 /¿gof chromatin as DNA. The mixture was stirred at 4°
overnight, and the chromatin was removed by centrifugation at 100,000
x gmaxfor 2 hr. The supernatant represented the absorbed antiserum.
Electrophoresis in polyacrylamide gels was performed as described
by Laemmli (16). To prepare the samples, chromatin was sonicated in
an ice bath using six 10-sec bursts each followed by a 30-sec cooling
period. The sonicate was mixed with 0.9 volume of 4.44% sodium
dodecyl sulfate:22.2%
glycerol:Pyronin
Y (25 /ig/ml):0.139
M TrisHCI, pH 6.8, and 0.1 volume of concentrated
2-mercaptoethanol,
peroxidase:antiperoxidase
(diluted 1:100) for 30 min and washed with
10 mM sodium phosphate-buffered
0.14 M NaCI, pH 7.2, and the
antigens were detected by staining with 0.03% diaminobenzidine
0.005% H2O2 in 50 mM Tris-HCI buffer, pH 7.5.
and
RESULTS
Three rabbits were immunized, each with a different amount
of the same preparation of dehistonized HeLa chromatin. All 3
antisera fixed complement at 1:200 dilution in the presence of
HeLa chromatin. However, as can be seen in Chart 1, their
activity was inversely related to the quantity of the dehistonized
chromatin used for immunization. All the 3 antisera reacted
only marginally with human placenta chromatin.
Electrophoretic separation of the HeLa chromatin proteins is
shown in Fig. 1/4. Because of the relatively low polyacrylamide
concentration (7.5%) used to emphasize resolution of highermolecular-weight proteins, the core histones and proteins of
similar molecular weights are not resolved at the leading edge
of the gel. Our previous results with amido black staining of the
proteins transferred to nitrocellulose sheets (13) indicated that
such transfers are representative of the original separations in
the acrylamide gel. Immunochemical localization of reactive
antigens on nitrocellulose transfers (Fig. 1B) showed that many
of the chromosomal protein species were immunogenic in
rabbits. In addition to a number of bands recognized by all 3
antisera (e.g., prominent proteins at approximate molecular
weights of 53,000 to 55,000, 81,000, 100,000, 112,000, and
145,000), several other proteins stained with intensities differ
ent for each antiserum. It is noteworthy that at least 2 bands
(approximate molecular weights of 62,000 and 100,000) de
creased in their intensities proportionally with the loss of com
plement-fixing activity (Chart 1; Fig. 16). Other differences in
staining intensity (e.g., in the low-molecular-weight area and at
approximate molecular weights of 55,000, 69,000 to 70,000,
and 90,000 to 96,000) varied from one antiserum to another
without a predictable pattern. The most reactive antiserum (by
complement fixation) was used in all subsequent experiments.
IOO -
sonicated again for 90 sec without cooling, and left to stand at room
temperature overnight. Solubilized chromatin samples were applied to
vertical polyacrylamide slab gels (1.5 mm thick) containing 10-cm-long
separating gel (7.5%) and 1-cm-long stacking gel (3%). The separated
proteins (50 ma for 3 to 4 hr) were either stained with Coomassie
Brilliant Blue or transferred electrophoretically
to nitrocellulose sheets
(13).
The nitrocellulose transfers were either stained with amido black to
visualize the transferred proteins, or the antigens were detected by the
peroxidaseiantiperoxidase
reaction of Sternberger (25). Briefly, the
transfers were placed in a 10 mM sodium phosphate-buffered
0.14 M
NaCI, pH 7.2, containing 3% bovine serum albumin and 10% heat
inactivated calf serum. After gentle shaking at 40°for 1 hr, the sheets
were incubated at 4°overnight with diluted (1:100) rabbit antiserum to
dehistonized HeLa chromatin. Next, the nitrocellulose sheets were
washed with 4 changes of phosphate-buffered
saline (50 ml for 30 min
each) and incubated for 30 min with antiserum to rabbit immunoglobulins (Serasource)
6 The abbreviation
NOVEMBER
1982
diluted
1:40. Finally, the sheets were placed into
used is: RNP, ribonucleoprotein.
