Origin of microcells in the human sarcoma cell line HT-1080.

The aim of this study was to investigate the development of microcells in the human sarcoma cell line HT-1080 after interference with thiophosphamidum. We found that damaged interphase macrocells located at the projection of the nucleolus may form one or several microcells. The micronuclei of the microcells intensively incorporate the thymidine analogue 5-bromo-2'-deoxyuridine and strongly express argyrophilic nucleolar organiser region proteins. At an early phase of the development, the micronuclei contain fragmented DNA, but in subsequent phases, the micronuclei accumulate polymeric DNA, simultaneously with an increase in their size. After desintegration of the damaged macrocell, the microcells appear in the intercellular space. The microcells can enter mitosis and they strongly express the lung resistance protein. Electron microscopic observations suggest that coiled bodies are involved in the development of the microcells. Since the observed path of microcell formation differs from apoptotic cell fragmentation into apoptotic bodies, we propose a new term for this microcell development: sporosis. We suggest that self-renewal of the tumour stem cells is likely based on sporosis.     

一种适用于生产转染色体动物的微细胞制备方法

微细胞介导的染色体转移技术(MMCT)是一项将外源染色体转入哺乳动物细胞的技术,具有广阔的应用前景.与体细胞核移植技术结合,MMCT可用于生产具有重要医学药用价值和优良农业生产性状的转染色体动物.制备高质量的微细胞是关系MMCT技术成功的关键步骤之一.通过荧光染色和吉姆萨染色分析,结果表明,A9(neo12)细胞经0.2mg/L秋水仙素酰胺处理48h后,89%的细胞产生微核化,每个细胞平均形成10个微核.微核化的细胞在含有20mg/L细胞松弛B的Percoll密度梯度介质中,经39000g高速离心后,包含微细胞、完整细胞、细胞核和细胞碎片的混合液,依次通过8μm和5μm孔径的滤膜过滤后可获得纯化的微细胞溶液.通过光学显微镜和吉姆萨染色观察,可见微细胞为一群直径约为3～5μm的类细胞核的球形物质.微细胞PCR技术首次用于检测微细胞溶液的质量,检测结果显示,所制备的溶液中均匀分布着带有目的染色体的微细胞,适用于进一步作转染色体动物实验.     

Cell size, DNA, and cytokeratin analysis of human head and neck tumors by flow cytometry

Cell subsets have been discriminated in cell suspensions derived from 37 human head and neck tumors by means of light scatter, DNA, and cytokeratin flow cytometry (FCM). Cell dispersion was performed overnight at 4 degrees C in two different enzyme mixtures, i.e., trypsin/dithioerythritol and collagenase/DNase, under slight agitation of sliced tumor tissue. Cells were examined before and after fractionation on a discontinuous low-density bovine serum albumin (BSA) gradient. Forward and right-angle light scatter FCM of 23 tumor specimens revealed four main subpopulations with different size and structure. Fractionation of primary cell suspensions on a BSA gradient at unit gravity separated debris, small cells and large cells. DNA FCM of the enriched populations demonstrated a relation between large cells and DNA aneuploidy. Epithelial cells, as recognized by cytokeratin antibodies, were also related with large cells. The results demonstrated the usefulness of light scatter, DNA, and cytokeratin analysis of crude and fractionated tumor cell suspensions for assessment of the efficacy of a particular dispersion technique and to obtain information of the cell subsets dispersed.     

Modal DNA values of normal and malignant urothelial cells of the bladder in relation to nuclear size

In a study of the correlation between mean nuclear size and DNA content in urinary bladder carcinoma, the modal DNA values of cell suspensions from 125 biopsies, obtained from 86 patients with malignant or normal urinary bladder epithelium, were analyzed by flow cytometry (FCM). Light microscopic measurements of nuclear size were carried out on smears from the same material. The results were correlated to the histopathologic stage and grade. The mean nuclear volumes were significantly larger in diploid tumor cells than in cells of normal epithelium. Aneuploid tumors showed significantly larger nuclei than did diploid tumors. Although there was a significant correlation between increases in the nuclear volume and in the DNA content, there was some overlapping between various grades of malignancy: mean nuclear volumes in aneuploid grade 2 tumors did not differ from those in aneuploid grade 3 tumors. A combination of FCM and morphometry discriminated all but 16% of the tumors from the normal cases. It is concluded that FCM and morphometry are complementary and can be used for the objective characterization of urinary bladder carcinomas.     

