In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors

Summary Methods for single- and double-target in situ hybridization (ISH) to, cells isolated from solid transitional cell carcinomas (TCC's) of the urinary bladder are described. Single cell suspensions were prepared from solid tumors of the urinary bladder by mechanical disaggregation and fixed in 70% ethanol. Using two DNA probes specific for the centromeres of chromosomes #1 and #18, ISH procedures were optimized for these samples. Human lymphocytes and cells from the T24 bladder tumor cell line were used as controls. In lymphocyte nuclei and metaphase chromosome spreads, ISH showed two major spots for each of the probes. About 80% of the nuclei from T24 cells showed three spots for both the chromosome #1 and #18 specific probes. When nuclei from TCC's were analyzed, often the number of spots for chromosome #1, and to a lesser extent for chromosome #18, differed from the number expected on basis of flow cytometric ploidy measurements. The double target-ISH method in all cases allowed the correlation of numerical aberrations for chromosomes #1 and #18 in one and the same cell. By such analyses a profound heterogeneity in chromosome number was detected in most tumors. In order to optimize the reproductbility of the method and the interpretation of the ISH-signals, criteria for their analysis have been determined. This procedure can now be applied on a routine basis to solid tumor specimens.     

In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors

Methods for single- and double-target in situ hybridization (ISH) to, cells isolated from solid transitional cell carcinomas (TCC's) of the urinary bladder are described. Single cell suspensions were prepared from solid tumors of the urinary bladder by mechanical disaggregation and fixed in 70% ethanol. Using two DNA probes specific for the centromeres of chromosomes #1 and #18, ISH procedures were optimized for these samples. Human lymphocytes and cells from the T24 bladder tumor cell line were used as controls. In lymphocyte nuclei and metaphase chromosome spreads, ISH showed two major spots for each of the probes. About 80% of the nuclei from T24 cells showed three spots for both the chromosome #1 and #18 specific probes. When nuclei from TCC's were analyzed, often the number of spots for chromosome #1, and to a lesser extent for chromosome #18, differed from the number expected on basis of flow cytometric ploidy measurements. The double target-ISH method in all cases allowed the correlation of numerical aberrations for chromosomes #1 and #18 in one and the same cell. By such analyses a profound heterogeneity in chromosome number was detected in most tumors. In order to optimize the reproducibility of the method and the interpretation of the ISH-signals, criteria for their analysis have been determined. This procedure can now be applied on a routine basis to solid tumor specimens.     

Expression of the nuclear protein mitotin in differentiating in vitro HL 60 cells.

Mitotin is a 125 kDa/pI 6.5 nuclear protein specific for proliferating cells and markedly increased prior to and during mitosis. This study presents evidence for the expression of this protein during dimethylsulfoxide (DMSO) induced differentiation of human promyelocytic leukemia HL 60 cells. The expression had been followed at two levels: as antigen, using a specific antimitotin monoclonal antibody and as mRNA, using a specific cDNA probe. The results from the immunofluorescent study show a gradual disappearance of mitotin in differentiating HL 60 cells starting from the fourth day after DMSO induction. On the other hand, the changes in the expression of mitotin mRNA were much more dramatic. This mRNA is expressed at a high level during the first three days of differentiation but shows a striking decrease after the fourth day. This correlates with the rapid changes in the number of blast cells in the differentiating HL 60 cell population. Therefore, the expression of mitotin mRNA can serve as a marker for the changes accompanying the termination of cell proliferation in differentiating cells.     

Flow cytometry to sort mammalian cells in cytokinesis.

