Budded karyoplasts from multinucleated fibroblast cells contain centrosomes and change their morphology to mitotic cells

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.     

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 senescence involves stochastic processes causing loss of expression of differentiated function genes: transfection with SV40 as a means for dissociating effects of senescence on growth and on differentiated function gene expression

In the accompanying work we demonstrated that the decline in expression of steroid 17 alpha-hydroxylase in mass cultures and clones of adrenocortical cells is the result of a stochastic switching process which yields mixtures of expressing and nonexpressing cells. There is an apparent positive correlation between the replicative potential of adrenocortical cell cultures and the number of cells in the culture that can express 17 alpha-hydroxylase. We investigated this by extending the cells' replicative potential by transfecting them with cloned SV40 virus. Cells from a senescent subclone, with very limited remaining replicative potential, were transfected. The cell population showed a progressive increase in growth rate and gave rise to a line of cells that expressed T antigen and which was apparently immortalized. Induction of mRNA for 17 alpha-hydroxylase by cyclic AMP was absent in this line of cells, as it was in the senescent cells prior to transfection. The cells remained responsive to gene induction by cyclic AMP as evidenced by increases in mRNA and activity for cholesterol side-chain cleavage. The absence of 17 alpha-hydroxylase expression in this line was not the result of interference by SV40 T antigen. When early passage cells were transfected with pSV3neo, which contains the early region of SV40 and neo, and were selected with G418, SV40 T antigen-expressing lines were derived which showed high levels of expression of 17 alpha-hydroxylase after induction with cyclic AMP. These cells maintained high levels of expression of 17 alpha-hydroxylase through four successive recloning events, over a period of replication much longer than that achievable by nontransfected cells. Thus, transfection by SV40 can be used to dissociate effects of senescence on growth and differentiated gene expression. T antigen expression selectively affects growth, but preserves the state of expression of a differentiated function gene as it was prior to transfection.     

SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.     

Surface T-antigen expression in simian virus 40-transformed mouse cells: correlation with cell growth rate.

Cell growth control appears to be drastically altered as a consequence of transformation. Because the cell surface appears to have a role in modulating cell growth and simian virus 40 (SV40)-transformed cells express large T antigen (T-Ag) in the plasma membrane, we investigated whether surface T-Ag expression varies according to cell growth rate. Different growth states were obtained by various combinations of seeding density, serum concentration, and temperature, and cell cycle distributions were determined by flow microcytofluorometry. Actively dividing SV40-transformed mouse cell cultures were consistently found to express higher levels of surface T-Ag and T-Ag/p53 complex than cultures in which cells were mostly resting. In addition, the T-Ag/p53 complex disappeared from the surface of tsA7-transformed cells cultured under restrictive conditions known to induce complete growth arrest (39.5 degrees C), although the surface complex did not disappear from other tsA transformants able to keep cycling at 39.5 degrees C. These results suggest that surface SV40 T-Ag or surface T-Ag/p53 complex, or both, are involved in determining the growth characteristics of SV40-transformed cells.     

Single‐cell analysis of p16INK4a and p21WAF1 expression suggests distinct mechanisms of senescence in normal human and Li‐Fraumeni Syndrome fibroblasts

Herein we used single‐cell observation methods to gain insight into the roles of p16^INK4A and p21^WAF1 (hereafter p16 and p21) in replicative senescence and ionizing radiation‐induced accelerated senescence in human [normal, ataxia telangiectasia (AT) and Li‐Fraumeni syndrome (LFS)] fibroblast strains. Cultures of all strains entered a state of replicative senescence at late passages, as evident from inhibition of growth, acquisition of flattened and enlarged cell morphology, and positive staining for senescence‐associated β‐galactosidase. In addition, proliferating early‐passage cultures of these strains exhibited accelerated senescence in response to ionizing radiation. Immunofluorescence microscopy revealed the heterogeneous expression of p16 in normal and AT fibroblast strains, with the majority of the cells exhibiting undetectable levels of p16 irrespective of in vitro culture age. Importantly, replicative senescence as well as accelerated senescence triggered by ionizing radiation were accompanied by sustained nuclear accumulation of p21, but did not correlate with p16 expression in p53‐proficient (normal and AT) fibroblasts. In p53‐deficient (LFS) fibroblasts, on the other hand, replicative senescence and ionizing radiation‐triggered accelerated senescence strongly correlated with expression of p16 but not of p21. Furthermore, senescence in LFS fibroblasts was associated with genomic instability encompassing polyploidy. Our findings are compatible with a model in which p16 serves as a backup regulator of senescence, triggering this response preferentially in the absence of wild‐type p53 activity. The possibility that one of the tumor‐suppressor functions of p16 may be associated with genomic instability, preventing the emergence of malignant progeny from polyploid giant cells, is also supported by these results. J. Cell. Physiol. 223: 57–67, 2010. © 2009 Wiley‐Liss, Inc.     

