Studies on N-linked glycosylation in proliferation and viability of normal and tumor cells in vitro

Abstract: STUDIES ON N-LINKED GLYCOSYLATION IN PROLIFERATION ANDVIABILITY OF NORMAL AND TUMOR CELLS IN VITROby Magdnlena CarlbergUnit of Tumor Pathology, Department of Oncology and Pathology, Karolinska Institute,S-l 71 77 Stockholm, Sweden.A number of studies suggest a critical role of N-linked glycosylation for cellularproliferation. In addition, many transformed and tumor cells have an altered 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and an increased N-linkedglycosylation. This thesis aimed to investigate the relationship between N-linkedglycosylation and initiation of DNA synthesis, and to compare the role of N-linkedglycosylation for the proliferation and viability of normal and tumor cells. It was found that tunicamycin (TM), an inhibitor of N-linked glycosylation,selectively killed Simian virus 40 (SV40)-transformed as opposed to normal fibroblasts,in a cell cycle-specific manner. A study of the role of N-linked glycosylation for DNA synthesis in serum-stimulated normal fibroblasts demonstrated that N-linked glycosylation was mevalonate(MVA)-dependent and was required for initiation of DNA synthesis. The synthesis of N-linked glycoproteins, which were determined to be 90-240 kDa, was confined to the mid-prereplicative period (4-8 h after serum stimulation). High doses of insulin-like growth factor-1 (IGF-1) and insulin could overcomethe inhibited DNA synthesis due to MVA-depletion, both in normal fibroblasts and inmelanoma cells. The rescue of DNA synthesis was abrogated by a monoclonal antibodyagainst the alpha subunit of the IGF-1 receptor (IGF-lR), suggesting that the IGF-1/insulin-mediated effect was due to interaction with the IGF-lR. By binding and cross-linkingassays with 125I-IGF-l, and immunoprecipitation of radiolabelled IGF-lRs, it wasconfirmed that N-linked glycosylation of the IGF- lR was critical for its expression on thecell surface. Furthermore, the expression of the IGF-lR at the plasma membrane was aprerequisite for initiation of DNA synthesis. As a result of experimental inhibition of HMG-CoA reductase activity, the level ofintracellular IGF-lRs was only slightly depressed, while the membrane-bound IGF-lRswere drastically suppressed. The membrane IGF-lR expression on the cell surface wasrestored by MVA and re-suppressed by TM, suggesting that MVA regulates IGF-lRexpression at the co-translational level. These results suggest that N-linked glycosylationof the IGF-lR is necessary for its translocation to the cell surface. Like serum, platelet-derived growth factor (PDGF) also stimulated DNAsynthesis in normal fibroblasts in a MVA-dependent manner. The prereplicative periodpreceding PDGF-induced DNA synthesis was demonstrated to include an increase in thenumber of IGF-lRs on the cell surface. In support of earlier studies, these resultsconfirm the role of PDGF as a competence factor, making cells responsive to progressionfactors like IGF-1 by increasing the number of IGF-lRs on the cell surface. TM was found to also kill human SV40-transformed, but not normal, fibroblasts.TM-induced cell death was demonstrated to be due to apoptosis, as assayed by studies oncell morphology, nuclear condensation and TM-induced DNA-degradation. Furthermore,investigations of the role of Ca2+ in this process indicated that a cytoplasmic increase incalcium is a part of the TM-evoked apoptotic response.Key Words: Apoptosis, DNA synthesis, IGF-1 receptor, HMG-CoA reductase,lovastatin, mevalonate, N-linked glycosylation, PDGF, SV40-transformation, tumor,tunicamycin.ISBN 91-628-2051-6