[CANCER RESEARCH 51. 4570-4574. September 1. 1991]
Antibodies to Proliferating Cell Nuclear Antigen as S-Phase Probes in Flow
Cytometric Cell Cycle Analysis1
GöranLandberg and Goran Rons2
Department of Pathology, University ofUmeà , S-90Ì87 Umeâ,Sweden
ABSTRACT
MATERIALS
The usefulness of different anti-proliferating cell nuclear antigen mono
clonal antibodies as S-phase probes in flow cytometric analysis was
evaluated after various fixation procedures on human cell lines. With a
newly developed detergent extraction/fixation method the monoclonal
antibody PC10 acted as a selective S-phase marker. A total of 27 human
hematopoietic tumors were analyzed using PC10, and all exhibited the
characteristic S-phase recognition pattern. The percentage of PC10positive cells was easily calculated and showed strong correlation to the
fraction of S-phase cells determined from DNA histograms. Using alter
native fixation procedures PC10, on the other hand, could recognize all
actively cycling cells, a feature also observed for the monoclonal antibod
ies, 19A2 and TOB7.
Cells. Thirteen human cell lines were used in the present study:
Daudi (14); MN-60 (15); RPMI-8226 (16); Jurkat (17); MOLT-4 (18);
CCRF-CEM (19); KG-1 (20); HL-60 (21); U937 (22); Hela (23); HT29
(24); 158-B4; and UM-42. The cell lines are described in the references
given, except for the 158-B4 and UM-42 lines which are diploid
lymphoblastoid B-cell lines not previously described. Cells were grown
in Ham's FIO or RPMI 1640 medium (Gibco, Scotland) supplemented
with 10% fetal calf serum and antibiotics. KG-1 cells were grown in
20% fetal calf serum.
Cells growing as monolayers (Hela, HT29) were detached with
trypsin treatment or by adding a lysing buffer (see below, Fixation D).
Cell suspensions from 24 fresh lymph nodes and three bone marrow
samples were prepared. Eighteen cases were non-Hodgkin's lymphomas
classified according to the Kiel classification (25), 10 were of low-grade
malignancy, and 8 were of high-grade malignancy. Two cases were
Hodgkin's lymphomas, and 4 were diagnosed as benign lymphadenitis.
INTRODUCTION
PCNA-' is involved in DNA replication and necessary for
AND METHODS
Also, 3 acute leukemias (2 acute myeloblastic, one acute lymphocytic)
were included in the study.
PCNA Antibodies. A human anti-PCNA serum was obtained from a
patient (A. K.) with systemic lupus erythematosus. This serum has been
used in previous studies (26-29) and is well defined.
19F4 (IgGl,x) and 19A2 (IgM) are MoAbs generated from mice
immunized with purified PCNA from rabbit thymus (8). I9F4 was
obtained as purified antibody and 19A2 as ascites fluid. The human
anti-PCNA serum and 19F4 and 19A2 antibodies were kindly provided
by Dr. E. M. Tan.
TOB7 (IgG 1,K)was a kind gift from Dr. Y. Takasaki, who established
it using purified calf thymus PCNA as immunogen (9).
PC 10 (IgG2) is a MoAb established after immunization with a
recombinant PCNA protein, and it was a kind gift from Dakopatts A/
S, Denmark (10).
The PCNA serum was diluted 1:400; PC 10, 1:50 to 1:100; 19A2,
1:200; and the MoAbs 19F4 and TOB7 were used at a concentration
of 50 ¿ig/ml.As a negative control for the PCNA autoantibody a serum
from a healthy laboratory worker was used. Controls for the MoAbs
were: anti-IgE MoAb (IgGI,K), Dakopatts A/S (19F4 and TOB7); an
irrelevant IgG2 control MoAb, Becton-Dickinson (PC 10); and antiLeu Ml MoAb (IgM), Becton-Dickinson (19A2).
