Generation and maintenance of acentric stable double minutes from chromosome arms in inter-species hybrid cells.

BACKGROUND: Extrachromosomal acentric double minutes (DMs) contribute to human malignancy by carrying amplified oncogenes. Recent cancer genomics revealed that the pulverization of defined chromosome arms (chromothripsis) may generate DMs, however, nobody had actually generated DMs from chromosome arm in culture. Human chromosomes are lost in human-rodent hybrid cells. RESULTS: We found that human acentric DMs with amplified c-myc were stable in human-rodent hybrid cells, although the degree of stability depended on the specific rodent cell type. Based on this finding, stable human-rodent hybrids were efficiently generated by tagging human DMs with a plasmid with drug-resistance gene. After cell fusion, human chromosomes were specifically pulverised and lost. Consistent with chromothripsis, pulverization of human chromosome arms was accompanied by the incorporation into micronuclei. Such micronucleus showed different replication timing from the main nucleus. Surprisingly, we found that the hybrid cells retained not only the original DMs, but also new DMs without plasmid-tag and c-myc, but with human Alu. These DMs were devoid of telomeres and centromeres, and were stable in culture for more than 3 months. Microarray analysis showed that the new DMs were generated from several human chromosomal regions containing genes advantageous for cellular growth. Such regions were completely different from the original DMs. CONCLUSIONS: The inter-species hybrid mimics the chromothripsis in culture. This is the first report that experimentally demonstrates the generation of multiple stable acentric DMs from the chromosome arm.

DNA replication occurs in all lamina positive micronuclei, but never in lamina negative micronuclei.

A micronucleus is a small nucleus-like structure found in the cytoplasm of dividing cells that suffered from genotoxic stress. It is generally hypothesised that micronuclei content is eventually lost from cells, though the mechanism of how this occurs is unknown. If DNA located within the micronucleus is not replicated, it may explain the loss of micronuclei content. Because there had been no compelling evidence for this issue, we have addressed whether DNA located within the micronucleus is replicated this issue. Pulse labelling of bromodeoxyuridine revealed that DNA synthesis takes place in a portion of micronuclei that contain nuclear lamin B protein. By using iodine 3'-deoxyuridine/chlorodeoxyuridine double labelling, we found that all micronuclei containing lamin B are replicated during one cell cycle, whereas micronuclei lacking lamin B are never replicated. This result suggests that the content of lamin B-negative micronuclei is lost during cell division. Furthermore, we simultaneously visualised sites of DNA synthesis, lamin B and the extrachromosomal double minutes chromatin, which contain amplified oncogenes. We found that although the replication timing of double minutes was generally preserved in micronuclei, at times it differed greatly from the timing in the nucleus, which may perturb the expression of the amplified oncogenes. Taken together, these findings uncovered the DNA replication occurring inside micronuclei.

Generation of micronuclei during interphase by coupling between cytoplasmic membrane blebbing and nuclear budding.

Micronucleation, mediated by interphase nuclear budding, has been repeatedly suggested, but the process is still enigmatic. In the present study, we confirmed the previous observation that there are lamin B1-negative micronuclei in addition to the positive ones. A large cytoplasmic bleb was found to frequently entrap lamin B1-negative micronuclei, which were connected to the nucleus by a thin chromatin stalk. At the bottom of the stalk, the nuclear lamin B1 structure appeared broken. Chromatin extrusion through lamina breaks has been referred to as herniation or a blister of the nucleus, and has been observed after the expression of viral proteins. A cell line in which extrachromosomal double minutes and lamin B1 protein were simultaneously visualized in different colors in live cells was established. By using these cells, time-lapse microscopy revealed that cytoplasmic membrane blebbing occurred simultaneously with the extrusion of nuclear content, which generated lamin B1-negative micronuclei during interphase. Furthermore, activation of cytoplasmic membrane blebbing by the addition of fresh serum or camptothecin induced nuclear budding within 1 to 10 minutes, which suggested that blebbing might be the cause of the budding. After the induction of blebbing, the frequency of lamin-negative micronuclei increased. The budding was most frequent during S phase and more efficiently entrapped small extrachromosomal chromatin than the large chromosome arm. Based on these results, we suggest a novel mechanism in which cytoplasmic membrane dynamics pulls the chromatin out of the nucleus through the lamina break. Evidence for such a mechanism was obtained in certain cancer cell lines including human COLO 320 and HeLa. The mechanism could significantly perturb the genome and influence cancer cell phenotypes.

Emergence of micronuclei and their effects on the fate of cells under replication stress.

The presence of micronuclei in mammalian cells is related to several mutagenetic stresses. In order to understand how micronuclei emerge, behave in cells, and affect cell fate, we performed extensive time-lapse microscopy of HeLa H2B-GFP cells in the presence of hydroxyurea at low concentration. Micronuclei formed after mitosis from lagging chromatids or chromatin bridges between anaphase chromosomes and were stably maintained in the cells for up to one cell cycle. Nuclear buds also formed from chromatin bridges or during interphase. If the micronuclei-bearing cells entered mitosis, they either produced daughter cells without micronuclei or, more frequently, produced cells with additional micronuclei. Low concentrations of hydroxyurea efficiently induced multipolar mitosis, which generated lagging chromatids or chromatin bridges, and also generated multinuclear cells that were tightly linked to apoptosis. We found that the presence of micronuclei is related to apoptosis but not to multipolar mitosis. Furthermore, the structural heterogeneity among micronuclei, with respect to chromatin condensation or the presence of lamin B, derived from the mechanism of micronuclei formation. Our study reinforces the notion that micronucleation has important implications in the genomic plasticity of tumor cells.

