Understanding the role of DNA methylation in successful biological invasions: a review

Biological invasions provide a unique opportunity to investigate rapid adaptation and evolution as the introduced taxa adapt to biogeographic contexts or habitats in which they have not evolved. The capacity of populations to evolve is generally thought to be constrained by their existing heritable genetic variation, which is usually associated with variation in genomic DNA nucleotide sequences. However, there is increasing acceptance that a range of mechanisms—collectively termed ‘epigenetics’ can alter gene function and affect ecologically important traits. Epigenetic processes may mediate adaptive phenotypic plasticity and provide heritable variation on a finer timescale than DNA sequence-based mutations. This review focuses on DNA methylation, a well-studied epigenetic mechanism known to be associated with biological adaptation to environmental stress. We explore the role of DNA methylation in characterising the adaptive potential of invasive species. We also provide an overview of studies focused on DNA methylation and invasive species to date, and identify knowledge gaps and potential ways to advance understanding of epigenetic-based adaptation. A summary of the literature suggests that DNA methylation could play a key role in the success of invasive species. Introduced populations with reduced genetic diversity often display increased DNA methylation variation in comparison with native populations, which could create phenotypic diversity when it is most required. Recent data show that DNA methylation could contribute to adaptation through both phenotypic plasticity and heritable variation, particularly through clonal reproduction. From a methodological perspective, recent advances in molecular techniques provide an exciting opportunity to explore the functional relevance of DNA methylation to successful biological invasions. Gaining a greater understanding of the adaptive and evolutionary processes that contribute to invasion success is critical for preventing and managing the future introduction, establishment and spread of invasive species.     

In situ activation of tumoricidal properties in rat alveolar macrophages and rejection of experimental lung metastases by intravenous injections of Nocardia rubra cell wall skeleton

Summary The IV injection of squalene-treated cell wall skeleton of Nocardia rubra (N-CWS) into F344 rats rendered their alveolar macrophages (AM) tumoricidal. Maximum tumoricidal activity developed in AM by 24 h after the IV, but not IP or SC, injection of 300 g N-CWS. Tumoricidal activity of AM was maintained for 48–72 h after one IV injection of N-CWS. Experimental lung metastases were produced in female F344 rats by the IV injection of viable syngeneic mammary adenocarcinoma cells. Treatments twice weekly with Hank's balanced salt solution, N-CWS placebo or N-CWS began 3, 7, or 10 days later and were continued for 3 or 4 weeks for a total of six or eight treatments. Practically all the rats (>90%) treated with N-CWS beginning on either day 3 or day 7 after tumor cell challenge survived until day 210, when the experiment was terminated. In contrast, 90% of the rats treated with balanced salt solution or N-CWS placebo died by day 70 of the experiment. Therapy with N-CWS preparation was not successful when the first injection was administered 10 days after tumor cell challenge, suggesting that this therapeutic regimen is effective only against minimal tumor burden. We conclude that in this animal tumor model, the IV injection of N-CWS preparations can render AM tumoricidal and aid in the eradication of pulmonary micrometastases.     

Bcl-2 protects cells from cytokine-induced nitric-oxide-dependent apoptosis

 Cytokine-mediated cell death in tumor cells can be achieved through endogenous nitric oxide (NO) from within tumor cells or exogenous NO from either activated macrophages or endothelial cells. The purpose of this study was to determine the role of Bcl-2 in NO-mediated apoptosis. The incubation of murine L929 and NIH3T3 cells with interleukin-1α (IL-1α) and interferon γ (IFNγ) induced high endogenous NO production only in the L929 cells that also underwent apoptosis. NIH3T3 cells were not resistant to NO-mediated apoptosis. In fact, the incubation of L929 and NIH3T3 cells with exogenous NO derived from NO donors, sodium nitroprusside, or S-nitroso-N-acetyl-DL-penicillamine (SNAP) induced death, characterized by typical apoptotic morphology and DNA fragmentation, in both cell types, but to a higher degree in NIH3T3 cells than in the L929 cells. We then measured the effect of Bcl-2 expression on exogenous NO-induced apoptosis. At both the mRNA and protein levels, L929 fibroblasts expressed higher levels of endogenous mouse Bcl-2 than did NIH3T3 cells. At the same time, L929 cells were much more resistant to exogenous NO-induced cell death than were NIH3T3 cells. The inverse correlation between mouse Bcl-2 expression and sensitivity to exogenous NO-mediated cell death was also found in the murine K-1735 melanoma C-23 and X-21 clonal populations. Transfection of both NIH3T3 cells and L929 cells with the human bcl-2 gene led to resistance to both exogenous and endogenous NO-mediated apoptosis. These data demonstrate that NO-mediated apoptosis can be suppressed by expression of Bcl-2, suggesting that abnormal expression of Bcl-2 may influence the efficacy of tumor immunotherapy. Received: 28 June 1998 / Accepted: 23 August 1996     