Fixations. Six different fixation procedures (A to F), which have been
shown to be useful in detecting nuclear antigens with FCM or with
immunohistochemistry, were evaluated. These procedures are: A, 70%
ethanol for 10 min at -20°C; B, 100% methanol for 10 min at -20°C
followed by 0.5% NP-40 (in PBS) on ice for 5 min; C, 1% PF in PBS
for 2 min and then, after a PBS wash, fixation in 100% methanol for
10 min at -20°C(11); D, 500 ß\
of lysing buffer4 (0.5% Triton X-100;
adequate leading strand synthesis, acting as the auxiliary pro
tein of DNA polymerase 5(1,2). There are at least two different
forms of PCNA (3). One detergent-extractable form is present
in significant amounts in proliferating cells but is almost undetectable in resting cells. Another form of PCNA is detergent
resistant and closely related to DNA synthesis. The staining
pattern of the S-phase-related PCNA resembles the topograph
ical patterns of DNA replication sites (4). Thus, PCNA could
theoretically serve as a marker for cells actively replicating
DNA. This feature is, from a diagnostic point of view, an
important aspect, since the fraction of S-phase cells is an
independent prognostic parameter for human tumors, such as
malignant lymphoma and breast cancer (5-7). The possibility
of quantitating S-phase cells using an antigen-antibody reaction
would be of great value.
Several MoAbs to PCNA have been produced, reacting with
the protein in Western blots (8-10). After special fixation
procedures two of these MoAbs (19F4 and 19A2) could detect
S-phase cells in the MOLT-4 cell line using FCM (11). How
ever, in another FCM study no significant S-phase staining was
achieved using the 19F4 MoAb (12). The MoAb PC10 showed
by FCM strong reactivity with Hela cells without any obvious
cell cycle phase variation (13). To clarify the usefulness of
PCNA MoAbs as S-phase markers in FCM analysis, we have
tested four MoAbs (19F4, 19A2, TOB7, and PC 10) and eval
uated the cell cycle phase-specific reactivity in human cell lines
after various fixations. With a specific extraction/fixation
method, PC 10 was shown to be an excellent S-phase marker in
all tested cell lines as well as in lymphoma cells.
0.2 Mg/m' of EDTA:1% bovine serum albumin in PBS) for 15 min on
ice followed by addition of 3 ml of 100% methanol for 10 min at
-20°C;E, 0.1% Triton X-100 in PBS for 15 min and then addition of
Received 3/13/91; accepted 6/17/91.
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.
1This study was supported by grants from the Swedish Cancer Society (90
1127). the Lions Cancer Research Foundation, and the Medical Faculty, Univer
sity of Umeâ.
2To whom requests for reprints should be addressed.
' The abbreviations used are: PCNA, proliferating cell nuclear antigen: FCM,
flow cytometry; FITC, fluorescein isothiocyanate: PF, paraformaldehyde; MoAb,
monoclonal antibody; PI, propidium iodide; IgG, immunoglobulin G (other
immunoglobulins defined similarly); NP-40, Nonidet P-40; PBS. phosphatebuffered saline; BrdUrd, bromodeoxyuridine.
an equal volume of 2% paraformaldehyde for 15 min; and F, 1% PF
for 15 min followed by permeabilization in 0.1% Triton X-IOO.
The initial cell number in each sample was 2 x IO6 cells, and after
fixation the cells were washed twice in PBS before immunostaining.
Stainings. Fixed cells were incubated with the primary antibody for
30 min at room temperature. After two PBS washes, FITC-conjugated
anti-human antibody (1:50) or FITC-conjugated anti-mouse antibody
(1:20) was added, and the samples were kept in the dark for 30 min (all
FITC antibodies were from Dakopatts A/S). After two additional PBS
4 G. Landberg and G. Roos. Flow cytometric analysis of proliferation-associ
ated nuclear antigens using washless staining of unfixed cells, submitted for
publication.