BACKGROUND: Cell division or cytokinesis, which results from a series of events starting in metaphase, is the mechanism by which the mother cell cytoplasm is divided between the two daughter cells. Hence it is the final step of the cell division cycle. The aim of the present study was to demonstrate that mammalian cells undergoing cytokinesis can be sorted selectively by flow cytometry. MATERIALS AND METHODS: Cultures of HeLa cells were arrested in prometaphase by nocodazole, collected by mitotic shake-off and released for 90 min into fresh medium to enrich for cells undergoing cytokinesis. After ethanol fixation and DNA staining, cells were sorted based on DNA content and DNA fluorescence signal height. RESULTS: We define a cell population that transiently accumulates when synchronized cells exit mitosis before their entry into G1. We show that this population is highly enriched in cells undergoing cytokinesis. In addition, this population of cells can be sorted and analyzed by immunofluorescence and western blotting. CONCLUSIONS: This method of cell synchronization and sorting provides a simple means to isolate and biochemically analyze cells in cytokinesis, a period of the cell cycle that has been difficult to study by cell fractionation.     

Purification of the chromosomes of the Indian muntjac by flow sorting.

Metaphase chromosomes were isolated from a male Indian muntjac cell line, were stained with ethidium bromide and were analyzed by flow microfluorometry to establish a deoxyribonucleic acid (DNA)-based karyotype. Five major peaks were evident on the chromosomal DNA distribution corresponding to the five chromosome types in this species. The amount of DNA in each chromosome was confirmed by cytophotometric measurements of intact metaphase spreads. The five chromosome types were separated by flow sorting at rates up to several hundred chromosomes per second. The sorted chromosomes were identified by morphology and by Giemsa banding patterns. The automsomes, Numbers 1, 2 and 3, and the X + 3 composite chromosome were separated with a high degree of purity (90%). The centromere region of the X + 3 chromosome was fragile to mechanical shearing, and during isolation a small proportion of these chromosomes broke into four segiments: the long arm, the short arm, the short arm plus centromere and the centromere region. A large fraction of the constitutive heterochromatin of this species is present in the centromere region of the X + 3 chromosome and in the Y chromosome; these two regions possess similar amounts of DNA and therefore sort together. Chromosome flow sorting is rapid, reproducible and precise; it allows the collection of microgram quantities of purified chromosomes.     

Creation and characterization of temperature-sensitive CENP-C mutants in vertebrate cells

CENP-C is an evolutionarily conserved centromere protein that is thought to be an important component in kinetochore assembly in vertebrate cells. However, the functional role of CENP-C in cell cycle progression remains unclear. To further understand CENP-C function, we developed a method incorporating the hyper-recombinogenic chicken B lymphocyte cell line DT40 to create several temperature-sensitive CENP-C mutants in DT40 cells. We found that, under restrictive conditions, one temperature-sensitive mutant, ts4-11, displayed metaphase delay and chromosome missegregation but proceeded through the cell cycle until arrest at G₁ phase. Furthermore, ts4-11 cells were transfected with a human HeLa cell cDNA library maintained in a retroviral vector, and genes that suppressed the temperature-sensitive phenotype were identified. One of these suppressor genes encodes SUMO-1, which is a ubiquitin-like protein. This finding suggests that SUMO-1 may be involved in centromere function in vertebrate cells. The novel strategy reported here will be useful and applicable to a wide range of proteins that have general cell-autonomous function in vertebrate cells.     

Ultrastructural localization of the nuclear protein mitotin during the cell cycle.

The work presents the results of the immunoelectron microscopical localization of mitotin in different phases of the cell cycle. The distribution of the protein was studied using its specific monoclonal antibody and immunogold labeling in synchronized WISH cells. In S phase the antigen was found in the nucleoplasm usually over the interchromatin granules. In G2 phase the amount of mitotin increases and it can be found also in the nucleolus. In mitosis the immunogold granules are always out of the condensed chromosomes.     

Localization of the nuclear matrix protein mitotin in mouse cells with a mitotic or endomitotic cell cycle.

Immunofluorescence staining was used to study the precise subcellular distribution of the nuclear matrix antigen, mitotin, in mouse cells characterized by either a mitotic or an endomitotic organization of the cell cycle. In mitotically dividing cells, mitotin showed a speckled distribution within interphase nuclei. In addition, some interphase cells exhibited a weak, focused signal adjacent to the nucleus, reflecting a possible staining of the centrosome region. Using digital contrast-enhanced immunofluorescence microscopy, a distinct association of mitotin to the centrosome, pole microtubules, and midbody could be revealed in cells at different stages of mitosis. In parallel, trophoblast giant cells characterized by an endomitotic cell cycle were derived from blastocyst outgrowths and analyzed likewise. In all giant cells examined so far, mitotin was restricted to the nuclear compartment alone, although different patterns of intranuclear staining could be detected. The present study provides further information about the precise localization of mitotin in mitotic cells, especially during mitosis. In view of the results, the staining pattern observed in endomitotic cells may allow for a better understanding of the origin and the organization of the endomitotic cell cycle.     

