Mitosis in vertebrate somatic cells with two spindles: Implications for the metaphase/anaphase transition checkpoint and cleavage

During mitosis an inhibitory activity associated with unattached kinetochores prevents PtK1 cells from entering anaphase until all kinetochores become attached to the spindle. To gain a better understanding of how unattached kinetochores block the metaphase/anaphase transition we followed mitosis in PtK1 cells containing two independent spindles in a common cytoplasm. We found that unattached kinetochores on one spindle did not block anaphase onset in a neighboring mature metaphase spindle 20 μm away that lacked unattached kinetochores. As in cells containing a single spindle, anaphase onset occurred in the mature spindles x̄ = 24 min after the last kinetochore attached regardless of whether the adjacent immature spindle contained one or more unattached kinetochores. These findings reveal that the inhibitory activity associated with an unattached kinetochore is functionally limited to the vicinity of the spindle containing the unattached kinetochore. We also found that once a mature spindle entered anaphase the neighboring spindle also entered anaphase x̄ = 9 min later regardless of whether it contained monooriented chromosomes. Thus, anaphase onset in the mature spindle catalyzes a “start anaphase” reaction that spreads globally throughout the cytoplasm and overrides the inhibitory signal produced by unattached kinetochores in an adjacent spindle. Finally, we found that cleavage furrows often formed between the two independent spindles. This reveals that the presence of chromosomes and/or a spindle between two centrosomes is not a prerequisite for cleavage in vertebrate somatic cells.

Carcinogen-induced loss of heterozygosity at the Aprt locus in somatic cells of the mouse

Genetic events leading to the loss of heterozygosity (LOH) have been shown to play a crucial role in the development of cancer. However, LOH events do not occur only in genetically unstable cancer cells but also have been detected in normal somatic cells of mouse and man. Mice, in which one of the alleles for adenine phosphoribosyltransferase (Aprt) has been disrupted by gene targeting, were used to investigate the potency of carcinogens to induce LOH in vivo. After 7,12-dimethyl-1,2-benz[a]anthracene (DMBA) exposure, a 3-fold stronger mutagenic response was detected at the autosomal Aprt gene than at the X chromosomal hypoxantine-guanine phosphoribosyltransferase (Hprt) gene in splenic T-lymphocytes. Allele-specific PCR analysis showed that the normal, nontargeted Aprt allele was lost in 70% of the DMBA-induced Aprt mutants. Fluorescence in situ hybridization analysis demonstrated that the targeted allele had become duplicated in almost all DMBA-induced mutants that displayed LOH at Aprt. These results indicate that the main mechanisms by which DMBA caused LOH were mitotic recombination or chromosome loss and duplication but not deletion. However, after treatment with the alkylating agent N-ethyl-N-nitrosourea, Aprt had a similar mutagenic response to Hprt while the majority (90%) of N-ethyl-N-nitrosourea-induced Aprt mutants had retained both alleles. Unexpectedly, irradiation with x-rays, which induce primarily large deletions, resulted in a significant increase of the mutant frequency at Hprt but not at Aprt. This in vivo study clearly indicates that, in normal somatic cells, carcinogen exposure can result in the induction of LOH events that are compatible with cell survival and may represent an initiating event in tumorigenesis.

Dynamic methylation adjustment and counting as part of imprinting mechanisms.

Monoallelic expression in diploid mammalian cells appears to be a widespread phenomenon, with the most studied examples being X-chromosome inactivation in eutherian female cells and genomic imprinting in the mouse and human. Silencing and methylation of certain sites on one of the two alleles in somatic cells is specific with respect to parental source for imprinted genes and random for X-linked genes. We report here evidence indicating that: (i) differential methylation patterns of imprinted genes are not simply copied from the gametes, but rather established gradually after fertilization; (ii) very similar methylation patterns are observed for diploid, tetraploid, parthenogenic, and androgenic preimplantation mouse embryos, as well as parthenogenic and androgenic mouse embryonic stem cells; (iii) haploid parthenogenic embryos do not show methylation adjustment as seen in diploid or tetraploid embryos, but rather retain the maternal pattern. These observations suggest that differential methylation in imprinted genes is achieved by a dynamic process that senses gene dosage and adjusts methylation similar to X-chromosome inactivation.

