Microcomputer experience in analysis of flow cytometric DNA distributions

The program described analyses DNA histograms obtained in flow cytometry using the Gaussians method. The program is written in BASIC to run on a low-cost microcomputer. It utilizes a simple strategy to obtain good estimates of the parameters required for reducing the problem to a task solvable with linear least-squares methods. Features of the program are flexibility, since it is possible to choose different options for parametrization and spacing of Gaussians, and the fact that the operator is not required to provide interactive inspection or inputting parameter values. The capability and velocity of the program, in all its options, are tested and compared on a series of different (not computer-simulated) histograms obtained in our flow cytometry laboratory. Our results suggest that a fresh approach to parametrization may be useful.     

Cytologic and flow cytometric DNA analysis of multinucleated tumor cells and derived microcells

Microcell production by means of Colcemid-induced micronucleation and subsequent enucleation with the density gradient technique was adjusted for use with the murine T-lymphoma line ESb-M. Modification of the standard protocol for a cell type on which no experiments had previously been performed required careful monitoring of the multiple steps in the procedure in order to optimize the final microcell yield. Traditional microscopic verification may sometimes be ambiguous, due to the lack of a clear cutoff point between small whole cells and cell fragments; in these conditions, the level of variability increases, thus impairing quantitative estimations. Flow cytometric (FCM) analysis of DNA content and size of donor cells and microcells was therefore applied in parallel to provide additional quantitative information. The FCM results supplemented the microscopic data in assessing which fraction recovered from the gradient has the lowest percentage of contaminant whole cells; however, FCM analysis may provide more statistically significant data due to the large size of the sample examined. Moreover, FCM is of prospective use in providing the basis for subsequent sorting of either pure microcells or specific subpopulations of defined DNA content and size.     

DNA flow cytometric analysis of mouse seminiferous epithelium

In order to provide a basis for quantitative studies of murine spermatogenesis, we performed a DNA flow cytometric analysis on the mouse seminiferous tubules isolated at defined stages of the epithelial cycle by transillumination-assisted microdissection. Accurate stage identification was performed by examining spermatids in the adjacent tubule segments by phase-contrast microscopy. For flow cytometry, suspension of nuclei of spermatogenic cells was obtained by detergent treatment of isolated seminiferous tubules, and fresh samples were stained with propidium iodide. DNA histograms of the 12 stages of the mouse seminiferous epithelial cycle varied in a stage-specific manner. DNA histograms of stages I-VIII of the cycle were characterized by a hypofluorescent haploid peak, the location of which changed with the decreasing DNA dye (propidium iodide)-binding capacity of elongated spermatids. The absence of the hypohaploid peak and the high ratio of the cells with 4C amount of DNA to the cells with 1C amount of DNA characterized stages IX-XI of the cycle. Stage XII showed a high 2C peak, owing to a large population of secondary spermatocytes arisen from the first meiotic division. By using fluorescent beads as an internal volume standard cell numbers in defined stages were determined. These data provide a basis for quantitative studies of mouse spermatogenesis.     

Automatic analysis of flow cytometric DNA histograms from irradiated mouse male germ cells

An automatic procedure for recovering the DNA content distribution of mouse irradiated testis cells from flow cytometric histograms is presented. First, a suitable mathematical model is developed, to represent the pattern of DNA content and fluorescence distribution in the sample. Then a parameter estimation procedure, based on the maximum likelihood approach, is constructed by means of an optimization technique. This procedure has been applied to a set of DNA histograms relative to different doses of 0.4-MeV neutrons and to different time intervals after irradiation. In each case, a good agreement between the measured histograms and the corresponding fits has been obtained. The results indicate that the proposed method for the quantitative analysis of germ cell DNA histograms can be usefully applied to the study of the cytotoxic and mutagenic action of agents of toxicological interest such as ionizing radiations.     