Nuclear matrix protein mitotin messenger RNA is expressed at constant levels during the cell cycle.

During the course of an investigation on nuclear matrix protein cDNAs, we have isolated a cDNA clone hybridizing with the messenger RNA encoding mitotin. Mitotin is a 125 kDa/pI 6.5 nuclear matrix protein present in proliferating but not in resting cells. This protein was shown to have a marked increase and characteristic redistribution in G2/M phase of the cell cycle. In this report, using synchronized Raji and WISH cells, we demonstrate that mitotin messenger RNA is expressed at the same level throughout the cell cycle.     

CENP—B的基因表达与细胞周期关系的研究

本文以HeLa细胞为材料研究一种着丝粒蛋白CENP-B的基因表达与细胞周期及细胞核骨架的关系。将HeLa细胞同步在不同周期时相,以流式细胞光度术、同位素掺入和ACA着丝粒染色等方法检测细胞同步化效果。我们分别提取了各周期时相细胞的总RNA和Poly(A)~ RNA,用Dot blot和Northern blot杂交方法研究CENP-B在细胞周期中的表达。结果表明,CENP-B基因在细胞周期中的各个时相均有表达,但表达的强度差别很大:G2期表达最强,S期最弱,G1期中的表达介于二者之间;有意义的是CENP-B基因在M期仍然有较强的表达,表现出其在细胞周期中表达的持续性;这种表达的持续性反映了一种可能性:着丝粒、动粒蛋白不断合成,但直到S期后进入G2期时着丝粒、动粒蛋白到一定临界浓度时才开始组装新的动粒。另外,着丝粒、动粒蛋白的持续合成对着丝粒、动粒功能的发挥可能是必需的。用Bam H I限制性内切酶消化处于不同细胞周期时相的HeLa细胞核骨架,提取与核骨架紧密结合的DNA,用~(32)P标记的cDNA为探针研究CENP-B基因与细胞核骨架的结合与其表达的关系。结果证明,在G2期细胞中CENP-B基因表达最强,与细胞核骨架结合最为紧密,G1期细胞中次之,S期中CENP-B基因与核骨架结合最弱,说明CENP-B基因与细胞核骨架结合的紧密度影响其表达强度。     

Topoisomerase II alpha is associated with the mammalian centromere in a cell cycle- and species-specific manner and is required for proper centromere/kinetochore structure

A study of the distribution of Topoisomerase II alpha (Topo II) in cells of six tissue culture cell lines, human (HeLa), mouse (L929), rat, Indian muntjac, rat kangaroo (PTK-2), and wallaby revealed the following features: (1) There is a cell cycle association of a specific population of Topo II with the centromere. (2) The centromere is distinguished from the remainder of the chromosome by the intensity of its Topo II reactivity. (3) The first appearance of a detectable population of Topo II at the centromere varies between species but is correlated with the onset of centromeric heterochromatin condensation. (4) Detectable centromeric Topo II declines at the completion of cell division. (5) The distribution pattern of Topo II within the centromere is species- and stage-specific and is conserved only within the kinetochore domain. In addition, we report that the Topo II inhibitor ICRF-193 can prevent the normal accumulation of Topo II at the centromere. This results in the disruption of chromatin condensation sub-adjacent to the kinetochore as well as the perturbation of kinetochore structure. Taken together, our studies indicate that the distribution of Topo II at the centromere is unlike that reported for the remainder of the chromosome and is essential for proper formation of centromere/kinetochore structure.     

