Enzyme levels of normal human cells: Aging in culture

Abstract

Normal human embryonic lung fibroblasts, WI-38 cells are characterized by a limited number of population doublings when cultured in vitro. In the present study, the specific activities of four enzymes representing markers for four different subcellular particles, were measured during the in vitro lifespan of these cells. Serially subcultivated mother-daughter cell lines were used. Enzyme specific activities were measured either by pseudo-zero or first-order reactions of cytoplasmic extracts containing about 90% of the total subcellular particles. Enzyme specific activity exhibited significant fluctuations among adjacent passage levels (a measure of population doublings) even when cells were harvested within a fixed time interval in the cell proliferation cycle. By normalizing with cell count number, the fluctuations of enzyme specific activity were minimized and clear relationships between cell passage level and enzyme specific activities were established. N-acetyl-β-glucosaminidase specific activity increased about 200% between passage level 20–50, while 5′-nucleotidase and cytochrome oxidase increased about 300%. Catalase activity showed no obvious correlation with cell count number or passage level. During the passage levels immediately preceding the termination of cell cultures, 5′-nucleotidase specific activity increased sharply up to tenfold of the normal range of enzyme activity. The causes for this large increase and its effect on nucleotide metabolism in senescent cells could be important in the in vitro aging phenomenon.