Studies on endoreduplication. III. Endoreduplication induced by mitomycin C in PHA-stimulated tonsillar lymphocyte cultures

The activity of mitomycin C (MMC) to induce endoreduplication was examined in PHA-stimulated tonsillar lymphocyte cultures. Cellular kinetics of both diploid and endoreduplicated cells was studied by the combined techniques of thymidine and BrdU incorporation. The kinetic data showed that the increase of endoreduplicated cells induced by MMC correlated with the increase of the second-generation diploid cells. Also, it showed that the majority of endoreduplicated cells found at the time when the maximal number of endoreduplicated cells was obtained had incorporated BrdU only once into their diplochromosomes. This suggests that MMC-induced endoreduplicated cells originate from MMC-induced G2-arrested cells.     

Induction of endoreduplication in double minutes of the human neuroblastoma cells and the replication pattern.

Induction of endoreduplication (ERD) using Hoechst 33258 as well as colcemid was carried out in cultured neuroblastoma (NB) line cells. In these endoreduplicated cells, the majority of double minutes (DMs) appeared to take a diplochromosome like configuration to form a cluster consisting of four minute elements, assuming a complex DM. Sister chromatid differential staining (SCD) using 5-bromo-2'-deoxyuridine (BrdUrd) revealed the non-random distribution of the stained chromatids among four chromatids composing each diplochromosome, suggesting the occurrence of so-called "outside replication" of DNA strands during the process of ERD. The same pattern of differential staining was also found in the quadruple minutes of each endoreduplicated DM. Since DMs are acentric, the present results suggest that centromeres do not play any essential role in the formation of diplochromosomes observed in the conventional cytologic preparations and that centromeres are probably not responsible for the phenomenon of the "outside replication" of DNA strands.     

Origin of araC-induced endoreduplicated cells

To investigate the origin of endoreduplicated cells induced by DNA-synthesis inhibitors, V79 Chinese hamster cells were treated with 1-beta-D-arabinofuranosylcytosine (araC). During the treatment, BrUdR was present in the culture medium. If endoreduplicated cells originate from cells which have undergone rereplication of DNA segments randomly distributed over the genome during araC treatment, the diplochromosomes should exhibit lightly stained bands. The data indicate that endoreduplicated cells originate from cells blocked at the G2 stage of the cell cycle rather than from cells which have undergone an aberrant rereplication during araC treatment.     

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.     

Induction of endoreduplication by hydrazine in Chinese hamster V 79 cells and reduced incidence of sister chromatid exchanges in endoreduplicated mitoses

Hydrazine in high concentrations very effectively induces endoreduplication in Chinese hamster V 79 cells. The addition of 5-bromodeoxyuridine (BrdU) for the duration of one cell cycle prior to the induction of endoreduplication produces diplochromosomes with sister chromatid differentiation (SCD) after differential chromatid staining. The fact that diplochromosomes with complete SCD are obtained shows that endoreduplication was induced in cells that were in G2-phase. The analysis of sister chromatid exchanges (SCEs) showed that hydrazine treatment rarely led to increased SCE frequencies in mitoses after endoreduplication, but that it caused a strong SCE induction in diploid second division metaphases in the same culture. Neither catalase nor cysteine had an effect on the induction of endoreduplication or the incidence of SCEs. Treatment of the cells with mitomycin C prior to addition of BrdU led to increased SCE frequencies. Compared with the normal mitoses from the same preparation, the mitoses after endoreduplication showed a significantly reduced induction of SCEs. In contrast to these findings, SCE induction was not reduced in the common tetraploid V 79 cells after colcemid-induced polyploidization.     

Regulatory mechanism of cell division : I. Colchicine-induced endoreduplication

Experiments were carried out to study the induction of endoreduplication by colchicine in Chinese hamster cells cultivated in vitro. The cells that endoreduplicate are those that, at the moment of treatment, are in late-S and, in particular, in G2. The endoreduplication cycle consists of two periods of synthesis (S1 and S2), as already noted by Schwarzacher & Schnedl [42], separated by an intervening period that we call G? The S2 synthesis begins in a highly synchronous manner, without the cells having gone into a c-mitosis. The quantity of endoreduplicated cells induced is proportional to the 3.5th root of the colchicine concentration, above a threshold value, and does not depend on the duration of the treatment. When the cultures are treated twice with colchicine, the second treatment is also able to induce endoreduplication and, after it, there appear double endoreduplicated cells (with quadruplochromosomes).     

