Carcinogenesis in the GI tract: from morphology to genetics and back again.

The genetic alterations in colorectal cancer progression are determined by one of two separate and distinct underlying pathways of genomic instability. The first pathway, chromosomal instability, is characterized by allelic losses and aneuploidy. The second pathway, microsatellite instability, is characterized by an abundance of subtle DNA mutations and diploidy. Although the genes causing chromosomal instability remain unknown, microsatellite instability is caused by inactivation of a DNA mismatch repair gene (predominantly MLH1 or MSH2). Microsatellite instability is present in 15% of colorectal cancers, and is diagnosed by analysis of tumor DNA from paraffin blocks and by demonstration of loss of mismatch repair protein expression in cancers. In addition to the unique profile of genetic alterations, colorectal cancers with microsatellite instability have distinct pathologic features and improved survival. Finally, cancers from most patients with hereditary non-polyposis colorectal cancer (or Lynch syndrome) have microsatellite instability due to germline mutations in the DNA mismatch repair genes. Identification of the microsatellite instability pathway has enormous implications for the clinical investigation and management of colorectal cancer patients.     

Perspectives on microbes as oncogenic infectious agents and implications for breast cancer

Cancer management is partly based on weighing risk factors attributed to noninfectious agents, human genes and epigenetic factors. Infectious disease causation has largely been restricted to genes directly responsible for causing cancer after sustaining damage i.e. oncogenes. Lately, evidence has emerged linking infectious agents to a number of chronic diseases. These studies have recognized the influence that acute, atypical, latent and chronic infections may play in tricking the immune system and affecting disease etiology. Similar evidence is emerging in model systems with respect to the role of infectious agents in gastrointestinal, liver and lung cancers. Although viruses have been found in association with breast cancer, skepticism remains about a role for other infectious agents, notably microbes in the disease etiology. Improved experimental designs employed in different cancer studies and a less rigid definition of infectious causation may aid in confirming or refuting a microbe-breast cancer connection. Cancer recurrence could potentially be minimized and treatment options further tailored on a case by case basis if microbes/microbial components/strain variants associated with breast cancer are identified; probiotics are employed to reduce treatment side-effects and if microbes could effectively be harnessed in immunotherapy.     

Alterations of DNA methylation and clinicopathological diversity of human cancers

Alterations of DNA methylation can account for the histological heterogeneity, reflected in the stepwise progression and complex biological characteristics of human cancers, that genetic alterations alone cannot explain. Analysis of DNA methylation status in tissue samples can be an aid to understanding the molecular mechanisms of multistage carcinogenesis. Human cancer cells show a drastic change in DNA methylation status, that is, overall DNA hypomethylation and regional DNA hypermethylation, which results in chromosomal instability and silencing of tumor-suppressor genes. Overexpression of DNA methyltransferase (DNMT) 1 is not a secondary result of increased cell proliferative activity but may underline the CpG island methylator phenotype of cancers. Splicing alteration of DNMT3B may result in chromosomal instability through DNA hypomethylation of pericentromeric satellite regions. Alterations of DNA methylation are observed even in the precancerous stage frequently associated with chronic inflammation and/or persistent viral infection or with cigarette smoking. Precancerous conditions showing alterations of DNA methylation may generate more malignant cancers. Aberrant DNA methylation is significantly associated with aggressiveness of cancers and poorer outcome of cancer patients. Genome-wide analysis of DNA methylation status based on array-based technology may identify DNA methylation profiles that can be used as appropriate indicators for carcinogenetic risk estimation and prognostication.     

