Jacob A. Aten

A method to measure the degree of co-localization of objects in confocal dual-colour images has been developed. This image analysis produced two coefficients that represent the fraction of co-localizing objects in each component of a dual-channel image. The generation of test objects with a Gaussian intensity distribution, at well-defined positions in both components of dual-channel images, allowed an accurate investigation of the reliability of the procedure. To do that, the co-localization coefficients were determined before degrading the image with background, cross-talk and Poisson noise. These synthesized sources of image deterioration represent sources of deterioration that must be dealt with in practical confocal imaging, namely dark current, non-specific binding and cross-reactivity of fluorescent probes, optical cross-talk and photon noise. The degraded images were restored by filtering and cross-talk correction. The co-localization coefficients of the restored images were not significantly different from those of the original undegraded images. Finally, we tested the procedure on images of real biological specimens. The results of these tests correspond with data found in the literature. We conclude that the co-localization coefficients can provide relevant quantitative information about the positional relation between biological objects or processes.

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.

Interactions between ends from different DNA double-strand breaks (DSBs) can produce tumorigenic chromosome translocations. Two theories for the juxta-position of DSBs in translocations, the static "contact-first" and the dynamic "breakage-first" theory, differ fundamentally in their requirement for DSB mobility. To determine whether or not DSB-containing chromosome domains are mobile and can interact, we introduced linear tracks of DSBs in nuclei. We observed changes in track morphology within minutes after DSB induction, indicating movement of the domains. In a subpopulation of cells, the domains clustered. Juxtaposition of different DSB-containing chromosome domains through clustering, which was most extensive in G1 phase cells, suggests an adhesion process in which we implicate the Mre11 complex. Our results support the breakage-first theory to explain the origin of chromosomal translocations.

Defective homologous recombination (HR) DNA repair imposed by BRCA1 or BRCA2 deficiency sensitizes cells to poly (ADP-ribose) polymerase (PARP)-1 inhibition and is currently exploited in clinical treatment of HR-deficient tumors. Here we show that mild hyperthermia (41-42.5 °C) induces degradation of BRCA2 and inhibits HR. We demonstrate that hyperthermia can be used to sensitize innately HR-proficient tumor cells to PARP-1 inhibitors and that this effect can be enhanced by heat shock protein inhibition. Our results, obtained from cell lines and in vivo tumor models, enable the design of unique therapeutic strategies involving localized on-demand induction of HR deficiency, an approach that we term induced synthetic lethality.