Differences in the flow and absorption cytometric DNA distributions of mouse hepatocytes and tumor cells

We have determined the DNA content, the ploidy levels, and the percentages of different cell types present in small and large mouse mammary tumors as well as in young and old mouse livers by using absorption and flow cytometry. Absorption cytometry data indicated a significant increase in the proportion of transformed G0/G1 cells in the tumors as compared to that of the stromal G0/G1 cells with progressive tumor growth. This increase was not detected by flow cytometry. In both young and old mouse livers, a small number of cells of higher ploidy (8C and 16C) were detected by absorption cytometry but were not apparent in histograms obtained by flow cytometry. Furthermore, changes in the proportions of liver cells of different ploidy with age were apparent in absorption cytometry data but not in flow cytometry data. In one mouse liver experiment, a 6C cell peak appeared in the flow cytometry histogram, but a direct measurement of DNA content by absorption cytometry failed to detect cells with such a peak. We therefore believe that some caution may be warranted in the use of flow cytometry alone for evaluation of DNA distributions and of the proportions of different types of cells in complex solid tissues.     

Visual selection and maintenance of the cell lines with high plant regeneration ability and low ploidy level in Dianthus acicularis by monitoring with flow cytometry analysis

Efficient plant regeneration system from cell suspension cultures was established in D. acicularis (2n=90) by monitoring ploidy level and visual selection of the cultures. The ploidy level of the cell cultures closely related to the shoot regeneration ability. The cell lines comprising original ploidy levels (2C+4C cells corresponding to DNA contents of G1 and G2 cells of diploid plant, respectively) showed high regeneration ability, whereas those containing the cells with 8C or higher DNA C-values showed low or no regeneration ability. The highly regenerable cell lines thus selected consisted of compact cell clumps with yellowish color and relatively moderate growth, suggesting that it is possible to select visually the highly regenerable cell lines with the original ploidy level. All the regenerated plantlets from the highly regenerable cell cultures exhibited normal phenotypes and no variations in ploidy level were observed by flow cytometry (FCM) analysis.     

Image and flow cytometric analysis of DNA content in breast cancer. Relation to estrogen receptor content and lymph node involvement

A comparative flow-cytometric and image-cytometric study was performed on 166 human breast cancers. Parallel measurements of 67 cases showed a good correlation between the DNA indices measured with each of the techniques. However, minor ploidy abnormalities were detected with flow cytometry. Only with this technique about 70% of the tumors appeared to be aneuploid. DNA profiles obtained with image cytometry frequently contained DNA values above the modal G2 value of the tumor. At least part of these broadly scattered DNA profiles resulted from multiploid tumors, as was found with flow cytometry. The mean percentage of cells with DNA contents exceeding the modal G0G1 value in image-cytometric DNA profiles (non- G0G1 cells) appeared to be highest in aneuploid lymph-node-positive (N+) tumors and lowest in near-diploid lymph-node-negative (N-) tumors. Near-diploid tumors were more frequently (70%) estrogen receptor positive (ER+) than were aneuploid tumors (50%), whereas highly aneuploid tumors with low or negative ER contents tended to be N+. Tumors with a high percentage of non- G0G1 cells value were predominantly estrogen-receptor negative (ER-).     

Development and characterization of breast carcinoma cell lines as in vitro and in vivo models for breast cancer diagnosis and therapy

Summary To establish a model system for preclinical radioimmunotherapy studies, attempts were made to graft 16 different human breast carcinoma cell lines into BALB/c nu/nu (nude) mice. Nine produced serially transplantable tumors growing at a variable rate, whereas seven failed to do so. Conversely, three new cell lines were established in monolayer culture from transplantable human breast tumors in nude mice. Twelve selected tumors and their corresponding cell lines were characterized for DNA ploidy, % S-phase, and breast epithelial mucin expression by immunohistochemistry and flow cytometry. A wide diversity of these cellular characteristics were found in that each tumor was unique and distinct from the others. DNA ploidy differed among the tumors but was not affected by switching between in vitro to in vivo growth. Some tumors expressed similar levels of the breast mucin both in vitro and in vivo, whereas most expressed lower levels as transplantable tumors. There was a good correlation between immunohistochemical and flow cytometric determination of surface and cytoplasmic mucin expression, and with both techniques estrogen and progesterone receptor positive tumors had significantly higher levels of mucin expression than receptor negative tumors. These 12 transplantable breast tumors, with their corresponding cell lines, provide an excellent model system for testing radioimmunotherapy and other therapeutic reagents because they exhibit diverse phenotypic characteristics that represented a mini-population of breast cancer patients’ tumors, allowing assessment of the effect of therapy when confronted with different breast tumors’ genotype and phenotype.     

