Studies of multipolar mitoses in euploid tissue cultures

In tissue cultures of male Microtus agrestis, diploid mitoses with two X or two Y chromosomes were found. For identifiying the sex chromosomes in nonhypotonioally treated mitoses, the asynchrony of DNA replication of the sex chromosomes of both sexes was used. The constitutive heterochromatin of Y replicates later in the S period than X, and X₂ of the female replicates later than X₁. Autoradiographic studies of tetraploid tripolar mitoses showed that the diploid daughter nuclei contain either XX or YY in the male; in the female, X₁X₂ daughter nuclei were found less frequently than X₁X₁ and X₂X₂ cells.     

Occurrence and possible consequences of multipolar mitoses in primary cultures of adult rat hepatocytes

Proliferating primary cultures of adult rat hepatocytes are characterized by the occurrence of multipolar mitoses, and chromosome loss resulting in the formation of micronuclei at telophase. The percentage of multipolar mitotic figures was determined to be 12.76 ± 7.9%, 80% of which were tripolar. Multipolar mitotic stages showed a high incidence of chromosome loss, increasing from meta- (61.7 ± 16.6%) to telophase (72.1 ± 19.3%). Regular bipolar mitotic figures on the other hand also showed chromosome loss, however, to a lesser degree and decreasing from meta- (49.5 ± 10.4%) to telophase (34.9 ± 7.9%). The incidence of chromosome loss even in regular mitotic figures is very high compared to other cells and appears to depend on another special feature of hepatocytes: they remain flat and well attached during mitosis, so that shearing forces could be responsible for the separation of chromosomes from the mitotic spindle. Additionally this morphology creates a situation allowing for a maximal interaction of mitotic spindles of binucleated cells, leading to the high rate of multipolar mitoses observed. Both multipolar mitoses and chromosome loss could also explain the consecutive detachment of hepatocytes reported for proliferating primary cultures, since the aneuploid daughter cells generated can be expected to be nonviable in most cases and eventually detach. © 1993 Wiley-Liss, Inc.     

Role of multipolar mitoses in the proliferation of multinucleate cells induced by cytochalasin B

A prolonged action of cytochalasin B results in the formation of numerous multipolar mitoses (26%) in Chinese hamster cell cultures. The transition to multipolar mitoses in the presence of cytochalasin B is not accompanied by K-mitotic delay. It is shown that a multipolar mitosis without cytoplasmic division is one of the main causes of multinucleation development in cytochalasin B-treated cultures. After stopping the drug action the cytochalasin B-induced multinucleate cells continue to divide by multipolar mitosis. In this case it completes with cytokinesis and, probably, leads to a decrease in the number of nuclei per cell. The origin of multipolar mitotic apparatus after the action of cytochalasin B is discussed in addition to the role of multipolar mitosis in formation and proliferation of multinucleate cells.     

Euploid segregation through multipolar mitosis in mammalian cell cultures

Cultures were made of kidney cells of male Rhesus monkey and a karyological analysis of these cells was carried out at various times after plating using the chromosome banding method of Seabringht (1972), in order to study the segregation phenomena which take place in cell cultures in vitro through multipolar mitoses and to identify segregating cells. — Several segregating cells were found: haploid cells, diploid cells with two X chromosomes and triploid cells which were strictly euploid. Polyploidization-segregation cycles in the cell cultures and their mechanism and chronological sequence were analyzed. — On the basis of these results it was suggested that the genome is organized in haploid sets capable of segregating as units and of producing strictly euploid segregating daughter cells.     

Pattern of DNA segregation in multipolar anatelophases of different ploidy in euploid and aneuploid mammalian cells cultivated in vitro

A microdensitometrical analysis of multipolar anatolophases of cuploid and aneuploid mammalian cells in vitro has been made in order to study the quantitative distribution of DNA to the poles.In the tripolar anatelophases of aneuploid Chinese hamster cells (obtained using colchicine) DNA is distributed to poles in an almost random way. In the spontaneous tripolar anatelophases of euploid cells of Rhesus, DNA is distributed to the poles in whole number multiples of 1c. The following distributions were observed in tetraploid cells: 14 cases of 3 : 3 : 2 and 3 cases of 4 : 2 : 2. In triploid cells, 6 cases of 2 : 2 : 2 and 1 case of 3 : 2 : 1.Thus we have deduced that each ploidy degree has a preferential distribution of sets and in contrast to the original hypothesis, random segregation of the sets is not the common occurrence. Hypotheses have been made to explain the origin of multipolar mitosis and the preferential distribution of chromatid sets to the poles. The results would confirm the existence of haploid sets of chromatids and would explain the appearance of triploid, pentaploid and hexaploid cells in tissues and cell cultures.     

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     

Occurrence of multipolar mitoses and association with Aurora-A/-B kinases and p53 mutations in aneuploid esophageal carcinoma cells

Background  

Aurora kinases and loss of p53 function are implicated in the carcinogenesis of aneuploid esophageal cancers. Their association with occurrence of multipolar mitoses in the two main histotypes of aneuploid esophageal squamous cell carcinoma (ESCC) and Barrett's adenocarcinoma (BAC) remains unclear. Here, we investigated the occurrence of multipolar mitoses, Aurora-A/-B gene copy numbers and expression/activation as well as p53 alterations in aneuploid ESCC and BAC cancer cell lines.     

