We have reported that mitochondrial DNA-depleted rho(0) cells are resistant to cell death. Because aged cells have frequent mitochondrial DNA mutations, the resistance of rho(0) cells against cell death might be related to the apoptosis resistance of aged cells and frequent development of cancers in aged individuals. We studied if rho(0) cells have features simulating aged cells. SK-Hepl hepatoma rho(0) cells showed typical morphology associated with aging such as increased size and elongated appearance. They had increased senescence-associated beta-Gal activity, lipofuscin pigment, and plasminogen activator inhibitor-1 expression. Consistent with their decreased proliferation, the expression of mitotic cyclins was decreased and that of cdk inhibitors was increased. Rb hypophosphorylation and decreased telomerase activity were also noted. Features simulating aged cells were also observed in MDA-MB-435 rho(0) cells. These results support the mitochondrial theory of aging, and suggest that rho(0) cells could serve as an in vitro model for aged cells. (C) 2004 Elsevier Inc. All rights reserved.