ARTICLE |
Correspondence to: Ichiro Yamadori, Dept. of Pathology, Okayama U. Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan.
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Summary |
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We compared two methods to stain apoptotic cells, one using terminal deoxynucleotidyl transferase (TDT), the other DNA polymerase I, using leukemia cell lines treated with anti-Fas monoclonal antibody (MAb). Both TDT and polymerase I strongly reacted with fragmented nuclei of apoptotic MOLT-16 and Jurkat cells, but only polymerase I strongly reacted with nonfragmented nuclei of early apoptotic cells. Anti-Fas MAb-treated MOLT-4 cells showed morphological changes corresponding to early apoptosis and were strongly positive for polymerase I only. MOLT-16 and Jurkat cells treated with anti-Fas MAb and inhibitors of endonuclease and poly(ADP-ribose) polymerase showed the morphology of early apoptosis but were not strongly stained by TDT. Because DNA polymerase I has nick-translation activity, it is possible that DNA polymerase I reaction is positive in early apoptotic cells by detecting single-strand DNA cleavage, which occurs before extensive oligonucleosomal DNA cleavage and late morphological changes of apoptosis in leukemia cell lines. Although TDT is widely used to stain apoptotic cells, DNA polymerase I may be more applicable in special cases of apoptosis, in which cells undergo single-strand rather than double-strand DNA breaks. However, the procedure has limitations, such as the necessity to use cell smears for comparison with the TDT reaction. (J Histochem Cytochem 46:85-90, 1998)
Key Words: apoptosis, leukemia cell line, DNA cleavage, DNA polymerase I, terminal deoxynucleotidyl transferase
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Introduction |
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Apoptosis is a form of cell death and is characterized by two major markers. The first is composed of morphological features such as reduction in cell volume, chromatin condensation, and nuclear fragmentation, resulting in apoptotic bodies. The other marker is DNA cleavage by a Ca2+/Mg2+-dependent endonuclease into oligonucleosomal-length fragments detected as a ladder pattern on gel electrophoresis (
Many lymphoma/leukemia cell lines undergo apoptosis in the presence of anti-Fas monoclonal antibody (MAb) (
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Materials and Methods |
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Cells
The human leukemia cell lines, Jurkat, MOLT-16, and MOLT-4 have been maintained at Fujisaki Cell Center, Hayashibara Biochemical Laboratories (Okayama, Japan) and well characterized (
Morphological Studies
For light microscopic observation, cells were collected at 1, 3, 6, 12, and 24 hr after the start of the treatment, smeared on glass slides, air-dried, and stained with May-Grünwald-Giemsa staining. Several hundred cells on each smear were counted to evaluate apoptotic reactions. For electron microscopy, cell pellets were fixed in 3% glutaraldehyde and 1% osmium tetroxide and embedded in Epon resin. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a transmission electron microscope (JEM-1200 EX II; Japan Electron Optics Laboratory, Tokyo, Japan).
Staining of Apoptotic Cells
The TDT and DNA polymerase I reactions were performed as previously reported (
DNA Fragmentation Assay
DNA was extracted from cells by the phenol/chloroform method after digestion with proteinase K and was then electrophoresed on a 1.2% SeaKem ME agarose gel (FMC BioProduct; Rockland, ME).
Statistical Analysis
The mode of apoptosis was compared based on the ratio of apoptotic cells with fragmented nuclei to apoptotic cells with nonfragmented nuclei. The difference was tested by the 2 test.
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Results |
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Light Microscopy
Anti-Fas MAb induced apoptosis of typical morphology (
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MOLT-4 cells reacted differently to anti-Fas MAb. Only a few typical apoptotic cells with fragmented nuclei appeared. Most reacting cells had condensed chromatin, but nuclei had lucent areas and did not become small (Figure 1C). The morphology of these cells was similar to that of Jurkat or MOLT-16 cells in the early stage of apoptosis. At the same time, many dead cells appeared early (Figure 2). The percentage of early apoptotic cells was much higher than in MOLT-16 or Jurkat cells (p<0.01). The concentration of anti-Fas MAb affected the number of apoptotic cells, but the mode of apoptosis was the same in all three cell lines.
Electron Microscopy
Anti-Fas MAb-treated Jurkat and MOLT-16 cells showed cytoplasmic and nuclear condensation; nuclei were fragmented and filled with compacted chromatin (Figure 3A). Apoptotic bodies were also observed, discrete cellular fragments containing one or more round small nuclear fragments filled with condensed chromatin. MOLT-4 cells treated with anti-Fas MAb also showed cytoplasmic and nuclear condensation, but nuclear fragmentation was minimal, and dense masses of condensed chromatin were often observed around the circumference of the nuclear envelope (Figure 3B).
