Spontaneous cell transformation: karyoplasts derived from multinucleated cells produce new cell growth in senescent human epithelial cell cultures

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.     

Human diploid fibroblast cells in senescence; Cycling through polyploidy to mitotic cells

Summary 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.     

Cellular aging studied by the reconstruction of replicating cells from nuclei and cytoplasms isolated from normal human diploid cells

Cytochalasin B (CB) was used to enucleate cells (cytoplasts) and to obtain karyoplasts (nuclei) from the human diploid fetal lung fibroblast strain WI-38. Fusion of cytoplasts and nuclei from young and old cells was accomplished with the aid of inactivated Sendai virus. Viable nuclei may be obtained from the karyoplast pellet after passage through a layer of bovine albumin which retains any contamination cytoplasts. The majority of successful fusions forming “whole cells” occurred when cytoplast from “old” cultures (PDL 40–51) and karyoplasts from “young” cultures were used (PDL 12–22), but almost always resulted in limited division of the viable reconstructed cells. When successful fusion occurred between “young” cytoplasts and “young” karyoplasts the number of cell divisions obtained was comparable to control cells kept under similar conditions.     

Stem cells, senescence, neosis and self-renewal in cancer

We describe the basic tenets of the current concepts of cancer biology, and review the recent advances on the suppressor role of senescence in tumor growth and the breakdown of this barrier during the origin of tumor growth. Senescence phenotype can be induced by (1) telomere attrition-induced senescence at the end of the cellular mitotic life span (MLS*) and (2) also by replication history-independent, accelerated senescence due to inadvertent activation of oncogenes or by exposure of cells to genotoxins. Tumor suppressor genes p53/pRB/p16INK4A and related senescence checkpoints are involved in effecting the onset of senescence. However, senescence as a tumor suppressor mechanism is a leaky process and senescent cells with mutations or epimutations in these genes escape mitotic catastrophe-induced cell death by becoming polyploid cells. These polyploid giant cells, before they die, give rise to several cells with viable genomes via nuclear budding and asymmetric cytokinesis. This mode of cell division has been termed neosis and the immediate neotic offspring the Raju cells. The latter inherit genomic instability and transiently display stem cell properties in that they differentiate into tumor cells and display extended, but, limited MLS, at the end of which they enter senescent phase and can undergo secondary/tertiary neosis to produce the next generation of Raju cells. Neosis is repeated several times during tumor growth in a non-synchronized fashion, is the mode of origin of resistant tumor growth and contributes to tumor cell heterogeneity and continuity. The main event during neosis appears to be the production of mitotically viable daughter genome after epigenetic modulation from the non-viable polyploid genome of neosis mother cell (NMC). This leads to the growth of resistant tumor cells. Since during neosis, spindle checkpoint is not activated, this may give rise to aneuploidy. Thus, tumor cells also are destined to die due to senescence, but may escape senescence due to mutations or epimutations in the senescent checkpoint pathway. A historical review of neosis-like events is presented and implications of neosis in relation to the current dogmas of cancer biology are discussed. Genesis and repetitive re-genesis of Raju cells with transient "stemness" via neosis are of vital importance to the origin and continuous growth of tumors, a process that appears to be common to all types of tumors. We suggest that unlike current anti-mitotic therapy of cancers, anti-neotic therapy would not cause undesirable side effects. We propose a rational hypothesis for the origin and progression of tumors in which neosis plays a major role in the multistep carcinogenesis in different types of cancers. We define cancers as a single disease of uncontrolled neosis due to failure of senescent checkpoint controls.     

Apoptosis is involved in the senescence of endothelial cells induced by angiotensin II

Vascular endothelial cells have a finite cell lifespan and eventually enter an irreversible growth arrest, cellular senescence. The functional changes associated with cellular senescence are thought to contribute to human aging and age-related cardiovascular disorders, e.g. atherosclerosis. In this study, induction of Angiotensin II (Ang II) promoted a growth arrest with phenotypic characteristics of cell senescence, such as enlarged cell shapes, increased senescence-associated beta-galactosidase (SA-beta-gal) positive staining cell, and depressed cell proliferation. Apoptotic changes were increased in senescent cells, with a small subset of the senescent cells showing aberrant morphology such as pronounced nuclear fragmentation or multiple micronuclei. The results suggest cell apoptosis is possibly an important factor in the process of pathologic and physiologic senescence of endothelial cells as well as vascular aging.     