Spatial and temporal regulation of Condensins I and II in mitotic chromosome assembly in human cells

Two different condensin complexes make distinct contributions to metaphase chromosome architecture in vertebrate cells. We show here that the spatial and temporal distributions of condensins I and II are differentially regulated during the cell cycle in HeLa cells. Condensin II is predominantly nuclear during interphase and contributes to early stages of chromosome assembly in prophase. In contrast, condensin I is sequestered in the cytoplasm from interphase through prophase and gains access to chromosomes only after the nuclear envelope breaks down in prometaphase. The two complexes alternate along the axis of metaphase chromatids, but they are arranged into a unique geometry at the centromere/kinetochore region, with condensin II enriched near the inner kinetochore plate. This region-specific distribution of condensins I and II is severely disrupted upon depletion of Aurora B, although their association with the chromosome arm is not. Depletion of condensin subunits causes defects in kinetochore structure and function, leading to aberrant chromosome alignment and segregation. Our results suggest that the two condensin complexes act sequentially to initiate the assembly of mitotic chromosomes and that their specialized distribution at the centromere/kinetochore region may play a crucial role in placing sister kinetochores into the back-to-back orientation.     

Efficient enrichment of undifferentiated GFR alpha 1+ spermatogonia from immature rat testis by magnetic activated cell sorting

Spermatogonial stem cells (SSCs) are a documented source for adult multipotent stem cells. Thus, the isolation of SSCs is of great interest. However, the isolation of spermatogonia from mammalian testes is difficult because of their low total numbers and the lack of well-characterized cell surface markers. Glial-cell-derived neurotrophic factor family receptor alpha-1 (GFRα1) is expressed on undifferentiated mouse spermatogonia (including SSCs) and plays a crucial role, in rodents, for the maintenance of SSCs mediated by the Sertoli cell product GDNF. The present study has aimed to optimize the sorting efficiency and total cell yield of magnetic activated cell sorting (MACS) with anti-GFRα1 antibodies. Because of the technical limitations intrinsic to the magnetic columns, various sorting setups and strategies were compared. Use of Mini-MACS (MS) columns for single cell suspensions from 7-day-old rat testes resulted in a three-fold enrichment of GFRα1-positive cells in sorted fractions versus presorted fractions. However, with this method, only 1.77% of cells loaded onto the column were recovered in the sorted fraction. A sequential two-step sorting approach did not improve this poor yield. We therefore evaluated cell separation by using larger volume Midi-MACS (LS) columns. Enrichment of GFRα1-positive cells in sorted fractions was four-fold, and 14.5% of cells loaded onto the column were directed to the sorted fraction. With this method, approximately half of all GFRα1-positive cells present in the sample were found in the sorted fraction. We conclude that GFRα1 serves as a suitable surface marker for the enrichment of rat spermatogonia, and that the large-volume Midi-MACS separation system is superior to the routinely used small-volume Mini-MACS separation system. This work was financially supported by startup funds from the University Münster, NIH grant U54 HD 008610, Center grant, project 1 (to S.S.), a doctoral scholarship from the Ernst Schering Research Foundation (to K.G.), and a Young Investigator Grant from the Lance Armstrong Foundation (to J.E.).     

Cell sorting analysis of cell cycle-dependent X-ray sensitivity in end joining-deficient human cells

Non-homologous end joining (NHEJ) plays a major role in the repair of ionizing radiation-induced DNA double-strand breaks (DSBs), especially during the G1-phase of the cell cycle. Using a flow cytometric cell sorter, we fractionated G1- and S/G2-phase cells based on size to assess the DSB-repair activity in NHEJ factor-deficient DT40 and Nalm-6 cell lines. Colony formation assays revealed that the X-ray sensitivities of the G1-enriched populations correctly reflected the DSB-repair activities of both the DT40 and Nalm-6 cell lines. Furthermore, as assessed by γ-H2AX foci formation, the sorted cells exhibited less DNA damage than chemically synchronized cells. Given that it does not use fluorescent labeling or chemical agents, this method of cell sorting is simpler and less toxic than other methods, making it applicable to a variety of cell lines, including those that cannot be synchronized by standard chemical treatments.     

