Chromosome Polyploidy Research Articles

In-depth analysis of genomes and functional genomics of orchid using cutting-edge high-throughput sequencing.

High-throughput sequencing technology has been facilitated the development of new methodologies and approaches for studying the origin and evolution of plant genomes and subgenomes, population domestication, and functional genomics. Orchids have tens of thousands of members in nature. Many of them have promising application potential in the extension and conservation of the ecological chain, the horticultural use of ornamental blossoms, and the utilization of botanical medicines. However, a large-scale gene knockout mutant library and a sophisticated genetic transformation system are still lacking in the improvement of orchid germplasm resources. New gene editing tools, such as the favored CRISPR-Cas9 or some base editors, have not yet been widely applied in orchids. In addition to a large variety of orchid cultivars, the high-precision, high-throughput genome sequencing technology is also required for the mining of trait-related functional genes. Nowadays, the focus of orchid genomics research has been directed to the origin and classification of species, genome evolution and deletion, gene duplication and chromosomal polyploidy, and flower morphogenesis-related regulation. Here, the progressing achieved in orchid molecular biology and genomics over the past few decades have been discussed, including the evolution of genome size and polyploidization. The frequent incorporation of LTR retrotransposons play important role in the expansion and structural variation of the orchid genome. The large-scale gene duplication event of the nuclear genome generated plenty of recently tandem duplicated genes, which drove the evolution and functional divergency of new genes. The evolution and loss of the plastid genome, which mostly affected genes related to photosynthesis and autotrophy, demonstrated that orchids have experienced more separate transitions to heterotrophy than any other terrestrial plant. Moreover, large-scale resequencing provide useful SNP markers for constructing genetic maps, which will facilitate the breeding of novel orchid varieties. The significance of high-throughput sequencing and gene editing technologies in the identification and molecular breeding of the trait-related genes in orchids provides us with a representative trait-improving gene as well as some mechanisms worthy of further investigation. In addition, gene editing has promise for the improvement of orchid genetic transformation and the investigation of gene function. This knowledge may provide a scientific reference and theoretical basis for orchid genome studies.

Epigenetic regulation of AURKA by miR-4715-3p in upper gastrointestinal cancers

Aurora kinase A (AURKA) is frequently overexpressed in several cancers. miRNA sequencing and bioinformatics analysis indicated significant downregulation of miR-4715-3p. We found that miR-4715-3p has putative binding sites on the 3UTR region of AURKA. Upper gastrointestinal adenocarcinoma (UGC) tissue samples and cell models demonstrated significant overexpression of AURKA with downregulation of miR-4715-3p. Luciferase reporter assays confirmed binding of miR-4715-3p on the 3UTR region of AURKA. miR-4715-3p mediated a reduction in AURKA levels leading to G2/M delay, chromosomal polyploidy, and cell death. We also detected a remarkable decrease in GPX4, an inhibitor of ferroptosis, with an increase in cleaved PARP and caspase-3. Inhibition of AURKA using siRNA produced similar results, suggesting a possible link between AURKA and GPX4. Analysis of UGC samples and cell models demonstrated increased methylation levels of several CpG nucleotides upstream of miR-4715-3p. 5-Aza-2′-deoxycytidine induced demethylation of several CpG nucleotides, restoring miR-4715-3p expression, leading to downregulation of AURKA. In conclusion, our data identified a novel epigenetic mechanism mediating silencing of miR-4715-3p and induction of AURKA in UGCs. Inhibition of AURKA or reconstitution of miR-4715-3p inhibited GPX4 and induced cell death, suggesting a link between AURKA and ferroptosis.

