Evidence that filopodia outgrowth is a common final pathway for fibroblast growth inhibition in vitro.

To identify events associated with fibroblast growth inhibition, the effect of two known inhibitors, interferon-alpha and all-trans retinoic acid, on the growth and surface morphology of cultured fibroblasts was examined. Interferon-alpha administered at seeding reduced both growth rate and saturation density; all-trans retinoic acid reduced only saturation density. However, both negative growth modulators were associated with an increase in filopodia outgrowth and an increase in intracellular filamentous actin in a time course corresponding to onset of growth inhibition by these agents.

Effect of donor age and prior sun exposure on growth inhibition of cultured human dermal fibroblasts by all trans-retinoic acid.

The effect of retinoic acid on human fibroblasts was studied in a cell culture model of chronologic aging and photoaging. During early exponential phase, all trans-retinoic acid significantly stimulated growth rate of adult arm-derived dermal fibroblasts but not of newborn or adult foreskin-derived fibroblasts. Retinoic acid also significantly reduced saturation density in most young adult arm-derived lines and all 24 lines derived from old adult arm and foreskin.

In-vitro studies of aging.

Research has conclusively established that normal diploid cells in culture have a limited replicative life span, and that cells from adult organisms have a shorter life span than cells from young organisms or embryos. Although it is unlikely that death of the organism is caused by failure in cellular proliferative capacity, the changes that accompany alterations in proliferative capacity may play significant roles in organismic aging. The causes of the decreased proliferative vigor of cells with age are not known, but one factor may reside in the fact that with increasing in-vitro or donor age, cells become less able to respond to mitogens.

An extract of bovine thymus stimulates human keratinocyte growth in vitro.

An extract prepared from newborn calf thymus stimulated proliferation of human keratinocytes cultured from newborn foreskins and from skin biopsies of 26 adult volunteers aged 19 to 70 years. Growth over the 7-day assay period in the basal medium was age-dependent, with newborn cultures achieving a 10-fold increase in cell number over seeding density, old adult cultures barely maintaining their seeding density and young adult cultures intermediate in proliferative capacity. Maximally stimulatory extract concentration was 5-fold higher for newborn than for adult keratinocytes, with adult cultures experiencing toxicity at doses still growth-promoting for newborn cultures.

Autocrine growth stimulation of human keratinocytes by epidermal cell-derived thymocyte-activating factor: implications for skin aging.

The monocyte-derived cytokine interleukin-1 (IL-1) has growth-promoting activity for a variety of cell types, including lymphocytes and fibroblasts. We have previously shown that the epidermal cell-derived thymocyte-activating factor (ETAF) strongly resembles IL-1 in terms of biological, biochemical, and molecular biological properties. Because some lymphokines are known ot alter epidermal cell growth and differentiation and because cultured epidermal keratinocytes are capable of autocrine growth stimulation in vitro through "conditioning" of their culture medium, we sought to evaluate the effect of ETAF on keratinocyte growth.

Use of strontium to separate calcium-dependent pathways for proliferation and differentiation in human keratinocytes.

The role of extracellular calcium (Caex) in modulating keratinocyte differentiation has been well documented, but its role in proliferation has been harder to define due to the confounding effect of terminal differentiation. Because strontium (Sr) does not induce terminal differentiation in murine keratinocytes but does mimic the stimulatory effect of Caex on DNA synthesis in chick fibroblasts, experiments were undertaken to determine if Sr could be used to separate the presumably opposing effects of Caex on the proliferation and differentiation of cultured human keratinocytes. In response to additions of SrCl2, keratinocytes in a serum-free hormone-supplemented basal medium containing 0.

Cellular senescence revisited: a review.

The field of cellular senescence (cytogerontology) is reviewed. The historical precedence for investigation in this field is summarized, and placed in the context of more recent studies of the regulation of cellular proliferation and differentiation. The now-classical embryonic lung fibroblast model is compared to models utilizing other cell types as well as cells from donors of different ages and phenotypes.

Hydrocortisone is associated with cell surface blebbing in cultures of human embryonic lung fibroblasts.

Treatment with hydrocortisone (HC) is associated with increased cell surface blebbing of proliferatively active WI-38 and IMR-90 fibroblasts at low and high population doubling levels within the first 24 h after seeding. Time course studies show a marked decrease in blebbing after 2 days. In contrast, very old cells near the end of the population life span (greater than 96% life span completed) show blebbing which is independent of HC treatment.

Growth factor responsiveness declines during adulthood for human skin-derived cells.

Keratinocytes and fibroblasts were obtained from upper arm biopsies of young (22-27 years) and old (60-82 years) adult donors. Keratinocytes were grown in serum-free medium containing variable quantities of either epidermal growth factor (EGF) or a bovine hypothalamic extract known to contain keratinocyte growth factor (KGF). Fibroblasts were grown in serum-free medium containing variable quantities of EGF or insulin.

Filopodia number increases with age and quiescence in populations of normal WI-38 cells, and is correlated with drug-induced changes in proliferation in both normal and transformed populations.

Filopodia in log and stationary phase populations of human fetal lung fibroblasts (WI-38) at low and high population doubling levels (PDLs) and of SV40 transformed WI-38 cells (VA13A), were observed and counted under different conditions of in vitro growth by scanning electron microscopy. Cells from old non-vigorously growing WI-38 populations (those at a high PDL) had more filopodia than younger populations (those at a lower PDL) at all times after seeding, and for any given population stationary phase cells (those entering, or in, quiescence), had more than log phase cells. Hydrocortisone (HC, 14 microM), which stimulates proliferation and increases life span of WI-38 cells, was associated with a marked decrease in filopodia.

Hydrocortisone modulates RA-induced growth inhibition of normal and transformed human embryonic lung fibroblasts.

All-trans retinoic acid (10(-5) M) added at seeding reduces the growth rate and saturation density of normal human embryonic lung fibroblasts of two lines (WI-38 and IMR-90) and similarly inhibits growth of SV40-transformed WI-38 cells (VA13A). The growth inhibitory effects of retinoic acid do not show serum dependency, and the viability of treated cells is 95-99% of controls. Old populations of WI-38 cells (cells at high population doubling levels) are more sensitive to the effects of retinoic acid than are young populations (cells at low population doubling levels), and population life span is reduced by continuous exposure to retinoic acid.