Systemic activation of tumoricidal properties in mouse macrophages and inhibition of melanoma metastases by the oral administration of MTP-PE, a lipophilic muramyl dipeptide

The purpose of these studies was to determine whether the oral administration of a lipophilic analog of muramyl dipeptide, MTP-PE, can produce in situ activation of tumoricidal properties in mouse macrophages. MTP-PE was dissolved in a phosphate-buffered saline to produce micelles. Single or multiple oral administrations of MTP-PE produced tumoricidal activation in both lung and peritoneal macrophages. This was in direct contrast to the i.v. or i.p. administrations of MTP-PE incorporated in liposomes, which produced activation in only lung or only peritoneal macrophages, respectively. The distribution and fate of [3H]-labeled MTP-PE subsequent to oral administration revealed that MTP-PE was found in various organs independent of reticuloendothelial activity. Finally, the repeated twice-weekly oral administrations of MTP-PE inhibited lung and lymph node metastasis in C57BL/6 mice by syngeneic B16 melanoma cells. The oral administration of MTP-PE, however, was not effective in eradicating well-established melanoma metastases. We conclude that the oral administration of a lipophilic muramyl dipeptide produces systemic activation of macrophages. The feasibility of enhancing host defense against infections and cancer by the oral administration of an immunomodulator has obvious clinical advantages.     

Natural and recombinant human interleukin 1-beta is cytotoxic for human melanoma cells

Human peripheral blood monocytes stimulated with LPS were found to release an activity that was cytotoxic for the A375 melanoma. Biochemical and immunological characterization of the activity indicated that IL 1-beta was the cytotoxic agent. Human recombinant IL 1-beta, purified to homogeneity, was directly cytotoxic for A375. Tumor necrosis factor, also released by activated monocytes, was not cytotoxic for the A375 melanoma.     

Cell surface receptors for lymphokines. I. The possible role of glycolipids as receptors for macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF).

Incubation of culture supernatants from concanavalin A-stimulated guinea pig and rat lymphocytes with protein-free preparations of bovine brain gangliosides abolished their macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF) activity. The identity of the MIF/MAF-binding component(s) present in these glycolipid mixtures has yet to be established, but adsorption experiments using purified preparations of mono- (GM₁, GM₂, and GM₃), di- (GD_1a), and trisialogangliosides (GT₁) were negative. Since these gangliosides account for over 90% of the glycolipid content in brain ganglioside mixtures it appears that the MIF-binding component(s) is present only in very small amounts. Treatment of guinea pig peritoneal macrophages with liposomes containing similar brain gangliosides or water-soluble glycolipids extracted from guinea pig macrophages enhanced their responsiveness to MIF. The enhanced response to MIF of liposome-treated macrophages was abolished by incubation of the treated macrophages with fucose-binding lectins (Lotus agglutinin and Ulex europaeus agglutinin I) before exposure to MIF, suggesting that the MIF-binding component donated by the liposomes may be a fucose-containing glycolipid. The possible role of glycolipids as surface receptors for MIF and MAF is discussed.     