Establishment of a SV40-transformed cell line from primary culture of rat dorsolateral prostatic epithelial cells

A permanent cell line of the rat dorsolateral prostate epithelium was established after transformation of primary cultured cells with simian virus 40 (SV40). The established cells had SV40 T-antigen in their nuclei but lacked such typical characteristics of transformed cells as piled-up growth. They grew in a monolayer with an epithelial morphology, were stained with antikeratin antisera, and also retained an ability to form dome-like structure at a confluent state.     

Possible role of SV40 T antigen in cell transformation]

The effect of purified SV40 T antigen on DNA synthesis in isolated nuclei from the confluent culture of CV-1 cells was studied. In the presence of T antigen the incorporation of [3H]TTP into DNA was found to be 2 to 3 times as high as in the control nuclei. The resulting labelled DNA was subjected to alkaline sucrose gradient centrifugation, which revealed the presence of 4S DNA species, corresponding to Okazaki fragments of animal cells. The latter finding suggests a replicative mode of DNA synthesis induced by T antigen. T antigen isolated from the cells infected with SV40 tsA-mutant and kept at a nonpermissive (41 degrees) temperature fails to stimulate DNA synthesis in isolated nuclei from resting cells. On storage at 4 degrees SV40 T antigen gradually loses its ability to stimulate DNA synthesis and by the 8th day even suppresses it when tested on isolated nuclei from a growing cell culture. No effect of T antigen on the endonuclease-induced reparative synthesis of DNA could be observed. The data described suggest that T antigen is directly involved in the control of DNA synthesis in the cells infected or transformed with SV40.     

Putative role for EPC-1/PEDF in the G0 growth arrest of human diploid fibroblasts

EPC-1/PEDF expression is closely associated with reversible growth arrest in normal human diploid fibroblast-like (HDF) cells and is diminished with proliferative senescence in vitro. EPC-1 expression in HDF cells is induced under conditions of density-dependent contact inhibition and growth factor deprivation. Antiserum generated against EPC-1 recognizes a secreted protein of approximately 50 kDa from medium conditioned by early passage HDF cells, but not from senescent cells. The addition of EPC-1 antiserum to early population doubling level (PDL) cultures near the plateau phase of growth significantly increases the number of cells entering DNA synthesis. Affinity purified EPC-1 antibodies alone enhance the ability of near plateau-phase early PDL WI-38 cells to synthesize DNA by as much as threefold. Further, the addition of recombinant EPC-1 (rEPC-1) to logarithmically growing cells resulted in a marked decrease in the ability of these cells to enter DNA synthesis. We also demonstrate the loss of EPC-1 expression in WI-38 and IMR-90 HDF cell lines with both senescence and simian virus 40 (SV40) transformation. The loss of EPC-1 expression with SV40 transformation occurs at the level of steady-state mRNA and protein accumulation with genomic EPC-1 sequences grossly intact. Taken together, these results suggest that EPC-1 may play a role in the entry of early passage fibroblasts into a G(0) state or the maintenance of such a state once reached.     

Establishment and characterization of a tamoxifen-mediated reversible immortalized mouse dental papilla cell line

Odontoblasts are a type of non-proliferating and terminally differentiated cells that play an important role in the pulpo–dentinal complex. Mouse dental papilla cells (mDPCs), which can differentiate into odontoblast-like cells in vitro, have a limited life span. We combined the traditional strategy of “Cre/LoxP-based reversible immortalization” with a tamoxifen-regulated Cre recombination system to generate a tamoxifen-mediated reversibly immortalized mouse dental papilla cell line, mDPCET. mDPCs were sequentially transduced with a floxed SV40 T antigen-TK (SV40Tag-TK) and an ^ERT2Cre^ERT2-expressing plasmid. Clonal-isolated SV40Tag- and Cre-positive cells showed modified growth characteristics and a significantly extended life span. When mDPCET cells were treated with 4-hydroxytamoxifen, ^ERT2Cre^ERT2 translocated from the cytoplasm to the nucleus and caused the excision of SV40Tag-TK, which led to the reversion of mDPCETs. After the immortalization was reversed, the cells underwent replicative senescence and transitioned into a more differentiated state. Tamoxifen-mediated reversible immortalization, therefore, allows for the expansion of primary mDPCs, leads to the production of odontoblast-like cells that retain most odontoblast-specific properties, and can represent a safe and ready-to-use method due to its simple manipulation.     

