Reduction of DNA synthesis in aging but still proliferating cells

Abstract

It is well known that cell proliferation (and hence, DNA synthesis) declines in human diploid fibroblast-like cells with increasing passage number. It is not clear whether D N A synthesis declines in t h e remaining cells that are still actively proliferating. Estimations of cell kinetic parameters permitted extrapolations to be made that reflected the declining numbers of cells still capable of D N A replication. D N A synthesis declined with culture age in intact cells, permeabilized cells, and in the isolated nuclear matrix even when corrected for declining numbers of proliferating cells. With age, DNA polymerase alpha and beta activity in cell lysates declined, but when corrected for the remaining proliferating cells, only polymerase alpha activity declined; D N A polymerase alpha and beta activity bound to the nuclear matrix declined, but when corrected for declining proliferation, no decline was apparent for either enzyme. There was an increase in the number of S1-nuclease sensitive sites and breaks in the parental DNA of the dividing cells in older cultures. It is suggested that in aging cultures, not only does overall DNA synthesis decline owing to decreasing cell proliferation, but also that D N A synthesis declines in t h e remaining proliferating cells, that this decline is not due to decreasing amounts of D N A polymerase bound to the nuclear matrix, and that alterations in D N A structure occur.

Reduction of DNA primase activity in aging but still proliferating cells

“…[3H]thymidine (54 Ci/mmol) and [methy13HlTTP (80 Ciimmol) were obtained from New England Nuclear. Ribonucleoside and deoxyribonucleoside triphosphates, dithiothreitol (D'IT), dideoxyadenosine triphosphate (ddATP), 5-Bromodeoxyuridine (BrdU), cx-aman-Cell culture IMR 90 cultures, reported to be "passage 10" were obtained from the American Tissue Culture Collection and were maintained on Basal Medium Eagle with 10% fetal calf serum and monitored for mycoplasma contamination, each exactly as previously described for WI-38 (Collins and Chu, 1985).…”

“…Culture doubling times. The theoretical doubling times were determined from the slopes of growth curves as described by Macieira-Coelho et al, 1966), as previously described for WI-38 (Collins and Chu, 1985).…”

“…Thus, it would seem reasonable to speculate that the age-associated decline in DNA replication might be altered at the level of the nuclear matrix or the nucleoid. However, it was observed that the amounts of activity of WI-38 DNA polymerase cx (and polymerase bound to the nuclear matrix did not decline with age of WI-38 cells when corrected for the reduced number of dividing cells (Collins and Chu, 1985). Thus, as a corrected replicative enzyme deficiency had not been identified, and as no replicative enzymes other than DNA polymerase a or p had been examined for age-associated effects, we decided to scrutinize DNA primase, the enzyme responsible for the synthesis of RNA primers prior to DNA polymerase a-catalyzed chain elongation.…”

“…Although this reduced number of S-phase cells alone would account for the well-known decrease in total DNA synthesis by aged cultures, it seems clear that the remaining Sphase cells synthesize DNA at slower rates. That is4 in vivo, phase I11 cultures of MRC 5 exhibited slower Hautoradiographic chain growth (Petes et al, 19741, and, in vitro, 3H-thymidine incorporation rates of permeabilized cells were lower, even when corrected for the reduced numbers of S-phase cells (Griffths and Carpenter, 1980;Collins and Chu, 1985).…”

Impaired nuclear responsiveness to cytoplasmic signals in lymphocytes from elderly humans with depressed proliferative responses.

“…Wright and Hayflick (18) and Muggleton-Harris and Hayflick (19) fused whole cells, cytoplasts (enucleated cells), and karyoplasts (nuclei) in various stages of their lifespan and found that the mechanism controlling the proliferative potential of a cell may be located in the nucleus. In other studies on in vitro aging, Collins and Chu found that DNA synthesis was reduced in proliferating cells ofaging cultures at the level of the nuclear matrix, probably due to alterations in DNA structure (20 (26). Furthermore, B cells from the C3H/HeJ mice became responsive to LPS after being microinjected with cytosol from LPS-stimulated responder cells (10).…”