Detecting novel mutations and combined Klinefelter syndrome in Usher syndrome cases

Background: Usher syndrome (USH) is an autosomal recessive disease characterized by hearing loss, vision loss, and occasionally vestibular dysfunction. Klinefelter syndrome (KS) is an X chromosome polyploidy characterized by one or more additional X chromosomes in males. To date, there has been no report of USH combined with KS.Objectives: This study examined the causative genes in three Chinese probands with congenital hearing loss.Material and methods: Targeted next-generation sequencing (NGS) was performed to identify mutations in three probands with hearing loss. Low-coverage whole-genome sequencing (WGS) analysis of aneuploidy was used to verify the chromosome aneuploidy.Results: Four novel MYO7A mutations were identified in two USH1 probands who were initially diagnosed with nonsyndromic hearing loss until the onset of vision loss. Another case was initially diagnosed with nonsyndromic hearing loss and USH2 and KS were discovered incidentally after the genetic analysis.Conclusions: Our findings expand the mutation spectrum of MYO7A. This is also the first report of concomitant USH and KS. Genetic testing can help with clinical management, particularly if an unrecognized syndromic disorder is identified before the onset of additional symptoms. A clinical genetic evaluation is recommended as part of the diagnostic work-up in congenital hearing loss.

Application of next-generation sequencing technique in genetic analysis of spontaneous abortion

Objective To investigate the value of next-generation sequencing (NGS) technique for genetic analysis of spontaneous abortion. Methods From January to June 2017, 154 patients who visited the First Affiliated Hospital of Zhengzhou University for spontaneous abortion were enrolled. All abortion tissue samples were analyzed by both NGS combined with short tandem repeat (STR) and single nucleotide polymorphism array (SNP-array). Results of the two methods were compared by Chi-square or Fisher's exact test. Results (1) Chromosomal abnormalities were detected in 109 of the 154 cases (70.7%), including 52 (47.7%) of numerical chromosomal abnormalities, 49 (45.0%) of structural chromosomal abnormalities, six (5.5%) of mosaicism, and two (1.8%) of uniparental disomy (UPD). In those 52 cases of numerical chromosome abnormalities, there were 45 of chromosome aneuploidy and seven of polyploidy. The top three numerical chromosomal abnormalities were 45,X (27.0%, 14/52), trisomy 22 (9.6%, 5/52) and trisomy 16 (7.7%, 4/52). Forty-nine structural abnormality cases carried 67 copy number variations (CNV), including 13 pathogenic CNV (pCNV, 19.4%), 24 variants of unknown clinical significance (35.8%) and 30 benign CNV (44.8%). In those 13 pCNVs, two were responsible for microdeletion and microduplication syndromes. (2) SNP-array was successful in 152 cases, but failed in two (1.3%) due to genomic DNA <200 ng. However, NGS technology was successful in all 154 cases and identified chromosomal abnormalities in the two cases that SNP-array had failed. No statistically significant difference was shown in the detection rate of chromosomal abnormalities between SNP-array and NGS technology [70.4% (107/152) vs 67.5% (104/154), χ2=0.293, P=0.588]. (3) No significant difference in the detection of chromosome aneuploidy (six cases in each group, 3.9% vs 3.9%) and mosaicism (45 cases in each group, 29.2% vs 29.6%) was found between NGS technology and SNP-array. Three cases of polyploidy (69, XXX) and two of UPD were identified by SNP-array, but not by NGS. When combined with STR, NGS was able to detect all three cases of polyploidy (69, XXX). (4) Forty-seven structural abnormality cases detected by SNP-array carried 53 CNVs, and 49 detected by NGS carried 67 CNVs. (5) NGS detected ten, three and one more CNVs than SNP-array did when the genome lengths were 100-<500, 500-<1 000 and ≥1 000 kb, respectively. Conclusions NGS can be used to detect chromosomal aneuploidy and mosaicism that can be identified by SNP-array with fewer limitations on total amount of genome. Moreover, CNVs that fail to be identified by SNP-array can also be detected by NGS. When combined with STR, NGS can effectively detect chromosomal polyploidy. Therefore, NGS could be a potential genetic analysis method for spontaneous abortion and of importance for genetic counseling. Key words: Abortion, spontaneous; High-throughput nucleotide sequencing; Chromosome aberrations; Polymorphism, single nucleotide

Regulation of DNA replication and chromosomal polyploidy by the MLL-WDR5-RBBP5 methyltransferases.