人类主要Cyclins在MOLT-4细胞阻断动力学下的表达规律

细胞周期素与相应的细胞周期素依赖性蛋白激酶相结合,驱动着细胞通过细胞周期各时相,而细胞周期素时相性规律大多来自酵母研究或同步化细胞的分析。本研究着重于人类非同步化细胞的细胞周期素时相性规律的揭示。采用人类白血病细胞株MOLT-4,使其处于对数生长期,加以有丝分裂中期阻滞法,应用多参数流式细胞术分析人类主要细胞周期素B1、A和E。分析发现,细胞周期素B1峰值在M期,降解于M期后,细胞周期素A峰值在G2期,降解于M期,细胞周期素E峰值在G1晚期,降解于S期。以上结果,使我们首次在人类非同步化培养细胞展现了主要细胞周期素的时相性表达规律。     

Study of the heterogeneity of a human non-small cell bronchopulmonary epidermoid primary carcinoma: determination of the DNA content of NSCLCN6L2 cells and of cloned sub-populations

A continuous cell line, NSCLCN6L2, was established in vitro from a human bronchopulmonary epidermoid carcinoma and then cloned on agar gel and by selective media. The DNA content of each sub-population was compared with that of the parent line by flow cytometry. This study showed the heterogeneity of the NSCLCN6L2 line and the possibility of selection by cloning distinct ploidy sub-populations (group 1: diploid lines; group 2: hypotetraploid lines; group 3: a diploid and tetraploid line); it also allowed the time-course of the evolving lines to be followed. Correlation of these results with other properties of the different sub-populations will provide a better understanding of their biological behavior, particularly of their chemosensitivity.     

Assessment of DNA flow cytometry as a surrogate end point biomarker in a bladder cancer chemoprevention trial

Although conventional cytology represents the most widely performed cytometric analysis of bladder cancer cells, DNA flow cytometry has, over the past decade, been increasingly used to evaluate cell proliferation and DNA ploidy in cells from bladder washings. We have investigated whether DNA flow cytometry and conventional cytology of epithelial cells obtained from bladder washings provide reliable surrogate endpoint biomarkers in clinical chemoprevention trials. We used cytometric and clinical data from a chemoprevention trial of the synthetic retinoid Fenretinide on 99 patients with superficial bladder cancer. A total of 642 bladder washing specimens obtained from the patients at 4 month intervals was analyzed. Intra-individual agreement and correlation of flow cytometric DNA ploidy (diploid vs. aneuploid), DNA Index, Hyper-Diploid-Fraction (proportion of cells with DNA content higher than 2C), and conventional cytologic examination, as assessed by kappa statistics and Spearman's correlation test, were poor from baseline through 24 months. Moreover, no correlation was found between DNA ploidy and cytology at each time point. The same results were obtained when the analyses were stratified by treatment group. In addition, the association between the results of bladder washing (by either DNA flow cytometry or cytology) and concomitant tumor recurrence was significant only for abnormal cytology, while neither biomarker was predictive of tumor recurrence at the subsequent visit. During the time of this study only four patients progressed to muscle-invasive bladder cancer, indicating the "low-risk" features of the patient population. We conclude that DNA flow cytometry and conventional cytology on epithelial cells obtained from bladder washings do not appear to provide suitable surrogate endpoint biomarkers during the early stages of bladder carcinogenesis.     

Correlating cell cycle with metabolism in single cells: combination of image and metabolic cytometry.

BACKGROUND: We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. METHODS: A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. RESULTS: Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells. CONCLUSIONS: This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.     