Nuclear control of tumorigenicity in cells reconstructed by PEG-induced fusion of cell fragments

The techniques of somatic cell hybridization have provided a valuable means of studying mechanisms of regulation of mammalian cell differentiation and transformation. Most previous studies have indicated that fusions between tumorigenic and nontumorigenic cells result in hybrid cells that are usually tumorigenic. In recent years it has been demonstrated that the phenotypic expression of tumorigenicity is at least partially due to the extensive chromosome loss that occurs in most interspecific and some intraspecific hybrid cells. In the present study we have utilized enucleation techniques that permit cells to be divided into nuclear (karyoplast) and cytoplasmic (cytoplast) cell fragments. Even though these nuclear and cytoplasmic fragments are metabolically stable for short periods of time, in our hands they ultimately degenerate. Viable cells can be reconstructed by PEG-induced fusion of karyoplasts to cytoplasts. Since reconstructed cells apparently do not segregate chromosomes, they may provide a clearer understanding of the interactions between the nucleus and the cytoplasm in the control of the expression of tumorigenicity. We have reconstructed cells using karyoplasts from the tumorigenic Y-1 cell line and cytoplasts from a nontumorigenic cell line, A-MT-BU-A1. In addition we have reconstructed cells containing Y-1 cytoplasts and A-MT-BU-A1 karyoplasts. The reconstructed cells porduced were assayed for tumorigenicity by their ability to grow in soft agar and in nude mice. The results of these experiments indicate that the reconstructed cells containing a tumorigenic nucleus and a nontumorigenic cytoplasm ultimately are tumorigenic and conversely the reconstructed cells containing a nontumorigenic nucleus and a tumorigenic cytoplasm are nontumorigenic. These experiments support the concept that with these cell lines the nucleus (karyoplast) is sufficient to control the phenotypic expression of tumorigenicity.     

Increase in DNA synthesis in Werner''s syndrome cells by hybridization with normal human diploid and HeLa cells

The dominance or recessiveness of the senescent phenotype in cells from patients with Werner's syndrome (WS cells) was investigated using cell fusion. The [³H]thymidine labeling index of normal human diploid fibroblast cell X WS cell heterodikaryons was considerably lower than that of normal homodikaryons, but was significantly higher than that of WS homodikaryons. The labeling index of WS cell X HeLa cell heterodikaryons was the same as that of HeLa homodikaryons. The labeling indices of heterodikaryons obtained by fusion between various strains of premature aging cells were as low as those of parental homodikaryons. These results indicate: (1) the senescent phenotype of WS cells appears to be partially recessive to the phenotype of normal cells and completely recessive to that of HeLa cells; (2) the marked inhibition of DNA synthesis in normal nuclei in heterodikaryons with WS cells could be due to ‘senescent factor(s)’ in WS cells; and (3) no complementation phenomenon was observed among genetically different premature aging cells, probably due to ‘senescent factor(s)’.     

Bovine oocyte cytoplasm supports nuclear remodeling but not reprogramming of murine fibroblast cells

Nuclear transfer (NT) is used to elucidate fundamental biological issues such as reversibility of cell differentiation and interactions between the cytoplasm and nucleus. To obtain an insight into interactions between the somatic cell nucleus and oocyte cytoplasm, nuclear remodeling and gene expression were compared in bovine oocytes that had received nuclei from bovine and mouse fibroblast cells. While the embryos that received nuclei from bovine fibroblast cells developed into blastocysts, those that received nuclei from mouse fibroblasts did not develop beyond the 8-cell stage. Similar nuclear remodeling procedures were observed in oocytes reconstructed with mouse and bovine fibroblast cells. Foreign centrosomes during NT were introduced into embryos reconstructed with both fibroblast cell types. A number of housekeeping mouse genes (hsp70, bax, and glt-1) were abnormally expressed in embryos that had received nuclei from mouse fibroblast cells. However, development-related genes, such as Oct-4 and E-cad, were not expressed. The results collectively suggest that the bovine oocyte cytoplasm supports nuclear remodeling, but not reprogramming of mouse fibroblast cells.     

