Understanding cancer as a formless phenomenon.

Complex living organisms possess qualities that cannot be reduced to the simple addition of quantities. Among such qualities are a specific form and a specific organization. Thinking about morphological aspects is a prime example of the qualitative approach to biological matters. Such a morphogenetic perspective has been continuously developed, both theoretically and experimentally, along the past century, even though it is now rather marginal within a mainstream dominated by molecular biology. However, the morphogenetic outlook can be applied to the understanding of complex biological phenomena, such as cancer. This phenomenon is currently explained as a cellular problem caused by specific gene mutations and/or specific loss of gene regulation. Nevertheless, cancer is a problem that affects the whole organism. Contemporary research based on the genetic paradigm of cancer causation has led to paradoxes and anomalies that cannot be explained within such a reductionist paradigm. Here it is proposed that real, non-experimental, sporadic cancer may be understood as a conflict between an organized morphology (the organism) and a part of such a morphology that drifts towards an amorphous state (the tumour). Thus, rare, sporadic cancer in children can be the result of early disruption of the developmental constraints before the organism has achieved its morphological maturity. While common sporadic cancer in aged individuals may ensue as a result of the weakening or exhaustion of the developmental constraints that determine the morphological stability of the organism, once the organism is past its reproductive prime.

Cancer development and progression: a non-adaptive process driven by genetic drift.

The current mainstream in cancer research favours the idea that malignant tumour initiation is the result of a genetic mutation. Tumour development and progression is then explained as a sort of micro-evolutionary process, whereby an initial genetic alteration leads to abnormal proliferation of a single cell that leads to a population of clonally derived cells. It is widely claimed that tumour progression is driven by natural selection, based on the assumption that the initial tumour cells acquire some properties that endow such cells with a selective advantage over the normal cells from which the tumour cells are derived. The standard view assumes that the transformed bodily cell somehow acquires "responsiveness" to natural selection independently of the whole organism to which the cell belongs. Yet, it is never explained where such an acquired capacity to respond to natural selection by the individual bodily cell comes from. This situation poses many difficult questions that so far have been left unanswered. For example, there is no explanation why some cells belonging to an organised whole and as such having no independent capacity for survival, apparently become 'independent' entities, able to respond to selective pressures in an autonomous fashion and then to be evaluated by natural selection. Hereunder it is argued that such a qualitative change cannot be the consequence of specific genetic mutations. Moreover, it is shown that natural selection is unlikely to be acting within the organism during tumour development and progression and that tumour evolution is a random, non-adaptive process, driven by no fundamental biological principle. Thus, mutations in the so-called oncogenes and tumour suppressor genes observed in epithelial cancers (that constitute more than 90% of all cancers) are not the result of selection for better cellular growth or survival under restrictive conditions. Instead, here it is suggested that they are the consequence of genetic drift acting upon gene functions that become non-relevant, either for the individual or the species fitness, once the organism is past its reproductive prime and as such, they also become superfluous for cell survival in the short term. It is proposed that the origin of cancer is epigenetic and it is a consequence of the need for a continued turnover of the individuals that constitute a species.

The Hox-gene research programme and the shortcomings of molecular preformationism.

The study of the so-called HOM/Hox genes has provided many important insights on the control, at the molecular level, of developmental processes in a variety of model systems such as the fly and the mouse. Yet, in the specialised literature on the subject abound the claims that such genes, and the products coded by them, are the true morphogens responsible for determining the actual form of a particular organism. According to this view, morphogenesis results from the expression of specific 'master control' genes and thus, organic form is somehow pre-established (i.e., preformed) as an assembly programme encoded within the genome. Or in other words, it is claimed that the complex spatio-temporal order that leads to the achievement and maintenance of organic form is implied in a two-dimensional organisation of the genome. Moreover, some authors have claimed that the success of the Hox-gene research programme strongly suggests that morphological evolution is a direct result of evolution at the genetic level. Hereunder I discuss recent evidence that falsifies the basic preformationist tenets of molecular developmental biology. Thus suggesting that the problem of the origin of organic form is left untouched by the Hox-gene research programme and therefore, there is a need to reconsider alternative approaches, such as the structuralist morphogenetic outlook, that are better suited to eventually explain the origin of organic form.

p53 is a rate-limiting factor in the repair of higher-order DNA structure.

The product of the p53 tumor suppressor gene has been implicated in safeguarding genomic stability by transactivating genes involved in cell cycle arrest, repair of DNA damage or induction of apoptosis. Several properties of p53 suggest that it might be directly involved in DNA repair processes. Eukaryotic DNA is highly organized in supercoiled loops anchored to the nuclear matrix. This organization is very important for cell function and survival, suggesting that repair of DNA damage must include both, the integrity of the double helix and the complex DNA topology. In this work, we studied the kinetics and efficiency of higher-order DNA structure repair in cells with normal and reduced levels of p53, and present evidence suggesting that p53 may be involved in the stabilization and/or repair of higher-order DNA structure.

