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Cytometry in Service of Cell Biology and Cancer Research

Laboratory of Zbigniew (Zbyszek) Darzynkiewicz

The association of Dr. Darzynkiewicz with cytometry has long history. During his post-graduate studies at the Medical Nobel Institute at the Karolinska Institute in Stockholm, Sweden (1967-69), he had an opportunity to use the very early cytometry instruments developed there by Torbjörn Caspersson, the “grandfather” of cytometry. At the time this laboratory had the most advanced microspectrophotometers, microfluorometers and microinterferometers. These instruments were homemade and their development and maintenance required close collaboration of biologists with optical and mechanic engineers and significant investment. The measurements were very burdensome and even after experience was gained no more that 40 cells could be measured per hour. In parallel, autoradiography has been used to quantify incorporation of radioisotopes, generally precursors of DNA or RNA, by individual cells. This was also extremely cumbersome and slow procedure that required extreme patience and perseverance. These early attempts to extend applications of cytometry in studies of cell biology, in particular cell cycle, and the results of these attempts, are described in the attached review article.

Darzynkiewicz Z, Crissman HA, Jacobberger JW. Cytometry of the cell cycle: Cycling through history. Cytometry, 58A; 21-32, 2004.

The close association of Dr. Darzynkiewicz with flow cytometry started when early in 1974 he teamed with Dr. Myron R. Melamed at the Memorial Sloan-Kettering Cancer Institute. Drs. Melamed and Louis A. Kamentsky pioneered the development of the flow cytometry instrumentation, and the instruments constructed by Dr. Kamentsky (Cytograph, then followed by the second generation Cytofluorograph) have been already in use in Dr. Melamed’s Pathology Department. Excited by the analytical capabilities of these instruments, especially when compared with the early instrumentation or autoradiography, Dr. Darzynkiewicz extended their use to various fields of cell biology. Collaborating with Drs Frank Traganos, Tom Sharpless and Lisa Staiano-Coico, he also developed many cytochemical probes and techniques to measure particular attributes of the cell. In 1990 Dr. Darzynkiewicz, with his laboratory, moved to the New York Medical College (NYMC) where he, together with Dr. Traganos, continued research that involved expanding the development of probes and applications of cytometry in various fields of cell biology and cancer research. While at NYMC he collaborated with Dr. Kamentsky on the development of the laser scanning cytometry (LSC), testing the beta-models of LSC and developing new applications for it. His contribution in the field of cytometry is reflected in the number of publications which received wide attention and many citations. Among most significant strides were the following:

  1. Development of the assay for differential staining of DNA and RNA by cytometry exploiting metachromatic property of acridine orange and application of this assay to distinguish quiescent G0 cells from cycling cells. This approach had been widely accepted and is being considered one of the unquestionable markers of G0 cells.

    Darzynkiewicz Z, Traganos F, Sharpless T, Melamed MR. Conformation of RNA in situ as studied by acridine orange staining and automated cytofluorometry. Exp Cell Res 95:143-153, 1975; cited over 200 times.

    Darzynkiewicz Z, Traganos F, Sharpless T, Melamed MR.Lymphocyte stimulation: A rapid multiparameter analysis. Proc Natl Acad Sci USA 73:2881-2884, 1976; cited over 440 times.

    Darzynkiewicz Z, Sharpless T, Staiano-Coico L, Melamed MR. Subcompartments of the G1 phase of cell cycle detected by flow cytometry. Proc Natl Acad Sci USA 77:6696-6700, 1980; cited 237 times.

  2. The use of RNA/DNA content assay to discriminate different leukemia types.

    Andreeff M, Darzynkiewicz Z, Sharpless TK, Clarkson BD, Melamed MR. Discrimination of human leukemia subtypes by flow cytometric analysis of cellular DNA and RNA. Blood 55:282-293, 1980; cited 242 times

  3. The development of the assay of susceptibility of DNA in situ to denaturation. This cytometric assay, which is worldwide accepted, allows one to distinguish cells based on the degree of chromatin condensation such as G0 from G1, G2 from M, or identify apoptotic cells.