Analysis of DNA synthesis rate of cultured cells from flow cytometric data

The rate of DNA synthesis along S phase is estimated from flow cytometric histograms on the basis of a mathematical model of a cell population. In the absence of loss, the model expresses the population kinetics in terms of DNA synthesis rate, S-phase influx, and population size. A single histogram is sufficient to determine the DNA synthesis rate when the population is in balanced exponential growth. Two suitably chosen histograms are necessary if the S-phase influx is exponential in a time interval longer than the S-phase duration. The analysis procedure was tested on published autoradiographic data and applied to three cultured cell lines (CM-S, 3LL, and M14 cells) that show various patterns of DNA distribution. In each case the cell-cycle fractions, the DNA synthesis rate, and the S-phase duration were obtained.     

DNA ligase from mouse Ehrlich ascites tumor cells

The molecular (Mr = 120,000; s20, w = 5S) and catalytic properties (Km (ATP) = 3 microM; Km (nicked DNA) = 0.2 microM; Km (Mg2+) = 3 mM) of DNA ligase from mouse Ehrlich ascites tumor cells are similar to those of the enzymes from calf thymus and rodent liver. The activity level of DNA ligase from the tumor cells is about 10-fold higher than that from mouse liver. Immunochemical titration of DNA ligase with antibodies against the calf thymus enzyme showed that the higher level of DNA ligase activity in the tumor cells is due to an increase in enzyme quantity and not to elevation of the catalytic efficiency of the enzyme molecule. These results suggest that there is little apparent difference between the qualities of DNA ligases from the tumor cells and normal tissues of rodents and calf.     

Method to improve the sensitivity of flow cytometric membrane potential measurements in mouse spinal cord cells.

We have developed a technique to improve the sensitivity of relative membrane potential measurements in mouse spinal cord cells using the fluorescent, anionic, voltage sensitive dye, DiBa-C4(3) (Oxonol) and flow cytometry. In order to attribute cellular fluorescence primarily to membrane potential, signal variability due to cell size and shape was reduced by dividing the log fluorescence signal from each cell by either its log forward angle light scatter or log side scatter signals. The use of these ratios in place of log oxonol fluorescence reduced the coefficient of variation of the distributions while leaving the changes in mean fluorescence largely unaffected. Kolmogorov-Smirnov analysis of pre- vs. postkainate stimulation (an excitatory amino acid) showed improved sensitivity of the assay with the use of this ratio technique.     

Preparation of tissues for DNA flow cytometric analysis

A method for measuring DNA in tissue cells by flow cytometry utilizing a one step combination nuclear isolation-DNA fluorochrome staining procedure is described. A variety of cells and tissues, both in vivo and in vitro, was used to illustrate the universal nature of this technique. These included murine bone marrow, liver testicle, sarcoma brain tumor, rat pancreatic islets, human peripheral blood, colon mucosa, colon cancer, sarcoma and brain tumor tissues. A special nuclear isolation medium, which contained either of the DNA fluorochromes, 4',6-diamidino-2 phenylindole-2 HCl or propidium iodide, was utilized successfully to isolate single suspensions of DNA fluorochrome stained nuclei in a rapid (5-10 min), consistent manner from a variety of tissues and cells. Multiple sampling of the same tissue or comparison between whole tissues and their single cell isolates showed that a representative sample was being obtained.     

Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Monoclonal antibody F7-26 was obtained by fusion of mouse myeloma cells with spleen cells isolated from a mouse immunized with DNA treated by nitrogen mustard (HN2). Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to the drug dose. Two-parameter analysis for the antibody binding and DNA content showed no binding of antibody to replicating DNA in control cells. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Binding of monoclonal antibody specific for the determinants expressed on ssDNA to the cells treated with alkylating agents may be attributed to local DNA denaturation. Potentiation of L-phenylalanine mustard cytotoxicity by buthionine sulfoximine or hyperthermia was accompanied by increased antibody binding to cellular DNA. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.     

