Cell-matrix and cell-cell adhesive interactions play important roles in the normal organization and stabilization of the cell layer in epithelial tissue. Alterations in the expression and function of ...these adhesion systems that cause a switch to a migratory phenotype in tumor invasion and metastasis are critical for the malignant conversion of epithelial cells. Thymosin beta-4 (Tbeta-4) is the major actin-sequestering protein that has been shown to be upregulated in a wide variety of human carcinomas and has been implicated to be involved in altering the motility of certain tumors. We have recently demonstrated that the growth rate, colony formation in soft agar, and motility, all good indicators for malignant progression, of SW480 colon carcinoma cells are dramatically increased by enforced Tbeta-4 expression. To test the hypothesis that overexpression of this G-actin sequestering peptide also promotes tumor invasion, we examined not only the invasion capability of Tbeta-4-overexpressing SW480 cells, but also the expression levels of Tbeta-4 as well as several proteins that participate in different stages of tumor progression in matched samples of human primary colorectal adenocarcinoma and liver metastases from several patients. A marked increase on the invasiveness in Tbeta-4-overexpressing SW480 cells with increased levels and activity of matrix metalloproteinase-7 (MMP-7) was observed. Furthermore, the levels of Fas as well as the susceptibility to Fas ligand-mediated apoptosis in Tbeta-4-overexpressing cells were significantly decreased. Interestingly, the levels of Tbeta-4 mRNA, beta-catenin, c-Myc, and MMP-7 in metastatic liver lesions were relatively higher, whereas the levels of E-cadherin and Fas were significantly lower than those in the matched primary colorectal tumors. These results suggest that upregulation of Tbeta-4, by promoting the disruption of cell-cell adhesion and a consequential activation of the beta-catenin signaling, could be a key event in the acquisition of growth advantages as well as invasive phenotypes in human colorectal carcinomas.
Thymosin beta-4 (Tbeta-4), a small peptide originally isolated from calf thymus, modulates the formation of F-actin microfilaments by sequestering the monomeric G-actin. Recent studies have shown ...that overexpression of the Tbeta-4 gene occurs not only in many human carcinomas but also in the highly metastatic melanomas and fibrosarcomas. However, little is known about the specific growth advantages acquired by different tumors from this genetic abnormality. To address the above questions, Tbeta-4-overexpressing human colon carcinoma (SW480) cells were established by stable transfection and their phenotypic changes were monitored. We found that both the morphology and the cortical actin cytoskeleton of SW480 cells were altered by Tbeta-4 overexpression. Moreover, both cellular level and that distributed over the intercellular junctions of the E-cadherin were decreased in the Tbeta-4 overexpressers, which were accompanied by a twofold increase in their saturation densities. Meanwhile, these cells also exhibited an increased ability to form colonies in soft agar. Interestingly, a dramatic increase of growth rate was detected in the Tbeta-4 overexpressers, which might be attributed to an accelerated proliferation induced by c-Myc that was activated by nuclear beta-catenin. Finally, a motility increase of these cells was demonstrated by two independent migration assays, which was accompanied by an enhanced focal contact. Taken together, our data suggest that the drastic growth property and motility changes of the SW480 cells overexpressing Tbeta-4 gene are due mainly to a deregulated cell-cell adhesion arisen from the downregulation of E-cadherin, plus uncontrolled cell proliferation owing to the upregulation of beta-catenin, both resulted from a breakdown of actin microfilaments caused by the overexpression of this G-actin sequestering peptide.
The polar glycolipids were isolated from the thermophilic bacteria Meiothermus taiwanensis ATCC BAA‐400 by ethanol extraction and purified by Sephadex LH‐20 and silica gel column chromatography. The ...fatty acid composition of O‐acyl groups in the glycolipids was obtained by gas chromatography mass spectroscopy analysis on their methyl esters derived from methanolysis and was made mainly of C15:0 (34.0%) and C17:0 (42.3%) fatty acids, with the majority as branched fatty acids (over 80%). Removal of O‐acyl groups under mild basic conditions provided two glycolipids, which differ only in N‐acyl substitution on a hexosamine. Electrospray mass spectroscopy analysis revealed that one has a C17:0 N‐acyl group and the other hydroxy C17:0 in a ratio of about 1 : 3.5. Furthermore, complete de‐lipidation with strong base followed by selective N‐acetylation resulted in a homogeneous tetraglycosyl glycerol. The linkages and configurations of the carbohydrate moiety were then elucidated by MS and various NMR analyses. Thus, the major glycolipid from M. taiwanensis ATCC BAA‐400 was determined with the following structure: α‐Galp(1‐6)‐β‐Galp(1‐6)‐β‐GalNAcyl(1,2)‐α‐Glc(1,1)‐Gro diester, where N‐acyl is C17:0 or hydroxy C17:0 fatty acid and the glycerol esters were mainly iso‐ and anteisobranched C15:0 and C17:0.
