A H Wyllie

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.

Mechanisms governing the normal resolution processes of inflammation are poorly understood, yet their elucidation may lead to a greater understanding of the pathogenesis of chronic inflammation. The removal of neutrophils and their potentially histotoxic contents is one prerequisite of resolution. Engulfment by macrophages is an important disposal route, and changes in the senescent neutrophil that are associated with their recognition by macrophages are the subject of this investigation. Over 24 h in culture an increasing proportion of human neutrophils from peripheral blood or acutely inflamed joints underwent morphological changes characteristic of programmed cell death or apoptosis. Time-related chromatin cleavage in an internucleosomal pattern indicative of the endogenous endonuclease activation associated with programmed cell death was also demonstrated. A close correlation was observed between the increasing properties of apoptosis in neutrophils and the degree of macrophage recognition of the aging neutrophil population, and a direct relationship between these parameters was confirmed within aged neutrophil populations separated by counterflow centrifugation into fractions with varying proportions of apoptosis. Macrophages from acutely inflamed joints preferentially ingested apoptotic neutrophils and histological evidence was presented for occurrence of the process in situ. Programmed cell death is a phenomenon of widespread biological importance and has not previously been described in a cell of the myeloid line. Because it leads to recognition of intact senescent neutrophils that have not necessarily disgorged their granule contents, these processes may represent a mechanism for the removal of neutrophils during inflammation that also serves to limit the degree of tissue injury.

We have investigated the influence of genetic instability [replication error (RER) phenotype] on APC (adenomatous polyposis coli), a gene thought to initiate colorectal tumorigenesis. The prevalence of APC mutations was similar in RER and non-RER tumors, indicating that both tumor types share this step in neoplastic transformation. However, in a total of 101 sequenced mutations, we noted a substantial excess of APC frameshift mutations in the RER cases (70% in RER tumors versus 47% in non-RER tumors, P < 0.04). These frameshifts were characteristic of mutations arising in cells deficient in DNA mismatch repair, with a predilection for mononucleotide repeats in the RER tumors (P < 0.0002), particularly (A)n tracts (P < 0.00007). These findings suggest that the genetic instability that is reflected by the RER phenotype precedes, and is responsible for, APC mutation in RER large bowel tumors and have important implications for understanding the very earliest stages of neoplasia in patients with tumors deficient in mismatch repair.

In human tumourigenesis the tumour suppressor gene most commonly affected by mutation, inactivation or allele loss is p53. Loss of p53 function is associated both with failure to maintain a normal diploid status and inability to delete cells by apoptosis following DNA damage. To investigate further the role of p53 we have generated mice carrying a large deletion within the gene. All animals homozygous for this deletion develop spontaneous tumours, predominantly lymphomas, by the age of 6 months. 10% of heterozygotes develop a range of neoplasms, with a lower predisposition towards lymphoma, by 9 months. Both tumour incidence and spectrum in heterozygotes differ from those previously reported in another p53 mutant stock, suggesting either difference in exposure to carcinogens between the two stocks, or a role for modulating genes within different genetic backgrounds. Tumours showed frequent loss of diploid status, and the majority of those arising in heterozygotes showed loss of the wild type allele. These findings are consistent with the concept that p53 acts as a tumour suppressor by preventing the propagation of DNA damage to daughter cells.

The expression of the nuclear phosphoprotein p53 was studied immunohistochemically in a series of 150 benign and malignant colorectal tumors. Using monoclonal antibody PAb1801, tumors divided unequivocally into two groups on the basis of immunohistochemistry. Forty of the carcinomas (46.5%) showed positive staining but only 4 of the adenomas (8.7%) were positive (P less than 0.001). The few positive adenomas always showed moderate or severe dysplasia. Metaplastic polyps (n = 9) and small familial adenomatous polyposis-related adenomas (n = 9) were uniformly negative. Carcinomas with p53 expression did not differ from those without in terms of site, differentiation or the prognostic indicators of Dukes' stage, DNA ploidy, or tumor histology. The improved morphologic resolution available in periodate lysine paraformaldehyde dichromate (PLPD)-fixed, paraffin-embedded tissue permitted several conclusions to be made: p53 is confined to neoplastic nuclei; staining in positive tumors is heterogeneous and often more marked at the infiltrative margins; and staining intensity is dramatically reduced in mitotic cells. It is concluded that expression of immunohistochemically detectable p53 (probably representing mutated forms of the protein) occurs in some adenomas around the time of transition to carcinoma. Therefore there is an association with the appearance of infiltrative behavior but not with degree of tumor progression (including metastasis) at the time of resection.