Mechanisms of clastogen-induced chromosomal aberrations: a critical review and description of a model based on failures of tethering of DNA strand ends to strand-breaking enzymes.

Most theories of the mechanisms of chromosomal aberrations involve the concepts of clastogens directly acting on DNA to produce strand breaks, and subsequently, the survival of these directly caused DNA strand breaks - or misrepairs of them - through to metaphase when they appear as chromosomal 'breaks' or translocations. Nevertheless, various observations are inconsistent with these theories such as the fact that many chemical clastogens (e.g. caffeine, acridines) do not covalently react with DNA, while almost all of the chemical clastogens (e.g. alkylating agents) which do react covalently with DNA, do not directly cause DNA strand breaks. This paper reviews the 'direct-clastogen damage to DNA' theories, and the phenomenology of chromosomal aberrations which are inconsistent with them. Then the theory is considered that the breaks in chromosomes seen at metaphase and anaphase are not the survivors of DNA breaks directly induced by clastogens, but rather derive from breaks created by the enzymes which repair damaged DNA. After that, newer knowledge is reviewed that (i) strand breaks are created during normal DNA unravelling (by topoisomerases), during DNA synthesis, and during DNA repairs, and these breaks can be single- or double-stranded, (ii) breaks variously associated with unravelling, synthesis and repair can occur 'anywhere, anytime' (pre-synthesis, synthesis or post-synthesis) in the cell cycle, and (iii) the enzyme assemblies for DNA unravelling, synthesis and repair which make and religate the breaks must be non-covalently tethered to the ends of the DNA strands while the breaks created by the enzymes are in existence. It is then suggested that all the morphological types and other phenomena of chromosomal aberrations can be explained by aspects, mechanisms and effects of failures of this tethering function. Circumstances involving the basic mechanism (failure of DNA-end-tethering function while enzyme-created breaks are in existence) are described which might result in 'gaps', translocations ('exchanges'), complex lesions such as 'triradials', as well as in 'minutes', amplifications and inversions. Predictions are made concerning likely results in various suggested studies including those involving sensitive assays for DNA-end-to-enzyme tethering functions in vitro.

Variation, "evolution", immortality and genetic instabilities in tumour cells.

The pathological characteristics of tumour cells often include variation of their histopathological features (i.e. "degrees of de-differentiation") between cases of the same tumour type and between different foci within individual tumours. Usually, only a few cell lines from tumours are immortal. Currently, somatic mutation, replicative infidelity of DNA and aneuploidy are suggested as alternative mechanisms of genomic disturbance underlying tumours. Nevertheless, apart from Hansemann's ideas of "anaplasia" and "de-differentiation" (proposed in the 1890s), and supposed "evolutionary themes" in cancer cell biology, little has been published concerning how histopathologic variation and immortality in tumour cells might arise. This paper reviews applications of the concepts of "variation" to tumours, including concepts of "evolution" and "cellular Darwinism". It is proposed that combinations of somatic mutation, DNA replicative infidelity and aneuploidy may explain the variabilities in tumours, and provide immortality in occasional tumour cells. A possible model involves (i) an initial somatic mutation causing reduced replicative fidelity of DNA, which could be variable in intensity, and thus give rise to variations between cases; (ii) a phase of replicative infidelity of DNA causing daughter cells lines to develop various abnormalities to different degrees, and hence provide for variation between areas of the same tumour. As a last event (iii) occasional asymmetric chromosomal distributions (aneuploidy) might "refresh" the ability of a daughter cell to replicate DNA faithfully causing them to become immortal. Thus extensively mutant and variable, hyperploid, and occasionally immortal cells might arise.

Mutation, replicative infidelity of DNA and aneuploidy sequentially in the formation of malignant pleomorphic tumors.

