This paper addresses the problem of detecting weak incorporation of BrdUrd and the related efficiency of the denaturation protocols used to unmask this thymidine analog. Evidence is presented that measuring the distribution of BrdUrd-tagged fluorescence intensities by image cytometry generates a standard deviation threshold that discriminates between positive and negative MRC5 cells in vitro. A comparison of the thresholding by standard deviation (SDT) with the usual thresholding by the nuclear total fluorescence intensity (FIT) demonstrated that SDT has a significantly higher sensitivity (99.4--100%, depending on the denaturation protocols) than FIT (94.7 and 74.3% respectively), although both tests have a high specificity (93% and loo%, respectively) for detecting S cells. Since detecting the S cells is not only dependent on the test used, but also on the denaturation protocols, a quality index (QI) was derived from the standard deviation and the mean value of the non- (1,12,18,19) as well as kinetic changes induced by anticancer, cytotoxic, or cytostatic drugs (14,241.Flow cytometry makes it possible to analyze BrdUrcU PI-stained cell populations rapidly and is being used for some clinical applications (9,11,16). Nevertheless, flow cytometry is often inapplicable for routine pathology, especially when the cell population samples are small (fine needle aspiration biopsies) or the tissue is difficult to disaggregate. Moreover, the discrimination accuracy between BrdUrd-positive versus-negative cells is questionable, especially when a low level of BrdUrd substitution is obtained as a consequence of short pulse and/or inefficient BrdUrd transport, resulting in weakly labelled nuclei.Image analysis methods are being used successfully in some clinical applications (4,13,21) since it does not require large samples and it has advantages for both visual cell control and assessment of cell population 'This work was supported by Grants from ARC (Association pour la Recherche contre le cancer).
This paper describes the relationship between the BrdUrd replicating pattern of a cell and its localization within the S phase by means of topographical features and DNA content measurement. The present study follows an objective ranking of the BrdUrd patterns obtained from a spectral analysis of the BrdUrd images. The pattern ranking was consistent with the DNA content increase throughout the S phase. Five texture groups were arbitrarily set up for the purpose of multivariate analysis. Nine topographical parameters were computed for each BrdUrd-labelled nucleus. The descriptive quality of these parameters was assessed by means of factorial discriminant analysis. These parameters made it possible to characterize objectively the known pattern distributions of replication sites qualitatively described in the literature. o 1992 Wiley-Liss, Inc. Key terms: Anti-BrdUrd monoclonal antibodies, fluorescence, topographical parameters, image analysisThere is a body of evidence to support the hypothesis that eukaryotic DNA replication occurs as a nonrandom process in a reproducible temporal order (see 35,40 for review); the best known example is the late replication of the inactive X chromosome of mammalian female (5,211.The mechanisms responsible for this fixed replication sequence are not known, but factors such as chromatin condensation, DNA functional activity, or intranuclear arrangement may be related to the S phase ordering on the basis of many observations. It is generally conceded that euchromatin replicates early and heterochromatin replicates late. Chromosomal band analysis reveals that early DNA synthesis corresponds to the Giemsa R-band, while late DNA synthesis corresponds to the G-band (14,21). Holmquist suggested that late replication, which gradually appears during developmental process of embryonic cells along with facultative heterochromatinization, may actively determine gene repression (13).The DNA synthesized in late S might be less important for cell survival than that synthesized in early S. This hypothesis is supported by the data reviewed by Laird et al. (26) indicating that fragile sites in chromosomes of humans, Drosophila, and Microtus represent regions where DNA replicates late. Furthermore, a number of potentially active genes has been shown to replicate early (12). The relationship between gene activity, intra-nuclear arrangement, and replication timing remains unclear, but alteration of genes, as in translocation, is accompanied by changes in their replication time sequence (21). Iqbal et al. explored the question of "the relationship between the temporal replication of a proto-oncogene and its genomic organization" (18). The existence of a relationship between gene location, involving the nuclear matrix arrangement, and DNA replication has been the subject of a number of biochemical studies (8,19,33,41). However, no definitive answer is available yet, due to the variety of nuclear matrix isolation procedures used (8,381.To learn more of the DNA replication process as a function of gene ac...
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