Tetraploid DNA Content Research Articles

Reproductive mode and DNA content of Paspalum accessions from Plicatula group: Reproduction and DNA content of Paspalum

This study aims the reproductive and DNA content characterization of ten different Paspalum species from the Plicatula group (P. atratum, P. compressifolium, P. guenoarum, P. lenticulare, P. lepton, P. oteroi, P. plicatulum, P. rhodopedum, P. rojasii and P. wrightii), contributing to the determination of the best breeding and conservation method to be used. The DNA content of 150 accessions was estimated by flow cytometry using foliar tissues. The reproductive mode of 135 accessions was characterized by flow cytometric seed screening, and five accessions were determined by cytoembryological ovary analysis. DNA content ranged from 1.50 pg (BGP 380) to 4.27 pg (BGP 193), both from P. compressifolium species. Most of the accessions were compatible to the tetraploid level, except for BGP 281 (P. lenticulare), BGP 272 (P. rojasii), and BGP 380 (P. compressifolium), which were diploids. Accessions with tetraploid DNA content presented apomictic reproduction, whereas the three diploid accessions were sexual. These results contribute to the reproductive knowledge of species of the Plicatula group. The diploid sexual accessions identified represent important material to overcome the apomictic barrier in crosses and can be used after polyploidization as female parents in crosses with apomictic accessions in Paspalum breeding programs.

PO-040 Characterisation of CDK12 knocked out ovarian cancer cell lines

IntroductionWhile cyclin-dependent kinases (CDKs) have a key role in promoting/controlling transition between the different phases of the cell cycle, transcriptional kinases, like CDK12, are mainly involved in gene transcription. CDK12 has been shown to regulate the expression of genes involved in DNA damage and to maintain genomic stability. Impairment of CDK12 activity is synergic with PARP inhibitor and cisplatin treatments in different cellular systems. We here aimed to generate ovarian cancer cell lines knocked out (KO) for CDK12 to understand its role in ovarian cancer and in response to chemotherapy.Material and methodsA2780 and SKOV3 CDK12 KO clones were generated with CRISPR/Cas9 technology. Cell cycle analysis was evaluated by standard flow cytometric methods and DNA repair genes levels by Real Time PCR. Caspase 3 activity was measured to detect apoptosis with a luminescence-based assay. Cytotoxicity experiments were performed treating cells with different drug concentrations and evaluating cell survival after 72 hours by MTS assay. For in vivo studies 7.5 millions of cells were transplanted subcutaneously in nude mice and animals were monitored for tumour appearance and growth.Results and discussionsWe obtained 2 CDK12 KO ovarian cancer clones, A2780 KO and SKOV3 KO, out of more than 300 clones screened. The cell growth of both A2780 KO and SKOV3 KO cells is slower than the wild type (WT) cells, they have a less clonogenic ability and a tetraploid DNA content. Both CDK12 KO clones have a higher basal caspase activity than the WT cell lines, indicative of higher basal induction of apoptosis, while no increase in autophagy or senescence is observed. Both CDK12 KO clones show a decreased expression in BRCA1 and FANCD2 DNA repair genes than the WT cells. Cytotoxic experiments with anticancer agents with different mechanism of action show that both KO clones are less sensitive to ATM, CHK1 and WEE1 inhibitors treatment as compared to WT cells, while platinum and PARP inhibitors show similar cytotoxic activity in KO and WT cells. Interestingly enough, when KO clones were transplanted in nude mice, no tumour take was observed.ConclusionWe were able to obtain CDK12 KO cells. We think that these models could help in disclosing new roles of CDK12 in ovarian carcinoma and may represent a useful tool to study new combination therapies for tumours with CDK12 mutations.