4570
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PCNA ANTIBODIES AS S-PHASE PROBES
washes, the cells were resuspended in a DNA-staining solution contain
ing PI (10 Mg/ml) and RNase (1.8 Kunits/ml) and kept cold and dark
for at least 30 min until FCM analysis.
Standard DNA histograms were obtained from the clinical samples
after PI staining according to the method of Vindelövet al. (30). The
percentage of S-phase cells were calculated using a modified peak reflect
method (31).
In order to evaluate the number of cells actively replicating DNA,
Jurkat cells were incubated with 10 nM BrdUrd (Sigma Chemical Co.,
St. Louis, MO) for 20 min, whereafter the cells were ethanol fixed and
treated with 2 M HC1 containing 0.2 mg/ml of pepsin for 20 min (32).
After treatment with 0.1 M Borax and two PBS washes, IO ¿il
of antiBrdUrd MoAb (Becton-Dickinson) were added for 30 min in room
temperature, followed by an FITC-labeled anti-mouse MoAb. In the
last step the cells were resuspended in a PI/RNase solution.
Flow Cytometry. Cells were analyzed in a FACScan flow cytometer
equipped with an argon laser (488 nm) (Becton-Dickinson Immunocytometry Systems, Mountain View, CA), and data were registered and
stored in list mode. Debris and damaged cells were excluded by gating
on a forward and side scatter dot plot or on the DNA histogram. FITC
fluorescence was detected in the FL1 channel (530 ±15 nm) and stored
using logarithmic or linear amplification. DNA was recorded in the
FL2 channel (585 ±22 nm) using linear amplification. Data obtained
were evaluated with the FACScan software (Becton-Dickinson).
In some experiments the FITC fluorescence was expressed as a ratio
between the median fluorescence channel for the anti-PCNA antibodies
divided by the median fluorescence channel for the control antibody
fluorescence. These ratios were calculated in specific parts of the DNA
histograms for both PCNA antibodies and control antibodies. The five
DNA compartments analyzed are shown in Fig. 1 and were: early G0GI; late G0-Gi; S; early G2-M; and late G2-M. For example, a ratio of
2.0 means that the PCNA fluorescence was twice as high as the control
fluorescence in that DNA compartment. These calculations were per
formed on linear fluorescence values. The ratios obtained made it
possible to visualize the PCNA expression in different parts of the cell
cycle in a proper way.
Correlation between PClO-positive cells and S-phase fractions was
determined by linear regression analysis.
Ratio
r~is
DNA compartment
Fig. 2. Fluorescence ratios calculated for the human anti-PCNA antibody in
different DNA compartments of Jurkat cells. A to D denote the different fixation
procedures used: A, ethanol: B, methanol + NP-40: C, PF + methanol; and D,
lysing buffer + methanol. Further details concerning fixation procedures are given
in "Materials and Methods."
Ratio
15
1311 0
7
S
31
h
RESULTS
M2
r~l3
^4
die
DNA compartment
Three cell lines (Daudi, Jurkat, MN-60) were primarily in
vestigated after fixation Procedures A (ethanol), B (methanol
+ NP-40), C (PF + methanol), and D (lysing buffer + methanol)
concerning the cell cycle phase-specific reactivity of anti-PCNA
antibodies. Figs. 2 to 6 show the fluorescence ratios calculated
for each antibody in different DNA compartments of the Jurkat
cell line. The Daudi and MN-60 cell lines gave similar results
as for Jurkat.
With the human PCNA autoantibody (AK), S-phase-specific
staining was obtained with Fixations A, B, and D (Fig. 2). The
best S-phase separation was found for Fixation A (ethanol).
With Fixation C (PF + methanol), the autoantibody yielded
intermediate PCNA expression in all DNA compartments.
Low fluorescence signals were obtained for the 19F4 MoAb,
45I
3
i 11I
|
o5E3C^_"55Ü1»iI2
1I
|1
1||1
'1
'kT^^T
1L
lAL
DNA
Fig. 1. DNA compartments used to analyze the expression of PCNA: /. early
Go-G,: 2. late G0-G,: 3. S: 4. early G2-M; 5, late G2-M.
