Membrane Association of the Short Transglutaminase Type 2 Splice Variant (TG2-S) Modulates Cisplatin Resistance in a Human Hepatocellular Carcinoma (HepG2) Cell Line.

Hepatocellular carcinoma (HCC) is a heterogeneous malignancy with complex carcinogenesis. Although there has been significant progress in the treatment of HCC over the past decades, drug resistance to chemotherapy remains a major obstacle in its successful management. In this study, we were able to reduce chemoresistance in cisplatin-resistant HepG2 cells by either silencing the expression of transglutaminase type 2 (TG2) using siRNA or by the pre-treatment of cells with the TG2 enzyme inhibitor cystamine. Further analysis revealed that, whereas the full-length TG2 isoform (TG2-L) was almost completely cytoplasmic in its distribution, the majority of the short TG2 isoform (TG2-S) was membrane-associated in both parental and chemoresistant HepG2 cells. Following the induction of cisplatin toxicity in non-chemoresistant parental cells, TG2-S, together with cisplatin, quickly relocated to the cytosolic fraction. Conversely, no cytosolic relocalisation of TG2-S or nuclear accumulation cisplatin was observed, following the identical treatment of chemoresistant cells, where TG2-S remained predominantly membrane-associated. This suggests that the deficient subcellular relocalisation of TG2-S from membranous structures into the cytoplasm may limit the apoptic response to cisplatin toxicity in chemoresistant cells. Structural analysis of TG2 revealed the presence of binding motifs for interaction of TG2-S with the membrane scaffold protein LC3/LC3 homologue that could contribute to a novel mechanism of chemotherapeutic resistance in HepG2 cells.

Agouti signalling protein is an inverse agonist to the wildtype and agonist to the melanic variant of the melanocortin-1 receptor in the grey squirrel (Sciurus carolinensis).

The melanocortin-1 receptor (MC1R) is a key regulator of mammalian pigmentation. Melanism in the grey squirrel is associated with an eight amino acid deletion in the mutant melanocortin-1 receptor with 24 base pair deletion (MC1RΔ24) variant. We demonstrate that the MC1RΔ24 exhibits a higher basal activity than the wildtype MC1R (MC1R-wt). We demonstrate that agouti signalling protein (ASIP) is an inverse agonist to the MC1R-wt but is an agonist to the MC1RΔ24. We conclude that the deletion in the MC1RΔ24 leads to a receptor with a high basal activity which is further activated by ASIP. This is the first report of ASIP acting as an agonist to MC1R.

Melanocortin 1 receptor (MC1R) gene sequence variation and melanism in the gray (Sciurus carolinensis), fox (Sciurus niger), and red (Sciurus vulgaris) squirrel.

Sequence variations in the melanocortin 1 receptor (MC1R) gene are associated with melanism in many different species of mammals, birds, and reptiles. The gray squirrel (Sciurus carolinensis), found in the British Isles, was introduced from North America in the late 19th century. Melanism in the British gray squirrel is associated with a 24-bp deletion in the MC1R. To investigate the origin of this mutation, we sequenced the MC1R of 95 individuals including 44 melanic gray squirrels from both the British Isles and North America. Melanic gray squirrels of both populations had the same 24-bp deletion associated with melanism. Given the significant deletion associated with melanism in the gray squirrel, we sequenced the MC1R of both wild-type and melanic fox squirrels (Sciurus niger) (9 individuals) and red squirrels (Sciurus vulgaris) (39 individuals). Unlike the gray squirrel, no association between sequence variation in the MC1R and melanism was found in these 2 species. We conclude that the melanic gray squirrel found in the British Isles originated from one or more introductions of melanic gray squirrels from North America. We also conclude that variations in the MC1R are not associated with melanism in the fox and red squirrels.

Processing of filamentous bacteriophage virions in antigen-presenting cells targets both HLA class I and class II peptide loading compartments.

Virions of filamentous bacteriophage fd are capable of displaying multiple copies of peptide epitopes and generating powerful immune responses to them. To investigate the antigen processing mechanisms in human B cell lines used as antigen presenting cells, the major coat protein (pVIII) in intact virions was fluorescently labeled, and its localization in various intracellular compartments was followed using confocal microscopy. We show that the virions were taken up and processed to yield peptides that reach both the major histocompatibility complex (MHC) class II compartment and the endoplasmic reticulum. Moreover, when exposed to bacteriophages displaying a cytotoxic T lymphocyte (CTL) epitope from the reverse transcriptase of human immunodeficiency virus type-1 (HIV-1), B cells were lysed by specific cytotoxic lymphocytes. This confirms that filamentous bacteriophage virions are capable of being taken up and processed efficiently by MHC class I and class II pathways, even in nonprofessional antigen presenting cells. These remarkable features explain, at least in part, the unexpected ability of virions displaying foreign T-cell epitopes to prime strong T-helper-dependent CTL responses. These findings have important implications for the development of peptide-based vaccines, using filamentous bacteriophage virions as scaffolds.

Detection of anti-p53 antibodies by ELISA using p53 synthetic or phage-displayed peptides.

Anti-p53 antibodies have been detected in the sera of patients with various types of cancers. In this report, we describe the development of a new ELISA aimed at detecting anti-p53 antibodies using two peptides belonging to immunodominant epitopes of the p53 N-terminal region. We first tested the reactivity of the sera by an indirect ELISA using the peptides as a capture system. Then, the specificity of the reaction was confirmed by an inhibition assay. Two systems of peptide presentation, phage display and the streptavidin/biotin system, were evaluated. Using a panel of sera from cancer patients, both systems were found to be equally reliable, demonstrating that both peptide-based ELISAs can be used for the specific detection of anti-p53 antibodies. The presence of anti-p53 antibodies was associated with p53 alteration whether it be mutation or accumulation.