ABSTRACTDNA replication licensing occurs on chromatin, but how the chromatin template is regulated for replication remains mostly unclear. Here, we have analyzed the requirement of histone methyltransferases for a specific type of replication: the DNA re-replication induced by the downregulation of either Geminin, an inhibitor of replication licensing protein CDT1, or the CRL4CDT2 ubiquitin E3 ligase. We found that siRNA-mediated reduction of essential components of the MLL-WDR5-RBBP5 methyltransferase complexes including WDR5 or RBBP5, which transfer methyl groups to histone H3 at K4 (H3K4), suppressed DNA re-replication and chromosomal polyploidy. Reduction of WDR5/RBBP5 also prevented the activation of H2AX checkpoint caused by re-replication, but not by ultraviolet or X-ray irradiation; and the components of MLL complexes co-localized with the origin recognition complex (ORC) and MCM2-7 replicative helicase complexes at replication origins to control the levels of methylated H3K4. Downregulation of WDR5 or RBBP5 reduced the methylated H3K4 and suppressed the recruitment of MCM2-7 complexes onto replication origins. Our studies indicate that the MLL complexes and H3K4 methylation are required for DNA replication but not for DNA damage repair.

Lack of topoisomerase copy number changes in patients with de novo and relapsed diffuse large B-cell lymphoma

Topoisomerase (TOP) gene copy number changes may predict response to treatment with TOP-targeting drugs in cancer treatment. This was first described in patients with breast cancer and is currently being investigated in other malignant diseases. TOP-targeting drugs may induce TOP gene copy number changes at relapse, with possible implications for relapse therapy efficacy. TOP gene alterations in lymphoma are poorly investigated. In this study, TOP1 and TOP2A gene alterations were investigated in patients with de novo diffuse large B-cell lymphoma (DLBCL) (n = 33) and relapsed DLBCL treated with chemotherapy regimens including TOP2-targeting drugs (n = 16). No TOP1 or TOP2A copy number changes were found. Polysomy of chromosomes 20 and 17 was seen in 3 of 25 patients (12%) and 2 of 32 patients (6%) with de novo DLBCL. Among relapsed patients, chromosome polysomy was more frequently observed in 5 of 13 patients (38%) and 4 of 16 patients (25%) harboring chromosome 20 and 17 polysomy, respectively; however, these differences only tended to be significant (p = 0.09 and p = 0.09, respectively). The results suggest that TOP gene copy number changes are very infrequent in DLBCL and not likely induced by TOP2-targeting drugs. Increased polyploidy of chromosomes 17 and 20 among patients with relapsed DLBCL may reflect genetic compensation in the tumor cells after TOP2 inhibition, but is more likely due to the increased genetic instability often seen in progressed cancers. Therefore, it is unlikely that TOP1 and TOP2A gene alterations can be used as predictive markers for response to treatment with TOP2-targeting drugs in patients with DLBCL.

Doubling the Chromosome Number of Black Poplar Trees (Populus nigra L.) by Colchicine In vitro

The possibility of inducing chromosomal polyploidy was studied for Black poplar (Populus nigra L.) which micropropagated from stem cutting by exposure to different colchicine concentrations 125, 250, 500 and 1000 mg / l and different exposure periods (0.0, 2, 4, 6, 8 days) in MS medium supplemented with 1mg/l BA and 0.3 mg/l IBA. After 4 weeks of exposure results showed the significant superiority of 125 mg / l colchicine treatment for stem cutting survival percentage transplant survival percentage after aclimitization and diploid plants percentage characteristics, also the results of polyploidy showed that the highest percentage of tetraploid plant (18%) was recorded for both treatments 125 and 250 mg/l colchicine, when 50.66% of plants treated with 500 mg/l colchicine has a mixed polyploidy and it was significantly different from the rest treatments. but the interaction effect of colchicine concentrations and exposure periods showed that the highest survival percentage was recorded for the treatments of non exposure and exposure to 125 mg/l colchicines for 2 days. After 16 weeks of aclimitization and field growing the results showed the significant superiority for both treatments of exposure with 125 and 250 mg/l colchicine for stem length, stem diameter, leaves number, vegetative system dry weight, stomata length, stomata width and total chlorophyll contents characteristics, this in comparison with 500 mg/l colchicine treatments, and this is in combined with finally the interaction effects of colchicine for the treatment of 250 mg/l colchicine for 2 days.