PO-044 Possible involvement of G2/M block defects in cell sensitivity towards ATR inhibitors

IntroductionWhile cyclin-dependent kinases (CDKs) have a key role in promoting/controlling transition between the different phases of the cell cycle, transcriptional kinases, like CDK12, are mainly involved in gene transcription. CDK12 has been shown to regulate the expression of genes involved in DNA damage and to maintain genomic stability. Impairment of CDK12 activity is synergic with PARP inhibitor and cisplatin treatments in different cellular systems. We here aimed to generate ovarian cancer cell lines knocked out (KO) for CDK12 to understand its role in ovarian cancer and in response to chemotherapy.Material and methodsA2780 and SKOV3 CDK12 KO clones were generated with CRISPR/Cas9 technology. Cell cycle analysis was evaluated by standard flow cytometric methods and DNA repair genes levels by Real Time PCR. Caspase 3 activity was measured to detect apoptosis with a luminescence-based assay. Cytotoxicity experiments were performed treating cells with different drug concentrations and evaluating cell survival after 72 hours by MTS assay. For in vivo studies 7.5 millions of cells were transplanted subcutaneously in nude mice and animals were monitored for tumour appearance and growth.Results and discussionsWe obtained 2 CDK12 KO ovarian cancer clones, A2780 KO and SKOV3 KO, out of more than 300 clones screened. The cell growth of both A2780 KO and SKOV3 KO cells is slower than the wild type (WT) cells, they have a less clonogenic ability and a tetraploid DNA content. Both CDK12 KO clones have a higher basal caspase activity than the WT cell lines, indicative of higher basal induction of apoptosis, while no increase in autophagy or senescence is observed. Both CDK12 KO clones show a decreased expression in BRCA1 and FANCD2 DNA repair genes than the WT cells. Cytotoxic experiments with anticancer agents with different mechanism of action show that both KO clones are less sensitive to ATM, CHK1 and WEE1 inhibitors treatment as compared to WT cells, while platinum and PARP inhibitors show similar cytotoxic activity in KO and WT cells. Interestingly enough, when KO clones were transplanted in nude mice, no tumour take was observed.ConclusionWe were able to obtain CDK12 KO cells. We think that these models could help in disclosing new roles of CDK12 in ovarian carcinoma and may represent a useful tool to study new combination therapies for tumours with CDK12 mutations.

Establishment of proliferative tetraploid cells from telomerase-immortalized normal human fibroblasts.

Aneuploidy is observed in the majority of human cancers and is considered to be causally related to carcinogenesis. Although malignant aneuploid cells are suggested to develop from polyploid cells formed in precancerous lesions, the mechanisms of this process remain elusive. This is partly because no experimental model is available where nontransformed polyploid human cells propagate in vitro. We previously showed that proliferative tetraploid cells can be established from normal human fibroblasts by treatment with the spindle poison demecolcine (DC). However, the limited lifespan of these cells hampered detailed analysis of a link between chromosomal instability and the oncogenic transformation of polyploid cells. Here, we report the establishment of proliferative tetraploid cells from the telomerase-immortalized normal human fibroblast cell line TIG-1. Treatment of immortalized diploid cells with DC for 4 days resulted in proliferation of cells with tetraploid DNA content and near-tetraploid/tetraploid chromosome counts. Established tetraploid cells had functional TP53 despite growing at almost the same rate as diploid cells. The frequency of clonal and sporadic chromosome aberrations in tetraploid cells was higher than in diploid cells and in one experiment, gradually increased with repeated subculture. This study suggests that tetraploid cells established from telomerase-immortalized normal human fibroblasts can be a valuable model for studying chromosomal instability and the oncogenic potential of polyploid cells. © 2016 Wiley Periodicals, Inc.

Analysis of Residual DSBs in Ataxia-Telangiectasia Lymphoblast Cells Initiating Apoptosis.

In order to examine the relationship between accumulation of residual DNA double-strand breaks (DSBs) and cell death, we have used a control and an ATM (Ataxia-Telangiectasia Mutated) defective cell line, as Ataxia-Telangiectasia (AT) cells tend to accumulate residual DSBs at long times after damage infliction. After irradiation, AT cells showed checkpoint impairment and a fraction of cells displayed an abnormal centrosome number and tetraploid DNA content, and this fraction increased along with apoptosis rates. At all times analyzed, AT cells displayed a significantly higher rate of radiation-induced apoptosis than normal cells. Besides apoptosis, 70–85% of the AT viable cells (TUNEL-negative) carried ≥10 γH2AX foci/cell, while only 12–27% of normal cells did. The fraction of AT and normal cells undergoing early and late apoptosis were isolated by flow cytometry and residual DSBs were concretely scored in these populations. Half of the γH2AX-positive AT cells undergoing early apoptosis carried ≥10 γH2AX foci/cell and this fraction increased to 75% in late apoptosis. The results suggest that retention of DNA damage-induced γH2AX foci is an indicative of lethal DNA damage, as cells undergoing apoptosis are those accumulating more DSBs. Scoring of residual γH2AX foci might function as a predictive tool to assess radiation-induced apoptosis.