    Darzynkiewicz Z, Traganos F, Sharpless T, Melamed MR. Cell cycle related changes in nuclear chromatin of stimulated lymphocytes as measured by flow cytometry. Cancer Res 37:4635-4640, 1977 (cited 108 times).

    Darzynkiewicz, Z, Traganos F, Sharpless T, Melamed MR. Different sensitivity of DNA in situ in interphase and metaphase chromatin to heat denaturation. J Cell Biol 73:128-138, 1977

  4. The demonstration that the rate of cell progression through S or throughout whole cell cycle is correlated with cellular RNA content.

    Darzynkiewicz Z, Evenson D, Staiano-Coico L, Sharpless T, Melamed MR. Relationship between RNA content and progression of lymphocytes through the S phase of the cell cycle. Proc Natl Acad Sci USA 76:358-362, 1979; cited over 125 times;

    Darzynkiewicz Z, Evenson DP, Staiano-Coico L, Sharpless T, Melamed MR. Correlation between cell cycle duration and RNA content. J Cell Physiol 100:425-438, 1979; cited 132 times.

  5. Based on the observation that DNA in chromatin of abnormal sperm cells has higher propensity to DNA denaturation the cytometric assay of DNA denaturation was adapted to identify abnormal sperm cells. This assay (“sperm chromatin structure assay;” SCSA) has been accepted by FDA and WHO and is being used worldwide as the primary male fertility assay.

    Ringertz NR, Gledhill BL, Darzynkiewicz Z. Changes in deoxyri-bonucleoprotein during spermiogenesis in the bull. II. Sensitivity of DNA to heat denaturation. Exp Cell Res 62:204-218, 1970.

    Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science 210:1131-1133, 1980; cited over 270 times; U.S. Patent No. 4,559,309 Issued Dec. 17, 1985: “Flow Cytometry-Fluorescence Measurements for Characterizing Sperm”.

  6. Identification of 12 functionally distinct cell cycle sub-compartments of the cell cycle based on the use of the DNA/RNA content, susceptibility of DNA to denaturation and BrdU incorporation assays.

    Darzynkiewicz Z, Traganos F, Melamed MR. New cell cycle compartments identified by multiparameter flow cytometry. Cytometry 1:98-108, 1980; cited over 280 times; Darzynkiewicz Z, Sharpless T, Staiano-Coico L, Melamed MR. Sub-compartments of the G1 phase of cell cycle detected by flow cytometry. Proc Natl Acad Sci USA 77:6696-6700, 1980; cited over 230 times.

  7. Rhodamine 123 has been used for the first time as a marker of mitochondrial electrochemical potential probe to identify G0 and apoptotic cells in cytometry.

    Darzynkiewicz,, Staiano-Coico L, Melamed MR. Increased mitochondrial uptake of rhodamine 123 during lymphocyte stimulation. Proc Natl Acad Sci USA 78:2383-2387, 1981, cited over 140 times.

    Darzynkiewicz Z, Traganos F, Staiano-Coico L, Kapuscinski J,.Melamed MR. Interactions of rhodamine 123 with living cells studied by flow cytometry. Cancer Res 42:799-806, 1982; cited over 150 times.

  8. For the first time flow cytometry has been used to demonstrate asymmetry of cytokinesis which leads to an increase in heterogeneity of the postmitotic cell population; the cell population however becomes more uniform at the restriction point in G1 when the small cells are hold in progression until they “catch up” in growth of their size (RNA, protein content) with the larger ones.

    Darzynkiewicz Z, Crissman H, Traganos F, Stainkamp J. Cell heterogeneity during the cell cycle. J Cell Physiol 112:465-474, 1982; cited over 115 times.

  9. The detection of cell cycle specific effects (arrest in the cycle, apoptosis) of TNF alpha on target cells with the use of multiparameter cytometry.

