The micronucleus (MN) assay on exfoliated buccal cells is a useful and minimally invasive method for monitoring genetic damage in humans. To determine the genotoxic effects of calcite dust that forms during processing, MN assay was carried out in exfoliated buccal cells of 50 (25 smokers and 25 non-smokers) calcite factory workers and 50 (25 smokers and 25 non-smokers) age- and sex-matched control subjects. Frequencies of nuclear abnormalities (NA) other than micronuclei, such as binucleates, karyorrhexis, karyolysis and ‘broken eggs', were also evaluated. Micronuclei and the other aforementioned anomalies were analysed by two way analysis of covariance. The linear correlations between the types of micronucleus and nuclear abnormalities were determined by Spearman's Rho. There was a positive correlation between micronuclei and other types of nuclear abnormalities in accordance with the Spearman's Rho test. Results showed statistically significant difference between calcite fabric workers and control groups. MN and NA frequencies in calcite fabric workers were significantly higher than those in control groups (p < 0.05). The results of this study indicate that calcite fabric workers are under risk of significant cytogenetic damage.
calcite; exfoliated buccal cells; micronucleus (MN); genotoxicity
The imaging analytical capabilities of laser scanning cytometer (LSC) have been used to assess morphological features considered to be typical of the senescent phenotype. The characteristic “flattening” of senescent cells was reflected by the decline in the density of staining (intensity of maximal pixel) of DNA-associated fluorescence [4,6-diamidino-2-phenylindole (DAPI)] paralleled by an increase in nuclear size (area). The decrease in ratio of maximal pixel to nuclear area was even more sensitive senescence biomarker than the change in maximal pixel or nuclear area, each alone. The saturation cell density at plateau phase of growth recorded by LSC was found to be dramatically decreased in cultures of senescent cells, thereby also serving as an additional marker. The induction of cyclin dependent kinase inhibitors p21WAF1 and p27KIP1 and γH2AX and activation of ATM markers of DNA damage response were measured in parallel with DNA/DAPI maximal pixel and nuclear area. These biomarker indices were expressed in quantitative terms by reporting them as a fraction of the respective controls. The effect of treatment of A549 and WI-38 cells with different concentrations of mitoxantrone (Mxt) and trichostatin A for various time periods was studied to assess the degree (depth) of cell senescence. Also assessed was the effect of 2-deoxy-d-glucose, the agent attenuating metabolic cell activity, on the depth of senescence induced by Mxt. A relationship between the ability of cells to synthesize RNA (incorporate 5-ethynyluridine) that leads to growth imbalance and induction of cell senescence was also studied. The data show that morphometric analysis of cellular attributes by LSC offers an attractive tool to detect cell senescence and measure its degree particularly in assessing effects of the factors that enhance or attenuate this process. This methodology is of importance in light of the evidence that cellular senescence is not only a biological process that is fundamental for organismal aging but also impedes formation of induced-pluripotent stem cells providing the barrier for neoplastic transformation and is the major mechanism of induction of reproductive cell death during treatment of solid tumors. © 2010 International Society for Advancement of Cytometry.
image analysis; trichostatin A; maximal pixel of fluorescence intensity; nuclear area; EU incorporation; growth imbalance; cell morphometry; chromatin condensation; cell cycle; apoptosis; p21WAF1
The imaging analytical capabilities of laser scanning cytometer (LSC) have been used to assess morphological features considered to be typical of the senescent phenotype. The characteristic “flattening” of senescing cells was reflected by the decline in the density of staining (intensity of maximal pixel) of DNA-associated fluorescence (DAPI) paralleled by an increase in nuclear size (area). The decrease in ratio of maximal pixel to nuclear area was even more sensitive senescence biomarker than the change in maximal pixel or nuclear area, each alone. The saturation density at plateau phase of growth recorded by LSC was found to be dramatically decreased in cultures of senescent cells thereby also serving as a convenient marker. The induction of cyclin dependent kinase inhibitors p21WAF1 and p27KIP1 as well as γH2AX and activation of ATM, markers of DNA damage response were measured in parallel with DNA/DAPI maximal pixel and nuclear area. These biomarker indices were expressed in quantitative terms by reporting them as a fraction of the respective markers in control cultures. The effect of treatment of A549 cells with different concentrations of mitoxantrone and trichostatin A for various time periods was studied to assess the degree (depth) of induction of senescence by these biomarkers. Induction of senescence in WI-38 fibroblasts by mitoxantrone and in HT-p21-a cells by enforced expression of p21WAF1 was also studied. The effects of 2-deoxy-D-glucose and rapamycin, the factors attenuating metabolic cell activity, were found to decrease the depth of senescence. The morphometric analysis of cellular attributes by LSC offers an attractive tool to detect cell senescence and measure its degree particularly in assessing effects of the factors that enhance or attenuate this process.