Endoreduplication preferentially occurs at the proximal side of the abscission zone during abscission of tomato leaf

Endoreduplication is a cell cycle variant in which multiple rounds of DNA replication occur without subsequent mitosis, resulting in polyploid cells. Although cells with endoreduplicated nuclei were ubiquitously distributed throughout the abscission zone (AZ) of tomato leaf before abscission induction by ethylene, endoreduplication was detected mostly on the proximal side of the AZ after induction. The possible association between endoreduplication and intensive membrane trafficking in cells at the proximal side of the AZ is discussed.     

Failure of sister chromatid separation leads to formation of diplochromosomes in colcemid treated PtK1 cells

The origin of diplochromosomes has been traced in multinucleate rat kangaroo cells (PtK1) obtained after colcemid treatment. In these cells the diplochromosomes were shown to originate from restitution nuclei, indicating that they were formed due to the omission or failure of sister chromatid separation and not due to endoreduplication. In this context the mechanism of sister chromatid separation has been discussed. The independence of this mitotic event from other associated processes, such as chromosome condensation, nuclear envelope breakdown or spindle formation has been stressed.     

Endoreduplication in cultured mammalian cells treated with 4-nitroquinoline I-oxide

Chinese hamster cells treated with 4-nitroquinoline 1-oxide (4NQO) developed diplochromosomes, indicating the induction of endoreduplication. The maximum ratio of diplochromosomes, about 3% of total mitoses, was reached 27 h after treatment with a concentration of 0.5 μg/ml (2.6·10^?6M) for 6 h. Various chromosomal aberrations other than changes in ploidy were observed in diplochromosomes. Spiral structures observed in diplochromosomes and the binding of 4NQO to proteins are discussed here.     

Identification of quantitative trait loci that affect endoreduplication in maize endosperm

Endoreduplication in maize endosperm precedes the onset of starch and storage protein synthesis, and it is generally thought to influence grain filling. We created four backcross populations by reciprocally crossing the F₁ progeny of a cross between Sg18 and Mo17 to the parental inbreds, which differ in endoreduplication by two parameters—mean ploidy and percentage of endoreduplicated nuclei. This four-backcross design allowed us to estimate and test the additive and dominant genetic effects of quantitative trait loci (QTLs) affecting endoreduplication. An analysis of endosperm from the four backcross populations at 16 days after pollination using a modified triploid mapping approach identified three endosperm QTLs influencing mean ploidy and two endosperm QTLs affecting the percentage of endoreduplicated nuclei. Some of these QTLs may manifest their effects on endoreduplication via expression in the embryo. The QTLs detected display strong dominance or over-dominance and interacted epistatically with an embryo-expressed QTL. This helps to explain the genetic basis for transgressive segregation in the backcross progeny. Although the favorable alleles that increase mean ploidy and percentage of endoreduplicated nuclei can be contributed by both parents, the Mo17-derived alleles for endoreduplication were often dominant or over-dominant to the Sg18-derived allele. One QTL on chromosome 7 that may be expressed in both the embryo and endosperm exerted a pleiotropic effect on two different parameters of endoreduplication. The results from this study shed light on the regulation of endoreduplication in maize endosperm and provide a marker-assisted selection strategy for potentially improving grain yield. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. C. M. Coelho and S. Wu contributed equally to this work and should be considered as first authors.     

Changes in radical scavenging activity of normal,endoreduplicated and depolyploid root tip cells of Allium cepa

The plant cell responds to abiotic stress conditions by adjusting its cellular metabolism and various defensive mechanisms. Cellular metabolism involves changes in the cell cycle, in which the cell undergoes repeated rounds of endocycles leading to polyploidization. Defense mechanisms such as role of antioxidants are a key to understand plant adaptation. The present work describes endoreduplication and radical scavenging activity as two different defense mechanisms adapted by plants for their survival under stress condition. The work describes linkage of these two processes with each other under abiotic stress. Endoreduplicated root tip cells of Allium cepa were depolyploidized by exogenous phytohormones. Further, free radical scavenging activity from normal, endoreduplicated and depolyploidized root tips cells was observed to understand the role of phytohormones. Elevated free radical scavenging potential was observed in endoreduplicated cells compared to normal and depolyploidized cells. Based on these results, it was concluded that endoreduplication and antioxidant pathways are linked with each other through phytohormonal activities. The concentration of auxin and cytokinin regulates the activity of ascorbate oxidase enzyme, which in turn maintains the concentration of AsA within the cell. AsA level directs the prolyl-hydroxylation process of cell division proteins in quiescent center cells either toward endoreduplication process or cell division process.     

