Abstract
Heparin affin regulatory peptide (HARP) is an heparin-binding growth factor, highly expressed in several primary human tumors and considered as a rate-limiting angiogenic factor in tumor growth, invasion, and metastasis. Implication of this protein in carcinogenesis is linked to its mitogenic, angiogenic, and transforming activities. Recently, we have demonstrated that the C-terminal residues 111-136 of HARP are required for its mitogenic and transforming activities (Bernard-Pierrot, I., Delbe, J., Caruelle, D., Barritault, D., Courty, J., and Milhiet, P. E. (2001) J. Biol. Chem. 276, 12228-12234). In this paper, HARP deleted of its last 26 amino acids was shown to act as a dominant negative effector for its mitogenic, angiogenic, transforming, and tumor-formation activities by heterodimerizing with the wild type protein. Similarly, the synthetic corresponding peptide P111-136 displayed in vitro inhibition of wild type HARP activities, but in this case, the inhibition was mainly explained by the competition of the peptide with HARP for the binding to the extracellular domain of the high affinity ALK receptor.
Highlights
The expression of polypeptide growth factors is tightly regulated and contributes to the timely development of tissues during embryonic and neonatal growth
Heparin affin regulatory peptide (HARP) is expressed in many human tumors and tumoral cell lines including neuroblastoma, glioblastoma, melanoma, pancreatic, and breast cancers (6 –10) and can be an in vivo ratelimiting angiogenic factor in tumor growth and metastasis [29, 30]
We have demonstrated the involvement of the C-terminal 111–136 amino acid of HARP in the mitogenic and in the transforming activities of this growth factor
Summary
Materials—Culture medium, fetal calf serum (FCS), and G418 were supplied by Invitrogen. For these clones, the presence of both HARP and mutant proteins in the culture medium was further investigated by Western blotting after heparin-Sepharose purification. Chemical Cross-linking Experiment—Purified recombinant HARP or H⌬111–136 (15 pmol) were pre-incubated for 60 min at 25 °C in the presence of 10 g/ml heparin and treated with 0.5 mM disuccinimidyl suberate according to the manufacturer’s protocol, and products were analyzed using a 15% acrylamide SDS-PAGE and anti-HARP Western blotting experiment. The resulting RECA-His cDNA was further subcloned into the pCEP4 vector (Invitrogen) to generate pCEP4-RECA-His. The human embryonic kidney HEK-293 cell line transfected with the EBNA-1 gene (Invitrogen) was cultured in DMEM containing 10% FCS and 0.4 mg/ml geneticin at 37 °C in 5% CO2. Bound antibodies were visualized using a peroxidase-labeled goat antirabbit antibodies at a 1:5000 dilution in PBS-T containing 1% BSA, and the peroxidase activity was detected with 3,3Ј,5,5Ј-tetramethyl benzidine dihydrochloride substrate according to the supplier
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