Fig. 3. Fluorescence ratios calculated for 19F4 using the same approach as in
Fig. 2.
but both Fixations B and C gave higher fluorescence in S-phase
cells compared with the other cell cycle compartments (Fig. 3).
19A2 showed intermediate to strong fluorescence with
Fixations A, B, and C without any cell cycle phase preference
(Fig. 4). With Fixation D, a general weak fluorescence was
observed with slightly higher levels in late Go-G] and S.
TOB7 and PC 10 yielded large differences in reactivity de
pending on the fixation used (Figs. 5 and 6). A weak S-phase
preference with Fixations B and C was observed for TOB7,
whereas PC 10 preferentially stained S-phase cells after Fixation
D (lysing buffer + methanol). The S-phase pattern for PC10
was very similar to that obtained with the human autoantibody
(Fig. 2, Fixation D).
All four MoAbs were almost unreactive after Fixations E
(Triton X-100 + PF) and F (PF + Triton X-100). The human
autoantibody, however, showed S-phase-specific reactivity with
Fixation E and intermediate fluorescence without cycle phase
specificity after Fixation F (not shown in figures).
After the initial testing, 19F4 (methanol + NP-40) and PC 10
(lysing buffer + methanol) were selected for further investiga
tions concerning S-phase specificity. Ten human cell lines were
tested with 19F4 and showed variable cell cycle phase reactivity.
Some cell lines exhibited a fairly nice S-phase recognition (e.g.,
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PCNA ANTIBODIES AS S-PHASE PROBES
Ratio
PI stainings are shown in Fig. 8. All samples tested showed the
characteristic S-phase expression, and the fraction of PC 10positive cells was easily determined in each case. We found a
highly significant correlation between S-phase cells determined
from DNA histograms and the percentage of PClO-positive
cells in the tumor material as shown in Fig. 9.
DISCUSSION
• 1
ESS2
CÜ3 »4
CDs
DNA compartment
Fig. 4. Fluorescence ratios calculated for 19A2 using the same approach as in
Fig. 2.
Ratio
The different forms of PCNA existing in resting and in
cycling cells have been explored in detail previously (3, 33). It
has been suggested that PCNA is present in high concentrations
in cycling cells in order to form a complex with DNA polymerase 5 in cells entering S phase (33). The complex which is
active in DNA replication is tightly bound to DNA, and this
form of PCNA is not extractable with detergent. With the
detergent extraction/fixation method described here, the antiPCNA MoAb PC 10 can act as an S-phase marker. The method
seemed reliable and could recognize S-phase cells in all different
cell lines and human hematopoietic tumors tested. When fixa
tion with lysing buffer + methanol was used, the fluorescence
intensity for PC10 diminished to approximately 25% of the
values observed after methanol fixation only. This is in agree
ment with the finding that the S-phase-associated PCNA form
constitutes about one-fourth of the total PCNA content (3). It
also indicates that, after the detergent extraction/fixation pro
cedure, only the DNA replicon-complexed PCNA was left in
the cell nuclei.
One question is whether PC 10 MoAbs using lysing buffer
Ratio
•il
Å’2
r~l3
^4
I
Ifi
DNA compartment
Fig. 5. Fluorescence ratios calculated for TOB7 using the same approach as in
Fig. 2.
Jurkat), whereas other cell lines gave more equally distributed
fluorescence signals (not shown in figures).
In contrast, PC 10 was S-phase specific in all cell lines tested
(Daudi, Jurkat, 158B4, UM-42, HL-60, Molt-4, MN-60, Hela,
and HT29). In Fig. 1A, we show an example of a PC 10 and PI
double staining of Jurkat cells. Thus, the best and most repro
ducible S-phase separation seemed to be achieved with Fixation
D (lysing buffer + methanol) and the PC 10 antibody.
