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Protease‐activated receptor 2 (PAR‐2) is a G protein‐coupled receptor suggested to play an important role in the proliferation and migration of tumor cells of epithelial origin. However, the role of PAR‐2 in the setting of pancreatic cancer remains largely unexplored.

A number of structurally unrelated DNA intercalators have been studied as stabilizers of mitotic chromosomes during isolation from rodent and human metaphase cells. Seven out of the nine intercalators tested were found to be useful as chromosome stabilizing agents. Chromosome suspensions prepared in this way could be preserved for long periods of time. After isolation the chromosomal DNA was longer than 150 kb. With intercalated chromosomes high resolution flow karyotypes could be obtained as jllustrated for the non-fluorescent intercalators 9-methylene-(1,3-dimethyl-2,4-dionepyrimidine-5-yl)-phenanthridiniumchloride and 4'-aminomethyl-4,5', 8-trimethylpsoralen combined with DAPI and 33258 Hoechst for fluorescent staining and for the fluorescent intercalator propidium iodide used as a stabilizer and as a fluorochrome. Passage of the intercalated chromosomes through the laser beam had no measureble effect on the length of the chromosomal DNA subsequently isolated. After flow analysis and collection on slides human chromosomes could easily be banded by Giemsa staining methods with the same resolution as obtained in conventional metaphase spreads. This allowed a ready indentification of about 80 percent of all chromosomes in the unfractionated suspension collected after passage through the laser beam.

Like many nuclear processes, DNA replication takes place in distinct domains that are scattered throughout the S-phase nucleus. Recently we have developed a fluorescent double-labeling procedure that allows us to visualize nascent DNA simultaneously with “newborn” DNA that had replicated earlier in the same nucleus during the same S-phase. Using this procedure we have shown that all DNA in a replication domain is replicated within 1 h (Manderset al.,1992,J. Cell Sci.103, 857–862). Here we extend these studies by analyzing the behavior of replication domains on a time scale of less than 1 h. We have carried out a series of double-labeling experiments in which we varied the time interval between nascent DNA and newborn DNA from 0 to 60 min. Subsequently, we determined from the confocal, 3D images the spatial position of replicated DNA domains and identified pairs of nearest neighbor domains containing newborn and nascent DNA, respectively. The distance between the centers of the two domains in a pair gradually increases. Accurate measurements show that domains containing nascent DNA and domains containing newborn DNA gradually separate from each other at a rate that is on the order of 0.5 μm/h. This indicates that either newly synthesized DNA moves away from sites of replication activity or the replication machinery is moving itself. This rate is essentially the same during early and late S-phase.

Exponentially growing cells of an established line derived from a mouse osteosarcoma (MOS) have been studied by time-lapse cinematography after irradiation with 3 Gy of 200 kV X- rays or 1·5 Gy of 14 MeV neutrons. Cell cycle times (Tc) of individual cells and their progeny in three subsequent generations as well as the occurrence of aberrant mitosis have been determined to evaluate the variation in expression of damage in relation to the stage in the intermitotic cycle and the radiation quality. The results show that the radiation doses applied cause an equal elongation of the mean Tc, which is largest in the irradiated cells but persists in the three subsequent generations. After 3 Gy of X-rays, mitotic delay is largest in cells irradiated in later stages of the cycle, but this difference is not observed after 1·5 Gy of 14 MeV neutrons. In subsequent generations the Tc values show larger variations among descendents of cells treated in the same stage of the cycle as compared to controls but this variation is equal for the doses of X-rays and neutrons applied.Division probability was significantly reduced in irradiated cells as well as in subsequent generations, whereby with neutrons as compared to X-rays the damage is expressed in earlier generations, with less variation as a function of the cell cycle.