Detection of cells resistant to alkylating agents by flow cytometric analysis of DNA damage

DNA damage was measured by flow cytometric analysis of cells sensitive and resistant to alkylating agents. Human ovarian carcinoma cell line A2780 and a subline which is 7 times more resistant to L-phenylalanine mustard (L-PAM) were treated with the drug, fixed, and stained with monoclonal antibody (MOAB) F7-26 which detects single-stranded regions in alkylated DNA. Mean fluorescent intensity was measured on a flow cytometer. Cells were heated before staining to amplify single-strandedness in alkylated DNA. Significantly larger amount of MOAB was bound to DNA in sensitive than in resistant cells. Fluorescence increased by 80 channels per micrograms L-PAM insensitive cells and only by 17 channels in resistant cells. Sensitive and resistant cells were treated with L-PAM, mixed in different proportions, and stained with MOAB. Populations of sensitive and resistant cells were clearly separated on fluorescence histograms by more than a decade difference in fluorescence intensity. Presence of 2-5% resistant cells was detected among sensitive cells as a separate cell subset. We conclude that staining with MOAB F7-26 can be used as an indicator of cell sensitivity or resistance to alkylating agents. Detection of minor subsets of resistant cells in heterogeneous populations by FCM analysis may be useful for monitoring emerging drug resistance.     

Synthesis of DNA and alpha-fetoprotein in the monolayer culture of mouse hepatocytes

Relation between processes of proliferation and synthesis of the embryonal serum protein alpha-fetoprotein (AFP), the influence on these processes of polyelectrolyte dextran sulfate (DS) and dimethyl-sulfoxide (DMSO) has been studied in the monolayer culture of mouse hepatocytes. In control cultures the correlations between the time of appearance and the level of DNA and AFP synthesis were observed. DS and DMSO were found to inhibit both processes. Cell proliferation could be reestablished by addition of epidermal growth factor. In case of the influence of DMSO, it wasn't followed by the induction of AFP synthesis. This the processes of DNA and AFP synthesis in monolayer cultures of mouse hepatocytes can be separated. The elongated incubation of hepatocytes with collagenase during their obtaining, abolished the effects of DS. This shows that surface components of hepatocytes, lost upon enzyme degradation, may be involved in the mechanism of DS effect.     

Nuclear DNA but not mtDNA controls tumor phenotypes in mouse cells

Recent studies showed high frequencies of homoplasmic mtDNA mutations in various human tumor types, suggesting that the mutated mtDNA haplotypes somehow contribute to expression of tumor phenotypes. We directly addressed this issue by isolating mouse mtDNA-less (rho(0)) cells for complete mtDNA replacement between normal cells and their carcinogen-induced transformants, and examined the effect of the mtDNA replacement on expression of tumorigenicity, a phenotype forming tumors in nude mice. The results showed that genome chimera cells carrying nuclear DNA from tumor cells and mtDNA from normal cells expressed tumorigenicity, whereas those carrying nuclear DNA from normal cells and mtDNA from tumor cells did not. These observations provided direct evidence that nuclear DNA, but not mtDNA, is responsible for carcinogen-induced malignant transformation, although it remains possible that mtDNA mutations and resultant respiration defects may influence the degree of malignancy, such as invasive or metastatic properties.     

DNA mapping of gastric cancers using flow cytometric analysis

Although numerous studies of gastric cancers on DNA ploidy have been reported, differences in the degree of aneuploidy (DNA index, DI) during progression have not been identified. We attempted to chart the differences in DIs during progression to clarify the role of aneuploidy in gastric cancers. We classified the gastric cancers examined into intestinal (n = 88) and diffuse (n = 48) types, and then analyzed 136 gastric cancers (intramucosal cancer, 42; submucosal cancer, 39; advanced cancer, 55) by flow cytometry using multiple sampling. In addition, we examined the DNA ploidy pattern of mucosal and submucosal lesions using the same submucosal cancers to study the tumor progression in individual cancers. Intratumoral DNA differences in DNA ploidy were observed in both types of gastric cancers. In intestinal-type cancers, multiple subclones indicated by a different DI occurred during the early stage of gastric cancers, whereas in diffuse-type cancers, multiple subclones were found primarily in advanced cancers. Although the DI varied widely in early intestinal-type cancers between 1.0 and 2.0, in early diffuse-type cancers, the DI tended to be less than 1.2. However, in advanced stage gastric cancers, the DI distribution was similar for both histological types. In intestinal-type cancers, high DI (>1.3) aneuploidy was frequently found in mucosal lesions. In contrast, only low DI (<1.2) aneuploid clones were observed in mucosal lesions of diffuse-type cancers. The present results suggest that high DI aneuploid tumor clones in intramucosal cancers acquire invasive ability when they progress to submucosal cancers, whereas DNA aneuploidy itself plays an important role in submucosal invasion of diffuse-type cancers.     