1.25 2.5 5.0
10.0
20.0
CHROMATIN (>jg DNA)
Chart 1. Complement fixation of 3 antisera to dehistonized HeLa chromatin
obtained by immunization with different amounts of immunogen. The assays were
performed in the presence of increasing concentrations of HeLa (O, A, D) or
human placenta (•,A, •¿)
chromatins. The antiserum dilution was 1:200. The
animals were immunized with: 250 ¡ig(as DNA) of chromatin (O, •¿);
500 /xg (as
DNA) of chromatin (A, A); and 750 ng (as DNA) of chromatin d, •¿).
4547
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Z. Wojtkowiak et al.
Polyacrylamide gel electrophoretic analysis showed consid
erable similarity of HeLa and HT-29 chromosomal protein pat
terns, while both were quite different from that of the GW-39
chromatin (Fig. 2A). In the latter, 2 prominent protein bands
can be seen at approximate molecular weights of 40,000 and
180,000. The HeLa antiserum did not contain antibodies to
these 2 proteins, indicating that they may be specific for the
GW-39 adenocarcinoma cell line. As can be seen in Fig. 26,
not all the proteins present in HeLa chromatin were equally
antigenic. At least 6 major chromatin antigens common to HeLa
and HT-29 cells can be observed, all present in much lesser
quantities in the GW-39 chromatin. These are bands at ap
proximate molecular weights of 145,000, 100,000, 81,000,
62,000, 55,000, and 53,000. Two of them, the M, 145,000
and 100,000 proteins, were found in chromatins of all human
tissues examined. The M, 81,000, 55,000, and 53,000 protein
antigens appeared absent from human placenta chromatin as
indicated by the immunoabsorption studies (Fig. 2C). In the
GW-39 cells, there is a relatively strong reacting antigen at an
approximate molecular weight of 50,000 which is staining only
weakly in HeLa and not at all in HT-29 chromatins.
Fig. 2C shows the same chromosomal proteins reacted with
antiserum to dehistonized HeLa chromatin which was absorbed
twice with human placenta chromatin. A number of bands resist
the absorption (e.g., bands at approximate molecular weights
of 145,000, 112,000, 100,000, 88,000, 81,000, 62,000,
55,000, and 53,000). In addition, the staining of M, 50,000
antigen in GW-39 cells also resisted the absorption attempts.
There was a general decrease of reactivity of the multiply
absorbed antisera due to the nonspecific absorption with chro
matin. This phenomenon was apparently responsible for the
disappearance of the M, 145,000 band from the GW-39 chro
matin. There was no staining of the placenta chromatin with
this absorbed antiserum (Fig. 2C).
The reaction of antiserum to dehistonized HeLa chromatin
with electrophoretic transfers of chromosomal proteins from
several human tumors is illustrated in Fig. 3. While the M,
145,000 and 100,000 protein antigens are present in a normal
tissue (i.e., kidney, as shown in this figure), the M, 81,000
protein can be seen only in chromatins from human cancer or
leukemic cells. This protein antigen was also found absent from
normal human lymphocytes (data not shown). Additionally, a
M, 112,000 protein antigen also appears to be significantly
enhanced in the tumors. The M, 53,000 to 62,000 proteins not
seen in the placenta chromatin are strongly represented.
Nuclear localization of the M, 81,000 protein antigen is
documented in Fig. 4. While absent from the cytoplasm, it is
strongly represented in the nuclei. Furthermore, nuclear fractionation indicates that the M, 81,000 antigen is associated
with nuclear matrix. Other particulate nuclear fractions com
pared in Fig. 4 show only small amounts of this protein, prob
ably resulting from contamination of the preparations with
nuclear matrix. This is especially true for the inner nuclear
envelope and RNP particles, both of which are regarded as
possible extensions of the nuclear matrix proper.
DISCUSSION
The nitrocellulose transfer method of Towbin ef a/. (27) for
the detection of individual, electrophoretically
separated anti
gens offers a sensitive tool for rapid screening of polyclonal
4548
antisera for specific antibodies. Although nuclear proteins and
chromatin are considered to be relatively weak immunogens,
our results show that even traces of chromosomal nonhistone
proteins, which are barely detectable by standard electropho
retic methods, are potent immunogens. As indicated by the
complement fixation assays, the immunological response de
pends more on the quantity of the administered immunogen
than on the sensitivity of individual animals. Curiously, this
response, as detected by complement fixation assays, was
inversely related to the amount of administered immunogen.
