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PubMed1998

Intratumoral cytogenetic heterogeneity detected by comparative genomic hybridization and laser scanning cytometry in human gliomas.

Although it is well-known that cancers show intratumoral phenotypic heterogeneity, genotypic studies have been scarce. Using comparative genomic hybridization and laser scanning cytometric analyses, we investigated intratumoral cytogenetic heterogeneity in 21 surgically removed gliomas including 11 glioblastomas (GBMs), 8 anaplastic astrocytomas (AAs) and 2 low-grade astrocytomas. Comparative genomic hybridization analysis revealed gain or amplification of 7p in 63%, gain of 7q in 73%, loss of 9p in 53%, loss of 10p in 47%, loss of 10q in 47%, loss of 13q in 53%, and loss of 22q in 37% of high-grade astrocytomas. Because these aberrations were region-independent within the same tumor, they did not contribute to intratumoral cytogenetic heterogeneity. Such heterogeneity was due to cytogenetic changes other than the above region-independent aberrations. Intratumoral cytogenetic heterogeneity was detected in 8 of 11 GBMs, 4 of 8 AAs, and none of the 2 low-grade astrocytomas. These observations suggest that cytogenetic changes at chromosomes 7, 9p, 10, 13, and 22 are primary events in high-grade astrocytomas and that subsequent cytogenetic changes involving increases in copy number provide intratumoral heterogeneity. DNA aneuploidy was detected by laser scanning cytometry in 5 of 11 GBMs and 1 of 8 AAs. All tumors with DNA aneuploidy exhibited intratumoral cytogenetic heterogeneity, and there was a significant correlation between DNA aneuploidy and intratumoral cytogenetic heterogeneity. These results support the notion that cytogenetic heterogeneity results from genetic instability within a tumor.

Adult↗
PubMed1999

Objective quantitative analysis of eosinophils and bronchial epithelial cells in induced sputum by laser scanning cytometry.

BACKGROUND: Sputum induction is an important non-invasive technique for measuring airway inflammation in asthma. Cell numbers are often too low for flow cytometric analysis. Laser scanning cytometry (LSC) is a novel technique that allows objective multicolour fluorescence analysis of cells on a microscope slide. METHODS: LSC was used to determine sputum eosinophil and bronchial epithelial cell counts. We first confirmed that we could measure eosinophil counts accurately in peripheral blood using alpha-major basic protein (MBP) immunofluorescent staining. Sputum induction was performed according to standard protocols. Sputum samples from eight normal controls and 12 asthmatic patients were analysed by LSC and manual counting by two independent observers. Octospot cytospins were fixed and stained with mouse-alpha-human-MBP monoclonal antibody or mouse-alpha-human-cytokeratin antibody and goat-alpha-mouse Oregon Green conjugated second antibody. RESULTS: Sputum induction provided a mean (SE) of 0.99 (0.2) x 10(6) cells per donor. More than 3000 cells on three cytospins per slide were analysed per cell type. The intraclass correlation coefficient (R) and standard deviation (SD) of differences in eosinophils determined by manual counting and LSC were 0.9 and 2.1, respectively, and for bronchial epithelial cell counts they were 0.7 and 2.0. Selective detection of labelled cells was confirmed visually after relocation. CONCLUSION: Eosinophils and bronchial epithelial cells can be accurately and reproducibly counted in an objective manner. LSC is therefore a potentially powerful new method for immunophenotyping leucocytes and epithelial cells objectively in induced sputum in patients with asthma.

Asthma↗
PubMed2000

Proliferation and apoptosis in solid tumors. Analysis by laser scanning cytometry.

