Acta Oncologica
ISSN: 0284-186X (Print) 1651-226X (Online) Journal homepage: http://www.tandfonline.com/loi/ionc20
Expression of Proliferating Cell Nuclear Antigen (PCNA) and Ki-67 Antigen in Human Malignant Hematopoietic Cells G. Landberg & G. Roos To cite this article: G. Landberg & G. Roos (1991) Expression of Proliferating Cell Nuclear Antigen (PCNA) and Ki-67 Antigen in Human Malignant Hematopoietic Cells, Acta Oncologica, 30:8, 917-921, DOI: 10.3109/02841869109088244 To link to this article: https://doi.org/10.3109/02841869109088244
Published online: 08 Jul 2009.
Submit your article to this journal
Article views: 186
View related articles
Citing articles: 17 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ionc20
Acta Oncologica Vol. 30 No. 8 1991
FROM THE DEPARTMENT OF PATHOLOGY, UNIVERSITY OF UMEA, UMEA, SWEDEN.
EXPRESSION OF PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA) AND Ki-67 ANTIGEN IN HUMAN MALIGNANT HEMATOPOIETIC CELLS G. LANDBERGand G. Roos
Abstract Human hematopoietic cell lines and fresh lymphoma cells were investigated for their expression of proliferating cell nuclear antigen (PCNA) and Ki-67 antigen using flow cytometry. Ki-67 was detected with a monoclonal antibody and the PCNA content was determined using a human autoantiserum and in some cases a monoclonal antibody (PCIO). The autoantibody to PCNA was found to recognize S-phase cells and the number of positively stained cells was significantly correlated with the S-phase fraction determined from DNA-histograms. There was also a strong association between Ki-67 positivity and the fraction of S-phase cells. By dual parameter analysis of Ki-67 and PCNA detected by the autoantibody a detailed cell cycle analysis could be performed of the lymphoma samples. The recently described PClO monoclonal antibody was found by FCM to be a good proliferation marker recognizing cycling cells after methanol and detergent treatment, however without any specific cell cycle phase preference. Key words: Non-Hodgkin’s lymphoma, cell cycle, PCNA, Ki67, monoclonal antibodies.
’
Tumor cell proliferation is an important prognostic parameter for non-Hodgkin’s lymphoma regardless of the technique used to assess cell growth ( 1 -S), and the fraction of S-phase cells determined by flow cytometric (FCM) DNA histogram analysis has been found to be an independent prognostic indicator (6,7). The DNA-FCM technique gives reproducible results in a majority of cases, but aneuploidy and cell debris can make the analysis less accurate. Furthermore, resting ( G o )cells cannot be discriminated from cycling G, cells. The total fraction of actively cycling cells can be assessed by Ki-67 antigen expression as a parameter (8). Using a monoclonal antibody (moab) this nuclear antigen can be detected by immunohistochemistry as well as by flow cytometry of freshly prepared cells (9, 10). The expression of proliferating cell nuclear antigen (PCNA), another growth-related
antigen, can after proper cell preparation serve as an S-phase marker (11-13). We have shown that dual parameter FCM analysis of Ki-67 and PCNA can give detailed cell kinetic information on cell lines (14). In the present paper we present data using this approach on a number of non-Hodgkin’s lymphoma. Recently a series of moabs to PCNA were described (15), including PClO which is commercially available. PCIO, in contrast to Ki-67, can be used on paraffin sections by immunohistochemistry and recent data indicate that PClO can act as a useful proliferation marker (16). We here present FCM data showing that PClO can recognize a form of PCNA present in cycling cells. Material and Methods Lymphoma specimens. Twenty-two fresh lymphoma samples were examined. Imprints and cell suspensions were prepared from fresh tissues as well as paraffin sections after formalin fixation. Cell surface phenotyping was performed using FCM and a series of antibodies detecting Tand B-cell antigens. The cases were classified according to the Kiel classification (17). Eight cases were low-grade lymphomas (LGM) and 14 were high-grade lymphomas (HGM). Cell lines. The following cell lines were studied: Jurkat (18), Daudi (19), MOLT4 (20) and HL-60 (21). Cell lines were cultured in Ham’s F10 medium supplemented with 10% fetal calf serum and antibiotics. In order to evaluate
Paper presented at Symposium on Flow and Static Cytometry in Umei, 10-11 September, 1990. Accepted for publication 24 May 1991.
917
918
G. LANDBERG AND G. ROOS
differences between Go and G I cells normal, purified T lymphocytes were mixed with MOLT4 cells before analysis. Also, nuclear antigen expression after DMSO ( 1.2%)induced granulocytic differentiation of HL-60 cells was studied. Antibodies. The human anti-PCNA serum (AK) was derived from a patient with SLE and is well characterized in previous studies ( 11, 12,22). It was used in a final dilution of 1 :200. The PClO moab was a kind gift from Dakopatts A/S, Denmark, and a 1 : 100 dilution was found optimal for FCM detection. The Ki-67 moab ( 1 : 20) and FITC-conjugated rabbit anti-human IgG ( 1 :25) were obtained from Dakopatts. Phycoerythrin-labelled goat antimouse IgG ( 1 :20) was from Tag0 Inc., Burlingame, CA. As a control to the autoantiserum a normal human serum was used. For Ki-67 an anti-IgE moab (IgG,) (Dakopatts A/S) and for PClO an lgGz moab ( Becton-Dickinson, CA) were used as controls. Flon cytornetry. Standard DNA-histograms were obtained from the lymphoma samples after propidium iodide staining according to the protocol of Vindelw et al. (23). Histogram evaluation was performed according to a modified peak reflect method (24). For double stainings of antigen expression and DNA-content a solution of propidium iodide ( 10 pg/ml) and RNAse (20 pg/ml) was used. Nuclear antigen expression was studied in cells fixed in 70% ethanol (human autoantiserum, Ki-67) or methanol followed by NP-40 (PCIO). The cells were incubated with the primary antibody in phosphate-buffered saline (PBS) for 30 min at room temperature followed by PBS washes and the secondary antibody for 30min. After additional PBS washes propidium iodide solution was added and the samples were kept dark on ice until FCM analysis. We have in a separate study shown that also a washless staining technique using a detergent for membrane permeabilization can be applied to the PCNA/Ki-67 staining (Landberg & Roos, unpublished study). The different cell preparation methods gave very similar results in the dual PCNA/Ki-67 analysis. In the present study most of the lymphoma samples (20/22) were prepared according to the detergent method. Shortly, a mixture of human autoantibody and Ki-67 moab in a buffer containing Triton X-100 (0.5%) was used in the first step followed by a mixture of the secondary antibodies without any intermediate washings. The FCM analyses were performed with a FACScan flow cytometer ( Becton-Dickinson lmmunocytometry Systems, Sunnyvale, CA). Data were stored in list mode and evaluated with the FACScan software ( Becton-Dickinson). Correlation between different parameters was determined by linear regression analysis.
