Survival-related DNA repair phenomena in cultured rat-kangaroo cells

Cultured cells (line PtK-2) from the marsupial mammal rat-kangaroo, or potoroo (Potorous tridactylis), which photoreactivate (PR) both UV-induced dimers and lethality, excise few dimers, and are only slightly sensitized by post-UV exposure to caffeine, were subjected to caffeine and hydroxyurea (HU) treatments during the 30-min PR period. It was found that neither caffeine nor HU inhibited PR of lethality as measured by colony-forming ability. Further, the cells exhibited no photoprotective properties and 3 mM caffeine potentiated the same slight survival decrease in both photoreactivated and unphotoreactivated cells. It is evident that caffeine does not inhibit PR or the survival-related dark repair systems to any great extent, and hence the caffeine-sensitive post-UV dark repair found in this (and possibly other) mammalian cell lines may not be related directly to survival-dependent pyrimidine dimer removal, but instead to lesions or repair processes other than, but not excluding, pyrimidine dimers.     

In situ visualization of telomere elongation patterns in human cells

The telomerase enzyme plays a critical role in human aging and cancer biology by maintaining telomere length and extending the proliferative lifespan of most stem cells and cancer cells. Despite the importance of this enzyme, our understanding of the mechanisms that regulate its activity and establish telomere length homeostasis in mammalian cells is incomplete, in part because the perfect repetitive nature of telomeric sequence hampers in situ detection of telomere elongation patterns. Here, we describe a novel assay using a mutant telomerase that adds a well-tolerated variant telomeric repeat sequence to telomere ends. By specifically detecting the addition of these variant repeats, we can directly visualize telomere elongation events in human cells. We validate this approach by in situ mapping of telomere elongation patterns within individual nuclei and across a population of cells.     

Canonical analysis of cells in normal and abnormal cervical smears

Over 4,000 cells from 105 normal and 96 abnormal uterine cervical scrapes were prepared according to the UCLA monolayer procedure, stained by a routine Papanicolaou method and visually classified by two cytopathologists and a technologist into seven classes: parabasal, metaplastic, mild dysplasia, moderate dysplasia, severe dysplasia, carcinoma in situ and invasive carcinoma. Canonical analysis was used to correlate effects-coded class membership variables with 23 cell features derived from digital image analysis. In general, nuclear texture measures derived from linear combinations of run-length correlations along with features derived from a Markov transitional probability matrix provided the best predictors of cell class. After cells were divided into benign (moderate dysplasia or less) and malignant (severe dysplasia or worse) groups, discriminant analysis correctly classified 84% of the benign cells and 91% of the malignant cells.     

Induction of multipolar mitoses in cultured cells: Decay and restructuring of the mitotic apparatus and distribution of centrioles

During recovery after a long (up to 12 h) treatment of pig embryo culture cells (PK) with nocodazole at concentrations of 0.02 g/ml and 0.2 g/ml all c-metaphase cells divide normally into two daughter cells. During recovery after a short (1–4 h) treatment with 0.6 g/ml nocodazole only multipolar mitoses (as a rule tripolar) arise. At the ultrastructural level, the increasing nocodazole concentration leads to progressive disruption of the mitotic spindle. At a nocodazole concentration of 0.2 g/ml kinetochores are not associated with microtubules. At a nocodazole concentration of 0.6 g/ml there are no microtubules around the centrosomes, and in every cell one of the two diplosomes disintegrates. In tripolar telophase centrioles are distributed among the spindle poles generally in a 2:2:0 pattern. Mother and daughter centrioles are always disoriented but not separated. The centriole-free pole contains a cloud of electron-dense material. During tripolar division two of the three daughter cells mainly fuse shortly after telophase forming one binucleate cell. Thus a multipolar mitosis arises as a result of the uncoupling of mother centrioles and spindle microtubules, but not of the duration of the c-mitotic arrest. Centriole-free poles account for the divergence of chromosomes, but mainly they are unable to ensure the normal cytokinesis of daughter cells.by M. Trendelenburg     

Heavy meromyosin-binding filaments in the mitotic apparatus of mammaliam cells

Guinea pig spermatozoa fail to fertilize eggs in Ca²⁺-free media primarily because of specific inhibition of the acrosome reaction and activation of the spermatozoa. In Ca²⁺-free media the spermatozoa undergo capacitation at the same rate as in Ca²⁺-containing media, but are arrested in the capacitated state. If Ca²⁺ is made available after the spermatozoa have reached the capacitated state, the spermatozoa immediately undergo the acrosome reaction and activation. The minimum concentration of Ca²⁺ necessary for the initiation of the acrosome reaction and activation is about 0.2 mM. Mg²⁺ cannot substitute for Ca²⁺ in initiating these processes. Possible mechanisms by which Ca²⁺ triggers the acrosome reaction and activation of guinea pig spermatozoa are discussed.     