Characterization of the population of the sulfur-oxidizing symbiont of Codakia orbicularis (Bivalvia, Lucinidae) by single-cell analyses

We investigated the characteristics of the sulfur-oxidizing symbiont hosted in the gills of Codakia orbicularis, a bivalve living in shallow marine tropical environments. Special attention was paid to describing the heterogeneity of the population by using single-cell approaches including flow cytometry (FCM) and different microscopic techniques and by analyzing a cell size fractionation experiment. Up to seven different subpopulations were distinguished by FCM based on nucleic acid content and light side scattering of the cells. The cell size analysis of symbionts showed that the symbiotic population was very heterogeneous in size, i.e., ranging from 0.5 to 5 mum in length, with variable amounts of intracellular sulfur. The side-scatter signal analyzed by FCM, which is often taken as a proxy of cell size, was greatly influenced by the sulfur content of the symbionts. FCM revealed an important heterogeneity in the relative nucleic acid content among the subclasses. The larger cells contained exceptionally high levels of nucleic acids, suggesting that these cells contained multiple copies of their genome, i.e., ranging from one copy for the smaller cells to more than four copies for the larger cells. The proportion of respiring symbionts (5-cyano-2,3-ditolyl-terazolium chloride positive) in the bacteriocytes of Codakia revealed that around 80% of the symbionts hosted by Codakia maintain respiratory activity throughout the year. These data allowed us to gain insight into the functioning of the symbionts within the host and to propose some hypotheses on how the growth of the symbionts is controlled by the host.     

DNA content of microcells prepared from rat kangaroo and mouse cells

Enucleation of animal cells in which nuclear fragmentation (micronucleation) has been induced by treatment with mitotic inhibitors results in the formation of subdiploid microcells consisting of one or several micronuclei, some cytoplasm and surrounded by a plasma membrane. Microcells were prepared from rat kangaroo cells (12 chromosomes) and a polyoma virus transformed mouse cell line (61 chromosomes) and analysed for DNA content. Microspectrophotometric DNA measurements and the appearance of micronuclei at mitosis show that small micronuclei contain genetic information equivalent to single chromosomes. A large proportion of the micronuclei and the microcells, however, contains DNA corresponding to several chromosomes. Heterogeneous mixtures of microcells can be fractionated by a unit gravity sedimentation procedure so as to isolate the small microcells. These can afterwards be fused with intact normal or mutant cells.     

Evaluation of the effects of cryopreservation of isolated erythrocytes and leukocytes of largemouth bass by flow cytometry

We examined the effects of separation and freezing on fish leukocyte and erythrocyte morphology by light microscopy and on DNA content as measured by flow cytometry (FCM). Leukocytes and erythrocytes of largemouth bass Micropterus salmoides were isolated by density gradient centrifugation of whole blood, and frozen in liquid nitrogen in a buffer containing DMSO as a cryopreservative. The coefficient of variation (CV) of the G₀/G₁ peak of the cells was used to assess variation in nuclear DNA content within cell populations before and after separation and freezing treatments. In erythrocytes, the CV did not change significantly (P>0.05) when nuclei were isolated and stained without freezing or when erythrocytes were frozen prior to nuclear isolation and staining. In leukocytes, freezing and thawing prior to isolation and staining of nuclei significantly increased the CV (P<0.05), and produced hyperdiploid shoulders of the G₀/G₁ peak. However, the CV of leukocyte nuclei that were isolated and stained prior to freezing and the CV of non-frozen leukocyte nuclei did not differ (P>0.05). Microscopy showed that the freezing protocol had little effect on erythrocyte morphology, but caused irregular swelling in leukocytes. Freezing intact leukocytes also significantly (p<0.05) altered the apparent distribution of cells among the phases of the cell cycle as measured by FCM. The distributions of leukocyte nuclei that were isolated and stained prior to freezing were not different to non-frozen leukocytes. DNA measurements of nucleated blood cells are widely used in physiological, genetic and toxicological studies. Our results suggest that whole blood and erythrocytes for use in such studies can be frozen whole using a simple protocol, but leukocyte nuclei must be isolated and stained before freezing to avoid serious artifacts.     