In vitro activation of tumoricidal properties in mouse macrophages using the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) incorporated in liposomes

Summary We investigated the ability of free or liposome-incorporated synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) to generate tumoricidal properties in mouse macrophages. As FMLP contains three hydrophobic amino acid residues, it can readily be incorporated into multilamellar vesicles (MLV) consisting of phosphatidylcholine (PC) and phosphatidylserine (PS). The incorporation of FMLP into MLV with a PC:PS ratio of 7:3 mol at FMLP concentrations of up to 10^–4 M did not affect the phagocytosis of liposomes by mouse peritoneal macrophages. Studies with radioactive FMLP revealed that higher levels of FMLP can be delivered to macrophages by liposomes than in the free, nonencapsulated form. Treatment of mouse macrophages with liposome-encapsulated FMLP, but not with free FMLP, generated tumoricidal properties in the macrophages. The mechanism appears to involve an intracellular site since 100-fold concentrations of free FMLP or free N-acetyl-methionyl-leucyl-phenylalanine, the FMLP agonist, failed to competitively inhibit the macrophage's tumoricidal properties generated by liposome-encapsulated FMLP.     

Heat Shock Protein-Derived T-Cell Epitopes Contribute to Autoimmune Inflammation in Pediatric Crohn's Disease

Pediatric Crohn''s disease is a chronic auto inflammatory bowel disorder affecting children under the age of 17 years. A putative etiopathogenesis of Crohn''s disease (CD) is associated with disregulation of immune response to antigens commonly present in the gut microenvironment. Heat shock proteins (HSP) have been identified as ubiquitous antigens with the ability to modulate inflammatory responses associated with several autoimmune diseases. The present study tested the contribution of immune responses to HSP in the amplification of autoimmune inflammation in chronically inflamed mucosa of pediatric CD patients. Colonic biopsies obtained from normal and CD mucosa were stimulated with pairs of Pan HLA-DR binder HSP60-derived peptides (human/bacterial homologues). The modulation of RNA and protein levels of induced proinflammatory cytokines were measured. We identified two epitopes capable of sustaining proinflammatory responses, specifically TNF〈 and IFN© induction, in the inflamed intestinal mucosa in CD patients. The responses correlated positively with clinical and histological measurements of disease activity, thus suggesting a contribution of immune responses to HSP in pediatric CD site-specific mucosal inflammation.     

Abnormal connexin43 in arrhythmogenic right ventricular cardiomyopathy caused by plakophilin-2 mutations

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a disorder of cardiomyocyte intercalated disk proteins causing sudden death. Heterozygous mutations of the desmosomal protein plakophilin-2 (PKP-2) are the commonest genetic cause of ARVC. Abnormal gap junction connexin43 expression has been reported in autosomal dominant forms of ARVC (Naxos and Carvajal disease) caused by homozygous mutations of desmosomal plakoglobin and desmoplakin. In tissue culture, suppression of PKP-2 results in decreased expression of connexin43. We sought to characterize the expression and localization of connexin43 in patients with ARVC secondary to heterozygous PKP-2 mutations. Complete PKP-2 gene sequencing of 27 ARVC patients was utilized to identify mutant genotypes. Endomyocardial biopsies of identified carriers were then assessed by immunofluorescence to visualize intercalated disk proteins. N-cadherin was targeted to highlight intercalated disks, followed by counterstaining for PKP-2 or connexin43 using confocal double immunofluorescence microscopy. Immunofluorescence was quantified using an AdobeA Photoshop protocol, and colocalization coefficients were determined. PKP-2 siRNA experiments were performed in mouse cardiomyocyte (HL1) cell culture with Western blot analysis to assess connexin43 expression following PKP-2 suppression. Missense and frameshift mutations of the PKP-2 gene were found in four patients with biopsy material available for analysis. Immunofluorescent studies showed PKP-2 localization to the intercalated disk despite mutations, but associated with decreased connexin43 expression and abnormal colocalization. PKP-2 siRNA in HL1 culture confirmed decreased connexin43 expression. Reduced connexin43 expression and localization to the intercalated disk occurs in heterozygous human PKP-2 mutations, potentially explaining the delayed conduction and propensity to develop arrhythmias seen in this disease.