Studies on tissue cultures of the genus Cinchona L. alkaloid production in cell suspension cultures

Initiation and culture of callus and cell suspensions of Cinchona ledgeriana and C. succirubra as well as the successful isolation and selection of a high-yielding alkaloid-forming strain derived from the leaf rachis of a C. succirubra plant are described. Results of feeding experiments with L-tryptophan using two different culture procedures are presented and discussed. Maximum alkaloid yields of up to 0.9% (based on dry weight) or 6.35 mg/l have been obtained.     

Studies on tissue cultures of the genus Cinchona L.

A procedure for the in vitro clonal mass propagation of shoots and plants of two Cinchona species derived from single shoot tips is described. Special attention is given to rooting problems of the shoots under in vitro conditions.     

When the genome plays dice: circumvention of the spindle assembly checkpoint and near-random chromosome segregation in multipolar cancer cell mitoses

Background

Normal cell division is coordinated by a bipolar mitotic spindle, ensuring symmetrical segregation of chromosomes. Cancer cells, however, occasionally divide into three or more directions. Such multipolar mitoses have been proposed to generate genetic diversity and thereby contribute to clonal evolution. However, this notion has been little validated experimentally.

Principal Findings

Chromosome segregation and DNA content in daughter cells from multipolar mitoses were assessed by multiphoton cross sectioning and fluorescence in situ hybridization in cancer cells and non-neoplastic transformed cells. The DNA distribution resulting from multipolar cell division was found to be highly variable, with frequent nullisomies in the daughter cells. Time-lapse imaging of H2B/GFP-labelled multipolar mitoses revealed that the time from the initiation of metaphase to the beginning of anaphase was prolonged and that the metaphase plates often switched polarity several times before metaphase-anaphase transition. The multipolar metaphase-anaphase transition was accompanied by a normal reduction of cellular cyclin B levels, but typically occurred before completion of the normal separase activity cycle. Centromeric AURKB and MAD2 foci were observed frequently to remain on the centromeres of multipolar ana-telophase chromosomes, indicating that multipolar mitoses were able to circumvent the spindle assembly checkpoint with some sister chromatids remaining unseparated after anaphase. Accordingly, scoring the distribution of individual chromosomes in multipolar daughter nuclei revealed a high frequency of nondisjunction events, resulting in a near-binomial allotment of sister chromatids to the daughter cells.

Conclusion

The capability of multipolar mitoses to circumvent the spindle assembly checkpoint system typically results in a near-random distribution of chromosomes to daughter cells. Spindle multipolarity could thus be a highly efficient generator of genetically diverse minority clones in transformed cell populations.     

Random position of human heterochromatin-bearing chromosomes in first and third mitoses of lymphocyte cultures

Summary The position of chromosomes 1, 9, and 16 in first and third mitoses of lymphocyte cultures was measured in BUdR-labelled air-dried chromosome preparations. No significant deviation from a random distance could be found between the two homologues of each chromosome, either in the first or in the third mitoses after phytohemagglutinin stimulation. Colchicine treatment also had no influence.     

Studies on growth and cardenolide production of Digitalis lanata tissue cultures

Digitalis lanata cell cultures grown as small undifferentiated aggregates in suspension culture can be redifferentiated into green embryos that produce cardenolides. The possibility of using a statistical (Box-Wilson) experimental design to study the effects of four different variables on growth, differentiation, and cardenolide production of D. lanata tissue cultures are investigated. The results of the analyses were processed by linear regression analysis. Mathematical models explaining the effects of the variables were developed. The concentration of maltose and the NO(3) (-)--NH(4) (+) ratio were found to be significant variables for both growth and cardenolide production. The size of the inoculum was also important.     

Studies on some factors affecting solasodine contents in tissue cultures of Solanum nigrum

Tissue cultures of Solanum nigrum L. were initiated from leaf explants on a solid medium containing inorganic salts [Murashige and Skoog (1962), Physiol. Plant. 15: 473–497], vitamins [Gamborg et al. (1968) Exp. Cell Res. 50:151–158], 3% sucrose and combinations of indoleacetic acid and benzyladenine. Solasodine content was determined in differentiated and undifferentiated (callus) tissues by a colorimetric technique and thin layer chromatography. Indoleacetic acid and sucrose in the medium markedly stimulated the production of solasodine in the tissue cultures. In the cultures grown in darkness the differentiated tissues produced significantly more (anywhere from 1.5 to 10 times) solasodine than the callus in several media. When sucrose concentration was increased to 4, 6 and 10% level in the medium which contained 10 μ M benzyladenine as the sole growth regulator, a significant increase of solasodine production in cultures was found.     

Studies on the morphogenetic response of maize tissue cultures of different origin

The participation of the genotype and of organ specifity effect in the quality of morphogenetic response (callogenesis, bud and root formation) of primary maize explants has been investigated. The presence of synthetic auxins — especially 2,4-D at 1 to 5 mg 1^?1 conc. - in cultivation medium was essential for both callus formation and continuous growth of tissue and suspension cultures. Anatomic structure of callus cultures is permanently heterogeneous, their growth is ensured by the action of meristems of the type found in root tips, and by repeated callogenesis from malformed roots. Adventive buds and plants could be regenerated only from cultures of embryonal origin (of one line). The presence or absence of the endosperm gene “opaque” did not influence callogenesis intensity in cultures of isolated embryos; however the morphogenetic response was clearly “line specific”.