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.     

Evidence for the presence of inhibitors of mitotic factors during G1 period in mammalian cells

Our earlier studies indicated that the mitotic factors, which induce germinal vesicle breakdown and chromosome condensation when injected into fully grown Xenopus oocytes, are preferentially associated with metaphase chromosomes and that they bind to chromatin as soon as they are synthesized during the G2 phase. In this study, we attempted to determine the fate of these factors as the cell completes mitosis and enters G1. Extracts from HeLa cells at different points during G1, S, and G2 periods were mixed with mitotic extracts in various proportions, incubated, and then injected into Xenopus oocytes to determine their maturation-promoting activity. The maturation-promoting activity of the mitotic extracts was neutralized by extracts of G1 cells during all stages of G1 but not by those of late S and G2 phase cells. Extracts of quiescent (G0) human diploid fibroblasts exhibited very little inhibitory activity. However, UV irradiation of G0 cells, which is known to cause decondensation of chromatin, significantly enhanced the inhibitory activity of extracts of these cells. These factors are termed inhibitors of mitotic factors (IMF). They seem to be activated, rather than newly synthesized, as the cell enters telophase when chromosomes begin to decondense. The IMF are nondialyzable, nonhistone proteins with a molecular weight of greater than 12,000. Since mitotic factors are known to induce chromosome condensation, it is possible that IMF, which are antagonistic to mitotic factors, may serve the reverse function of the mitotic factors, i.e., regulation of chromosome decondensation.     

Initiation of DNA synthesis in the prematurely condensed chromosomes of G1 cells

The objective of this study was to investigate whether G1 cells could enter S phase after premature chromosome condensation resulting from fusion with mitotic cells. HeLa cell synchronized in early G1, mid-G1, late G1, and G2 and human diploid fibroblasts synchronized in G0 and G1 phases were separately fused by use of UV-inactivated Sendai virus with mitotic HeLa cells. After cell fusion and premature chromosome condensation, the fused cells were incubated in culture medium containing Colcemid (0.05 micrograms/ml) and [3H]thymidine ([3H]ThdR) (0.5 microCi/ml; sp act, 6.7 Ci/mM). At 0, 2, 4, and 6 h after fusion, cell samples were taken to determine the initation of DNA synthesis in the prematurely condensed chromosomes (PCC) on the basis of their morphology and labeling index. The results of this study indicate that PCC from G0, G1, and G2 cells reach the maximum degree of compaction or condensation at 2 h after PCC induction. In addition, the G1-PCC from normal and transformed cells initiated DNA synthesis, as indicated by their "pulverized" appearance and incorporation of [3H]ThdR. Further, the initiation of DNA synthesis in G1-PCC occurred significantly earlier than in the mononucleate G1 cells. Neither pulverization nor incorporation of label was observed in the PCC of G0 and G2 cells. These findings suggest that chromosome decondensation, although not controlling the timing of a cell's entry into S phase, is an important step for the initiation of DNA synthesis. These data also suggest that the entry of a S phase may be regulated by cell cycle phase-specific changes in the permeability of the nuclear envelope to the inducers of DNA synthesis present in the cytoplasm.     

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.     

A high incidence of mitotic chiasmata in endoreduplicated Bloom's syndrome cells

Summary The frequency of mitotic chiasmata is compared in endoreduplicated and non-endoreduplicated Bloom's syndrome fibroblasts and in endoreduplicated Fanconi's anemia lymphocytes. The incidence of mitotic chiasmata in BS diplochromosomes is greatly increased over that in diploid BS cells and is much higher than in FA or normal diplochromosomes. The distribution of chiasmata among the BS diplochromosomes is not significantly different from that expected if crossing-over occurs at random along the chromosomes. This is in contrast to the distribution of chiasmata in chromosomes of diploid BS cells which is highly non-random among chromosomes and chromosome regions (Kuhn 1976). Mitotic crossing-over is increased in endoreduplicated cells from all sources compared to diploid cells, but the incidence is highest in endoreduplicated BS cells. This provides evidence against the idea that the high rate of mitotic crossing-over in diploid BS lymphocytes is primarily due to an increase in chromosome pairing. BS chromosomes apparently have a greater tendency to undergo mitotic exchange than normal or FA cells, both in diplo-chromosomes and in accidentally paired homologous segments in diploid cells.     

