¡CANCERRESEARCH 47, 3266-3272, June 15, 1987]
Novel Cell Cycle-related Nuclear Proteins Found in Rat and Human Cells with
Monoclonal Antibodies1
Ann Black, James W. Freeman, Guohao Zhou, and Harris Busch
Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030
five monoclonal antibodies which exhibit a cell cycle-dependent
distribution pattern; the molecular weights of these proteins
were not reported.
Monoclonal antibodies to other PCNA-like antigens have
recently been reported including, a A/r 86,000 nuclear protein
(12, 19) and a nuclear protein recognized by the Ki 67 antibody
(20-22). The M, 86,000 antigen has been shown to be associated
with a M, 70,000 peptide and binds to DNA (19). The M,
86,000-70,000 proteins are very similar to the Ku antigen (M,
80,000) recognized by autoimmune sera (23, 24). The antigen
recognized by the Ki 67 antibody is present in S and G2 phases
and remains bound to the mitotic chromosomes. The Ki 67
antibody has been proposed as a prognostic tool in evaluating
the proliferative degree of malignant non-Hodgkin's lymphoma
ABSTRACT
Monoclonal antibodies which react specifically with the nuclei of
interphase cells recognized three nuclear antigens with molecular weights
of 110,000 (pi 10), 85,000 (p85), and 18,000 (pl8). pi 10 and p85 were
found in eight tumor cell lines but were not found in resting lymphocytes.
pl8 was found in resting lymphocytes as well as the tumor cell lines.
Protein p85 appeared in phytohemagglutinin-stimulated lymphocytes in
the GI phase and protein pi 10 appeared in the S phase, pi 10 and p85
were localized to the extranucleolar chromatin while p IS was distributed
throughout the nucleus and was determined by microscopic and DNase
digestion studies to be DNA associated. The anti-pi 10 antibody recog
nized a component of the DNA polymerase a2 complex. Three novel
nuclear proteins were identified using monoclonal antibodies. Two of
these proteins (pi 10 and p85) are proliferating cell nuclear and nucleolar
antigen-like while the third (pl8) is not cell cycle dependent.
(21), as has a nucleolar PCNA recognized by a polyclonal serum
(9). A M, 145,000 nucleolar antigen has been identified (25)
which is associated with growing cells (26). The M, 145,000
nucleolar protein was readily detectable in growing and dividing
HL-60 human promyelocytic leukemia cells but not after retinoie acid-induced differentiation (27).
The present study was developed to identify potential PCNA
proteins using monoclonal antibodies developed against HeLa
nucleolar extracts. Several hybridoma clones from different
fusions produced bright nuclear immunofluorescence on HeLa
cells. These MAbs identified two novel nuclear antigens that
were expressed specifically in cycling cells.
INTRODUCTION
The G|-S phase of the cell cycle is crucial in determining
whether the cell proceeds from the resting G0 state to initiate
DNA synthesis and becomes committed to cell division (1, 2).
The precise mechanisms which permit cell proliferation to
proceed beyond the "restriction point(s)" are not known, but it
is suggested that an accumulation of "trigger proteins" must
occur to continue the mitogenic cascade (3). If such an accu
mulation of specific proteins occurs, then phase-specific pro
teins should be demonstrable in the d-S phase.
Phase-specific PCNA2 were detected with autoimmune sera
during the d-S phase of the cell cycle (4-9). With two-dimen
sional gel electrophoresis, the protein referred to as cyclin (A/r
36,000/pI 5.0) was found to be a PCNA which appears at the
d-S interface (10-12). Further studies have shown that the
synthesis of cyclin correlates directly with the proliferative state
of normal and transformed cells (13). Celis et al. (14) have
shown that some of these autoimmune sera react specifically
with cyclin. Celis et al. ( 13) suggest that cyclin may be important
in the identification of blast cells in leukemia patients and in
determination of nuclear synchrony.
Busch et al. (15,16) have developed rabbit polyclonal antisera
to nucleolar proteins. These antisera were shown to distinguish
malignant tissues from normal, resting tissues suggesting that
some nucleolar antigens may be associated with cell growth
(15, 16). Polyclonal antisera generated against a spectrum of
HeLa proteins revealed changes in the expression of particular
proteins during the cell cycle (17). Bhorjee et al. (18) developed
Received 11/10/86; revised 2/24/87; accepted 3/20/87.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1These studies were supported by Cancer Research Center Grant CA-10893,
PI, awarded by the National Cancer Institute, Department of Health and Human
Services, I Sl'l IS: the DeBakey Medical Foundation; the Pauline Sterne Wolff
Memorial Foundation; an H. Leland Kaplan Cancer Research Endowment; and
the Linda and Ronny Finger Cancer Research Endowment Fund.