OBJECTIVE: To examine the relationship between apoptosis and proliferation in a series of human solid malignant tumors, making use of objective, reproducible techniques newly developed for laser scanning cytometry (LSC). STUDY DESIGN: Apoptosis was detected by in situ end labeling of DNA strand breaks with FITC-conjugated nucleotide. Proliferation was detected by Ki-67 antibody. Two parameters were detected independently and simultaneously with DNA measurement on aliquots of cell suspensions obtained by mechanical dissociation of fresh tumors and placed on microscope slides. RESULTS: The number of cells undergoing apoptosis varied from 0.5% to 28.1% (average, 5.4 +/- 6.0). Aneuploid tumors showed a higher percentage of apoptotic cells (7.9 +/- 7.2) as compared to diploid tumors (3.4 +/- 4.0). Tumors with the greatest number of apoptotic cells on LSC also had the largest number of apoptotic cells on light microscopic examination. The number of cells labeled by Ki-67 ranged from 1.7% to 56.7% (average, 20.0 +/- 15.5). Aneuploid tumors were characterized by a higher Ki-67 index (average, 28.3 +/- 14.3%) than the diploid tumors (13.2 +/- 13.3%). CONCLUSION: Overall, there was a very weak or no correlation between apoptosis and proliferation. However, a subset of aneuploid tumors had a high percentage of cells positive for Ki-67 and low percentage of apoptotic cells. Diploid tumors did not show any correlation between apoptosis and proliferation, although many of those tumors had both low apoptotic and proliferative indices. Whether those differences are of prognostic significance remains to be determined in follow-up studies that include more cases and clinical data. Here we have shown that LSC is a powerful new tool of potential clinical value for fast, objective analysis of apoptosis, proliferation and DNA ploidy in solid malignant tumors.

Adolescent↗
PubMed2003

Use of laser scanning cytometry to study tumor microenvironment.

The study of phenomena occurring in the tumor microenvironment is a challenging task because of technical difficulties, particularly when dealing with hypocellular specimens. Laser scanning cytometry (LSC) is a new laboratory technology that has been recently introduced to overcome the limitations of other traditional technologies. By combining the properties and the advantages of flow cytometry (FC) and immunohistochemistry (IHC), LSC allows the investigator to obtain objective information on DNA content, protein expression and cellular localization is combination with morphological features. It has been already shown that LSC results are reliable compared to more traditional technologies, and its implementation in the clinical routine is under way. Its use in oncology, which is rapidly expanding, spans from apoptosis analysis to DNA content quantitation and tumor cell phenotyping. Here we describe the technology underlying this novel fluorescence-based device, review its use in oncology by dissecting the phenomena occurring in the tumor microenvironment and propose its application for the immunological follow-up of malignant lesions undergoing immunotherapeutic manipulation.

Animals↗
PubMed1999

Laser-scanning cytometry: A new instrumentation with many applications.

The laser-scanning cytometer (LSC) is a microscope-based cytofluorometer which has attributes of both flow and image cytometry. Laser-excited fluorescence emitted from fluorochromed individual cells on a microscope slide is measured at multiple wavelengths rapidly with high sensitivity and accuracy. Though the instrument has been available commercially for only 3 years, it is already used in a variety of different applications in many laboratories. This review focuses on the following unique analytical capabilities of LSC which complement those of flow cytometry and fluorescence image analysis: (a) the cells are positioned on slides during measurement so they may be examined repeatedly over time, a feature useful for studies of enzyme kinetics and other time-resolved processes; (b) sequential analysis of the same cells can be carried out using different immuno- or cytochemical stains or genetic probes, merging information on cell immunophenotype, cell functions, expression of particular proteins, DNA ploidy and cell cycle position, and/or cytogenetic profile for each measured cell; (c) any of the cells measured can be relocated to correlate with visual examination by fluorescence or brightfield microscopy or with any other parameter; (d) topographic distribution of fluorescence measurements within the cell, in cytoplasm vs nucleus, permits analysis of the translocation of regulatory molecules such as NFkappaB, p53, etc., and is essential for FISH analysis; (e) hyperchromicity of nuclear DNA as measured by maximal pixel fluorescence intensity allows one to identify cell types differing in degree of chromatin condensation such as mitotic or apoptotic cells; (f) analysis of tissue section architecture and of the constituents in transected cells within tissue sections by ratiometric assays normalized to DNA content extends applications of LSC in clinical pathology; (g) because cell loss during sample preparation and staining is minimal, samples with a paucity of cells can be analyzed; and (h) analyzed cells can be stored indefinitely, e.g., for archival preservation or additional analysis. Potential future applications of LSC are discussed.