The normal association between newly replicated DNA and the nuclear matrix is abolished in cells infected by herpes simplex virus type 1.

In cells infected by herpes simplex virus type 1 (HSV1), a series of nuclear changes can be observed in a temporal sequence. Such changes are related to important modifications in the higher-order structure of host cell chromatin, such as loss of DNA loop supercoiling and wholesale DNA loop disorganization. It is known that the topological relationship between DNA and the nuclear substructure is a critical factor for proper nuclear physiology. Here we report that the usual association between newly replicated DNA and the nuclear substructure, commonly known as nuclear matrix, is abrogated in cells infected by HSV1, thus establishing a correlation between the virus-induced modifications in chromatin higher-order structure and a major biochemical change within the infected cell nucleus.

Loss of DNA loop supercoiling and organization in cells infected by herpes simplex virus type 1.

In cells infected by herpesviruses, a sequence of nuclear changes during interphase, as well as chromosomal aberrations during mitosis, are commonly observed. These changes suggest the progressive modification of host-cell chromatin. Previous studies have shown that the early chromatin modifications in cells infected by herpes simplex virus type 1 (HSV1) are not due to extensive breakdown of host-cell DNA or disruption of the nucleosomal structure. We have previously shown that infection by HSV1 induces single-stranded breaks in the host-cell DNA early in the course of infection, and that such breaks lead to modifications in the higher-order structure of host-cell chromatin. Here we report that virus-induced DNA breaks produce permanent long-term effects on the state of supercoiling and organization of the nuclear DNA loops, comparable to the DNA loop disorganization produced by high (and irreparable) doses of ultraviolet radiation.

A role for the nucleotype in the pathogenesis of primary hepatocellular carcinoma.

The risk of developing primary hepatocellular carcinoma (HCC) is 200 times higher for chronic carriers of hepatitis B virus antigen (HBsAg) than for the rest of the population. There is experimental evidence which suggests that the expression of viral DNA sequences integrated into the host's genome directly contributes to HCC development. However, most evidence available suggests that development of HCC requires a second genetic event that results from the chronic hepatitis and cirrhosis observed in symptomatic HBsAg chronic carriers. Here it is argued that the nucleotype, defined as those non-genic characters of nuclear DNA that affect or control the phenotype, is the element that links the viral, cellular and host factors involved in the genesis of HCC.

Evidence for an altered kinetics of DNA excision-repair in cells infected by herpes simplex virus type 1.

In cells infected with herpesviruses a series of host cell nuclear changes can be observed in a temporal sequence. Such changes include chromosome aberrations. The precise mechanism by which virus infection produces chromosome damage is not known, but we have previously reported that herpes simplex virus type 1 (HSV-1) induces a significant number of single-stranded breaks in the host cell DNA at early hours of infection and in a time-dependent fashion. Here, it is reported that HSV-1-infected cells subjected to irradiation with ultraviolet light, show an altered kinetics in the normal process of DNA excision-repair at early hours of infection.

Human immunodeficiency virus type 1 productive infection in staurosporine-blocked quiescent cells.

Staurosporine, an antibiotic known to inhibit cellular protein kinases, can reversibly block the progress of normal and tumour cells into the cell cycle. The ability of HIV-1 to infect and replicate in cells blocked by staurosporine was investigated. The results show that blocked, non-cycling cells can be productively infected by HIV-1, steadily releasing infectious progeny virus for several weeks. This suggests that at least in some cases, HIV-1 can be found in a stable and active state in resting, non-proliferating T cells.

Possible cell-free prion replication.

Spongiform encephalopathies, such as scrapie or bovine spongiform encephalopathy in animals, or kuru, Creutzfeld-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker disease (GSS) in man, seem to be caused by a transmissible agent whose nature is still a matter of debate. The properties of this agent which has been designated as prion, differ from those of any other known infectious agents, including viruses and viroids. Several lines of evidence suggest that the prion is devoid of nucleic acid and is identical with a modified form of a normal host protein. This lack of nucleic acid poses the question of how a protein moiety can propagate itself as a transmissible agent. Here it is proposed that prion replication is a process similar to crystallization and as such, the propagation of prions can take place as a sort of chain-reaction in an in vitro cell-free system.

HHV-6 inhibition by two polar compounds.

Dimethyl sulphoxide and dimethyl formamide, two polar compounds and powerful cell differentiation inducers, inhibit HHV-6 infection when added to HHV-6-infected HSB2 cultures. This was established by a delay in the time-course of infection and in the development of virus-induced cytopathic effects. Furthermore, viral titration of supernatants showed a significant reduction (3 log10) of the number of infectious particles. Electron microscopy confirmed that viable cells and extracellular virions were present in the cultures containing the polar compounds, while in the non-treated cultures all cells were lysed and no extracellular virus was evident. The mode of action of these compounds is still unclear and warrants further investigation.