DNA ploidy assessment method applied to primary breast carcinoma FNA biopsies

The goal of our study was to assess suitability of FNA biopsy material as a source of samples (cell suspension) for DNA ploidy assessment in neoplastic tumors using flow cytometry. DNA ploidy is an established prognostic factor in many types of cancers. Aneuploid breast tumors are characterized by increased aggressiveness which manifests itself through rapid local progression and metastatic spread. Investigated specimens were breast cancer FNA biopsy cell suspensions. Measurements were performed using flow cytometry. Material studied comprised 143 cases analyzed in 1999-2000. We found in this group 101 carcinoma cases with aneuploid type and 42 cases of primary breast carcinoma with diploid type of cell cycle. Immunocytochemical assesssment of estrogen receptor and progesterone receptor status was performed in group of 105 cases. DNA ploidy was compared to receptor status of the investigated cells. DNA aneuploidy correlated with weak or no reaction for the presence of estrogen and progesterone receptors. Our study demonstrates the suitability of DNA ploidy assessment method applied to cytological material from FNA biopsies.     

Application of inter-simple sequence repeats to insect cell lines: identification at the clonal and tissue-specific level

Inter-simple sequence repeat (ISSR) primers designed to anneal to microsatellites were used to obtain deoxyribonucleic acid (DNA) fingerprint profiles to distinguish among 16 established insect cell lines derived from an assortment of lepidopteran, dipteran, and coleopteran species. Three different levels of cell line comparison were made: (1) between parents and their clones, (2) among cell lines derived from different tissues from the same species, and (3) among cell lines derived from different insect species. Of the 16 repeat oligonucleotide primers used in this study, nine primers generated several unique markers to distinguish between parental cell lines and their clones. Four of the 16 primers also generated DNA profiles with a number of unique bands, enabling the distinction among cell lines derived from specific tissues from the same species. In addition, ISSR-generated DNA profiles provided the greatest number of unique markers to distinguish easily among insect cell lines derived from different species.     

A Simple FCM Method to Avoid Misinterpretation in Saccharomyces cerevisiae Cell Cycle Assessment between G0 and Sub-G1

Extensively developed for medical and clinical applications, flow cytometry is now being used for diverse applications in food microbiology. Most uses of flow cytometry for yeast cells are derived from methods for mammalian cells, but yeast cells can present specificities that must be taken into account for rigorous analysis of the data output to avoid any misinterpretation. We report an analysis of Saccharomyces cerevisiae cell cycle progression that highlights possible errors. The cell cycle was analyzed using an intercalating fluorochrome to assess cell DNA content. In analyses of yeast cultures, the presence of a sub-G1 peak in the fluorescent signal is often interpreted as a loss of DNA due to its fragmentation associated with apoptosis. However, the cell wall and its stucture may interfere with the fluorescent signal recorded. These observations indicate that misinterpretation of yeast DNA profiles is possible in analyses based on some of the most common probes: cells in G0 appeared to have a lower DNA content and may have been mistaken as a sub-G1 population. However, careful selection of the fluorochrome for DNA quantification allowed a direct discrimination between G0 and G1 yeast cell cycle steps, without additional labeling. We present and discuss results obtained with five current fluorochromes. These observations led us to recommend to use SYTOX Green for cycle analysis of living cells and SYBR Green I for the identification of the apoptosis sub-G1 population identification or the DNA ploidy application.     

Flow cytometry DNA analysis on tumor cell subpopulation of human tumor specimens by exclusion of lymphohemopoietic cells

We describe a new flow cytometry procedure in which DNA analyses can be obtained selectively on pure, freshly obtained tumor cell subpopulations of human tumor specimens. This procedure is based on exclusion from analysis of the contaminating lymphohemopoietic cells mixed with tumor cells in tumor specimens. This exclusion is made possible by labeling all lymphohemopoietic cells with an antibody to HLe-1 (HLE), which is present on all lymphohemopoietic cells but on no other cells, and by gating against these labeled cells when analyzing for DNA. For the model system, a 1:1 mixture of normal human peripheral blood leukocytes and either of two human cancer cell lines, HEp-2 and MCF-7, normal leukocyte contamination can be reduced to 3.1% while retaining 94.7% of tumor cells for DNA analysis. Four examples of human tumor samples, two cases each of malignant effusions and lymph node metastases, were analyzed with this procedure. The results clearly indicate that this new method will improve ploidy analysis/aneuploidy detection and will make it possible to obtain more accurate cell-cycle analyses of tumor cells than have previously been possible. This new procedure will contribute to clinical and biological studies involving DNA of human tumors.     

