Cellular trafficking of thymosin beta-4 in HEPG2 cells following serum starvation.

Thymosin beta-4 (Tß4) is an ubiquitous multi-functional regenerative peptide, related to many critical biological processes, with a dynamic and flexible conformation which may influence its functions and its subcellular distribution. For these reasons, the intracellular localization and trafficking of Tß4 is still not completely defined and is still under investigation in in vivo as well as in vitro studies. In the current study we used HepG2 cells, a human hepatoma cell line; cells growing in normal conditions with fetal bovine serum expressed high levels of Tß4, restricted to the cytoplasm until 72 h. At 84 h, a diffuse Tß4 cytoplasmic immunostaining shifted to a focal perinuclear and nuclear reactivity. In the absence of serum, nuclear reactivity was localized in small granules, evenly dispersed throughout the entire nuclear envelop, and was observed as earlier as at 48 h. Cytoplasmic immunostaining for Tß4 in HepG2 cells under starvation appeared significantly lower at 48 h and decreased progressively at 72 and at 84 h. At these time points, the decrease in cytoplasmic staining was associated with a progressive increase in nuclear reactivity, suggesting a possible translocation of the peptide from the cytoplasm to the nuclear membrane. The normal immunocytochemical pattern was restored when culture cells submitted to starvation for 84 h received a new complete medium for 48 h. Mass spectrometry analysis, performed on the nuclear and cytosolic fractions of HepG2 growing with and without serum, showed that Tß4 was detectable only in the cytosolic and not in the intranuclear fraction. These data suggest that Tß4 is able to translocate from different cytoplasmic domains to the nuclear membrane and back, based on different stress conditions within the cell. The punctuate pattern of nuclear Tß4 immunostaining associated with Tß4 absence in the nucleoplasm suggest that this peptide might be localized in the nuclear pores, where it could regulate the pore permeability.

Thymosin ß4 expression reveals intriguing similarities between fetal and cancer cells.

Thymosin ß4 (Tß4) is highly expressed in saliva of human newborns but not in adults. Here preliminary immunohistochemical analyses on different human tissues are reported. Immunoreactivity for Tß4 in human salivary glands show high quantities of Tß4 before birth, followed by downregulation of expression in adulthood. In contrast, Tß4 is detected in tumors of salivary glands, suggesting that tumor cells might utilize fetal programs, including Tß4 synthesis. Immunohistochemical analyses in the gastrointestinal tract showed strong reactivity for Tß4 in enterocytes during development, but weak immunostaining in mature enterocytes. In colorectal cancer, the association of a high expression of Tß4 with epithelial-mesenchymal transition was observed. On the basis of these data, the process of epithelial-mesenchymal transition could represent the unifying process that explains the role of Tß4 during fetal development and in cancer progression.

Thymosin ß 4 in colorectal cancer is localized predominantly at the invasion front in tumor cells undergoing epithelial mesenchymal transition.

OBJECTIVE: Thymosin ß 4 (Tß(4)) is a ubiquitous peptide that plays pivotal roles in the cytoskeletal system and in cell differentiation during embryogenesis. Recently, a role for Tß(4) has been proposed in experimental and human carcinogenesis. This study was aimed at evaluating the correlation between Tß(4) immunoractivity and colorectal cancer, with particular attemption to tumor cells undergoing epithelial-mesenchymal transition. METHODS AND RESULTS: 86 intestinal biopsies were retrospectively analyzed including 76 colorectal adenocarcinomas with evident features of epithelial-mesenchymal transition, and 10 samples of normal colorectal mucosa. Paraffin sections were immunostained for Tß(4) and for E-cadherin. Total RNA was isolated from frozen specimens obtained, at surgery, from the normal colon mucosa, the deeper regions and the superficial tumor regions in four cases of colon cancer. Tß(4) immunoreactivity was detected in the vast majority (59/76) of colon carcinomas, showing a patchy distribution, with well differentiated areas significantly more reactive than the less differentiated tumor zones. We also noted a zonal pattern in the majority of tumors, characterized by a progressive increase in immunostaining for Tß(4) from the superficial toward the deepest tumor regions. The strongest expression for Tß(4) was frequently detected in invading tumor cells with features of epithelial-mesenchymal transition. The increase in reactivity for Tß(4) matched with a progressive decrease in E-cadherin expression in invading cancer cells. At mRNA level, the differences in Tß(4) expression between the surrounding colon mucosa and the tumors samples were not significant. CONCLUSIONS: Our data show that Tß(4) is expressed in the majority of colon cancers, with preferential immunoreactivity in deep tumor regions. The preferential expression of the peptide and the increase in intensity of the immunostaining at the invasion front suggests a possible link between the peptide and the process of epithelial mesenchymal transition, suggesting a role for Tß(4) in colorectal cancer invasion and metastasis.

Different thymosin Beta 4 immunoreactivity in foetal and adult gastrointestinal tract.

BACKGROUND: Thymosin beta 4 (Tbeta(4)) is a member of beta-thymosins, a family of peptides that play essential roles in many cellular functions. A recent study from our group suggested a role for Tbeta(4) in the development of human salivary glands. The aim of this study was to analyze the expression of Tbeta(4) in the human gut during development, and in the adult. METHODOLOGY/PRINCIPAL FINDINGS: Immunolocalization of Tbeta(4) was studied in autoptic samples of tongue, oesophagus, stomach, ileum, colon, liver and pancreas obtained from two human foetuses and two adults. Tbeta(4) appeared unevenly distributed, with marked differences between foetuses and adults. In the stomach, superficial epithelium was positive in foetuses and negative in adults. Ileal enterocytes were strongly positive in the adult and weakly positive in the foetuses. An increase in reactivity for Tbeta(4) was observed in superficial colon epithelium of adults as compared with the foetuses. Striking differences were found between foetal and adult liver: the former showed a very low reactivity for Tbeta(4) while in the adult we observed a strong reactivity in the vast majority of the hepatocytes. A peculiar pattern was found in the pancreas, with the strongest reactivity observed in foetal and adult islet cells. SIGNIFICANCE: Our data show a strong expression of Tbeta(4) in the human gut and in endocrine pancreas during development. The observed differential expression of Tbeta(4) suggests specific roles of the peptide in the gut of foetuses and adults. The observed heterogeneity of Tbeta(4) expression in the foetal life, ranging from a very rare detection in liver cells up to a diffuse reactivity in endocrine pancreas, should be taken into account when the role of Tbeta(4) in the development of human embryo is assessed. Future studies are needed to shed light on the link between Tbeta(4) and organogenesis.