Mutations are thought to be involved in tumor formation because (i) tumor cells transmit their abnormalities to their descendants; and (ii) many carcinogens are mutagens. Aneuploidy is thought to be involved in tumor formation because (i) it is a common phenomenon, especially among malignant neoplasms; (ii) certain particular types of tumors are associated with specific karyotypic changes; and (iii) many immortal tumor cell lines are hyperploid. In recent years, acquired somatic cell replicative infidelity of DNA ("mutator phenotype") has been suggested as a mechanism of tumor formation, because more somatic genomic events occur in malignant tumor cells than could be caused by repeated exogenous mutagenic insults. Previously, theories of the genomic pathogenesis of tumors have involved these mechanisms individually. Here it is suggested that all three mechanisms may play roles in the formation of certain tumor types. For example, a sequence could occur such that first, a mutation affects genomic elements for control of growth, and for replicative fidelity of DNA, leading to "mutator phenotype". Second, when replicative infidelity of DNA results in mutation of genomic elements for mitotic-and-chromosomal stability, aneuploidy develops. Third, an asymmetric mitosis (in the course of the aneuploid stage) could produce occasional cells in which the "bad copy" is lost (or an extra "good copy" is gained) of the original genomic element which had supported replicative fidelity of DNA. These resulting cells would regain fidelity of replication of DNA, and hence could give rise to populations which are relatively genomically stable, hyperploid and immortal.

Chaotic genomes make chaotic cells: the mutator phenotype theory of carcinogenesis applied to clinicopathological relationships of solid tumors.

Abnormalities of cell morphology and chromosomes have been identified as features of the pathology of tumors for more than 100 years. However, no theory of carcinogenesis until recently has provided a basis for relating them either to each other or to the clinical behavior of individual tumors in many patients. The mutator phenotype theory is based on large numbers of mutations occurring chaotically (randomly, unpredictably, and variably from cell to cell) in individual tumors. Chaotic mutation clearly parallels both the often-observed chaotic morphology of tumors and the frequently unpredictable relationship of these morphologies to clinical behavior. Possible implications of this concept for staging, grading, multifocality, and therapy of cancers are discussed.

Initiation of genetic instability and tumour formation: a review and hypothesis of a nongenotoxic mechanism.

Genetic instability in tumours results in cell-to-cell variability of genome which parallels the cell-to-cell variability of microscopic morphology and of behaviour (tumour cell heterogeneity) of these lesions. Genetic instability is therefore strongly supported as the fundamental process by which normal tissue cells become neoplastic. The commonest current suggestion for the mechanism of initiation of carcinogenesis is a 'direct hit' mutation of a 'cancer critical' gene in a somatic cell by carcinogenic agents. However, this mechanism does not account for the activity of carcinogens which are not mutagens, and does not explain why many mutagens are not carcinogens. This paper proposes a nonmutational (nongenotoxic) mechanism of initiation of genetic instability in previously normal cells as follows: 1) During S phase of local tissue stem cells, carcinogen binds to and disables the proofreading enzyme for a new DNA strand. 2) While it is disabled, the proofreading enzyme fails to correct illicit changes in the nucleotide sequence(s) for one or more genes for proofreading fidelity or repair of DNA in the new strand of DNA, which passes to one daughter cell. 3) When this daughter cell is a continuing stem cell, the resulting cell line remains immortal, and retains its prior differentiation commitment to produce daughter cells of a particular type. However, the acquired genetic instability in this cell line causes secondary mutations which lead to uncontrolled growth, and the heterogeneous morphologic and behavioural features of a tumour resembling the parent cell type.

The mutator phenotype theory of carcinogenesis and the complex histopathology of tumours: support for the theory from the independent occurrence of nuclear abnormality, loss of specialisation and invasiveness among occasional neoplastic lesions.

The mutator phenotype theory of carcinogenesis suggests that genetic instability is an early and essential part of tumour development. This instability provides for substantially random cell-to-cell genomic variation (genomic heterogeneity) to arise among cells of individual tumours. Genetically unstable cells then produce 'successful' clones of cells with the necessary mutations for malignant behaviour. In a previous paper (Bignold L. P., Cell. Mol. Life Sci. 2002; 59: 950-958), it was pointed out that a population of cells which is heterogeneous for behaviour-related genes may well also be heterogeneous for morphology-related genes. This would result in cellular pleomorphism among cells of individual tumours, and so explain this almost universal characteristic of solid malignancies. paragraph sign If the concept of random genomic variability applies fully to the histopathology of tumours, then most tumours should show a mixture of neoplastic features, especially nuclear atypia, loss of specialised function (such as loss of production of mucus by glandular cells) and invasiveness. However, occasional lesions might be expected to occur which show these characteristics independently. That is, lesions should exist which exhibit one or two of the three characteristics of neoplasms without the other(s). paragraph sign This paper identifies, among human tumours, lesions which show independence of these characteristics. Two of the examples discussed are a Bowenoid solar keratosis that shows severe nuclear atypia, but no apparent loss of specialisation and no invasiveness. On the other hand, anaplastic small cell carcinoma of the lung often exhibits marked loss of differentiation, very aggressive invasion and metastasis, but little nuclear pleomorphism. paragraph sign These examples are considered to provide further support for the importance of the mutator phenotype to the pathogenesis of neoplasia.