Sensitive period for the induction of endoreduplication by rotenone in cultured Chinese hamster cells

Rotenone-induced endoreduplication was investigated in Chinese hamster CHL cells. Cell cycle analyses, using 5-bromo-2-deoxyuridine (BrdU) labeling, revealed that endoreduplicaiton was induced between the G₂-phase and mitotic metaphase. Morphological studies indicated that the chromosomes of cells in metaphase at the time of rotenone exposure immediately aggregated. Within 1 h, however, the aggregated chromosomes began to decondense forming telophase nuclei. Cells with aggregated chromosomes were collected by mitotic selection using the mitotic arrestant TN-16 and then cultured for 30 h following rotenone administration. This population of cells demonstrated an extremely high frequency of endoreduplicated metaphases. Further analysis by BrdU labeling indicated that the aggregated metaphases underwent only one round of DNA replication before endoreduplicated metaphases were formed. The most sensitive period for the induction of endoreduplication by rotenone occurs during mitotic metaphase.by M.F. Trendelenburg     

The nature of DNA plays a role in chromosome segregation: endoreduplication in halogen-substituted chromosomes

AA8 Chinese hamster ovary cells were treated with halogenated nucleosides analogues of thymidine, namely CldU, 5-iodo-2'-deoxyuridine (IdU), and 5-bromo-2'-deoxyuridine (BrdU), following different experimental protocols. The purpose was to see whether incorporation of exogenous pyrimidine analogues into DNA could interfere with normal chromosome segregation. The endpoint chosen was endoreduplication, that arises after aberrant mitosis when daughter chromatids segregation fails. Treatment with any of the halogenated nucleosides for two consecutive cell cycles resulted in endoreduplication, with a highest yield for CldU, intermediate for IdU, and lowest for BrdU. The frequency of endoreduplicated cells paralleled in all cases the level of analogue substitution into DNA. Our results seem to support that thymidine analogue substitution into DNA is responsible for the triggering of endoreduplication. Besides, the lack of any effect on endoreduplication when CldU was present for only one S-period strongly suggest that it is the nature of template, and not nascent DNA, that plays a major role in chromosome segregation. Taking into account that topoisomerase II cleaves DNA at preferred sequences within its recognition/binding sites, the likely involvement of the enzyme is discussed.     

Do the frequencies of sister chromatid exchanges in endoreduplieated mitoses provide a measure for lesion persistence and repair?

Endoreduplication was induced in V 79 cells using Colcemid. The concentration of Colcemid necessary to induce endoreduplication is about 1000 times higher than that needed to arrest mitoses or to induce ordinary tetraploid cells. Diplochromosomes with sister chromatid differentiation were obtained by adding BrdU for the duration of one cell cycle prior to the induction of endoreduplication. The induction of endoreduplication with Colcemid had no influence on the frequency of sister chromatid exchanges (SCEs). Treating the cultures with mitomycin C (MMC) before adding BrdU increased the percentage of endoreduplieated mitoses and also led to marked SCE induction. In the diplochromosomes, the frequencies of both twin SCEs (first cycle) as well as single SCEs (second cycle) were increased. It was also found that the SCE frequencies in mitoses after endoreduplication were lower than the values found in diploid and ordinary tetraploid metaphases of the same preparation. The possible conclusions concerning the lifetime of SCE-inducing lesions and the influence of repair processes are discussed.     

Flow cytometric and karyological analysis of polysomaty and polyploidization during callus formation from leaf segments of various potato genotypes

Summary Flow cytometry and karyological analysis were used to study polysomaty and polyploidization during the first 15 days of callus formation in leaf segments from shoot cultures and greenhouse-grown plants of various lines and genotypes of Solanum tuberosum and S. phureja. The greenhouse-grown plants showed a higher degree of polysomaty (77% and 49% of polyploidized nuclei) than the shoot cultures (< 3%). During the in vitro culture period, polyploidization occurred through endoreduplication. Segments of the five shoot cultures showed up to 87%, 53%, 59%, 45% and 56% polyploidization, respectively; the DNA content of corresponding interphase nuclei amounted to 8C, 16C, 16C, 16C and 8C, and the chromosome numbers to 96. Segments from the two greenhouse-grown genotypes showed up to 87% and 84% polyploidization; the DNA content amounted to 32C and 16C, and the chromosome numbers to 192 and 96. The number of reduplication cycles was species-dependent; the degree of polyploidization was dependent on the initial ploidy level of the genotypes. Cell proliferation did not take place at a constant rate. The maximum frequencies of metaphases (52–171 per segment) occurred after 1 week of culture and were correlated with the ploidy level of the genotypes. Cells were triggered to mitosis rather than to endoreduplication. Cell cycles with normal monochromosomes could be shorter than 1 day, and those with diplochromosomes lasted at least 1 day. Polysomaty, degree of polyploidization and abnormal nuclear processes are discussed in relation to the origin of genetic instability early in culture.     