Results The human autoantiserum recognized PCNA in ethanolfixed cells with a preferential reactivity in cells with S-
IB
G a A
W
a
Z
0
n
1
I
q
IL
DNA
A
DNA
Fig. 1 . PCNA expression in relation to DNA content in lymphocytes mixed with MOLT4 cells. A. Human autoantibody (AK), ethanol-fixed cells, B. PClO moab. met1 8
A
a Z
8
I
\
0
n
B
1
Q2lM
t Ql
Ki - 67
Ki-67
Fig. 2. Dual parameter PCNA/Ki-67 FCM analysis of two nonHodgkin's lymphomas. PCNA was detected with a human autoantiserum. The assignment of cells to different cell cycle phases has been detailed previously ( 14). A. Low-grade malignant lymphoma, B. High-grade malignant lymphoma.
phase DNA content (Fig. IA). Unfixed, detergent-treated cells gave a similar staining pattern and the reactivity was identical to the pattern obtained after BrdUrd staining of pulse labelled cells (not shown in figures). Dual parameter analysis of PCNA, detected with the autoantibody, and Ki-67 expression was performed on a series of non-Hodgkin's lymphomas and showed obvious differences between lymphomas of low- and high-grade malignancy (Fig. 2A and B). The low-grade lymphomas had as a rule a low fraction of Ki-67 positive, i.e. cycling, cells, whereas the high-grade malignant cases showed higher numbers of cycling cells. A summary of the findings are shown in Table I . There were highly significant correlations between S-phase cells and PCNA positivity, Sphase cells and Ki-67 reactive cells and accordingly also between Ki-67 and PCNA positive cells (Fig. 3A. B and C). With the PCNA/Ki-67 staining method the different cell cycle compartments within the cycling cell population can be evaluated. The fraction of PCNA-positive cells within the cycling (i.e. Ki-67 positive) cell compartment varied considerably from case to case, but there was no obvious difference between LGM and HGM cases (Table I). The mean cell cycle phase distribution within the Ki-67-positive population was similar for LGM and HGM (Table 2). Furthermore, no association between total cell S-phase
919
CELL CYCLE ANALYSIS OF MALIGNANT LYMPHOMA
Table 1 Dual parameter PCNA IKi-67flow cytometric analysis of 22 non-Hodgkin's lymphomas in relation to diagnostic subgroups and S-phases determined from DNA -histograms. PCNA was detected using a human autoantibody
S(%)
Diagnosis
PCNA+(%)
PCNA: Ki-67
Ki-67+(%)
33.0 37. I 37.0 60.7 61.9 68.3 63.1 29.0
3.7 2.4 2.7 3.8 3.8 2.0 2.8 10.2
LGM case I 2 3 4 5 6 7 8
2.4 2. I
I .2 0.9
1.4 2.0 2.2 2.2 AN
2.3 2.4 I .3
Mean _+ SD
1.9 f 0.5
1.7 f 0.8
48.8 f 16.1
3.9 f 2.6
25.0 25.3 26.5 14.4 6.7 5.6 17.0 6.2 4.3 5.3 AN 3.2 17.6 ND
58.5 39.3 34.7 11.7 7.6 6.4 19.4 7.0 4.2 11.2 13.2 3.9 18.6 19.2
65.0 52.5 45.9 40.3 47.8 27.5 50.3 31.6 31.4 52.9 46.4 24.5 42.2 41.1
90.0 74.9 75.7 28.9 16.0 23.2 38.6 22.1 13.2 21.2 28.4
13.1 k9.0
18.2k 15.8
42.8 f 15.8
1 .o
HGM case 9 10
I1 12 13 14 15
16 17 18
19 20 21 22 Mean k S D
1 .o
1.8
3.0
15.8 44.1
46.7 38.5 f 25.0
LGM = low-grade malignant lymphoma; HGM = high-grade malignant lymphoma; PCNA: Ki-67 = % PCNA-positive cells within the cycling population; A N = Aneuploid; N D = Not done.
c Q) 3Q I
v)
::r'j
( I ' r ' j
II.-
Q
I.
.
.a
,I
I,
6,
.*
I, I
z
..:
a 8
"
a
."
I,
,' I
3
Q
v)
.: ,I
.I
.I
Ki-67
.6
6.
*
10
I.
(I..
*I
11,.
9.
I.
I.
D
..
I.
:8
8
I
.0
Ki-67
*' I.
o *
I.
;:;:rI v)
PCNA
2
fractions and PCNA-positive cells within the cycling compartment seemed to be present (Fig. 3D). More detailed analysis indicated that for HGM such a positive correlation existed, but not for LGM. However, a larger material is needed in order to better clarify this point. In methanol-fixed and NP-40-treated cells the PClO moab gave a strong signal in the FCM analysis. In a mixture of normal lymphocytes and proliferating cells the latter were clearly positive whereas the lymphocytes were negative (Fig. 1B). No cell cycle phase preference was seen since G , , S and GJM cells gave equally strong fluorescence signals. HL-60 cells induced to differentiate to mature granulocytic cells showed a large proportion of PC10-negative cells, whereas the control HL-60 cells were positive (Fig. 4A). DNA-histogram analysis revealed a low S-phase fraction in DMSO-treated cells compared with control cells (Fig. 4B). Microscopic evaluation showed about 15% in the DMSo treated Thus, a fraction Of the HL-60 Cells Were resistant to the DMSO COnCentratiOn used, explaining that some cells were still PC 10-positive.
