Moghaddam A, Bicknell RExpression of platelet-derived endothelial cell growth factor in Escherichia coli and confirmation of its thymidine phosphorylase activity. Biochemistry 31: 12141-12146

University of Oxford, Oxford, England, United Kingdom
Biochemistry (Impact Factor: 3.02). 12/1992; 31(48):12141-6. DOI: 10.1021/bi00163a024
Source: PubMed


Platelet-derived endothelial cell growth factor (PD-ECGF) has been expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The fusion protein was purified by one-step affinity chromatography on glutathione-agarose beads, and recombinant PD-ECGF was proteolytically cleaved with thrombin from its GST leader peptide to yield pure protein. Recombinant PD-ECGF stimulated [3H]methylthymidine uptake by endothelial cells in vitro; however, we were unable to detect stimulation of cell proliferation under a wide variety of conditions. We confirm that in accord with the recent report that PD-ECGF and human thymidine phosphorylase are products of the same gene [Furukawa, T., Yoshimura, A., Sumizawa, T., Haraguchi, M., & Akiyama, S. I. (1992) Nature 356, 668] recombinant PD-ECGF has thymidine phosphorylase activity comparable to that of E. coli thymidine phosphorylase. Further, E. coli thymidine phosphorylase was able to mimic the activity of recombinant PD-ECGF in the [3H]methylthymidine uptake assay, and it appears that recombinant PD-ECGF's effect on the uptake of thymidine by endothelial cells may be due to modulation of cellular thymidine pools. The mechanism by which PD-ECGF stimulates angiogenesis remains to be elucidated.

3 Reads
  • Source
    • "PD-ECGF, an endothelial cell mitogen that was initially purified to homogeneity from human platelets, has chemotactic activity for endothelial cells in vitro and is angiogenic in vivo [34]. PD-ECGF was shown to be identical to thymidine phosphorylase, an enzyme involved in pyrimidine nucleoside metabolism [35]. PD-ECGF expression is elevated in several types of solid tumor including colon cancer [36, 37]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Tumor angiogenesis is the result of an imbalance between positive and negative angiogenic factors released by tumor and host cells into the microenvironment of the neoplastic tissue. The stroma constitutes a large part of most solid tumors, and cancer-stromal cell interactions contribute functionally to tumor growth and metastasis. Activated fibroblasts and macrophages in tumor stroma play important roles in angiogenesis and tumor progression. In gastric cancer, tumor cells and stromal cells produce various angiogenic factors, including vascular endothelial growth factor, interleukin-8, platelet-derived endothelial cell growth factor, and angiopoietin. In addition, Helicobacter pylori infection increases tumor cell expression of metastasis-related genes including those encoding several angiogenic factors. We review the current understanding of molecular mechanisms involved in angiogenesis and lymphangiogenesis of human gastric cancer.
    Journal of Oncology 03/2010; 2010(2):468725. DOI:10.1155/2010/468725
  • Source
    • "Sequence analysis of the gene revealed a stretch of 120 amino acids to be identical to thymidine phosphorylase (TP), an enzyme catalysing the reversible phosphorolysis of thymidine to thymine and 2-deoxyribose-1-phos- phate (dR-1-P) (Furukawa et al, 1992). Subsequently, this enzymatic activity was identified for PD-ECGF (Moghaddam and Bicknell, 1992; Usuki et al, 1992). The two enzymes are considered to be identical and are designated as TP. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Platelet-derived endothelial cell growth factor (PD-ECGF)/thymidine phosphorylase (TP) catalyses the reversible phosphorolysis of thymidine to thymine and 2-deoxyribose-1-phosphate and is involved in the metabolism of fluoropyrimidines. It can also activate 5'-deoxyfluorouridine (5'DFUR) and possibly 5-fluorouracil (5FU) and Ftorafur (Ft), but inactivates trifluorothymidine (TFT). We studied the contribution of TP activity to the sensitivity for these fluoropyrimidines by modulating its activity and/or expression level in colon and lung cancer cells using a specific inhibitor of TP (TPI) or by overproduction of TP via stable transfection of human TP. Expression was analysed using competitive template-RT-PCR (CT-RT-PCR), Western blot and an activity assay. TP activity ranged from nondetectable to 70678 pmol h(-1) 10(-6) cells, in Colo320 and a TP overexpressing clone Colo320TP1, respectively. We found a good correlation between TP activity and mRNA expression (r=0.964, P&<0.01) in our cell panel. To determine the role of TP in the sensitivity to 5FU, 5'DFUR, Ft and TFT, cells were cultured with the various fluoropyrimidines with or without TPI and differences in IC(50)'s were established. TPI modified 5'DFUR, increasing the IC(50)'s 2.5- to 1396-fold in WiDR and Colo320TP1, respectively. 5-Fluorouracil could be modified by inhibiting TP but to a lesser extent than 5'DFUR: IC(50)'s increased 1.9- to 14.7-fold for WiDR and Colo320TP1, respectively. There was no effect on TFT or Ft. There appears to be a threshold level of TP activity to influence the 5'DFUR and 5FU sensitivity, which is higher for 5FU. Even high levels of TP overexpression only had a moderate effect on 5FU sensitivity.
    British Journal of Cancer 03/2003; 88(6):957-64. DOI:10.1038/sj.bjc.6600808 · 4.84 Impact Factor
  • Source
    • "BALB/c mice were immunized with 50 ␮ g of recombinant dThdPase, prepared as described elsewhere (Moghaddam and Bicknell 1992), in complete Freund's adjuvant. Boosters containing 50 ␮ g of recombinant dThdPase in complete Freund's adjuvant were given twice at 4-week intervals. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Thymidine phosphorylase (dThdPase) is an essential enzyme for activation of the oral cytostatic drug capecitabine and its intermediate metabolite, doxifluridine, to 5-fluorouracil in tumors. Methods to estimate dThdPase expression in tumor tissue might be useful to predict the efficacy of capecitabine and doxifluridine in cancer patients. We established a new monoclonal antibody (MAb), 1C6-203, applicable for dThdPase immunohistochemistry and compared its staining characteristics with those of a previously established MAb, 654-1. In 4% paraformaldehyde-fixed colorectal carcinoma, 1C6-203 and 654-1 stained cancer cells in 19/30 and 9/30 patients, respectively. In 10% formalin-fixed colorectal carcinoma, 1C6-203 and 654-1 stained cancer cells in 18/30 and 6/30 patients, respectively. In negative 10% formalin-fixed tissues, microwave treatment improved the positivity of 654-1-stained cancer cells. These results suggest that an epitope recognized by 1C6-203 is resistant to epitope masking by formaldehyde fixation, whereas that for MAb 654-1 is sensitive. Therefore, MAb 1C6-203 might be more suitable than MAb 654-1 for evaluating dThdPase expression in colorectal carcinoma.
    Journal of Histochemistry and Cytochemistry 02/2001; 49(1):131-8. DOI:10.1177/002215540104900113 · 1.96 Impact Factor
Show more

Similar Publications