Non-motile tetraploid spermatozoa of Misgurnus loach hybrids

We have compared various properties of spermatozoa from the wild diploid male pond loach Misgurnus anguillicaudatus to those from the interspecific male hybrid of the cross between a female M. anguillicaudatus and a male mud loach M. mizolepis. Our results show that spermatozoa from this interspecific hybrid had poor motility, low viability, abnormal morphology, a larger volume of mitochondrial mass per cell and higher ATP content of spermatozoa with tetraploid DNA content, and they were present at a low concentration. The interspecific hybrid males produced spermatozoa with a larger head, with either no flagellum (36.4 %), one flagellum (46.7 %) or two flagella (16.9 %). These flagella were shorter than those of the normal wild-type male M. anguillicaudatus and often presented with abnormalities in microtubule structure. An abnormally shorter flagellum has difficulty in propelling tetraploid spermatozoa with an increased head size in normal progressive motility, although they had higher energy, as shown by their larger volume of mitochondrial mass and higher ATP content. These tetraploid spermatozoa are likely produced by the arrest of the regular meiotic division after chromosomal replication, followed by abnormal spermiogenesis.

Abstract 2066: Aberrant upregulation of 14-3-3 gamma promotes mononucleated polyploidization in human lung cancers

Abstract Advanced non-small cell lung cancers (NSCLC), frequently display increased expression of 14-3-3 gamma (14-3-3γ); a scaffolding protein shown to have oncogenic capacity. This aberrant increase in expression has also been correlated with poorer survival, indicating that upregulation of 14-3-3γ results in a more aggressive tumor phenotype. Our lab is in pursuit of characterizing the mechanisms driving this more advanced cancer phenotype. One such mechanism currently being investigated is 14-3-3γ's ability to promote polyploidization in lung cancer cells. Polyploidization is a known mechanism observed to increase tumorigenicity, resistance to conventional therapies, and theorized to promote chromosomal instability. Our data shows that overexpression of 14-3-3γ results in a subpopulation of cells that harbor mono-nucleated tetraploid DNA content. This suggests that elevated expression of 14-3-3γ promotes aberrant cell cycle progression, either by mechanisms of an abortive mitosis known as endomitosis, or by means of endoreplication, a re-replication event occurring in the absence of mitosis. Further analyses have confirmed that these cells, in the absence of tumor suppressor p53, remain viable and undergo cellular division; supporting our hypothesis that upregulation of 14-3-3γ is resulting in an unstable, tumorigenic tetraploid cell state. Taken together, our data elucidates one role by which 14-3-3γ may be contributing to a more aggressive tumor phenotype in patients with advanced NSCLC. Citation Format: Cecil J. Gomes, Michael Harman, Jesse Martinez, Sara Centuori. Aberrant upregulation of 14-3-3 gamma promotes mononucleated polyploidization in human lung cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2066. doi:10.1158/1538-7445.AM2015-2066

Abstract LB-082: FLJ25439, a novel cytokinesis-associated protein, induces tetraploidization and maintains chromosomal stability via enhancing expression of endoplasmic reticulum stress chaperones