LO
I.
@
.I
'
.I 1
PCNA:Ki- 6 7
Fig. 3. Correlation between different proliferation parameters. A. S-phase versus PCNA expression evaluated by the autoantibody ( r = 0.92), B, S-phase versus Ki-67 expression ( r = 0.97). C. pCNA versus Ki-67 ( r = 0.97), D. !&phase versus PCNA-positive cells of cycling (Ki-67 +) cells ( r = 0.17).
920
G . LANDBERG AND G. ROOS
Table 2 Cell cycle distribution nithin the cycling cell population
evuluuted from the PCNAlKi-61 analysis Percentage of cycling ( Ki-67 + ) cells
Diagnosis
LG M HGM All cases
GI
S
G2/M
n
45.4 52.0 49.6
48.8 42.8 45.0
5.8 5.2 5.4
8 14 22
PCIO
DNA
Fig. 4. Effects of DMSO induced HL-60 differentiation. A. PClO expression in HL-60 cells, 8. DNA histogram of HL-60 cells ' ' ' control cells, DMSO-treated cells (6d.) ~
IA
6
ih h PCIO
PCIO
Fig. 5. PClO expression in non-Hodgkin's lymphomas. A. Low-grade malignant lymphoma, B. High-grade malignant lymphoma.
Also in fresh lymphomas it was possible to detect PClO by FCM. Fig. 5A illustrates an LGM case with less than 5% PCIO-positive cells in contrast to Fig. SB, showing a high PCIO-positive cell fraction in a case of HGM
Discussion In the present study we analysed the expression of two different growth-associated nuclear antigens ( PCNA and Ki-67) in human lymphoma cells. The fractions of cells, positive for a human anti-PCNA serum, and the moab Ki-67 were strongly correlated to each other as well as to the number of S-phase cells determined by DNA histogram analysis. All three parameters discriminated between LGM and HGM cases as defined by morphologic appearance. In conclusion, any of these parameters can be chosen for crude cell proliferation assessments using FCM .
The Ki-67 positive cell fraction determined by immunohistochemical staining is a significant prognostic parameter for non-Hodgkin's lymphoma (25.26). Our FCM-derived Ki-67 values are comparable to those obtained by Hall et al. (25) using immunostaining of cryosections. In both studies the majority of LGM cases had 20%. By dual parameter analysis of PCNA and Ki-67 more detailed cell kinetic information was achieved, since actively cycling cells could be discriminated from resting cells and characterized concerning their cell cycle phase distribution. The fraction of S-phase cells in the cycling compartment showed rather large variations (24-68%. Table I ) in both the LGM and HGM group. By comparative analysis of bromodeoxyuridine (or thymidine) and Ki-67 labelling indices similar calculations can be made. In a study of various malignant tumors the S-phase size within the proliferating cell fraction showed a mean value of 58'% (27) compared with our mean value of 45%. We did not find any significant difference between LGM and HGM cases concerning this feature, which contrasts to the study of Veneroni et al. (28) showing only lo'%,S-phase cells within the cycling cell population in LGM cases. The reason for this discrepancy might be due to the different staining and detection techniques used. By comparison with data obtained through in vivo iododeoxyuridine incorporation and FCM analysis our PCNA/Ki-67 results can be further substantiated, and such studies are in progress. It is well established that the overall cell proliferation in non-Hodgkin's lymphomas is an independent prognostic factor. Perhaps the fraction of S-phase cells in the cycling compartment might give additional information. It is possible that, e.g., response to cytostatic treatment is related to such a factor. Future clinical follow-up will show the possible clinical significance of these studies. The PClO moab was established after immunization with a recombinant protein product and it detects PCNA in Western blots and by immunoprecipitation (15). PCNA immunoreactivity using PClO was highly correlated to Ki-67 positivity in non-Hodgkin's lymphomas estimated by immunohistochemistry stainings ( 16). Our data generated by FCM support the findings that PClO can be used as a good marker for cell proliferation in hematopoietic cells. For other neoplasms the relation between proliferation and PCNA expression detected by PClO has been reported to be lost, and deregulation of PCNA in tumor adjacent tissues seems to have been present (16). These findings indicate that further studies are needed to clarify the value of this antibody for diagnostic purposes. In contrast to the autoantibody, PClO recognized all cycling cells without any S-phase predominance. This is probably due to epitope differences. It is known that epitopes recognized by autoantibodies are highly dependent on protein conformation whereas monoclonal anti-PCNA antibodies recognized linear epitopes (29). Pre-
CELL CYCLE ANALYSIS OF MALIGNANT LYMPHOMA
liminary studies using synthetic peptides indicate that the PClO epitope is also linear (Roos et al., to be published). The different cell cycle reactivity seen for the PCNA antibodies is not contradictory since there are at least two different populations of PCNA, one of which is associated with the DNA replication machinery (30). These PCNA populations can be affected in different ways during the staining procedure leading to differential epitope expression. In order to clarify this we are presently investigating cell cycle phase reactivity after different fixation and extraction procedures. Preliminary results indicate that under certain circumstances the PC 10 antibody can also preferentially yield staining of S-phase cells.
ACKNOWLEDGEMENTS This study was supported by grants from the Swedish Cancer Society (No. 90 1127). Lions Cancer Research Foundation and the Medical Faculty, University of U m e i The authors are grateful to Miss Bodil Backlund for skillful technical assistance. Corresponding nutltor: Dr Goran Roos. Department of Pathology, University of Umei, S-90187 Umei, Sweden.
