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A H Wyllie

Publications and source records attributed to A H Wyllie.

At least 91 records · Page 5Linked to original sources
PubMed1994

Apoptosis is inversely related to necrosis and determines net growth in tumors bearing constitutively expressed myc, ras, and HPV oncogenes.

Immortalized rat fibroblasts were transfected with expression plasmids containing a mutated human Ha-ras (T24) oncogene, human c-myc, HPV 16 or 18 genomes, or combinations of these. Cell proliferation rates in vitro of the resulting 13 transformed lines were closely similar but apoptotic rates in vitro varied over a 60-fold range and correlated inversely with rates of population expansion in culture. To determine whether such differences in susceptibility to apoptosis affected the pattern of tumor growth in vivo, the transfected lines were injected subcutaneously into immunesuppressed mice producing fibrosarcomas in which prevalence of apoptosis and mitosis, extent of necrosis, and net growth rate were measured. Cell lines with high apoptotic rates in vitro tended to generate slowly growing tumors with high ratios of apoptosis to mitosis and little necrosis. The three most extreme examples of this phenotype all resulted from single transfections with c-myc. Lines with low apoptotic rates in vitro generated rapidly expanding tumors with high mitotic rates, extensive necrosis, and little apoptosis relative to mitosis, even in the compromised zone at the edge of necrotic regions. The four fastest-growing tumors all contained a T24-ras oncogene. The results suggest that oncogene expression determines intrinsic apoptotic rates and in this way may significantly influence the net growth rate and extent of necrosis in tumors.

Animals↗
PubMed1994

p53 dependence of early apoptotic and proliferative responses within the mouse intestinal epithelium following gamma-irradiation.

p53 is now well characterized as a tumour suppressor gene, with loss of normal p53 function being recorded as the commonest genetic event associated with human malignancy. In particular, its involvement with tumorigenesis within the intestine is well established. Normal p53 function has been shown to be crucial for the induction of apoptosis in tumour cell lines, murine thymocytes and murine haematopoietic cells following DNA damage. To elucidate further the role of p53 in the cellular response to DNA damage we have investigated the response to gamma-irradiation of crypt cells in vivo from the small and large intestine of mice bearing a constitutive p53 deletion. Four hours after gamma-irradiation, a time point at which wild type crypt cells show abundant apoptosis, crypt cells from p53-deficient mice differed in that they were completely resistant to the induction of apoptosis. The p53 dose dependence of this phenomenon was clearly shown by the intermediate level of apoptosis observed in p53 heterozygotes. Analysis of the mitotic index and the bromodeoxyuridine labelling index showed that two other responses of wild type crypts to gamma-irradiation, namely the G2 block and the reduction in bromodeoxyuridine incorporation, were both largely intact in p53 deficient animals. These observations demonstrate that p53 function is essential for a major component of the normal response to gamma-irradiation induced DNA damage in intestinal mucosal cells, and suggest that p53 deficiency permits a population of cells bearing DNA damage to escape the normal process of deletion.

Animals↗
PubMed1994

Tumour incidence, spectrum and ploidy in mice with a large deletion in the p53 gene.

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.

Alleles↗
PubMed1993

Thymocyte apoptosis induced by p53-dependent and independent pathways.

Death by apoptosis is characteristic of cells undergoing deletion during embryonic development, T- and B-cell maturation and endocrine-induced atrophy. Apoptosis can be initiated by various agents and may be a result of expression of the oncosuppressor gene p53 (refs 6-8). Here we study the dependence of apoptosis on p53 expression in cells from the thymus cortex. Short-term thymocyte cultures were prepared from mice constitutively heterozygous or homozygous for a deletion in the p53 gene introduced into the germ line after gene targeting. Wild-type thymocytes readily undergo apoptosis after treatment with ionizing radiation, the glucocorticoid methylprednisolone, or etoposide (an inhibitor of topoisomerase II), or after Ca(2+)-dependent activation by phorbol ester and a calcium ionophore. In contrast, homozygous null p53 thymocytes are resistant to induction of apoptosis by radiation or etoposide, but retain normal sensitivity to glucocorticoid and calcium. The time-dependent apoptosis that occurs in untreated cultures is unaffected by p53 status. Cells heterozygous for p53 deletion are partially resistant to radiation and etoposide. Our results show that p53 exerts a significant and dose-dependent effect in the initiation of apoptosis, but only when it is induced by agents that cause DNA-strand breakage.