Concanavalin A induces endoreduplication in cultured mammalian cells.

Concanavalin A (Con A) induced endoreduplication in an established cell line, Don, of the Chinese hamster. The inducibility of Con A was inhibited by α-methyl-D-mannoside. When a secondary culture of kidney cells (CHK), which showed the contact-inhibition of growth, was used, there was an increase in spontaneous endoreduplication. CHK cells or some of them were more sensitive to Con A than Don cells, in which few spontaneous endoreduplications were observed. Mitotic shake-off after Con A treatment led to the higher ratio of endoreduplicated cells to normal mitoses, suggesting that endoreduplicating cells do not “round-up” and probably do not condense chromosomes through the cell cycle until M is reached.     

Halogen substitution of DNA protects from poisoning of topoisomerase II that results in DNA double-strand breaks

DNA topoisomerase II (topo II), a fundamental nuclear enzyme, cleaves the double-stranded DNA molecule at preferred sequences within its recognition/binding sites. We have recently reported [F. Cortés, N. Pastor, S. Mateos, I. Domínguez, The nature of DNA plays a role in chromosome segregation: endoreduplication in halogen-substituted chromosomes, DNA Repair 2 (2003) 719-726] that when cells incorporate halogenated nucleosides analogues of thymidine into DNA, it interferes with normal chromosome segregation, as shown by an extraordinarily high yield of endoreduplication. The frequency of endoreduplicated cells paralleled the level of analogue substitution into DNA, lending support to the idea that thymidine analogue substitution into DNA is most likely responsible for the triggering of endoreduplication. Using the pulsed-field gel electrophoresis (PFGE) technique, we have now analyzed a possible protection provided by the incorporation of exogenous halogenated nucleosides against DNA breakage induced by the topo II poison m-AMSA. The result was that the different halogenated nucleosides were shown as able to protect DNA from double-strand breaks induced by m-AMSA depending such a protection upon the relative percent of incorporation of a given thymidine analogue into DNA. Our results clearly indicate that the presence of halogenated nucleosides in DNA diminishes the frequency of interaction of topo II with DNA and thus the frequency with which cleavage can occur.     

Effect of sucrose on polyploidization in early callus cultures of Solanum tuberosum

Leaf segments of a monohaploid, dihaploid and tetraploid genotype of the potato (Solanum tuberosum; x = 12) were cultured on callus-inducing medium with 10, 20, 30 or 40 gl^–1 sucrose. After 5 and 7 days of culture, metaphases contained the somatic or polyploidized number of mono- or diplochromosomes. The percentages of polyploidized metaphases were inversely correlated with the number of chromosome sets of the genotypes. In monohaploid leaf segments the percentages of polyploidized metaphases and of metaphases with diplochromosomes increased when the sucrose concentration was raised from 10 or 20 to 30 gl^–1 and remained constant or decreased from 30 to 40 gl^–1. Higher concentrations of sucrose but not higher osmolalities of the medium due to mannitol induced endoreduplication in more cells. The frequency of polyploidized metaphases and metaphases with diplochromosomes in dihaploid and tetraploid leaf segments remained constant through increases in sucrose concentrations.     

Induction of high frequencies of endoreduplication in mammalian cell cultures with 33258 Hoechst and rubidazone.

Cells of the mouse L strain and the Chinese hamster CHO line were treated with 33258 Hoechst, rubidazone, and a combination of these. Recovering cell populations following the drug removal exhibited a high frequency (20--50%) of metaphases with diplochromosomes (endoreduplication), especially in the combination treatment series (up to more than 70% in the L strain). Such a procedure should be useful in probing the mechanisms of the endoreduplication process.     

The structure and position of mouse centromeric heterochromatin in BrdU-labelled chromosomes and diplochromosomes stained by the pH 10.4 method

A mouse cell line of C57B1/6J spontaneous melanoma (clone PG 19), and a C-type virus transformed cell line (G-8 clone 124) originating from normal Balb/c mice were used in a study of the centromeric heterochromatin region of BrdU-labelled chromosomes stained by the Giemsa pH 10.4 method. Three possible explanations for the generation of compound lateral asymmetry within the centromeric heterochromatin region of the laboratory mouse are discussed: 1) inverted translocation; 2) centric fusion followed by paracentromeric fission and 3) inversion of part of the centromeric satellite DNA. These processes could be of considerable genetic and evolutionary significance. The non-random spatial position of unstained and dark stained C-bands in BrdU-labelled diplochromosomes of endoreduplicated cells can be explained as being due to the localization of the old and new DNA chains in a unineme chromatid model. The late replicating regions are shown to be located on the inside of the half-chromatid close to the axial symmetry axis of the metaphase chromosome.