The present work was conducted to further examine the effects of thymosin β-4 (Tβ4) upregulation on the apoptosis of SW480 colon cancer cells induced by T cells and various chemotherapeutic agents ...because reduced susceptibility to the cytotoxicity of an anti-Fas IgM (CH-11) in Tβ4-overexpressing cells has previously been reported by us. As expected, Tβ4 overexpressers were also more resistant to the killing effect of FasL-bearing Jurkat T cells. On the other hand, pretreating these cells with an MMP inhibitor restored not only their Fas levels but also their sensitivity to CH-11, suggesting a pivotal role of MMP in downregulating Fas in Tβ4 overexpressers. Interestingly, while the susceptibilities of Tβ4 overexpressers to 5-FU and irinotecan remained unchanged, they were more resistant to doxorubicin and etoposide which triggered apoptosis via a mitochondrial pathway. Concordantly, activation of both caspases 9 and 3 in Tβ4 overexpressers by the two aforementioned topoisomerase II inhibitors was dramatically abrogated which could be accounted mainly by an increased expression of Survivin, a critical anti-apoptotic factor. Finally, poor survival was found in stage III colon cancer patients whose tumors were stained positively by the anti-Survivin antibody. Thus, advantages such as immune evasion and resistance to anticancer drug-induced apoptosis acquired by colon cancer cells through Tβ4 overexpression might facilitate their survival during metastasis and chemotherapy.
We previously showed that the -278 to +410 region of mouse thymosin β^sub 4^ (mTβ^sub 4^) gene supports high levels of reporter gene expression in NIH3T3 cells. This region contains part of the ...5'-flanking sequences (-278 to -1), the intact first exon (+1 to +133), and portion of the first intron (+134 to +410). However, the size of this exon is much longer than those of its rat and human counterparts. To resolve the question regarding this size discrepancy, transcription start site for the mTβ^sub 4^ gene was re-examined by primer extension and bioinformatics analyses. We found that the first exon of mTβ^sub 4^ gene spans 56 bp with its cap site situated in a putative initiator highly similar to the consensus mammalian sequence. In addition, a TATA box-like motif and two consecutive downstream promoter elements were also found. To delineate the cis-elements involved in modulating the constitutive expression of mTβ^sub 4^ gene, transient transfection assay was performed. Interestingly, expression level of the reporter gene driven by the -117 to +56 region of mTβ^sub 4^ gene was approximately 8-fold higher than that directed by the SV40 promoter and significant promoter activity was found to be associated with the smaller (-56 to +56) fragment. A nuclear protein-bound silencer was located in the region between the -167 and -118 and an enhancer whose effect did not seem to be dependent on protein binding was identified in the downstream (-117 to -88) region. However, neither of these cis-elements affected reporter expression driven by a SV40 promoter. Intriguingly, mTβ^sub 4^ promoter functioned well in human colorectal (SW480) and cervical (HeLa) carcinoma cells. Taken together, our findings not only provide crucial information for further elucidation of the transcriptional regulation of mTβ^sub 4^ gene but also raise the possibility of utilizing its promoter to produce large quantity of recombinant proteins in mammalian cells.PUBLICATION ABSTRACT
We previously showed that the -278 to +410 region of mouse thymosin beta sub(4) (mT beta sub(4)) gene supports high levels of reporter gene expression in NIH3T3 cells. This region contains part of ...the 5'-flanking sequences (-278 to -1), the intact first exon (+1 to +133), and portion of the first intron (+134 to +410). However, the size of this exon is much longer than those of its rat and human counterparts. To resolve the question regarding this size discrepancy, transcription start site for the mT beta sub(4) gene was re-examined by primer extension and bioinformatics analyses. We found that the first exon of mT beta sub(4) gene spans 56 bp with its cap site situated in a putative initiator highly similar to the consensus mammalian sequence. In addition, a TATA box-like motif and two consecutive downstream promoter elements were also found. To delineate the cis-elements involved in modulating the constitutive expression of mT beta sub(4) gene, transient transfection assay was performed. Interestingly, expression level of the reporter gene driven by the -117 to +56 region of mT beta sub(4) gene was approximately 8-fold higher than that directed by the SV40 promoter and significant promoter activity was found to be associated with the smaller (-56 to +56) fragment. A nuclear protein-bound silencer was located in the region between the -167 and -118 and an enhancer whose effect did not seem to be dependent on protein binding was identified in the downstream (-117 to -88) region. However, neither of these cis-elements affected reporter expression driven by a SV40 promoter. Intriguingly, mT beta sub(4) promoter functioned well in human colorectal (SW480) and cervical (HeLa) carcinoma cells. Taken together, our findings not only provide crucial information for further elucidation of the transcriptional regulation of mT beta sub(4) gene but also raise the possibility of utilizing its promoter to produce large quantity of recombinant proteins in mammalian cells.