Mice cloned from embryonic stem cells

Cloning allows the asexual reproduction of selected individuals such that the offspring have an essentially identical nuclear genome. Cloning by nuclear transfer thus far has been reported only with freshly isolated cells and cells from primary cultures. We previously reported a method of cloning mice from adult somatic cells after nuclear transfer by microinjection. Here, we apply this method to clone mice from widely available, established embryonic stem (ES) cell lines at late passage. With the ES cell line R1, 29% of reconstructed oocytes developed in vitro to the morula/blastocyst stage, and 8% of these embryos developed to live-born pups when transferred to surrogate mothers. We thus cloned 26 mice from R1 cells. Nuclei from the ES cell line E14 also were shown to direct development to term. We present evidence that the nuclei of ES cells at G1- or G2/M-phases are efficiently able to support full development. Our findings demonstrate that late-passage ES cells can be used to produce viable cloned mice and provide a link between the technologies of ES cells and animal cloning. It thus may be possible to clone from a single cell a large number of individuals over an extended period.

Fluorescent RNA cytochemistry: tracking gene transcripts in living cells

The advent of jellyfish green fluorescent protein and its spectral variants, together with promising new fluorescent proteins from other classes of the Cnidarian phylum (coral and anemones), has greatly enhanced and promises to further boost the detection and localization of proteins in cell biology. It has been less widely appreciated that highly sensitive methods have also recently been developed for detecting the movement and localization in living cells of the very molecules that precede proteins in the gene expression pathway, i.e. RNAs. These approaches include the microinjection of fluorescent RNAs into living cells, the in vivo hybridization of fluorescent oligonucleotides to endogenous RNAs and the expression in cells of fluorescent RNA-binding proteins. This new field of ‘fluorescent RNA cytochemistry’ is summarized in this article, with emphasis on the biological insights it has already provided. These new techniques are likely to soon collaborate with other emerging approaches to advance the investigation of RNA birth, RNA–protein assembly and ribonucleoprotein particle transport in systems such as oocytes, embryos, neurons and other somatic cells, and may even permit the observation of viral replication and transcription pathways as they proceed in living cells, ushering in a new era of nucleic acids research in vivo.

Nucleotide excision repair in rat male germ cells: low level of repair in intact cells contrasts with high dual incision activity in vitro

The acquisition of genotoxin-induced mutations in the mammalian germline is detrimental to the stable transfer of genomic information. In somatic cells, nucleotide excision repair (NER) is a major pathway to counteract the mutagenic effects of DNA damage. Two NER subpathways have been identified, global genome repair (GGR) and transcription-coupled repair (TCR). In contrast to somatic cells, little is known regarding the expression of these pathways in germ cells. To address this basic question, we have studied NER in rat spermatogenic cells in crude cell suspension, in enriched cell stages and within seminiferous tubules after exposure to UV or N-acetoxy-2-acetylaminofluorene. Surprisingly, repair in spermatogenic cells was inefficient in the genome overall and in transcriptionally active genes indicating non-functional GGR and TCR. In contrast, extracts from early/mid pachytene cells displayed dual incision activity in vitro as high as extracts from somatic cells, demonstrating that the proteins involved in incision are present and functional in premeiotic cells. However, incision activities of extracts from diplotene cells and round spermatids were low, indicating a stage-dependent expression of incision activity. We hypothesize that sequestering of NER proteins by mispaired regions in DNA involved in synapsis and recombination may underlie the lack of NER activity in premeiotic cells.