Contemporaneous fluctuations in T cell responses to persistent herpes virus infections

The classical paradigm for T cell dynamics suggests that the resolution of a primary acute virus infection is followed by the generation of a long-lived pool of memory T cells that is thought to be highly stable. Very limited alteration in this repertoire is expected until the immune system is re-challenged by reactivation of latent viruses or by cross-reactive pathogens. Contradicting this view, we show here that the T cell repertoire specific for two different latent herpes viruses in the peripheral blood displayed significant contemporaneous co-fluctuations of virus-specific CD8(+) T cells. The coordinated responses to two different viruses suggest that the fluctuations within the T cell repertoire may be driven by sub-clinical viral reactivation or a more generalized 'bystander' effect. The later contention was supported by the observation that, while absolute number of CD3(+) T cells and their subsets and also the cell surface phenotype of antigen-specific T cells remained relatively constant, a loss of CD62L expression in the total CD8(+) T cell population was coincident with the expansion of tetramer-positive virus-specific T cells. This study demonstrates that the dynamic process of T cell expansion and contractions in persistent viral infections is not limited to the acute phase of infection, but also continues during the latent phase of infection.     

Phenotypic mixing between murine oncoviruses and murine cytomegalovirus.

In vitro interactions between murine cytomegalovirus (MCMV) and murine leukaemia viruses (MuLV), two groups of enveloped viruses capable of causing persistent or latent infections in vivo, were examined for evidence of phenotypic mixing. The growth of MCMV in murine cells productively infected with ecotropic MuLV was shown to result regularly in the production of phenotypically mixed particles having the envelope antigens of MuLV and the genome of MCMV [MCMV(MuLV) pseudotypes]. The identity of such pseudotype particles was confirmed by the use of specific anti-MuLV serum and by the demonstration of restriction due to viral interference of penetration of these particles on MuLV-infected murine cells. This restriction was independent of N- or B-tropism. The production of reverse pseudotypes could not be examined because of the lytic effects of MCMV on the requisite assay cells.     

Persistent genomic instability in peripheral blood lymphocytes from hodgkin lymphoma survivors

Advances in cancer treatment have led to an increase in patient survival. However, exposure to genotoxic chemotherapeutic agents and ionizing radiation may induce persistent genetic damage in cancer survivors. In this study, we detected genomic instability in chromosomes of peripheral blood lymphocytes from Hodgkin lymphoma patients, 2–17 years after MOPP (nitrogen mustard, Oncovin, procarbazine, and prednisone) chemotherapy with or without radiotherapy. Samples were obtained from 11 healthy individuals, 5 pretreatment patients, and 20 posttreatment patients. Cytogenetic analysis with GTG banding was performed on 1,000 lymphocyte metaphases per donor to identify genomic instability, including numerical and structural chromosomal aberrations, at a resolution of 10 Mb across the entire genome. Our results showed that anticancer treatment did not induce significant differences in the frequency of aneuploidy among the three study groups. However, 1 of the 11 healthy individuals, and 13 of the 20 posttreatment patients had a high frequency of chromosomal breaks and gross chromosomal rearrangements. The types of aberrations observed were random and complex, consistent with persistent genomic instability that was induced by cancer treatment. Clonal expansion of cells with chromosomal lesions was observed in one posttreatment patient only. These findings show that anticancer treatments induce persistent genomic instability, but not aneuploidy. Chemotherapy may affect genes with a role in DNA damage surveillance or repair, which in turn allows the accumulation of nontargeted structural chromosomal damage in future generations of cells. This genomic instability may facilitate the development of second malignancies in Hodgkin lymphoma survivors. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

Modes of infection and oncogenesis by the Epstein–Barr virus

The EBV is a human γ‐herpesvirus associated with various neoplasms. It is responsible for causing cancers of B, T, and NK cells as well as cells of epithelial origin. Such diversity in target cells and the complicated steps of oncogenesis are perplexing when we speculate about the mechanisms of action of EBV‐positive cancers. Here, we first note three common features that contribute to the development and maintenance of EBV‐positive cancers: effects of EBV oncogenes, immunosuppression and evasion/exploitation of the immune system, and genetic and epigenetic predisposition/alteration of the host genome. Then, we demonstrate the mechanisms of oncogenesis and the means by which each EBV‐positive cancer develops, with particular focus on the mode of EBV infection. The EBV has two alternative life cycles: lytic and latent. The latter is categorized into four programs (latency types 0–III) in which latent viral genes are expressed differentially depending on the tissue of origin and state of cells. The production of viral latent genes tends to decrease with an increase in time, and, in an approximate manner, the expression levels of viral genes are inversely correlated with the degree of abnormalities in the host genome. Occasional execution of the viral lytic cycle also contributes to oncogenesis. Understanding this life cycle of the EBV and its relevance in oncogenesis may provide valuable clues to the development of effective therapies for the associated cancers. Copyright © 2014 John Wiley & Sons, Ltd.     