REFERENCES I . Costa A. Bonadonna G, Villa E, Valagussa P, Silvestrini R. Labeling index as a prognostic marker in non-Hodgkin's lymphomas. J Natl Cancer lnst 1981; 66: 1-5. 2. Kvalsi S. Marton P, Kaalhus 0, Hsie J, Foss-Abrahamsen A. Godal T. 'H-thymidine uptake in B cell lymphomas-relationship to treatment response and survival. Scand J Haemato1 1985; 34: 429-35. 3. Wooldridge TN. Grierson HL, Weisenburger DD, et al. Association of DNA content and proliferative activity with clinical outcome in patients with diffuse mixed cell and large cell non-Hodgkin's lymphoma. Cancer Res 1988; 48: 6608- 13. 4. Brandt L, Johnson A. Olsson H, Akerman M. Mitotic activity and survival in advanced non-Hodgkin's lymphoma of unfiavourable histology. Eur J Cancer 1990; 26: 227-30. 5. Joensuu H, Klemi PJ, Jalkanen S. Biologic progression in non-Hodgkin's lymphoma. A flow cytometric study. Cancer 1990; 65: 2564-71. 6. Lenner P. Roos G . Johansson H, Lindh J. Dige U. NonHodgkin lymphoma. Multivariate analysis of prognostic factors including fraction ofS-phasecells. Acta Oncol 1987; 26: 179-83. 7. Rehn S, Glimelius B, Strang P, Sundstrom C, Tribukait B. Prognostic significance of flow cytometry studies in B-cell non-Hodgkin lymphoma. Hematol Oncol 1990; 8: 1 - 12. 8. Gerdes J, Lemke H. Baisch H, Wacker H-H. Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J lmmunol 1984; 133: 1710-15. 9. Brown DC. Gatter KC. Monoclonal antibody Ki-67: its use in histopathology. Histopathology 1990; 17: 489-503. 10. Sasaki K. Murakami T. Kawasaki M. Takahashi M. The cell cycle associated change of the Ki-67 reactive nuclear antigen expression. J Cell Physiol 1987; 133: 579-84. I I . Takasaki Y, Fishwild D, Tan EM. Characterization of proliferating d l nuclear antigen recognized by autoantibodies in Lupus sera. J Exp Med 1984; 159: 981 -92. 12. Kurki P, Vanderlaan M. Dolbeare F. Gray J. Tan EM.
92 1
Expression of proliferating cell nuclear antigen ( PCNA/cyclin during the cell cycle. Exp Cell Res 1986; 166: 209- 19. 13. Kurki P, Ogata K, Tan EM. Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry. J lmmunol Methods 1988; 109: 49-59. 14. Landberg G, Tan EM, Roos G . Flow cytometric multiparameter analysis of proliferating cell nuclear antigen/cyclin and Ki-67 antigen: A new view of the cell cycle. Exp Cell Res 1990; 187: 111-8. 15. Waseem NH, Lane DP. Monoclonal antibody analysis of the proliferating cell nuclear antigen (PCNA). Structural conservation and the detection of a nucleolar form. J Cell Sci 1990; 96: 121 -9. 16. Hall PA, Levison DA. Woods AL. et al. Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: An index of cell proliferation with evidence or deregulated expression in some neoplasms. J Pathol 1990; 162: 285-94. 17. Gerard-Marchant R, Hamlin 1. Lennert K. Rilke F, Stansfeld AG, van Unnik JAM. Classification of non-Hodgkin's lymphomas. Lancet 1974; 2: 406-8. 18. Kaplan J, Tilton J, Peterson WD. Identification of T-cell lymphoma tumor antigens on human T-cell lines. Am J Hematol 1976; I : 219-23. 19. Klein E, Klein G . Nadkarni JS, Nadkarni JJ, Wigzell H, Clifford P. Surface IgM kappa specificity on a Burkitt lymphoma cell in vivo and in derived culture lines. Cancer Res 1968; 28: 1300-10. 20. Minowada J, Ohnuma T. Moore GE. Rosette-forming human lymphoid cell lines. I. Establishment and evidence for origin of thymus-derived lymphocytes. J Natl Cancer lnst 1972; 49: 891 -5. 21. Collins SJ, Gallo RC, Gallagher RE. Continuous growth and differentiation of human myeloid leukemic cells in suspension culture. Nature 1977; 270: 347-9. 22. Takasaki Y. Robinson WA, Tan EM. Proliferating cell nuclear antigen in blast crisis cell of patients with chronic myeloid leukemia. J Natl Cancer lnst 1984; 73: 655-61. 23, Vindelnv LL. Christensen IJ. Nissen NI. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 1983; 3: 323-7. 24. Barlogie B, Drewinko B, Johnston DA. Buchner T. Hauss WH, Freidreich EJ. Pulse cytophotometric analysis of synchronized cells in vitro. Cancer Res 1976: 36: 1176-81. 25. Hall PA, Richards MA, Gregory WM. d'Ardenne AJ, Lister TA, Stansfeld AG. The prognostic value of Ki-67 immunostaining in non-Hodgkin's lymphoma. J Pathol 1988; 154: 223-35. 26. Gerdes J, Stein H, Pileri S, et al. Prognostic relevance of tumor-cell growth fraction in malignant non-Hodgkin's lymphomas. Lancet 1987; 2: 448-9. 27. Sasaki K, Matsumura K, Tsuji T, Shinozaki F, Takahashi M. Relationship between labeling indices of Ki-67 and BrdUrd in human malignant tumors. Cancer 1988; 62: 989-93. 28. Veneroni S , Costa A, Molta R. Giardini R, Rilke F, Silvestrini R. Comparative analysis of [3H]-thymidine labelling index and monoclonal antibody Ki-67 in non-Hodgkin's lymphomas. Hematol Oncol 1988; 6: 21 -8. 29. Huff JP. Roos G . Peebles CL, Houghten R. Sullivan KF, Tan EM. Insights into native epitopes of proliferating cell nuclear antigen using recombinant DNA protein products. J Exp Med 1990; 172: 419-29. 30. Bravo R, MacDonald-Bravo H. Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J Cell Biol 1987; 105: 1 549 - 54.