We investigated the outcome of the interaction of Cryptococcus neoformans with murine macrophages using laser scanning cytometry (LSC). Previous results in our lab had shown that phagocytosis of C. neoformans promoted cell cycle progression. LSC allowed us to simultaneously measure the phagocytic index, macrophage DNA content, and 5-ethynyl-2′-deoxyuridine (EdU) incorporation such that it was possible to study host cell division as a function of phagocytosis. LSC proved to be a robust, reliable, and high-throughput method for quantifying phagocytosis. Phagocytosis of C. neoformans promoted cell cycle progression, but infected macrophages were significantly less likely to complete mitosis. Hence, we report a new cytotoxic effect associated with intracellular C. neoformans residence that manifested itself in impaired cell cycle completion as a consequence of a block in the G2/M stage of the mitotic cell cycle. Cell cycle arrest was not due to increased cell membrane permeability or DNA damage. We investigated alveolar macrophage replication in vivo and demonstrated that these cells are capable of low levels of cell division in the presence or absence of C. neoformans infection. In summary, we simultaneously studied phagocytosis, the cell cycle state of the host cell and pathogen-mediated cytotoxicity, and our results demonstrate a new cytotoxic effect of C. neoformans infection on murine macrophages: fungus-induced cell cycle arrest. Finally, we provide evidence for alveolar macrophage proliferation in vivo.
This study evaluated the clastogenic and/or aneugenic potential of three nucleoside reverse transcriptase inhibitors (zidovudine - AZT, lamivudine - 3TC and stavudine - d4T) using the cytokinesis-block micronucleus (CBMN) assay in human lymphocyte cultures. All three inhibitors produced a positive response when tested in binucleated cells. The genotoxicity of AZT and 3TC was restricted to binucleated cells since there was no significant increase in the frequency of micronuclei in mononucleated cells. This finding indicated that AZT and 3TC caused chromosomal breakage and that their genotoxicity was related to a clastogenic action. In addition to the positive response observed with d4T in binucleated cells, this drug also increased the frequency of micronuclei in mononucleated cells, indicating clastogenic and aneugenic actions. Since the structural differences between AZT and 3TC and AZT and d4T involve the 3' position in the 2'-deoxyribonucleoside and in an unsaturated 2',3',dideoxyribose, respectively, we suggest that an unsaturated 2', 3', dideoxyribose is responsible for the clastogenic and aneugenic actions of d4T.
lamivudine; micronucleus assay; stavudine; transcriptase inhibitors; zidovudine
The multi-endpoint cytokinesis-blocked micronucleus assay is used for assessing chromosome aberrations. We have recently reported that this assay is extremely sensitive to genetic damage caused by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyr-idyl)-1-butanone (NNK) and that the binucleated cells with micronuclei, nucleoplasmic bridges, and nuclear buds in lymphocytes (chromosome damage endpoints measured by the assay) are strong predictors of lung cancer risk. In the current study, we refined our analysis to include toxicity endpoints (micronuclei in mononucleated cells, apoptosis, necrosis, and nuclear division index) to investigate the benefit of including these variables on improving the predictive value of the assay. Baseline and NNK-induced micronuclei in mononucleated cells were significantly higher in patients (n = 139) than controls (n = 130; P < 0.001). Baseline apoptosis was higher among cases; however, the controls showed a significant higher fold increase in NNK-induced apoptosis compared with baseline (P < 0.001). Principal components analysis was used to derive a summary measure for all endpoints and calculate the positive predictive value (PPV) and negative predictive value (NPV) for disease status. First principal component for NNK-induced chromosome damage endpoints (binucleated cells with micronuclei, nucleoplasmic bridges, and nuclear buds) had an area under the curve = 97.9 (95% confidence interval, 95.9-99.0), PPV = 94.8, and NPV = 92.6. The discriminatory power improved when micronuclei in mononucleated cells were included: area under the curve = 99.1 (95% confidence interval, 97.9- 100.0), PPV = 98.7 and NPV = 95.6. The simplicity, rapidity, and sensitivity of the assay together with potential for automation make it a valuable tool for screening and prioritizing potential cases for intensive screening.