Animals↗
PubMed2001

Whole blood microvolume laser scanning cytometry for monitoring resting and activated platelets.

Enumerating and phenotyping of platelets, resting and activated, from whole blood is important for both the identification and verification of many disease states. Microvolume laser scanning cytometry (MLSC) has been shown to be a simple method for enumerating and phenotyping peripheral blood cells. Here, the utility of MLSC, in conjunction with an anticoagulant containing platelet activation inhibitors, for simultaneously measuring platelet count, phenotype and responsiveness directly from non-fixed whole blood was examined. CTAD or EDTA anticoagulated blood was collected from five to 20 healthy volunteers, stained with fluorescence-labeled antibodies specific for platelet antigens, and run on an in-house modified MSLC device. MLSC was able to measure antigens CD9, CD29, CD36, CD41, CD42a, CD42b, and CD61 on platelets and determine an average of 2.3 x 10(5) +/- 7 x 10(4) platelets per microliter. Counts correlated well with those obtained from the Cell-Dyn 3500 (r(2)=0.84). Agreeing with previous data, less than 2% of platelets from peripheral blood of normal individuals expressed the activation markers CD62P or CD63. After in vitro thrombin activation, >93% of the platelets expressed activation markers. Data presented here shows the benefits of using MLSC in combination with platelet inhibitors to quantitate and phenotype platelets while maintaining a viable responsive state.

Anticoagulants↗
PubMed2004

Laser scanning cytometry and tissue microarray analysis of salinity effects on killifish chloride cells.

The effects of salinity on chloride cells (CC) and Na(+)/K(+)-ATPase content in gill epithelium of euryhaline killifish Fundulus heteroclitus were analyzed using laser scanning cytometry (LSC) and tissue microarrays (TMAs). Salinity acclimations consisted of acute transfer from freshwater (FW) to 1x seawater (SW) and gradual transfer from FW to 2.4x SW. Suspensions of dissociated gill epithelial cells were stained with DASPMI and evaluated using LSC. CC number and volume are proportional to external salinity, being lower in FW (0.5+/-0.2 x 10(5) and 405+/-32 micro m(3), respectively) and higher after 5 weeks in 2.4x SW (3.7+/-0.9 x 10(5) and 2697+/-146 micro m(3), respectively). TMAs were constructed from fixed gill tissues and developed using antibody for Na(+)/K(+)-ATPase to visualize CCs in situ and compare their characteristics with isolated CCs. Na(+)/K(+)-ATPase content per CC increases transiently (from 2.2+/-0.5 x 10(6) to 4.8+/-1.1 x 10(6) relative fluorescence units, RFU) after 1 week of acute acclimation to 1x SW but returns to baseline values (2.4+/-0.5 x 10(6) RFU) within 5 weeks. In contrast, gradual acclimation to 2.4x SW permanently increases Na(+)/K(+)-ATPase content per CC (from 2.0+/-0.8 x 10(6) to 6.7+/-2.7 x 10(6) RFU after 5 weeks). CC size in situ did not correlate well to salinity because of basolateral membrane infoldings. Taken together, these data suggest that euryhaline fishes are capable of sensing environmental salinity to utilize transient short-term and permanent long-term adaptations for coping with salinity changes. These results also demonstrate the power of LSC and TMA for comparative biology.

Acclimatization↗
PubMed2003

Measurement of proliferation marker Ki67 in breast tumour FNAs using laser scanning cytometry in comparison to conventional immunocytochemistry.