ISSN: 0284-186X (Print) 1651-226X (Online) Journal homepage: http://www.tandfonline.com/loi/ionc20
Expression of Proliferating Cell Nuclear Antigen (PCNA) and Ki-67 Antigen in Human Malignant Hematopoietic Cells G. Landberg & G. Roos To cite this article: G. Landberg & G. Roos (1991) Expression of Proliferating Cell Nuclear Antigen (PCNA) and Ki-67 Antigen in Human Malignant Hematopoietic Cells, Acta Oncologica, 30:8, 917-921, DOI: 10.3109/02841869109088244 To link to this article: https://doi.org/10.3109/02841869109088244
Published online: 08 Jul 2009.
Submit your article to this journal
Article views: 186
View related articles
Citing articles: 17 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ionc20
Acta Oncologica Vol. 30 No. 8 1991
FROM THE DEPARTMENT OF PATHOLOGY, UNIVERSITY OF UMEA, UMEA, SWEDEN.
EXPRESSION OF PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA) AND Ki-67 ANTIGEN IN HUMAN MALIGNANT HEMATOPOIETIC CELLS G. LANDBERGand G. Roos
Abstract Human hematopoietic cell lines and fresh lymphoma cells were investigated for their expression of proliferating cell nuclear antigen (PCNA) and Ki-67 antigen using flow cytometry. Ki-67 was detected with a monoclonal antibody and the PCNA content was determined using a human autoantiserum and in some cases a monoclonal antibody (PCIO). The autoantibody to PCNA was found to recognize S-phase cells and the number of positively stained cells was significantly correlated with the S-phase fraction determined from DNA-histograms. There was also a strong association between Ki-67 positivity and the fraction of S-phase cells. By dual parameter analysis of Ki-67 and PCNA detected by the autoantibody a detailed cell cycle analysis could be performed of the lymphoma samples. The recently described PClO monoclonal antibody was found by FCM to be a good proliferation marker recognizing cycling cells after methanol and detergent treatment, however without any specific cell cycle phase preference. Key words: Non-Hodgkin’s lymphoma, cell cycle, PCNA, Ki67, monoclonal antibodies.
’
Tumor cell proliferation is an important prognostic parameter for non-Hodgkin’s lymphoma regardless of the technique used to assess cell growth ( 1 -S), and the fraction of S-phase cells determined by flow cytometric (FCM) DNA histogram analysis has been found to be an independent prognostic indicator (6,7). The DNA-FCM technique gives reproducible results in a majority of cases, but aneuploidy and cell debris can make the analysis less accurate. Furthermore, resting ( G o )cells cannot be discriminated from cycling G, cells. The total fraction of actively cycling cells can be assessed by Ki-67 antigen expression as a parameter (8). Using a monoclonal antibody (moab) this nuclear antigen can be detected by immunohistochemistry as well as by flow cytometry of freshly prepared cells (9, 10). The expression of proliferating cell nuclear antigen (PCNA), another growth-related
antigen, can after proper cell preparation serve as an S-phase marker (11-13). We have shown that dual parameter FCM analysis of Ki-67 and PCNA can give detailed cell kinetic information on cell lines (14). In the present paper we present data using this approach on a number of non-Hodgkin’s lymphoma. Recently a series of moabs to PCNA were described (15), including PClO which is commercially available. PCIO, in contrast to Ki-67, can be used on paraffin sections by immunohistochemistry and recent data indicate that PClO can act as a useful proliferation marker (16). We here present FCM data showing that PClO can recognize a form of PCNA present in cycling cells. Material and Methods Lymphoma specimens. Twenty-two fresh lymphoma samples were examined. Imprints and cell suspensions were prepared from fresh tissues as well as paraffin sections after formalin fixation. Cell surface phenotyping was performed using FCM and a series of antibodies detecting Tand B-cell antigens. The cases were classified according to the Kiel classification (17). Eight cases were low-grade lymphomas (LGM) and 14 were high-grade lymphomas (HGM). Cell lines. The following cell lines were studied: Jurkat (18), Daudi (19), MOLT4 (20) and HL-60 (21). Cell lines were cultured in Ham’s F10 medium supplemented with 10% fetal calf serum and antibiotics. In order to evaluate
Paper presented at Symposium on Flow and Static Cytometry in Umei, 10-11 September, 1990. Accepted for publication 24 May 1991.
917
918
G. LANDBERG AND G. ROOS
differences between Go and G I cells normal, purified T lymphocytes were mixed with MOLT4 cells before analysis. Also, nuclear antigen expression after DMSO ( 1.2%)induced granulocytic differentiation of HL-60 cells was studied. Antibodies. The human anti-PCNA serum (AK) was derived from a patient with SLE and is well characterized in previous studies ( 11, 12,22). It was used in a final dilution of 1 :200. The PClO moab was a kind gift from Dakopatts A/S, Denmark, and a 1 : 100 dilution was found optimal for FCM detection. The Ki-67 moab ( 1 : 20) and FITC-conjugated rabbit anti-human IgG ( 1 :25) were obtained from Dakopatts. Phycoerythrin-labelled goat antimouse IgG ( 1 :20) was from Tag0 Inc., Burlingame, CA. As a control to the autoantiserum a normal human serum was used. For Ki-67 an anti-IgE moab (IgG,) (Dakopatts A/S) and for PClO an lgGz moab ( Becton-Dickinson, CA) were used as controls. Flon cytornetry. Standard DNA-histograms were obtained from the lymphoma samples after propidium iodide staining according to the protocol of Vindelw et al. (23). Histogram evaluation was performed according to a modified peak reflect method (24). For double stainings of antigen expression and DNA-content a solution of propidium iodide ( 10 pg/ml) and RNAse (20 pg/ml) was used. Nuclear antigen expression was studied in cells fixed in 70% ethanol (human autoantiserum, Ki-67) or methanol followed by NP-40 (PCIO). The cells were incubated with the primary antibody in phosphate-buffered saline (PBS) for 30 min at room temperature followed by PBS washes and the secondary antibody for 30min. After additional PBS washes propidium iodide solution was added and the samples were kept dark on ice until FCM analysis. We have in a separate study shown that also a washless staining technique using a detergent for membrane permeabilization can be applied to the PCNA/Ki-67 staining (Landberg & Roos, unpublished study). The different cell preparation methods gave very similar results in the dual PCNA/Ki-67 analysis. In the present study most of the lymphoma samples (20/22) were prepared according to the detergent method. Shortly, a mixture of human autoantibody and Ki-67 moab in a buffer containing Triton X-100 (0.5%) was used in the first step followed by a mixture of the secondary antibodies without any intermediate washings. The FCM analyses were performed with a FACScan flow cytometer ( Becton-Dickinson lmmunocytometry Systems, Sunnyvale, CA). Data were stored in list mode and evaluated with the FACScan software ( Becton-Dickinson). Correlation between different parameters was determined by linear regression analysis.
