Protein turnover in serum-deprived mammalian cell cultures
The aim of this project is to test the hypothesis that the rate of turnover of newly synthesized proteins plays a major part in the regulation of cell growth. Normal and transformed cells have been used to compare their responses to serum levels. When the cells are cultured in 0.2% serum medium, increase of cell number is slower than in normal cultures. Cell volume is reduced, while cell mass may be maintained or decreased depending on the rate of protein accumulation and cell proliferation, so unbalanced growth may occur in certain circumstances. During serum deprivation, accumulation of newly synthesized proteins slows down due to both aspects of reduced protein synthesis rate and increased degradation of proteins, mainly, medium- and long-lived proteins. After serum restoration, their rate of protein synthesis increased, and the rate of long-lived protein degradation was reduced to a lower level than in cells kept continuously in 10&37 serum, indicating that medium- and long-lived proteins are more important than short-lived proteins in the regulation of cell growth. After serum step-down, untransformed 3T3 cells did not enter a truly G0 state, but showed a very slow increase in cell number; transformed 3T3 and HeLa cells showed 'unbalanced growth', i.e. cell number increased faster than proteins accumulated. Serum deprivation quickly and substantially affected growth of untransformed 3T3 cells, but transformed 3T3 and HeLa cells retain their growth rate higher than that of untransformed 3T3 cells. Contrary to general belief, the transformed 3T3 cells and HeLa cells were quite responsive in many respects to serum withdrawal. This fact indicates that stimulating growth by 'autocrine' mechanism is limited. Investigation of the pathways of protein degration and rate of RNA synthesis were undertaken. Attention was paid to basic problems associated with the serum deprivation, and the serum-deprived cell culture system as a model or paradigm for studies of this nature is discussed.