Highly efficient base excision repair (BER) in human and rat male germ cells

The quality of germ cell DNA is critical for the fate of the offspring, yet there is limited knowledge of the DNA repair capabilities of such cells. One of the main DNA repair pathways is base excision repair (BER) which is initiated by DNA glycosylases that excise damaged bases, followed by incision of the generated abasic (AP) sites. We have studied human and rat methylpurine-DNA glycosylase (MPG), uracil-DNA glycosylase (UNG), and the major AP endonuclease (HAP1/APEX) in male germ cells. Enzymatic activities and western analyses indicate that these enzymes are present in human and rat male germ cells in amounts that are at least as high as in somatic cells. Minor differences were observed between different cellular stages of rat spermatogenesis and spermiogenesis. Repair of methylated DNA was also studied at the cellular level using the Comet assay. The repair was highly efficient in both human and rat male germ cells, in primary spermatocytes as well as round spermatids, compared to rat mononuclear blood cells or hepatocytes. This efficient BER removes frequently occurring DNA lesions that arise spontaneously or via environmental agents, thereby minimising the number of potential mutations transferred to the next generation.

Can ends justify the means?: Telomeres and the mechanisms of replicative senescence and immortalization in mammalian cells

Finite replicative lifespan, or senescence, of mammalian cells in culture is a phenomenon that has generated much curiosity since its description. The obvious significance of senescence to organismal aging and the development of cancer has engendered a long-lasting and lively debate about its mechanisms. Recent discoveries concerning the phenotypes of telomerase knockout mice, the consequences of telomerase reexpression in somatic cells, and genes that regulate senescence have provided striking molecular insights but also have uncovered important new questions. The objective of this review is to reconcile old observations with new molecular details and to focus attention on the key remaining puzzles.

Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination.

A procedure of reversible immortalization of primary cells was devised by retrovirus-mediated transfer of an oncogene that could be subsequently excised by site-specific recombination. This study focused on the early stages of immortalization: global induction of proliferation and life span extension of cell populations. Comparative analysis of Cre/LoxP and FLP/FRT recombination in this system indicated that only Cre/LoxP operates efficiently in primary cells. Pure populations of cells in which the oncogene is permanently excised were obtained, following differential selection of the cells. Cells reverted to their preimmortalized state, as indicated by changes in growth characteristics and p53 levels, and their fate conformed to the telomere hypothesis of replicative cell senescence. By permitting temporary and controlled expansion of primary cell populations without retaining the transferred oncogene, this strategy may facilitate gene therapy manipulations of cells unresponsive to exogenous growth factors and make practical gene targeting by homologous recombination in somatic cells. The combination of retroviral transfer and site-specific recombination should also extend gene expression studies to situations previously inaccessible to experimentation.

Differentiation of immortal cells inhibits telomerase activity.

Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5'-TTAGGG-3' to the ends of mammalian chromosomal DNA (telomeres) to compensate for the progressive loss that occurs with successive rounds of DNA replication. Although somatic cells do not express telomerase, germ cells and immortalized cells, including neoplastic cells, express this activity. To determine whether the phenotypic differentiation of immortalized cells is linked to the regulation of telomerase activity, terminal differentiation was induced in leukemic cell lines by diverse agents. A pronounced downregulation of telomerase activity was produced as a consequence of the differentiated status. The differentiation-inducing agents did not directly inhibit telomerase activity, suggesting that the inhibition of telomerase activity is in response to induction of differentiation. The loss of telomerase activity was not due to the production of an inhibitor, since extracts from differentiated cells did not cause inhibition of telomerase activity. By using additional cell lineages including epithelial and embryonal stem cells, down-regulation of telomerase activity was found to be a general response to the induction of differentiation. These findings provide the first direct link between telomerase activity and terminal differentiation and may provide a model to study regulation of telomerase activity.

A biomarker that identifies senescent human cells in culture and in aging skin in vivo.