Results The human autoantiserum recognized PCNA in ethanolfixed cells with a preferential reactivity in cells with S-
IB
G a A
W
a
Z
0
n
1
I
q
IL
DNA
A
DNA
Fig. 1 . PCNA expression in relation to DNA content in lymphocytes mixed with MOLT4 cells. A. Human autoantibody (AK), ethanol-fixed cells, B. PClO moab. met1 8
A
a Z
8
I
\
0
n
B
1
Q2lM
t Ql
Ki - 67
Ki-67
Fig. 2. Dual parameter PCNA/Ki-67 FCM analysis of two nonHodgkin's lymphomas. PCNA was detected with a human autoantiserum. The assignment of cells to different cell cycle phases has been detailed previously ( 14). A. Low-grade malignant lymphoma, B. High-grade malignant lymphoma.
phase DNA content (Fig. IA). Unfixed, detergent-treated cells gave a similar staining pattern and the reactivity was identical to the pattern obtained after BrdUrd staining of pulse labelled cells (not shown in figures). Dual parameter analysis of PCNA, detected with the autoantibody, and Ki-67 expression was performed on a series of non-Hodgkin's lymphomas and showed obvious differences between lymphomas of low- and high-grade malignancy (Fig. 2A and B). The low-grade lymphomas had as a rule a low fraction of Ki-67 positive, i.e. cycling, cells, whereas the high-grade malignant cases showed higher numbers of cycling cells. A summary of the findings are shown in Table I . There were highly significant correlations between S-phase cells and PCNA positivity, Sphase cells and Ki-67 reactive cells and accordingly also between Ki-67 and PCNA positive cells (Fig. 3A. B and C). With the PCNA/Ki-67 staining method the different cell cycle compartments within the cycling cell population can be evaluated. The fraction of PCNA-positive cells within the cycling (i.e. Ki-67 positive) cell compartment varied considerably from case to case, but there was no obvious difference between LGM and HGM cases (Table I). The mean cell cycle phase distribution within the Ki-67-positive population was similar for LGM and HGM (Table 2). Furthermore, no association between total cell S-phase
919
CELL CYCLE ANALYSIS OF MALIGNANT LYMPHOMA
Table 1 Dual parameter PCNA IKi-67flow cytometric analysis of 22 non-Hodgkin's lymphomas in relation to diagnostic subgroups and S-phases determined from DNA -histograms. PCNA was detected using a human autoantibody
S(%)
Diagnosis
PCNA+(%)
PCNA: Ki-67
Ki-67+(%)
33.0 37. I 37.0 60.7 61.9 68.3 63.1 29.0
3.7 2.4 2.7 3.8 3.8 2.0 2.8 10.2
LGM case I 2 3 4 5 6 7 8
2.4 2. I
I .2 0.9
1.4 2.0 2.2 2.2 AN
2.3 2.4 I .3
Mean _+ SD
1.9 f 0.5
1.7 f 0.8
48.8 f 16.1
3.9 f 2.6
25.0 25.3 26.5 14.4 6.7 5.6 17.0 6.2 4.3 5.3 AN 3.2 17.6 ND
58.5 39.3 34.7 11.7 7.6 6.4 19.4 7.0 4.2 11.2 13.2 3.9 18.6 19.2
65.0 52.5 45.9 40.3 47.8 27.5 50.3 31.6 31.4 52.9 46.4 24.5 42.2 41.1
90.0 74.9 75.7 28.9 16.0 23.2 38.6 22.1 13.2 21.2 28.4
13.1 k9.0
18.2k 15.8
42.8 f 15.8
1 .o
HGM case 9 10
I1 12 13 14 15
16 17 18
19 20 21 22 Mean k S D
1 .o
1.8
3.0
15.8 44.1
46.7 38.5 f 25.0
LGM = low-grade malignant lymphoma; HGM = high-grade malignant lymphoma; PCNA: Ki-67 = % PCNA-positive cells within the cycling population; A N = Aneuploid; N D = Not done.
c Q) 3Q I
v)
::r'j
( I ' r ' j
II.-
Q
I.
.
.a
,I
I,
6,
.*
I, I
z
..:
a 8
"
a
."
I,
,' I
3
Q
v)
.: ,I
.I
.I
Ki-67
.6
6.
*
10
I.
(I..
*I
11,.
9.
I.
I.
D
..
I.
:8
8
I
.0
Ki-67
*' I.
o *
I.
;:;:rI v)
PCNA
2
fractions and PCNA-positive cells within the cycling compartment seemed to be present (Fig. 3D). More detailed analysis indicated that for HGM such a positive correlation existed, but not for LGM. However, a larger material is needed in order to better clarify this point. In methanol-fixed and NP-40-treated cells the PClO moab gave a strong signal in the FCM analysis. In a mixture of normal lymphocytes and proliferating cells the latter were clearly positive whereas the lymphocytes were negative (Fig. 1B). No cell cycle phase preference was seen since G , , S and GJM cells gave equally strong fluorescence signals. HL-60 cells induced to differentiate to mature granulocytic cells showed a large proportion of PC10-negative cells, whereas the control HL-60 cells were positive (Fig. 4A). DNA-histogram analysis revealed a low S-phase fraction in DMSO-treated cells compared with control cells (Fig. 4B). Microscopic evaluation showed about 15% in the DMSo treated Thus, a fraction Of the HL-60 Cells Were resistant to the DMSO COnCentratiOn used, explaining that some cells were still PC 10-positive.
