In vitro senescence of human keratinocyte cultures.

Abstract

ABSTRACT. Human keratinocytes have been serially cultivated in low (0.015 mM) and high (1.8 mM) calcium containing medium. The calcium concentration of the growth medium significantly influenced the cell growth period in vitro. Cells grown in low calcium medium underwent 35-40 population doublings over 16-17 passages, while cells grown in high calcium medium ceased to proliferate after 20 population doublings over 7 passages. Changing the keratinocytes from one in vitro environment to the other drastically altered the lifespan in culture of populations derived from the same primary tissue. The degree of DNA methylation of human keratinocytes was shown to decrease with age in both high and low calcium culture conditions but does not appear to be associated with differentiation.

Differential inheritance modes of DNA methylation between euchromatic and heterochromatic DNA sequences in ageing fetal bovine fibroblasts

“…In aged cells, methylation was changed such that a larger proportion of PCR products of both sequences was shown to be resistant to enzyme digestions. Thus in ageing bovine fibroblasts, all the examined euchromatic sequences constantly showed a decrease in methylation level, which agrees well with previous reports describing a reduction in DNA methylation level with passages of cultured human and mouse cells [9–11]. This suggests that a loss of 5‐methylcytosine with passages of culture is a general phenomenon shown in cultured mammalian cells.…”

Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control

“…In cell culture, KCs are uncoupled from their tissue environment that naturally provides a network of homeostatic control signals; they are induced to either retain an active proliferative state or to differentiate. However, the prolonged KC culturing leads to the induction of cellular senescence [ 4 ] and therefore not only primary KCs but also immortalized KC lines, such as HaCaT (a spontaneously immortalized cell line) [ 5 ], have been widely studied to understand various normal and altered functions of the skin. HaCaT cells represent a highly popular model system since despite some UV-inducible mutations in TP53 alleles [ 6 , 7 ] they are non-tumorigenic and have retained their capacity to differentiate [ 5 , 7 , 8 ].…”

Ultraviolet B irradiation-induced keratinocyte senescence and impaired development of 3D epidermal reconstruct

“…2b,c). NHK in this experiment rapidly reached replicative senesce due to the high calcium concentration (more than 0.1 mmol L -1 ) of the growth medium which induced the cease of cell proliferation earlier than the cultures grown under low calcium medium (less than 0.1 mmol L -1 ) (15,16). Thus, the control of calcium levels is crucial for triggering cell division or stress-induced senescence.…”