Study on the biological characteristics of polyploid cervical cancer HeLa cells induced by radiation / 国际肿瘤学杂志

Objective:To investigate the biological characteristics of proliferation, apoptosis, migration and invasion of radiation-induced polyploid cervical cancer HeLa cells, and to analyze the potential facilitation of polyploid HeLa cells in cervical cancer recurrence after radiotherapy.Methods:HeLa cells were irradiated by 6 MV-X ray with 7 Gy and 14 Gy, the cells were cultured until the third day, and then they were recorded as 7 Gy group and 14 Gy group respectively. The unirradiated HeLa cells were recognized as the control group. The cell morphology was checked under optical microscope. Flow cytometry was used to determine cell ploidy. MTT assay was applied to detect cell proliferation. Flow cytometry by AnnexinⅤ-FITC/PI double labeling was used to detect apoptosis. The ability of migration and invasion was detected by Transwell assay. The expression levels of STAT3 signal pathway-related proteins were analyzed by Western blotting.Results:Compared with the control group, the volume of HeLa cells in 7 Gy group and 14 Gy group increased significantly. The percentages of polyploid HeLa cell subsets in the control group, 7 Gy group and 14 Gy group were (6.33±1.26) %, (21.13±0.50) % and (46.07±1.68) % respectively, with a statistically significant difference ( F=780.47, P<0.001) . The absorbance values in the control group, 7 Gy group and 14 Gy group of polyploidy HeLa cells were 0.21±0.01, 0.23±0.02, 0.16±0.01 at 24 h, 0.37±0.03, 0.38±0.06, 0.21±0.00 at 48 h, 0.66±0.02, 0.55±0.01, 0.28±0.01 at 72 h, and there were statistically significant differences ( F=31.62, P=0.001; F=20.10, P=0.002; F=708.52, P<0.001) . Further pairwise comparison showed that the proliferation abilities of polyploidy HeLa cells of the 14 Gy group at 24, 48 and 72 h were significantly lower than those of the control group and the 7 Gy group (all P<0.05) . The proportions of apoptotic cell subset in the control group, 7 Gy group and 14 Gy group were (3.67±1.16) %, (3.07±0.81) %, (3.83±0.91) %, the proportions of early apoptotic subset were (2.33±0.35) %, (2.13±0.61) %, (2.23±0.32) %, and the proportions of late apoptotic subset were (1.33±0.81) %, (0.93±0.31) %, (1.60±0.60) % respectively. There were no statistically significant differences ( F=0.52, P=0.620; F=0.15, P=0.864; F=0.92, P=0.450) . The migrated numbers of cells in the control group, 7 Gy group and 14 Gy group were 297.40±26.53, 121.33±15.16, 18.40±4.79, and the invaded numbers were 195.67±20.26, 63.60±6.91, 9.47±3.23 respectively, with statistically significant differences ( F=647.28, P<0.001; F=213.94, P<0.001) . Compared with the control group, the migration and invasion abilities of polyploid HeLa cells in the 7 Gy and the 14 Gy groups were significantly decreased, and the migration and invasion abilities of polyploid HeLa cells in the 14 Gy group were significantly lower than those in the 7 Gy group (all P<0.001) . The expression levels of P-STAT3 (Tyr 705) and Bcl-2 in radiation-induced polyploidy HeLa cells were higher than those in the control group, and the expression levels were further increased with the increase of radiation dose. Compared with the control group, the expression levels of Survivin and Mcl-1 in polyploid HeLa cells in the 14 Gy group were up-regulated (both P<0.05) . There was no significant difference in Bcl-xL expression among the three groups ( F=0.52, P=0.618) . Conclusion:The proliferation, migration and invasion abilities of polyploid HeLa cells are reduced by radiation, and the proportion of apoptotic subset is not significantly changed, but the activation of STAT3 signaling pathway is accompanied by up-regulation of downstream anti-apoptotic related proteins, which is favorable for the polyploid tumor cells to be the potential risk factor of recurrent cervical cancer after radiotherapy.

Endoreplication and its role in tumor development and progression / 中国肿瘤临床

Most diploid cells proliferate by proceeding through the canonical G1 (DNA pre-synthesis), S (DNA synthesis), G2 (DNA post-synthesis), and M(mitosis) phases of the cell cycle. However, there is another type of cell cycle that occurs frequently in both plants and animals, known as endoreplication. Endoreplication consists of alternating periods of G and S phases without cytokinesis, which results in polyploidy. It is indispensable for normal development, organ formation, and wound healing in humans. In recent years, con-siderable attention has been paid to delineating the connections of endoreplication with tumorigenesis and tumor progression. Here, we review the role of endoreplication in normal human development and discuss its possible role in tumor development and the un-derlying molecular mechanisms.