LO
I.
@
.I
'
.I 1
PCNA:Ki- 6 7
Fig. 3. Correlation between different proliferation parameters. A. S-phase versus PCNA expression evaluated by the autoantibody ( r = 0.92), B, S-phase versus Ki-67 expression ( r = 0.97). C. pCNA versus Ki-67 ( r = 0.97), D. !&phase versus PCNA-positive cells of cycling (Ki-67 +) cells ( r = 0.17).
920
G . LANDBERG AND G. ROOS
Table 2 Cell cycle distribution nithin the cycling cell population
evuluuted from the PCNAlKi-61 analysis Percentage of cycling ( Ki-67 + ) cells
Diagnosis
LG M HGM All cases
GI
S
G2/M
n
45.4 52.0 49.6
48.8 42.8 45.0
5.8 5.2 5.4
8 14 22
PCIO
DNA
Fig. 4. Effects of DMSO induced HL-60 differentiation. A. PClO expression in HL-60 cells, 8. DNA histogram of HL-60 cells ' ' ' control cells, DMSO-treated cells (6d.) ~
IA
6
ih h PCIO
PCIO
Fig. 5. PClO expression in non-Hodgkin's lymphomas. A. Low-grade malignant lymphoma, B. High-grade malignant lymphoma.
Also in fresh lymphomas it was possible to detect PClO by FCM. Fig. 5A illustrates an LGM case with less than 5% PCIO-positive cells in contrast to Fig. SB, showing a high PCIO-positive cell fraction in a case of HGM
Discussion In the present study we analysed the expression of two different growth-associated nuclear antigens ( PCNA and Ki-67) in human lymphoma cells. The fractions of cells, positive for a human anti-PCNA serum, and the moab Ki-67 were strongly correlated to each other as well as to the number of S-phase cells determined by DNA histogram analysis. All three parameters discriminated between LGM and HGM cases as defined by morphologic appearance. In conclusion, any of these parameters can be chosen for crude cell proliferation assessments using FCM .
The Ki-67 positive cell fraction determined by immunohistochemical staining is a significant prognostic parameter for non-Hodgkin's lymphoma (25.26). Our FCM-derived Ki-67 values are comparable to those obtained by Hall et al. (25) using immunostaining of cryosections. In both studies the majority of LGM cases had 20%. By dual parameter analysis of PCNA and Ki-67 more detailed cell kinetic information was achieved, since actively cycling cells could be discriminated from resting cells and characterized concerning their cell cycle phase distribution. The fraction of S-phase cells in the cycling compartment showed rather large variations (24-68%. Table I ) in both the LGM and HGM group. By comparative analysis of bromodeoxyuridine (or thymidine) and Ki-67 labelling indices similar calculations can be made. In a study of various malignant tumors the S-phase size within the proliferating cell fraction showed a mean value of 58'% (27) compared with our mean value of 45%. We did not find any significant difference between LGM and HGM cases concerning this feature, which contrasts to the study of Veneroni et al. (28) showing only lo'%,S-phase cells within the cycling cell population in LGM cases. The reason for this discrepancy might be due to the different staining and detection techniques used. By comparison with data obtained through in vivo iododeoxyuridine incorporation and FCM analysis our PCNA/Ki-67 results can be further substantiated, and such studies are in progress. It is well established that the overall cell proliferation in non-Hodgkin's lymphomas is an independent prognostic factor. Perhaps the fraction of S-phase cells in the cycling compartment might give additional information. It is possible that, e.g., response to cytostatic treatment is related to such a factor. Future clinical follow-up will show the possible clinical significance of these studies. The PClO moab was established after immunization with a recombinant protein product and it detects PCNA in Western blots and by immunoprecipitation (15). PCNA immunoreactivity using PClO was highly correlated to Ki-67 positivity in non-Hodgkin's lymphomas estimated by immunohistochemistry stainings ( 16). Our data generated by FCM support the findings that PClO can be used as a good marker for cell proliferation in hematopoietic cells. For other neoplasms the relation between proliferation and PCNA expression detected by PClO has been reported to be lost, and deregulation of PCNA in tumor adjacent tissues seems to have been present (16). These findings indicate that further studies are needed to clarify the value of this antibody for diagnostic purposes. In contrast to the autoantibody, PClO recognized all cycling cells without any S-phase predominance. This is probably due to epitope differences. It is known that epitopes recognized by autoantibodies are highly dependent on protein conformation whereas monoclonal anti-PCNA antibodies recognized linear epitopes (29). Pre-
CELL CYCLE ANALYSIS OF MALIGNANT LYMPHOMA
liminary studies using synthetic peptides indicate that the PClO epitope is also linear (Roos et al., to be published). The different cell cycle reactivity seen for the PCNA antibodies is not contradictory since there are at least two different populations of PCNA, one of which is associated with the DNA replication machinery (30). These PCNA populations can be affected in different ways during the staining procedure leading to differential epitope expression. In order to clarify this we are presently investigating cell cycle phase reactivity after different fixation and extraction procedures. Preliminary results indicate that under certain circumstances the PC 10 antibody can also preferentially yield staining of S-phase cells.
ACKNOWLEDGEMENTS This study was supported by grants from the Swedish Cancer Society (No. 90 1127). Lions Cancer Research Foundation and the Medical Faculty, University of U m e i The authors are grateful to Miss Bodil Backlund for skillful technical assistance. Corresponding nutltor: Dr Goran Roos. Department of Pathology, University of Umei, S-90187 Umei, Sweden.