    Darzynkiewicz Z, Williamson B, Carswell EA, Old LJ. The cell cycle specific effects of tumor necrosis factor. Cancer Res 44:83-90, 1984; cited over 210 times.

  10. The demonstration that accessibility of DNA in situ to different fluorochromes varies depending on chromatin structure and therefore cell stainability with these dyes not always reflects DNA content (ploidy).

    Darzynkiewicz Z, Traganos F, Kapuscinski J, Staiano-Coico L, Melamed MR. Accessibility of DNA in situ to various fluorochromes: Relationship to chromatin changes during erythroid differentiation of Friend leukemia cells. Cytometry, 5:355-363, 1984; cited over 190 times.

  11. The first demonstration of the molecular mechanism of differential staining of DNA and RNA with pyronin Y.

    Kapuscinski J, Darzynkiewicz, Z. Interactions of pyronin Y (G) with nucleic acids. Cytometry 8:129-137, 1987; Darzynkiewicz Z, Kapuscinski J, Traganos F, Crissman HA. Application of pyronin Y (G) in cytochemistry of nucleic acids. Cytometry 8:138-145, 1987.

    Traganos F, Crissman HA, Darzynkiewicz Z. Staining with pyronin Y detects changes in conformation of RNA during mitosis and hyperthermia of CHO cells. Exp Cell Res 179:535-544, 1988.

  12. Perhaps the most important in the career of Dr. Darzynkiewicz was the development of the assay to label DNA strand breaks with fluoresceinated deoxynucleotides in the reaction catalyzed by terminal deoxynucleotidyl transferase commonly named as the TUNEL assay. This assay is widely used to identify apoptotic cells, primarily by cytometry. It was initially used to demonstrate for the first time that activation of serine protease is required for internucleosomal DNA fragmentation during apoptosis. Numerous variants of this assay are commercially available (e.g. APO-DIRECT, APO-BrdU TUNEL).

    Gorczyca W, Bruno S, Darzynkiewicz RJ, Gong J, Darzynkiewicz, Z. DNA strand breaks occurring during apoptosis: Their early in situ detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors. Int J Onc 1:639-648, 1992; cited over 210 times.

    Gorczyca W, Gong J, Darzynkiewicz Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res 53:1945-1951, 1993; cited over 760 times.

    Gorczyca W, Gong JP, Ardelt B, Traganos F, Darzynkiewicz Z. The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents. Cancer Res 53:3186-3192, 1993 (cited 386 times).

    U.S. Patent No. 5,912,126, Issued June 15, 1999: “Methods for Labeling DNA Ends with Halogenated Nucleotides and Detecting the Same with Antibodies”.

  13. The first demonstration that during chemotherapy leukemic cells are killed by the mechanism of apoptosis.

    Gorczyca W, Bigman K, Mittelman A, Ahmed T, Gong J, Melamed MR, Darzynkiewic, Z. Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. Leukemia 7: 659-670, 1993; cited over 246 times.

  14. The first demonstration of the presence of a multitude of double-strand DNA breaks in human abnormal sperm cells and to propose that the apoptosis-like mechanism leads to elimination of defective sperm cells from reproductive pool. This assay is now used in different institutions to test male fertility.

    Gorczyca, W., Traganos, F., Jesionowska, H., Darzynkiewicz, Z.: Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells. Analogy to apoptosis of somatic cells. Exp Cell Res 207:202-205, 1993; cited over 160 times.

  15. The development of the method to selectively extract fragmented DNA from apoptotic cells. This method is now widely used to analyze DNA fragmentation during apoptosis and to identify apoptotic cells as well as to demonstrated the cells with fractional DNA content (“sub-G1;” “hypodiploid”) by cytometry as well as to demonstrate “DNA laddering” by gel electrophoresis.

    Gong J, Traganos F, Darzynkiewicz Z. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal Biochem 218:314-319,1994, cited over 400 times.