Morphological and molecular characterization of a new record of Isoëtes coromandelina subsp. coromandelina from Cambodia

Plants of a heterosporous fern, Isoetes, were collected from a wetland in Pu Rodet Leu, Mondulkiri, Cambodia, in which the flora has not yet been well studied. The characteristics of this quillwort, for example, dimorphic megaspores and small tuberculate ornamentation, were congruent with those of Isoetes coromandelina L. f. subsp. coromandelina. Unlike most Isoetes endemic species, this subspecies has a wide geographical range across India and the neighboring regions, and another subspecies, I. coromandelina subsp. macrotuberculata Marsden, grows in Australia. We have provided detailed characteristics for the first record of an Isoetes species in Cambodia. Moreover, megaspore morphological features and the sequence of the chloroplast trnL intron were investigated to verify infraspecific and regional variation within this species, using specimens from India (Rajasthan and Varanasi), Sri Lanka, and Australia. Dimorphic megaspores were observed in similar proportions in a megasporangium of the Cambodian plant; the large spherical type averaged 378.5 μm in height in 409 megaspores, and the small flattened type averaged 227.7 μm in height in 544 megaspores. Microspores were not observed. The Cambodian Isoetes plant is a tetrapolyploid (2n = 44 + 1). The morphological characteristics of the Cambodian quillwort are closer to those of a plant from Varanasi, India, than to those of plants from other regions in terms of tubercle size on the megaspore surface and polyploidy of chromosomes. Three haplotypes of the trnL intron showed regional specificity according to the country. However, the phylogenetic monophyly of all the plants examined was supported among the phylogeny of the entire Isoetes genus. This close molecular relationship indicates that a recent dispersal of I. coromandelina may be related to its present distribution.

Aberrations of Chromosomes 9 and 22 in Acute Lymphoblastic Leukemia Cases Detected by ES-FluorescenceIn SituHybridization

A reciprocal translocation between chromosomes 9 and 22 creates oncogenic BCR/ABL fusion in the breakpoint region of the derivative chromosome 22. The aim of this study was to evaluate the importance of atypical fluorescence in situ hybridization (FISH) signal patterns in pediatric and adult acute lymphoblastic leukemia (ALL) cases. We evaluated t(9;22) translocation in 208 cases with ALL (294 tests), including 139 childhood and 69 adult cases by FISH technique using BCR/ABL extra signal (ES) probe. FISH signal patterns observed in pediatric ALL cases were as follows; Major-BCR/ABL (M-BCR/ABL) (1.4%), minor-BCR/ABL (m-BCR/ABL) (3.6%), trisomy 9 (4.3%), trisomy 22 (4.3%), trisomy or tetrasomy of both chromosomes 9 and 22 (2.9%), monosomy 9 (1.4%), monosomy 22 (0.7%), ABL gene amplification (1.4%), derivative chromosome 9 deletion (1.4%), and extra copies of the Philadelphia chromosome (1.4%). FISH signal patterns observed in adult ALL cases were as follows; M-BCR/ABL (5.8%), m-BCR/ABL (11.6%), two different cell clones with major and minor BCR/ABL signal pattern (2.9%), extra copies of Philadelphia chromosome (4.3%), derivative chromosome 9 deletion (1.4%), trisomy 9 (2.9%), tetraploidy (1.4%), monosomy 9 (1.4%), trisomy 22 (1.4%), and coexistence of both trisomy 22 and monosomy 9 (1.4%). Trisomy 9, trisomy 22, and polyploidy of chromosomes 9 and 22 were specific atypical FISH signal patterns for childhood B cell acute lymphoblastic leukemia (B-ALL) patients. However, monosomy 9 and ABL gene amplification were highly specific for childhood T cell acute lymphoblastic leukemia (T-ALL) patients. Our report presents the correlation between atypical FISH signal patterns and clinical findings of a large group of ALL cases.