Establishment of a tetraploid Meth-A cell line through polyploidization by demecolcine but not by staurosporine, K-252A and paclitaxel

Polyploid cells are made by DNA reduplication without cell division, however, it is not easy to establish polyploid mammalian cell lines. It is worth studying the difference in cell character between hyperploid and parent cell lines. Meth-A cells were polyploidized by demecolcine, K-252a, staurosporine and paclitaxel. The cell-cycle responses of highly polyploid Meth-A cells after the removal of the drugs were examined by flow cytometry (FCM). Meth-A cells were highly polyploidized by these drugs. The polyploid Meth-A cells gradually decreased in ploidy after the drug release. A tetraploid Meth-A cell line was established only from the demecolcine-induced polyploid Meth-A cells. The duration of G1, S and G2/M phases of the tetraploid cell line were mostly the same as those of the parent diploid cells, except that the G2/M phase was 1.5 h longer. The chromosome number of tetraploid Meth-A cell line was about twice of the diploidy. A tetraploid Meth-A cell line was established.     

四倍体不结球白菜的诱导及染色体倍性鉴定

用不同浓度秋水仙素处理子叶期不结球白菜生长点对其进行染色体倍性操作,根据形态解剖学、细胞学特征和流式细胞仪进行倍性鉴定.结果表明,浓度为0.2%的秋水仙素处理4次的效果最好,四倍体诱变率为8.42%.与二倍体相比,四倍体植株叶片、花器官、气孔等均表现巨大性;气孔密度和结实率降低;抽薹较晚.用流式细胞仪进行倍性鉴定,对照DNA相对含量为100,疑似株为200,表明是四倍体;疑似株有2个值与对照的比值约为1和2,表明是嵌合体(2x 4x).流式细胞仪鉴定结果与染色体计数法鉴定结果一致,表明流式细胞仪可以较准确地检测不结球白菜突变株倍性.     

Image analysis software for automatic DNA ploidy assessment of archival solid tumours.

BACKGROUND: Image cytometry has proved to provide a good alternative to flow cytometry for DNA ploidy measurement of archival tumors. However, when interactively done this technique is unable to give statistically valuable results within an acceptable time for clinical oncology. METHODS: An image cytometer was developed for fully automatic DNA ploidy quantitation, focusing efforts on speed and accuracy. Software functionalities include systematic acquisition of fields on a microscopic slide, detection, localization and sorting of nuclei, computation of the DNA content together with post-processing tools, for a deeper analysis of the DNA ploidy diagram. RESULTS: DNA ploidy analysis of archival breast carcinoma samples illustrates the accuracy of DNA ploidy measurements and the sensitivity in the detection of DNA ploidy abnormalities as a result of cell sorting. CONCLUSIONS: Fully automatic image cytometry is able to combine qualities of flow cytometry (automatic analysis of a statistically significant collection of cell nuclei) with additional advantages: sorting of unwanted events (debris, stromal and inflammatory cell nuclei) and facilities for an a posteriori control of the quality of cell selection. This method is well suited to DNA ploidy analysis of archival cancer samples.     

Cell-cycle studies by multiparametric automatic scanning of topographically preserved cells in culture