The radiation-induced genotoxic damage in three established cell lines and 15 primary cultures of human malignant melanoma and ovarian carcinoma showing different radiosensitivity was tested by the cytokinesis-block micronucleus assay. A dose-related increase in micronucleus frequency was observed in all the cell systems. The mean number of micronuclei per Gy of ionising radiation per binucleated cell was respectively 0.44 +/- 0.0075 and 0.43 +/- 0.04 for M14 and JR8 malignant melanoma cell lines and 0.19 +/- 0.013 for the A2780 ovarian cancer cell line. The number of micronuclei did not rank the cell lines in the same order of radiosensitivity as clonogenic cell survival, which showed a surviving fraction at 2 Gy of 0.38 +/- 0.02 for JR8, 0.34 +/- 0.05 for M14 and 0.22 +/- 0.007 for A2780. As regards primary tumour cultures, no correlation was observed between micronucleus induction and surviving fraction at 2 Gy. In conclusion, the discrepancy we observed between micronucleus formation and cell death raises doubts about the potential of the micronucleus assay as a preclinical means to predict radiosensitivity.
We established an in vitro cytokinesis-block micronucleus assay of human tumours for estimation of the proportion of cells undergoing mitosis (the dividing fraction, DF), the time for the number of nuclei to double and the radiosensitivity in terms of the micronucleus frequency, based on a concept described previously. Under certain conditions, the nuclear number doubling time (NNDT) was considered to represent the potential doubling time. Tumour specimens obtained at surgery were disaggregated into single-cell suspensions and were directly cultured in the presence of cytochalasin B with or without irradiation. At various intervals, the percentage of multinucleate cells (the plateau value represented the DF), the average number of nuclei per cell and the number of micronuclei in binucleate cells were determined. DF and NNDT values were obtained in 58 of the 73 tumours investigated, and the micronucleus frequency was obtained in 54 of these 58 tumours. The DF ranged from 4.1% to 71% and the NNDT ranged from 3.1 to 83 days. A DF > or = 20% was associated with a higher recurrence rate in patients undergoing curative operation. A correlation was found between the NNDT and the time to relapse in patients with recurrent disease. The average number of micronuclei per binucleate cell at 2 Gy of irradiation (after subtraction of the value at 0 Gy) ranged from 0.052 to 0.35. Tumours which produced more micronuclei after irradiation showed a better response to radiotherapy. This assay can be readily performed on human tumours and appears to have promise as a predictive assay for radiation therapy.
Quantifying phenotypic variation at the level of protein expression (variegation) within populations of retinal pigment epithelium (RPE) cells may be important in the study of pathologies associated with this variation. The lack of quantitative methods for examining single cells, however, and the variable presence of pigment and/or lipofuscin complicate this experimental goal. We have applied the technique of laser scanning cytometry (LSC) to paraffin sections of mouse and human eyes to evaluate the utility of LSC for these measurements.
Mouse eyes were perfusion fixed in 4% paraformaldehyde and embedded in paraffin. Postmortem human eyes were fixed and dissected to obtain a 9-mm punch, which was then embedded in paraffin. A laser scanning cytometer equipped with violet, argon, and helium-neon lasers and the detectors for blue, green, and long red were used to record the fluorescence of each individual cell at all three wavelengths. Raw data were recorded and processed using the WinCyte software. Individual nuclei were identified by the fluorescence of the 4’,6-diamidino-2-phenylindole (DAPI) nuclear counterstain. Next, RPE cells were uniquely identified in the green channel using an anti-retinal pigment epithelium-specific protein 65 kDa (anti-RPE65) monoclonal antibody with an Alexa Fluor 488-labeled secondary antibody. Mn-superoxide dismutase (MnSOD) was quantified in the long-red channel using an anti-MnSOD antibody and an Alexa Fluor 647-labeled secondary antibody. MnSOD+ and RPE65+ cells exhibited peaks in the plot of fluorescence intensity versus cell number, which could be characterized by the mean fluorescence intensity (MFI), the coefficient of variation (CV), and the percentage of total RPE cells that were also labeled for MnSOD.