2The abbreviations used are: PCNA, proliferating cell nuclear and nucleolar
antigens; MAb, monoclonal antibody; FCS, fetal calf serum; PMFS, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel
electrophoresis; ELISA, enzyme-linked immunosorbent assay; PHA, phytohemagglutinin; pi 10, M, 110,000 protein; p85, M, 85.000 protein; pl8, M, 18,000
protein; HMG, high mobility group.
MATERIALS
AND METHODS
Cells. Human tumor cell lines HeLa S3 and Hep-2 (obtained from
the American Type Culture Collection, Rockville, MD), HL-60 (human
promyelocytic leukemia, obtained from B. Dowell), human colon tumor
lines HCT 116, GEO, JVC, and Moser (obtained from M. Brattain)
and rat Novikoff cells (obtained from Bob Ochs) were grown in Dulbecco's minimum essential medium containing pyruvate, nonessential
amino acids, and glutamine supplemented with 10% FCS. Hybridoma
cells were grown in Dulbecco's minimum essential medium supple
mented with 20% FCS. Cells were maintained at 37°Cin a humidified
5% CO2 incubator.
Isolation of Nuclear Proteins. Nuclei were isolated from HeLa S3
cells as described by Yaneva et al. (19). Nuclear proteins were extracted
from the isolated nuclei in 10 mM Tris-HCl (pH 8.0), 0.2 HIMPMSF,
1 Me/ml leupeptin, and 1 /jg/ml aprotinin. The mixture was centrifuged
at 12,000 x g for 15 min after a 2-h incubation at 4*C, and the
supernatant was collected as the source of nuclear antigen.
Isolation of Nucleolar Proteins. HeLa S3 nucleoli were isolated as
described by Busch and Smetana (28). Nucleolar proteins were ex
tracted from the isolated nucleoli in 10 mM Tris-HCl (pH 7.5), 20 mM
dithiothreitol, 0.2% deoxycholate, 10 mM KC1, 0.5 mM MgCl2,0.5 mM
PMSF, and 1 ¿ig/mlleupeptin and aprotinin. The extracted nucleolar
proteins were partially purified from protein C23 by 30% (NH^iSCX
precipitation, as described by Freeman et al. (25). The precipitated
proteins were dissolved in 2 M urea/1 M NaCI and were used as the
source of antigen for immunization.
Immunization. Female BALB/c mice were immunized with extracted
nucleolar proteins as described by Freeman et al. (29). Subsequent cell
fusion and cloning were also as described by Freeman et al. (29). Initial
screening of clones from several fusions revealed clones which reacted
with whole nuclei and nucleolar fractions of HeLa cells. Antibodies to
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CELL CYCLE-RELATED
NUCLEAR
nuclear proteins could be the result of common nucleolar and nuclear
elements or nuclear "contaminants" of the nucleolar extracts.
Antibody Typing. The class and subclass identity of each mouse
monoclonal antibody was determined by Ouchterlony immunodiffusion
using a typing kit obtained from Miles Scientific (ICN Immunobiologicals, Irvine, CA). All four antibodies are IgGs. 5C2, 9F9, and 4F8 are
IgGl; 5C7 is an IgG2b (Table 1).
Immunofluorescence. HeLa S3 cells were grown on 10-well microscope slides (Roboz Surgical). The cells were fixed in 2% paraformaldehyde in PBS, pH 7.4, for 30 min at room temperature and then
washed 3 times in PBS for 15 min each. The fixed cells were permeabilized with acetone at -20°Cfor 10 min, followed by 3 washes in PBS.
The cells were incubated with monoclonal antibody culture supernatants for l h at 37°Cin a humidified chamber, followed by 3 washes
for 15 min in PBS. The cells were incubated with a 1/50 dilution of
fiuorescein isothiocyanate-conjugated goat anti-mouse immunoglobulins (Boehringer-Mannheim) at 37°Cfor 30 min, washed 3 times in
PBS for 15 min each, and observed with a Zeiss fluorescent microscope.