Transient G2M arrest and subsequent release of apoptotic and mitotic cells in vanadyl(4)-prepulsed human Chang liver cells

The relationship between cell cycling and apoptosis/programmed cell death has been perceived as either checkpoint arrests or mitotic aberration where common pathways between mitosis and apoptosis seem suggested. We show here evidence implicating both perceptions of cell cycle involvement. The process was initiated by hydroxyl free radicals (OH*) generated intracellularly from internalized vanadyl(4). Intranuclear sequestration of vanadyl(4) was verified by nuclear microscopy. Resultant high oxidative reactivity in the nucleus was shown by the redox indicator methylene blue, suggesting direct oxidative damage to genomic DNA. Oxidative stress was further enhanced by depletion of glutathione which is the main cellular reducing agent. Genomic degradation and fragmentation was confirmed by flow cytometric evaluation of terminal deoxynucleotidyl transferase (TdT)-mediated 3'OH end-labelling (TUNEL) of DNA nicks, and cell cycle DNA profiling demonstrating sub-G1 (sub-2N) accumulation. With DNA degradation, there was a G2M transient with hyperdiploid right-shifting, consistent with G2 arrest. G2 arrest was subsequently 'released' with abolition of G2M and all other cell cycle phases except for a solitary sub-G1 (apoptotic) peak. The cytological profile of this 'release' phenomenon was initially marked by the appearance of clusters of mitotic and apoptotic cells. At later stages, the cell population was composed exclusively of nuclear ghosts, apoptotic cells, mitotic cells, and mitotic cells with both chromosomes and apoptotic condensations. Concurrent and conjoint expression of cell death and cell division as the exclusive process of an entire cell population refuted the notion of mutual exclusivity between life and death. Zn2+, an endonuclease inhibitor, abolished all observed cytological and DNA profile changes.     

Neosis--a paradigm of self-renewal in cancer

We recently described a novel form of cell division termed neosis, which appears to be the mode of escape of cells from senescence and is involved in the neoplastic transformation and progression of tumors (Cancer Biol & Therap 2004;3:207-18). Neosis is a parasexual somatic reduction division and is characterized by (1) DNA damage-induced senescence/mitotic crisis and polyploidization, (2) followed by production of aneuploid daughter cells via nuclear budding, (3) asymmetric cytokinesis and cellularization conferring extended, but, limited mitotic life span to the offspring, and (4) is repeated several times during tumor growth. The immediate neotic progeny are termed the Raju cells, which seem to transiently display stem cell properties. The Raju cells immediately undergo symmetric mitotic division and become mature tumor cells. Exposure of tumor cells to genotoxic agents yields neosis-derived Raju cell progenies that are resistant to genotoxins, thus contributing to the recurrence of drug-resistant tumor growth. Similar events have been described in the literature under different names through several decades, but have been neglected due to the lack of appreciation of the significance of this process in cancer biology. Here we review and interpret the literature in the light of our observations and the recent advances in self-renewal in cancer. Neosis paradigm of self-renewal of cancer growth is consistent with the telomere attrition, aging and origin of cancer cells after reactivation of telomerase, and constitutes an alternative to the cancer stem cell hypothesis. We summarize the arguments favoring Raju cells and not cancer stem cells, as the source of self-renewal in cancer and present a comprehensive hypothesis of carcinogenesis, encompassing various aspects of cancer biology including senescence, tumor suppressor genes, oncogenes, cell cycle checkpoints, genomic instability, polyploidy and aneuploidy, natural selection, apoptosis, endoapoptosis, development of resistance to radiotherapy and chemotherapy leading tumor progression into malignancy.     