Enucleation of human endometrial cells: nucleo-cytoplasmic distribution of DNA polymerase alpha and estrogen receptor

Nucleo-cytoplasmic distribution of estrogen receptors and DNA polymerase alpha activity in human endometrial adenocarcinoma cells (HEC-50 line) was evaluated after separation of nuclei following either homogenization or enucleation with cytochalasin B. About 30% of the estrogen receptor was found in the nuclear fraction after homogenization whereas 86% was found in the karyoplasts after enucleation. The total amounts of estrogen receptor per cell after homogenization and enucleation were not significantly different (14,000-17,000 binding sites/cell). Receptor measurements were carried out using the hydroxylapatite method after labeling with [3H]estradiol (5 nM [3H]E2 +/- 500 nM E2) at 30 degrees C for 3 h. About 20% of the DNA polymerase alpha activity was found in the nuclear fraction after homogenization, whereas 96% was found in the karyoplasts after enucleation. The average total activity (0.84 Units/10(6) cells) in homogenized cells was about 1/8 of the activity in karyoplasts. These results indicate that estrogen receptor and DNA polymerase alpha activity reside in the nucleus in intact HEC-50 cells. DNA polymerase alpha is translocated to the cytoplasmic fraction and inactivated after homogenization.     

A NOVEL PATTERN OF APICAL CELL POLYPLOIDY,SEQUENTIAL POLYPLOIDY REDUCTION AND INTERCELLULAR NUCLEAR TRANSFER IN THE RED ALGA POLYSIPHONIA

Nearly a century ago, Rosenvinge published a now-classic paper reporting nuclear transfer between cells of Polysiphonia during secondary pit connection (SPC) formation. While reinvestigating this phenomenon, we discovered that the uninucleate apical cell, which is the progenitor of all cells in the plant, has many times (ca. 64–128 ×) the level of nuclear DNA characteristic of nuclei of gametes or mature pericentral cells. Via a regular sequence of cell divisions, the polyploid apical cell gives rise to tiers of cells, each composed of a number of pericentral cells which surround a single central cell. A large proportion of the nuclear divisions are not accompanied by DNA replication. Thus, as the number of nuclei within elongating pericentral cells increases, the DNA level of nuclei in these cells “cascades” down to the DNA level expected for the particular life history generation (i.e., gametophyte or tetrasporophyte). In mature pericentral cells, the number of nuclei is proportional to the volume of the cell. The pattern of nuclear division, reduction in ploidy level and the timing of intercellular nuclear transfer via SPC formation is regular and characteristic of a species. Nuclei transferred from one cell to an adjacent cell participate in the further nuclear divisions of the recipient cell. The degree of polyploidy in apical cells may determine the number of cells in a “determinant” branch or even the number of cells in “indeterminant” axes. In addition, the highly polyploid state of the germinating spore and its pattern of development may provide for the rapid initial growth so characteristic of this taxon.     

Construction of viable mouse-human hybrid cells by nuclear transplantation

Viable interspecies cytoplasmic-nuclear hybrid cells were constructed by fusion of karyoplasts prepared from the highly tumorigenic A9 mouse fibroblast cell line and cytoplasts prepared from the Detroit 532 normal human diploid cell strain. The identity of the hybrid cells was ascertained using a variety of morphological, immunological, and genetic criteria, including: nuclear pattern of staining with the fluorochrome Hoechst 33258, appearance of the actin-myosin containing cytoskeleton, presence of fibronectin, and resistance to azaguanine and diphtheria toxin. About 90% of the hybrid cells were viable, that is, capable of division. Changes in the morphology of the hybrid cells, apparently nuclear directed, were observed before cell division occurred. Using the techniques described here, large numbers of interspecies hybrid cells suitable for many types of biochemical analyses can be routinely produced.     