How transcription proceeds in a large artificial heterochromatin in human cells.

Heterochromatin is critical for genome integrity, and recent studies have suggested the importance of transcription in heterochromatin for maintaining its silent state. We previously developed a method to generate a large homogeneously staining region (HSR) composed of tandem plasmid sequences in human cells that showed typical heterochromatin characteristics. In this study, we examined transcription in the HSR. We found that transcription of genes downstream to no-inducible SRalpha promoter was restricted to a few specific points inside the large HSR domain. Furthermore, the HSR localized to either to the surface or to the interior of the nucleolus, where it was more actively transcribed. The perinucleolar or intranucleolar locations were biased to late or early S-phase, and the location depended on either RNA polymerase II/III or I transcription, respectively. Strong activation of the inducible TRE promoter resulted in the reversible loosening of the HSR domain and the appearance of transcripts downstream of not only the TRE promoters, but also the SRalpha promoters. During this process, detection of HP1alpha or H3K9Me3 suggested that transcription was activated at many specific points dispersed inside large heterochromatin. The transcriptional rules obtained from studying artificial heterochromatin should be useful for understanding natural heterochromatin.

Nonselective DNA damage induced by a replication inhibitor results in the selective elimination of extrachromosomal double minutes from human cancer cells.

Gene amplification plays a pivotal role in human malignancy. Highly amplified genes frequently localize to extrachromosomal double minutes (dmin), which usually segregate to daughter cells in association with mitotic chromosomes. We and others had shown that treatment with low-dose hydroxyurea (HU) results in the elimination of dmin and reversion of the cancer cell phenotype. HU treatment in early S-phase, when dmin are replicated, results in their detachment from chromosomes at the next M-phase, leading to the appearance of micronuclei enriched in dmin, followed by their elimination. In this article, we examined the effect of low-dose HU on the behavior of dmin in relation to DNA damage induction by simultaneously monitoring LacO-tagged dmin and phosphorylated histone H2AX (gammaH2AX). As expected, treatment with low-dose HU induced numerous gammaH2AX foci throughout the nucleus in early S-phase, and these rarely coincided with dmin. Most chromosomal gammaH2AX foci disappeared by metaphase, whereas, unexpectedly, those that persisted frequently associated with dmin. We found that these dmin aggregated, detached from anaphase chromosomes, and apparently formed micronuclei. Because gammaH2AX foci likely represent DNA double strand breaks (DSBs), the response to DSBs sustained by extrachromosomal dmin appears to be different from that sustained by chromosomal loci, which may explain why DSB-inducing agents cause the selective elimination of dmin.

Micronuclei bearing acentric extrachromosomal chromatin are transcriptionally competent and may perturb the cancer cell phenotype.

Extrachromosomal double minutes (DM) bear amplified genes that contribute to the malignancy of human cancer cells. A novel intracellular behavior of DMs resulted in their selective entrapment within micronuclei; opening the vista, this could perturb the cancer cell phenotype if genes located on DMs were expressed in micronuclei. Here, using fluorescence in situ hybridization, we detected transcripts in DM-enriched micronuclei. Visualization of DMs and their transcripts in live cells showed that DMs are as actively transcribed in the micronuclei and nuclei. Moreover, pulse-incorporated bromouridine was detected in the micronuclei, and the transcripts eventually exited from the micronuclei, similar to the behavior of nuclear transcripts. This apparently normal pattern of gene expression in DM-enriched micronuclei was restricted to micronuclei associated with lamin B, and lamin B association was more frequent for micronuclei that incorporated DMs than for those that incorporated a chromosome arm. The frequency of lamin B-associated micronuclei increased after entry into S phase, and accordingly, there was a concomitant increase in transcription in micronuclei. Taken together, these results indicate that the expression of genes on DMs can be temporally altered by their incorporation into micronuclei. This may be relevant for a broad spectrum of other extrachromosomal elements.

Regulation of c-myc through intranuclear localization of its RNA subspecies.

We used fluorescence in situ hybridization (FISH) to detect c-myc RNA subspecies in human COLO 320DM tumor cells. Although the FISH procedure removed the majority of RNAs from the nucleolus, c-myc RNA continued to be detected in both the nucleoplasm and nucleolus. This finding suggests stable association between c-myc RNA and the nucleolus. Nucleolar accumulation of c-myc RNA appeared to be temporally regulated by cell-cycle progression. Hybridization with exon- and strand-specific RNA probes indicated that the non-protein coding exon 1 plays a novel role in determining the subnuclear localization of c-myc RNA. Antisense RNA targeting exon 2 localized only with nucleoplasmic foci, where it might interact with the sense strand. Thus, c-myc gene expression may be regulated by intranuclear localization of its RNA.