Animals↗
PubMed1993

Human papillomavirus type 18 associates with more advanced cervical neoplasia than human papillomavirus type 16.

A type-specific, sensitive, polymerase chain reaction-based assay for human papillomavirus (HPV) types 6b, 11, 16, 18, and 33 was applied to 47 cervical carcinomas, 60 cases of cervical intraepithelial neoplasia (CIN), and 24 samples of histologically normal cervix. As expected, the combined incidence of the common high-risk genital HPVs (types 16 and 18) was high in carcinomas (79%) and CIN 2/3 (60%), low in CIN 1 (25%), and nonexistent in the normal controls. Analysis of the data by viral type and pathology revealed statistically significant differences that consistently pointed to an association of HPV 18 with more advanced disease than HPV 16. This was exemplified by calculation of the relative HPV frequency in squamous cancers and CIN 2/3 lesions, which gave cancer to CIN prevalence ratios of 1.2 for HPV 16 and 2.3 for HPV 18, a twofold difference suggesting the possibility that there is a greater risk of progression or a more rapid transition to malignancy associated with HPV 18. Furthermore, HPV 16 was associated with 2.5-fold more cancers showing squamous differentiation (58%) than HPV 18 (23%), but both types showed an identical prevalence of 41% in the clinically more sinister adenocarcinomas, indicating that there may be an association between HPV type and cancer cell differentiation.

Adenocarcinoma↗
PubMed1993

Allele loss from 5q21 (APC/MCC) and 18q21 (DCC) and DCC mRNA expression in breast cancer.

Thirty-four primary, untreated sporadic breast cancers were examined for loss of heterozygosity (LOH) at tumour suppressor loci involved in colorectal cancer: APC/MCC at 5q21 and DCC at 18q21. LOH was identified in 28% informative patients at 5q21 and 31% at 18q21. LOH at 5q21 and 18q21 was compared with allele loss at 17p13 and concurrent LOH at two or more of the loci was noted in 24% of tumours. Expression of a 12 kb DCC mRNA was demonstrated in 14/34 (42%) of the cancers and in all five tumours with LOH at the DCC locus there was an additional 11 kb DCC mRNA. Abnormalities of three loci involved in colorectal cancer (5q21, 17p13 and 18q21) therefore also occur in sporadic breast cancer. The accumulation of such genetic abnormalities may confer a growth advantage important in the development of breast cancer.

Alleles↗
PubMed1993

Apoptosis (the 1992 Frank Rose Memorial Lecture).

Apoptosis is a mode of cell death with characteristic structural features. These appear to result from a set of discrete cellular events that are regulated by gene expression. Oncogenesis and oncosuppressor genes are involved in this regulation. The role of c-myc is of particular interest, as it can act as a bivalent regulator, determining either cell proliferation or apoptosis, depending on whether free movement around the cell cycle is supported (by growth factors) or is limited by growth factor deprivation or treatment with other cycle-blocking agents. In vivo, c-myc expression may be associated with a 'high-turnover' state in which cell proliferation and apoptosis co-exist. Certain other oncogenes (e.g. ras, bcl-2) rescue cells from susceptibility to apoptosis and so convert this high-turnover state into rapid population expansion. One role of the oncosuppressor gene p53 may be to initiate apoptosis by causing G 1/S arrest in cells expressing c-myc. Some aspects of resistance and sensitivity to chemotherapeutic agents can be explained on the basis of movement between the population-expansion and the high-turnover states, perhaps through modulation of the expression of these and other genes.

Apoptosis↗
PubMed1993

Susceptibility to apoptosis is differentially regulated by c-myc and mutated Ha-ras oncogenes and is associated with endonuclease availability.