RPE cells can be uniquely identified in human and mouse paraffin sections by immunolabeling with anti-RPE65 antibody. A second antigen, such as MnSOD, can then be probed only within this set of RPE. Results are plotted primarily with the population frequency diagram, which can be subdivided into multiple regions. The data collected for each region include the MFI, the CV, and the number of cells that are immunolabeled in that region. Background interference from pigment or autofluorescent material can be successfully overcome by elevating the concentrations of fluorescent secondary antibodies. In the human and mouse eyes, age-related changes in MFI, CV, and percent RPE cells immunolabeled for MnSOD were observed.
The extent of the variability of gene expression in RPE cells at the protein level can be quantified by LSC. Relative changes in the MFI, the CV, and/or percentage of RPE cells double labeled for a second antigen quantify the changes observed. The analysis of these data also suggest whether the effects observed are related to local changes in transcription (alterations of CV) or major changes of protein expression (MFI), which are likely to be due to changes in the chromatin structure. The changes of these variables with age suggest that the observed age-related variegation is primarily due to changes in the chromatin structure in individual cells.
Toxoplasma gondii is among the most common protozoan parasites of humans. Both attachment to and invasion of host cells by T.gondii are necessary for infection, yet little is known about the molecular mechanisms underlying these processes. T.gondii’s etiological importance and its role as a model organism for studying invasion in related parasites necessitate a means to quantitatively assay host cell attachment and invasion.
We present here Laser Scanning Cytometer (LSC)-based assays of T.gondii invasion and attachment. The invasion assay involves automated counting of invaded and non-invaded parasites, differentially labeled with distinct fluorochromes. The attachment assay compares the relative binding of differentially labeled parasites. The assays were evaluated using treatments known to decrease invasion or attachment.
The LSC-based assays are robust and reproducible, remove operator bias, and significantly increase the sample size that can be feasibly counted compared to other currently available microscope-based methods. In the first application of the new assays, we have shown that parasites attach to fixed and unfixed host cells using different mechanisms.
The LSC-based assays represent useful new methods for quantitatively measuring attachment and invasion by T.gondii, and can be readily adapted to study similar processes in other host-pathogen systems.
Toxoplasma gondii; invasion; attachment; laser scanning cytometer
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 α-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-α-human-MBP monoclonal antibody or mouse-α-human-cytokeratin
antibody and goat-α-mouse Oregon Green conjugated second antibody.
induction provided a mean (SE) of 0.99 (0.2) × 106 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.
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.
Micronuclei (MN) are extra-nuclear bodies that contain damaged chromosome fragments and/or whole chromosomes that were not incorporated into the nucleus after cell division. MN can be induced by defects in the cell repair machinery and accumulation of DNA damages and chromosomal aberrations. A variety of genotoxic agents may induce MN formation leading to cell death, genomic instability, or cancer development. In this review, the genetic and epigenetic mechanisms of MN formation after various clastogenic and aneugenic effects on cell division and cell cycle are described. The knowledge accumulated in literature on cytotoxicity of various genotoxins is precisely reflected and individual sensitivity to MN formation due to single gene polymorphisms is discussed. The importance of rapid MN scoring with respect to the cytokinesis-block micronucleus assay is also evaluated.