Reduction of exportin 6 activity leads to actin accumulation via failure of RanGTP restoration and NTF2 sequestration in the nuclei of senescent cells

We have previously reported that G-actin accumulation in nuclei is a universal phenomenon of cellular senescence. By employing primary culture of human diploid fibroblast (HDF) and stress-induced premature senescence (SIPS), we explored whether the failure of actin export to cytoplasm is responsible for actin accumulation in nuclei of senescent cells. Expression of exportin 6 (Exp6) and small G-protein, Ran, was significantly reduced in the replicative senescence, but not yet in SIPS, whereas nuclear import of actin by cofilin was already increased in SIPS. After treatment of young HDF cells with H₂O₂, rapid reduction of nuclear RanGTP was observed along with cytoplasmic increase of RanGDP. Furthermore, significantly reduced interaction of Exp6 with RanGTP was found by GST-Exp6 pull-down analysis. Failure of RanGTP restoration was accompanied with inhibition of ATP synthesis and NTF2 sequestration in the nuclei along with accordant change of senescence morphology. Indeed, knockdown of Exp6 expression significantly increased actin molecule in the nuclei of young HDF cells. Therefore, actin accumulation in nuclei of senescent cells is most likely due to the failure of RanGTP restoration with ATP deficiency and NTF2 accumulation in nuclei, which result in the decrease of actin export via Exp6 inactivation, in addition to actin import by cofilin activation.     

Replicative potentials of various fusion products between WI-38 and SV40 transformed WI-38 cells and their components

Hybrid cells derived from whole-cell fusions of replicating phase-II normal fibroblast cells (WI-38s) with SV40 transformed WI-38 fibroblast cells (CL-1s) demonstrated that the majority of the hybrid experimental cells still maintained a finite life-span. Approximately 2% demonstrated sustained and possibly indefinite replication. Experimental binucleate cells and subsequent hybrid synkaryons were also formed by fusing CL-1 karyoplasts into phase-II WI-38 replicating normal fibroblasts. In addition, viable cells were constructed from WI-38 fibroblast cytoplasts with CL-1 karyoplasts. Sustained replication was not observed in these crosses.     

in vitro development of porcine enucleated oocytes reconstructed by the transfer of porcine fetal fibroblasts and cumulus cells

In the pig little information is available on cytoplasmic events during the reprogramming of oocytes reconstructed with somatic nuclei. The present study was conducted to determine the developmental potential of porcine cumulus cells (CC) and fetal fibroblasts (FF) after they were transferred into enucleated oocytes. Non-quiescent FF were fused to the enucleated oocytes using electrical pulse, whereas CC were directly injected into the oocytes. Transferred nuclei from both CC and FF underwent premature chromosome condensation (PCC), nuclear swelling and pronucleus formation. The remodeled oocytes developed to the mitotic and 2-cell stage at 18 to 24 h after nuclear transfer. The pattern of nuclear remodeling was similar regardless of the sources of karyoplasts or nuclear transfer methods. However, using FF, 24% of nuclear transferred embryos developed to the morula or blastocyst stage, whereas only 8% of those using CC developed to the morula or blastocyst stage. These results suggest that porcine oocyte cytoplasm can successfully reprogram somatic cell nuclei and support the development of nuclear transferred embryos to the blastocyst stage.     

Interaction of vinblastine with the secretagogue action of db-cAMP in the rat exocrine pancreas,.

Developmentally pluripotent embryonal carcinoma cells isolated from mouse teratocarcinomas were fused to whole cells, to cytoplasts, and to karyoplasts of 3T3 fibroblasts. The cybrids (cell X cytoplast fusion product) retained the developmental potency of the embryonal carcinoma cell parent. On the other hand, the karyobrids (cell X karyoplast fusion product) and the hybrids resembled the fibroblast parent cell and were incapable of differentiation. These experiments, therefore, failed to reveal the presence of cytoplasmic regulators of nuclear gene expression.     

Cytotoxic lymphocyte-derived lytic granules do not induce DNA fragmentation in target cells

When target cells are exposed to CTL, they very quickly sustain nuclear damage, including DNA cleavage, and then they lyse. Nuclear damage of this type is not seen when cells are killed by antibody and C. The role of nuclear damage in the T cell-mediated killing process as well as the mechanism by which the killer cell induces this damage are unknown; however, accumulating evidence suggests that cytolysis may depend on induction of nuclear damage. The exocytosed contents of CTL granules are thought by many workers to mediate target cell lysis. We have now determined whether lytic granules also induce nuclear damage (DNA fragmentation) in cells which they lyse. They do not. In addition, no DNA fragmentation was detected in nuclei incubated with lytic granules or activated CTL. In summary, our results suggest that target cell DNA fragmentation induced by CTL is mediated neither by lytic granules nor by a CTL-derived endonuclease and support the view that the target cell is itself responsible for the internal damage it sustains.     