Neosis – a paradigm of self-renewal in cancer

We recently described a novel form of cell division termed neosis, which appears to be the mode of escape of cells from senescence and is involved in the neoplastic transformation and progression of tumors (Cancer Biol & Therap 2004;3:207–18). Neosis is a parasexual somatic reduction division and is characterized by (1) DNA damage-induced senescence/mitotic crisis and polyploidization, (2) followed by production of aneuploid daughter cells via nuclear budding, (3) asymmetric cytokinesis and cellularization conferring extended, but, limited mitotic life span to the offspring, and (4) is repeated several times during tumor growth. The immediate neotic progeny are termed the Raju cells, which seem to transiently display stem cell properties. The Raju cells immediately undergo symmetric mitotic division and become mature tumor cells. Exposure of tumor cells to genotoxic agents yields neosis-derived Raju cell progenies that are resistant to genotoxins, thus contributing to the recurrence of drug-resistant tumor growth. Similar events have been described in the literature under different names through several decades, but have been neglected due to the lack of appreciation of the significance of this process in cancer biology. Here we review and interpret the literature in the light of our observations and the recent advances in self-renewal in cancer. Neosis paradigm of self-renewal of cancer growth is consistent with the telomere attrition, aging and origin of cancer cells after reactivation of telomerase, and constitutes an alternative to the cancer stem cell hypothesis. We summarize the arguments favoring Raju cells and not cancer stem cells, as the source of self-renewal in cancer and present a comprehensive hypothesis of carcinogenesis, encompassing various aspects of cancer biology including senescence, tumor suppressor genes, oncogenes, cell cycle checkpoints, genomic instability, polyploidy and aneuploidy, natural selection, apoptosis, endoapoptosis, development of resistance to radiotherapy and chemotherapy leading tumor progression into malignancy.     

Mitosis is not the only distributor of mutated cells: Non‐mitotic endopolyploid cells produce reproductive genome‐reduced cells

Giant endopolyploid nuclei (>16n) can spontaneously fragment by endomitosis (nuclear internal division) into near‐diploid cells with reproductive capacity (depolyploidization), and endotetra/octopolyploidy can undergo chromosome‐visible meiotic‐like genome reductional divisions also to replicative subcells. These unconventional divisions are associated with production of aneuploidy, which led to the question in this study of whether endopolyploidy, in general, can contribute genetic variability to tumorigenic potential. For this purpose, non‐proliferative endopolyploid cells (range: 4n–32n) in near‐senescence of normal diploid cell strains were analysed for nuclear–morphogenic changes associated with the presence of diploid‐sized nuclei in the cytoplasm. A one‐by‐one nuclear‐cutoff process gave rise to reproducing genome‐reduced cells. It was concluded that these unconventional cell divisions are, indeed, suspects of originating genetic variability. Details of these irregular mitoses were compared to ‘mitotic–meiosis’ in primitive organisms, which suggested activation of an ancestral trait in the mammalian cells.     

Expression of the calcium binding protein calretinin in WiDr cells and its correlation to their cell cycle

Ca2+ ions intervene during different phases of the progression of the cell cycle, but only one calcium-binding protein, calmodulin, has been shown to be associated with dividing cells. We therefore screened cancer cells for the presence of other related calcium-binding proteins. Using molecular biological and immunohistochemical techniques we show that human tumor cells of epithelial origin, express calretinin. Calretinin immunoreactivity can be demonstrated at precise moments of the cell cycle and, in particular, in phase G1 and during mitosis. During mitosis calretinin is localized both in the cytoplasm and in the mitotic spindle. In the cytoplasm we find calretinin after prophase and until telophase. In the spindle apparatus, calretinin is already present in cells in prometaphase and persists in all the succeeding mitotic phases. It is associated with the kinetochore microtubules but, in contrast to calmodulin, also with the polar microtubules. The role that calretinin plays in well-defined moments of the cell cycle of these cells is as yet unknown, but our results strongly suggest that, in collaboration with other molecules, calretinin intervenes in the dynamic phenomena regulating the separation of the chromosomes.     