Sef Downregulation by Ras Causes MEK1/2 to Become Aberrantly Nuclear Localized Leading to Polyploidy and Neoplastic Transformation

Subcellular trafficking of key oncogenic signal pathway components is likely to be crucial for neoplastic transformation, but little is known about how such trafficking processes are spatially controlled. In this study, we show how Ras activation causes aberrant nuclear localization of phosphorylated mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK; MEK) MEK1/2 to drive neoplastic transformation. Phosphorylated MEK1/2 was aberrantly located within the nucleus of primary colorectal tumors and human colon cancer cells, and oncogenic activation of Ras was sufficient to induce nuclear accumulation of phosphorylated MEK1/2 and ERK1/2 in intestinal epithelial cells. Enforced nuclear localization of MEK1 in epithelial cells or fibroblasts was sufficient for hyperactivation of ERK1/2, thereby driving cell proliferation, chromosomal polyploidy, and tumorigenesis. Notably, Ras-induced nuclear accumulation of activated MEK1/2 was reliant on downregulation of the spatial regulator Sef, the reexpression of which was sufficient to restore normal MEK1/2 localization and a reversal of Ras-induced proliferation and tumorigenesis. Taken together, our findings indicate that Ras-induced downregulation of Sef is an early oncogenic event that contributes to genetic instability and tumor progression by sustaining nuclear ERK1/2 signaling.

Microsatellite instability in metastatic colorectal cancer: a review of pathology, response to chemotherapy and clinical outcome

Approximately 10% of colorectal carcinomas demonstrate microsatellite instability (MSI). Distinct from the majority of colorectal cancers with chromosomal instability (CIN) which harbour allelic imbalance from chromosomal polyploidy and aneuploidy, MSI tumours retain intact chromosomal numbers but contain microsatellite repeats due to deficiency in mismatch repair which are thought to contribute to the early steps of tumourigenesis in colorectal cancer. While emerging clinical data has highlighted improved prognosis of tumours with MSI in early colorectal cancer and potentially circumventing the need for adjuvant chemotherapy, the implications of MSI deficiency in metastatic colorectal cancer (mCRC) remain uncertain. In order to assess the significance of MSI in mCRC, a broad literature review was carried out through online PubMed search on published articles encompassing pathological and clinical papers. This included pathological studies identifying the correlation with MSI status between primary sites and metastases, and chemotherapeutic studies assessing the impact of fluoropyrimidine-, irinotecan- and oxaliplatin-based regimens on mCRC with MSI.

Effects of chromosomal polyploidy on survival of colon cancer cells

Tetraploid cells are frequently observed in the inflamed mucosal epithelial cells of the patients with Barrett's esophagus or chronic ulcerative colitis. Polyploidy often occurs during cell fusion, abortive cell cycle, and endoreplication. Most tetraploid cells are engaged to apoptotic pathway, but some remaining stable tetraploid cells consequently cause aneuploidization and chromosomal instability. We investigated whether tetraploid cells could acquire survival advantage and hold a dominant position for natural selection. We established tetraploid cell line (HCT116GH) from parental diploid colorectal cancer cell line (HCT116) via PEG-mediated cell fusion and compared its cell viability, cell cycle response and apoptotic fractions responded to H₂O₂) with diploid HCT116 and p53 suppressed HCT116/H6 cell lines. Using MTT assay, plating efficiency and clonogenicity, we evaluated the survival of each cell line. Tetraploid cell line HCT116GH demonstrated an 83 fold greater resistance to 100 microM H₂O₂ than the parental diploid HCT116, and 6 fold greater than even the p53 negative diploid HCT116/E6. Cellular sensitivity, G2/M arrests, and apoptotic proportion were observed less in response to H₂O₂ in HCT116GH compared with HCT116 and HCT116/E6. HCT116GH expressed lower level of p53 and p21 than diploid HCT116. Stable tetraploid cell lines showed enhanced viability in comparison to parental diploid cell lines. The enhanced viability observed in tetraploidization surpassed that from downregulation of p53. Frequent appearance of tetraploid cells in stressful condition can be caused by natural selection owing to their enhanced viability and may consequently contribute to cancer cell transformation.