Study on Polyploid Induction and Identification of Nervilia fordii / 广州中医药大学学报

Objective To investigate the polyploid induction and identification of Nerviliae fordii for harvesting the polyploid plants. Methods The materials and methods for polyploid induction of Nerviliae fordii were screened separately by comparing the induction rates of rhizomes and bulbs under natural conditions and tissue culture environment, and by comparing soaking method with agar method. The effects of colchicine concentration ( 200, 300, 400, 300 mg/L), colchicine action time ( 7, 14, 21, 28 d), DMSO concentration ( 0, 10, 20, 40 mL/L) and KT concentration ( 0, 1.0, 2.0, 4.0 mg/L) on induction rate were observed by orthogonal design method. The polyploid induction in the treated plants was identified by morphology, cytology and chromosome methods. Results After the tissue culture rhizomes were treated with 300 mg/L colchicine, 10 mL/L DMSO, and 2.0 mg/L KT by agar method for 28 days, the polyploid induction rate arrived to 50%, showing better induction effect. The morphology of polyploid plants was characterized by giantism, and the leaf length, leaf width and plant height were respectively 152.17%, 158.67%and 60.90%of those of the diploid plants. The length, width and density of stoma of leaf epidermal cell as well as the number of chloroplast in the treated plants were 138.46%, 153.00%, 59.09% and 109.09% of those of the untreated plants. The results of chromosome identification showed that the amount of the tetraploid ( about 40) was 2 times of the diploid chromosome ( about 20) in the treated plants, proving that the achieved Nerviliae fordii was a tetraploid plant. Conclusion Polyploid plants of Nerviliae Fordii have been successfully obtained, which will supply evidence for improving species, richening seed-breeding resources, and selecting of improved seeds of Nerviliae fordii.

Effect of monocrotaline on induction of pancreatic cancer cell BxPC-3 into polyploid giant cells / 中草药

Objective: To observe the effect of monocrotaline (MCT) extract from Crotalaria sessiliflora on inducing human pancreatic cancer cell BxPC-3 into polyploid giant cells in vitro. Methods: BxPC-3 cells (1 × 104/mL) were inoculated with MCT (0, 5, 10, and 20 μg/mL) in RPMI-1640 for 72 h, respectively, then the cell morphology was observed by Giemsa staining, and the DNA content was measured by flow cytometry; BxPC-3 cells-induced apoptosis was checked by AnnexinV-FITC/PI double staining using flowcytometry, BxPC-3 polyploid giant cell genome was checked by chromosome spreading assay, and Cyclin B1 expression also was analysed by Western blotting. Results: Giemsa staining and DNA content by flow cytometry showed that MCT induced BxPC-3 cell into polyploid giant cells in vitro. MCT treatment for 72 h appeared 4N, and 8N polyploid giant of BxPC-3 cells, induction of apoptosis, decreased the expression of cyclin B1 in a dose dependent manner. Chromosome analysis demonstrated once again that polyploid giant cell was mainly in 8N. Conclusion: Monocrotaline might exert its antitumor effect by blocking the cell cycle, inducing apoptosis, and down-regulating Cyclin B1 protein.

Caracterização citogenética para identificação dos níveis de ploidia em cinco espécies do gênero Mentha L / Citogenetic characterization to determination of ploidy level on five Mentha L. species

Neste trabalho foi feita a caracterização citogenética da: microsporogênese, tétrades, estimativa da viabilidade do pólen pelo método de coloração e contagem do número máximo de nucléolos por célula interfásica, para identificação dos níveis de ploidia, em cinco espécies do gênero Mentha L. Foram coletadas inflorescências em 30 plantas de cada espécie, em duas florações sucessivas, nos anos 2006 e 2007. As inflorescências foram tratadas em etanol-ácido acético (3:1), em temperatura ambiente durante seis horas, transferidas para álcool 70% (v/v) e conservadas em geladeira até análise. Nas análises da microsporogênese, tétrades e pólen o corante usado foi carmin propiônico 2% e na identificação dos nucléolos nitrato de prata (AgNO3). Os resultados demonstraram que as cinco espécies são poliplóides. M. crispa heptaplóide (2n=7x=84) com 11 nucléolos, M. spicata tetraplóide (2n=4x=48) com 8 nucléolos, M.x gentilis pentaplóide (2n=5x=60) com 12 nucleólos, M. piperita e M.x piperita ambas hexaplóides (2n=6x=72) com 8 e 9 nucléolos respectivamente. M. spicata e M. crispa mantiveram as mais altas porcentagens de células normais na microsporogênese, na formação de tétrades e na estimativa da viabilidade do pólen por coloração, sugerindo maior estabilidade meiótica quando comparados aos demais poliplóides estudados.

