Role of dolichyl phosphate, N-linked glycosylation and cell membrane expression of insulin-like growth factor-1 receptor in maintenance of malignant cell growth

Several studies have demonstrated that mevalonate (MVA) synthesis is required for cell proliferation. The mechanisms underlying MVA-regulated cell growth are still not fully understood. One conceivable mechanism for MVA-dependent cell growth is N-linked glycosylation of growth regulatory proteins. It has for example been proposed that MVA may be important for N-linked glycosylation and function of the insulin-like growth factor 1 receptor (IGF-1R). A reduced rate of N-linked glycosylation, induced by tunicamycin (TM), has been shown to kill malignant cells. The aims of this thesis were to clarify the role of MVA in regulation of N-linked glycosylation and cell membrane expression of IGF-1R with special regard to growth control of malignant cells, and to elucidate the mechanisms involved in TM induced cell death.

Inhibition of MVA synthesis led to depression of N-linked glycosylation and growth arrest in melanoma cells. This was correlated to a drastic decrease in translocation of IGF-1R to the cell surface. By stimulating MVA-depleted arrested cells I found that IGF-1R expression occurred before initiation of DNA synthesis. Using alpha R-3, an antibody blocking the IGF-1R binding domain, it was confirmed that the IGF-1R expression was necessary for initiation of DNA synthesis. I could also provide evidence that dolichyl phosphate, by its participation in N-linked glycosylation of IGF-1R, comprises the MVA product involved in cell growth control. Furthermore, my results suggest that the low expression of membrane-bound IGF-1R in estrogen-negative breast cancer cells is due to a low rate of de novo synthesis of dolichyl phosphate and N-linked glycosylation of IGF-1R. Since these cells normally produce functional (ligand-binding) IGF-1R and synthesize IGF-1 my data raise the possibility that dolichyl phosphate maybe important for whether breast cancer cells use an extracellular or intracellular autocrine IGF-1 pathway.

Inhibition of N-linked glycosylation with tunicamycin induced apoptosis in melanoma cells and virus-transformed fibroblasts. In melanoma cells this effect was due to a down regulation of IGF-1R at the cell surface. In SV40-transformed cells the apoptotic effect, which was mediated through very rapid mechanisms, was related to an increase in cytoplasmic Ca2+.