Chapter 4 Flow Cytometry in the Study of Proliferation and Apoptosis Michael G. Ormerod, Michael G. Ormerod I Furze Place, Furze Hill, Redhill, United KingdomSearch for more papers by this authorRonald M. Hamelik, Ronald M. Hamelik Pathology Department, University of Miami Miller School of Medicine, Miami, FloridaSearch for more papers by this author Michael G. Ormerod, Michael G. Ormerod I Furze Place, Furze Hill, Redhill, United KingdomSearch for more papers by this authorRonald M. Hamelik, Ronald M. Hamelik Pathology Department, University of Miami Miller School of Medicine, Miami, FloridaSearch for more papers by this author Book Editor(s):Awtar Krishan, Awtar Krishan University of Miami Miller School of Medicine, Miami, FloridaSearch for more papers by this authorH. Krishnamurthy, H. Krishnamurthy Tata Institute of Fundamental Research, Bangalore, IndiaSearch for more papers by this authorSatish Totey, Satish Totey Advance Neuroscience Allies Pvt. Ltd., Bangalore, IndiaSearch for more papers by this author First published: 28 July 2010 https://doi.org/10.1002/9780470631119.ch4 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary This chapter contains sections titled: Introduction Cell Proliferation Apoptosis Applications of Flow Cytometry in Stem Cell Research and Tissue Regeneration RelatedInformation

The α‐folate receptor (α‐FR) is a folate transporter with restricted expression levels in normal tissues. It is over‐expressed in several cancers, particularly epithelial carcinomas, including nonmucinous ovarian carcinoma. It offers a novel therapeutic target for selective imaging and cytotoxic agents. Measurement of the receptor could be a valuable tool in selecting patients more likely to respond to new drugs that target the α‐FR, and monitoring them while on treatment. While tumor samples are often unavailable, a number of patients who relapse develop ascites, which are often rich in tumor cells. We have therefore developed a triple antibody flow cytometric method to assess α‐FR expression on tumor cells from ascites. An antibody to BerEP4, an epithelial cell marker expressed on >90% ovarian cancers, labeled with fluorescein, and an α‐FR antibody labeled with antimouse‐phycoerythrin have been used to label tumor cells, with a CD45‐phycoerythrin‐cyanine5 antibody used to exclude white blood cells from the analysis. The method was optimized using human carcinoma cell lines (JEG‐3, IGROV‐1, and KB cells). Calibrated beads were used to quantify the number of antibodies bound per cell. The triple antibody protocol successfully measured α‐FR expression levels in cell lines spiked with blood. Tumor cells were obtained from ascites in 25 patients with relapsed ovarian cancer. In each case sufficient cells were harvested to identify an epithelial cell population to estimate the number of binding sites/cell. All the samples contained a single population of BerEP4, α‐FR positive cells between 5 × 10 3 and 5 × 10 5 antibody binding sites/cell. The method can be used to determine the number of anti‐α‐FR antibodies bound per epithelial cell in ascites from patients with ovarian carcinoma. The results obtained were reproducible and the method could be applied to specimens that had been stored at −80°C. © 2007 International Society for Analytical Cytology.

Activation of the phosphatidylinositol-3-kinase (PI3K)/AKT survival pathway is a mechanism of cytotoxic drug resistance in ovarian cancer, and inhibitors of this pathway can sensitize to cytotoxic drugs. The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) depletes some proteins involved in PI3K/AKT signaling, e.g., ERBB2, epidermal growth factor receptor (EGFR), and phosphorylated AKT (p-AKT). 17-AAG and paclitaxel were combined (at a fixed 1:1 ratio of their IC50) in four ovarian cancer cell lines that differ in expression of p-AKT, EGFR, and ERBB2. The EGFR-overexpressing A431 and KB epidermoid cell lines were also included. Combination indices (CI) were calculated using the median-effect equation and interpreted in the context of 17-AAG-mediated inhibition of PI3K signaling. Synergy was observed in IGROV-1- and ERBB2-overexpressing SKOV-3 ovarian cancer cells that express a high level of constitutively activated p-AKT [CI at fraction unaffected (fu)0.5 = 0.50 and 0.53, respectively]. Slight synergy was observed in A431 cells (moderate p-AKT/overexpressed EGFR; CI at fu0.5 = 0.76) and antagonism in CH1 (moderate p-AKT), HX62 cells (low p-AKT), and KB cells (low p-AKT/overexpressed EGFR; CI at fu50 = 3.0, 3.5, and 2.0, respectively). The observed effects correlated with changes in the rate of apoptosis induction. 17-AAG induced a decrease in HSP90 client proteins (e.g., C-RAF, ERBB2, and p-AKT) or in downstream markers of their activity (e.g., phosphorylated extracellular signal-regulated kinase or p-AKT) in SKOV-3, IGROV-1, and CH1 cells at IC50 concentrations. A non–growth-inhibitory concentration (6 nmol/L) reduced the phosphorylation of AKT (but not extracellular signal-regulated kinase) and sensitized SKOV-3 cells to paclitaxel. In conclusion, 17-AAG may sensitize a subset of ovarian cancer to paclitaxel, particularly those tumors in which resistance is driven by ERBB2 and/or p-AKT. [Mol Cancer Ther 2006;5(5):1197–208]

