Oncogenically transnformed mammalian cells irrespective of their origin synthesize and secrete osteopontin (OPN), a sialic acid rich, adhesive, phosphoglycoprotein, not only in excessive amounts but also in different molecular forms, as compared to their non-transformed counterparts. It has been postulated that OPN has important functional roles in oncogenesis, but its mechanism of action remains obscure. In the present study this question was addressed by using Rat-1 cells transformed by a temperature-sensitive mutant of Rous sarcoma virus (tsB77 cells) which secrete two discrete molecular forms of OPN, a 69-kDa OPN at the non-permissive temperature (41°C and a 62-kDa form at the permissive temperature (34°C). Investigations were aimed to determine how the two isoforms of OPN secreted by transformed and non-transformed cells originate, whether the two forms have different functional properties, and the effects of specific inhibition of OPN synthesis on the transformed state of the cells. The latter was achieved by transfecting tsB77 cells with antisense OPN cDNA at the permissive temperature. Immunoprecipitation, V8 protease mapping, tryptic peptide analysis, and thrombin digestion confirmed that 62-kDa and 69-kDa proteins are two isoforms of OPN. It was also observed that tsB77 cells at both temperatures transcribe a single 1.6 kb OPN mRNA and contain only the 69-kDa OPN intracellularly, suggesting that 69-kDa OPN is modified to its 62-kDa form prior to or immediately after secretion by cells at 34ºC. Proteolytic cleavage, differential phosphorylation, or lack of N- or O-linked carbohydrates as the possible mechanism for the generation of 62-kDa OPN were ruled out, but it was observed that 62-kDa OPN contains significantly reduced levels of sialic acid residues, as compared to its 69-kDa form. The binding assays using 32P-labeled OPN revealed that only the 69-kDa OPN, not its 62-kDa form, undergoes receptor-mediated localization on the cell surface,
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.34681 |
| Date | January 1997 |
| Creators | Shanmugam, Vijayalakshmi. |
| Contributors | Mukherjee, Barid B. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001618393, proquestno: NQ37025, Theses scanned by UMI/ProQuest. |
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