High infectious burden, low cancer incidence, and early malignancy in developing countries: a molecular hypothesis in term of the role of nitric oxide

Nitric oxide (NO) is a neurotransmitter which plays a powerful role in the immune system: it kills bacteria, and, it also destroys the tumor cells. Specifically, immune system stimuli gamma interferon and lipopolysaccharide transmit signals to a macrophage nucleus causing the production of nitric oxide synthase, the enzyme that converts arginine to NO. The NO thus produced not only destroys bacteria but also attacks the tumor cells by inhibiting the energy-producing Krebs cycle, electron transport activity and DNA synthesis. People in developing countries who survive repeated childhood infections must be inferred to have robust microphage/NO systems and thus, also, a strong immunity against cancer--thence the low incidence of cancers in these countries. However, those unfortunate few in these countries who do develop cancer, despite a robust microphage/NO system, must be presumed to have a markedly virulent tumor development micro-environment (e.g., activation of tumor promotion genes, inactivation of tumor suppression genes, multiple mutations, etc.) that escapes even the particularly alert immune surveillance--thence the earlier (by about a decade) death by cancer in those countries. Thus the NO hypothesis put forward here simultaneously provides a mechanistic causation for (i) low cancer incidence in countries subjected to heavy infectious burdens, and (ii) the earlier occurrence (by about a decade) of major cancers in those countries when the immune surveillance, despite its robustness, fails to destroy the incipient formation of cancer cells.     

The FRA2C common fragile site maps to the borders of MYCN amplicons in neuroblastoma and is associated with gross chromosomal rearrangements in different cancers

Common fragile sites (cFS) represent chromosomal regions that are prone to breakage after partial inhibition of DNA synthesis. Activation of cFS is associated with various forms of DNA instability in cancer cells, and is thought to be an initiating event in the generation of DNA damage in early-stage tumorigenesis. Only a few cFS have been fully characterized despite the growing interest in cFS instability in cancer genomes. In this study, six-color fluorescence in situ hybridization revealed that FRA2C consists of two cFS spanning 747 kb FRA2Ctel and 746 kb FRA2Ccen at 2p24.3 and 2p24.2, respectively. Both cFS are separated by a 2.8 Mb non-fragile region containing MYCN. Fine-tiling array comparative genomic hybridization of MYCN amplicons from neuroblastoma (NB) cell lines and primary tumors revealed that 56.5% of the amplicons cluster in FRA2C. MYCN amplicons are either organized as double minutes or as homogeneously stained regions in addition to the single copy of MYCN retained at 2p24. We suggest that MYCN amplicons arise from extra replication rounds of unbroken DNA secondary structures that accumulate at FRA2C. This hypothesis implicates cFS in high-level gene amplification in cancer cells. Complex genomic rearrangements, including deletions, duplications and translocations, which originate from double-strand breaks, were detected at FRA2C in different cancers. These data propose a dual role for cFS in the generation of gross chromosomal rearrangements either after DNA breakage or by inducing extra replication rounds, and provide new insights into the highly recombinogenic nature of cFS in the human cancer genome.     

