Bernard L. Strehler

The limited growth span of cell strains isolated from the chick embryo

Abstract Fibroblasts isolated from embryonic chick skin and muscle have been studied with respect to the following properties, using standard cell culture techniques and measurement of metabolite flux as well as quantitative biochemical assay: (1) Under our culture conditions these cells can undergo a maximum of 25 divisions during a culture period of about 2 months. (2) Cells isolated from 10–12-day-old embryos did not differ significantly in growth span from cells obtained from 18–20-day-old embryos. (3) A marked decrease in the number of cells present at confluency occurs between the early and late stages of culture. This decrease amounts to about 3·5 per cent generation if we assume a linear decrease in cell number. (4) The amount of DNA per cell decreases slightly at late culture stages while the ratio of RNA to DNA and of protein to DNA remains essentially constant. The lipid content per DNA appears to increase significantly. (5) The influx of glycine, leucine, and glucose, and the efflux of radioactivity from cells grown in medium containing 14 C-glycine was studied, but no significant differences were observed as a function of passage number. (6) Evidence is presented indicating that the inoculation density is an important determinant of division limitation and survival. An hypothesis explaining the phenomenon of growth limitation is advanced which is consistent with this finding and with the observations that feeder cell systems or conditioned medium increase the growth span of euploid cells in vitro .

Enzymic activities of lipofuscin age pigments: comparative histochemical and biochemical studies.

Summary 1. A number of particulate fractions have been isolated in continuous density-gradients of sucrose from homogenates of human heart, human liver, and beef heart and assayed for cytochrome oxidase (EC 1.9.3.1), acid phosphatase (EC 3.1.3.2), β -glucuronidase (EC 3.2.1.31), cathepsin, and non-specific esterase. 2. These studies were supplemented by histochemical assays of sections made from frozen pellets of the isolated fractions. 3. Non-specific esterase and acid phosphatase, histochemical activities normally found associated with liver lipofuscin in histological sections, were not found to be concentrated in the bulk of the fluorescent, brown pigmented fractions isolated from human liver. Rather, these activities as well as leucine aminopeptidase (EC 3.4.1.1), were found in more slowly-sedimenting particles forming an overlying layer of the lipofuscin pellets. The lipofuscin fractions isolated from human and beef heart had low hydrolytic activities except for cathepsin and a non-specific esterase resistant to “A” and “B” esterase inhibitors. The esterase activity appeared to be associated with particles distributed throughout the pellets. 4. These results demonstrate the value of histochemical tests on sections of particle pellets as a means of determining the homogeneity of density gradient fractions. 5. Dispersion of lipofuscin particles in phenol has been found to facilitate quantitative measurements of absorption spectra and fluorescence.

Studies on the mechanism of cellular death—I. Enzymatic changes during early and late cardiac necroses

A battery of 15 different histoenzymological tests were employed to study the enzymatic changes which occur during the death of dog myocardial cells induced by local surgically induced cardiac ischemia and in autopsy specimens of infarcted human myocardium. The sequence of events was observed at intervals from 1 to 480 hr. Succinoxidase activity was apparent both in the longitudinal and transverse axes of the myofibers. In the early necrotic phases, the network arrangement was disorganized and discrete mitochondria were observed at the edge of the infarcts. In later stages these mitochondria were associated with fat droplets. Esterase activity was located on the same particulate elements as succinoxidase but no interconnecting network was evident. In older infarcts these particles increased in size and occurred both within and at the margin of the infarcts. Esterase activities persisted for a longer period of time than did the DPN diaphorase or succinoxidase activity. Cytochrome oxidase activity persisted much longer than did either succinoxidase or DPN diaphorase activity. Acid phosphatase activity using a standard azo dye coupling technique was also particulate and increased during the early stages of an infarct. The particulate acid phosphatase activity was replaced by a smooth, homogeneous staining of the cytoplasm at about 24 hr. Acid phosphatase activity revealed by ASBI as substrate was localized in the lipofuscin inclusions. Leucine aminopeptidase activity was undetectable in early stages of cell death but reached very high persistent levels in the most advanced infarcts. ATPase activity was localized at the blood vessels as well as myocardial tissue elements. The activity increased from 4 to 64 hr after occlusion and it disappeared thereafter. ADPase and alkaline phosphatase activities demonstrated a disruption of the blood vessels in the moderately advanced infarcts. ADPase tests revealed a greater number of vascular elements than did the alkaline phosphatase methods. Leukocyte infiltration was observed at about 24 hr. Cells having positive leucine aminopeptidase activity as well as having acid phosphatase activity by the ASBI method were observed after 24 hr of infarction. Histiocytes, rich in acid phosphatase and esterase activities, appeared at about 144 hr and persisted thereafter. Human material revealed a comparable sequence of histoenzymatic events.