Cytogenetic analysis of Astylus antis (Perty, 1830) (Coleoptera, Melyridae): Karyotype, heterochromatin and location of ribosomal genes

Cytogenetic analysis of Astylus antis using mitotic and meiotic cells was performed to characterize the haploid and diploid numbers, sex determination system, chromosome morphology, constitutive heterochromatin distribution pattern and chromosomes carrying nucleolus organizer regions (NORs). Analysis of spermatogonial metaphase cells revealed the diploid number 2n = 18, with mostly metacentric chromosomes. Metaphase I cells exhibited 2n = 8II+Xyp and a parachute configuration of the sex chromosomes. Spermatogonial metaphase cells submitted to C-banding showed the presence of small dots of constitutive heterochromatin in the centromeric regions of nearly all the autosomes and on the short arm of the X chromosome (Xp), as well as an additional band on one of the arms of pair 1. Mitotic cells submitted to double staining with base-specific fluorochromes (DAPI-CMA(3) ) revealed no regions rich in A+T or G+C sequences. Analysis of spermatogonial mitotic cells after sequential Giemsa/AgNO (3) staining did not reveal any specific mark on the chromosomes. Meiotic metaphase I cells stained with silver nitrate revealed a strong impregnation associated to the sex chromosomes, and in situ hybridization with an 18S rDNA probe showed ribosomal cistrons in an autosomal bivalent.     

Early chromosome condensation without cell fusion. I. Preliminary results with allogeneic and xenogeneic cells.

Early chromatin condensation in interphase cells (G1) of human peripheral blood lymphocytes has been induced without virus or cell fusion by exposure to allogeneic or xenogeneic mitotic cells. The event, although similar in some ways to the phenomenon described as "premature chromosome condensation," "chromosome pulverization," and "prophasing," differs in that it does not require the presence of viruses and cell fusion before mitosis proceeds in the G1 cell. Early chromatin condensation in interphase cells induced by mitotic cells only, consists of chromatids in the early or late G1 phase of the cell cycle that are not pulverized or fragmented at mitosis. Some of the chromosomes are twice as long as the metaphase chromosomes and exhibit natural bands. Almost twice as many of these bands are produced as by trypsin treatment of metaphase chromosomes. The nuclear membrane is intact and nucleoli are present, to which some chromosomes are attached. The DNA content of the precocious chromosomes in G1 is half the amount of the metaphase complement.     

Genome reversion process of endopolyploidy confers chromosome instability on the descendent diploid cells

Endotetraploidy with 4-chromatid chromosomes divides by a bipolar, 2-step meiotic-like division back to diploidy (subcells), which is chiefly achieved by co-segregation of whole genomes uncoupled from spindle participation. This study shows diploid subcell inheritance of endopolyploid-division traits: perpendicular division relative to the cytoskeleton axis, dysfunctional centromere/kinetochore regions and whole genomic separations from co-segregation. The assimilation of these traits into the innate mitotic machinery of the subcells resulted in diploid mitotic divisions that tolerated mild disturbances in cycling progression and in chromosomal distributions. The data were interpreted as demonstrating a blending together of endopolyploid and mitotic division traits with result of an endo-modified mitosis in subcell propagation. Additionally, chromosomal stickiness caused breakage in anaphase/telophase. The observations are discussed in regard to a potential for slowly developing aneuploidy with increasing genomic complexity, which is widely accepted to be the basic route in tumorigenesis.     

Misexpression of cyclin D1 in embryonic germ cells promotes testicular teratoma initiation

Testicular teratomas result from anomalies in embryonic germ cell development. In the 129 family of inbred mouse strains, teratomas arise during the same developmental period that male germ cells normally enter G1/G0 mitotic arrest and female germ cells initiate meiosis (the mitotic:meiotic switch). Dysregulation of this switch associates with teratoma susceptibility and involves three germ cell developmental abnormalities seemingly critical for tumor initiation: delayed G1/G0 mitotic arrest, retention of pluripotency, and misexpression of genes normally restricted to embryonic female and adult male germ cells. One misexpressed gene, cyclin D1 (Ccnd1), is a known regulator of cell cycle progression and an oncogene in many tissues. Here, we investigated whether Ccnd1 misexpression in embryonic germ cells is a determinant of teratoma susceptibility in mice. We found that CCND1 localizes to teratoma-susceptible germ cells that fail to enter G1/G0 arrest during the mitotic:meiotic switch and is the only D-type cyclin misexpressed during this critical developmental time frame. We discovered that Ccnd1 deficiency in teratoma-susceptible mice significantly reduced teratoma incidence and suppressed the germ cell proliferation and pluripotency abnormalities associated with tumor initiation. Importantly, Ccnd1 expression was dispensable for somatic cell development and male germ cell specification and maturation in tumor-susceptible mice, implying that the mechanisms by which Ccnd1 deficiency reduced teratoma incidence were germ cell autonomous and specific to tumorigenesis. We conclude that misexpression of Ccnd1 in male germ cells is a key component of a larger pro-proliferative program that disrupts the mitotic:meiotic switch and predisposes 129 inbred mice to testicular teratocarcinogenesis.     