micronuclei; chromosome-defective; epigenetics; genotoxicity; DNA methylation; histones; small RNAs
Okadaic Acid (OA) the major diarrheic shellfish poisoning (DSP) toxin is known as a tumor promoter and seems likely implicated in the genesis of digestive cancer. Little is known regarding genotoxicity and carcinogenicity of Domoic Acid (DA), the major Amnesic Shellfish Poisoning (ASP) toxin. Both OA and DA occur in seafood and are of human health concerns. Micronuclei (MN) arise from abnormalities in nuclear division during mitosis due to a failure of the mitotic spindle or by complex chromosomal configurations that pose problems during anaphase. In order to evaluate the ability of okadaic acid (OA) and domoic acid (DA) to induce DNA damage we performed the micronucleus assay using the Caco-2 cell line. To discriminate between a clastogenic or aneugenic effect of OA and DA, the micronucleus assay was conducted by cytokinesis-block micronucleus assay using cytochalasin B with Giemsa staining and/or acridine orange staining, in parallel to fluorescence in situ hybridization (FISH) using a concentrated human pan-centromeric chromosome paint probe. Our results showed that OA and DA significantly increased the frequency of MN in Caco-2 cells. The MN caused by OA are found in mononucleated cells and binucleated cells, whereas those caused by DA are mainly in binucleated cells. The results of FISH analysis showed that OA induced centromere-positive micronuclei and DA increased the percentage of MN without a centromeric signal. In conclusion, both OA and DA bear mutagenic potential as revealed in Caco-2 cells by induction of MN formation. Moreover, OA induced whole chromosome loss suggesting a specific aneugenic potential, whereas DA seems simply clastogenic. At present, one cannot rule out possible DNA damage of intestinal cells if concentrations studied are reached in vivo, since this may happen with concentrations of toxins just below regulatory limits in case of frequent consumption of contaminated shell fishes.
Okadaic acid; Domoic acid; Micronuclei; Clastogenicity; Aneugenicity
The aim of this study was to investigate the radioprotective effect of green tea against genotoxicity induced by gamma irradiation in cultured blood lymphocytes from 5 human volunteers.
Materials and Methods
Peripheral blood samples were collected from volunteers before and 1, 3 and 5 hr after drinking a decoction 4 g green tea in 280 ml boiling water for 5 constitutive days with the same quantity. At each time point, the whole blood samples were exposed to 200 cGy of 60 Co gamma irradiation and then were cultured with mitogenic stimulation to determine the chromosomal aberration in micronucleus assay on cytokinesis-blocked binucleated cells.
As expected, for each volunteer, the results showed a significant increase in the incidence of micronuclei after exposure to gamma irradiation as compared to non-irradiated control samples. Only lymphocytes blood sample collected 3 hr after drinking green tea exhibited a significant decrease in incidence of micronuclei compared to non-treated irradiated samples.
These results suggest the radioprotective ability of green tea against ionizing radiation in human lymphocytes, at specified time after consumptior.
Gamma irradiation; Green tea; Lymphocytes; Micronucleus; Natural radioprotective
The long-term-treatment of glaucoma with topical medications is associated with side effects involving cornea damage. We examined the effect of glaucoma topical medications (bimatoprost, travoprost, latanoprost, timolol, betaxolol, dorzolamide, brinzolamide, brimonidine) on growth of cells of three human epithelial corneal lines.
The cells were cultured in 8-chamber slides, treated with different concentrations of the medications, and fixed at 24, 48, and 72 h. Cell number on slides to estimate viability and growth curves, frequency of apoptosis (FLICA and caspase-3 activation probes), and proliferation (BrdU incorporation assay) were measured by laser scanning cytometry (LSC).
Depending on concentration all examined medications induced cell necrosis or apoptosis and suppressed proliferation. Significant variability in proliferation and apoptosis was observed within the same cultures depending on local cell density, with cells in high density areas being more resistant. The data indicate that commonly used topical medications exert cytostatic and cytotoxic effects in cultures of corneal cells and suggest that caution should be exercised in their use, particularly, when the corneal diseases are accompanied by cell proliferation and regeneration, in long-term-treatment.
The present approach of using LSC makes it possible to assess and compare cytostatic and cytotoxic effects of different topical medications on the respective target cells.
cell proliferation; cell death; cell cycle; apoptosis; antiglaucoma topical medications
Leukemia progression and relapse is fueled by leukemia stem cells (LSC) that are resistant to current treatments. In the progression of chronic myeloid leukemia (CML), blast crisis progenitors are capable of adopting more primitive but deregulated stem cell features with acquired resistance to targeted therapies. This in turn promotes LSC behavior characterized by aberrant self-renewal, differentiation, and survival capacity. Multiple reports suggest that cell cycle alterations, activation of critical signaling pathways, aberrant microenvironmental cues from the hematopoietic niche, and aberrant epigenetic events and deregulation of RNA processing may facilitate the enhanced survival and malignant transformation of CML progenitors. Here we review the molecular evolution of CML LSC that promotes CML progression and relapse. Recent advances in these areas have identified novel targets that represent important avenues for future therapeutic approaches aimed at selectively eradicating the LSC population while sparing normal hematopoietic progenitors in patients suffering from chronic myeloid malignancies.