Pathogenetic mechanisms of nuclear pleomorphism of tumour cells based on the mutator phenotype theory of carcinogenesis.

The nuclei of the cells of most solid tumours in histopathologic preparations vary in size, shape and chromatin pattern, both from normal nuclei and from each other. These features have not been explained in terms of conventional concepts of nuclear structure and theories of carcinogenesis. In recent years, the unfolded chromosomes have been shown to occupy "domains" in the nucleus during interphase, providing a relatively uniform density of fine chromatin fibres throughout the nucleus in the living state. This is in contrast to the appearances of interphase chromatin existing as coarse clumps and fibres (heterochromatin and euchromatin respectively) as are seen in histologic preparations. Additionally, the binding of chromatin to nuclear membrane, the possible existence of a nuclear matrix, the functions of nuclear pores, and the attachments of cytoskeletal structures to the outer nuclear membrane are now recognised. Studies of genetic instability of cancer cells (many random mutations are present in the genome, which vary from nucleus-to-nucleus in individual tumours) have shown that this phenomenon occurs early in tumour formation, can be present in morphologically-normal cells adjacent to tumours, and can result in thousands of genomic events per tumour cell. These observations form the basis for the mutator phenotype/clonal selection theory of carcinogenesis, which proposes that genetic instability is an essential early part of carcinogenesis. Genetic instability has been used to explain significant cell-to-cell variability of behaviour (tumour cell heterogeneity) among cells of individual tumours. This paper proposes that a high incidence of nucleus-to-nucleus-variable mutation of the genes for factors controlling nuclear morphology in tumours can explain nucleus-to-nucleus variations of histopathologic appearance of these nuclei when some additional effects of histological processing are taken into account.

The mutator phenotype theory can explain the complex morphology and behaviour of cancers.

Almost all solid malignancies exhibit complex cytological and architectural abnormalities, which vary from cell to cell and area to area within the same tumour, and between tumours of the same type. The degrees of these abnormalities do not correlate perfectly with the biological behaviour (especially growth rate and metastatic potential) among the various tumour types. These features of tumours have long been considered to invalidate simple mutational or 'abnormal gene expression' (epigenetic) theories of carcinogenesis. The 'mutator phenotype/clonal selection' hypothesis is based on the now well-established phenomenon of genetic instability of cancer cells, and proposes that this instability is an essential requirement for the development of tumours, and not an irrelevant side-effect of some other process. This paper argues that this hypothesis can provide a satisfactory explanation for the diverse histological and biological features of solid malignancies. Further, because virtually all solid tumours are histologically abnormal, genetic instability is likely to be essential for the malignant process. The concepts of mutator phenotype and clonal selection are therefore supported.

Hypothesis for the influence of fixatives on the chromatin patterns of interphase nuclei, based on shrinkage and retraction of nuclear and perinuclear structures.

Nuclear chromatin patterns are used to distinguish normal and abnormal cells in histopathology and cytopathology. However, many chromatin pattern features are affected by aspects of tissue processing, especially fixation. Major effects of aldehyde and/or ethanol fixation on nuclei in the living state include shrinkage, chromatin aggregation and production of a 'chromatinic rim'. The mechanisms of these effects are poorly understood. In the past, possible mechanisms of fixation-induced morphological change have been considered only in terms of the theoretical model of the nucleus, which involves only a random tangle of partly unfolded chromosomes contained within the nuclear membrane. Such a model provides no basis for chromatin to be associated with the nuclear envelope, and hence no obvious clue to a mechanism for the formation of the 'chromatinic rim' in fixed nuclei. In recent years, two new models of nuclear structure have been described. The nuclear membrane-bound, chromosomal-domain model is based on the discoveries of chromatin-nuclear membrane attachments and of the localisation of the chromatin of each chromosome within discrete, exclusive parts of the nucleus (the 'domain' of each partly unfolded chromosome). The nuclear matrix/scaffold model is based on the discovery of relatively insoluble proteins in nuclei, which it suggests forms a 'matrix' and modulates gene expression by affecting transcription of DNA. Here, a hypothesis for fixation-associated chromatin pattern formation based mainly on the first model but partially relying on the second, is presented. The hypothesis offers explanations of the variations of appearance of nuclei according to fixation (especially air-drying versus wet-fixation with formaldehyde, glutaraldehyde or ethanol); the appearances of the nuclei of more metabolically active versus less metabolically active cells of the same type; the appearances of nuclei after fixation with osmium tetroxide; and of the marked central clearing ('egg-shell' or 'orphan Annie' appearance) of tumour nuclei of papillary carcinoma of the thyroid gland. A similar process may underlie the phenomenon of 'chromatin margination' seen in apoptosis. Various tests of the hypothesis, such as time-lapse confocal microscopy of living nuclei during fixation, are suggested. The significance of the theory is that it suggests that chromatin patterns could be investigated in terms of qualitative and quantitative aspects of nuclear components, and hence be related to the results of studies of the structure and function of nuclei in health and disease.