BACKGROUND: A variety of markers, including Ki67, estrogen receptors (ER), and progesterone receptors (PgR), are frequently measured in fine needle aspirates from human breast carcinomas. Previously, we demonstrated the use of laser scanning cytometry (LSC) for the measurement of Ki67, ER, and PgR in a human breast carcinoma cell line, MCF7 (21). In the present study, we investigated the expression of Ki67 in breast tumour fine needle aspirates (FNAs) using LSC and compared the results to the data obtained using conventional immunocytochemistry (ICC). METHODS: A total of 11 sets of cytospins of FNAs taken from breast tumours at various stages of tamoxifen treatment were used. For LSC, the cytospins were stained for Ki67 with fluorescein using immunofluorescence; the nuclei were counterstained with propidium iodide. A parallel set of cytospins was stained using horseradish peroxidase and diaminobenzidine and scored manually by conventional light microscopy. RESULTS: Values for Ki67 obtained using the LSC were generally lower than ICC scores. The changes in Ki67 measured by the LSC were almost all parallel to those obtained by manual scoring of immunocytochemical stains. CONCLUSIONS: It should be possible to use LSC for the routine measurement of Ki67 marker in FNAs from human breast carcinomas.

Biomarkers, Tumor↗
PubMed2005

Analysis of hypoxia-inducible factor-1alpha accumulation and cell cycle in geldanamycin-treated human cervical carcinoma cells by laser scanning cytometry.

BACKGROUND: Tumor hypoxia has been linked to increased disease aggressiveness and poorer treatment outcomes, and the transcription factor hypoxia-inducible factor-1 (HIF-1) has been identified as the key molecule mediating the cellular response to hypoxic microenvironments. The alpha-subunit of this factor is accumulated under hypoxia and rapidly degraded during re-oxygenation, rendering the reliable measurement of HIF-1alpha a difficult task. Heat shock protein 90 (Hsp90) is an essential protein that controls the activity, turnover, and trafficking of a variety of other proteins including HIF-1alpha and cell cycle regulators. Hsp90 inhibitors like geldanamycin therefore have the potential to target tumor-cell survival by at least two mechanisms, compromising the accumulation of HIF-1alpha and cell proliferation. METHODS: We describe here the simultaneous measurement of HIF-1alpha and cell cycle parameters by laser scanning cytometry (LSC) after exposure of two different human cervical carcinoma cell lines to hypoxia and geldanamycin. RESULTS: Our analysis demonstrates that the cell lines react to hypoxia and drug treatment in a distinct way, with SiHa being more affected by low oxygen concentrations than is ME180, which was more sensitive to geldanamycin treatment. Both cell lines respond to geldanamycin with a G(2)/M-phase arrest and a decrease in HIF-1alpha accumulation. Cell death due to geldanamycin occurs in association with mitosis, presumably through mitotic catastrophe. CONCLUSION: Our results indicate that LSC can significantly contribute to the evaluation of in vitro drug effects particularly with respect to tumor hypoxia and the measurement of HIF-1alpha.

Antibiotics, Antineoplastic↗
PubMed2001

Automation of mouse micronucleus genotoxicity assay by laser scanning cytometry.

BACKGROUND: The evaluation of the safety of drugs and other chemicals is an important aspect of toxicology work. The mouse micronucleus assay is a standard in vivo genotoxicity assay. Chromosomal damage is an indicator of genotoxicity, which manifests in the formation of micronuclei in polychromatic erythrocytes from bone marrow and in peripheral blood erythrocytes. The assay is laborious to perform by manual counting. The laser scanning cytometer allows automated and rapid quantitation of cellular and subcellular fluorescence in monodisperse cell samples on a microscope slide. The object of this study was to evaluate the application of this new technology in the mouse micronucleus genotoxicity assay. Materials and Methods One hundred forty-four mice of various strains were dosed with combinations of carcinogens and antioxidants. Duplicate blood films were prepared 3 days later. One set of slides was stained with acridine orange, and the proportion of micronucleated erythrocytes was counted in 5,000 cells per slide. The duplicates were stained with propidium iodide (40 microg/ml). Five thousand cells per sample were examined using a laser scanning cytometer. The proportion of micronucleated erythrocytes was measured. RESULTS: A coefficient of correlation of 0.96 was found between the data from the two assays. The automation of the assay on the LSC produced a considerable time saving and efficiency gain. CONCLUSIONS: We conclude that with further development, laser scanning cytometry is likely to become the preferred modality for the performance of standard genotoxicity assays.

Animals↗
PubMed2000

Measurement of markers for breast cancer in a model system using laser scanning cytometry.

