Changes in Lysosomal Associated Structures in Human Fibroblasts Kept in Resting Phase

Abstract

Cell division can be induced within 24 hr after the addition of fresh medium to several different cell types in the resting phase (1-7). Human fibroblasts respond in the same fashion if stimulated soon after the cells reach the resting phase (5). However, when the cells are kept for a few days in the resting phase before stimulation, fewer cells enter the division cycle and eventually the whole cell population fails to divide. The present work investigates the physiological and structural changes associated with the delay between stimulation and initiation of cell division.

Materials and Methods. Cell culture. The WI-38 human embryonic diploid line was maintained in Eagle's essential medium supplemented with 10% calf serum as described by Hayflick (8). Cultures were considered to be in the resting phase when cell division had virtually ceased following proliferation in the same medium after a 1:2 split (9). For each experiment, cells were pooled and distributed evenly into culture flasks.

Biochemical analyses. Deoxyribonucleic acid (DNA) synthesis was measured adding 0.1 μC/ml of tritium-labeled thymidine (³H-TdR) to the cultures which were treated with trichloroacetic acid (TCA) 24 hr later as described by Levine et al. (10). The acid precipitate was dissolved in 1 ml of soluene (Packard Co.) added to 12 ml of Liquifluor (Packard Co.) and the radioactivity was measured in a liquid scintillation spectrometer. Quenching corrections were made when necessary. Protein determinations were performed by the method of Lowry et al. (11) and acid phosphatase measurements were made according to De Mars (12). Enzyme activity is expressed as micromoles of p-nitrophenol produced per hour. In all measurements, each value represents the mean of duplicate samples.

Electron microscopy. Cell monolayers grown in 30 ml plastic Falcon flasks were fixed in situ with glutaraldehyde (5% in cacodylate buffer) for 1 hr; treated with osmium tetroxide (2% in cacodylate buffer) for 30 min; and dehydrated with 50, 75, 95%, and finally absolute ethanol.