Inhibition of C5a-induced neutrophil chemotaxis and macrophage cytokine production in vitro by a new C5a receptor antagonist.

A cyclic peptide, Phe-[Orn-Pro-D-Cyclohexylalanine-Trp-Arg] (F-[OPdChaWR]), was recently shown in vitro to antagonise the binding of C5a to its receptor (CD88) on human polymorphonuclear leukocytes (PMNs) and in vivo to inhibit the neutropenia associated with septic shock induced by lipopolysaccharide (LPS) in rats. The aim of this study was to investigate whether F-[OPdChaWR] inhibits C5a-mediated chemotaxis of human PMNs using a modified Boyden chamber and C5a-stimulated release of cytokines from human monocytes in vitro. Approximately 50% of the chemotactic activity induced by 10 nM C5a was inhibited by 76 nM F-[OPdChaWR]. This correlated with inhibition of C5a-induced polarisation of PMNs by F-[OPdChaWR]. C5a alone failed to induce release of the inflammatory cytokines interleukin(IL)-1beta, tumour necrosis factor (TNF)-alpha, and IL-6 from human monocytes at concentrations up to 100 nM. However, in the presence of low concentrations of LPS (50 ng/mL), both IL-1beta and TNF-alpha were stimulated by 1 nM C5a. This co-stimulation was inhibited by F-[OPdChaWR] with IC(50)s of 0.8 and 6.9 nM for release of TNF-alpha and IL-1beta, respectively. No agonist activity was detected for F-[OPdChaWR] in either the chemotaxis or cytokine release assays at concentrations up to 50 microM. These results show that F-[OPdChaWR] inhibits several important inflammatory activities of C5a and suggest that C5a receptor antagonists may be effective in the treatment of inflammatory diseases mediated by C5a.

Interleukin-5 transgenic mice show enhanced resistance to primary infections with Nippostrongylus brasiliensis but not primary infections with Toxocara canis.

In this study, interleukin-5 (IL-5) transgenic mice with lifelong eosinophilia were assessed for resistance to primary infections with two tissue-invading nematodes, Nippostrongylus brasiliensis and Toxocara canis. Relative to nontransgenic littermates, three lines of IL-5 transgenic mice with varying degrees of eosinophilia all displayed enhanced resistance to N. brasiliensis. Although the timing of final worm expulsion was similar in transgenic and nontransgenic hosts, intestinal worms in transgenic mice were fewer in number throughout infection, failed to increase in size over the course of the infection, and were much less fecund. In contrast, T. canis larvae were recovered in similar numbers from tissues of transgenic mice with "low" or "high" eosinophilia and from nontransgenic mice. These results and other data suggest that eosinophils can contribute to host resistance to some parasite species. Parasite transit time through the host may correlate with relative sensitivity to eosinophils.

Eosinophilic interleukin 5 (IL-5) transgenic mice: eosinophil activity and impaired clearance of Schistosoma mansoni.