The vesicular compartment of the mitotic apparatus in mammalian cells

Intracellular membranes might play an eminent role in regulating several events during mitosis: In this paper the appearance and changing configurations of the vesicular compartment of the mitotic apparatus of HeLa cells was studied from anaphase to telophase. In early prophase electron opaque and transparent membranous vesicles are found in the pericentriolar region outside the nucleus. During prometaphase when the nuclear envelope opens and starts to disappear, an increasing number of these vesicles appears in the mitotic apparatus near the chromosomes. During metaphase vesicles are spread all over the mitotic apparatus, the number of electron opaque vesicles decreases while the total amount of vesicles does not change significantly. Anaphase shows the same pattern of distribution in the half-spindle and in the midbody. In telophase the amount of electron opaque vesicles increases again. They are now found around vacuoles and near the newly appearing Golgi-cisternae. We assume that the electron opaque vesicles are derived from the Golgi- apparatus which disintegrates during prophase and reappears in late telophase. The change in the appearance of the different types of vesicles during metaphase coincides with drastic changes in the ionic milieu in the mitotic apparatus (Wolniak et al., 1983).     

In vivo hybridisation of cultured melanoma cells and isogenic normal mouse cells.

Somatic cell hybrids were derived by fusing tumourigenic and melanogenic melanoma (PAZG) cells with normal diploid male mouse cells in vivo. Their chromosomal composition was equivalent to the sum of both parental genomes and included a Y chromosome lacking in the melanoma parent. Our study showed that in PAZG X C57BL hybrids (MP), tumourigenicity was suppressed but pigmentation was expressed.     

Organization of mitotic apparatus poles in etoposide-treated CHO-K1 cells

Analyzed in this study is the organization of mitotic spindle poles in CHO-K1 cells dividing after treatment with etoposide (1 h, 25 μM). At various periods after the treatment, we studied the following: (1) the distribution of γ-tubulin in mitotic cells by immunofluorescent staining, (2) the level of post-translational modification of α-tubulin in spindle microtubules by immunoelectron microscopy, and (3) the ultrastructure of mitotic apparatus poles by standard electron microscopy. 48 h after the addition of etoposide, disturbances in the ultrastructure of mitotic spindle poles were observed in etoposide-treated CHO-K1 cells with both bipolar and with multipolar mitotic apparatuses. The increased number of centrioles was unevenly distributed between the mitotic spindle poles; some centrioles did not take an obvious part in the mitotic spindle organization and differed in their number of outgrowing microtubules. Most centrioles were without fibrillar halos. Immunoelectron microscopy showed the differences in the staining of the poles of a multipolar spindle within one cell with antibodies to tyrosinated α-tubulin, whereas the staining of cells with antibodies to acetylated α-tubulin did not reveal such differences. Immunofluorescence staining for γ-tubulin also indicated differing organizations of poles in the same spindle. Our data findings provided the first evidence that the pattern of immunostaining and ultrastructure of mitotic apparatus poles can differ in cells dividing at various time periods after the action of etoposide.     

Romancing mitosis and the mitotic apparatus

One of the earliest lessons students learn in biology is the process of mitosis and how cells divide to produce daughter cells. Although first described more than a century ago by early investigators such as E. B. Wilson, many aspects of mitosis and cell division remain the subject of considerable research today. My personal investigations and research contributions to the study of mitosis were made possible by recent developments in the field when I began my career, including access to novel mammalian cell culture models and electron and fluorescence microscopy. Building upon those innovations, my laboratory and other contemporary investigators first charted the ultrastructure and molecular organization of mitosis and chromosome movement and the assembly and function of the cytoskeleton. This field of research remains a significant challenge for future investigators in cell biology and medicine.     

The organization of the mitotic apparatus poles in etoposide-treated CHO-K1 cells

In this study, we have examined the organization of the mitotic spindle poles in CHO-K1 cells dividing after treatment with the etoposide (1 h, 25 microM). We studied at various periods after the treatment: 1) the distribution of gamma-tubulin in mitotic cells by immunofluorescent staining; 2) the level of posttranslational modification of a-tubulin in the spindle microtubules by immunoelectron microscopy; 3) the ultrastructure of the mitotic apparatus poles by standard electron microscopy. In 48 h after the addition of the agent we identified considerable changes in the ultrastructure of poles in etoposide-treated CHO-K1 cells with bipolar and multipolar spindles. The number of centrioles increased. The centrioles were unevenly distributed among the poles, and some centrioles were not explicitly involved in the organization of mitotic spindle, furthermore they can differ in the number of outgrowing microtubules. Most centrioles were without fibrillar halo. In 48 h after the addition of etoposide, electron microscopy of cells after immunoperoxidase staining with antibodies to acetylated and tyrosinated alpha-tubulin has shown that different poles of a multipolar spindle within the same cell are stained differently for tyr-tubulin but not for acet-tubulin. Immunofluorescence staining for gamma-tubulin also points to different organization of poles in the same spindle. Our findings provide the first evidence that the pattern of immunostaning and the ultrastructure of mitotic apparatus poles differ in the cells dividing at various periods after etoposide treatment.     