Senescence in polyploid giant cancer cells: A road that leads to chemoresistance

Cellular senescence has been associated with age-related diseases, wound healing, fibrosis, diabetes and cancer. Senescent cells lack the capacity to proliferate, but are known to aggravate tumorigenesis. The polyploid giant cells arise from the cancer cell population mainly due to genotoxic stress caused by chemotherapy and/or radiotherapy. They exhibit features of senescence and have been reported to secrete an array of cytokines, chemokines and growth factors. These small molecules can bind to their receptors located on the surface of neighboring cells and activate/deactivate relevant signaling pathways, thereby modulating the tumor microenvironment. Some of these signaling cascade(s) might play a role in imparting therapy resistance to the cancer cells. This review throws light on the incidence of senescence and how the senescent polyploid giant cells affect the tumor microenvironment. Their role in giving rise to chemoresistant cancer cell population as well as acquired chemoresistance in the neighboring cancer cells along with various potential and established therapeutic avenues have also been discussed.     

Dominance of parental genomes in embryonic stem cell/fibroblast hybrid cells depends on the ploidy of the somatic partner

Two dozen hybrid clones were produced by fusion of diploid embryonic stem (ES) cells positive for green fluorescent protein (GFP) with tetraploid fibroblasts derived from DD/c and C57BL-I(I)1RK mice. Cytogenetic analysis demonstrated that most cells from these hybrid clones contained near-hexaploid chromosome sets. Additionally, the presence of chromosomes derived from both parental cells was confirmed by polymerase chain reaction (PCR) analysis of polymorphic microsatellites. All hybrid cells were positive for GFP and demonstrated growth characteristics and fibroblast-like morphology. In addition, most hybrid cells were positive for collagen type I, fibronectin, and lamin A/C but were negative for Oct4 and Nanog proteins. Methylation status of the Oct4 and Nanog gene promoters was evaluated by bisulfite genomic sequencing analysis. The methylation sites (CpG-sites) of the Oct4 and Nanog gene promoters were highly methylated in hybrid cells, whereas the CpG-sites were unmethylated in the parental ES cells. Thus, the fibroblast genome dominated the ES genome in the diploid ES cell/tetraploid fibroblast hybrid cells. Immunofluorescent analysis of the pluripotent and fibroblast markers demonstrated that establishment of the fibroblast phenotype occurred shortly after fusion and that the fibroblast phenotype was further maintained in the hybrid cells. Fusion of karyoplasts and cytoplast derived from tetraploid fibroblasts with whole ES cells demonstrated that karyoplasts were able to establish the fibroblast phenotype of the reconstructed cells but not fibroblast cytoplasts. Thus, these data suggest that the dominance of parental genomes in hybrid cells of ES cell/somatic cell type depends on the ploidy of the somatic partner.     

Effect of poly(ADP-ribose) formation on DNA synthesis and DNA fragmentation in nuclei of rat liver and rat ascites hepatoma AH-130 cells

To elucidate the role of poly(ADP-Rib) in the nucleus, DNA synthesis and DNA fragmentation were studied in isolated nuclei of rat liver and rat ascites hepatoma AH-130 cells. Liver and hepatoma cell nuclei formed the same amount of poly(ADP-Rib) per mg of nuclear DNA from NAD. Preincubation of liver nuclei with NAD repressed DNA polymerase activity to 30% of that of the control, but preincubation of hepatoma cell nuclei with NAD did not affect DNA polymerase activity. It was also found that incubation of liver nuclei with NAD prevented the fragmentation of nuclear DNA which occurred without NAD. Incubation of hepatoma cell nuclei with or without NAD did not result in fragmentation of DNA. The role of endonuclease in primer formation for DNA synthesis is discussed.