Immunochemical staining of the electrophoretically separated
total HeLa chromatin proteins points out the immunological
heterogeneity of our antisera. Among the many positive protein
bands, only a few decreased in intensity with the diminishing
trend in complement fixation (e.g., proteins with approximate
molecular weights of 100,000 and 62,000). Although proteins
present in these bands may represent the major complementfixing antigens, this evidence is only circumstantial. It is pos
sible that the majority of complement-fixing
antibodies are
directed to protein components which stain only marginally
with the peroxidaseiantiperoxidase
reaction or, perhaps more
likely, that they recognize large complexes between DNA and
chromosomal nonhistone proteins. Such complexes will dis
sociate in the presence of sodium dodecyl sulfates and the
electrophoretically
resolved components of these complexes
may not be individually reactive. Our work with antisera to
dehistonized Novikoff hepatoma chromatin points to the latter
possibility (22).
Regardless of the lack of major correlations between the
complement-fixing
activity and peroxidase:antiperoxidase
staining of HeLa chromatin and its protein components, the
comparison of electrophoretically separated chromosomal pro
teins from various human malignant cells is of interest. Again,
one is presented with the considerable heterogeneity of anti
bodies comprising the antisera to dehistonized chromatin. Al
though the resolution level is limited to only one dimension, a
considerable number of antigenic proteins can be discerned in
chromatins from HeLa, HT-29, and other malignant cells. When
the staining sensitivity of individual antigens was further en
hanced by fractionation of nuclei, many of the only weakly
detectable antigens increased in their staining intensities. Nu
clear fractionation also demonstrated that the M, 81,000 anti
gen, strongly represented in human malignant cells, is associ
ated with nuclear matrix.
On the present level of detection, several major protein
bands (at approximate molecular weights of 145,000, 112,000, 100,000, 88,000, 81,000, 62,000, 55,000, and 53,000)
resisted repeated absorption of the antiserum with human
placenta chromatin. This indicates that these proteins are
essentially absent from human placenta and that at least some
of them may be specific for HeLa cells. The staining pattern of
chromatin proteins from the human colon adenocarcinoma
GW-39 presented antigens comparable to those of the HeLa
or HT-29 chromatins. However, some of the bands, notably
those at approximate molecular weights of 145,000, 81,000,
and 55,000 to 53,000, were considerably weaker. An addi
tional protein with an approximate molecular weight of 50,000,
represented only weakly in HeLa chromatin, was much more
positive in the GW-39 tumor. Activity to this latter protein was
not absorbed with placenta chromatin. Since the GW-39 tumor
is maintained and harvested in Syrian hamsters, it is conceivCANCER
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Nuclear Antigens in HeLa Cells
able that the weaker staining of the GW-39 chromatin is the
result of antigen "dilution" by chromatin from hamster cells
present in the transplanted tumors.
Human placenta, because of the embryonic origin as well as
proliferative capacity of the trophoblast, is often used in com
parative studies on tumor markers. Our results comparing the
immunological specificity of chromosomal proteins from 5 var
ious human malignant cell types cast some doubt on the value
of such practice. With the exception of proteins with approxi
mate molecular weights of 81,000 and possibly 112,000, all
the other major HeLa antigens could be detected electrophoretically in human kidney chromatin.
The number of antigens detected by our antigens may appear
rather low in view of the anticipated extensive complexity of
chromosomal proteins assumed to participate in the transcriptional regulation of specific genes. However, when analyzed
electrophoretically,
the immunogen (i.e., dehistonized chro
matin) presented only 4 major bands at approximate molecular
weights of 55,000, 81,000, 145,000, and 200,000. However,
as documented by the staining of numerous protein bands in
total tumor chromatin protein patterns, even these antigens
present in dehistonized chromatin only in very low concentra
tion were antigenic. It is conceivable that, when chromatin
fractions instead of the total chromatin are subjected to im
munological analysis, many more specific proteins can be
detected.
The nuclear fractionation experiments established that the
M, 81,000 protein present in largely enhanced quantities in
several human cancers, although noticeable in all nuclear
fractions, is concentrated in the nuclear matrix. It is obvious
that even the most careful fractionation attempts will result in
variable degrees of cross-contamination between the individual
fractions. This is especially true for the nuclear matrix since it
is believed to permeate the entire nucleus. The somewhat
larger presence of the M, 81,000 antigenic protein in the inner
nuclear membrane and RNP fractions supports this interpre
tation since these 2 nuclear organelles are believed to be
associated with the nuclear matrix.
