CD43 is a cell-surface sialoglycoprotein expressed by a variety of haematopoietically derived cells, including T lymphocytes. Earlier observations of defective CD43 expression by T lymphocytes from boys with the X-chromosome-linked Wiskott-Aldrich syndrome suggested the importance of CD43 in lymphocyte function. Subsequent studies have suggested that CD43 facilitates leukocyte adhesion and has a co-stimulatory role during T-cell activation. To define the physiologically relevant function(s) of CD43, we have generated CD43-knockout mice. We report here that CD43-deficient T cells from such mice show a marked increase in their in vitro proliferative response to concanavalin A, anti-CD3, the superantigen SEB and allostimulation. Additionally, CD43-deficient T cells show a substantial enhancement in homotypic adhesion and in their ability to bind different ligands, including fibronectin and the intercellular adhesion molecule ICAM-1. Vaccinia-virus-infected CD43-knockout mice mounted an augmented anti-vaccinia cytotoxic T-cell response compared with their wild-type littermates, yet developed an increased virus load. We conclude that CD43 negatively regulates T-cell activation and adhesion and is important for viral clearance.

Both oxidative/nitrosative stress and alterations in DNA methylation are observed during carcinogenesis of different tumor types, but no clear correlation between these events has been demonstrated until now. Melanoma cell lines were previously established after submitting the nontumorigenicmelanocyte lineage, melan-a, to cycles of anchorage blockade. In this work, increased intracellular oxidative species and nitric oxide levels, as well as alterations in the DNA methylation, were observed after melan-a detachment, which were also associated with a decrease in intracellular homocysteine (Hcy), an element in the methionine (universal methyl donor) cycle. This alteration was accompanied by increase in glutathione (GSH) levels and methylated DNA content. Furthermore, a significant increase in dnmt1 and 3b expression was identified along melan-a anchorage blockade. L(G)-Nitro-L-arginine methyl esther (L-NAME), known as a nitric oxide synthase (NOS) inhibitor, and N-acetyl-L-cysteine (NAC) prevented the increase in global DNA methylation, as well as the increase in dnmt1 and 3b expression, observed during melan-a detachment. Interestingly, both L-NAME and NAC did not inhibit nitric oxide (NO) production in these cells, but abrogated superoxide anion production during anchorage blockade. In conclusion, oxidative stress observed during melanocyte anchorage blockade seems to modulate DNA methylation levels and may directly contribute to the acquisition of an anoikis-resistant phenotype through an epigenetic mechanism.

Exclude experimental models of malignant transformation employ chemical and physical carcinogens or genetic manipulations to study tumor progression. In this work, different melanoma cell lines were established after submitting a nontumorigenic melanocyte lineage (melan-a) to sequential cycles of forced anchorage impediment. The great majority of these cells underwent anoikis when maintained in suspension. After one deadhesion cycle, phenotypic alterations were noticeable in the few surviving cells, which became more numerous and showed progressive alterations after each adhesion impediment step. No significant differences in cell surface expression of integrins were detected, but a clear electrophoretic migration shift, compatible with an altered glycosylation pattern, was observed for beta1 chain in transformed cell lines. In parallel, a progressive enrichment of tri- and tetra-antennary N-glycans was apparent, suggesting increased N-acetylglucosaminyltransferase V activity. Alterations both in proteoglycan glycosylation pattern and core protein expression were detected during the transformation process. In conclusion, this model corroborates the role of adhesion state as a promoting agent in transformation process and demonstrates that cell adhesion disturbances may act as carcinogenic stimuli, at least for a nontumorigenic immortalized melanocyte lineage. These findings have intriguing implications for in vivo carcinogenesis, suggesting that anchorage independence may precede, and contribute to, neoplastic conversion.

The eukaryotic translation initiation factor 5A (eIF5A) undergoes a specific post-translational modification called hypusination. This modification is required for the functionality of this protein. The compound N1-guanyl-1,7-diaminoheptane (GC7) is a potent and selective inhibitor of deoxyhypusine synthase, which catalyses the first step of eIF5A hypusination process. In the present study, the effects of GC7 on cell death were investigated using two cell lines: melan-a murine melanocytes and Tm5 murine melanoma. In vitro treatment with GC7 increased by 3-fold the number of cells presenting DNA fragmentation in Tm5 cells. Exposure to GC7 also decreased viability to both cell lines. This study also describes, for the first time, the in vivo antitumour effect of GC7, as indicated by impaired melanoma growth in C57BL/6 mice.