Oncogenes and oncosuppressors can deregulate cell replication in tumours, and recently have been shown to influence the probability of apoptosis. The effects of human c-myc and mutated (T24) Ha-ras oncogenes on susceptibility to apoptosis were investigated by introducing them into immortalised rat fibroblasts. The resulting family of transfectants showed closely similar measures of proliferation, but widely divergent rates of apoptosis, differing by up to fifteen-fold, that correlated inversely with population expansion rates in vitro. T24-ras transfectants with moderate or high p21ras expression showed reduced apoptosis, and this was reversed by pharmacological inhibition of membrane localisation of p21ras by mevinolin. In contrast, c-myc stimulated apoptosis, and this was further enhanced by serum deprivation. Inducibility of effector proteins represents one possible mechanism of genetic control of the susceptibility to apoptosis, and its investigation showed that c-myc was associated with expression by viable cells of latent calcium/magnesium sensitive endonuclease activity characteristic of apoptosis. In contrast, endonuclease activity was not detected in viable cells of a T24-ras transfectant expressing high levels of p21ras. Thus, there appeared to be differential regulation of susceptibility to apoptosis, positively by c-myc and negatively by activated ras, and this was associated with availability of endonuclease activity. Genetic modulation of apoptosis in human neoplasms is likely to influence net growth rate, retention of cells acquiring new mutations and response to certain chemotherapeutic agents.

Analysis of Variance↗
PubMed1993

Relationship between the GSTM1 genetic polymorphism and susceptibility to bladder, breast and colon cancer.

Mammalian cytosolic glutathione S-transferases (GSTs; EC 2.5.1.18) form a supergene family consisting of four distinct families, named alpha, mu, pi and theta. In humans one member of the mu class gene family (GSTM1) has been shown to be polymorphic and is only expressed in 55-60% of individuals. Previous studies have shown a possible link with the null phenotype and susceptibility to cancer, in particular to lung cancer. In this study we genotyped individuals with breast, bladder and colorectal cancer. A total of 490 individuals with cancer were studied, and consisted of 97 bladder, 197 breast and 196 colorectal cancers. No significant differences were observed in the frequency of nulled individuals in bladder or breast cancer patients when compared with a control population of 225 individuals. However, a significant excess of nulled individuals were seen in colorectal cancer: 56.1% compared with the control group value of 41.8%. This was shown to be highly significant depending on the site of the tumours and > 70% of individuals with a tumour in the proximal colon were GSTM1 nulled. This is an approximately 2-fold increase in colon cancer risk in these individuals.

Base Sequence↗
PubMed1993

Deletion mapping in colorectal cancer of a putative tumour suppressor gene in 8p22-p21.3.

Although previous studies of acquired loss of heterozygosity (LOH) in colorectal tumours have suggested that a tumour suppressor gene may lie within the short arm of chromosome 8, its precise localisation remains to be determined. To obtain a more accurate positional map 120 colorectal cancers were examined with eight chromosome 8 polymorphic markers comprising both restriction fragment length polymorphisms and microsatellite polymorphisms based on (CA)n repeats. 91 cases were informative and LOH was detected in 47 (51%). The markers most commonly sited within the lost region mapped to the lipoprotein lipase gene (LPL) at chromosome 8p22. From study of tumours showing break-points within 8p, a common region of deletion was established extending centromerically from LPL to the ankyrin 1 gene (ANK1) which is mapped to 8p21.1-11.2. This overlaps with common deleted regions observed in other studies of colorectal tumours (8p23.1-p21.3) and bladder tumours (8p21-q11.2). Taken together, the results in colorectal cancer delineate a region in 8p22-p21.3 where the putative tumour suppressor gene must lie. The chromosome 8p deletions appear to be independent of those involving 5q and 17p in the same tumours. No relationship was found between the presence of 8p deletion and site or stage of the tumour, or the sex or age of the patient at diagnosis.

Animals↗
PubMed1993

Stabilised p53 facilitates aneuploid clonal divergence in colorectal cancer.

Mutations in the p53 tumour suppressor gene are amongst the most frequent genetic abnormalities acquired in tumours. Recent studies in vitro suggest that mutant p53 destabilises the genome and facilitates development of aneuploidy. Here, in a study of 83 colorectal carcinomas, we demonstrate that alterations in p53 (detected by immunocytochemical stabilisation) precede and apparently facilitate divergence of aneuploid sub-clones. Aneuploidy in these tumours (but not those with normal p53) is predominantly in the subtetraploid range, suggesting that endoreduplication is important in its origin. This association with a specific phase of carcinoma progression is not shared by other commonly acquired genetic abnormalities in these tumours. These observations highlight the critical role of p53 in the regulation of abnormal chromosome replication and afford an explanation for the association between p53 abnormalities, aneuploidy and biological aggression in cancer.

