In glucocorticoid-treated rat thymocytes and the murine lymphoid cell lines L5178 and S49 the morphology of apoptosis is associated with chromatin cleavage. The cleavage is at internucleosomal sites, apparently through activation of an endogenous endonuclease. In variants of the cell lines selected for resistance to glucocorticoid, neither apoptosis nor chromatin cleavage were observed after steroid treatment, and steroid receptors were undetectable. In thymocytes, both the morphological changes of apoptosis and chromatin cleavage were inhibited by cycloheximide and actinomycin D. The calcium-magnesium ionophore A23187 induced apoptosis and chromatin cleavage in thymocytes, and these effects were also inhibited by cycloheximide. The data confirm that the condensed chromatin which characterizes apoptosis morphologically consists of endogenously digested chromatin fragments. They also provide support for the view that at least some cells enter apoptosis by a process dependent upon macromolecular synthesis.

Sary To establish whether p53 mutation precedes or follows clonal divergence in human colorectal carcinomas, 17 tumours were analysed at multiple sites (2-5 each) for single-strand conformation polymorphisms (SSCP) within exons 5-8 of the p53 gene. A previous study had demonstrated subclones of differing DNA pkoidy in these tumours, but all showed immunocytochemical evidence for p53 stabilisation, using the monockal antibody PAb 1801. Mutations within exons 5-8 of p53 were identified by the presence of an abnormally migrating band in 10 of the 17 carcinomas: five in exon 5, four in exon 7 and one in exon 8. In each of these positive cases samples from different parts of the cacino showed identiclW gd migration patterns in SSCP analysis. Similarly, the rm_ainin seven tumours were concordant for absence of band shift across all samples of each tumour. Six SSCP-positive cases contained multiple populations differing in DNA ploidy, while four were homogeneously diploid or aneuploid throughout. Very similar proportions were observed in the SSCP-negtive cases. In four positive tumours the mutation was confirmed by sequencing or through alteration of nucleotide-specific restriction enzyme cleavage. Identical mutations appeared in every sample from the same tumour. The results provide unequivocal evidnce that the same mutant allele of p53 is present throughout each tumour bearing a mutation, regardless of the clonal variation identified by analysis of DNA ploidy. We conclude that in colorectal tumorigenesis mutation of p53 occurs as a single event which precedes and may facilitate the aneuploid clonal divergence of carcinomas.

No abstract

Promyelocytic leukaemia nuclear domains (PML-NDs) comprise a shell of PML protein and many labile cargo proteins. The nature of their cargo, their juxtaposition to foci of damaged DNA following ionizing radiation (IR), and the altered DNA damage responses in PML null cells all implicate PML-NDs in the DNA damage response. In this work, the propensity of PML-NDs to increase in number and decrease in size following IR has been studied. Serial quantitative studies of endogenous PML-NDs prove that the PML-ND response to IR is not the result of the asymmetry in cell cycle distribution that can follow IR, but reflects more directly the process of DNA damage. The response is swift, sensitive (evident after 1 Gy), and potentially reversible in untransformed fibroblasts. In these cells and in HCT116 colon cancer cells, failure to restore PML-ND number within 24 h correlates with later loss of growth potential--in fibroblasts, through prolonged cell cycle arrest and in HCT116 cells, through apoptosis. Failure to express an intact ATM/CHK2 DNA damage signalling pathway in either cell type leads to a delay in the PML-ND response to IR. Conversely, cell cycle progression following IR in cells that detect damaged DNA accelerates PML-ND reorganization. Collectively, these data show that the increase in PML-ND number seen after irradiation is, in part, triggered by the receipt of the DNA damage stimulus. The senescent cell state is also associated with chronic DNA damage and Hayflick-limited fibroblasts were found to express nuclei with elevated numbers of PML-NDs before IR that remained unresponsive to IR. Though the underlying reasons for damage-induced PML alteration remain obscure, it is noteworthy that significant numbers of PML-NDs juxtapose with ionizing radiation-induced foci after IR. The co-regulation of these structures may necessitate the stereotyped increases in PML-ND number following damage.

S_qy Overall, the causative APC mutation has been identified in only 30% of the patients with familial adenomatous polyposis (FAP) (Murday et al., 1989). The clinical manifestations of FAP are a result of mutations in APC, a gene located on chromosome 5q21-q22 and for which the entire coding sequence is now known (Groden et al., 1991;Kinzler et al., 1991; Nishishio et al., 1991). Nonethekss, of over 800 FAP patients reported in the world literature, the overall frequency of identifying the causative mutation is only 30% (Nagase et al., 1992a;Nagase & Nakamura, 1993;Mandl et al., 1994) and ranges from 21% (Mandl et al., 1994) to 67% (Nagase et al., 1992a). This may reflect any one or more of the following: the selection criteria for the diagnosis of FAP, the sensitivity and robustness of the mutation detection technique employed, the assiduousness of the search for mutations, the presence of causative gene alterations influencing APC expression outwith the coding sequence and the possibility of genetic heterogeneity in FAP.Presymptomatic colonic screening of at-risk relatives and appropriate prophylactic surgery considerably reduces morbidity and mortality from FAP (Jarvinen, 1992