SOS-PAGE and Immunoblotting. Proteins were separated by electrophoresis on 7.5 or 12.5% SDS-polyacrylamide gels according to the
procedure of Takacs (30). Gels were either stained with 0.3% Coomassie
brilliant blue or silver (31) or transferred to nitrocellulose paper (0.45
/¿m;Schleicher and Schuell, Keene, NH) as described by Towbin et al.
(32). Immunoblotting of the bound proteins with the monoclonal
antibodies was as described by Freeman et al. (29).
Protein Assay. Protein concentrations were determined by the BioRad protein assay (33).
ELISA Assay. Antigen was absorbed onto microtiter plates (Inumi
Ion I) overnight at 4"( '. Nonspecific binding was inhibited by blocking
in 10% chicken serum-3% bovine serum albumin in PBS, pH 7.5, for
1 h. The monoclonal antibodies (hybridoma culture supernatants) to
nuclear proteins were the primary antibodies, and goat anti-mouse
conjugated to peroxidase (Jackson Imunoresearch) diluted 1:1000 in
PBS:0.5% Tween 20 was the secondary antibody. The color was devel
oped by the addition of 0.02% H2O2 and 300 nM 2,2'-azino-di-[3'ethylbenzthizolin-sulfonate (6)]. The reaction was terminated after 30
min by the addition of l M NaF. The absorbance was read at 405 nm
on an ELISA reader (Fischer).
Sucrose Gradient Fractionation. HeLa cell nuclear extracts in 10 m\i
Tris-HCI, pH 8.0, with protease inhibitors (1 ng/m\ leupeptin, aprotinin, and 0.2 mM PMSF) were layered on top of linear 5-45% (w/v)
sucrose density gradients, made up in the same buffer. The gradients
were centrifuged for 16 h at 28,000 rpm on an SW28 rotor (Beckman,
Munich, Germany) at 4°C.Gradient fractions of 0.4 or 1.2 ml were
collected, and antigen was detected by ELISA assay and immunoblotting. Lactate dehydrogenase (7.3S), catatase (I I. IS), and apoferritin
( 17.6S) were used as markers.
Cell Cycle Analysis. Normal human peripheral blood lymphocytes
(obtained from four individuals at separate times) were isolated from
30 ml of peripheral blood using Ficoll type FP (Sigma; F8626). The
isolated lymphocytes were counted and 1 x 10*cells/ml were added to
Table 1 Comparison of the four nuclear monoclonal antibodies
5C7IgG
5C2
class
Antigen
weightImmunofluorescence
molecular
2b110,000
9F9
4F8
1
110,000
1
85.000
18,000
Tumor cell lines
HeLa
Hep-2
HL-60
HCT 116
GEO
JVC
Moser
NovikofT1
Normal human lymphocytes
Nonproliferating
Proliferating
°+, low level positive Immunofluorescence; ++, high positive immunofluorescence; +++, very intense immunofluorescence.
PROTEINS
FOUND WITH MAbs
RPMI 1640(K.C. Biological) supplemented with 10% PCS (Hazelton).
In PHA-stimulated cultures, the concentration of PHA was 5 tig/mi
(Sigma; L-8504); control cultures did not contain PHA. The cells were
incubated for 72 h at 37°Cin a humidified 5% CO2 incubator.
For cell cycle analysis, isolated lymphocytes were cultured as de
scribed above except autologous human serum was used instead of FCS.
Control cells were taken at 0 h, prior to addition of PHA. Cells were
collected after PHA stimulation at 6, 22, 44, and 68 h. These studies
were done in two independent experiments.
Cells were attached to a slide surface by cytocentrifugation at 1000
rpm for 5 min. The cells were prepared for immunofluorescence as
described above.
RESULTS
Immunofluorescence Localization. Those antigens exhibiting
nuclear localization in HeLa cells were tested for PCNA-like
properties by comparative immunofluorescence in resting and
PHA-stimulated lymphocytes and in tumor cell lines. Four
MAbs (5C2,9F9,4F8, and 5C7) were selected for further study
because three of these antigens possessed PCNA-like properties
(5C2, 9F9, and 4F8) and one antigen (5C7) was present in all
cells and was used in these studies as a control. MAbs 5C2 (Fig.
