Thymidine Labelling Index as a Criterion of Aging <i>in vitro</i>

Abstract

For logarithmically growing cultures of the human diploid cell lines WI-38 and WI-26, there is an exponential increase in the fraction of cells not incorporating [³H]dT under conditions of continuous labelling. Thymidine uptake, phosphorylation and incorporation into DNA can be correlated with cell proliferation. In addition, determination of the labelling index is reproducible within relatively broad limits of thymidine concentrations and specific activity. The plateauing phenomenon of the curve expressing percent-labelled nuclei versus time, which occurs in populations with less than 100% cycling cells, is largely due to radiation damage to the cells. The results of these studies provide insight into the population dynamics of aging fibroblast-like cell cultures. More importantly, however, the measurement of labelling index as described can be used as a reproducible and quantitative measure of the age of the diploid cell culture.

Modulation of Replicative Senescence of Diploid Human Cells by Nuclear ERK Signaling

“…Cell viability in response to apoptotic stimuli was measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Roche Applied Science) as per the manufacturer's instructions using a BioRad ELISA reader. [ 3 H]Thymidine incorporation as a measure of cell proliferation potential was performed as described previously (60).…”

Age Associated Variations in RNA Biosynthesis by Cells of the Periodontal Ligament: An Autoradiographic Study1

“…This pattern is characteristic of the uptake and utilization of 'H-U and has been extensively investigated in other mammalian cells including osteogenic and chondrogenic tissues [Owen, 1967;Prescott, 1964;Singh & Tonna, 1973, 1974Tonna & Singh, 1973, 1976. Cytoplasmic incorporation equalled nuclear values at about 8 to 16 hours, surpassed nuclear values subsequently and showed peak values at 1 to 2 days depending upon age.…”

“…The biosynthesis of ribonucleic acid (RNA) by osteogeneic and chondrogenic tissues during aging has recently been autoradiographically assessed by investigations of the utilizations and turnover of 'H-uridine by those tissues [Singh & Tonna, 1973, 1974, 1981Tonna & Singh, 1973, 1976. The present report constitutes an investigation of age-associated changes in RNA biosynthesis in four cell compartments representing the periodontal ligament of the mouse.…”

“…Similarly, the uptake of 'H-thymidine has been used to assess the response of the periodontal ligament to tooth movement [Baumrind & Buck 1970], as well as to differing consistencies of diet . The 'thymidine labelng index' has recently been recommended by Cristofalo [ 1976] as a measure of the age of diploid cells in vitro and several investigations have evaluated changes concomitant with age in the rate of cell proliferation in the oral and dental tissues [Meyer et al, 1956;Jensen, & Toto, 1968;Barakat et al, 1969;Lavelle, 1968Lavelle, , 1969Stahl et al, 1968Stahl et al, , 1969Stahl et al, 1969. Tonna, Weiss and Stahl [1972] have documented an age-associated decline in the proliferative activity of fibroblasts in the periodontal ligament of the mouse.…”

Protein synthetic errors do not increase during aging of cultured human fibroblasts.

“…Strains used are listed in Table 1. "Early passage" denotes cultures with less than 50% of the life span completed (4) and with greater than 80% of cells capable of proliferation by the thymidine labeling index (13). "Late passage" denotes cultures with greater than 90% of the life span completed (7) or with a thymidine labeling index of 20% or less.…”