If the lysing step in Fixation D was performed in room
temperature, similar results were obtained as on ice but with a
higher degree of debris and aggregates. The 15-min lysing time
was not critical (10 to 30 min tested). The coefficients of
variation of the Gìpeaks were low (regularly <4%) for all
fixation procedures except for those containing PF.
The PC10 cell cycle phase recognition pattern was found to
be similar to the pattern obtained with the human anti-PCNA
autoantibody (not shown in figures). Cells pulse labeled with
BrdUrd also revealed a comparable staining pattern after vis
ualization with an anti-BrdUrd antibody (Fig. 7). One difference
between the PC 10 and BrdUrd patterns was observed in late Sphase, in which the PC 10 reactivity declined in contrast to the
BrdUrd staining.
To verify the usefulness of PC 10 as an S-phase marker we
performed dual-parameter analysis of PCNA expression and
DNA content in a series of human hematopoietic tumors, and
the results are presented in Table 1. Four examples of PC 10-
i
ES32
i
la
M4
CDs
DNA compartment
Fig. 6. Fluorescence ratios calculated for PC10 using the same approach as in
Fig. 2.
B
T3
m
DNA
DNA
Fig. 7. Jurkat cells labeled with 10 nM BrdUrd (BrtiL') for 20 min and stained
with PC10 and PI (lysing buffer + methanol) (A) and anti-BrdUrd and PI (B).
The antibody fluorescence is presented in logarithmic scale and the DNA content
in linear scale.
4572
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PCNA ANTIBODIES AS S-PHASE PROBES
A
B
DNA
DNA
O)
.o
E
D
Fig. 8. PCIO-PI stainings of human hematopoietic tumor cells fixed according to Method
D (lysing buffer + methanol). PC 10 fluorescence
is presented in logarithmic scale and DNA in
linear scale. A, non-Hodgkin's lymphoma. low-
"35
ü
grade malignancy; B, acute lymphatic leukemia;
C, non-Hodgkin's lymphoma, high-grade malig
nancy; and D, non-Hodgkin's lymphoma, highgrade malignant case with aneuploidy.
and methanol fixation exclusively recognize S-phase cells. Sev
eral authors have shown that the staining pattern obtained with
anti-PCNA antibodies is similar to that obtained with thymidine labeling or anti-BrdUrd stainings (Footnote 5; Refs. 3, 4,
34, and 35). Data from PC 10 stainings of cells blocked in late
G, with mimosin, in early S with aphidicolin, or in G2 with
mitoxantron also indicate that PC 10 MoAbs selectively recog
nize cells in S phase.5 Furthermore, dual-parameter analysis of
human anti-PCNA serum and PC 10 (lysing buffer + methanol)
indicates that these antibodies recognize the same cells (not
shown in figures). Thus, we conclude that PC 10 can be used as
an S-phase marker.
One advantage in estimating S-phase cells from PC 10 reac
tivity instead of from DNA histograms is that it might give
more exact S-phase values. In a few PC10-DNA stainings of
malignant lymphomas, we noticed cells with S-phase DNA
content negative for PC 10. This could be attributed to debris,
quiescent S-phase cells, or aneuploid non-S-phase cells. These
cells were, however, included in the S-phase fraction determined
from a DNA histogram. This problem is often encountered in
material from solid tumors and especially those with much
debris giving false positive signals in the DNA histograms.
In lymphomas, the number of PClO-positive cells correlated
well with the S-phase fractions determined from DNA histo
grams. In a previous study (36), the PCNA expression measured
with the human autoantibody correlated well with S-phase
percentages as well as with the number of cells positively stained
for the proliferation-associated
antigen Ki-67. In the same
report, PC 10 was shown to distinguish cycling cells from resting
cells after methanol fixation and NP-40 treatment. In the
present study it seemed that PC 10, 19A2, and TOB7 were
strongly reactive with cycling cells after methanol + NP-40 or
PF + methanol treatment. Any of these MoAbs could theoret
ically serve as a proliferation marker using the proper fixation.