ÃŒA)
and 9F9 (Fig. IB) which recognized a M, 110,000 protein
(see below) and M Ab 4F8 (Fig. 1C) which recognized a A/r
85,000 protein each produced bright nuclear immunofluorescent staining in HeLa S3 cells; in each case the nucleoli were
negative and appeared as dark spots. With these MAbs, the
immunofluorescence staining at mitosis was found throughout
the cell and was not associated with the chromosomes. On the
other hand, M Ab 5C7 which recognized a M, \ 8,000 antigen
produced bright homogeneous nuclear immunofluorescent
staining in HeLa S3 cells and the nucleoli were included in the
immunostaining. At mitosis, the immunofluoresence remained
associated with the condensed chromosomes. Other human
tumor cell lines and NovikofT hepatoma cells had the same
immunofluorescent staining pattern as seen in HeLa S3 for all
four monoclonal antibodies (Table 1).
Immunoblot Analysis. Immunoblots prepared with these
MAbs using rabbit anti-mouse antibody as a second antibody
and I25l-Protein A, were used to determine the molecular
weights of the antigens. Nuclear extracts (Fig. 2, Lane A) from
HeLa S3 cells were the source of antigen. MAbs 5C2 and 9F9
(Fig. 2, Lanes B and C) recognized an approximately A/r
110,000 polypeptide. Competitive immunoblots revealed that
these two MAbs recognize the same antigen (data not shown).
A A/r 85,000 polypeptide was recognized by MAb 4F8 (Fig. 2,
Lane D)\ and MAb 5C7 (Fig. 2, Lane E) recognized a polypep
tide with a molecular weight of approximately 18,000.
Sucrose Density Gradient Analysis. HeLa S3 nuclei extracted
in 0.01 M Tris, pH 8.0, were subjected to linear sucrose density
gradient fractionation to determine whether the polypeptides
recognized by the individual antibodies were associated with
any high molecular weight particles (deoxyribonucleoprotein or
ribonucleoprotein). Peak ELISA activity of MAb 5C2 and 9F9
(pi 10) was found in fraction 20 (Fig. 3, a and b, respectively)
which corresponds to a particle of 10-15S. Analysis for MAb
4F8 (p85) showed immunoreactivity in the ELISA assay in
fraction 16 (Fig. 3c), which corresponds to an approximately
IOS particle.
Every fourth fraction was applied to a 7.5% SDS gel, and the
proteins were separated by electrophoresis. Silver staining re
vealed that most fractions contained many proteins (Fig. 4)
when compared with the original nuclear extract (Fig. 4J).
Fractions 12,16, and 20 (Fig. 4, Lanes G, F, and E respectively)
contained protein bands which corresponded to the M, 110,000
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CELL CYCLE-RELATED
NUCLEAR
PROTEINS
FOUND
WITH MAbs
Fig. I. Immunofluorescence of HeLa cells
fixed in 2% paraformaldehyde and permeabilized with acetone. (A) MAb 5C2 labels the
nuclei of interphase cells but does not label the
nucleoli. In mitotic cells, the label is in on the
cytoplasm and is not bound to the chromo
somes, seen here in an anaphase cell (arrow).
(B) MAb 9F9 labels the interphase nuclei but
is not found in the nucleoli. In mitotic cells the
chromosomes are not labeled while the cyto
plasm has a low level of reactivity. (C) MAb
4F8 also labels interphase nuclei but not nu
cleoli. As with MAb 5C2 and 9F9. mitotic
chromosomes were not stained with MAb 4F8.
(D) MAb 5C7 has a homogeneous nuclear
staining pattern and is found associated with
the chromosomes at mitosis (arrows). (E) Hybridoma-negative control shows background
staining. (F) Phase-contrast microscopy off.
and the M, 85,000 proteins. When gels were transferred to
nitrocellulose and immunoblotted, the M,\ 10,000 protein was
mainly detected in fractions 16 and 20 (Fig. 5A) and the M,
85,000 protein was detected in fractions 16 and 20 (Fig. 5B).
The immunoblot data on pi 10 and p85 confirmed the results
obtained by ELISA on sucrose density gradient fractionation.
When the ELISA activity of MAb 5C7 (pi 8) was tested with
0.01 M Tris nuclear extracts subjected to sucrose density gra
dient fractionation, no definitive peak was found (Fig. 6/0;
instead, a gradual increase was found across the gradient.
Immunoblot analysis of the gradient fractions with MAb 5C7
did not show detectable levels of pl8.