Tumor cells can escape DNA-damaging cisplatin through DNA endoreduplication and reversible polyploidy

Cancer chemotherapy can induce tumor regression followed, in many cases, by relapse in the long-term. Thus this study was performed to assess the determinants of such phenomenon using an in vivo cancer model and in vitro approaches. When animals bearing an established tumor are treated by cisplatin, the tumor initially undergoes a dramatic shrinkage and is characterized by giant tumor cells that do not proliferate but maintain DNA synthesis. After several weeks of latency, the tumor resumes its progression and consists of small proliferating cells. Similarly, when tumor cells are exposed in vitro to pharmacological concentrations of cisplatin, mitotic activity stops initially but cells maintain DNA duplication. This DNA endoreduplication generates giant polyploid cells that then initiate abortive mitoses and can die through mitotic catastrophe. However, many polyploid cells survive for weeks as non-proliferating mono- or multi-nucleated giant cells which acquire a senescence phenotype. Prolonged observation of these cells sheds light on the delayed emergence of a limited number of extensive colonies which originate from polyploid cells, as demonstrated by cell sorting analysis. Theses colonies are made of small diploid cells which differ from parental cells by stereotyped chromosomal aberrations and an increased resistance to cytotoxic drugs. These data suggest that a multistep pathway, including DNA endoreduplication, polyploidy, then depolyploidization and generation of clonogenic escape cells can account for tumor relapse after initial efficient chemotherapy.     

Cell cycle-dependent 3D distribution of telomeres and telomere repeat-binding factor 2 (TRF2) in HaCaT and HaCaT-myc cells

Telomeres are specialized structures at the ends of the chromosomes that, with the help of proteins--such as the telomere repeat-binding factor TRF2 -, form protective caps which are essential for chromosomal integrity. Investigating the structure and three-dimensional (3D) distribution of the telomeres and TRF2 in the nucleus, we now show that the telomeres of the immortal HaCaT keratinocytes are distributed in distinct non-overlapping territories within the inner third of the nuclear space in interphase cells, while they extend more widely during mitosis. TRF2 is present at the telomeres at all cell cycle phases. During mitosis additional TRF2 protein concentrates all around the chromosomes. This change in staining pattern correlates with a significant increase in TRF2 protein at the S/G2 transition as seen in Western blots of synchronized cells and is paralleled by a cell cycle-dependent regulation of TRF2 mRNA, arguing for a specific role of TRF2 during mitosis. The distinct territorial localization of telomeres is abrogated in a HaCaT variant that constitutively expresses c-Myc--a protein known to contribute to genomic instability. These cells are characterized by overlapping telomere territories, telomeric aggregates (TAs), that are accompanied by an overall irregular telomere distribution and a reduced level in TRF2 protein. These TAs which are readily detectable in interphase nuclei, are similarly present in mitotic cells, including cells in telophase. Thus, we propose that TAs, which subsequently also cluster their respective chromosomes, contribute to genomic instability by forcing an abnormal chromosome segregation during mitosis.     

Transformation of normal diploid cells by isolated metaphase chromosomes of virus-transformed or spontaneous tumor cells.