Transfusion MedicineVolume 12, Issue 3 p. 223-224 Cytometric Analysis of Cell Phenotype and Function D. A. McCarthy, D. A. McCarthySearch for more papers by this authorM. G. Macey, M. G. MaceySearch for more papers by this author D. A. McCarthy, D. A. McCarthySearch for more papers by this authorM. G. Macey, M. G. MaceySearch for more papers by this author First published: 18 June 2002 https://doi.org/10.1046/j.1365-3148.2002.00379.xRead the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume12, Issue3June 2002Pages 223-224 RelatedInformation

We have studied the effects of the chemotherapeutic drug cis-diamminedichloroplatinum(II) (cisplatin) on three human ovarian carcinoma cell lines one sensitive to the drug (CHI), one with acquired resistance (CHlcisR) and one with intrinsic resistance (SKOV-3). Previous work has shown that the 50% inhibitory concentrations (IC50 values) after a 2-h exposure to the drug are: CHI, 2.5 (µM; CHlcisR, 7.5 µM; and SKOV-3, 33 µM. Despite the variation in sensitivity, the amount of Pt bound to DNA and the rate of removal of Pt was similar for the three lines. There were significant differences in the rates of formation of DNA cross-links but these were not large enough to account for the high resistance of the SKOV-3 line. We have reported that in the L1210 murine leukaemia cell line there are two mechanisms of cisplatin-induced cell death — one of which involves apoptosis. In this paper, we report on an investigation into whether sensitivity to apoptosis played a role in the resistance of these ovarian lines towards cisplatin. After a 2-h incubation with the drug, cells from the three lines showed evidence of death through apoptosis. The cells detached from the culture dish in a time- and dose-dependent fashion. These cells morphologically were quite distinctive from the attached cells and showed changes in their chromatin structure indicative of apoptosis. Their DNA had not been degraded into oligonucleosomal fragments (200 bp and multiples thereof) but had been cut into larger fragments (30 kilobase pairs, kbp) of a size associated with chromatin domains (chromatin loops). At equitoxic doses of drug, the quantity of cells undergoing apoptosis was similar for the three cell lines. The most prominent effect on cell-cycle kinetics was a slowdown in S-phase transit during which the cells underwent apoptosis. Cells that successfully completed the S phase subsequently suffered a temporary G2 block. We propose that the sensitivity of these cell lines to cisplatin was governed by their ability to handle damage caused by platination of the DNA and that the major mechanism of cisplatin-induced cell death in all three cell lines was the induction of apoptosis.

Dexamethasone and etoposide both induce apoptosis in immature rat thymocytes. We investigated the dependence of apoptosis on the phase of the cell cycle after incubation with these drugs. Cell cycle progression was followed by a combination of pulse labelling with 5-bromo-2'-deoxyuridine (BrdU), labelling fixed cells with an anti-BrdU antibody and flow cytometry. Dexamethasone had little effect on the cell cycle progression of proliferating thymocytes, while etoposide caused cell cycle arrest. Normal and apoptotic thymocytes were separated by centrifugation on discontinuous Percoll gradients into four fractions (F1-F4). It was found that both dexamethasone and etoposide induced apoptosis in cells in G0/G1 and G2/M of the cell cycle, whereas only etoposide induced apoptosis of cells in S phase. These results demonstrated that dexamethasone induced apoptosis in quiescent cells while only etoposide could induce apoptosis in cells from the proliferative compartment. Following treatment of thymocytes with etoposide, some of the proliferating thymocytes (F1) were converted to cells with intermediate size and density (F3). We have recently identified these cells as a population of preapoptotic thymocytes, at an early stage of apoptosis. These cells then further progressed to fully apoptotic cells (F4). These data support the hypothesis that normal thymocytes (F1) became apoptotic (F4) via an intermediate population (F3).