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DNA Fragmentation Assay
Anti-Fas MAb-treated MOLT-16 and Jurkat cells showed a clear ladder pattern of DNA fragmentation, but anti-Fas MAb-treated MOLT-4 cells showed minimal DNA fragmentation and no ladder pattern (Figure 4).
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TDT and DNA Polymerase I Reactions
Jurkat and MOLT-16 cells showing typical apoptotic morphology with fragmented nuclei were strongly positive for both TDT and DNA polymerase I (Figure 5A and Figure 6A). Cells at the early stage of apoptosis without nuclear fragmentation were weakly positive for TDT but clearly positive for DNA polymerase I (Figure 5A and Figure 6A). Apoptotic MOLT-4 cells were strongly positive for DNA polymerase I but only weakly positive for TDT (Figure 5B and Figure 6B).
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Effect of Inhibitors of Endonuclease and PARP
The addition of Zn2+, an inhibitor of Ca2+/Mg2+-dependent endonuclease, to the cell suspension caused some degree of cytoplasmic condensation and chromatin aggregation even without treatment with anti-Fas MAb. None of the usual apoptotic changes induced by anti-Fas MAb were observed in Jurkat and MOLT-16 cells on addition of 1 mM Zn2+. Very few of the apoptotic cells appeared typical, with most showing nuclear morphology consistent with early apoptosis (lacking nuclear fragmentation) (Figure 7A and Figure 8). The percentage of early apoptotic cells was much higher than in cells treated only with anti-Fas MAb (p<0.01). The addition of Zn2+ did not prevent cell death. In all three cell lines treated with anti-Fas MAb and Zn2+, very few apoptotic cells were positive for TDT. Zn2+ was not effective in anti-Fas MAb-treated MOLT-4 cells.
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Treatment with inhibitors of PARP, aminobenzamide, benzamide, or nicotinamide, in addition to anti-Fas MAb, in MOLT-16 and Jurkat cells remarkably reduced the percentage of typical apoptotic cells (p< 0.01), and produced many early apoptotic cells without nuclear fragmentation (Figure 7B and Figure 8). Many dead cells also appeared. No such effect occurred in MOLT-4 cells treated with anti-Fas MAb. Cells treated only with PARP inhibitors were morphologically unchanged. No cells positive for TDT were detected on combined treatment with anti-Fas MAb and PARP inhibitors.
The effect of inhibitors of endonuclease and PARP on anti-Fas MAb-treated cells was basically the same as the effect of anti-Fas MAb and Zn2+ treatment. The cells were shrunken as with Zn2+ treatment, the nuclear morphology was basically that of early apoptosis, and the appearance of dead cells was not prevented.
DNA polymerase I did not react well in experiments with inhibitors. The reaction was weak overall and could not be judged as positive or negative.
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Discussion |
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The TUNEL method is widely used to demonstrate apoptosis in histology sections, but we showed in this experiment that TDT and DNA polymerase I reactions give different staining results in leukemia cell lines. These two procedures utilize different enzymatic reactions detecting different kinds of DNA breaks.
In addition to oligonucleosomal cleavage by endonuclease, other forms of DNA cleavage have been observed during apoptosis (
It has been reported that thymocytes undergo apoptosis when incubated with dexamethasone, but in the presence of Zn2+ cell death morphologically resembles early apoptosis but without the DNA laddering (
Because DNA polymerase I has nick-translation activity, it is possible that DNA polymerase I reaction is positive in early apoptotic cells by detecting single-strand DNA cleavage, which occurs before extensive oligonucleosomal DNA cleavage and late morphological changes of apoptosis in leukemia cell lines. Our staining results were consistent with the idea that activation of endonuclease is a later event in the apoptotic process (
The MOLT-4 cell line responded to anti-Fas MAb in a manner different from the other leukemia cell lines examined. Cells died with the morphology of early stage apoptosis. Apoptotic cells were weakly positive for TDT but strongly positive for DNA polymerase I, and did not show a ladder pattern of DNA fragmentation on gel electrophoresis. Similar examples of cell death have been documented in the literature. Although surface IgM crosslinking or treatment with anti-Fas MAb induced apoptosis in several IgM+ B-leukemia and Fas antigen-positive leukemia cell lines, the cell death in B104 cells treated with anti-IgM or anti-Fas MAb more closely resembles necrosis than apoptosis (
As pointed out by
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Acknowledgments |
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We thank Tadao Zouda for technical assistance with electron microscopy and Yoshiko Senda for secretarial work.