Chemical inactivation of human immunodeficiency virus in vitro.

Most chemicals with potential virucidal activity are extremely cytotoxic even at very small concentrations, thus introducing a number of technical problems and uncertainties in the evaluation of the net virucidal effect. In the present study, an attempt was made to confirm the reported virucidal activity of certain well-known chemicals and a number of new compounds were investigated. The results suggest that HIV inactivation is dependent on the viral concentration, the time of incubation in presence of the putative disinfectant and the degree of virucidal activity of the latter. The data illustrate methodological problems arising from residual cytotoxicity of the chemical which may mask or mimic the presence of a true virucidal activity and lead to erroneous conclusions. Alcohol, the most commonly used disinfectant, was found to be ineffective for high viral concentrations, whilst sodium hypochlorite was the most efficient.

Early induction of DNA single-stranded breaks in cells infected by herpes simplex virus type 1.

In cells infected with herpesviruses a series of host-cell nuclear changes can be observed in a temporal sequence, such changes include chromosome aberrations. The precise mechanism by which virus infection produces chromosome damage is not known. Previous studies have revealed modifications in the properties of chromatin from infected cells, but such modifications are not due to extensive breakdown of host DNA or alteration of the nucleosomal structure in bulk host chromatin. We have adapted and modified a fluorescence enhancement assay for DNA damage in order to study the effects of herpes simplex virus type 1 infection on the integrity of the host-cell DNA. Here it is reported that HSV-1 induces a significant number of single-stranded breaks in the host-cell DNA at early hours post-infection. Such breaks seem not to be directly related to the major breakdown of host-cell DNA seen at later times post-infection.

Altered chromatin higher-order structure in cells infected by herpes simplex virus type 1.

In cells infected by herpesviruses chromosomal aberrations such as breaks and constrictions are commonly observed. Previous studies have shown that chromatin modifications in cells infected by herpes simplex virus type 1 (HSV-1) are not due to extensive breakdown of host-cell DNA or disruption of nucleosomal structure. We have previously shown that infection by HSV-1 induces single-stranded breaks in the host-cell DNA in a time-dependent fashion. Here we report that the early DNA damage observed in virus-infected cells is related to modifications in the higher-order structure of host-cell chromatin. Such modifications seem to be of a more permanent nature than the DNA single-stranded breaks.

A role for CD8+ T lymphocytes in the pathogenesis of AIDS.

Experimental and clinical evidence is presented which supports the hypothesis that CD8+ T lymphocytes aimed at suppressing HIV replication in infected CD4+ T cells may have an important role in the pathogenesis of AIDS by directly causing a decrease in CD4+ T lymphocyte numbers. Possible models to test this hypothesis are discussed.

On the regulation of DNA methylation by higher-order structure in the cell nucleus.

Several years ago it was postulated that DNA methylation may play a role in the regulation of gene activity in animal cells. Progress has been made in establishing the mechanisms by which methylation influences gene expression, but little is known about the mechanisms that control methylation itself. Here is proposed a model, susceptible of experimental confirmation, which suggests that chromatin higher-order structure is a determinant factor for the regulation of DNA methylation. The background for such a model as well as current evidence in support of it are discussed.

Dimethyl sulfoxide inhibits human immunodeficiency virus production in vitro.

LDV/7, H9, and MOLT-4, three cell lines infectible by human immunodeficiency virus were incubated with dimethyl sulfoxide, an inducer of cell differentiation. It was shown that this is a powerful inhibitor of viral production, but its effect is transient: viral production resumes when the compound is removed from the culture medium. It does not inactivate the virus, and it fails to prevent viral infection or to inhibit expression of p24 on the surface of the infected cells.

Is immune oversuppression the direct cause of AIDS?

AIDS is presumably caused by HIV, a retrovirus. The main support for this view comes from the consistent epidemiological correlation between AIDS and the presence of antibody against HIV. Several HIV-associated mechanisms have been described to explain the cytopathic effects on helper-T-lymphocytes observed in vitro but so far, none of these mechanisms has been confirmed in vivo. On the other hand, there is virtually no free virus and HIV RNA synthesis is very low both in AIDS patients and in asymptomatic carriers. Thus it is unlikely that HIV causes AIDS by acting as a conventional cytocidal virus. Here it is proposed, on the basis of current experimental evidence, that an active immune suppression mediated by T8+ cells is the direct culprit for a gradual decline of helper-T-lymphocyte numbers leading to an immunocompromised state in AIDS. The role of HIV in triggering and perpetuating a state of immune oversuppression is discussed.