Abstract Purpose of the study: Identify and characterize a novel D box-containing protein, FLJ25439 for tetraploid induction and maintenance of genomic stability. Pertinent experimental procedures: Immunofluorescent microscopy and flow cytometry for cell cycle determination. Proteomics and bioinformatics approaches for protein category profiling. Summary of the data: Investigation of the mechanisms leading to aneuploidy and polyploidy is critical to cancer research. Previous studies have provided strong evidence of the importance of tetraploidization as an early step in tumorigenesis. In cancer cells, tetraploid cells may contribute to abnormal mitotic progression, which may be associated with cytokinesis failure. Tetraploidy leads to genomic instability due to centrosome and chromosome over-replication. Until now, the mechanism by which cells maintain tetraploid status has been unknown. Here, we identified a novel D box-containing protein, FLJ25439, which displays a dynamic expression profile during mitosis/cytokinesis with the midbody as the most prominent associated structure. To understand the function of FLJ25439, we established stable cell lines overexpressing FLJ25439. FLJ25439-overexpression cells grew slower and displayed a tetraploid DNA content in comparison with diploid parental cells. They also showed aberrant mitosis and dysregulated expression of p53, pRb and p21, suggesting a defect in cell cycle progression. To explore the molecular mechanisms responsible for FLJ25439-induced tetraploidization, we conducted a comparative analysis of the global protein expression patterns of wild type and overexpressors using proteomics and bioinformatics approaches. Protein category profiling indicated that FLJ25439 is involved in pathways related to anti-apoptosis, protein folding, the cell cycle, and cytoskeleton regulation. Specifically, genotoxic-stress- and ER stress-related chaperone proteins greatly contributed to the FLJ25439 overexpression phenotypes. Conclusion: The results of this study pave the way to our further understanding of the role of FLJ25439, a novel cytokinesis-related protein in protecting cells from environmental stress and tetraploid formation. Note: This abstract was not presented at the meeting. Citation Format: Pin Ouyang. FLJ25439, a novel cytokinesis-associated protein, induces tetraploidization and maintains chromosomal stability via enhancing expression of endoplasmic reticulum stress chaperones. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-082. doi:10.1158/1538-7445.AM2015-LB-082

Expression of the chicken GDNF family receptor α-1 as a marker of spermatogonial stem cells

The identification, enrichment and subsequent isolation of spermatogonial stem cells (SSCs) are integral to the success of SCC transplants between fertile donor and sterilized recipient males. In birds generally and particularly in chicken, SSC-specific has yet to be identified. The receptor for glial cell-derived neurotrophic factor (GDNF), i.e. GDNF family receptor alpha-1 (GFRα1), has been identified as a potential marker for different mouse spermatogonial subtypes. In the present study, we characterized the chicken cGFRα1 receptor and compared its predicted amino-acid sequence with mouse, rat and human GFRα1 proteins. Using specific polyclonal mouse anti-cGFRα1 serum, a total of 2.8% cells were recognized as cGFRα1-positive among isolated testicular cells recovered from sexually mature cockerels. The percentages of cGFRα1-positive testicular cells with haploid, diploid, tetraploid and SP DNA content were 1.6%, 2.5%, 39.3% and 76.8%, respectively. The presence of cGFRα1 protein on the surfaces of all cells of the seminiferous epithelium was confirmed by immunocytochemical and immunohistochemical analyses. Tissue specificity of cGFRα1 mRNA expression was significantly higher in adult testes compared to brain tissue which itself was several times higher than tissues prepared from the spleen, liver and heart. No expression was observed in muscular tissue. At last, we demonstrated the successful repopulation of sterilized recipient's testes with transplanted cGFRα1-positive donor testicular cells. Recipient males subsequently produced functional heterologous spermatozoa capable of fertilizing an ovum and obtaining chicks with donor cell genotypes.

MiR-675 Mediates Downregulation of Twist1 and Rb in AFP-Secreting Hepatocellular Carcinoma

Alpha-fetoprotein (AFP)-secreting hepatocellular carcinomas (HCC) represent a genetically distinct subset of tumors often associated with a worse prognosis. However, the molecular mechanisms that underlie these phenotypic differences remain poorly understood. HCC tumor samples from 27 patients were profiled using the Affymetrix 133 Plus 2.0 GeneChips. GeneGO Metacore software was used to identify altered biologic pathways. Expression validation was confirmed by RT-PCR. Manipulation of miR-675 by overexpression and antagomir-mediated knockdown was carried out with subsequent evaluation of effects on cell behavior by cell cycle, proliferation, invasion, and growth in soft agar assays. We identified a strong relationship between primary tumor H19 gene expression and elevated serum AFP. H19 has recently been identified to encode microRNA-675 (miR-675), and we confirmed the relationship in an independent sample of patients. Pathway analyses of the effect of miR-675 overexpression in hepatoma cells revealed a predominant upregulation of cell adhesion and cell cycle initiation pathways. We have demonstrated that miR-675 mediates increases in proliferation and an accumulation of cells with tetraploid DNA content associated with a repression of Rb. We also demonstrated that overexpression of miR-675 alters cellular morphology, reduces invasive potential, and increases anchorage-independent growth capacity. These findings are consistent with a mesenchymal-to-epithelial transition, associated with a reduction in the expression of the key EMT mediator, Twist1. Expression of the miR-675 in hepatocellular carcinoma links a dramatic upregulation of proliferative and growth capacity with inhibition of motility in HCC cells.