Flow cytometric DNA content analysis of neoplastic cells in haemolymph of the cockle Cerastoderma edule

Epizootiological outbreaks of disseminated neoplasia (DN) have been reported in association with mass mortalities in various bivalve species including the cockle Cerastoderma edule. A flow cytometric (FCM) procedure to study DNA content was successfully adapted and tested in haemolymph cells (haemocytes and neoplastic cells) of the cockle. The FCM results were similar to those obtained by histological analysis (DN diagnosis and haemolymph cell features). FCM analysis revealed differences in DNA content among normal haemocytes (diploid) and neoplastic cells. Four types of cells with abnormal DNA content were found in the haemolymph of affected animals: hypodiploid, hyperdiploid, triploid-sesploid and pentaploid. Our results suggest that the flow cytometric DNA content analysis can be applied to identify neoplastic cell types and to study the association between different cell types and the DN progression or remission in this edible and commercially important bivalve species.     

Flow-cytometric analysis of mixed cell populations using intermediate filament antibodies

Using two human tumour cell lines, T24 bladder carcinoma and Molt-4 leukemia, flow-cytometric DNA analysis of pure and mixed cell populations was performed using cellular cytokeratin content to distinguish cytokeratin-containing carcinoma cells from leukemia cells which do not contain cytokeratin. Using cytokeratin content to gate DNA analysis, the same specificity and sensitivity of cellular DNA content and distribution measurement could be achieved by single-pass FCM analysis of a mixture of the two cell types as was seen when analysing pure populations of the two cell lines. This technique has broad applicability to FCM analysis of mixed populations composed of cells from different tissues of origin.     

Cytochemistry and C-values: the less-well-known world of nuclear DNA amounts

BACKGROUND: In the plant sciences there are two widely applied technologies for measuring nuclear DNA content: Feulgen absorbance cytophotometry and flow cytometry (FCM). While FCM is, with good reasons, increasingly popular among plant scientists, absorbance-cytophotometric techniques lose ground. This results in a narrowing of the methodological repertoire, which is neither desirable nor beneficial. Both approaches have their advantages, but static cytophotometry seems to pose more instrumental difficulties and material-based problems than FCM, so that Feulgen-based data in the literature are often less reliable than one would expect. SCOPE: The purpose of this article is to present a selective overview of the field of nuclear DNA content measurement, and C-values in particular, with a focus on the technical difficulties imposed by the characteristics of the biological material and with some comments on the photometrical aspects of the work. For over 20 years it has been known that plant polyphenols cause problems in Feulgen DNA cytophotometry, since they act as major staining inhibitors leading to unreliable results. However, little information is available about the chemical classes of plant metabolites capable of DNA staining interference and the mechanisms of their inhibition. Plant slimes are another source of concern. CONCLUSIONS: In FCM research to uncover the effects of secondary metabolites on measurement results has begun only recently. In particular, the analysis of intraspecific genome size variation demands a stringent methodology which accounts for inhibitors. FCM tests for inhibitory effects of endogenous metabolites should become obligatory. The use of dry seeds for harvesting embryo and endosperm nuclei for FCM and Feulgen densitometry may often provide a means of circumventing staining inhibitors. The importance of internal standardization is highlighted. Our goal is a better understanding of phytochemical/cytochemical interactions in plant DNA photometry for the benefit of an ever-growing list of plant genome sizes.     