Chronic myeloid leukemia; CML; Leukemia stem cell; LSC; BCR-ABL; Hematopoiesis; Hematopoietic progenitors; Blast crisis; Malignant; Hematologic malignancies; Tyrosine kinase inhibitors; TKI; Imatinib; Therapeutic resistance; RNA; Splicing; Microenvironment; Bone marrow niche; Quiescence; Cell cycle; Signal transduction; Shh; BCL2; Survival; Self-renewal; Medicine & Public Health; Oncology; Hematology; Geriatrics/Gerontology
Malignant tumors have telomerase activity, which is thought to play a critical role in tumor growth. However, the relation between telomerase activity and genomic DNA status in tumor cells is poorly understood. In the present study, we examined telomerase activity in 13 clear cell type renal cell carcinomas (CRCCs) with similar clinicopathologic features by telomeric repeat amplification protocol assay (TRAP). Based on TRAP assay results, we divided the CRCCs into two groups: a high telomerase activity group and a low/no telomerase activity group. We then analyzed genomic aberration, DNA ploidy, and telomere status in these two groups by comparative genomic hybridization (CGH), laser scanning cytometry (LSC), and telomere-specific fluorescence in situ hybridization (T-FISH), respectively. CGH showed the high telomerase activity group to have fewer genomic changes than the low/no telomerase activity group, which had many genomic aberrations. Moreover, with LSC, DNA diploid cells were found more frequently in the high telomerase activity group than in the low/no telomerase activity group. In addition, T-FISH revealed strong telomere signal intensity in the high telomerase activity group compared with that of the low/no telomerase activity group. These results suggest that telomerase activity is linked to genomic DNA status and that high telomerase activity is associated with genomic stability, DNA ploidy, and telomere length in CRCC.
Telomerase; CGH; DNA ploidy; cancer; genomic stability
Combining the technologies of protein tag labeling and optical microscopy allows sensitive analysis of protein function in cells.
Here, we describe development of applications using protein tag technology (HaloTag (HT)-based) for flow and laser scanning cytometry (LSC). Cell lines, expressing recombinant surface β1-integrin-HT and HT-p65 fusion protein, and a CD4 T cell line (Jurkat) infected with human immunodeficiency virus type 1 (HIV-1) reporter virus expressing the unfused HT (HIV-1Lai-Halo), were stained with different HT ligands and successfully detected by flow cytometers equipped with 488 and 561 nm lasers as well as a laser scanning cytometer (equipped with 488 and 405 nm lasers) alone or combined with cell cycle and viability markers.
Use of HT technology for cytometric applications has advantages over its use in microscopy as it allows for the statistical measurement of protein expression levels in individual cells within a heterogeneous cell population in combination with cell cycle analysis. Another advantage is the ability of the HaloTag to withstand long fixation and high concentration of fixative, which can be useful in research of infectious agents like HIV and/or mycobacteria.
This study compares the sensitivities and specificities of three techniques for the detection of circulating epithelial cells in the blood of patients with breast cancer. The number of circulating epithelial cells present in the blood of 40 patients with metastatic breast cancer and 20 healthy volunteers was determined by: immunomagnetic separation (IMS) and laser scanning cytometry (LSC), cell filtration and LSC and a multimarker real-time RT–PCR assay. Numbers of cytokeratin-positive cells identified and expression of three PCR markers were significantly higher in the blood of patients with breast cancer than in healthy volunteers. Using the upper 95% confidence interval of cells detected in controls to determine positive patient samples: 30% of patients with metastatic breast cancer were positive following cell filtration, 48% following IMS, and 60, 45 and 35% using real-time RT–PCR for cytokeratin 19, mammaglobin and prolactin-inducible peptide. Samples were significantly more likely to be positive for at least one PCR marker than by cell filtration (83 vs 30%, P<0.001) or IMS (83 vs 48%, P<0.001).The use of a multimarker real-time RT–PCR assay was therefore found to be the most sensitive technique for the detection of circulating epithelial cells in the blood of patients with breast cancer.