Accordingly, PC 10 can act as an S-phase probe or, dependent
on the fixation procedure used, a marker for all proliferating
cells. A similar finding has been reported for 19A2 using
immunohistochemistry (37).
Human anti-PCNA autoantibodies preferentially react with
S-phase cells after alcohol fixations, whereas formaldehyde
treatment can reveal the non-S-phase form of PCNA probably
by unmasking antibody recognition sites (3). The masked
PCNA form is still present after methanol fixation, since it can
be visualized with the autoantibody after postfixation in PF.5
5 Unpublished results.
DNA
DNA
This is further substantiated in the present study with the
MoAbs, especially PC 10, showing strong signals in all DNA
compartments after methanol fixation.
The differences in reactivity found for the autoantibody and
the MoAbs can be explained by epitope differences. It has been
shown that the human autoantibody identifies epitopes which
are highly dependent on protein conformation, whereas the
anti-PCNA MoAbs recognize linear epitopes (38). Further
more, using synthetic peptides we have found that the 19A2,
19F4, and PC 10 MoAbs recognize closely related, although not
identical, epitopes.5
Our results were mainly generated from studies of hematopoietic cells, but it was also possible to detect S-phase cells with
PC 10 in two epithelial cell lines. Thus, it might be possible to
use PC 10 as an S-phase marker in solid tumors. However,
preliminary studies on colon, renal, and breast carcinomas have
been unsuccessful so far. There might be differences between in
vivo- and in vitro-grown epithelial cells concerning fixation
effects and the availability of PCNA epitopes. It is obvious that
Table 1 Flow cytomelric analysis of PCI 0 and S phase determined from DNA
histograms on 27 hematopoietic tumors
of S-phase % of PClO-positive
Patient
Diagnosis123456789101112131415161718192021222324252627LGM°LGMLGMLGMLGMLGML
Age/sex
Phenotype46/M51/M55/M73/M63/F45/M68/F46/M44/M44/M62/M69/M54/M77/F
cells1.61.92.11.01.32.22.21.33.12.45.411.8
cells
CD10)B(k)B(CD19,
CDS)B(k)B(k)B(k)B(l)B
CDS)B(k)B(k)B(k)B(k)B(k)B(k)TB(k,
(CD 19,
An14.7
An17.04.720.324.0
CD10)B(k)B(k)Pre-B(CD19)%
An6.71.22.43.217.24.54.03.61.52.81.70.80.60.40.90.
" LGM, non-Hodgkin's lymphoma of low-grade malignancy; HGM, nonHodgkin's lymphoma of high-grade malignancy; An. aneuploid; ALL, acute
lymphocytic leukemia; AML. acute myeloblastic leukemia.
4573
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PCNA ANTIBODIES AS S-PHASE PROBES
O
Q_
20
20
25
S-phase
Fig. 9. Correlation between the percentage of PClO-positive cells and S-phase
cells evaluated from DNA histograms of 21 hematopoietic tumors (r = 0.936. P
< 0.001).
further studies are needed to clarify the value of PC 10 as a
proliferation marker for nonhematopoietic cells. This is further
stressed by the reported deregulated PCNA expression detected
with PC10 in normal, tumor-adjacent tissues (13).
The procedure presented here to determine S-phase cells
using the PC 10 Mo Ab and FCM is easy to perform and evaluate
and can be of potential value in characterizing hematopoietic
tumors. Since the S-phase fraction of non-Hodgkin's lymphomas is an independent prognostic predictor (5, 6), it can be
assumed that this property of tumors might influence the choice
of therapy in the future.
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Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1991 American Association for Cancer Research.
Antibodies to Proliferating Cell Nuclear Antigen as S-Phase
Probes in Flow Cytometric Cell Cycle Analysis
Göran Landberg and Göran Roos
Cancer Res 1991;51:4570-4574.
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