(Circulation. 2000;101:e239-a.)
© 2000 American Heart Association, Inc.
Circulation Electronic Pages |
The Significance of Expression of Proliferating Cell Nuclear Antigen in the Cardiovascular System: Mitosis or DNA Repair?
Department of Medicine
Multi-Imaging Centre Department of Anatomy, University of Cambridge, Cambridge, United Kingdom
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To the Editor:
We read the article by Kanoh et al1 with great interest. They showed that the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick-end labeling (TUNEL) technique is not specific for apoptosis, which was pointed out previously.2 3 Although not specific, TUNEL is a selective method3 and, within the limits imposed by its high sensitivity, the TUNEL technique stains apoptotic nuclei.2 3
Kanoh et al found that a number of TUNEL-positive nuclei expressed
proliferating cell nuclear antigen (PCNA) in myocytes from hearts with
dilated cardiomyopathy.1 A few years
ago, we found that TUNEL-positive nuclei can express PCNA at the edge
of the lipid core of human advanced atherosclerotic
lesions.2 This region, the periphery of the acellular core
of atheroma, is chiefly occupied by lipid-laden
macrophage foam cells.2 We have come to the same
conclusion as Kanoh et al: the coexpression of PCNA and TUNEL (PCNA
being the accessory protein of DNA polymerase 
4 )
indicates DNA repair synthesis. We also suggested that this repair may
presage apoptosis in human atherosclerotic
lesions.2 Indeed, the presence of TUNEL-positive
PCNA-negative nuclei (or nuclear fragments) in the
lesions2 may suggest late stages of apoptosis.
These observations underline the most likely possibility: the expression of PCNA and certain other markers of DNA synthesis, which are frequently used to estimate the fraction of proliferative cells, may also show DNA repair synthesis.1 2 5 The positivity of nick-end labeling techniques, including TUNEL, proves extensive DNA damage and large numbers of DNA breaks exist in these nuclei.
Taken together, the expression of PCNA in TUNEL-positive nuclei suggests that the positivity of some markers of DNA synthesis in certain (probably terminally differentiated) cells, such as macrophages in atherosclerotic lesions and myocytes in the heart, is due to DNA repair rather than mitosis. This view is supported by our finding that PCNA expression without cytological evidence of mitosis was seen in some human macrophages incubated in the presence of oxidized LDL (unpublished data). Any results obtained using markers of DNA synthesis, therefore, should clearly be interpreted with caution.
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References |
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1. Kanoh M, Takemura G, Misao J, et al. Significance of myocytes with positive DNA in situ nick end labeling (TUNEL) in hearts with dilated cardiomyopathy. Circulation.. 1999;99:2757–2764.
2. Hegyi L, Skepper JN, Cary NRB, et al. Foam cell apoptosis and the development of the lipid core of human atherosclerosis. J Pathol. 1996;180:423–429.
3. Kockx MM, Muhring J, Knaapen MWM, et al. RNA synthesis and splicing interferes with DNA in situ end labeling techniques used to detect apoptosis. Am J Pathol.. 1998;152:885–888.
4.
Bravo R, Frank R, Blundell PA, et al. Cyclin/PCNA is
the auxiliary protein of DNA polymerase-. Nature. 1987;326:515–517.
5. Shivji MKK, Kenny MK, Wood RD. Proliferating cell nuclear antigen is required for DNA excision repair. Cell. 1992;69:367–374.
Response
Second Department of Internal Medicine, Gifu University School of Medicine, Gifu, Japan
Department of Oriental Medicine Gifu University School of Medicine
Department of Food Science Kyoto Women’s University, Kyoto, Japan
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Drs Hegyi and Skepper came to the same conclusion that we did: the coexpression of proliferating cell nuclear antigen (PCNA) and DNA in situ nick-end labeling (TUNEL) suggests severe DNA repair in macrophages and cardiac myocytes.R1 R2 In addition, they suggested that this DNA repair may presage apoptosis in human atherosclerotic lesions. In our study on dilated cardiomyopathy (DCM), each myocyte with positive TUNEL (indicating the presence of both single- and double-stranded DNA fragments) was not ultrastructurally apoptotic or necrotic, but living and PCNA-positive. These cells were negative for the Taq polymerase-based in situ ligation assay, which detects double-stranded DNA fragments with single-base, 3' overhangs and is specific for apoptotic mechanisms. Therefore, we do not think that TUNEL- and PCNA-positive myocytes themselves are in the process of apoptosis. However, we assume that these myocytes with severe DNA repair may proceed to apoptosis in the future, because we think that myocyte loss in DCM may be due to apoptosis.