Mitogen-activated protein/extracellular signal-regulated kinase kinase 1act/tubulin interaction is an important determinant of mitotic stability in cultured HT1080 human fibrosarcoma cells.

Activation of the mitogen-activated protein kinase (MAPK) pathway plays a major role in neoplastic cell transformation. Using a proteomics approach, we identified alpha tubulin and beta tubulin as proteins that interact with activated MAP/extracellular signal-regulated kinase kinase 1 (MEK1), a central MAPK regulatory kinase. Confocal analysis revealed spatiotemporal control of MEK1-tubulin colocalization that was most prominent in the mitotic spindle apparatus in variant HT1080 human fibrosarcoma cells. Peptide arrays identified the critical role of positively charged amino acids R108, R113, R160, and K157 on the surface of MEK1 for tubulin interaction. Overexpression of activated MEK1 caused defects in spindle arrangement, chromosome segregation, and ploidy. In contrast, chromosome polyploidy was reduced in the presence of an activated MEK1 mutant (R108A, R113A) that disrupted interactions with tubulin. Our findings indicate the importance of signaling by activated MEK1-tubulin in spindle organization and chromosomal instability.

Hepatitis C Virus Causes Uncoupling of Mitotic Checkpoint and Chromosomal Polyploidy through the Rb Pathway

Hepatitis C virus (HCV) infection is associated with the development of hepatocellular carcinoma and probably also non-Hodgkin's B-cell lymphoma. The molecular mechanisms of HCV-associated carcinogenesis are unknown. Here we demonstrated that peripheral blood mononuclear cells obtained from hepatitis C patients and hepatocytes infected with HCV in vitro showed frequent chromosomal polyploidy. HCV infection or the expression of viral core protein alone in hepatocyte culture or transgenic mice inhibited mitotic spindle checkpoint function because of reduced Rb transcription and enhanced E2F-1 and Mad2 expression. The silencing of E2F-1 by RNA interference technology restored the function of mitotic checkpoint in core-expressing cells. Taken together, these data suggest that HCV infection may inhibit the mitotic checkpoint to induce polyploidy, which likely contributes to neoplastic transformation.

PwRn1, a novel Ty3/gypsy-like retrotransposon of Paragonimus westermani: molecular characters and its differentially preserved mobile potential according to host chromosomal polyploidy

BackgroundRetrotransposons have been known to involve in the remodeling and evolution of host genome. These reverse transcribing elements, which show a complex evolutionary pathway with diverse intermediate forms, have been comprehensively analyzed from a wide range of host genomes, while the information remains limited to only a few species in the phylum Platyhelminthes.ResultsA LTR retrotransposon and its homologs with a strong phylogenetic affinity toward CsRn1 of Clonorchis sinensis were isolated from a trematode parasite Paragonimus westermani via a degenerate PCR method and from an insect species Anopheles gambiae by in silico analysis of the whole mosquito genome, respectively. These elements, designated PwRn1 and AgCR-1 – AgCR-14 conserved unique features including a t-RNATrp primer binding site and the unusual CHCC signature of Gag proteins. Their flanking LTRs displayed >97% nucleotide identities and thus, these elements were likely to have expanded recently in the trematode and insect genomes. They evolved heterogeneous expression strategies: a single fused ORF, two separate ORFs with an identical reading frame and two ORFs overlapped by -1 frameshifting. Phylogenetic analyses suggested that the elements with the separate ORFs had evolved from an ancestral form(s) with the overlapped ORFs. The mobile potential of PwRn1 was likely to be maintained differentially in association with the karyotype of host genomes, as was examined by the presence/absence of intergenomic polymorphism and mRNA transcripts.ConclusionOur results on the structural diversity of CsRn1-like elements can provide a molecular tool to dissect a more detailed evolutionary episode of LTR retrotransposons. The PwRn1-associated genomic polymorphism, which is substantial in diploids, will also be informative in addressing genomic diversification following inter-/intra-specific hybridization in P. westermani populations.