Etoposide, a DNA topoisomerase II inhibitor, caused a concentration-dependent induction of apoptosis in immature thymocytes. Using a flow cytometric method to separate and quantify normal and apoptotic cells, etoposide-induced apoptosis was inhibited by cycloheximide and actinomycin D but not by zinc. Etoposide induced a marked cleavage of DNA into nucleosomal length fragments or multiples thereof, which was completely inhibited if the thymocytes were also incubated in the presence of zinc. Etoposide, alone, induced the classical ultrastructural features of apoptosis, but in the presence of zinc, the morphological pattern was markedly different and dominated by discrete clumps of condensed chromatin abutting the nuclear membrane. These latter changes resemble those described as the earliest changes in apoptosis. These results support the hypothesis that, in the induction of apoptosis, critical alterations in nuclear chromatin occur prior to endonuclease cleavage of DNA into nucleosomal fragments.

We have recently developed a method for the separation and quantification of viable apoptotic cells without the need for permeabilisation or fixation of the cells. The method is based on the observation that apoptotic rat thymocytes fluoresce more brightly than normal cells after a brief incubation with the DNA binding dye, Hoechst 33342. In order to understand these differences, we have investigated the uptake of Hoechst 33342 by normal and apoptotic thymocytes. By staining with fluorescein diacetate, we have shown that the efflux of fluorescein from apoptotic cells is more rapid than that from normal thymocytes. This result demonstrated an increase in the permeability of the plasma membrane of the apoptotic thymocytes and it is this change which probably results in the more rapid uptake of Hoechst 33342. The data also revealed two populations of apoptotic thymocytes.

Apoptosis and necrosis are two important mechanisms of cell death. Several methods have recently been described for quantifying apoptotic cells by flow cytometry. We report a novel method for the quantification and separation of viable normal and apoptotic cells. We have applied this method both to immature rat thymocytes treated with a variety of agents and to a murine haemopoetic cell line after withdrawal of a growth factor. The cells were incubated with two dyes which give fluorescent complexes when bound to DNA, the bis-benzimidazole, Hoechst 33342, and propidium iodide. Three populations were identified and characterized. On excitation with UV radiation, dead cells fluoresced red due to the uptake of propidium iodide whereas apoptotic cells fluoresced bright blue; normal cells showed low blue, low red fluorescence. In this paper, we demonstrate how this method may be used to help to distinguish between cell death by apoptosis and necrosis.

Continuous labelling of cells with deoxybromouridine (BrdUrd) followed by staining with a bis-benzimidazole (Hoechst 33258) and a phenanthridinium (propidium iodide or ethidium bromide) allows the cells to be separated by flow cytometry according to the extent of their DNA replication. This BrdUrd-Hoechst/PI method has been used mainly to observe perturbations of the cell cycle in synchronously growing cells. In this paper we demonstrate that, when the method is applied to asynchronously dividing cells, more extensive information can be derived about the effects of cytotoxic and other treatments on the kinetics of the cell cycle. The interpretation of the data is explained, the effects of different types of cytotoxic agent are described, and the method is compared briefly to other methods for following cell cycle kinetics.

For flow cytometry, a suspension of single cells, free of large clumps and excess debris, is essential. The quality of the data obtained depends as much on the quality of the preparation as on that of the flow cytometer.

Using flow cytometry, we describe a method for separating and quantifying normal and apoptotic thymocytes. Apoptosis was induced in isolated thymocytes from immature rats by treatment with the glucocorticoid dexamethasone or the antitumor agent etoposide. Subsequent incubation with the vital bisbenzimidazole dye Hoechst 33342 and the DNA intercalating agent propidium iodide enabled three distinct populations of cells to be identified and sorted by flow cytometry. Dead cells fluoresced red due to propidium iodide whereas normal and apoptotic cells fluoresced blue due to Hoechst 33342. Apoptotic cells were distinguished from normal thymocytes both by their higher intensity of blue fluorescence and by their smaller size as determined by a reduction in forward light scatter. The larger cells, with low blue fluorescence, showed normal thymocyte morphology by electron microscopy and the absence of any DNA fragmentation as measured by agarose gel electrophoresis. In contrast, the smaller cells showed both the morphological characteristics of apoptosis and extensive internucleosomal fragmentation of DNA to multiples of approximately 180 bp. Using this method, a time-dependent induction of apoptosis by dexamethasone, which was inhibited by cycloheximide, actinomycin D, and aurin tricarboxylate, was observed. The method should facilitate mechanistic studies on the induction of apoptosis in thymocytes.