breast cancer; micrometastases; circulating tumour cells
There is abundant evidence that immune cells infiltrating into a transplanted organ play a critical role for destructive inflammatory or regulatory immune reactions. Quantitative in situ analysis (i.e. in tissue sections) of immune cells remains challenging due to a lack of objective methodology. Laser scanning cytometry (LSC/iCys) is a recently developed methodology that utilizes fluorescence-based quantitative measurements on tissue sections or other cellular preparations at a single-cell level. In this study, we have developed a novel objective method for analysis of immune cells, including Foxp3+ T regulatory cell (T reg), on formalin-fixed / paraffin embedded (FFPE) transplant biopsy sections using LSC/iCys.
The development of multiple immunofluorescent staining was established using FFPE human tonsil sample. The CD4/CD8 ratio and the population of T reg among CD4+ cells were analyzed using LSC/iCys and compared with the results from conventional flow cytometry analysis (FCM).
Our multiple immunofluorescent staining techniques allow obtaining clear staining on FFPE sections. The CD4/CD8 ratio analyzed by LSC/iCys was concordant with those obtained by FCM. This method was also applicable for liver, small intestine, kidney, pancreas and heart transplant biopsy sections and provide an objective quantification of T regs within the grafts.
T regulatory cell; Foxp3; laser scanning cytometry; transplant biopsy; quantification
Assays for assessing human islet cell quality which provide results prior to transplantation would be very beneficial to improving outcomes for islet transplantation therapy. Parameters such as percent beta cell apoptosis and cell composition are found to vary markedly between different islet preparations, and may serve as markers of islet quality. We have developed fluorescence-based assays using laser scanning cytometry (LSC) for assessing beta cell apoptosis and islet cell composition on serial sections of intact isolated islets.
Isolated human islets were fixed in formalin and embedded in paraffin. Serial sections were immunostained for the pancreatic hormones, acinar and ductal cell markers. DNA fragmentation was used to label apoptotic cells. Stained cells were quantified using an iCys laser scanning cytometer.
Islet preparations from 102 human pancreatic islet isolations were analyzed. For the whole set of islet preparations we found a mean islet cell composition of 54.5±1.2% insulin positive; 33.9±1.2% glucagon; 12.1±0.7% somatostatin and 1.5±0.2% pancreatic polypeptide positive cells. The apoptotic beta cells were 2.85±0.4% with a range of 0.27% to 18.3%. The percentage of apoptotic beta cells correlated well (p<0.0001, n=59) with results obtained in vivo by transplantation of the corresponding islets in diabetic NODscid mice.
The analysis of whole, non-dissociated islets for cell composition and beta cell apoptosis using LSC is giving reliable and reproducible results and could be done both before islet transplantation, as well as on preserved cell blocks at any future time. Thus, they can be a powerful tool for islet quality assessment.
Laser scanning cytometry; human pancreatic islets; islet cell composition; β cell apoptosis
This laboratory has developed a flow cytometric approach for scoring in vitro micronuclei (In Vitro MicroFlow®) whose characteristics are expected to benefit studies designed to comprehensively investigate genotoxicity dose-response relationships. In particular, new experimental designs become possible when automated scoring is combined with treatment, processing and sampling that all occur in microtiter plates. To test this premise, experiments described herein investigated micronucleus (MN) formation in TK6 cells treated with genotoxic agents applied at 22 closely-spaced concentrations in quadruplicate, with 10,000 cells analyzed per replicate. The genotoxicants colchicine, vinblastine sulfate, ethyl methanesulfonate, methyl methanesulfonate, ethyl nitrosourea, methyl nitrosourea, and bleomycin were applied continuously for 24 – 30 hrs. Following treatment, all cell processing, sampling and data acquisition steps were accomplished in the same 96-well plate. Data acquisition occurred in a walk-away mode via the use of a high throughput sampling device. The resulting flow cytometric MN values were evaluated with a statistical model that indicated non-linear relationships describe the data better than linear fits. The one exception was bleomycin, where MN induction was consistently best described by a linear dose-response relationship. Collectively, these results suggest that flow cytometry represents a practical and efficient approach for thoroughly examining the dose-response relationship, and clearly benefits studies that seek to characterize no observable genotoxic effect levels, lowest observable genotoxic effect levels, and/or benchmark doses.