Abstract IA17: The bacterial world and the genome.

Abstract In part due to the Human Microbiome Project and related large scale efforts, there is increasing awareness that the microorganisms that live on and in us can modify risk for a number of chronic diseases including cancer. While Helicobacter pylori was the first bacterium to listed as a carcinogen, recent studies have begun to associate additional bacteria with cancers. Moving beyond association requires establishment of causality and biological mechanism. I will discuss these issues broadly and in the context of our recent efforts to query the role of the upper-GI microbiome in modifying risk for developing esophageal adenocarcinoma. Esophageal cancer is the fastest growing cancer in the US, rising nearly 600% from 1972 – 2002, suggesting a new environmental exposure. Esophageal adenocarcinoma is highly lethal with five year survival of less than 15%. Barrett's esophagus is a precursor to esophageal adenocarcinoma and H. pylori is a Proteobacterium colonizing the stomach of 50% of the world's population. Infection with H. pylori can cause peptic ulcers and gastric cancers but also shows a protective association for Barrett's esophagus and esophageal adenocarcinoma. Utilizing a large Seattle based prospective cohort of Barrett's esophagus participants, we investigated associations of H. pylori infection with development of cancer or genomic instability defined as tetraploid or aneuploid DNA content measured by flow cytometry and found a significant association of H. pylori infection with less aneuploidy. These results indicate H. pylori carriage has protective effects in the Seattle population and may act at the level of preventing genomic instability, an important step in cancer progression. Investigation of bacterial communities present in multiple niches within the upper GI tract of Seattle Barrett's Study participants (squamous esophagus, Barrett's esophagus, stomach body, stomach antrum) suggest relationships between these communities that may influence disease progression. Citation Format: Nina R. Salama. The bacterial world and the genome. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr IA17.

Ascorbic acid has superior antiviral and antiproliferative effects over IFN-alpha in HAM/TSP PBMC ex vivo

IFN-alpha and high dose ascorbic acid (AA) have a modest clinical benefit in HAM/TSP (Nakagawa, 1996). We investigated the effect of ex vivo and in vitro AA and IFN-alpha treatment on HAM/TSP PBMC and HTLV-1-infected cell lines, respectively. We treated cells for 48-72h ex vivo and in vitro with low-high (10-100µg/ml) dose of AA and 1000U/ml of IFN-alpha and quantified lymphoproliferation by [3H]thymidine incorporation, tetraploid DNA content and PCNA expression (flow cytometry). Viral expression was measured at the RNA (tax, LTR) and protein (Tax, p19) level by RT-PCR, Western blot and ELISA, respectively. Apoptosis was quantified by subdiploid DNA content (flow cytometry). Th1/Th2/Th17 cytokines were quantified by cytometric bead array. AA induced a dramatic 95% decrease (control 3689±755 cpm vs. AA 121±52 cpm, p=0.001) in spontaneous, virus-driven lymphoproliferation and a decrease in tax and LTR transcription in HAM/TSP PBMC. In addition, AA decreased the exacerbated ex vivo IFN-gamma production in HAM/TSP PBMC. In HTLV-1 infected cell lines (MT-2 and MT-4), AA induced a dose-dependent increase in DNA fragmentation (p=0.02, p=0.005), paralleled by a decrease in PCNA (p=0.003), as well as p19 (p<0.001) and Tax levels. These effects appear virus-specific, since high-dose (100µg/ml) AA did not exert a significant antiproliferative or pro-apoptotic effect on PBMC of normal donors. On top, AA displayed a superior antiproliferative, antiviral and immunomodulatory effect over IFN-alpha in both cell lines and HAM/TSP PBMC. Considering the selective antiproliferative and antiviral effects of AA ex vivo and in vitro, the therapeutic potential of combination therapy with high dose AA in HAM/TSP should be further explored.