Flow cytometric measurements of cell volumes and DNA contents during culture of indigenous soil bacteria

Indigenous soil bacteria were released from a clay loam soil by repeated washing and centrifugation followed by density gradient centrifugation to remove enough soil particles to allow a flow cytometric (FC) study of cell numbers, cell sizes, and DNA content in single cells. The bacteria were suspended in liquid soil extract medium and incubated at 15°C for 60 h, during which direct fluorescence microscopic counts (acridine orange direct counts, AODC) were done along with the FC measurements. Cells of Escherichia coli with a known number of whole genomes per cell (rifampicin treated) were used as a calibration standard both for the DNA measurements (mitramycin-ethidium bromide stain) and cell volumes (light scatter). In response to the nutrients in the soil extract medium, the indigenous soil bacteria increased in numbers and respiration rate after a lag period of about 17 h. The onset of growth was seen first as an increase in respiration rate, numbers of large cells, and the amounts of DNA per cell in the large cells. Respiration and direct microscopical determination of biovolume was used to calculate the average growth yield on the basis of cell carbon, which was found to be 20–30% during the period of active growth. For separate volume groups of the indigenous cells, the DNA content ranged from 1.5 to 15 fg DNA per cell, the majority being below 4 fg DNA. During growth in soil extract medium, the numbers of large cells (volume > 0.18 m³) increased, and the frequency of cells with high DNA contents increased as well for this group. For the smallest sized cells (volumes < 0.065 m³) it was not possible to detect any increase in numbers during the 60-h incubation, and the DNA contents of these cells remained virtually unchanged. Compared with cell volumes based on microscopy (AODC), the FC-light scatter data grossly overestimated the volume for indigenous cells but apparently not for the newly formed cells during growth in the suspension. This probably reflects differences in light scatter properties due to adsorbed materials on the indigenous cells. The FC-DNA measurements confirmed earlier findings in that the average DNA content per cell was low (around 2 fg DNA per cell), but demonstrated a positive relationship between cell size and DNA content for indigenous cells.     

Cancer chemo-endocrine therapy and its cell biological basis

The aim of our cell kinetic studies is to better understand the effects of chemo-endocrine therapy at the cell biological and molecular level. Cancer cell growth is characterized by uncontrolled proliferation, resulting in DNA distribution pattern in which, at any time, more cells are not G1 phase but in S, G2 and M phase of a shortened cycle. In a recent progress, flow cytometry (FCM) has become a powerful tool for the quantitative analysis of cell cycle parameters by measuring nuclear DNA content in large cell population with high speed. With the aid of FCM in earlier work about 60-80% of ovarian cancers were found to contain aneuploid cells. Now, multi-parameter FCM linked to a computer is available to measure fluorescent intensities not only no base total DNA (Propidium iodide) but also A-T (Hoechst 33342) and G-C (Mithramycin) base pairs in solid cancer nuclei. Since cisplatinum (CDDP) is the most important drug in the treatment of ovarian cancer, we have studied the relationship of CDDP cytotoxicity, pertubations cell cycle kinetis and DNA damage in ovarian adenocarcinoma cells in vitro & in vivo. We employed both CDDP sensitive cell line (KFt) and resistant cell line (KFr) derived from human serous cystoadenocarcinoma of the ovary by Kikuchi et al (JNCI 1986). Comparing cell kinetic pertubations of experimental cells demonstrates a decrease in G1 phase cells concomitant increase in S phase cells. The KFr cells had distinctly a shorter S-phase block up to 24 hrs not A-T but G-C preference in a quick response followed repairing of DNA damage to 48 hrs. However, some fractions of CDDP resistant cell population showed a later onset of G2, M phase accumulation. Comparison with the increase in early S phase cells of KFr in detailed analysis suggests only those damaged cells that are not killed immediately may proceed to G1 phase and start into DNA synthesis in S phase. Measurement of labeling index (L. I.) with Bromodeoxyuridine (BrdU) support our interpretation of differences between sensitivity and resistance to anti-cancer drug. Additionally, we discuss a targeting chemotherapy by coupling cytotoxic drugs with estrogen based on increasing DNA damage into apoptosis and interfares with DNA repair process.     

