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Results for “Laser Scanning Cytometry”

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PubMed2000

Pressure related apoptosis in neuronal cell lines.

Pressure is a crucial component of the cellular environment, and can lead to pathology if it varies beyond its normal range. The increased intra-ocular pressures in acute glaucoma are associated with the loss of neurons by apoptosis. Little is known regarding the interaction between pressure and apoptosis at the level of the cell. The model developed in this study examines the effects of elevated ambient hydrostatic pressure directly upon cultured neuronal lines. Conditions were selected to be within physiological limits: 100 mmHg over and above atmospheric pressure for a period of 2 hr, as seen clinically in acute glaucoma. This system can be used to investigate pressure relatively independently of other variables. Neuronal cell line cultures (B35 and PC12) were subjected to pressure conditions in specially designed pressure chambers. Controls were treated identically, except for the application of pressure, and positive controls were treated with a known apoptotic stimulus. Apoptosis was detected by cell morphology changes and by 2 specific apoptotic markers: TUNEL (Terminal transferase dUTP Nick-End Labeling) and Annexin V. These fluorescent markers were detected and quantified by automated Laser Scanning Cytometry. All techniques showed that increased pressure was associated with a greater level of apoptosis compared to equivalent controls. Our results suggest that pressure alone may act as a stimulus for apoptosis in neuronal cell cultures. This raises the possibility of a more direct relationship at the cellular level between pressure and neuronal loss.

Acute Disease↗

PubMed2005

Automated in situ measurement of cell-specific antibody secretion and laser-mediated purification for rapid cloning of highly-secreting producers.

Cloning of highly-secreting recombinant cells is critical for biopharmaceutical manufacturing, but faces numerous challenges including the fact that secreted protein does not remain associated with the producing cell. A fundamentally new approach was developed combining in situ capture and measurement of individual cell protein secretion followed by laser-mediated elimination of all non- and poorly-secreting cells, leaving only the highest-secreting cell in a well. Recombinant cells producing humanized antibody were cultured serum-free on a capture matrix, followed by staining with fluorescently-labeled anti-human antibody fragment. A novel, automated, high-throughput instrument (called LEAP) was used to image and locate every cell, quantify the cell-associated and secreted antibody (surrounding each cell), eliminate all undesired cells from a well via targeted laser irradiation, and then track clone outgrowth and stability. Temporarily sparing an island of helper cells around the clone of interest improved cloning efficiency (particularly when using serum-free medium), and helper cells were easily eliminated with the laser after several days. The in situ nature of this process allowed several serial sub-cloning steps to be performed within days of one another, resulting in rapid generation of clonal populations with significantly increased and more stable, homogeneous antibody secretion. Cell lines with specific antibody secretion rates of > 50 pg/cell per day (in static batch culture) were routinely obtained as a result of this cloning approach, often times representing up to 20% of the clones screened.

Animals↗

PubMed2003

Human papillomavirus typing and DNA ploidy determination of squamous intraepithelial lesions in liquid-based cytologic samples.

BACKGROUND: Infection by high-risk human papillomavirus (HPV) plays a role in the evolution of cervical carcinoma. Cellular atypia and consecutive DNA content alterations in cytologic samples are consequences of a preexisting viral infection. METHODS: We analyzed the frequency and association of HPV types and the presence of rare cells with abnormally high DNA content. We also evaluated whether these findings support the cytologic diagnosis in 112 routine cases with low and high-grade squamous intraepithelial lesions (LSIL/HSIL) when performed from liquid-based cytologic samples (ThinPrep). For DNA content measurements, laser scanning cytometry was applied and at least 10,000 cells were analyzed. HPV typing was performed by a direct sequencing approach using the consensus primers GP5+/GP6+ and MY09/MY11. RESULTS: Of 112 SIL cases, 110 (98.2%) were HPV positive and 95 (84.8%) had a high-risk type HPV infection. Almost one-half of the cases (46 of 95, 48.4%) with a high-risk HPV infection presented aneuploid squamous cells with greater than 9c DNA content, whereas none of the low-risk HPV-positive or HPV-negative SIL cases showed any aneuploid cells in this range. Although 91.8% of the HSIL cases displayed greater than 9c aneuploid cells, only 7.9% of the LSIL cases were positive for such cells with abnormally high DNA content. CONCLUSIONS: HPV typing and DNA measurements help in the objectivation of cytologic atypia and both can be performed efficiently from the same liquid-based cytologic samples.