Purification of mitotic spindles from cultured human cells

In eukaryotes, both chromosome segregation and the determination of the cell division cleavage plane depend on the mitotic spindle apparatus. Spindle malfunctioning can lead to chromosome mis-segregation and cytokinesis defects and hence result in aneuploidy. Thus, the understanding of the structure and function of mitotic spindles is of interest not only from the perspective of basic science, but has implications also for human health and disease. Until recently, this complex microtubule-based structure was studied mainly by cell biological techniques in mammalian cells, by biochemical assays in Xenopus egg extracts, and by genetic approaches in genetically tractable organisms such as yeast, flies, and nematodes. With the rapid development of mass spectrometry and its increasing application to biological problems, it has become possible to subject highly complex structures, such as the mitotic spindle apparatus, to proteomics approaches. Such studies require the isolation of the mitotic spindle, or its substructures, in sufficient amounts and free of excessive contaminants. A number of methods for the isolation of mitotic spindles from mammalian tissue culture cells have been developed in the past. We have compared these methods and found that protocols based on the stabilization of microtubules by taxol were most efficient and reproducible. Here, we describe the further optimization of a taxol-based method, originally developed by Zieve and Solomon [Cell 28 (1982) 233-242], and its application to the isolation of human mitotic spindles at a scale suitable for mass spectrometric analysis [G. Sauer, R. Korner, A. Hanisch, A. Ries, E.A. Nigg, H.H.W. Sillje, Mol. Cell. Proteomics 4 (2005) 35-43].     

In situ analysis of single-stranded and duplex siRNA integrity in living cells

To attain the full therapeutic promise of short interfering RNA (siRNA), it is believed that improvements such as increased biostability are critical. Regrettably, thus far, insufficient in situ data are on hand regarding the intracellular stability of siRNAs. We report on the use of an advanced fluorescence-based method to probe the nucleolytic decay of double labeled siRNAs, which are subject to fluorescence resonance energy transfer (FRET). In vitro measurements with RNAse A and cellular extracts demonstrate that the ratio of acceptor (5'-Cy5) to donor (3'-rhodamine green) fluorescence can be used to study the degradation of the labeled siRNA substrates upon donor excitation. Intracellular FRET analysis showed substantial degradation of single-stranded siRNA, whereas duplex siRNA stayed intact during the measured time period. These data underline the high intrinsic nuclease resistance of unmodified duplex siRNA and prove that cellular persistence is much more critical for the single-stranded structure. For the first time, the stability of siRNA is investigated in real-time inside living cells. The fluorescence-based method presented here is a straightforward technique to gain direct information on siRNA integrity inside living cells and provides a bright outlook to learn more about the intracellular fate of siRNA therapeutics.     

Diphenylhydantoin and mitotic spindle abnormalities in cultured mouse and human cells

The present study demonstrates that the antiepileptic drug diphenylhydantoin (DPH) is capable of inducing aneuploidy but not structural aberrations in cultured mouse embryonic fibroblasts. A high concentration of 200 micrograms/ml was found to increase the percentage of hyperdiploidy from 4.8 (control) to 16.0. The treatment was found to increase mitotic indices as a consequence of a mitotic-arresting action of the drug. These effects are probably due to the effect of the drug on the structure of the mitotic apparatus. Abnormal cell divisions and mitotic disturbance were found to increase in a dose-dependent manner after DPH treatment. In a parallel study, human amnion cells were found to show similar response to DPH treatment.     

Reorganization of mitotic apparatus in the etoposide-treated CHO-K1 cells precedes apoptotic death

In a culture of CHO-K1 cells, etoposide (1 h, 25 μM) has been shown to produce interphase arrest, after which the cells resume mitotic division and, after some time, are submitted to apoptotic death. Accumulation of apoptotic cells in the culture follows a gradual increase in the number of multipolar mitoses. Our findings provide the first evidence for differences in the pattern of immunofluorescent staining of multipolar mitotic spindle microtubules with antibodies to α-tubulin, acetylated α-tubulin, and tyrosinated α-tubulin in mitotic cells dividing in the period preceding apoptosis. Moreover, some parts of the multipolar mitotic spindle can differ by the presence of antigenic determinants accessible to anti-tyrosinated α-tubulin antibodies. These abnormalities of the mitotic apparatus are aggravated immediately before the increase in the number of cells submitted to apoptosis. Our data have also shown that some cells pass through at least two mitotic cycles prior to a sharp increase in the number of apoptotic cells in the cell culture.