  16. The development of the method to reveal DNA replication based on the induction of DNA strand breaks by photolysis at the sites of BrdU incorporation (Strand Break-Induction by Photolysis; SBIP). Unlike the conventional method to detect the incorporated BrdU that requires acid or heat treatment the SBIP approach is fully compatible with the concurrent detection of surface or intracellular antigens. The kits based on this procedure (ABSOLUTE-S™) are provided by many vendors and widely used.

    Li X, Traganos F, Melamed MR. Darzynkiewicz Z. Detection of 5-bromo-2-deoxyuridine incorporated into DNA by labeling strand breaks induced by photolysis (SBIP). Int J Oncol 4:1157-1161, 1994.

    Li X, Melamed MR, Darzynkiewicz Z. Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color. Exp Cell Res 222: 28-37, 1996; U.S. Patent No. 5,747,258, Issued May 5, 1998 “Detection of Halogenated Precursors Incorporation by Selective Photolysis of Halogenate-Substituted Bases”.

  17. The first demonstration, by cytometry, that most tumor cell lines express cyclins D, A and B1 in an unscheduled manner. Such unscheduled cyclin expression may contribute to loss of control on cell cycle progression in cancer. In fact, only multiparameter cytometry can detect this phenomenon.

    Darzynkiewicz Z, Gong J, Juan G, Ardelt B, Traganos, F.: Cytometry of cyclin proteins. Cytometry 25: 1-13, 1996; cited over 110 times.

  18. The first demonstration that phosphorylation status and conformation of protein can be monitored in individual cells by cytometry (Ser-10 histone H3 and hypophosphorylated pRB).

    (Juan G, Gruenwald S, Darzynkiewicz, Z. Phosphorylation of retinoblastoma susceptibility gene protein assayed in individual lymphocytes during their mitogenic stimulation. Exp Cell Res 239: 104-110, 1998

    Juan G, Traganos F, James WM, Ray JM, Roberge M, Sauve DM, Anderson H, Darzynkiewicz Z. Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis. Cytometry 32: 71-77; 1998

    US patent No. 6,821740 “Flow cytometric methods for the concurrent detection of discrete functional conformations of pRB in single cells”.

  19. The adaptation of LSC to measure kinetics of enzymatic reactions and fluorochrome uptake in the same individual cells by cytometry.

    Bedner E, Melamed MR, Darzynkiewicz Z. Time resolved kinetic reactions measured in individual cells by laser scanning cytometry (LSC) Cytometry 33: 1-9, 1998

  20. The adaptation of LSC to measure, within the same cells, the attributes that can be studied only in live cells and correlate them with the attributes that require prior cell fixation and permeabilization. This approach was used to reveal that caspases activation during apoptosis may occur in the cells that have the mitochondrial potential still preserved.

    Li X, Darzynkiewicz Z. The Schrödinger’s cat quandary in cell biology: Integration of live cell functional assays with measurements of fixed cells in analysis of apoptosis. Exp Cell Res 249: 404-412, 1999.

    Li X, Du L, Darzynkiewicz, Z. Caspases are activated during apoptosis independently of dissipation of mitochondrial electrochemical potential. Exp Cell Res 257: 290-297, 2000.

  21. The development of a simple assay to detect translocation of Bax to mitochondria during initiation step of apoptosis, based on measurement of maximal pixel intensity of Bax immunofluorescence by LSC.

    Bedner E, Li X, Kunicki J, Darzynkiewicz Z. Translocation of Bax to mitochondria during apoptosis measured by laser scanning cytometry. Cytometry 41: 83-88, 2000.

  22. The development of an assay to semi-automatically measure micronucleation e.g. induced by genotoxic agents, by LSC.

    Smolewski P, Ruan Q, Vellon L, Darzynkiewicz Z. The micronuclei assay by laser scanning cytometry. Cytometry, 45: 19-26, 2001.

  23. The development of the in vitro model of wound healing adapted to LSC that can be used to study effects of exogenous agents on the healing (cell proliferation, migration). Using this assay the beneficial effect of hyaluronate (by decreasing incidence of apoptosis and enhancing cell migration) on epithelial cells “wound healing” was demonstrated.

