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

Abstract

A number of studies suggest a critical role of N-linked glycosylation for cellular proliferation. In addition, many transformed and tumor cells have an altered 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and an increased N-linked glycosylation. This thesis aimed to investigate the relationship between N-linked glycosylation and initiation of DNA synthesis, and to compare the role of N-linked glycosylation 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 overcome the inhibited DNA synthesis due to MVA-depletion, both in normal fibroblasts and in melanoma cells. The rescue of DNA synthesis was abrogated by a monoclonal antibody against 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-linking assays with 125I-IGF-l, and immunoprecipitation of radiolabelled IGF-lRs, it was confirmed that N-linked glycosylation of the IGF- lR was critical for its expression on the cell surface. Furthermore, the expression of the IGF-lR at the plasma membrane was a prerequisite for initiation of DNA synthesis. As a result of experimental inhibition of HMG-CoA reductase activity, the level of intracellular IGF-lRs was only slightly depressed, while the membrane-bound IGF-lRs were drastically suppressed. The membrane IGF-lR expression on the cell surface was restored by MVA and re-suppressed by TM, suggesting that MVA regulates IGF-lR expression at the co-translational level. These results suggest that N-linked glycosylation of the IGF-lR is necessary for its translocation to the cell surface. Like serum, platelet-derived growth factor (PDGF) also stimulated DNA synthesis in normal fibroblasts in a MVA-dependent manner. The prereplicative period preceding PDGF-induced DNA synthesis was demonstrated to include an increase in the number of IGF-lRs on the cell surface.

In support of earlier studies, these results confirm the role of PDGF as a competence factor, making cells responsive to progression factors 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 on cell morphology, nuclear condensation and TM-induced DNA-degradation. Furthermore, investigations of the role of Ca2+ in this process indicated that a cytoplasmic increase in calcium is a part of the TM-evoked apoptotic response.