DNA, Viral↗

PubMed2002

Exposure of cells to static magnetic field accelerates loss of integrity of plasma membrane during apoptosis.

BACKGROUND: Much attention is being paid to the biologic effects of magnetic fields (MFs). Although MFs enhance tumorigenesis, they are neither mutagenic nor tumorigenic. The mechanism of their tumorigenic effect has not been elucidated. METHODS: To investigate the effect of MFs on apoptosis in HL-60 cells, we exposed the cells to static MFs of 6 mT generated by a magnetic disk of known intensity. Apoptosis was triggered by the DNA topoisomerase I inhibitor, camptothecin (CPT). Activation of caspases in situ using the fluorochrome-labeled inhibitor (FLICA) method and determination of plasma membrane integrity by excluding propidium iodide (PI) were measured by both laser scanning cytometry (LSC) and flow cytometry (FC). LSC and FC identified cells at three sequential stages of their demise: early apoptosis (cells with activated caspases and PI negative); late apoptosis (cells with activated caspases but unable to exclude PI); secondary necrosis (cells with apoptotic morphology no longer stained with FLICA, not excluding PI). RESULTS: MF alone did not induce any apoptogenic or necrogenic effect. CPT exposure led to the sequential appearance of apoptotic cells. In the presence of CPT and MF, the overall proportion of cells undergoing apoptosis was not significantly changed. However, we consistently observed a significant increase in the frequency of late apoptotic/necrotic cells when compared with samples treated with CPT alone (P < 0.001), as well as a decrease in the percentage of early apoptotic cells (P = 0.013). The data obtained by FC and LSC were consistent with each other, showing a similar phenomenon. CONCLUSION: Whereas MF alone or with CPT did not affect overall cell viability, it accelerated the rate of cell transition from apoptosis to secondary necrosis after induction of apoptosis by the DNA-damaging agent, CPT. Modulation of the kinetics of the transition from apoptosis to secondary necrosis by MF in vivo may play a role in inflammation and tumorigenesis.

Apoptosis↗

PubMed2002

Separation of live cells in different phases of the cell cycle for gene expression analysis.

BACKGROUND: Homogeneity of cell populations is a basic requirement for gene expression analyses of the cell cycle, such as those based on microarrays. The most common approach to obtain specific populations is the use of synchronization methods that increase the number of cells representing a certain cell cycle stage. On the one hand, conventional synchronization usually causes undesirable effects. On the other hand, cell separation methods may imply loss of RNA quality, another limiting factor for expression profiling. We describe a new strategy to specifically separate live cells in different phases of the cell cycle (G(1) and G(2)/M) to obtain good quality RNA for gene expression analyses. METHODS: The experimental design included sorting G(1) and G(2)/M cells with the vital fluorochrome Hoechst 33342, followed by RNA isolation from the sorted cells. RESULTS: Sorted living G(1) and G(2)/M cells, analyzed by immunocytochemistry and laser scanning cytometry, showed strong enrichment. The quality and specificity of the isolated RNA were demonstrated by northern blot. CONCLUSIONS: This new approach has many potential applications, such as expression profiling of specific cell populations after eliminating the irrelevant data produced by cells in other stages of the cycle.

Benzimidazoles↗

PubMed2004

Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by the DNA cross-linking agent cisplatin.