When HeLa S3 nuclear extracts were treated with DNase 1
(Cooper Biomedicai; 50 /ig/ml/120 units) in the presence of
1.5 mM MgCl2, pi8 was released and was found by ELISA
assay at the top of the gradient (Fig. 6Ä).The pi 10 and p85
polypeptides were unaffected by DNase I.
PCNA. To evaluate whether the polypeptides were associated
specifically with proliferating cells, immunofluorescent local
ization was done on resting and PHA-stimulated human pe
ripheral blood lymphocytes. In control resting lymphocytes,
MAb 5C7 (pi8) immunostained the nuclei; the pattern was
similar to that obtained for human cancer cell lines (Table 1).
Proteins p 110 and p85 were not observed in control, resting
lymphocytes by immunofluorescence. All four antibodies la
beled PHA-stimulated lymphocytes in a pattern consistent with
that obtained in HeLa S3 cells (Table 1).
Cell Cycle Analysis. To determine the time of expression of
the antigens, a time course study was performed on PHAstimulated normal human lymphocytes. The M, 18,000 polypeptide was detectable throughout the time course studied. The
M, 85,000 protein was undetectable at 0 h; it was detected at 6
h and remained so throughout the time course. The M, 110,000
protein was undetectable at 0 and 6 h: it was weakly detectable
at 22 and 44 h and was more evident at 68 h. Cells (1 x IO5)
from each time point were dissolved in Laemmli buffer, sepa
rated by SDS-PAGE (Fig. 1A), and transferred to nitrocellulose.
Immunoblot analysis detected the M, 110,000 protein at 68 h
(Fig. IB, Panel a, Lane E); the M, 85,000 protein was detected
at 44 and 68 h (Fig. IB, Panel b, Lanes D and £);and the M,
18,000 protein was detected at 22, 44, and 68 h (Fig. IB, Panel
c, Lanes C, D, and E).
DNA Polymerase a2 Complex. To determine whether the
growth-related polypeptides (i.e., pi 10 or p85) were associated
with the DNA polymerase a2 complex of HeLa cells, purified
DNA polymerase a2, obtained from Earl F. Baril, Worcester
Foundation, was fractionated on a 7.5% polyacrylamide gel and
transferred to nitrocellulose. When reacted with the antibodies,
p85 was not detectable in the fractionated complex. However,
pi 10 appears as a prominent band in the fractionated complex
(Fig. 8).
DISCUSSION
Of the proteins detected with the four monoclonal antibodies,
the M, 18,000 peptide was located exclusively in the nuclei of
3268
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CELL CYCLE-RELATED
D
NUCLEAR
PROTEINS
FOUND WITH MAbs
A
E
B
C
D
E
F
G
H
I
J
I
200-
200-
-43 S
116;
92.5-
•
'. 68»
4543-
31-
21.5Fig. 2. SDS-PAGE and immunoblotting. Proteins extracted from HeLa nuclei
were fractionated by 7.5% (Lanes A-D; molecular weight markers on left) or
12.5% (Lane E: molecular weight markers on right) SDS-PAGE, transferred to
nitrocellulose and immunoblotted. (A) Coomassie blue stain gel of nuclear extract.
(B) A M, I10,000 protein was immunostained with M Ab 522. (C) A similar M,
110,000 protein was immunoslained with M Ab 9F9. (D) A M, 85.000 protein
was immunostained with MAb 4F8. (E) A M, 18.000 protein was immunostained
with MAb 5C7.
Fig. 4. Silver-stained SDS-PAGE of sucrose density gradient fractions; 100 ¡t\
of each fraction or 50 MÕ
of original 0.01 M Tris nuclear extract were diluted in
2x Laemmli buffer and applied to a 7.5% gel. Lane A, fraction 36; Lane B,
fraction 32; Lane C, fraction 28; Lane 1). fraction 24; Lane E, fraction 20; Lane
F. fraction 16; Lane G, fraction 12; Lane H, fraction 8; Lane I, fraction 4; Lane
J, original extract. Arrowhead, position of pi 10; arrow, position of p85.
at the top of a linear sucrose density gradient following DNase
I treatment of the nuclear extract, suggesting that it is probably
bound to the DNA. Immunoprecipitation
experiments using
32P in vivo labeled DNA confirmed that pi8 binds DNA (data
X
50
60
Friction Numbir
Fig. 3. ELISA analysis of sucrose density gradient fractions. Nuclear extracts
were applied to a 5-45% (w/v) linear sucrose density gradient and centrifuged at
28,000 rpm for 16 h. After fractionation, ELISA activity and protein concentra
tion were determined for each fraction, (a) Peak ELISA activity of MAb 5C2
(pi 10) occurs in fractions 20-22 which corresponds to the second protein peak.