    Haider AS, Grabarek J, Eng B, Pedraza P, Ferreri NR, Balazs EA, Darzynkiewicz Z. In vitro wound healing analyzed by laser scanning cytometry. Accelerated healing of epithelial cell monolayers in the presence of hyaluronate. Cytometry 53A:1-8, 2003; the article received “The best paper in CYTOMETRY 2003” award.

  24. The development of the assay to estimate DNA damage (double-strand breaks) induced by DNA topoisomerase I and II inhibitors or UV light in individual cells by multiparameter cytometry, based on measuring expression of Ser139-phosphorylated histone H2AX and phosphorylation of ATM on Ser1981, in relation with cell cycle phase and initiation of apoptosis (caspase-3 activation).

    Huang X, Traganos F, Darzynkiewicz Z. DNA damage induced by DNA topoisomerase I- and topoisomerase II- inhibitors detected by histone H2AX phosphorylation in relation to the cell cycle phase and apoptosis. Cell Cycle 2: 614-619; 2003.

    Halicka HD, Huang X, Traganos F, King MA, Dai W, Darzynkiewicz Z. Histone H2AX phosphorylation after cell irradiation with UV-B: Relationship to cell cycle phase and induction of apoptosis. Cell Cycle 4: 339-345, 2005

    Huang, X., Halicka, H.D., Traganos, F., Tanaka, T., Kurose, A., and Darzynkiewicz, Z. Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis. Cell Prolif 38: 223-243, 2005.

Dr. Darzynkiewicz is Principal Investigator on one of the longest-lasting RO1 NIH-NCI grants devoted to the development of cytometric probes and assays (RO1 CA 28-704, “Probes for Cytometry,” funded since 1980 – and still continuing). Most of his peer reviewed papers (>510), book chapters (>90), and edited books (>12), are devoted to different aspects of cytometry, particularly in developing and expanding cytometric assays to explore cell proliferation, apoptosis or antitumor drug effects. The overview of his career is given in the article by Z. Demidenko, G. Studzinski and M. Blagosklonny (“From Cytometry to Cell Cycle, A Portrait of Zbigniew Darzynkiewicz; published in Cell Cycle 3:525-8, 2004).

Attached are also four most cited papers from his laboratory published in Cytometry. They become “classics” in the literature, not only in the field of cytometry as pure methodology but also in the field of cell biology, particularly cell cycle and apoptosis. The earliest one (“New Cell Cycle Compartments…” published in 1980 revolutionized the methodology of the cell cycle analysis by introducing the concept of sub-compartments of the cell cycle in addition to the traditionally identified four phases, and demonstrating the capability to detect these 12 distinct compartments, based on differences in cellular RNA content, chromatin structure and proliferation characteristics. This article was cited over 290 times.

With the advent of the discoveries of cyclins and cyclin-dependent kinases (CDKs), the key components of the cell cycle progression machinery, his laboratory was the first to use multiparameter cytometry to relate expression of these subdivide the cell cycle with the traditional cell cycle phases. The paper (“Cytometry of cyclin proteins”, published in Cytometry in 1996, has also become one of the citation classics.

Another paper (“Accessibility of DNA in situ…”, Cytometry, ’84) was the first to provide evidence of non-stoichiometric DNA stainability with nine different fluorochromes, resulting from differences in chromatin structure. This paper was cited over 200 times.

The most cited Cytometry paper is the classic on apoptosis – the description of the methods to detect apoptosis – it was already cited over 1,330 times (“Features of Apoptotic Cells…”, Cytometry ’92). The follow-up article on cytometric detection of apoptosis (“Cytometry in Cell Necrobiology…” Cytometry 97) has similar rate of citations as the first one (already cited over 540 times). In this article new word “cell necrobiology” that defines biology of cell death has been coined. This word and the article has become an inspiration to the artist Julie Newdoll who created a series of paintings devoted to cell death (www.brushwithscience.com) and the use of the term cell necrobiology in the literature is expanding.