Simultaneous flow cytometric detection of nuclear DNA and tumor-associated antigens in lung cancers

Flow cytometric (FCM) determinations of DNA index were found to be insufficient to distinguish the presence of tumor cells from normal ones in neoplastic tissues obtained from 29 patients with lung cancer. Therefore, the DNA and tumor-associated antigen (TAA) contents of cultured human lung cancer cells were simultaneously analyzed using FCM to assess whether this dual technique would help in distinguishing tumor cells from normal ones. For the study, cells from PC-10 (a squamous cell carcinoma line), PC-3 (an adenocarcinoma line) and PC-6 (a small cell carcinoma line) were mixed with normal peripheral lymphocytes. The TAAs studied were carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCCA) and neuron-specific enolase (NSE). The alcohol-fixed cells were treated with the respective primary TAA, followed by fluorescein-isothiocyanate-conjugated secondary antibody; the cellular DNA was then stained using propidium iodide. Red and green fluorescences were measured simultaneously by FCM. The results showed CEA mainly in PC-3 cells, SCC in PC-10 cells and NSE in PC-6 cells; thus, each cell type had a relatively specific TAA. DNA content and cell size analyses differentiated neoplastic cells from normal lymphocytes for PC-3 and PC-10 cells, but not for PC-6 cells. Simultaneous FCM analyses of DNA and the TAA specific for the individual cell type made it possible to distinguish all tumor cell types from normal lymphocytes.     

DNA quantification in colorectal carcinoma using flow and image analysis cytometry

Numerous studies using flow cytometry (FCM) have shown that DNA quantification and ploidy classification can provide information of prognostic significance for patients with colorectal carcinoma; recent advances in image analysis cytometry (image cytometry, ICM) provide a new, alternative technique for DNA quantification. This study investigated whether (1) patients with colorectal carcinomas that exhibit a diploid pattern of DNA distribution have improved five-year survival statistics as compared to their non-diploid counterparts and (2) ICM provides quantitative data comparable to that obtained by FCM. DNA quantification and ploidy classification of 27 cases of primary colorectal carcinoma was performed on archival paraffin-embedded tissue by both FCM and ICM; 70% (19) of the tumors were classified as nondiploid by ICM while 56% (15) were similarly classified by FCM. Diploid tumors were associated with Dukes' stage A while nondiploid tumors were associated with Dukes' stage D. The overall five-year survival rate was 75% for patients with ICM diploid tumors and 67% for patients with FCM diploid tumors. The five-year survival was only 53% for patients with nondiploid tumors identified by both techniques. This study confirmed that DNA quantification is an important prognostic indicator for patients with colorectal carcinoma. It also showed that ICM provides data comparable to that of FCM and may be more sensitive.     

Flow cytometric analysis and cytopathology of body cavity fluids

A total of 75 samples of body cavity fluids from 71 patients were analyzed by both flow cytometry (FCM), to detect cells with an abnormal DNA content (aneuploidy), and by conventional cytopathology. Samples included 27 pleural fluids, 35 peritoneal fluids, 11 peritoneal washings and 2 pericardial fluids. For cytologic examination, the samples were prepared using standard techniques. Samples for FCM analysis were centrifuged and exposed to a hypotonic solution containing detergent and propidium iodide, a DNA intercalating fluorescent stain. Aneuploidy as well as cytologic malignancy were found in 17 samples. Forty-seven samples had normal DNA histograms by FCM and were also cytologically negative. Four samples suspicious by cytology but normal by FCM were from patients with renal-cell carcinoma (two samples from the same patient), endometrial adenocarcinoma without metastasis and chronic lymphocytic leukemia. Three samples abnormal by FCM but negative by cytology were from patients with ovarian cystadenoma, cirrhosis and uterine leiomyoma. FCM showed aneuploidy in four cytologically negative samples from patients with histologically proven malignancy (lymphoma, colonic adenocarcinoma, cervical squamous cell carcinoma, and endometrial adenosquamous carcinoma). Based on these results, FCM analysis combined with conventional cytopathology yielded 100% sensitivity, 100% predictive value of a negative result and 94% specificity. This rapid and quantitative FCM analysis of body cavity fluids can be a very useful adjunct to conventional diagnostic cytopathology.     