MiR-675 Mediated Deregulation Of The Rb-E2F Pathway In Hepatocellular Carcinoma

Introduction: Although up to 50-75% of hepatocellular carcinomas (HCC) are associated with elevated serum AFP levels, it is unclear whether these tumors represent a genetically distinct subset of HCC. To better understand potential biologic differences, we sought to compare gene expression profiles between AFP-secreting (AFP+) and non-secreting (AFP-) tumors. Methods: From 2000-2009, we identified 27 patients having undergone resection for HCC and also consented to our tissue banking protocol. Tumors (14 AFP+ and 13 AFP-) were profiled using the Affymetrix 133 Plus 2.0 GeneChip. MetaCore gene ontology software was used to identify altered biologic pathways. Expression validation was performed on a subset of patients, using quantitative real-time PCR. Overexpressing transfections of miR-675 were carried out in the non-expressing HepG2 cell line. Functional assays of proliferation (MTT assay), cell cycle distribution (flow cytometry) and anchorage independence (growth in soft agar) were performed. Results: At a false discovery rate of 0.2, we identified 418 differentially regulated genes between AFP+ and AFP- tumors, which were independent of cirrhosis or hepatitis viral activity. H19, a maternally imprinted gene encoding the precursor for miR-675, a novel microRNA, was found to be the most highly upregulated gene (+14 fold change) in AFP+ tumors. In AFP+ HCC, we identified 23 downregulated biological pathways (p<0.01) that were predominantly involved in substrate metabolism, and two upregulated pathways (p<0.03) involved in cell adhesion and cell cycle regulation. We validated the expression of miR-675 in clinical specimens, demonstrating an average fold change of +12.5 for patients with AFP+ versus AFP- HCC. The endogenous expression levels of the miR-675 in a panel of hepatoma cell lines were determined using RT-PCR and were found to correlate with AFP expression. We transfected miR-675 into the HepG2 cell line and confirmed successful transfection using RT-PCR. Overexpression of miR-675 resulted in a significant 2-fold upregulation of proliferation (p<0.01), an accumulation of cells with tetraploid DNA content, and a dramatic increase in colony formation in soft agar as compared to control (p<0.0001). There was no effect on AFP secretion (p=NS). Investigation of known proliferative pathways demonstrated that miR-675 overexpression resulted in a marked abrogation of pRb expression. Conclusions: HCC associated with elevated serum AFP demonstrates distinct alterations in gene expression and biologic pathways as compared to AFP- tumors. Ectopic expression of miR-675 resulted in augmentation of tumor cell oncogenicity, which may be mediated by deregulation of the Rb-E2F pathway. MiR-675 may play a critical role in the pathogenesis of AFP+ HCCs and further investigation into its downstream effects and its potential as a therapeutic target are clearly warranted.

Reproductive and genetic capacity of spermatozoa of inter-populational hybrid males in the loach, Misgurnus anguillicaudatus

Summary Reproductive capacity was analyzed in inter-populational hybrid males between geographically different populations of the loach Misgurnus anguillicaudatus, which were suggested to be genetically distinct by previous studies. Hybrid males exhibited high percentages of spermatozoa with poor or no motility, when compared with wild type diploid males. Sperm volume was apparently less than that from wild type. Scanning electron microscopy (SEM) revealed that spermatozoa of hybrid males showed abnormal morphology such as no flagellum, double or more flagella, ill-developed mid-piece and significantly larger head sizes. Three different cell populations with haploid (1C), diploid (2C) and tetraploid (4C) DNA content were flow-cytometrically detected in sperm samples collected from hybrid males, whereas diploid males showed only haploid spermatozoa. Fertilization of eggs of wild type females with sperm of hybrid males gave very few diploid and triploid survivors. Microsatellite analyses showed that haploid spermatozoa of hybrids were produced by meiotic segregation, while diploid spermatozoa were formed by unreduced spermatogenesis. The present results showed that inter-populational loach hybrid males were almost sterile, but a few fertile haploid and diploid spermatozoa were generated. Such traits of inter-populational hybrid males are similar to those reported in inter-specific hybrids. Hybridization disrupted regular meiosis and normal spermatogenesis in the loach from the cross between different

Identification of various testicular cell populations in pubertal and adult cockerels