Flow cytometric analysis of unbalanced control of protein accumulation and DNA synthesis in differentiating mouse myeloid leukemia cells

The protein and DNA contents of mouse myeloid leukemia M1 (clone B24) cells were determined by flow cytometry (FCM) after double fluorescent staining of the cells with fluorescein isothiocyanate and propidium iodide. FCM analysis showed that there was a linear relationship between the DNA and protein contents in logarithmically growing cells, although the protein content showed some variation. B24 cells can be induced to differentiate into macrophage-like cells by treatment with a protein inducer(s) in conditioned medium (CM) of hamster embryo cells. When the cells were treated with various concentrations of CM, cells with a 2C DNA content, G1/0 cells, increased and protein accumulated in these G1/0 cells. The increases in the number of G1/0 cells and in their protein content per cell were proportional to the concentration of CM. Serial analysis of changes in the contents of DNA and protein in differentiating B24 cells showed that DNA synthesis was suppressed by differentiation-induced block of the cell cycle at the G1/0 phase, whereas increase in the protein content was not completely suppressed by block of the cell cycle. These results suggest that unbalanced control of the DNA and protein contents of B24 cells is involved in the mechanisms of the morphological changes during differentiation into macrophages.     

Diagnostic significance of flow cytometric DNA analysis applied for the detection of cancer cells in bronchial washing fluid

Since both DNA aneuploidy and increased proliferative activity are important characteristics of malignant neoplasms, flow cytometric (FCM) analysis was used to examine the cell content in bronchial washing samples obtained via fiberoptic bronchoscopy from 73 patients. The results were compared with the results of histology and conventional cytology. The patients included 30 with bronchial carcinomas, 12 with bronchopneumonia and 31 with no evidence of lung disease. Of the 30 patients with histologically confirmed lung cancers, 25 showed either aneuploid stem lines (19 cases) or high levels of proliferation (6 cases) as determined by analysis of cell-cycle stages. The same rate of cancer cell detection was obtained by cytology. In the 43 cases with neither histologic nor clinical evidence of malignancy, FCM data yielded 5 false-positive results, as compared to 4 erroneous suspicions of cancer by cytology. From these data, it is concluded that FCM measurements of both DNA ploidy and proliferative activity may complement conventional cytology in the recognition of bronchial carcinomas.     

Single-colour flow cytometric assay to determine NK cell-mediated cytotoxicity and viability against non-adherent human tumor cells

A flow cytometry-based cytotoxicity (FCC) assay was developed using a single fluorophore, calcein-acetoxymethyl diacetylester (calcein-AM), to measure NK cell-mediated cytotoxicity. Non-adherent human K562 and U937 target cells were individually labelled with calcein-AM and co-incubated with effector NK cells to measure calcein loss, and therefore calculate target cell cytotoxicity. This FCC assay also provided a measure of sample viability. Notably, cell viability measured by traditional calcein/7-amino-actinomycin D (7-AAD) double labelling and Trypan Blue methods were comparable to the viability calculated using calcein-loss FCC. This FCC assay may also be used with various effector and target cell types and as a multi-parameter tool to measure viability and immunophenotype cells for tissue engineering purposes.     

Comparison of three commercially available antibodies for flow cytometric monitoring of P-glycoprotein expression in tumor cells.

Cellular drug resistance to natural products is often due to the presence of an efflux pump which reduces intracellular drug content and chemosensitivity. A 170 kD cell surface resident P-glycoprotein is believed to act as the efflux pump. In the present report, we have compared three commercially available antibodies C-219, JSB-1, and mdr(Ab-1) for use in flow cytometric detection of P-glycoprotein positive cells. Our data show that C219 gives uniformly good results in a variety of murine and human tumor cell lines for detection of P-glycoprotein positive cells. We have also compared data of C219 stained cells analyzed in parallel on a flow cytometer equipped with a small laser (15 mW) and a large laser (5 watt) cell sorter. Data obtained on these two instruments are comparable. A staining protocol and data on dual staining of cells for DNA content by propidium iodide and P-glycoprotein expression after FITC labeling are also presented.