BACKGROUND: DNA double-strand breaks (DSBs) in chromatin, whether induced by radiation, antitumor drugs, or by apoptosis-associated (AA) DNA fragmentation, provide a signal for histone H2AX phosphorylation on Ser-139; the phosphorylated H2AX is denoted gammaH2AX. The intensity of immunofluorescence (IF) of gammaH2AX was reported to reveal the frequency of DSBs in chromatin induced by radiation or by DNA topoisomerase I (topo 1) and II (topo 2) inhibitors. The purpose of this study was to further characterize the drug-induced (DI) IF of gammaH2AX, and in particular to distinguish it from AA gammaH2AX IF triggered by DNA breaks that occur in the course of AA DNA fragmentation. METHODS: HL-60 cells in cultures were treated with topotecan (TPT), mitoxantrone (MTX), or with DNA cross-linking drug cisplatin (CP); using multiparameter flow and laser-scanning cytometry, induction of gammaH2AX was correlated with: 1) caspase-3 activation; 2) chromatin condensation, 3) cell cycle phase, and 4) AA DNA fragmentation. The intensity of gammaH2AX IF was compensated for by an increase in histone/DNA content, which doubles during the cell cycle, and for the "programmed" H2AX phosphorylation, which occurs in untreated cells. RESULTS: In cells treated with TPT or MTX, the increase in DI-gammaH2AX IF peaked at 1.5 or 2 h, and was maximal in S- or G(1)-phase cells, respectively, for each drug. In cells treated with CP, compared with TPT, the gammaH2AX IF was less intense, peaked later (3 h) and showed no cell cycle-phase specificity. In the presence of phosphatase inhibitor calyculin A, a continuous increase in the TPT-induced gammaH2AX IF was still seen past 1.5 h, and after 3 h gammaH2AX IF was 2.7- to 3.4-fold higher than in the absence of the inhibitor. The AA gammaH2AX IF was distinguished from the DI-gammaH2AX IF by: 1) its greater intensity; 2) its prevention by caspase inhibitor zVAD-FMK; and 3) the concurrent activation of caspase-3 in the same cells. A decrease in AA gammaH2AX IF coinciding with AA chromatin condensation was seen in the late stages of apoptosis. CONCLUSIONS: Multiparameter analysis of gammaH2AX IF, caspase-3 activation, cellular DNA content, and chromatin condensation allowed us to distinguish the DI from AA H2AX phosphorylation and relate them to the cell cycle phase and stage of apoptosis. With a comparable degree of ds DNA breaks, the cells arrested at the G1 or G2/M checkpoint were less prone to undergo apoptosis than the cells replicating DNA. H2AX phosphorylation seen in CP-treated cells may be associated with DNA repair that involves nucleotide excision repair (NER) and nonhomologous end joining (NHEJ). When the primary drug-induced lesions do not involve ds DNA breaks, but ds DNA breaks are formed during DNA repair, as in the case of CP, analysis of H2AX phosphorylation may reflect extent of the repair process.

Antineoplastic Agents↗

PubMed2004

High-throughput laser-mediated in situ cell purification with high purity and yield.

BACKGROUND: Technologies for purification of living cells have significantly advanced basic and applied research in many settings. Nevertheless, certain challenges remain, including the robust and efficient purification (e.g., high purity, yield, and sterility) of adherent and/or fragile cells and small cell samples, efficient cell cloning, and safe purification of biohazardous cells. In addition, existing purification methods are generally open loop and exhibit an inverse relation between cell purity and yield. METHODS: An automated closed-loop (i.e., employing feedback control) cell purification technology was developed by building upon medical laser applications and laser-based semiconductor manufacturing equipment. Laser-enabled analysis and processing has combined high-throughput in situ cell imaging with laser-mediated cell manipulation via large field-of-view optics and galvanometer steering. Laser parameters were determined for cell purification using three mechanisms (photothermal, photochemical, and photomechanical), followed by demonstration of system performance and utility. RESULTS: Photothermal purification required approximately 10(8) W/cm(2) at 523 nm in the presence of Allura Red, resulting in immediate protein coagulation and cell necrosis. Photochemical purification required approximately 10(9) W/cm(2) at 355 nm, resulting in apoptosis induction over 4 to 24 h. Photomechanical purification required more than 10(10) W/cm(2) independent of wavelength, resulting in immediate cell lysis. Each approach resulted in high efficiency purification (>99%) after a single operation, as demonstrated with eight cell types. An automated closed-loop process to re-image and irradiate remaining targets in situ was implemented, resulting in improved purification (99.5-100%) without decreasing cell yield or affecting sterility in this closed system. Efficient purification was demonstrated with B- and T-cell mixtures over a wide range of contaminating cell percentages (0.1-99%) and cell densities (10(4)-10(6)/cm(2)). Efficient cloning of 293T cells based on fluorescence with green fluorescent protein after plasmid transfection was also demonstrated. CONCLUSIONS: In situ laser-mediated purification was achieved with nonadherent and adherent cells on the automated laser-enabled analysis and processing platform. Closed-loop processing routinely enabled greater than 99.5% purity with a greater than 90% cell yield in sample sizes ranging from 10(1) to 10(8) cells. Throughput ranged from approximately 10(3) to 10(5) total cells/s for contaminating percentages ranging from 99% to 0.1%, respectively.