We previously showed that the -278 to +410 region of mouse thymosin beta4 (mT,beta4) gene supports high levels of reporter gene expression in NIH3T3 cells. This region contains part of the ...5'-flanking sequences (-278 to -1), the intact first exon (+1 to +133), and portion of the first intron (+134 to +410). However, the size of this exon is much longer than those of its rat and human counterparts. To resolve the question regarding this size discrepancy, transcription start site for the mTbeta4 gene was re-examined by primer extension and bioinformatics analyses. We found that the first exon of mTbeta4 gene spans 56 bp with its cap site situated in a putative initiator highly similar to the consensus mammalian sequence. In addition, a TATA box-like motif and two consecutive downstream promoter elements were also found. To delineate the cis-elements involved in modulating the constitutive expression of mTbeta4 gene, transient transfection assay was performed. Interestingly, expression level of the reporter gene driven by the -117 to +56 region of mTbeta4 gene was approximately 8-fold higher than that directed by the SV40 promoter and significant promoter activity was found to be associated with the smaller (-56 to +56) fragment. A nuclear protein-bound silencer was located in the region between the -167 and -118 and an enhancer whose effect did not seem to be dependent on protein binding was identified in the downstream (-117 to -88) region. However, neither of these cis-elements affected reporter expression driven by a SV40 promoter. Intriguingly, mTbeta4 promoter functioned well in human colorectal (SW480) and cervical (HeLa) carcinoma cells. Taken together, our findings not only provide crucial information for further elucidation of the transcriptional regulation of mTbeta4 gene but also raise the possibility of utilizing its promoter to produce large quantity of recombinant proteins in mammalian cells.
We previously showed that the -278 to +410 region of mouse thymosin b sub(4) (mTb sub(4)) gene supports high levels of reporter gene expression in NIH3T3 cells. This region contains part of the ...5'-flanking sequences (-278 to -1), the intact first exon (+1 to +133), and portion of the first intron (+134 to +410). However, the size of this exon is much longer than those of its rat and human counterparts. To resolve the question regarding this size discrepancy, transcription start site for the mTb sub(4) gene was re-examined by primer extension and bioinformatics analyses. We found that the first exon of mTb sub(4) gene spans 56 bp with its cap site situated in a putative initiator highly similar to the consensus mammalian sequence. In addition, a TATA box-like motif and two consecutive downstream promoter elements were also found. To delineate the cis-elements involved in modulating the constitutive expression of mTb sub(4) gene, transient transfection assay was performed. Interestingly, expression level of the reporter gene driven by the -117 to +56 region of mTb sub(4) gene was approximately 8-fold higher than that directed by the SV40 promoter and significant promoter activity was found to be associated with the smaller (-56 to +56) fragment. A nuclear protein-bound silencer was located in the region between the -167 and -118 and an enhancer whose effect did not seem to be dependent on protein binding was identified in the downstream (-117 to -88) region. However, neither of these cis-elements affected reporter expression driven by a SV40 promoter. Intriguingly, mTb sub(4) promoter functioned well in human colorectal (SW480) and cervical (HeLa) carcinoma cells. Taken together, our findings not only provide crucial information for further elucidation of the transcriptional regulation of mTb sub(4) gene but also raise the possibility of utilizing its promoter to produce large quantity of recombinant proteins in mammalian cells.
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