BACKGROUND: A variety of markers, including Ki67, estrogen receptors (ER), and progesterone receptors (PgR), are frequently measured in fine needle aspirates (FNA) from human breast carcinomas. We used a human breast carcinoma cell line, MCF7, as a model system to investigate the use of laser scanning cytometry (LSC) for the measurement of these markers. Additionally, we measured the number of apoptotic cells. METHODS: Cells were treated with drugs to vary the expression of markers and the number of apoptotic cells. They were then fixed on microscope slides. For LSC, the cells were stained for the different markers with fluorescein using immunofluorescence and for apoptotic cells using the TUNEL assay. The nuclei were counterstained with propidium iodide. A parallel set of slides was stained using horseradish peroxidase and diaminobenzidine and scored manually by conventional light microscopy. RESULTS: The results from the LSC closely paralleled those obtained by manual scoring of immunohistochemical stains. CONCLUSIONS: It should be possible to use LSC for the routine measurement of nuclear markers in FNAs from human breast carcinomas.

Apoptosis↗
PubMed2002

Detection of localized caspase activity in early apoptotic cells by laser scanning cytometry.

BACKGROUND: Caspase activation is a critical early step in the onset of apoptosis. Cell-permeable fluorogenic caspase substrates have proven valuable in detecting caspase activation by flow cytometry. Nevertheless, detection of early low-level caspase activation has been difficult using conventional area or peak fluorescence analysis by flow cytometry, despite the apparent presence of these cells as observed by microscopy. We describe a method utilizing maximum fluorescence pixel analysis by laser scanning cytometry (LSC) to detect early apoptotic cells. METHODS: The PhiPhiLux-G(1)D(2) caspase 3/7 substrate was used in combination with DNA dye exclusion and annexin V binding to identify several stages of apoptosis in EL4 murine thymoma cells by both traditional flow and LSC. LSC analysis of maximum pixel brightness in individual cells demonstrated an intermediate caspase-low subpopulation not detectable by flow or LSC integral analysis. LSC analysis of caspase activity was then carried out using the larger UMR-106 rat osteosarcoma cell line to determine if this apparent early caspase activity could be correlated with localized, punctate caspase activity observed by microscopy. RESULTS: The caspase-low subpopulation found in apoptotic EL4 cells was also observable in UMR-106 cells. Relocation to cells with low fluorescence due to caspase activity and subsequent examination by microscopy demonstrated that these latter cells indeed show punctate, highly localized caspase activation foci that might represent an early stage in caspase activation. CONCLUSIONS: Cells with low-level, localized caspase expression can be detected using maximum pixel analysis by LSC. This methodology allows an early step of apoptotic activation to be resolved for further analysis.

Animals↗
PubMed2000

Relationship between cytogenetic aberrations by CGH coupled with tissue microdissection and DNA ploidy by laser scanning cytometry in head and neck squamous cell carcinoma.

BACKGROUND: The relationship between DNA sequence copy number aberrations (DSCNAs) and DNA ploidy in head and neck squamous cell carcinomas (HNSCCs) is still controversial. Materials and Methods We analyzed DSCNAs by comparative genomic hybridization (CGH) combined with microdissection and DNA ploidy by laser scanning cytometry (LSC) in 18 surgically removed HNSCCs and compared the data. RESULTS: Copy number increases were most frequently observed on chromosomes 3q (16 cases), 8q (13 cases), and 12p (11 cases). Copy number decreases were observed on chromosome 3p (14 cases). LSC revealed DNA aneuploidy in 10 of the 18 cases. All DNA aneuploid tumors exhibited gain or amplification of DNA copy number at 12p11-12.1, whereas gain of DNA copy number was found in only 1 of 8 diploid tumors. DSCNAs were more frequent in DNA aneuploid tumors than in diploid tumors (P < 0.005). CONCLUSIONS: The present observations indicate a close relationship between DSCNAs and DNA ploidy in HNSCCs.

Aged↗
PubMed1999

Functional assay of NF-kappaB translocation into nuclei by laser scanning cytometry: inhibitory effect by dexamethasone or theophylline.