Initiation of DNA synthesis in the prematurely condensed chromosomes of G1 cells

The objective of this study was to investigate whether G1 cells could enter S phase after premature chromosome condensation resulting from fusion with mitotic cells. HeLa cell synchronized in early G1, mid-G1, late G1, and G2 and human diploid fibroblasts synchronized in G0 and G1 phases were separately fused by use of UV-inactivated Sendai virus with mitotic HeLa cells. After cell fusion and premature chromosome condensation, the fused cells were incubated in culture medium containing Colcemid (0.05 micrograms/ml) and [3H]thymidine ([3H]ThdR) (0.5 microCi/ml; sp act, 6.7 Ci/mM). At 0, 2, 4, and 6 h after fusion, cell samples were taken to determine the initation of DNA synthesis in the prematurely condensed chromosomes (PCC) on the basis of their morphology and labeling index. The results of this study indicate that PCC from G0, G1, and G2 cells reach the maximum degree of compaction or condensation at 2 h after PCC induction. In addition, the G1-PCC from normal and transformed cells initiated DNA synthesis, as indicated by their "pulverized" appearance and incorporation of [3H]ThdR. Further, the initiation of DNA synthesis in G1-PCC occurred significantly earlier than in the mononucleate G1 cells. Neither pulverization nor incorporation of label was observed in the PCC of G0 and G2 cells. These findings suggest that chromosome decondensation, although not controlling the timing of a cell's entry into S phase, is an important step for the initiation of DNA synthesis. These data also suggest that the entry of a S phase may be regulated by cell cycle phase-specific changes in the permeability of the nuclear envelope to the inducers of DNA synthesis present in the cytoplasm.     

Short-term physiologic effects of mechanical flow sorting and the Becton-Dickinson cell concentrator in cultures of the marine phytoflagellata Emiliania huxleyi and Micromonas pusilla.

BACKGROUND: In contrast to large, high-efficiency cytometers, mechanically sorting benchtop instruments provide a feasible alternative for shipboard cell sorting of oceanic microbial communities. However, sorting efficiency of these instruments is constrained by their maximum sorting rate of approximately 300 cells/s and by constant dilution of sorted samples by sheath flow. These factors often render too low sorted cell concentrations for postsorting experiments of oceanic phytoplankton populations of low natural abundance. A Cell Concentrator module has been marketed to overcome these dilution effects. Postsorting experiments also have to consider potential physiologic effects of cell sorting. Short-term physiologic effects on phytoplankton photosynthetic rates and esterase activities by mechanical flow sorting and cell concentration and on the efficiency of the Cell Concentrator module are evaluated. METHODS: Increasing numbers of the oceanic phytoflagellates Micromonas pusilla and Emiliania huxleyi were sorted and concentrated, and recovery in the concentrated samples was compared with the sorted-only samples (concentration rate) and the total number of sorted cells (recovery rate). Photosynthetic rates and metabolic activities of sorted and sorted/concentrated cells were compared with unsorted cells. Photosynthetic rates were estimated from 14CO2 uptake experiments and metabolic activity quantified cytometrically after cleavage of fluorescein diacetate. RESULTS: Irrespective of the total number of sorted cells, concentration rates between concentrated and sorted cells remained mostly below 10-fold and did not increase with the number of concentrated cells. Recovery rates in the concentrated samples amounted to fewer than 10% of total sorted cells, except for forceful resuspension attempts in the Concentrator insert (25-44%), which might be unsuitable for delicate species. Cell sorting resulted in a 24-49% decrease in photosynthetic rates. Metabolic activity within metabolically active cells was not affected by cell sorting, but the share of metabolically active cells decreased by 32-37%. Cell concentration did not affect metabolic activity or the fraction of active cells but did increase photosynthetic rate several-fold compared with unsorted cells. CONCLUSION: Low recovery of concentrated cells, probably due to cell adhesion to the filer bottom of the Concentrator insert, render the Cell Concentrator of limited use to overcome dilution problems of mechanical flow sorting, particularly when results are extrapolated to natural, low-abundance populations. Severe changes in photosynthetic rates also render concentrated cells suspicious for subsequent physiologic experiments. Mechanical sorting alone also exhibited significant physiologic effects on sorted cells, some of which might not be temporary. Comparable effects between mechanical sorting and droplet sorting as previously reported confirm that physiologic effects might be caused predominantly by shear stress and laser exposure during cytometric analysis rather than the sorting process. Sufficient recovery time must be allowed before postsorting experiments, but potential changes in cell physiology from the natural conditions during postsorting recovery must be considered.     

CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase.

We have identified a novel human centromere-associated protein by preparing monoclonal antibodies against a fraction of HeLa chromosome scaffold proteins enriched for centromere/kinetochore components. One monoclonal antibody (mAb177) specifically stains the centromere region of mitotic human chromosomes and binds to a novel, approximately 250-300 kd chromosome scaffold associated protein named CENP-E. In cells progressing through different parts of the cell cycle, the localization of CENP-E differed markedly from that observed for the previously identified centromere proteins CENP-A, CENP-B, CENP-C and CENP-D. In contrast to these antigens, no mAb177 staining is detected during interphase, and staining first appears at the centromere region of chromosomes during prometaphase. This association with chromosomes remains throughout metaphase but is redistributed to the midplate at or just after the onset of anaphase. By telophase, the staining is localized exclusively to the midbody. Microinjection of the mAb177 into metaphase cells blocks or significantly delays progression into anaphase, although the morphology of the spindle and the configuration of the metaphase chromosomes appear normal in these metaphase arrested cells. This demonstrates that CENP-E function is required for the transition from metaphase to anaphase.     

Mitochondrial Staining Allows Robust Elimination of Apoptotic and Damaged Cells during Cell Sorting

High-speed fluorescence-activated cell sorting is relevant for a plethora of applications, such as PCR-based techniques, microarrays, cloning, and propagation of selected cell populations. We suggest a simple cell-sorting technique to eliminate early and late apoptotic and necrotic cells, with good signal-to-noise ratio and a high-purity yield. The mitochondrial potential dye, TMRE (tetramethylrhodamine ethyl ester perchlorate), was used to separate viable and non-apoptotic cells from the cell sorting samples. TMRE staining is reversible and does not affect cell proliferation and viability. Sorted TMRE⁺ cells contained a negligible percentage of apoptotic and damaged cells and had a higher proliferative potential as compared with their counterpart cells, sorted on the basis of staining with DNA viability dye. This novel sorting technique using TMRE does not interfere with subsequent functional assays and is a method of choice for the enrichment of functionally active, unbiased cell populations.     

Centromere position in budding yeast: evidence for anaphase A.

Although general features of chromosome movement during the cell cycle are conserved among all eukaryotic cells, particular aspects vary between organisms. Understanding the basis for these variations should provide significant insight into the mechanism of chromosome movement. In this context, establishing the types of chromosome movement in the budding yeast Saccharomyces cerevisiae is important since the complexes that mediate chromosome movement (microtubule organizing centers, spindles, and kinetochores) appear much simpler in this organism than in many other eukaryotic cells. We have used fluorescence in situ hybridization to begin an analysis of chromosome movement in budding yeast. Our results demonstrate that the position of yeast centromeres changes as a function of the cell cycle in a manner similar to other eukaryotes. Centromeres are skewed to the side of the nucleus containing the spindle pole in G1; away from the poles in mid-M and clustered near the poles in anaphase and telophase. The change in position of the centromeres relative to the spindle poles supports the existence of anaphase A in budding yeast. In addition, an anaphase A-like activity independent of anaphase B was demonstrated by following the change in centromere position in telophase-arrested cells upon depolymerization and subsequent repolymerization of microtubules. The roles of anaphase A activity and G1 centromere positioning in the segregation of budding yeast chromosomes are discussed. The fluorescence in situ hybridization methodology and experimental strategies described in this study provide powerful new tools to analyze mutants defective in specific kinesin-like molecules, spindle components, and centromere factors, thereby elucidating the mechanism of chromosome movement.