Quantitative high-resolution genomic analysis of single cancer cells

During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics.     

Interrelation of formalin fixation, chromatin compactness and DNA values as measured by flow and image cytometry

The severity and consistency of the effect of formalin fixation on the quantitation of DNA by flow cytometry (FCM) and image cytometry (ICM) were studied. As compared to ethanol, formalin fixation substantially decreased the propidium iodide fluorescence from mouse hepatocyte nuclei analyzed by FCM; it was also associated with an altered 4n-to-2n signal ratio and with false aneuploid peaks by FCM, but not by ICM (microspectrophotometry). ICM, on the other hand, suffered from a dependence of the DNA signal on nuclear size, which was not seen with FCM. The DNA signal variation was related to variations in the chromatin state, as shown by differences between monocytes and lymphocytes, and between RAJI cells fixed under various ionic strengths. The dependence of the DNA signal on the chromatin state indicates a need for caution in interpreting aneuploidy in formalin-fixed cells. For FCM, pseudoaneuploidy appears avoidable by using a Feulgen fluorescence staining technique. New imaging modes may be necessary to solve the problem of cell size dependence for ICM DNA determination.     

Perturbation of DNA replication and cell cycle progression by commonly used [3H]thymidine labeling protocols.

The effect of tritiated thymidine incorporation on DNA replication was studied in Chinese hamster ovary cells. Rapidly eluting (small) DNA from cells labeled with 2 microCi of [3H]thymidine per ml (200 microCi/mmol) for 60 min matured to a large nonelutable size within approximately 2 to 4 h, as measured by the alkaline elution technique. However, DNA from cells exposed to 10 microCi of [3H]thymidine per ml (66 microCi/mmol) was more rapidly eluting initially and did not mature to a nonelutable size during subsequent incubation. Semiconservative DNA replication measured by cesium chloride gradient analysis of bromodeoxyuridine-substituted DNA was also found to be affected by the final specific activity of the [3H]thymidine used in the labeling protocol. Dramatic cell cycle perturbations accompanied these effects on DNA replication, suggesting that labeling protocols commonly used to study DNA metabolism produce aberrant DNA replication and subsequent cell cycle perturbations.     

Isolation and physicochemical characterization of mitochondrial DNA from cultured cells ofPetunia hybrida

Summary Mitochondrial DNA ofPetunia hybrida was purified from cell suspension cultures. Up to 50% of the DNA could be isolated as supercoiled DNA molecules by CsCl-ethidium bromide density gradient centrifugation. The DNA purified from DNase-treated mitochondria bands at a single buoyant density of 1.760 gcm^–3 in neutral density gradients and runs on agarose gels as a single band with an apparent molecular weight exceeding 30 megadaltons (Md). Summing of the restriction endonuclease fragment lengths indicates a mitochondrial genome size of at least 190 Md. Electron microscopic analysis reveals the presence of a heterogeneous population of circular DNA molecules, up to 60 Md in size. Small circular DNA molecules, ranging in size from 2–30 Md are present, but unlike in cultured cells of other plant species they do not form discrete size classes and furthermore, they constitute less than 5% of the total DNA content of the mitochondria. The restriction endonuclease patterns of mitochondrial DNA do not qualitatively alter upon prolonged culture periods (up to at least two years).