Eosinophilia is a feature common to many invasive helminth infections and eosinophils are often considered to be effector cells in immunity to helminths. This study examined the possible influence of constitutive eosinophilia on the clearance of Schistosoma mansoni infections in mice. Eosinophils from interleukin-5 transgenic mice exhibit normal ultrastructure and function with regard to phagocytosis and killing of bacteria and responses to chemotactic stimuli. IL-5 transgenics and non-transgenic littermates were immunized once or four (hyperimmunization) times with irradiated cercariae of S. mansoni. Animals were challenged percutaneously with unirradiated cercariae one month after their last exposure to irradiated parasites. One month after challenge transgenic animals, whether unimmunized, vaccinated or hypervaccinated, carried significantly more liver-stage parasites than non-transgenic animals. These results suggest that although eosinophils from IL-5 transgenic mice are functional for a number of key parameters, large numbers of eosinophils and/or high levels of IL-5 may in some way impair clearance of S. mansoni. A re-evaluation of the roles of eosinophils and IL-5 in infections with this and other parasites may therefore be warranted.

Phlegmonous enterocolitis: a rare form of inflammatory bowel disease.

Phlegmonous enterocolitis is a rarely encountered and almost universally fatal bacterial infection seen most often in the alcoholic population. Despite being first described in 1842, fewer than 60 cases have been reported. The role of surgical management in this condition is unknown. We present a case of phlegmonous enterocolitis with a successful outcome after total colectomy.

The eosinophil leukocyte: controversies of recruitment and function.

Eosinophil leukocytes have been studied for over 100 years, with various theories being advanced of the mechanism of their recruitment and function, especially in relation to the lesions of allergy, asthma and parasitism. Early notions of recruitment and function depended on observations of the cells in inflammatory lesions, while later theories have used additional information from in vitro studies. Many issues are still unresolved. This review aims to cover the older and more recent literature of the mechanisms of accumulation of eosinophil leukocytes and their functions, with a view to illuminating the controversies and difficulties of research in the area.

Chemotaxis of polymorphonuclear leukocytes towards human pre-ovulatory follicular fluid and serum using a 'sparse-pore' polycarbonate filtration membrane.

The chemotactic responses of polymorphonuclear leukocytes (PMN) towards pre-ovulatory follicular fluid and serum from patients participating in an in vitro fertilization program were tested in a modified Boyden chamber using a 'sparse-pore' polycarbonate filtration membrane method. The chemotactic activities of follicular fluids were generally low, but were significantly higher in conceptual cycles than in non-conceptual cycles. The chemotactic activities of sera were generally high, but were significantly lower in conceptual cycles. The chemotactic activity of few follicular fluids ever exceeded that of serum, regardless of the occurrence of conception. These findings demonstrate for the first time using an appropriate technique, that pre-ovulatory follicular fluids can be chemotactic for PMN, but the relationship between this activity, serum chemotactic activity and successful pregnancy is not clear.

Neutrophil polarisation in plasma differs to that induced by endogenous chemoattractants with regard to frequency of uropod formation and requirement for divalent cations.

Human neutrophils suspended in Hanks' balanced salt solution (37 degrees C, 20 mM Hepes, pH 7.2) produced extensions, elongated and developed a polarised morphology with both a pseudopod and uropod when exposed to C5a (10 nM), leukotriene B4 (10 nM), platelet activating factor (40 nM) or interleukin-8 (12.5 nM). Responses to each mediator were generally enhanced or unaffected by chelators of extracellular Ca2+ and Mg2+. Neutrophils suspended in heparinised plasma (90-10% v/v in Hanks' balanced salt solution) produced extensions, elongated and developed a pseudopod, but rarely developed a uropod unless additional Mg2+ ions (0.5-5 mM) were added. These findings demonstrate that the polarisation of neutrophils in plasma is significantly different to that induced by endogenous chemoattractants with regard to the frequency of uropod formation and requirement for extracellular divalent cations.

Comparison of techniques for the assessment of polymorphonuclear leukocyte polarisation in suspension.

Polarisation of polymorphonuclear leukocytes (PMN) in suspension was assessed using three techniques: 1) visual classification; 2) computerised morphometry; and 3) flow cytometry. While visual classification detected the formation, polarisation and type of cytoplasmic extensions produced by PMN, morphometry and flow cytometry detected only the formation of extensions. The area, perimeter and ellipticity were, in general, statistically different for each subtype of PMN-shape identified by visual classification. Furthermore, the magnitude and direction of changes detected by flow cytometry were affected by the use of erythrocyte lysis (during isolation of the cells) and the fixative used prior to analysis. The findings of this study demonstrate that visual classification is a more sensitive, reliable and appropriate assay of PMN polarisation than current morphometric and flow cytometric methods.