The distribution of other nuclear antigens in the individual
nuclear fractions further indicates that although not absolutely
efficient, the fractionation procedures used in our experiments
yielded fractions considerably enriched in the individual organ
elles. Our experiments also demonstrate that a large majority
of the observed antigenic proteins are indeed nuclear since
they are virtually absent from the ribosomal pellets and cytosol.
No attempts were made to further purify the crude mitochondrial fraction (the 10,000 x g precipitate). Consequently, it
was contaminated with nuclear debris. This contamination is
documented by the presence, although in reduced quantities,
of nuclear antigens in this fraction.
Several nuclear proteins, similar in molecular weights to
some of the antigens described here, were reported in the
literature. The antigen with an approximate molecular weight
of 112,000 may be identical to the 110/8.4 DNA-binding
phosphoprotein identified by Durban et al. (12) in the nuclei of
rat hepatoma as well as HeLa and Namalwa cells (both human).
It is noteworthy that, when chromatin from 32P-labeled HeLa
cells was electrophoresed and subjected to autoradiography
(results not shown), both antigens with approximate molecular
weights of 100,000 and 112,000 were found extensively phosphorylated. Also phosphorylated to a considerable extent were
NOVEMBER 1982
the major HeLa antigenic proteins with approximate molecular
weights of 81,000, 62,000, and 53,000 to 55,000.
A protein antigen of M, 54,000 has been identified with the
specific immunohistochemical staining of nucleoli in many hu
man tumors (5, 6,10). This 54/6.3 antigen is readily solubilized
by the extraction of chromatin or nuclei with low-ionic-strength
buffers (e.g., 10 mw Tris, pH 8.0) and is probably lost during
the isolation of HeLa chromatin and its dehistonization.
Using 2-dimensional electrophoresis, Wu et al. (35) com
pared nuclear proteins from 7 human tumors and normal cells.
Two proteins spots, 54/6.6 and 140/7.7, were found in all
tumor cells but not in the normal cells studied. Since we have
compared our chromatin only by one-dimensional electropho
resis, we cannot comment with confidence on whether, similar
to the nuclear 54/6.3 antigen, the 54/6.6 protein is removed
by dehistonization. The M, 145,000 protein antigen detected
by the antisera to dehistonized HeLa chromatin in our chro
matin preparations may be identical to the 140/7.7 protein of
Wuefa/. (35). This protein appears to be strongly immunogenic
since it can be detected only with difficulty in chromatin pat
terns stained with Coomassie brilliant blue. Its immunochemical
staining in nitrocellulose transfers of such patterns is quite
strong, and consequently it is possible that we were able to
detect its presence in normal cells, even if present in very small
amounts.
Cellular heterogeneity, both in terms of phenotypic expres
sion and cell cycle kinetics, complicates direct comparative
studies on nonhistone proteins in normal tissues and malignant
tumors. These difficulties were discussed in detail by Allfrey
and Boffa (1 ), who cautioned that many of the findings reported
in the literature may reflect differences in cell populations
rather than tumor specificity. With this in mind, we have raised
antisera to one cell type, the HeLa S3 cell chromatin. This
permitted us to address the question of whether selected
antigens found in HeLa cells are present also in other cell types
or tissues. However, because the log phase population used in
our experiments contained cells at all stages of the cell cycle,
we cannot exclude the possibility that the M, 81,000 antigen,
found in several lines of human malignant cells, reflects only
rapid cell division instead of being characteristic for the malig
nant phenotype. The absence of this antigen in nondividing
cells such as normal human lymphocytes or in tissue with slow
turnover (kidney or term placenta) may support this possibility.
Although several authors have used 2-dimensional polyacrylamide gel electrophoresis to compare proteins of normal and
malignant cells, we have elected to use the single dimension
system only. The exceptional specificity of antigen-antibody
interactions circumvents some of the problems of protein de
tection by conventional staining procedures and, as indicated
by our recent work on nuclear antigens in Novikoff hepatoma
(22), it can lead to a rapid selection of cell-specific protein
species. Only after the specificity has been established by the
convenient and rapid unidirectional analysis, the more elabo
rate 2-dimensional analysis of the immunoreactive band pro
teins offers specific advantage by allowing detailed analysis of
the immunoreactive species. As a rule, a more specific antiserum raised to proteins comprising the indicated antigenic band
is necessary for the 2-dimensional analysis (21).