micronuclei; flow cytometry; genotoxicity; threshold; risk assessment; TK6 cells
Laser scanning cytometry has been proven as a powerful technology for high-content, high-throughput quantitative analysis of cellular functions in a fully automated manner. It utilizes a large-area fluorescence imaging scheme and rigorous image quantitation algorithms to enable informative analysis of cell samples attached to solid substrates. While this technology represents a powerful approach for high-content screening using cell lines, it has not been applied to the study of tumor-cell behaviors on these solid nanopatterned substrates after several hours of incubation. Herein, we statistically demonstrated functional cellular morphology information, including size, shape, and distribution of the captured cells after 0.5 to 45 h of incubation on nanopatterned substrates, such as silicon nanowires and quartz nanopillars, along with planar glass substrates. With increasing incubation time up to 45 h, we observed that the nanopatterned substrates could have not only increased adhesion and traction forces between cells and nanopatterned substrates, but also limited cell spreading on the substrates compared to the planar glass substrates. On the basis of our results, we suggest that the most important factors to influence the cell behaviors on the three solid substrates are the degree of dimension on cell behaviors and cell traction force.
Nanowire arrays; Cell adhesion; Circulating tumor cells; Filopodia; Cell migration; Cell capture efficiency
Mechanisms that couple protein turnover to cell cycle progression are critical for coordinating the events of cell duplication and division. Despite the importance of cell cycle-regulated proteolysis, however, technologies to measure this phenomenon are limited, and typically involve monitoring cells that are released back into the cell cycle after synchronization. We describe here the use of laser scanning cytometry (LSC), a technical merger between fluorescence microscopy and flow cytometry, to determine cell cycle-dependent changes in protein stability in unperturbed, asynchronous, cultures of mammalian cells. In this method, the ability of the LSC to accurately measure whole cell fluorescence is used, together with RNA fluorescence in situ hybridization and immunofluorescence, to relate abundance of a particular RNA and protein in a cell to its point at the cell cycle. Parallel monitoring of RNA and protein levels is used, together with protein synthesis inhibitors, to reveal cell cycle-specific changes in protein turnover. We demonstrate the viability of this method by analyzing the proteolysis of two prominent human oncoproteins, Myc and Cyclin E, and argue that this LSC-based approach offers several practical advantages over traditional cell synchronization methods.
Laser scanning cytometry; cell cycle; proteolysis; Myc; Cyclin E
The chromosomal radiosensitivity of breast cancer patients with a known or putative genetic predisposition was investigated and compared to a group of healthy women. The chromosomal radiosensitivity was assessed with the G2 and the G0-micronucleus assay. For the G2 assay lymphocytes were irradiated in vitro with a dose of 0.4 Gy 60Co γ-rays after 71 h incubation, and chromatid breaks were scored in 50 metaphases. For the micronucleus assay lymphocytes were exposed in vitro to 3.5 Gy 60Co γ-rays at a high dose rate or low dose rate. 70 h post-irradiation cultures were arrested and micronuclei were scored in 1000 binucleate cells. The results demonstrated that the group of breast cancer patients with a known or putative genetic predisposition was on the average more radiosensitive than a population of healthy women, and this with the G2 as well as with the high dose rate and low dose rate micronucleus assay. With the G2 assay 43% of the patients were found to be radiosensitive. A higher proportion of the patients were radiosensitive with the micronucleus assay (45% with high dose rate and 61% with low dose rate). No correlation was found between the G2 and the G0-micronucleus chromosomal radiosensitivity. Out of the different subgroups considered, the group of the young breast cancer patients without family history showed the highest percentage of radiosensitive cases in the G2 (50%) as well as in the micronucleus assay (75–78%).
British Journal of Cancer (2002) 87, 1379–1385. doi:10.1038/sj.bjc.6600628 www.bjcancer.com
© 2002 Cancer Research UK
breast cancer; genetic predisposition; chromosomal radiosensitivity; G2 assay; micronucleus (MN) assay; peripheral blood lymphocytes