Normal diploid human cells with a limited life-span in culture, as well as primary or secondary cell cultures of mouse or rat embryos, can be transformed in vitro (i.e. grow in soft-agar or low-serum medium) after a single exposure to metaphase chromosomes from SV40-transformed human or rat cells, Ad5-transformed human cells and several spontaneous human or mouse tumor cells. Chromosomes from normal diploid cells do not show any such transforming activity. As judged from the number of colonies formed in selective medium, the efficiency of transformation is, with some exceptions, of the order of 10(-5)--10(-6) and is generally higher for homologous than for heterologous transfers. A fraction of the colonies demonstrate abortive transformation. Nevertheless, using chromosomes from all but one donor cell population, at least one transferent cell line expressing a stable transformed phenotype has been established. Our results demonstrate that transformation of normal diploid cells by a presumptive chromosome-mediated gene transfer can be obtained with a variety of donor and recipient cells.     

Cell cycle related behavior of a chromosomal scaffold protein in MDCK epithelial cells

Because the mechanisms that govern mitosis are a key to the understanding of cell growth, the proteins associated with chromosomes specifically during this phase have received thorough attention. In the present work we report an Mr 58000 protein in MDCK epithelial cells, recognized by a monoclonal antibody (LFM-1) that decorates chromosomes during M-phase. Cell fractionation methods followed by immunoblotting and immunofluorescence showed that this protein is associated with the nuclear fraction. Biochemical extraction procedures on isolated metaphase chromosomes from nocodazole-synchronized cells indicated that the Mr 58000 protein behaves as a chromosomal scaffold protein, that is, it remains in the pellets after high salt (2 M NaCl) or 3–5 diiodosalicylic acid treatments, even in DNAse pre-digested samples. In addition, confocal microscopy of those chromosomes revealed the LFM-1 epitopes distributed on the external surface and the axis of chromatids. Parallel analysis of interphase nuclei revealed LFM-1 epitopes inside G1-, but excluded from G2-phase nuclei. These results were independently confirmed on nuclei sorted by flow cytometry and in cell populations synchronized by release of G1-/S-phase hydroxyurea arrest. The Mr 58000 and a minor Mr 38000 protein (which was enriched only in mitotic chromosomes of synchronized cells) were analyzed by Edman degradation. They shared the sequence at the amino-terminal end but failed to show total homology with known proteins. These results suggest that LFM-1 antigens fit some of the predictions of the licensing factor model, and may have a role in cell cycle dependent events.     

Hyperproliferation and p53 status of lens epithelial cells derived from alphaB-crystallin knockout mice

alphaB-Crystallin, a major protein of lens fiber cells, is a stress-induced chaperone expressed at low levels in the lens epithelium and numerous other tissues, and its expression is enhanced in certain pathological conditions. However, the function of alphaB in these tissues is not known. Lenses of alphaB-/- mice develop degeneration of specific skeletal muscles but do not develop cataracts. Recent work in our laboratory indicates that primary cultures of alphaB-/- lens epithelial cells demonstrate genomic instability and undergo hyperproliferation at a frequency 4 orders of magnitude greater than that predicted by spontaneous immortalization of rodent cells. We now demonstrate that the hyperproliferative alphaB-/- lens epithelial cells undergo phenotypic changes that include the appearance of the p53 protein as shown by immunoblot analysis. Sequence analysis showed a lack of mutations in the p53 coding region of hyperproliferative alphaB-/- cells. However, the reentry of hyperproliferative alphaB-/- cells into S phase and mitosis after DNA damage by gamma-irradiation were consistent with impaired p53 checkpoint function in these cells. The results demonstrate that expression of functionally impaired p53 is one of the factors that promote immortalization of lens epithelial cells derived from alphaB-/- mice. Fluorescence in situ hybridization using probes prepared from centromere-specific mouse P1 clones of chromosomes 1 and 9 demonstrated that the hyperproliferative alphaB-/- cells were 30% diploid and 70% tetraploid, whereas wild type cells were 83% diploid. Further evidence of genomic instability was obtained when the hyperproliferative alphaB-/- cells were labeled with anti-beta-tubulin antibodies. Examination of the hyperproliferative alphaB-/- mitotic profiles revealed the presence of cells that failed to round up for mitosis, or arrested in cytokinesis, and binucleated cells in which nuclear division had occurred without cell division. These results suggest that the stress protein and molecular chaperone alphaB-crystallin protects cells from acquiring impaired p53 protein and genomic instability.