Genomic organization of the immunoglobulin light chain gene loci in Xenopus tropicalis: Evolutionary implications

Based on presently available genome data, we characterized the genomic organization of all three light chain gene ( ρ, σ and type III) loci in Xenopus tropicalis. The ρ gene locus in X. tropicalis, structurally similar to the κ gene loci in mammals, was shown to contain a single C ρ gene and nine J ρ segments. The σ locus also contains a single C gene, although two distinct C σ genes have previously been found in Xenopus laevis (most likely due to chromosome polyploidy). Four J σ gene segments were identified upstream of the C σ . The type III light chain gene locus, spanning approximately 170 kb DNA, structurally resembles the topology of mammalian λ gene loci, containing three C genes (C III1–3). C III2 and C III3 are both preceded by single, unique, J genes, whereas C III1 contains three J gene segments. Furthermore, two additional J gene segments, termed J IIIx1 and J IIIx2, were found in the intron separating V III2 and pV III1 (a pseudogene). Based on BLAST searches against the X. tropicalis EST database, all the C genes identified in this study were shown to be functional. On the basis of similarity of protein sequences, genomic organization and chromosomal location of the light chain genes among frogs and mammals, our data strongly support the previous suggestions that the ρ genes belong to the κ gene lineage, whereas the type III genes share a common origin with the λ genes.

Thioredoxin overexpression in HT-1080 cells induced cellular senescence and sensitization to gamma radiation

An increment of thioredoxin-1 (TRX) is observed in many human primary cancers and appears to contribute to an increase of cell growth and a resistance to chemotherapy. On the contrary, when TRX was overexpressed in the HT-1080 fibrosarcoma cells, the cell growth was retarded and chromosomal polyploidy and cellular senescence were induced. TRX-overexpression made HT-1080 cells resistant to an oxidative stress caused by H 2O 2 or paraquat. But these cells were significantly sensitive to ionizing radiation, showing an abrogation of the G 2 checkpoint. Their DNA contents were twice of the controls and they expressed typical senescence markers. Their expression levels of p53 and cyclin-dependent kinase inhibitors (CDKI) were about 2–3-fold higher than the control. Nevertheless, cyclin D1 and D3, which are negatively regulated by CDKIs, were also increased. Overall, in HT-1080 cells the TRX-overexpression created a state of cellular senescence caused by a simultaneous stimulation of the mitogen-activated pathways and an inhibition of the cyclin-dependent kinases, which is known as a hypermitogenic arrest.

Triploidy and Tetraploidy Induction in the Chinese Mitten-Handed Crab (Eriocheir sinensis) by Cytochalasin B

Induction of triploidy and tetraploidy in the Chinese mitten-handed crab, Eriocheir sinensis, was successfully carried out for the first time by administration of cytochalasin B (CB) treatment to zygotes in January-April 1992 and January-May 1993, in Shanghai, China. Results of studies showed that CB treatment in chromosome manipulation was feasible for this species. In the treatment with 0.5-2.0 mg• 1·1 CB, begun 10-20 minutes after insemination and lasting 10-20 minutes, the proportion of triploid embryos as judged from chromosome counts was 14.29-58.18%. Following the results of triploidy induction, tetraploids were also produced by 18-minute treatments with 1.5 mg· I·1 CB prior to first cleavage. The optimum CB treatment for inducing tetraploidy appeared to be 9.2 and 9.5 h post-fertilization at 20 °C, which yielded 56.94 and 57.89% tetraploids, respectively. The ef­fects of CB treatment on the extent of embryo development are also discussed. Sterile trip­loids may continue to moult and grow after reaching maturity as diploid crabs. The suc­cessful induction of chromosome polyploidy in the crab has great scientific significance on conservation and modern breeding programs for this species.