Precise identification of the male germinal stem cell population is important for their practical use in programs dedicated to the integration of exogenous genetic material in testicular tissues. In the present study, our aim was to identify germinal cell populations in the testes of pubertal and adult cockerels based on the detection of the nuclear DNA content by fluorescence-activated cell sorting (FACS) and on the expression of the Dazl and Stra8 genes in single-cell suspensions of testicular tissues. Cells with a tetraploid DNA content (4c) represent a small and equal fraction of the total germinal cell population in both pubertal and adult males. In contrast, the diploid (2c) and haploid (c) subpopulations differ significantly between ages as a consequence of different degrees of sexual maturation. A specific subpopulation of testicular cells, the side-scatter subpopulation of cells, or side population (SP), was identified at the junction between the haploid and diploid cell populations. The percentage of this cell subpopulation differs significantly in pubertal and adult cockerels, accounting for 4.1% and 1.3% of the total cell population, respectively. These four testicular cell populations were also tested for the expression of Dazl and Stra8 genes known to be expressed in premeiotic cells including stem spermatogonia. Both genes were expressed in SP, whereas the expression of either Dazl or Stra8 genes was detected only in the 4c and in the 2c testicular cell subpopulations, respectively. The correlation between the cell ploidy and Dazl/ Stra8 expression was the same at both male ages. We conclude that SP cells might represent a subpopulation of germinal cells enriched in stem spermatogonia, which can be of great importance for transgenesis in chicken.

Immunophenotyping of Mya arenaria neoplastic hemocytes using propidium iodide and a specific monoclonal antibody by flow cytometry

Disseminated neoplasia (DN) is a disorder referred to as hemic neoplasia (HN) in the soft-shell clam Mya arenaria. Traditionally, diagnosis is performed by hematocytology or histology. The intensity of the disease is generally given as the percentage of transformed neoplastic cells out of total number of hemocytes. Flow cytometry techniques have found a field of application in diagnosis of HN with analysis of ploidy. Hemocytes of the soft-shell clams with HN display tetraploid DNA content, as shown by propidium iodide staining. This feature makes difficult HN diagnosis in the soft-shell clam, especially for early stages of the condition, since the percentage of normal circulating cells undergoing mitosis, which also are tetraploid, remains unknown in molluscs. Use of specific monoclonal antibodies in a flow cytometry assay was foreseen as a way to overcome the difficulty. The purpose of this study was to develop a double staining protocol using propidium iodide for hemocyte cycle analysis and the MAb 1E10 for staining of HN cells. Our results showed a correlation between tetraploid and MAb 1E10-stained hemocytes in a single clam with moderate HN. This protocol offers some potential for further investigation of this cell disorder. However, a validation step will be necessary to confirm our preliminary results.

Analysis of neuroblastoma tumour progression; loss of PHOX2B on 4p13 and 17q gain are early events in neuroblastoma tumourigenesis

Neuroblastomas are biologically and clinically heterogeneous tumours that most often occur sporadically in children at median age of 2 years. The PHOX2B gene is implicated in the development of the autonomic nervous system and has been found to be infrequently mutated in sporadic neuroblastoma tumours and in some patients with hereditary neuroblastoma. We have screened a selected series of 36 paediatric tumours with presumed genetic predisposition, 34 of them neuroblastomas, for mutations in PHOX2B. A constitutional heterozygous missense mutation was found in a boy who developed bilateral adrenal tumours and stage 4 disease during infancy. The second allele of the PHOX2B locus was lost in the tumour DNA. Histopathological evaluation of the tumours suggested growth of two primary tumours, one with diploid DNA content and the other with tetraploid DNA content, i.e. a case of neuroblastoma stage 4M (multifocal tumour). However, array CGH (comparative genomic hybridization) data performed on both tumour masses from the patient instead supported a model where a common malignant precursor gave rise to the diploid tumour and subsequently the tetraploid tumour have progressed from the common precursor or by metastasis from the diploid tumour with additional genetic changes. The whole genome dosage analysis showed that the remaining alleles of PHOX2B had been lost in both tumours together with a specific 17q gain pattern. The tetraploid tumour had these features together with additional whole chromosomal loss of chromosomes 3, 9, 14 and 15. Based on the data presented here we suggest that loss of PHOX2B and 17q gain are early events in neuroblastoma tumourigenesis. We also propose investigators to re-analyze the rare cases of multifocal neuroblastomas with the array CGH technique for better understanding of the origin of these tumours.

