Previously, it was found that senescent cells can undergo a modified cell cycle with mitotic cells as the end results. The major cycling events started with polyploidization, followed by depolyploidization to multinucleated cells (MNCs). These latter cells produced mononuclear offspring cells that could express mitotic cell divisions. In this report the emphasis is on late senescent fibroblasts that exhibited the senescence-associated change in cell morphology to large flat cells. Prior to live cell photography, flat cell cultures were maintained for months in the same culture flasks and therefore judged to be in a late senescent phase. All of the cellular events outlined above were present in these old cell cultures. Time lapse pictures showed movements of mitotic daughter cells away from each other and alignment of the chromosomes on the metaphase plate was visible in other mitotic cells. These data challenge the common view that cell senescence is irreversible and, therefore, an antitumor mechanism. A new finding was that the spike in polyploid cells in the near senescent phase consisted of cells with pairs of sister chromosomes from endoreduplication of DNA (two rounds of DNA synthesis and no mitosis). The lack of cells with 92 single chromosomes (e.g., G2 tetraploid cells) suggested that these polyploid cells also went through a changed cell cycle. The question now is whether these atypical polyploid cells are a subpopulation in senescence that can undergo the cycling from polyploidy to genome-reduced mitotic cells.

Previously, it was shown that SV40-induced cell transformation of human diploid (2N), epithelial cells was a dynamic process of nuclear and cellular events. In this process, nuclei of polyploid (above 2N) cells broke down into multinucleated cells (MNCs) by amitotic division. An induced mass karyoplast (i.e., small cell with reduced amount of cytoplasm) budding process from the MNCs produced transformed cells with extended life span (EL) and altered morphology. In this study, without the use of SV40 and no induction of karyoplast budding, the same sequence of cellular events was found to occur spontaneously for the same type of cells at replicative senescence (no mitosis). These cell transformation events were followed by phase-contrast photography of living cell cultures. Primary, diploid, epithelial cell cultures grew for two to three passages and then entered senescence. Cells remaining in the cultures after widespread cell death (mortality stage 1; M1) developed the typical large, flat-cell morphology of senescence with increased cytoplasmic volume. Some of these cells were MNCs, mostly with two to four nuclei. Cytokinesis in MNCs and spontaneous karyoplast budding from MNCs were observed, and new, limited EL cell growth was present either in foci of cells or as prolonged cell growth over one to two passages. At the end of their replicative phase, the EL cells entered another death crisis (M2) from which no cells survived. In M2-crisis, rarely transformed cells appear with immortal cell growth characteristics (i.e., cell lines). Numerous examples of fragmentation or amitosis of polyploid nuclei in the production of multinucleated cells (MNCs) are presented. Such nuclear divisions produced nuclei with unequal sizes, which suggest unbalanced chromosomal segregations. The nuclear and cellular events in cell transformation are compared with a natural (no induction) occurrence of MNC-offspring cells in mammalian placentas. The possibility of a connection between these two processes is discussed. And finally the difference in the duration of EL cell growth from SV40-MNCs versus from senescent-MNCs is ascribed to increased mutational load in SV40-induced MNCs as compared with that in senescence MNCs.

No abstract

Cell changes to accelerated rates of growth are determined by random, new gene mutations that have their origin in vitro in a morphologically visible process of cell alterations. It is a continuous process that, step by step, transforms the normal cell into extended life (EL) cells. These latter cells with limited life spans can further transform to immortalized cells (i.e., cell lines) by the same sequence of morphological cell changes. In contrast to human epithelial cells, fibroblasts in culture have not given rise spontaneously to EL cells. Therefore, it was assumed that some of the cell changes (i.e., cell indicators of the process of transformation) might not be present in near senescent fibroblast cell cultures. As a positive control to normal fibroblast expansion to senescence, the same cells were stressed by inadequate nutrition and confluence. Another positive control was cell indicators induced by SV40 infections. The consecutive sequence of the cell indicators in the transformation process reported previously were: (1) large polyploid cells with nuclei that contained more than two genomes, (2) fragmentation/amitosis of the polyploid nuclei to bi- and multinucleated cells (MNCs), and (3) nuclear budding (i.e., karyoplasts) from MNCs that gave rise to EL cell colonies with various longevities beyond the senescent phase. The present study shows that MNCs and karyoplasts were present in the near-senescent fibroblast cell cultures. Furthermore, new data on the following aspect of the cell transformation process are presented: (1) association of the nuclear fragmentation process with death of cells, (2) cytological markers that distinguish between fragmentation-MNCs versus MNCs from cell fusion, (3) cytological changes of karyoplasts that go through mitotic division to produce daughter cells, and (4) presence of two centrosomes (spindle polar bodies) in the budded karyoplasts. These new findings are discussed in regard to the nuclear fragmentation process in polyploid cells which gives rise to smaller, viable nuclei in MNCs with reduced numbers of whole genomes.

No abstract