Isolation and characterization of cloned DNA sequences that hybridize to the human X chromosome

A partial library of cloned human DNA was screened for sequences represented on and specific to the X chromosome. The library was constructed from Bam HI-digested human DNA from cells with X chromosome polyploidy, and was cloned in pBR322. The screening was performed by individually hybridizing 32P-labeled cloned plasmids to Southern blots containing Bam HI-digested DNA from mouse-human hybrid cells having the human X chromosome and from derivative hybrids lacking the human X. Of 45 clones assayed, 33 contained sequences homologous to ones represented many times on the X. In situ hybridization to metaphase chromosomes demonstrated that at least four of these clones were homologous to autosomes as well. Only one of the 18 clones of this kind tested cross-hybridized with another. Two recombinant plasmids were shown to be derived from the X chromosome and to be X chromosome-specific by three criteria: they hybridized to a single band in the Southern blots of Bam HI-digested DNA from hybrid cells containing the X chromosome; they hybridized to a band of the same molecular weight as did the inserted DNA fragment; and they showed a dosage effect when hybridized to Southern blots of Bam HI-digested DNA from XY and XXX cells. One of these hybridized as a single-copy or low-order reiterated sequence in a Cot analysis using male DNA as driver. Our methods can be applied to the identification of any chromosome-specific clone. The two X-specific clones identified here should be useful in investigating the mechanism of X inactivation and in isolating a Barr body.

CHROMOSOME POLYPLOIDIZATION IN HUMAN LEUKOCYTES INDUCED BY RADIATION

A great quantity of literature has accumulated on the effects of radiation and radiomimetic agents upon cells. Much of this work has been performed on mammalian cells both in vivo and in tissue cultures. Some of these damaging effects can be seen in dividing cells. After the development of a technique for tissue culture of human peripheral blood, we were able to demonstrate abnormal patterns and chromosomal polyploidy in cultures from patients who had been treated with A-8103 (a piperazine derivative with antitumor activity) , l nitrogen mustard, 6-mercaptopurine, and A-649 (another antitumor agent).2 During treatment and for some time after treatment in these patients, structural changes could be seen in the chromosomes as either chromatid or chromosome deletions and chromosomal breakage. Abnormally shaped chromosomes were also seen, particularly ring forms and dicentrics. Although aneuploidy was observed, the major numerical variation consisted of metaphases showing polyploidy with random distribution of chromosomes and diplochromosomes. When normal human leukocytes were tested in vitro with the chemotherapeutic agents already mentioned, as well as with streptonigrin and cycl~phosphamide,~ similar results were obtained. From these studies we postulated that two different types of damage were induced in the cells by the chemicals. One type of damage is apparent in the hereditary material, and it can be characterized by the structural chromosomal changes. The second type of damage is interpreted as damage to the mitotic apparatus of the cells, as evidenced by the induction of polyploidy. Simultaneously, initial studies from patients treated with x-rays and cobalt-60 demonstrated that the same type of chromosomal anomalies were present in their peripheral leukocytes as those induced with chemotherapeutic agents. Generally, these were structural variations and chromosome polyploidization. Certainly, chromosomal changes have been described in association with x r a d i a t i ~ n ~ , ~ and 1311 therapye,' and, rarely, following diagnostic x-radiation. However, in most of these reports, the occurrence of polyploidy was overlooked, and when polyploidy was mentioned, it was not characterized. Since both structural damage as well as polyploidy lead to genetic transformation or to somatic cell death, we thought it of paramount interest to determine whether or not induction of chromosomal polyploidy by radiation was a constant feature in human somatic cells. Such effects may have a bearing on both the induction as well as the treatment of cancer. For the present study, we determined not only polyploidy, but also the amount of aneuploidy, chromosomal breakage, ring forms, translocations, dicentrics, and tricentrics as well as other rearrangements appearing in patients exposed to radiation therapy. In addition, normal human leukocytes were exposed to x-rays in cultures, and information of induced damage was obtained.