A Tetraploid Amazon Molly, Poecilia formosa

Polyploidization is thought to be an important driving force in evolution as it increases the genetic material on which mutation and selection can act. In the Amazon molly, Poecilia formosa, triploid genotypes can be found in the field and frequently arise from diploid breeding stocks, a tetraploid individual, however, was so far never documented. Here, we report the first tetraploid Amazon molly. Flow cytometry clearly showed the tetraploid DNA content, whereas microsatellite analysis not only confirmed the tetraploidy but also pointed to allotetraploidy. Most likely the fourth genome was received through paternal leakage, namely, by fertilization of a triploid egg with a haploid sperm. The existence of tetraploid individuals offers new explanations for the enormous clonal diversity observed in wild populations of P. formosa.

Aneuploidy and DNA replication in the normal human brain and Alzheimer's disease.

Reactivation of the cell cycle, including DNA replication, might play a major role in Alzheimer's disease (AD). A more than diploid DNA content in differentiated neurons might alternatively result from chromosome mis-segregation during mitosis in neuronal progenitor cells. It was our objective to distinguish between these two mechanisms for aneuploidy and to provide evidence for a functional cell cycle in AD. Using slide-based cytometry, chromogenic in situ hybridization, and PCR amplification of alu-repeats, we quantified the DNA amount of identified cortical neurons in normal human brain and AD and analyzed the link between a tetraploid DNA content and expression of the early mitotic marker cyclin B1. In the normal brain, the number of neurons with a more than diploid content amounts to approximately 10%. Less than 1% of neurons contains a tetraploid DNA content. These neurons do not express cyclin B1, most likely representing constitutional tetraploidy. This population of cyclin B1-negative tetraploid neurons, at a reduced number, is also present in AD. In addition, a population of cyclin B1-positive tetraploid neurons of approximately 2% of all neurons was observed in AD. Our results indicate that at least two different mechanisms need to be distinguished giving rise to a tetraploid DNA content in the adult brain. Constitutional aneuploidy in differentiated neurons might be more frequent than previously thought. It is, however, not elevated in AD. In addition, in AD some neurons have re-entered the cell cycle and entirely passed through a functional interphase with a complete DNA replication.

Micro-RNA deletion in neuroblastoma: A possible alternative mechanism of MYCN overexpression

9052 Background: Micro-RNAs (miRNAs) are small noncoding RNAs of 18 to 25 nucleotides that regulate protein expression. The biological functions of miRNAs are not yet fully understood. miRNA genes were recently found to be abnormally expressed in several types of cancer. By negatively regulating proto-oncogenes, miRNAs could act as tumor suppressors and conversely, by inhibiting tumor suppressors function as oncogenes. Neuroblastoma (NB) is the most common solid tumor in children under 5 years of age. Major adverse prognostic factors include: age over 1 year, advanced stages, adrenal primary site, MYCN amplification, diploid or tetraploid DNA content (DI), and chromosomal aberrations - including 1p deletions. We studied a possible involvement of miRNAs in neuroblastoma. Methods: The cohort consisted of 72 patients: 65% of them were above 1 year of age, 72% with advanced stages, 33% of the patients progressed. Median follow up was 60 months (ranging 1–221). Genomic DNA from 72 tumors were analyzed for the presence of 7 miRNAs located on chromosome 1p, ranging from 1p36.33 to 1p31.1 by PCR. Results: Deletion of one or more of the miRNAs was identified in 50% of the tumors.The most prevalent deletions were of two miRNAs: miR 30e located on 1p34.2 (29%) and miR 34a on 1p 36.23 (22%), accounting for 40% of the patients with a deletion of one or both of these miRs. Interestingly, miR 30e and miR 34a are known to negatively regulate the oncogene MYCN. MYCN protein expression is currently being evaluated in this cohort. Conclusions: The deletions of these miRNAs in neuroblastoma tumors suggest their involvement as tumor suppressor genes. This implies a new mechanism for the overexpression of MYCN. Discovery of the miRNAs and their targets involved in neuroblastoma has a therapeutic potential, for the development of new targeted therapies. No significant financial relationships to disclose.