Automation↗

PubMed2005

Regenerative and predictive medicine of cardiovascular disease: the 9th Leipziger Workshop and the 2nd International Workshop on slide based cytometry.

Slide-based cytometry (SBC) and related techniques offer unique tools to perform complex immunophenotyping, thereby enabling diagnostic procedures at very early disease stages. Multicolor or polychromatic analysis of cells by SBC is of special importance, not only as a cytomics technology platform but also for patients with low blood volume such as neonates. The exact knowledge of the location of each cell on the slide allows restaining and subsequent reanalysis of the specimen. These separate measurements of the same specimen can be fused to one data file (merging), thus increasing the information obtained per cell. Relocalization and optical evaluation of the cells, a feature typical of SBC, can be of integral importance for cytometric analysis. Due to this feature, artifacts can be excluded and morphology of measured cells can be documented. Predictive medicine aims at the detection of changes in patient's state before the manifestation of the disease or its complications. Such instances concern multiorgan failure in sepsis or noninfectious posttraumatic shock in intensive care patients or the pretherapeutic identification of high-risk patients undergoing cancer cytostatic therapy. Early anti-infectious or antishock therapy and curative chemotherapy in combination with stem cell transplantation may provide better chances of patients' survival at concomitant cost containment. Predictive medicine that guides early individualized decrease or cessation of therapy may lower or abrogate potential therapeutic side effects (individualized medicine). Regenerative medicine concerns patients who have diseased and injured organs and may be treated with transplanted organs. However, there is a severe shortage of donor organs that is worsening yearly given the aging population. Regenerative medicine and tissue engineering apply the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Neovascularization is promoted by bone marrow-derived endothelial progenitor cells that lead to the formation of entirely new vessels into ischemic tissue. With this knowledge, many therapeutical borders can be skipped. Diseases formerly uncontrolled can be corrected with stem cells to provide causal healing with regeneration processes. The 9th Leipziger Workshop combined with the 2nd International Workshop on SBC aimed to offer new methods in image cytometry and SBC for solutions in clinical research. It moved toward practical applications in clinics and the clinical laboratory. This development will be continued in 2005 at the upcoming Leipziger Workshop and the 3rd International Workshop on SBC.

Cardiovascular Diseases↗

PubMed2006

Sequential phosphorylation of Ser-10 on histone H3 and ser-139 on histone H2AX and ATM activation during premature chromosome condensation: relationship to cell-cycle phase and apoptosis.