Nuclear factor-kappa B (NF-kappaB)/Rel transcription factors play an important role in the inducible regulation of a variety of cytokine genes in epithelial cells. We assessed accumulation of fluorescence-stained NF-kappaB into propidium iodide-stained nuclei using laser scanning cytometry. "Activity-specific" antibodies to the Rel A (p65) and NF-kappaB1 (p50) subunits of NF-kappaB were detected in the nuclei of A549 cells (an immortalized human type II alveolar epithelial cell line). Exposure to TNF-alpha caused p65 and p50 to translocate into nuclei in a dose- and time-dependent manner. Preincubation with dexamethasone for 24 h or 30 min, or with theophylline for 30 min before TNF-alpha stimulation attenuated the nuclear translocation of Rel A and p50. These findings suggest that theophylline, as well as dexamethasone, may inhibit translocation of NF-kappaB.

Antineoplastic Agents, Hormonal↗
PubMed2000

Laser scanning cytometry for comet assay analysis.

BACKGROUND: The comet assay (single-cell gel electrophoresis) is a sensitive method for evaluating nuclear DNA damage. Previously used evaluation methods for the comet assay are time consuming and have an inherent risk of biased selection of comets due to manual selection and categorization of comet images. Laser scanning cytometry (LSC), the principle of which is equivalent to flow cytometry, enables quantification of fluorescence emitted from the cells on a microscope slide. In the present study, we explored whether LSC could be used to determine the degree of DNA damage demonstrated by the comet assay. METHODS: DNA damage was induced by ultraviolet A irradiation of keratinocytes and visualized by the comet assay. The evaluation included (a) LSC determination of DNA-specific fluorescence in 1,000 comet heads (undamaged DNA), (b) image acquisition of comets by rescanning of the microscope slide, and (c) digital image analysis and computation of tail moment and DNA content in the comet tails. RESULTS: Cells with damaged DNA were observed in a sub-G(1) area because the comet head loses DNA to the tail. We found a strong inverse correlation between tail moment and DNA content per nucleus. CONCLUSIONS: LSC enables an automated method for cell recognition and evaluation of the comets, thus providing quantitative information about nuclear DNA damage without subjective selection of analyzed comets.

Cells, Cultured↗
PubMed2000

Multiparameter analysis of progeny of individual cells by laser scanning cytometry.

BACKGROUND: Effectiveness of antitumor drugs to suppress unrestricted proliferation of cancer cells is commonly measured by cell clonogenicity assays. Assays of clonogenicity are also used in studies of stem/progenitor cells and in analysis of carcinogenic transformation. The conventional assays are limited to providing information about frequency of colonies (cloning efficiency) and do not reveal the qualitative (phenotype) attributes of individual colonies that may yield clues on mechanisms by which cell proliferation was affected by the studied agent. METHODS: Laser scanning cytometry (LSC) was adapted to identify and characterize size and phenotype of colonies of MCF-7 cells growing in microscope slide chambers, untreated and treated with the cytotoxic ribonuclease, onconase (Onc). Individual colonies were located and data representing each colony were segmented based on >650-nm fluorescence excited by a He-Ne laser of the cells whose protein was stained with BODIPY 630/650-X. The DNA of the cells was stained with propidium iodide (red fluorescence) whereas specific proteins (estrogen receptor [ER] or tumor suppressor p53) were detected immunocytochemically (green fluorescence), each excited by an Ar ion laser. RESULTS: A plethora of attributes of individual colonies were measured, such as (a) morphometric features (area, circumference, area/circumference ratio, DNA or protein content per area ratio), (b) number of cells (nuclei), (c) DNA content, (d) protein content and protein/DNA ratio, and (e) expression of ER or p53 per colony, per total protein, per nucleus or per DNA, within a colony. Also cell cycle distribution within individual colonies and heterogeneity of colonies with respect to all the measured features could be assessed. The colonies growing in the presence of Onc had many of the above attributes different than the colonies from the untreated cultures. CONCLUSIONS: Analysis of the features of cell colonies by LSC provides a wealth of information about the progeny of individual cells. Changes in colony size and phenotype, reflecting altered cell shape, cell size, colony protein/DNA ratio, and expression of individual proteins, may reveal mechanisms by which drugs suppress the proliferative capacity of the cells. This may include inducing growth imbalance and differentiation and modulating expression of the genes that may be associated with cell cycle, apoptosis, or differentiation in a progeny of individual cells. Extensions of LSC may make it applicable for automatic analysis of cloning efficiency and multiparameter analysis of cell colonies in soft agar. Such analyses may be useful in studies of the mechanisms and effectiveness of antitumor drugs, in the field of carcinogenesis, and for analyzing primary cultures and assessing tumor prognosis and drug sensitivity. The assay can also be adapted to analysis of microbial colonies.