A mother's sacrifice: what is she keeping for herself?

Individual cells of the budding yeast, Saccharomyces cerevisiae, have a limited life span and undergo a form of senescence termed replicative aging. Replicative life span is defined as the number of daughter cells produced by a yeast mother cell before she ceases dividing. Replicative aging is asymmetric: a mother cell ages but the age of her daughter cells is 'reset' to zero. Thus, one or more senescence factors have been proposed to accumulate asymmetrically between mother and daughter yeast cells and lead to mother-specific replicative senescence once a crucial threshold has been reached. Here we evaluate potential candidates for senescence factors and age-associated phenotypes and discuss potential mechanisms underlying the asymmetry of replicative aging in budding yeast.     

Human telomerase can immortalize Indian muntjac cells

The mechanisms of replicative senescence by telomere shortening are not fully understood. The Indian muntjac has the fewest chromosomes of all mammals, greatly simplifying the analysis of each telomere over time. In this study, telomere shortening was observed throughout the life span of cultured normal muntjac cells by quantitative fluorescence in situ hybridization and terminal restriction fragment analysis. Ectopic expression of the human telomerase catalytic subunit in these cells reconstituted telomerase activity, extended telomere lengths, and immortalized the cells, demonstrating that the Indian muntjac cells can serve as a telomere-based replicative senescence model for human cells. In one strain, two chromosome ends had significantly shorter telomeres than the other ends, which led to a variety of chromosome abnormalities. Near senescence, additional ends became telomere signal free, and chromosome aberrancies increased dramatically. Interstitial telomere sequences coincided with fragile sites, suggesting that these remnants of chromosome fusion events might contribute to genome instability. One SV40-immortalized cell line lacked telomerase, and its genetic instability was corrected by the ectopic expression of telomerase, confirming that too-short telomeres were the source of abnormalities. Indian muntjac cells provide an excellent system for understanding the mechanism of replicative senescence and the role of telomerase in the elongation of individual telomeres.     

Enumeration of the simian virus 40 early region elements necessary for human cell transformation

While it is clear that cancer arises from the accumulation of genetic mutations that endow the malignant cell with the properties of uncontrolled growth and proliferation, the precise combinations of mutations that program human tumor cell growth remain unknown. The study of the transforming proteins derived from DNA tumor viruses in experimental models of transformation has provided fundamental insights into the process of cell transformation. We recently reported that coexpression of the simian virus 40 (SV40) early region (ER), the gene encoding the telomerase catalytic subunit (hTERT), and an oncogenic allele of the H-ras gene in normal human fibroblast, kidney epithelial, and mammary epithelial cells converted these cells to a tumorigenic state. Here we show that the SV40 ER contributes to tumorigenic transformation in the presence of hTERT and oncogenic H-ras by perturbing three intracellular pathways through the actions of the SV40 large T antigen (LT) and the SV40 small t antigen (ST). LT simultaneously disables the retinoblastoma (pRB) and p53 tumor suppressor pathways; however, complete transformation of human cells requires the additional perturbation of protein phosphatase 2A by ST. Expression of ST in this setting stimulates cell proliferation, permits anchorage-independent growth, and confers increased resistance to nutrient deprivation. Taken together, these observations define the elements of the SV40 ER required for the transformation of human cells and begin to delineate a set of intracellular pathways whose disruption, in aggregate, appears to be necessary to generate tumorigenic human cells.     

Differentiation between senescence (M1) and crisis (M2) in human fibroblast cultures