Presently, we do not know the significance of the M, 81,000
phosphoprotein found in rapidly proliferating human cells. It
was shown that nuclear matrix plays an important role in DMA
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Z. Wojtkowiak et al.
replication (19). According to our observations, this protein
antigen is significantly enriched in nuclear matrix of HeLa cells.
It is conceivable that the M, 81,000 antigen plays a role in the
process of DMA synthesis.
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Fig. 1. Electrophoretic separation of proteins reacting with 3 antisera to dehistonized HeLa chromatin. A. polyacrylamide gels (stained with Coomassie brilliant
blue). Lane M, molecular weight standards: myosin (200,000); /i-galactosidase (116.500); phosphorylase B (94,000); bovine serum albumin (68,000); and ovalbumin
(43,000). tañes / to 3. HeLa cell chromatin. B, localization of immunoreactive antigens transferred to nitrocellulose from polyacrylamide gels with 3 different antisera
to dehistonized HeLa chromatin. The antisera were: 1. to 250 ^g; 2, to 500 ;ig; and 3, to 750 /jg of dehistonized chromatin (as DNA). All antisera were diluted 1:100.
The approximate molecular weights of the major antigenic protein bands are indicated in the margin.
Fig. 2. Comparison of antigenic proteins transferred to nitrocellulose sheets. Antiserum 1 (Chart 1; Fig. 1) to dehistonized HeLa chromatin (250 /ig of immunogen)
was used in all experiments at a dilution of 1:100. A. polyacrylamide gel electrophoresis (stained with Coomassie brilliant blue). Lane M, molecular weight standards
(same as in Fig. 1); Lane 1. human placenta; Lane 2. HeLa; Lane 3, HT-29: and Lane 4, GW-39 chromatins. B. proteins shown in A after electrophoretic transfer to
nitrocellulose sheets and immunological staining with Antiserum 1 and peroxidase:antiperoxidase.
C. proteins shown in A, transferred to nitrocellulose sheets and
stained with Antiserum 1 which was absorbed twice with human placenta chromatin. Approximate molecular weights of the major antigens are indicated in the
margins.
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VOL. 42
Nuclear Antigens in HeLa Cells
M
I
2
3
I
2
3
—¿145,000
112,000
—¿100,000
81,000
62,000
—¿55,000
B
M
234
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-<_
_II2,000
_IOO,000
—¿145,000
112,000
—¿100,000
—¿81,000
—¿81,000
62,000
—¿55,000
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A
NOVEMBER 1982
62,000
55,000
. 50,000
B
4551
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Z. Wojtkowiak et al.
l
23456
145,000
112,000
100,000
Fig. 3. Immunochemical comparison of chromosomal proteins from various
human cells. Electrophoretically separated proteins were transferred to nitrocel
lulose sheets and stained with Antiserum 1 (Fig. 1) to HeLa dehistonized chromatin. Lane 1. human kidney; Lane 2. chronic myelocytic leukemia; Lane 3,
chronic lymphocytic leukemia; Lane 4, HeLa; Lane 5, HT-29; Lane 6, GW-39
chromatins. Approximate molecular weights of the major antigens are indicated
on the margin. A pattern similar to that of human kidney was also obtained for
human lymphocyte chromatin.
M
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62,000
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Fig. 4. Electrophoretic comparison of proteins from various fractions of HeLa nuclei and cytoplasm. The separated proteins were transferred to nitrocellulose
sheets and stained with Antiserum 1 (Fig. 1) to HeLa dehistonized chromatin. Lane M, molecular weight standards; Lane /, whole nuclear envelope; Lane 2, nuclear
envelope from nuclei washed with Triton X-100; Lane 3, total chromatin; Lane 4, nuclear matrix; Lane 5, nucleoli; Lane 6, RNP particles; Lane 7. monomeric
nucleosomes; Lane 8, whole nuclei washed with Triton X-100; Lane 9, 10,000 x g cytoplasmic pellet; Lane 10, 100,000 x g cytoplasmic pellet; and Lane /),
100,000 x g cytoplasmic supernatant. A, polyacrylamide gels (stained with Coomassie brilliant blue), ß,localization of immunoreactive antigens transferred to
nitrocellulose from polyacrylamide gels.
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VOL. 42
A Nuclear Matrix Antigen in HeLa and Other Human Malignant
Cells
Zbigniew Wojtkowiak, David M. Duhl, Robert C. Briggs, et al.
Cancer Res 1982;42:4546-4552.
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