BACKGROUND: Histone H1 and H3 phosphorylation associated with chromatin condensation during mitosis has been studied extensively. Less is known on histone modifications that occur during premature chromosome condensation (PCC). The aim of the present study was to reveal the status of histone H3 and H2AX phosphorylation on Ser-10 and Ser-139, respectively, as well as ATM activation through phosphorylation on Ser-1981, during PCC, and relate these events to cell-cycle phase and to initiation of apoptosis. MATERIALS AND METHODS: To induce PCC, A549 and HL-60 cells were exposed to the phosphatase inhibitor calyculin A (Cal A). Phosphorylation of histone H3 and H2AX as well as ATM activation were detected immunocytochemically concurrent with analysis of cellular DNA content and activation of caspase-3, a marker of apoptosis. The intensity of cellular fluorescence was measured by flow- or laser scanning cytometry. RESULTS: Induction of PCC led to rapid histone H3 phosphorylation, followed by activation of ATM and then H2AX phosphorylation in both, HL-60 and A549 cells. All these events occurred sequentially, prior to caspase-3 activation, and affected cells in all phases of the cell cycle. ATM activation and H2AX phosphorylation was seen during mitosis of A549 but not HL-60 cells. CONCLUSIONS: Because the Cal A-induced phosphorylation of histone H3 and H2AX, and of ATM, precede caspase-3 activation these modifications are pertinent to PCC and not to apoptosis-associated chromatin condensation. The sequence of histone H3 and H2AX phosphorylation and ATM activation during PCC is compatible with a role of ATM in mediating phosphorylation of H2AX but not H3. Mitosis in some cell types may proceed without ATM activation and H2AX phosphorylation.

Apoptosis↗

PubMed2006

Signalling responses linked to betulinic acid-induced apoptosis are antagonized by MEK inhibitor U0126 in adherent or 3D spheroid melanoma irrespective of p53 status.

MEK1/2 inhibitors like U0126 can potentiate or antagonize the antitumor activity of cytotoxic agents such as cisplatin, paclitaxel or vinblastine, depending on the drug or the target cells. We now investigated whether U0126, differentially regulates melanoma signaling in response to UV radiation or betulinic acid, a drug lethal against melanoma. This report shows that U0126 inhibits early response (ERK) kinase activation and cyclin A expression in wt p53 C8161 melanoma exposed to either UV radiation or betulinic acid. However, U0126 does not protect from UV damage, but counteracts betulinic acid-mediated apoptosis in the same cells. Protection from the latter drug by joint treatment with U0126 was also evident in wt p53 MelJuso melanoma and mutant p53 WM164 melanoma. The latter cells were the most responsive to betulinic acid, showing a selective decline in the cdk4 protein, without a comparable change in other key cell cycle proteins like cdc2, cdk2, cdk7 or cyclin A, prior to apoptosis-associated PARP fragmentation. Laser scanning cytometry also showed that betulinic acid induced a significant increase in chromatin condensation in WM164 melanoma irrespective of whether they were in adherent form or as multicellular spheroids. All these betulinic acid-induced changes were counteracted by U0126. Our data show for the first time that (a) cdk4 protein is an early target of betulinic acid-induced apoptosis and (b) unrestricted ERK signaling favours betulinic acid-induced apoptosis, but this is counteracted by U0126, partly through counteracting chromatin condensation and restoring Akt activation decreased by betulinic acid treatment.

Apoptosis↗

PubMed2005

Ectodomain shedding of membrane-anchored heparin-binding EGF like growth factor and subcellular localization of the C-terminal fragment in the cell cycle.

Heparin-binding EGF-like growth factor (HB-EGF) is initially synthesized as a type I transmembrane protein (proHB-EGF). The proHB-EGF is shed by specific metalloproteases, releasing the N-terminal fragment into the extracellular space as a soluble growth factor (HB-EGF) and the C-terminal fragment (HB-EGF-C) into the intracellular space, where it prevents transcriptional repression by the promyelocytic leukemia zinc finger protein (PLZF). The goal of the present study was to characterize regulation of proHB-EGF shedding and study its temporal variations in HB-EGF-C localization throughout the cell cycle. Quantitative combination analyses of cell surface proHB-EGF and HB-EGF in conditioned medium showed that proHB-EGF shedding occurred during the G(1) cell cycle phase. Laser scanning cytometry (LSC) revealed that HB-EGF-C was internalized into the cytoplasm during the late G1 phase and accumulated in the nucleus beginning in the S phase. Subsequent nuclear export of PLZF occurred during the late S phase. Further, HB-EGF-C was localized around the centrosome following breakdown of the nuclear envelope and was localized to the interzonal space with chromosome segregation in the late M phase. Temporal variations in HB-EGF localization throughout the cell cycle were also characterized by time-lapse imaging of cells expressing YFP-tagged proHB-EGF, and these results were consistent with those obtained in cytometry studies. These results indicate that proHB-EGF shedding and subsequent HB-EGF-C signaling are related with progression of the cell cycle and may provide a clue to understand the unique biological significance of non-receptor-mediated signaling of proHB-EGF in cell growth.