REFERENCES I . Costa A. Bonadonna G, Villa E, Valagussa P, Silvestrini R. Labeling index as a prognostic marker in non-Hodgkin's lymphomas. J Natl Cancer lnst 1981; 66: 1-5. 2. Kvalsi S. Marton P, Kaalhus 0, Hsie J, Foss-Abrahamsen A. Godal T. 'H-thymidine uptake in B cell lymphomas-relationship to treatment response and survival. Scand J Haemato1 1985; 34: 429-35. 3. Wooldridge TN. Grierson HL, Weisenburger DD, et al. Association of DNA content and proliferative activity with clinical outcome in patients with diffuse mixed cell and large cell non-Hodgkin's lymphoma. Cancer Res 1988; 48: 6608- 13. 4. Brandt L, Johnson A. Olsson H, Akerman M. Mitotic activity and survival in advanced non-Hodgkin's lymphoma of unfiavourable histology. Eur J Cancer 1990; 26: 227-30. 5. Joensuu H, Klemi PJ, Jalkanen S. Biologic progression in non-Hodgkin's lymphoma. A flow cytometric study. Cancer 1990; 65: 2564-71. 6. Lenner P. Roos G . Johansson H, Lindh J. Dige U. NonHodgkin lymphoma. Multivariate analysis of prognostic factors including fraction ofS-phasecells. Acta Oncol 1987; 26: 179-83. 7. Rehn S, Glimelius B, Strang P, Sundstrom C, Tribukait B. Prognostic significance of flow cytometry studies in B-cell non-Hodgkin lymphoma. Hematol Oncol 1990; 8: 1 - 12. 8. Gerdes J, Lemke H. Baisch H, Wacker H-H. Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J lmmunol 1984; 133: 1710-15. 9. Brown DC. Gatter KC. Monoclonal antibody Ki-67: its use in histopathology. Histopathology 1990; 17: 489-503. 10. Sasaki K. Murakami T. Kawasaki M. Takahashi M. The cell cycle associated change of the Ki-67 reactive nuclear antigen expression. J Cell Physiol 1987; 133: 579-84. I I . Takasaki Y, Fishwild D, Tan EM. Characterization of proliferating d l nuclear antigen recognized by autoantibodies in Lupus sera. J Exp Med 1984; 159: 981 -92. 12. Kurki P, Vanderlaan M. Dolbeare F. Gray J. Tan EM.
92 1
Expression of proliferating cell nuclear antigen ( PCNA/cyclin during the cell cycle. Exp Cell Res 1986; 166: 209- 19. 13. Kurki P, Ogata K, Tan EM. Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry. J lmmunol Methods 1988; 109: 49-59. 14. Landberg G, Tan EM, Roos G . Flow cytometric multiparameter analysis of proliferating cell nuclear antigen/cyclin and Ki-67 antigen: A new view of the cell cycle. Exp Cell Res 1990; 187: 111-8. 15. Waseem NH, Lane DP. Monoclonal antibody analysis of the proliferating cell nuclear antigen (PCNA). Structural conservation and the detection of a nucleolar form. J Cell Sci 1990; 96: 121 -9. 16. Hall PA, Levison DA. Woods AL. et al. Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: An index of cell proliferation with evidence or deregulated expression in some neoplasms. J Pathol 1990; 162: 285-94. 17. Gerard-Marchant R, Hamlin 1. Lennert K. Rilke F, Stansfeld AG, van Unnik JAM. Classification of non-Hodgkin's lymphomas. Lancet 1974; 2: 406-8. 18. Kaplan J, Tilton J, Peterson WD. Identification of T-cell lymphoma tumor antigens on human T-cell lines. Am J Hematol 1976; I : 219-23. 19. Klein E, Klein G . Nadkarni JS, Nadkarni JJ, Wigzell H, Clifford P. Surface IgM kappa specificity on a Burkitt lymphoma cell in vivo and in derived culture lines. Cancer Res 1968; 28: 1300-10. 20. Minowada J, Ohnuma T. Moore GE. Rosette-forming human lymphoid cell lines. I. Establishment and evidence for origin of thymus-derived lymphocytes. J Natl Cancer lnst 1972; 49: 891 -5. 21. Collins SJ, Gallo RC, Gallagher RE. Continuous growth and differentiation of human myeloid leukemic cells in suspension culture. Nature 1977; 270: 347-9. 22. Takasaki Y. Robinson WA, Tan EM. Proliferating cell nuclear antigen in blast crisis cell of patients with chronic myeloid leukemia. J Natl Cancer lnst 1984; 73: 655-61. 23, Vindelnv LL. Christensen IJ. Nissen NI. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 1983; 3: 323-7. 24. Barlogie B, Drewinko B, Johnston DA. Buchner T. Hauss WH, Freidreich EJ. Pulse cytophotometric analysis of synchronized cells in vitro. Cancer Res 1976: 36: 1176-81. 25. Hall PA, Richards MA, Gregory WM. d'Ardenne AJ, Lister TA, Stansfeld AG. The prognostic value of Ki-67 immunostaining in non-Hodgkin's lymphoma. J Pathol 1988; 154: 223-35. 26. Gerdes J, Stein H, Pileri S, et al. Prognostic relevance of tumor-cell growth fraction in malignant non-Hodgkin's lymphomas. Lancet 1987; 2: 448-9. 27. Sasaki K, Matsumura K, Tsuji T, Shinozaki F, Takahashi M. Relationship between labeling indices of Ki-67 and BrdUrd in human malignant tumors. Cancer 1988; 62: 989-93. 28. Veneroni S , Costa A, Molta R. Giardini R, Rilke F, Silvestrini R. Comparative analysis of [3H]-thymidine labelling index and monoclonal antibody Ki-67 in non-Hodgkin's lymphomas. Hematol Oncol 1988; 6: 21 -8. 29. Huff JP. Roos G . Peebles CL, Houghten R. Sullivan KF, Tan EM. Insights into native epitopes of proliferating cell nuclear antigen using recombinant DNA protein products. J Exp Med 1990; 172: 419-29. 30. Bravo R, MacDonald-Bravo H. Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J Cell Biol 1987; 105: 1 549 - 54.