The murine haemopoietic cell line, BAF3, undergoes apoptosis when the growth factor IL-3 is withdrawn. Two flow cytometric methods for quantifying the apoptotic cells are described. Cell sorting followed by DNA gel electrophoresis, and both light and electron microscopy have been used to identify the apoptotic cells. In the first method the cells are fixed in ethanol, stained with propidium iodide and a DNA histogram recorded. The apoptotic cells give a ‘sub-G1’ peak. In the second method unfixed cells are incubated with the bis-benzimidazole, Hoechst 33342. The apoptotic cells take up this dye more rapidly. In this latter method, the non-viable cells can also be enumerated by addition of propidium iodide. The value of the method has been demonstrated in a brief study of the effects of a panel of cytokines on growth and apoptosis.

Identifying a subset of cells, particularly peripheral blood lymphocytes, by means of a fluorescently-tagged antibody is one of the commonest applications of flow cytometry. There are many methods, all of them basically similar, for labeling antigens on cell surfaces. Two slightly different procedures are given in this chapter; these can be adapted to meet most needs.

Conference Article| June 01 1989 Differentiation and growth in the human breast parenchyma MICHAEL J. O'HARE; MICHAEL J. O'HARE *Section of Pathology, Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton, Surrey SM2 5NG, U.K. Search for other works by this author on: This Site PubMed Google Scholar MICHAEL G. ORMEROD; MICHAEL G. ORMEROD *Section of Pathology, Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton, Surrey SM2 5NG, U.K. Search for other works by this author on: This Site PubMed Google Scholar PAUL MONAGHAN; PAUL MONAGHAN *Section of Pathology, Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton, Surrey SM2 5NG, U.K. Search for other works by this author on: This Site PubMed Google Scholar COLIN S. COOPER; COLIN S. COOPER †Section of Chemical Carcinogenesis, Institute of Cancer Research, Chester Beatty Laboratories, Fulham Road, London SW6, U.K. Search for other works by this author on: This Site PubMed Google Scholar BARRY A. GUSTERSON BARRY A. GUSTERSON ‡ *Section of Pathology, Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton, Surrey SM2 5NG, U.K. ‡To whom correspondence should be addressed Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1989) 17 (3): 589–591. https://doi.org/10.1042/bst0170589 Article history Received: October 11 1987 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation MICHAEL J. O'HARE, MICHAEL G. ORMEROD, PAUL MONAGHAN, COLIN S. COOPER, BARRY A. GUSTERSON; Differentiation and growth in the human breast parenchyma. Biochem Soc Trans 1 June 1989; 17 (3): 589–591. doi: https://doi.org/10.1042/bst0170589 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: EMA, epithelial membrane antigen, CALLA, common acute lymphoblastic leukaemia antigen This content is only available as a PDF. © 1989 Biochemical Society1989 Article PDF first page preview Close Modal You do not currently have access to this content.

A murine monoclonal antibody, LICR-LON-23.10, which had been raised against a well-differentiated squamous cell carcinoma cell line (LICR-LON-HN5), recognises an antigen which is present on the membrane of basal cells of the epidermis. The tissue distribution of the antigen, as defined using immunohistochemical techniques, suggests that it is expressed preferentially on cells which are adjacent to a basement membrane. In squamous cell carcinomas, the antigen is expressed uniformly on undifferentiated cells, but in areas of keratinisation, the antigen is absent. The antigen recognised by the antibody was characterised as being a pair of glycoproteins with molecular masses of 120 and 135 daltons. The antibody was used for flow-cytometric analyses of epidermal keratinocyte preparations. Together with other basal cell markers, this antibody may be useful in the characterisation of the epidermal basal cell population as well as in broadening our understanding of the interaction between epithelial cell populations and their relationship with basement-membrane components.