Animals↗

PubMed2001

Oxidative DNA damage and 8-hydroxy-2-deoxyguanosine DNA glycosylase/apurinic lyase in human breast cancer.

To test the hypothesis that oxidative stress is involved in breast cancer, we compared the levels of 8-hydroxy-2-deoxyguanosine (8-oxo-dG), an oxidized DNA base common in cells undergoing oxidative stress, in normal breast tissues from women with or without breast cancer. We found that breast cancer patients (N = 76) had a significantly higher level of 8-oxo-dG than control subjects (N = 49). The mean ( +/- SD) values of 8-oxo-dG/10(5) dG, as measured by high-performance liquid chromatography electrochemical detection, were 10.7 +/- 15.5 and 6.3 +/- 6.8 for cases and controls, respectively (P = 0.035). This difference also was found by immunohistochemistry with double-fluorescence labeling and laser-scanning cytometry. The average ratios (x10(6)) of the signal intensity of antibody staining to that of DNA content were 3.9 +/- 7.2 and 1.1 +/- 1.4 for cases (N = 57) and controls (N = 34), respectively (P = 0.008). There was no correlation between the ages of the study subjects and the levels of 8-oxo-dG. Cases also had a significantly higher level of 8-hydroxy-2-deoxyguanosine DNA glycosylase/apurinic lyase (hOGG1) protein expression in normal breast tissues than controls (P = 0.008). There was no significant correlation between hOGG1 expression and 8-oxo-dG. Polymorphism of the hOGG1 gene was very rare in this study population. The previously reported exon 1 polymorphism and two novel mutations of the hOGG1 gene were found in three of 168 cases and two of 55 controls. In conclusion, normal breast tissues from cancer patients had a significantly higher level of oxidative DNA damage. The elevated level of 8-oxo-dG in cancer patients was not related to age or to deficiency of the hOGG1 repair gene.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2001

Expression of BAX in cell nucleus after experimentally induced apoptosis revealed by immunogold and embedment-free electron microscopy.

The unique combination of immunocytochemistry with embedment-free electron microscopy was applied for precise and specific localisation of BAX in the human colon adenocarcinoma COLO 205 cell line stimulated to undergo apoptosis by camptothecin (DNA topoisomerase I inhibitor). Camptothecin-induced apoptosis was associated with redistribution of BAX from cytosol to organelle membranes: mitochondria, Golgi apparatus, endoplasmic reticulum and via nuclear envelope pores to the nucleus, occurring within 60-180 min of cell exposure to the drug. An increase in BAX immunoreactivity on fine filaments and the lamina-pore complex of the nuclear matrix was also observed. The increase in BAX expression in the nuclear area of camptothecin-treated COLO 205 cells was confirmed by quantitative analysis using laser scanning cytometry. The subcellular translocations of BAX preceded the appearance of any morphological symptoms of apoptosis.

Apoptosis↗

PubMed2000

Cleavage of Poly(ADP-ribose) polymerase measured in situ in individual cells: relationship to DNA fragmentation and cell cycle position during apoptosis.

Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a target of caspases during apoptosis: its cleavage onto 89- and 24-kDa fragments is considered to be a hallmark of the apoptotic mode of cell death. Another hallmark is the activation of endonuclease which targets internucleosomal DNA. The aim of the present study was to reveal cell cycle phase specificity as well as the temporal and sequence relationships of PARP cleavage vis-à-vis DNA fragmentation in two model systems of apoptosis, one induced by DNA damage via cell treatment with camptothecin (CPT) (mitochondria-induced pathway) and another by the cytotoxic ligand tumor necrosis factor alpha (TNF-alpha) (cell surface death receptor pathway). PARP cleavage was detected immunocytochemically using antibody which recognizes its 89-kDa fragment (PARP p89) while DNA fragmentation was assayed by in situ labeling of DNA strand breaks. The frequency and extent of PARP cleavage as well as DNA fragmentation were measured by mutiparameter flow and laser scanning cytometry. PARP cleavage, selective to S phase cells, was detected 90 min after administration of CPT. PARP cleavage in the cells treated with TNF-alpha was not selective to any cell cycle phase and was seen already after 30 min. DNA fragmentation trailed PARP cleavage by about 30 min and showed a similar pattern of cell cycle specificity. PARP p89 was present in nuclear chromatin but at least in the early phase of apoptosis it did not colocalize with DNA strand breaks. The rate of cleavage of PARP molecules in individual cells whether induced by CPT or TNF-alpha was rapid as reflected by the paucity of cells with a mixture of cleaved and noncleaved PARP molecules. In contrast, DNA fragmentation proceeded stepwise before reaching the maximal number of DNA strand breaks. Although time windows for PARP cleavage vs DNA fragmentation were different at early stages of apoptosis, a good overall correlation between the cytometric assays of apoptotic cells identification based on these events was observed in both CPT- and TNF-alpha-treated cultures.

Apoptosis↗

PubMed2000

Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation.

Activation of caspases is the key event of apoptosis and new methods are needed to assay this event, particularly in situ, in individual cells. To measure in situ caspases activation in the present study we employed fam-VAD-fmk and fam-VEID-fmk, the fluorochrome (fam)-labeled inhibitors of caspases (FLICA), which through the fluoromethylketone (fmk) moiety bind to active center of the activated enzymes. The peptide moiety of these inhibitors defines their specificity; VAD is generic to most caspases and VEID is caspase-6 specific. The frequencies of cells showing caspases activation were compared with those showing DNA fragmentation (detected by the TUNEL assay) in the same cultures. Apoptosis of HL-60 cells was induced by DNA topoisomerase I inhibitor camptothecin (CPT) or tumor necrosis factor-alpha combined with cycloheximide (TNF-alpha + CHX). The cells that bound FLICA had morphological changes typical of apoptosis. The intensity of their fluorescence was measured by laser scanning cytometry. Maximal rate of activation of the caspases, measured by the increase in frequency of the cells that bound fam-VAD-fmk, occurred between 30 and 90 min after the administration of TNF-alpha + CHX and between 2 and 4 h after the administration of CPT. In the CPT-treated cultures about 30% fewer cells bound fam-VEID-fmk than fam-VAD-fmk which suggests that the activation of caspase-6 was delayed or was not induced in some cells. A strong overall correlation between the cytometric assays of the apoptotic index based on the detection of caspases activation by the FLICA and the TUNEL assay was observed. The data indicate that FLICA offers a rapid and convenient method of assessing caspase's activation in individual cells and can also be used to estimate the frequency of apoptosis.

Amino Acid Chloromethyl Ketones↗

PubMed2002

Specific lectins map the distribution of fibronectin and beta 1-integrin on living MDCK cells.

The expression and dynamics of bound fibronectin and the sialylated integral membrane protein, beta 1-integrin, were analyzed on the apical membrane of living MDCK cells. Fibronectin was identified by its specific binding of fluorescent peanut agglutinin and sialylated beta 1-integrin by its binding of Sambucus nigra agglutinin. Confocal epifluorescence microscopy and laser scanning cytometry determined the distribution and abundance of binding sites of the two fluorescently labeled lectins. Both fibronectin and beta 1-integrin were restricted to specific regions uniformly distributed over the entire apical surface. Apical-surface fibronectin binding varied much more between cells than did the expression of beta 1-integrin. Sialylated beta 1-integrin colocalized >92% with membrane microplicae while fibronectin was unrelated to these surface structures. This lack of colocalization of the proteins was confirmed by double-labeling experiments. From the maturation dependence of the fibronectin-binding capacity and the differences in protein turnover times, it was evident that fibronectin did not bind to sialylated beta 1-integrin. Furthermore, desialylation of beta 1-integrin uncovered additional fibronectin receptors on the apical membrane. We conclude that these lectins permit tracking of two membrane-associated glycoproteins in living cells and that fibronectin binds only to desialylated beta 1-integrin on MDCK cells.