Adenoma↗
PubMed1992

Induction of apoptosis in fibroblasts by c-myc protein.

Although Rat-1 fibroblasts expressing c-myc constitutively are unable to arrest growth in low serum, their numbers do not increase in culture because of substantial cell death. We show this cell death to be dependent upon expression of c-myc protein and to occur by apoptosis. Regions of the c-myc protein required for induction of apoptosis overlap with regions necessary for cotransformation, autoregulation, and inhibition of differentiation, suggesting that the apoptotic function of c-myc protein is related to its other functions. Moreover, cells with higher levels of c-myc protein are more prone to cell death upon serum deprivation. Finally, we demonstrate that deregulated c-myc expression induces apoptosis in cells growth arrested by a variety of means and at various points in the cell cycle.

Animals↗
PubMed1992

Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry.

Necrosis and apoptosis are two distinct modes of cell death which differ in morphology, mechanism and incidence. Membrane disruptants, respiratory poisons and hypoxia cause ATP depletion, metabolic collapse, cell swelling and rupture leading to inflammation. These are typical features of necrosis. Apoptosis plays a crucial role in embryogenesis and development and is also prevalent in tumours. It is characterised by cell shrinkage, chromatin condensation and systematic DNA cleavage. Apoptotic cells are rapidly engulfed by phagocytes, thus preventing inflammatory reaction to degradative cell contents. In vivo, apoptosis is almost impossible to quantify due to problems of heterogeneity and the short half-life of an apoptotic cell. In vitro, mechanistic studies are further complicated by a late phase of apoptosis where the cell membrane becomes permeable to vital dyes and which occurs in the absence of phagocytes. Here we describe a novel and rapid multiparameter flow cytometric assay which discriminates and quantifies viable, apoptotic and necrotic cells via measurement of forward and side light scatter (proportional to cell diameter and internal granularity, respectively) and the DNA-binding fluorophores Hoechst 33342 and propidium. It is anticipated that mechanistic studies of apoptosis in a variety of cell types will greatly benefit from this mode of analysis.

Animals↗
PubMed1992

Ras p21 in breast tissue: associations with pathology and cellular localisation.

Immunocytochemistry with monoclonal antibody Y13-259 demonstrated p21 ras in paraffin sections of breast tissue from 171 women: 85 with invasive breast carcinoma, 14 with non-invasive carcinoma and 72 with benign changes only. Many different tissue elements contributed to ras expression. Semiquantitative assessment showed that intensity of immunostaining in the normal epithelium of large ducts, small extralobular ducts and terminal duct lobular units (TDLU) was usually exceeded by that of myoepithelial cells. Vascular smooth muscle and apocrine epithelium also stained strongly, but the flat epithelial cells lining cysts did not express detectable p21 ras. There was a progressive increase from normal epithelium through epithelial hyperplasia of usual type and atypical hyperplasia to carcinoma in situ, without further increase in invasive carcinoma. Expression in carcinomas was inversely related to oestrogen receptor content but independent of the prognosis-associated variables of size, histological type, vascular invasion or lymph node metastasis.

Antibodies, Monoclonal↗
PubMed1992

The apoptosis endonuclease and its regulation.

Activation of an endogenous endonuclease has been observed in conjunction with the structural changes of apoptosis in a wide variety of cell types and circumstances. The endonuclease is present constitutively in some cells (e.g. rodent cortical thymocytes) in which apoptosis is readily triggered by many unrelated stimuli, but is inducible in others. Purification of this enzyme is an objective of some importance in apoptosis research, as it might act as a marker of susceptibility to apoptosis and lead to better understanding of the regulation of the process as a whole. Early data suggest that the thymocyte endonuclease is an anionic protein of molecular weight greater than 110 kDa, with a pH optimum of 7.5 and a double-strand cleavage preference. Its activity, and the induction of apoptosis as a whole, is regulated by several familiar cellular proto-oncogenes and oncosuppressor genes, including c-myc, Ha-ras, bcl-2 and p53.

Animals↗