Thalamus in flame: targeting of infectious agents to thalamic nuclei

The involvement of the thalamus in infectious diseases of the nervous system has been hitherto rather neglected by investigators in clinical and basic neuroscience, despite numerous reports indicating that the thalamus, and territories within this region, can be attacked by different types of microbes. This topic is here reviewed. First, an overview is provided on general principles of spread of microbes to the brain (through peripheral nerves, or through the blood or cerebrospinal fluid) and their interactions with neurons and immune cells to cause acute, transient or persistent infections. Examples are given on how non-cytolytic infections can cause long-lasting disturbances in synaptic activities and neuronal networks as a result of a “hit-and-run” mechanism, or as an effect of factors released in the microenvironment to control the neuronal infection. Emerging data on how molecules functioning at the “immunological synapse” (the site of contact between immune cells and target infected cells) may affect nervous system synapses are pointed out. An account is then given of clinical and experimental infections of the thalamus caused by viruses (rabies and herpes viruses, influenza A virus, flaviviruses, HIV virus), the parasite Toxoplasma gondii, and prions. The implications and consequences of the attack of these microbes to the thalamus are discussed. Of special interest is the potential persistence of latent infections in thalamic neurons, which could cause disturbances of neuronal functions in the absence of overt structural lesions. Altogether these data recall attention on the pathogenesis and consequences of acute and persistent infections in the mammalian thalamus.     

Activated oncogenes promote and cooperate with chromosomal instability for neoplastic transformation

Most cancer cells are aneuploid. The chromosomal instability hypothesis contends that aneuploidy is the catalyst for transformation, whereas the gene mutation hypothesis asserts that cancer is driven by mutations to proto-oncogenes and tumor-suppressor genes, with the aneuploidy a side effect of tumorigenesis. Because genotoxic stress induced by “culture shock” can obscure the transforming potential of exogenous genes, we cultured wild-type and p53^-/- mouse embryo fibroblasts in a more physiological (serum-free) environment. Under these conditions, the cells were immortal and, more importantly, chromosomally stable. Expression of oncogenic H-RasV12 did not induce senescence, but sensitized these cells to p53-dependent apoptosis. In addition, H-RasV12 induced chromosomal instability, as well as accumulation and phosphorylation of p53. Significantly, whereas cells grown under standard conditions could be transformed by coexpression of H-RasV12 and E1A, the chromosomally stable cells were refractory to transformation, as measured by anchorage-independent growth and tumor formation in nude mice. These oncogenes required a third genetic alteration that abolished the p53 pathway to create a permissive environment that promotes rapid chromosomal instability and transformation. Oncogene-induced chromosomal instability and transformation was attenuated by antioxidants. These data indicate that chromosomal instability could be a catalyst for oncogenic transformation, and bring together aspects of the chromosomal instability hypothesis and the gene mutation hypothesis for tumorigenesis.     

Induced genome instability as a potential screening test for cancer susceptibility?

The variety of mutations associated with carcinogenesis, along with variations in penetrance and environmental factors, complicate the genetic screening for cancer predisposition. It is proposed here that the detection of inherent genome instability as determined by increased mutagen susceptibility may enhance the identification of populations at risk for cancer. In support for this hypothesis, our analysis reveals a strong association between mutagen-induced chromosomal instability in peripheral blood lymphocytes and the propensity for cancers of oral cavity, pharynx, larynx, and lung. DNA instability in response to a variety of mutagens identifies patients with gastrointestinal, brain, endocrine, breast, skin, and hematologic tumors as well as individuals with cancer family syndromes. Induced genome instability therefore appears to be strongly linked to cancer predisposition, and prospective studies may yield a screening test utilizing a panel of mutagens to better identify populations at risk.     

Reactivation of Epstein-Barr virus from latency

The general problem in cancer treatment centres on finding agents that specifically affect cancer cells without damaging normal cells. The differences between cancer cells and normal cells are usually very subtle but about 15% of all human cancers involve a virus infection, for example the Epstein-Barr virus associated cancers. In these cancers, every tumour cell carries the virus in a latent infection but the number of normal cells infected is very low. So a treatment that could somehow cause the elimination of EBV infected cells would be very specific for the cancer in such cases. One potential approach could involve finding ways to reactivate the latent virus in cancer cells into the early part of the lytic cycle, impeding cell proliferation, targeting chemotherapeutic agents to the cancer and causing the cancer cells to become targets for immune surveillance. This review considers the mechanisms by which EBV reactivation is controlled and discusses possible therapeutic approaches.     