Animals↗

PubMed1999

The role of glutathione in chronic adaptation to oxidative stress: studies in a normal rat kidney epithelial (NRK52E) cell model of sustained upregulation of glutathione biosynthesis.

Reduced glutathione (GSH) is considered to play a central role in protection of cells from oxidant injury. However, the question remains as to whether sustained elevation of intracellular GSH levels, as compared with the ability to rapidly upregulate GSH synthesis, is more important with respect to protection of cell constituents from oxidative stress. To address this question, we conducted studies to evaluate the direct influence of chronically increased endogenous GSH content on chemically induced intracellular free radical formation and oxidative stress using a kidney epithelial cell model adapted to sustain intracellular GSH concentrations in excess of eightfold that observed in unadapted parent kidney cells. Elevated GSH levels in adapted cells were found to be attributable, at least in part, to coordinately increased amounts of both the regulatory and catalytic subunits of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis. Studies using electron spin resonance (ESR) spectroscopy and scanning laser cytometry demonstrated that cells having sustained elevation of GSH levels did not attenuate free radical formation and associated oxidative stress compared with parent cells when treated with the prooxidant chemicals, menadione or potassium dichromate. In contrast, nonadapted kidney parent cells treated 18 h after initial prooxidant challenge displayed significantly attenuated free radical signals. Additionally, cells adapted to sustain excess GSH were somewhat more sensitive than parent cells in terms of resistance to prooxidant (chromate) toxicity, as determined by cell viability assays. These findings suggest that the capacity of cells to rapidly upregulate GSH synthesis, rather the ability to chronically sustain elevated intracellular GSH levels, may play a more important role in terms of protection from cytotoxicity associated with prooxidant chemical exposures.

Adaptation, Physiological↗

PubMed2004

Chromosomal aberrations accumulate in polyploid cells of high-grade squamous intraepithelial lesions (HSIL).

Persistant infection with human papillomavirus (HPV) of the uterine cervix is related with cytological atypia (SIL), the oncogenic potential of which is unclear in a given time point of monitoring. HPV-induced genetic instability result in polyploidization as well as in low frequency random chromosome aberrations in squamous cells. In the present work we analyzed whether highly polyploid/aneuploid cells reflect genomic changes at the chromosomal level. 13 samples with the cytological diagnosis of HSIL were analyzed for HPV type and nuclear DNA content measured by laser scanning cytometry (LSC). Hyperdiploid cells with >5c and with >9c DNA content were further analyzed for numerical aberrations of the chromosomes 3 and 17 by fluorescence in situ hybridization (FISH) following repositioning. Cells with >5c DNA content were found more frequently than cells with >9c DNA content (5-98 and 1-44 cells, respectively). The FISH analysis demonstrated frequent polysomies, however, the rate of aneusomy (other than 2, 4, 8 or 16 chromosome copies) was significantly higher in cells with >9c DNA content than in cells with >5c DNA content or the normal diploid cells. The imbalance of chromosome 3 and 17 copy number was also increased in cells with >9c DNA content. Moreover, in three out of the 13 analyzed HSIL samples, recurrent abnormal chromosome 3/17 ratio was demonstrated in a significant part of the cells, indicating a common origin of these cells. Highly polyploid/aneuploid cells in HSIL accumulate cytogenetic aberrations detectable by FISH analysis. These cells may reflect early changes with tumorigenic potential in a very concentrated fashion.

Chromosome Aberrations↗