(A) Peak ELISA activity of MAb 9F9 (pi 10) also occurs in fractions 20-22. (c)
Peak ELISA activity for MAb 4F8 occurs in fraction 16. A. protein concentration:
O, ELISA activity;
, absorbance at 254 nm; Fraction 0, top of gradient;
Fraction 60, bottom of gradient.
interphase cells and had a homogeneous nuclear staining pat
tern. At mitosis, pi8 was associated with the condensed chro
mosomes in all cell lines examined. The pi8 peptide was found
not shown). The M, 18,000 protein was present in normal
resting lymphocytes as well as PHA-stimulated lymphocytes.
The presence of pl8 on mitotic chromosomes and its pres
ence in noncycling cells suggests that it may be a structural
protein of the nucleus and/or chromosomes. The M, 18,000
antibody cross-reacts with Novikoff hepatoma and PTK2 cells,
as well as human cells. The antibody does not recognize any of
the histones based on immunoblot analysis of purified histones
(data not shown). Based on molecular weight, pi8 is too large
to be ubiquitin (M, 8500), HMG proteins HMG-17 (M, 9000)
or HMG-24 (M, 12,000). Therefore, pi8 is probably a novel
nuclear DNA-associated protein which may be a potentially
useful tool for studies on nuclear and chromosome structure
and function.
The M, 85,000 nuclear protein was localized exclusively in
the nuclei and was not found in the nucleoli. At mitosis, the
protein migrated to the cytoplasm and did not associate with
the chromosomes. The M, 85,000 protein was found in all
tumor cell lines examined, but was not present in normal resting
(Go) lymphocytes. Upon PHA stimulation, p85 appeared within
6 h after induction and was present thereafter at all time points
examined. The appearance of p85 within 6 h following PHA
stimulation of lymphocytes may correlate with initiation of
RNA synthesis (34). Although p85 may have a role in RNA
synthesis, further studies on this point are required.
Although p85 has PCNA-like properties, it is not the Mr
86.000 PCNA described by Zweig et al. (12) and Yaneva et al.
(19); its immunofluorescence distribution differs and on im
munoblot analysis this MAb did not react with purified M,
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CELL CYCLE-RELATED NUCLEAR PROTEINS FOUND WITH MAbs
FRACTIONNUMBER
*
5.0
12 16
20
24
28
32
36
40 44
Õ4.0
1 '•"
2.0 ¿
I 0.75
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= 0.5
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I 1.0 . I 0.25
a.
E
o S 0
200
o, I
o I
a
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6
8 10 12 14 16 18 20 22 24 26 28 30 32
Friction Number
25.7-
Fig. 6. Sucrose density gradient analysis of MAb 5C7 (p 18) localization. (A)
sucrose density gradient fractions of 0.01 M Tris nuclear extracts were assayed
for ELISA activity. No definitive peak was observable. Instead, the ELISA activity
appeared to increase across the gradient. (B) Sucrose density gradient fractionation of DNase I-treated nuclear extracts released pi8 to the top of the gradient,
as determined
, absorbancebyatELISA
254 nm;assay.
S markers
O, ELISA
are indicated
activity; byA,arrows.
protein concentration;
200B
C
200-
9768Ill
97
KI
43-
68-
25.7-.
68-
Fig. 5. Immunoblot analysis of gradient fractions shown in Fig. 4 for detection
of pi 10 (arrowhead). Most activity is seen infractions 16 and 20. (B) Immunoblot
analysis of gradient fractions shown in Fig. 4 for detection of p85. p8S (arrowhead)
is detected in fractions 16-24, with minor amounts detected in other fractions.
86,000 protein (obtained from M. Yaneva). As shown by DNase
treatment of nuclear extracts fractionated on linear sucrose
density gradients, the M, 85,000 antigen is not bound to DNA.
These results were confirmed for p85 and pi 10 by DNAbinding experiments (data not shown). The Ku antigen (23, 24)
is bound to DNA and thus also differs from the p85 described
here.
