Tadao Ohno

Strict relationship between dialyzed serum concentration and cellular life span in vitro

To determine the extent of the contribution of growth factor level on the life span of human diploid fibroblasts in vitro, the growth rate changes of IMR-90 cells under altered dialyzed serum (d-FBS) concentrations were investigated at different population doubling levels (PDL). As the PDL increased, the cells showed an accelerated requirement for d-FBS in order to maintain a constant growth rate, thus indicating a rapid loss of responsiveness of the cells to serum growth factors. A similar relationship was observed when the growth rate was extrapolated to zero and the cellular life span was predicted from this relationship. The cells cultured with 0.3% and 10% d-FBS ceased their growth at 54 and 76 PDL, respectively, while their predicted life span was 58 and 80--85 PDL, respectively. The cells cultured with 0.3% d-FBS responded poorly to an increase in the d-FBS concentration after entering phase III. These results suggest that the serum growth factor level is one of the determinants of cellular life span in vitro.

Differential Recovery from Potentially Lethal Damage in Normal Human Lung Fibroblasts after Irradiation with 60Co γ-rays and Accelerated N-ion Beam

PLD recovery after irradiation with accelerated N-ion was examined and compared with that after irradiation with 60Co gamma-rays in normal human lung fibroblasts (IMR-90). No shoulder region was observed in the survival curve of the cells after N-ion irradiation. Ratios of D0 values before and after a 6-hour incubation in plateau phase were 1.1 and 1.8 for N-ion irradiation and gamma-ray irradiation, respectively. The results show that recovery after irradiation with high-LET radiation is slow and much less than after low-LET irradiation.

Distinction of G0 cells from senescent cells in cultures of non-cycling human fetal lung fibroblasts by anti-MAP-1 monoclonal antibody staining.

On staining with a monoclonal antibody raised against microtubule-associated protein-1 (MAP-1), dot-like structures were seen in the nuclei of interphase cells, but not in those of non-cycling G0-arrested cells. Dots were also not seen in the nuclei of non-cycling senescent human cells (IMR-90). A SV40-DNA-transformed subline of IMR-90 with a limited growth potential showed progressive decrease of cells with nuclei containing dots in the final stage of their lifespan. The dots appeared in G0-arrested IMR-90 cells when these cells were incubated in medium of high osmotic pressure for 3 min. In contrast, no dots appeared in senescent cells or X-ray-irradiated young cells when they were incubated in medium of high osmotic pressure. Thus irreversibly non-cycling cells could be distinguished from G0-phase cells on the level of whole cultures. The results suggest that senescent cells lose their division potential by entering an irreversible cell-cycle stage differing from G0.

Growth promotion by preventing G0-arrest does not enhance the replicative life span of human diploid fibroblasts

Abstract Platelet-derived growth factor promotes cell growth by stimulating the potential of G0-arrested cells to synthesize DNA and by preventing replicating cells from entering G0 phase. Although platelet-derived growth factor markedly increased the growth rate of IMR-90 cells, it failed to enhance the replicative life span of the cells, thereby suggesting that G0 phase experienced in the replicative life is not involved in determining the life span and that stimulation of growth rate can be dissociated from increasing life span.

Factors controlling the appearance of the immunofluorescent nuclear dots revealed with monoclonal antibody against microtubule-associated protein-1

A monoclonal antibody raised against microtubule-associated protein-1 (MAP-1) binds to nuclei of normal human fibroblasts, its binding site being detected as intranuclear immunofluorescent dots. The percentage of cells showing these dots increased with insulin, hydrocortisone, epidermal growth factor (EGF), and transferrin in serum-free medium, their effects decreasing in this order. The appearance of dots induced by serum or insulin plus hydrocortisone was dependent on the Ca2+ concentration of the medium. The Ca2+ ionophore A23187 increased the rate of appearance of cells containing dots. Conversely, the calmodulin inhibitor W-7 blocked their appearance and decreased the number of cells containing dots. The dose-response curves of W-7 for inhibition of the appearance of dots and of DNA synthesis were essentially identical. These results suggest that the appearance of intranuclear MAP-1 antigen is controlled mainly by insulin, Ca2+ and calmodulin and is associated with DNA replication.

Change of responsiveness to growth stimulation of normal cells during aging.

The sole determinant of life span of normal diploid cells in vitro has been considered to be the intrinsic doubling number of the cells (1,2,3). Recent investigations, however, suggest that extracellular factors can affect the proliferative potential of the cells. For example, hydrocortisone (4) and epidermal growth factor (5) elongate the life span of fibroblasts and keratinocytes, respectively. In vivo experiments also suggest that the milieu in old animals reduces the growth potential of hematopoietic stem cells (6). However, quantitative relationships between cellular growth potential and factors which are supposed, or have been known, to influence cellular growth potential are obscure. This report describes the relationship between the amount of growth stimulating factors and cellular growth potential in the course of aging in vitro, and discusses a possible growth property of divergent cells in a cell population.