Detection of replicative integrity in small colonic biopsies using the BrdUrd comet assay.

The alkaline single-cell gel electrophoresis or comet assay is a relatively simple method of measuring DNA single-strand breaks and alkali-labile sites in individual cells. Previously, we have used a combination of this with bromodeoxyuridine labelling of DNA and immunolocalisation of the BrdUrd to show that DNA replicative integrity can be assessed in single cultured cells. This study demonstrates the application of the technique to single cells derived from small human colonic biopsies isolated at routine endoscopy. A high level of reproducibility within replicate comet slides and between comet slides prepared from various colonic sites within a single patient is shown. Preliminary results demonstrate that defects in replication can be detected in tumour and premalignant colonic tissue adjacent to the tumour, suggesting that alterations in replicative integrity are an early event in neoplasia, appearing in premalignant mucosal cells. This development deems the BrdUrd comet assay suitable as an ex vivo molecular end point that can be measured easily in tissue collected by biopsy at routine colonic endoscopy. Thus, the BrdUrd comet assay has the potential to facilitate trial investigations of diet- or environment-related factors that may affect replicative integrity in the colon and provides a novel biomarker for colon carcinogenesis.

Alternative effects of RAS and RAF oncogenes on the proliferation and apoptosis of factor-dependent FDC-P1 cells.

Despite the fact that RAF-1 lies immediately downstream of p21RAS in the MAP kinase-signalling cascade, recent evidence in non-haematopoietic environments suggest that RAS and RAF can transduce signals through alternative pathways specific to a particular cell type. Since mutational activation of RAS occurs at high frequency in human leukaemia, we have investigated the contribution of signalling from mutant RAF in mediating the transforming effects of the N-RAS oncogene in the growth factor-dependent cell line, FDC-P1. Independent activation of N-RAS extended the period of exponential growth leading to an increased saturating density under optimal growth conditions. Under conditions of growth factor withdrawal, cells expressing mutant RAS, but not control cells, demonstrated protection against apoptotic death. Although RAF promoted cell proliferation in a similar manner to that observed in FDCP-RAS cells, expression of mutant RAF was not as effective at protecting these cells against apoptotic death following growth factor withdrawal. The results suggest that RAS utilises RAF-dependent signals in promoting the proliferation of FDC-P1 cells but the anti-apoptotic effects of this oncogene are mediated through a RAF- and BCL-2-independent pathway.

The bromodeoxyuridine comet assay: detection of maturation of recently replicated DNA in individual cells.

The single-cell gel electrophoresis (Comet) assay is a relatively simple method of measuring DNA single strand breaks and alkali-labile sites in individual cells. We have combined this with bromodeoxyuridine (BrdUrd) labeling of DNA and immunolocalization of the BrdUrd to assess DNA replicative integrity on a single-cell basis. We show that the existence of strand discontinuities in recently replicated domains of DNA, caused during semiconservative replication or exacerbated by the arrest of replicative polymerases at UV irradiation- or chemical-induced lesions, can be detected in individual cells. Data obtained from BrdUrd-Comets are consistent with biochemical data derived with a range of techniques showing that DNA replication involves the creation of strand breaks or gaps adjacent to recently replicated material, and that DNA damage prolongs the duration of such discontinuities where DNA polymerases are stalled opposite lesions (R. T. Johnson et al, The Legacy of Cell Fusion, pp. 50-67, Oxford: Science Publications, 1994; R. B. Painter, J. Mol. Biol., 143: 289-301, 1980.). Compared with standard biochemical techniques, the BrdUrd-Comet assay is simple and suitable for the accurate and automatable assessment of replicative integrity in very small numbers of mammalian cells, such as may be obtained by biopsy.