Protooncogenes have been implicated as the regulators of
animal cell proliferation (35). Reed et al. (36) have shown that
protooncogenes may also be important regulators of T-lymphocyte production. Nuclear oncogenes such as c-myc (37) and cfos (38) are activated early in G, whereas c-myb is activated in
the middle of G, (39). While p85 does not correspond to any
known protooncogene products based on ¡mmunofluorescent
localization and/or molecular weight, it may also be a regulator
of proliferation and/or may have characteristics similar to those
of some oncogenes.
II-
Fig. 7. (A) Coomassie-stained
7.5% polyacrylamide
gel of control and PHAstimulated normal human lymphocytes.
Cells (1 x 10s) from each time point
were solubilized in Laemmli buffer and fractionated by SDS-PAGE.
Lane A, Oh
control; Lane B, 6 h following PHA stimulation;
Lane C, 22 h following PHA
stimulation; Lane D, 44 h following PHA stimulation;
Lane E, 68 h following
PHA stimulation. The large bands seen at M, 50,000 (arrowheads) are the heavy
chains of IgG contained in the samples. (B) Immunoblot
analysis of control and
PHA-stimulated
lymphocytes shown in A. pi 10 is observable following 68 h of
PHA stimulation
(arrowhead; Panel a). p85 appears following 44 h of PHA
stimulation (arrowhead; Panel b). Immunoblot analysis of pl8 (on 12.5% gel). In
Panel c, pl8 appears at 22 h following PHA stimulation
(arrowhead). Lane A, 0
h; Lane B, 6 h; Lane C, 22 h; Lane D, 44 h; Lane E, 68 h. The decreased
immunoreactivity
seen in those immunoblots
is due to the reduced amount of
specific protein available. The nuclei were not isolated from the lymphocytes;
instead, whole cells were used.
The pi 10 protein is localized to the nuclei of cycling cells
and is not found in the nucleoli based on immunofluorescent
studies. At mitosis, the protein is distributed throughout the
cell. It is not associated with the mitotic chromosomes. The M,
110,000 protein was found in all tumor cell lines investigated
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CELL CYCLE-RELATED
NUCLEAR
PROTEINS
FOUND WITH MAbs
may be in the prekinetochores (47). In human mitotic cells,
topoisomerase I is localized over the chromosomes (47). The
M, 110,000 antigen detected in these studies differs from the
previously reported M, 110,000 antigens.
A Ki 67 monoclonal antibody also recognizes a nuclear
PCNA but the molecular weight of the antigen has not been
reported (20-22, 48). Since the Ki 67 antibody remains asso
ciated with the chromosomes during mitosis (20), it is unlikely
that the PCNA-like antigens described here are related to the
antigen recognized by Ki 67. Only the A/r 18,000 antigen
remains associated with the chromosomes, and it does not have
PCNA-like properties.
These data show that both pi 10 and p85 are PCNA-like
proteins. The p85 protein appears in d (6 h after PHA stim
ulation), and pi 10 appears following 22 h of PHA stimulation
in S phase. Protein pi8 is not PCNA-like and may be a
structural protein.
B
200-
97-
ACKNOWLEDGMENTS
We would like to thank Dr. Earl F. Baril, Worcester Foundation,
for his kind gift of purified DNA polymerase «2complex isolated from
HeLa cells; Dr. M. Yaneva, Baylor College of Medicine, for her purified
M, 86,000 protein; Dr. M. Brattain, Bristol-Baylor Laboratory, Baylor
College of Medicine, for his kind of gift of human colon tumor cell
lines, HCT 116, GEO, JVC, and Moser; B. Dowell, for HL-60 cells;
and R. Reddy for his DNA-binding experiments using 32P-labeled DNA.
43-
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Fig. 8. Immunoblot analysis of pi 10 in DNA polymerasc «2complex. Six fig
of the purified DNA polymerase n¡complex were solubilized in Laemmli buffer,
fractionated in a 7.5% SDS-polyacrylamide gel, and transferred to nitrocellulose.
Lane A, silver-stained gel showing protein profile of the purified DNA polymerase
•
•
•
complex: Lane B. immunoblot using anti pi 10 antibody.
but not in normal resting (G0) lymphocytes. Upon PHA stim
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DNA polymerase «2from calf thymus is a IOS multiprotein
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Novel Cell Cycle-related Nuclear Proteins Found in Rat and
Human Cells with Monoclonal Antibodies
Ann Black, James W. Freeman, Guohao Zhou, et al.
Cancer Res 1987;47:3266-3272.
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