Normal somatic cells invariably enter a state of irreversibly arrested growth and altered function after a finite number of divisions. This process, termed replicative senescence, is thought to be a tumor-suppressive mechanism and an underlying cause of aging. There is ample evidence that escape from senescence, or immortality, is important for malignant transformation. By contrast, the role of replicative senescence in organismic aging is controversial. Studies on cells cultured from donors of different ages, genetic backgrounds, or species suggest that senescence occurs in vivo and that organismic lifespan and cell replicative lifespan are under common genetic control. However, senescent cells cannot be distinguished from quiescent or terminally differentiated cells in tissues. Thus, evidence that senescent cells exist and accumulate with age in vivo is lacking. We show that several human cells express a beta-galactosidase, histochemically detectable at pH 6, upon senescence in culture. This marker was expressed by senescent, but not presenescent, fibroblasts and keratinocytes but was absent from quiescent fibroblasts and terminally differentiated keratinocytes. It was also absent from immortal cells but was induced by genetic manipulations that reversed immortality. In skin samples from human donors of different age, there was an age-dependent increase in this marker in dermal fibroblasts and epidermal keratinocytes. This marker provides in situ evidence that senescent cells may exist and accumulate with age in vivo.

Mos overexpression in Swiss 3T3 cells induces meiotic-like alterations of the mitotic spindle.

High levels of mos protooncogene product are expressed during oocyte meiotic maturation and Mos has been implicated in formation of the spindle and spindle pole. Here, we show that in Swiss 3T3 cells with 4N DNA content, high levels of Mos lead to the production of binucleated cells. The Swiss 3T3 cells in mitosis, before binucleation occurs, are anastral and the spindle poles are juxtaposed to the cell membrane. These phenotypes may be related to the meiotic process of attachment of the spindle pole to the oocyte membrane during polar body formation. The production of binucleated somatic cells could result from attachment of the altered mitotic spindle pole to the cell membrane that interferes with cytokinesis but not karyokinesis. This can explain at least one form of genetic instability that leads to altered DNA content in tumor cells.

Ubiquitous, heritable damage in cell populations that survive treatment with methotrexate

A permanent line of mouse embryo fibroblasts was treated with concentrations of the anticancer drug methotrexate (MTX) that left 20–50% surviving colonies. The surviving population initially multiplied at a much slower rate than controls after subculture in the absence of the drug, and required 9–12 days of serial subculture, with selective growth of the faster growing cells, to approximate the control rate. To determine the distribution of growth rates of cells in the original posttreatment populations, many single cells were isolated in multiwell plates immediately after the treatment period, and the resulting clones were serially subcultured. Most of the control clones underwent about 2 population doublings per day (PD/D). Almost all the survivors of MTX treatment multiplied at heterogeneously reduced rates, ranging from 0.6 PD/D to as high as control rates for a very few clones. They maintained the reduced rates through many subcultivations. The heritability of the reduced growth rates indicates that most cells that retain proliferative capacity after treatment with MTX carry random genetic damage that is perpetuated through many divisions of their progeny. Similar results have been described for cells that survive x-irradiation, and suggest random genetic damage is a common occurrence among cells in rapidly growing tissues that survive cytotoxic treatment. It also occurs in serial subcultures of cells that had been held under the constraint of confluence for extended periods, which suggests that the accumulation of random genetic damage to somatic cells during aging of mammals underlies the reduction of growth rate and function of the cells that characterizes the aging process.

Identification of a small, very acidic constitutive nucleolar protein (NO29) as a member of the nucleoplasmin family

We report the discovery and molecular characterization of a small and very acidic nucleolar protein of an SDS/PAGE mobility corresponding to Mr 29,000 (NO29). The cDNA-deduced sequence of the Xenopus laevis protein defines a polypeptide of a calculated molecular mass of 20,121 and a pI of 3.75, with an extended acidic region near its C terminus, and is related to the major nucleolar protein, NO38, and the histone-binding protein, nucleoplasmin. This member of the nucleoplasmin family of proteins was immunolocalized to nucleoli in Xenopus oocytes and diverse somatic cells. Protein NO29 is associated with nuclear particles from Xenopus oocytes, partly complexed with protein NO38, and occurs in preribosomes but not in mature ribosomes. The location and the enormously high content of negatively charged amino acids lead to the hypothesis that NO29 might be involved in the nuclear and nucleolar accumulation of ribosomal proteins and the coordinated assembly of pre-ribosomal particles.