TUNEL is useful for screening apoptotic cells, but it is not specific for apoptosis.R2 R3 DNA ladders indicating the presence of double-stranded DNA fragmentation in the tissue do not differentiate which myocytes or interstitial cells are apoptotic in cardiac tissues, especially in the infarcted myocardium, which has rich infiltrating interstitial cells.R4 The apoptotic morphology of myocytes at the light microscopic level is not reliable, except for the finding of apoptotic bodies, because bizarre-shaped nuclei, which may look like apoptotic nuclei by light microscopy (but are not apoptotic by electron microscopy), are observed in hypertrophied and/or failing hearts.R2 Therefore, we think that both electron microscopic analysis (including electron microscopic TUNEL [EM-TUNEL]) and the Taq polymerase-based in situ ligation assay are necessary for the precise detection of apoptotic cells in vivo, as was shown in our study.
Saraste et al question the percentage of positive TUNEL myocytes in our study. However, this percentage was 0.1% to 1.0% in the autopsied and explanted hearts from the 10 patients with DCM and ischemic cardiomyopathy in their studyR5 and 1% in the 40 specimens from the 20 patients with DCM (7.9% in the 6 TUNEL-positive specimens) in our study.R1 Considering that the tissue size of an endomyocardial biopsy specimen is very small compared with that of tissue sections obtained from autopsied and explanted hearts and that the distribution of TUNEL-positive myocytes varies considerably in tissues from the same heart (as observed by the studies of autopsied and explanted hearts), our data are surprisingly similar to those of Saraste et al, rather than different. Although we agree that their percentages, which result from data obtained using large tissue sections, may be more precise than ours, which were determined from data obtained from very small tissue sections, the main purpose of our study was to examine whether the TUNEL-positive myocytes in DCM were apoptotic or not; we did not attempt to quantify TUNEL-positive myocytes in entire hearts with DCM. Therefore, we used all 6 specimens with light microscopic TUNEL-positive myocytes and 6 of 34 specimens with light microscopic TUNEL-negative myocytes for EM-TUNEL analysis. We found that the percentage of EM-TUNEL–positive myocytes was similar to that of the light-microscopic TUNEL-positive ones. Note that in each patient, the biopsied samples for light and electron microscopic examination were taken from similar portions. Thus, we could have observed many EM-TUNEL–positive myocytes.
Finally, as a positive control, we used prostate tissue from a rabbit castrated 2 days before the study in which many typical apoptotic cells were observed, as was discussed in the Methods section.R1 We found good agreement in incidence between TUNEL-positive cells and ultrastructurally apoptotic cells. The incubation time of DCM specimens was adjusted with the degree of staining in the positive control section. Saraste et al propose using adjacent cardiac tissue sections treated with DNase I as a positive control, without showing why their positive control (artificially fragmented DNA due to treatment with DNase I, which has not been proven to be the DNase responsible for the actual apoptotic process) is better than our control (true apoptotic DNA fragmentation). Further investigation is warranted regarding this issue.
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References |
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TopIntroductionReferencesIntroduction References |
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1. Hegyi L, Skepper JN, Cary NRB, et al. Foam cell apoptosis and the development of the lipid core of human atherosclerosis. J Pathol. 1996;180:423–429.
2. Kanoh M, Takemura G, Misao J, et al. Significance of myocytes with positive DNA in situ nick end-labeling (TUNEL) in hearts with dilated cardiomyopathy: not apoptosis but DNA repair. Circulation. 1999;99:2757–2764.
3. Ohno M, Takemura G, Ohno A, et al. "Apoptotic" myocytes in the infarct area in rabbit hearts may be oncotic myocytes with DNA fragmentation: analysis by immunogold electron microscopy combined with in situ nick end-labeling. Circulation. 1998;98:1422–1430.
4. Takemura G, Ohno M, Hayakawa Y, et al. Role of apoptosis in the disappearance of infiltrated and proliferated interstitial cells after myocardial infarction. Circ Res. 1998;81:1130–1138.
5. Saraste A, Pulkki K, Kallajoki M, et al. Cardiomyocyte apoptosis and progression of heart failure to transplantation. Eur J Clin Invest. 1999;29:380–386.
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