Boron Compounds↗
PubMed2001

Detection of caspases activation by fluorochrome-labeled inhibitors: Multiparameter analysis by laser scanning cytometry.

BACKGROUND: The fluorochrome-labeled inhibitors of caspases (FLICA) were recently used as markers of activation of these enzymes in live cells during apoptosis (Bedner et al.: Exp Cell Res 259:308-313, 2000). The aims of this study were to (a) explore if FLICA can be used to study intracellular localization of caspases; (b) combine the detection of caspase activation with analysis of the changes with cell morphology detected by microscopy and laser scanning cytometry (LSC); and (c) adapt the assay to fixed cells that would enable correlation, by multiparameter analysis, of caspase activation with the cell attributes that require cell permeabilization in order to be measured. METHODS: Apoptosis of human MCF-7, U-937, or HL-60 cells was induced by camptothecin (CPT) or tumor necrosis factor-alpha (TNF-alpha) combined with cycloheximide (CHX). Binding of FLICA to apoptotic versus nonapoptotic cells was studied in live cells as well as following their fixation and counterstaining of DNA. Intensity of cell labeling with FLICA and DNA-specific fluorochromes was measured by LSC. RESULTS: Exposure of live cells to FLICA led to selective labeling of cells that had morphological changes characteristic of apoptosis. The FLICA labeling withstood cell fixation and permeabilization, which made it possible to stain DNA and measure its content for identification of the cell cycle position of labeled cells. When fixed cells were treated with FLICA, both apoptotic and nonapoptotic cells became strongly labeled and the labeling pattern was consistent with the localization of caspases as reported in the literature. A translocation of the FLICA binding targets from mitochondria to cytosol was seen in the MCF-7 cells treated with CPT. FLICA binding was largely (> 90%) prevented by the substrates of the caspases or by the unlabeled caspase inhibitors having the same peptide moiety as the respective FLICA. CONCLUSIONS: The detection of caspase activation combined with cell permeabilization requires exposure of live cells to FLICA followed by their fixation. Cell reactivity with the respective FLICA, under these conditions, identifies the activated caspases and makes it possible to correlate their activation with the cell cycle position and other cell attributes that can be measured only after cell fixation/permeabilization. FLICA can also be used to study intracellular localization of caspases, including their translocation.

Amino Acid Chloromethyl Ketones↗
PubMed2000

[The relationships among DNA ploidy type determined by laser scanning cytometry, the overexpression of p53 protein and the numerical aberrations of chromosome 7 in bladder cancer].

The cellular DNA content of certain malignancies is regarded as a prognostic parameter. The mutant p53 is thought to destabilize centrosome replication, which leads to aberrant mitosis and chromosome instability. We investigated the relationship among DNA ploidy pattern type, numerical aberrations of chromosome 7 and p53 overexpression in 20 transitional cell carcinomas of the urinary bladder. The DNA ploidy pattern type was determined by laser scanning cytometry, while p53 overexpression was investigated immunohistochemically. Using fluorescence in situ hybridization with chromosome-specific probes, the copy number of chromosome 7 was counted by touch preparations of interphase nuclei. The cytometric analysis revealed that the DNA patterns were highly correlated with both the numerical aberrations of chromosome 7 (p = 0.0002) and the overexpression of p53. The incidences of p53 overexpression in DNA aneuploid tumors and DNA diploid ones were 78% and 10%, respectively (p = 0.0017). Our results suggest that the overexpression of abnormal p53 protein induces DNA aneuploidy in bladder cancer.

Aged↗