The temporal and spatial progression of DNA replication in interphase nuclei of eukaryotic cells has been investigated. Application of a recently developed technique for the immunofluorescence double staining of cell nuclei labelled first with iododeoxyuridine (IdUrd) and subsequently with chlorodeoxyuridine (CldUrd) allows the visualization of two replication patterns in the same nucleus originating from two different periods of the S-phase. We have analysed changes in the three-dimensional replication patterns during the S-phase. To record dual colour three-dimensional images of doubly stained nuclei, a confocal microscope is used. This CSLM is equipped with a specific laser/filter combination to collect both fluorescence signals (FITC and Texas Red) in a single scan, thus precluding pixel shift between the images. A method for the quantitative evaluation of the degree of overlap between DNA regions replicated in two different periods of the S-phase is applied. The results confirm the generally accepted theory that DNA is replicated coordinately in a specific temporal order during the S-phase. The replication time of a DNA domain (i.e. the time between initiation and termination of DNA replication within a domain) at the very beginning of the S-phase was known to be one hour (Nakamura et al., 1986). Our observations show that in the rest of the S-phase, the replication time of a DNA region is also about one hour. We conclude that replicon clusters located in the same region are replicated in the same relatively short period of time. After this period there is no unreplicated DNA left in this region.

The temporal and spatial progression of DNA replication in interphase nuclei of eukaryotic cells has been investigated. Application of a recently developed technique for the immunofluorescence double staining of cell nuclei labelled first with iododeoxyuridine (IdUrd) and subsequently with chlorodeoxyuridine (CldUrd) allows the visualization of two replication patterns in the same nucleus originating from two different periods of the S-phase. We have analysed changes in the three-dimensional replication patterns during the S-phase. To record dual colour three-dimensional images of doubly stained nuclei, a confocal microscope is used. This CSLM is equipped with a specific laser/filter combination to collect both fluorescence signals (FITC and Texas Red) in a single scan, thus precluding pixel shift between the images. A method for the quantitative evaluation of the degree of overlap between DNA regions replicated in two different periods of the S-phase is applied. The results confirm the generally accepted theory that DNA is replicated coordinately in a specific temporal order during the S-phase. The replication time of a DNA domain (i.e. the time between initiation and termination of DNA replication within a domain) at the very beginning of the S-phase was known to be one hour (Nakamura et al., 1986). Our observations show that in the rest of the S-phase, the replication time of a DNA region is also about one hour. We conclude that replicon clusters located in the same region are replicated in the same relatively short period of time. After this period there is no unreplicated DNA left in this region.

Analysis of time-lapse cinematographic film permitted the construction of pedigrees from 88 well oxygenated cells of a mouse osteosarcoma (MOS). These cells have been chronically treated with various concentrations of the hypoxic cell sensitizer misonidazole (MIS) over periods of up to 96 h. At concentrations of 0.5 and 7 mM there is a 2--3 h increase in cell-cycle time. Concentrations of 2 mM show an intermitotic time delay of 7.6--10.3 h. At 4 mM cells divided only once. With increasing drug concentration there was an increase in the number of abnormal mitoses. These results were compared with cloning efficiency (PE) experiments. PE at 0.5 mM is 80%, at 1 mM 40 and at 2 mM is reduced to 4%. Cells treated with 2mM MIS over a period of 28.6 h resume their normal cycle when the drug is washed from the culture. This may indicate that DNA is not a major target for MIS. It is concluded that this hypoxic cell sensitizer is also toxic for MOS cells in well oxygenated conditions.

A procedure was developed that very effectively distinguishes between IdUrd and CldUrd incorporated in the DNA of cell nuclei and chromosomes. For double staining we used the, rat anti-BrdUrd monoclonal antibody from Sera-lab that binds specifically to CldUrd and BrdUrd but not to IdUrd, in combination with the mouse anti-BrdUrd monoclonal antibody from Becton Dickinson. This antibody binds to all three halogenated deoxyuridines, but when the nuclei are washed in TRIS buffer with a high salt concentration the antibodies linked to CldUrd-labelled DNA are removed. When analysing the effect of the deoxyuridines on the cell cycle we found that the growth kinetics of Chinese hamster cells were not changed by adding IdUrd or CldUrd for 30 min at a concentration of 10 μm, whereas adequate double labelling required only 2 min pulses. The effectiveness of the technique was demonstrated in two model experiments. The first test concerned the assessment of cell recruitment in the central areas of slow-growing clones, after addition of fresh medium. The second experiment focussed on the spatial resolution of the method. Double-labelled metaphase chromosomes showed interspersed green and red replication bands with a spacing corresponding with medium resolution Giemsa banding patterns.