Tetranucleotide repeats in coding regions: no evidence for involvement in EMAST carcinogenesis

Genetic instability is a hallmark of malignancy. In the majority of malignant tumors, chromosomal instability leads to major numerical and structural chromosomal aberrations. In contrast, some tumors have a deficient DNA mismatch repair system and accumulate mutations particularly in repetitive mono- and dinucleotide sequences, a phenomenon referred to as microsatellite instability (MSI). Recently, a novel phenotype of tumors presenting with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) has been reported. To date, not much is known about the molecular mechanisms of EMAST tumorigenesis. In MSI tumors, instability at specific mono- and dinucleotide repeats leads to alteration of genes carrying these repeats and thus may contribute to MSI tumorigenesis. We hypothesized that, similarly to the MSI phenotype, development of EMAST cancers may be promoted by mutations affecting tetranucleotides located in coding regions of the genome. To test this hypothesis, we performed a genome-wide database search to identify tetranucleotides in gene-encoding regions. Only seven tetranucleotide repeats located in predicted gene-encoding regions were retrieved. Allele length analysis yielded three remaining candidates with a monomorphic pattern in healthy individuals. Mutation analysis revealed that none of these three candidates displayed mutations in EMAST-positive bladder cancers. These data suggest that mutational inactivation of tetranucleotide-containing genes is very unlikely to contribute to the progression of EMAST tumors.     

Modulation of dendritic cell function by persistent viruses

Worldwide, chronic viral infections cause major health problems with severe morbidity and mortality. HIV and hepatitis C virus (HCV) manifest themselves as persistent infections, but they are entirely distinct viruses with distinct replication mechanisms, tropism, and kinetics. Coinfections with HCV among people with HIV are emerging as a growing problem. Cellular immune responses play an important role in viral clearance and disease pathogenesis. However, cellular immunity to HIV and HCV is affected severely in chronic patients. Various hypotheses have been proposed to explain the dysfunctional T cell response, including viral escape mutations, exhaustion of the T cell compartment, and the activity of regulatory T cells. Also, modulation of the function of dendritic cells (DC) has been suggested as one of the mechanisms used by persistent viruses to evade the immune system. In this review, we will focus on DC interactions with one murine persistent virus (lymphocytic choriomeningitis virus clone 13) and two human persistent viruses (HIV-1 and HCV), intending to examine if general strategies are used by persistent viruses to modulate the function of DC to improve our understanding of the mechanisms underlying the development and maintenance of viral persistence.     

Mucosal immunology of HIV infection

Recent advances in the immunology, pathogenesis, and prevention of human immunodeficiency virus (HIV) infection continue to reveal clues to the mechanisms involved in the progressive immunodeficiency attributed to infection, but more importantly have shed light on the correlates of immunity to infection and disease progression. HIV selectively infects, eliminates, and/or dysregulates several key cells of the human immune system, thwarting multiple arms of the host immune response, and inflicting severe damage to mucosal barriers, resulting in tissue infiltration of ‘symbiotic’ intestinal bacteria and viruses that essentially become opportunistic infections promoting systemic immune activation. This leads to activation and recruitment or more target cells for perpetuating HIV infection, resulting in persistent, high-level viral replication in lymphoid tissues, rapid evolution of resistant strains, and continued evasion of immune responses. However, vaccine studies and studies of spontaneous controllers are finally providing correlates of immunity from protection and disease progression, including virus-specific CD4⁺ T-cell responses, binding anti-bodies, innate immune responses, and generation of antibodies with potent antibody-dependent cell-mediated cytotoxicity activity. Emerging correlates of immunity indicate that prevention of HIV infection may be possible through effective vaccine strategies that protect and stimulate key regulatory cells and immune responses in susceptible hosts. Furthermore, immune therapies specifically directed toward boosting specific aspects of the immune system may eventually lead to a cure for HIV-infected patients.