Normal human fibroblasts undergo only a limited number of divisions in culture and eventually enter a nonreplicative state designated senescence or mortality stage 1 (M1). Expression of certain viral oncogenes, such as the SV40 large T antigen (SV40 T-Ag), can elicit a significant extension of replicative life span, but these cultures eventually also cease dividing. This proliferative decline has been designated crisis or mortality stage 2 (M2). BrdU incorporation assays are commonly used to distinguish between senescence (<5% labeling index) and crisis (>30% labeling index). It has not been possible, however, to ascertain whether the high labeling index, indicative of ongoing DNA replication, was caused by the presence of T-Ag. We used gene targeting to knock out both copies of the p21(CIP1/WAF1) gene in presenescent human fibroblasts. p21 -/- cells displayed an extended life span but eventually entered a nonproliferative state. In their terminally nonproliferative state both p21 +/+ and p21 -/- cultures were positive for the senescence-associated beta-galactosidase (SA-beta-gal) activity; in contrast, the labeling index of p21 +/+ cells was low (<5%) whereas the labeling index of p21 -/- cells was high (>30%). The observation that p21 -/- and SV40 T-Ag-expressing cells behave identically with respect to life span extension as well as the high labeling index in the terminally nonproliferative state indicates that crisis is not a phenomenon induced solely by viral oncogenes, but a physiological state resulting from the bypass of normal senescence mechanisms. The widely used biomarker for senescence, SA-beta-gal, cannot distinguish between senescence and crisis. We propose that all SA-beta-gal-positive cultures should be further examined for their BrdU labeling index.     

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.     

Bcl-2 can promote p53-dependent senescence versus apoptosis without affecting the G1/S transition

With the aim to identify events involved in the determination of p53-dependent apoptosis versus growth arrest, we used rat embryo fibroblasts expressing a temperature-sensitive mutant (tsA58) of the SV40 large tumour antigen (LT). Heat-inactivation of LT leads to p53 activation and commitment to a senescent-like state (REtsA15 cell line) or apoptosis (REtsAF cell line). We report that senescence is associated with high levels of the anti-apoptotic Bcl-2 protein and a cell cycle arrest in G1 phase, whereas apoptosis is associated with low levels of Bcl-2 and a cell cycle arrest in G2 phase. Here we show that Bcl-2, which can inhibit apoptosis and proliferation, turns the apoptotic phenotype into a senescent-like phenotype in G2 phase. This result suggests that Bcl-2-dependent inhibition of apoptosis could be crucial for the commitment to replicative senescence, whereas its ability to inhibit G1 progression would not be required.     

Abrogation of simian virus 40 DNA-mediated transformation of primary C57BL/6 mouse embryo fibroblasts by exposure to a simian virus 40-specific cytotoxic T-lymphocyte clone.

Primary mouse embryo fibroblasts of C57BL/6 origin (B6/MEF) were transformed in vitro by transfection with simian virus 40 (SV40) DNA. The transformation frequency was markedly reduced if the SV40 DNA-transfected cultures were briefly exposed to K11 cells, an SV40-specific clone of cytotoxic T lymphocytes. This abrogation of SV40 transformation in vitro by cytotoxic T-lymphocyte clone K11 was specific, since transformation of B6/MEF cells by adenovirus type 5 DNA was not affected. The approach described here should serve as an ideal model of dissecting immunological events during in vivo tumorigenesis.     

Isolation and characterization of revertant cell lines. 3. Isolation of density-revertants of SV40-transformed 3T3 cells using colchicine

Treatment of the SV40 transformed 3T3 cell line SV101 with colchicine permits the isolation of polyploid revertant sublines Which have lower saturation densities than SV101. These low saturation density lines have also reverted to a high serum requirement for growth, and are unable to form colonies in methocel. Normal SV40 has been recovered from these revertants. 3T3 cells are more resistant to colchicine than SV3T3 cells at all cell densities. Colchicine revertants do not display a 3T3-like resistance to colchicine at low density, but do survive colchicine at confluent cell densities, presumably due to their increased contact inhibition.     

Isolation and characterization of defective simian virus 40 genomes which complement for infectivity.

A new variant of simian virus 40 (EL SV40), containing the complete viral DNA separated into two molecules, was isolated. One DNA species contains nearly all of the early (E) SV40 sequences, and the other DNA contains nearly all of the late (L) viral sequences. Each genome was encircled by reiterated viral origins and termini and migrated in agarose gels as covalently closed supercoiled circles. EL SV40 or its progenitor appears to have been generated in human A172 glioblastoma cells, as defective interfering genomes during acute lytic infections, but was selected during the establishment of persistently infected (PI) green monkey cells (TC-7). PI TC-7/SV40 cells contained EL SV40 as the predominant SV40 species. EL SV40 propagated efficiently and rapidly in BSC-1, another line of green monkey cells, where it also formed plaques. EL SV40 stocks generated in BSC-1 cells were shown to be free of wild-type SV40 by a number of criteria. E and L SV40 genomes were also cloned in the bacterial plasmid pBR322. When transfected into BSC-1 cell monolayers, only the combination of E and L genomes produced a lytic infection, followed by the synthesis of EL SV40. However, transfection with E SV40 DNA alone did produce T-antigen, although at reduced frequency.