Effects of hydrogen peroxide on slow- and fast-growing NIH/3T3-derived cultures: nuclear and cytoplasmic aspects related to senescence and transformation

Cellular senescence can occur with similar phenotypes in normal cells, during aging, and in tumor cells, spontaneously or after cytostasis. The fall or increase in proliferative activity are key aspects of the respective conditions, in which the levels of reactive oxygen species can vary, affecting the cellular redox homeostasis. This work aimed to study the relationships between senescence and transformation by comparing cells with different proliferative activities and phenotypes attributable to transformation (NIHs cultures) or senescence (NIHv cultures), before and after incubation with hydrogen peroxide. Both cultures were derived from the NIH/3T3 cell line, which was used here as a reference (NIHb), after the serum starvation. Our experimental model can be representative of the heterogeneity of cell subpopulations, with different degrees of transformation and senescence, found in some tumors. The characterization of the functional properties of NIHb, NIHs, and NIHv cells was performed by a morphocytometric analysis of the cell cycle progression, mitochondrial and lysosomal content/activity, and superoxide anion production. The efficiency of the lysosomal compartment was also assessed by estimating the autophagic activity and measuring lipofuscin autofluorescence. Comparisons of nuclear and cytoplasmic parameters before and after the incubation with hydrogen peroxide revealed differences in the expression and modulation of cellular senescence patterns. The treatment effects were very limited in the NIHb culture; the senescence condition was essentially maintained in the NIHv cells, while the most relevant changes were found in the NIHs cells. In the latter, the acquisition of the senescent phenotype, also demonstrated by the positivity of SA-β-galactosidase, was correlated with a decrease in proliferative activity and a change in the content/activity of the mitochondria and lysosomes, which showed similarities with the basal senescence conditions of NIHv cells. In NIHs cells, increased autophagy events and lipofuscin accumulation also indicate the establishment of cytoplasmic dynamics typical of senescence. The variable responses to hydrogen peroxide, besides depending on the different basal cytokinetic activity of the cultures examined, appeared to be related to the specific cell redox state resulting from the balance between endogenous ROS and those produced after treatment. Especially in NIHs cells, the slowing down of the cell cycle was linked to dynamic interconnections between the mitochondrial and lysosomal compartments. This would indicate that transformed cells, such as NIHs, may express morpho-functional aspects and markers typical of cellular senescence, as a consequence of the modulation of their redox state.