The authors have developed a new methodology for characterizing in situ the cell cycle of human mammary epithelial cell lines. Using a SAMBA 200 cell image processor (scanning cytometry), 15 densitometric and textural parameters were computed on each Feulgen-stained nucleus. Parameters computed from the grey level cooccurrence and run-length section matrices allowed assessment of the chromatin pattern. Multiparametric analysis of data defined: 1) the relative position of each cell; 2) the relative positions of groups of cells, each group corresponding to a definite phase of the cell cycle; and 3) the function of these parameters best separating these phases. Files then were constructed for each phase: G0/G1, S, G2/ and M. Using these three files as a reference to classify cells, it was possible to ascertain the phase of the cell cycle for each cell of a population. The MDA AG human cell line synchronized by mitotic selection was used as a model to develop this method. The criteria used to assign cells to G0/G1, S, or G2 was DNA content. Classification in M phase was achieved by visual identification of mitotic cells. This method was checked on unsynchronized MDA AG and then applied to other human cell lines (MDA MB231, MCF-7, T47D C111). Comparison of results obtained by scanning cytometry and flow cytometry showed the proportion of cells assigned to G0/G1, S, and G2/M by the two methods to be similar. This new method removes some of the limitations of flow cytometry by 1) allowing visual verification of each cell analyzed; 2) lowering the number of cells required for study; 3) discriminating between G2 and M; and 4) preserving cell topography.     

Ploidy stability of maize anther culture-derived callus lines

Summary Ploidy levels of 26Zea mays L. anther culture-derived callus lines of the F1 hybrids (H99 × Pa91, Pa91 × FR16, and H99 × FR16) were determined at various times after culture initiation using flow cytometry (for 21 lines) or chromosome counting of callus cells or regenerated plants (for the remaining 5 lines). Twenty of the lines remained haploid, whereas 6 were diploid. The results from flow cytometry, after examining the DNA content of 5000 nuclei of each callus line, show that each callus line consisted of homogenous haploid or diploid cells. Thus for diploid callus lines, spontaneous chromosome doubling must have occurred before or in the early stages of androgenesis, before the initiation of callus cultures. These long-term callus cultures (growing for up to 38 mo.) have stably maintained their ploidy levels so it is unlikely that the culture conditions have caused chromosome doubling. The restriction fragment length polymorphism pattern obtained with 52 to 58 markers for each diploid callus line shows that all the diploid lines are homozygous diploid so each originated from a microspore and not from diploid maternal F1 hybrid tissue.     

Cell cycle analysis of synchronized chinese hamster cells using bromodeoxyuridine labeling and flow cytometry

Summary Chinese hamster ovary cells were synchronized into purified populations of viable G1-, S-, G2-, and M-phase cells by a combination of methods, including growth arrest, aphidicolin block, cell cycle progression, mitotic shake-off, and centrifugal elutriation. The DNA content and bromodeoxyuridine (BrdUrd) labeling index were measured in each purified fraction by dual-parameter flow cytometry. The cell cycle distributions determined from the DNA measurements alone (single parameter) were compared with those calculated from both DNA and BrdUrd data (dual parameter). The results show that highly purified cells can be obtained using these methods, but the assessed purity depends on the method of cell cycle analysis. Using the single versus dual parameter measurement to determine cell cycle distributions gave similar results for most phases of the cell cycle, except for cells near the transition from G1- to S-phase and S- to G2-phase. There the BrdUrd labeling index determined by flow cytometry was more sensitive for detecting small amounts of DNA synthesis. As an alternative to flow cytometry, a simple method of measuring BrdUrd labeling index on cell smears was used and gave the same result as flow cytometry. Measuring both DNA content and DNA synthesis improves characterization of synchronized cell populations, especially at the transitions in and out of S-phase, when cells are undergoing dramatic shifts in biochemical activity.     

Limited cell attachment time as a method to synchronize cells grown in monolayer culture

Summary A novel method of synchronizing monolayer tissue culture cells is described. By limiting the period of attachment of trypsinized cells and the subsequent removal of unattached cells a G₁ population of cells is isolated. Evaluation of the degree of synchrony has been carried out by measuring the labeling index and incorporation of [³H]thymidine into DNA. Further conformation of synchrony, as well as a comparison with synchrony by isoleucine deprivation, was obtained by flow cytometry. The expected peak in DNA synthesis rate following limited attachment was observed. This peak becomes more prominent and shifts to earlier times with shorter attachment intervals. The synchronization method described is simple, rapid, yields a substantial number of cells, and is applicable to many cell lines. This research was supported by a Basic Science Research Grant and a grant from the American Heart Foundation to K. Janakidevi. Paul Held and Christian Sell were also supported by predoctoral training grant HL07194 from the National Institutes of Health, Bethesda, MD.