Received for publication December 23, 1996; accepted July 10, 1997.
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Literature Cited |
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Aftabuddin M, Yamadori I, Yoshino T, Kondo E, Akagi T (1995) Correlation between the number of apoptotic cells and expression of the apoptosis-related antigens Fas, Ley and bcl-2 in non-Hodgkin's lymphomas. Pathol Int 45:422-429[Medline]
Akagi Y, Ito K, Sawada S (1993) Radiation-induced apoptosis and necrosis in Molt-4 cells: a study of dose-effect relationships and their modification. Int J Radiat Biol 64:47-56[Medline]
Arends MJ, Morris RG, Wyllie AH (1990) Apoptosis. The role of the endonuclease. Am J Pathol 136:593-608[Abstract]
Bortner CD, Oldenburg NBE, Cidlowski JA (1995) The role of DNA fragmentation in apoptosis. Trends Cell Biol 5:21-26
Cohen GM, Sun X, Snowden RT, Dinsdale D, Skilleter DN (1992) Key morphological features of apoptosis may occur in the absence of internucleosomal DNA fragmentation. Biochem J 286:331-334[Medline]
Creissen D, Shall S (1982) Regulation of DNA ligase activity by poly(ADP-ribose). Nature 296:271-272[Medline]
Duke RC, Cohen JJ, Boehme SA, Lenardo MJ, Surh CD, Kishimoto H, Sprent J (1995) Morphological, biochemical, and flow cytometric assays of apoptosis. In Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W, eds. Current Protocols in Immunology. New York, Current Protocols, 3.17.1-3.17.3.17.33
Durkacz BW, Omidiji O, Gray DA, Shall S (1980) ADP-ribose)n participates in DNA excision repair. Nature 283:593-596[Medline]
Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493-501[Abstract]
Gold R, Schmied M, Rothe G, Zischler H, Breitschopf H, Wekerle H, Lassmann H (1993) Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections. J Histochem Cytochem 41:1023-1030
Ishigami T, Kim K, Horiguchi Y, Higaki Y, Hata D, Heike T, Katamura K, Mayumi M, Mikawa H (1992) Anti-IgM antibody-induced cell death in a human B lymphoma cell line, B104, represents a novel programmed cell death. J Immunol 148:360-368
Kerr JFR, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239-257[Medline]
Kondo E, Yoshino T, Yamadori I, Matsuo Y, Kawasaki N, Minowada J, Akagi T (1994) Expression of bcl-2 protein and Fas antigen in non-Hodgkin's lymphomas. Am J Pathol 145:330-337[Abstract]
Minowada J (1988) Leukemia cell lines. Cancer Rev 10:1-18
Oberhammer F, Wilson JW, Dive C, Morris ID, Hickman JA, Wakeling AE, Walker PR, Sikorska M (1993) Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J 12:3679-3684[Abstract]
Seto S, Carrera CJ, Kubota M, Wasson DB, Carson DA (1985) Mechanism of deoxyadenosine and 2-chlorodeoxyadenosine toxicity to nondividing human lymphocytes. J Clin Invest 75:377-383[Medline]
Sumimoto S, Ishigami T, Horiguchi Y, Yonehara S, Kanazashi S, Heike T, Katamura K, Mayumi M (1994) Anti-Fas antibody induces different types of cell death in the human histiocytic cell line, U937, and the human B cell line, B104: the role of single-strand DNA breaks and poly(ADP-ribosyl)ation in cell death. Cell Immunol 153:184-193[Medline]
Uemura S, Osamura RY, Tsutsumi Y (1996) 'Magentosis' in human lactating breast: a mode of cell death accumulating single-stranded DNA stretches or breaks. Pathol Int 46:122-129[Medline]
Wijsman JH, Jonker RR, Keijzer R, Van de Velde CJH, Cornelisse CJ, Van Dierendonck JH (1993) A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA. J Histochem Cytochem 41:7-12
Wright SC, Wei QS, Kinder DH, Larrick JW (1996) Biochemical pathways of apoptosis: nicotinamide adenine dinucleotide-deficient cells are resistant to tumor necrosis factor or ultraviolet light activation of the 24-kD apoptotic protease and DNA fragmentation. J Exp Med 183:463-471[Abstract]
Wyllie AH (1993) Apoptosis (The 1992 Frank Rose Memorial Lecture). Br J Cancer 67:205-208[Medline]
Yoon YS, Kim JW, Kang KW, Kim YS, Choi KH, Joe CO (1996) Poly(ADP-ribosyl)ation of histone H1 correlates with internucleosomal DNA fragmentation during apoptosis. J Biol Chem 271:9129-9134