The cytogenetic characterization of five species of Mentha L. genus, including the data: regularity of microsporogenesis and tetrads, and polen viability, using the coloration method and the counting of the maximum number of nucleolus by interphasic cell were carried out in this study to identify the ploid levels. These analyses were performed from inflorescences collected in 30 plants of each species, during two successive florations in 2006 and 2007. Inflorescences were treated in 3:1 ethanol:acetic acid mixture at room temperature during six hours, then transferred to 70%(v/v) ethanol solution and refrigerated until the analysis. For microsporogenis, tetrad and pollen analysis, we used carmine propionic 2% (m/v) and for nucleolus identification, we used AgNO3 solution. It was possible to observe that all five species were polyploids. M. crispa heptaploid (2n=7x=84) with 11 nucleolus, M. spicata tetraploid (2n=4x=48) with 8 nucleolus, M. x gentilis pentaploid (2n=5x=60) with 12 nucleolus, M. piperita and M. x piperita both hexaploid (2n=6x=72) with 8 and 9 nucleolus respectively. M. spicata and M. crispa kept the highest percentual values of normal cells in microsporogenesis as well as in tetrads formation and pollen viability, suggesting a higher meiotic stability when compared to the other polyploids studied.

Meiotic behavior during microsporogenesis of Alchornea triplinervia (Sprengel) Müller Argoviensis / Comportamento meiótico durante a microsporogênese de Alchornea triplinervia (Sprengel) Müller Argoviensis

The Alchornea triplinervia specie belongs to the Euphorbiaceae family, one of the main families of the Brazilian flora. In order to contribute to a better understanding of the specie, a counting of chromosome number and the microsporogenesis analysis of A. triplinervia were done. The inflorescences were collected in the municipalities of Paranavaí and Diamante do Norte, State of Paraná, Brazil, and the slides were prepared by squashing technique and staining with 1% acetic carmine. The analysis were performed using an optical microscope and showed a chromosome number for the specie equal to 2n=8x=72. Irregularities in the chromosome segregation process were the main meiotic abnormalities, presenting typical polyploid behavior. Other irregularities were observed; however, at low frequency without compromising the pollen grain formation of the analyzed plants.

A espécie Alchornea triplinervia pertence à família Euphorbiaceae, uma das principais famílias da flora brasileira. Visando a contribuir para um melhor entendimento da espécie, foi realizada a contagem do número de cromossomos e a análise da microsporogênese de A. triplinervia. As Inflorescências foram coletadas nos Municípios de Paranavaí e Diamante do Norte, no Estado do Paraná, Brasil, sendo as lâminas preparadas pela técnica de esmagamento e coradas com carmim acético 1%. As análises foram realizadas ao microscópio óptico, revelando um número cromossômico para a espécie igual a 2n=8x=72. Irregularidades no processo de segregação dos cromossomos foram as principais anormalidades meióticas, mostrando comportamento típico de poliploides. Outras irregularidades foram observadas, porém, em baixa frequência, não comprometendo a formação dos grãos de pólen das plantas analisadas.

Cytogenetic analyses in Paspalum L. reveal new diploid species and accessions / Análises citogenéticas em Paspalum L. revelam novas espécies e acessos diplóides

Chromosome numbers were counted in 126 new accessions of 50 Paspalum species from Brazil, Argentina, Paraguay and Bolivia. The chromosome numbers 2n=12, 20, 24, 30, 40, 50, 60, 80 were confirmed. Chromosome numbers for P. arenarium (2n=20), P. barretoi (2n=20), P. aff. ceresia (2n=40), P. corcovadense (2n=20), P. crispulum (2n=20), P. flaccidum (2n=40), P. nummularium (2n=20), P. scalare (2n=20), P. vescum (2n=20) and P. rectum (2n=20) and a diploid cytotype of P. malacophyllum are reported for the first time. The predominance of tetraploid accessions (43.6 percent) was confirmed, but an unusually high number of diploid species (44 percent) and accessions (35.7 percent) was found. These results open new perspectives for breeding programs, phylogenetic studies, and for research on apomixis control, since diploids of Paspalum are typically sexual.

O número cromossômico foi determinado para 126 novos acessos de 50 espécies de Paspalum do Brasil, Argentina, Paraguai e Bolívia. Foram verificados os números somáticos 2n=12, 20, 24, 30, 40, 50, 60 e 80. Estas são as primeiras contagens para P. arenarium (2n=20), P. barretoi (2n=20), P. aff. ceresia (2n=40), P. corcovadense (2n=20), P. crispulum (2n=20), P. flaccidum (2n=40), P. nummularium (2n=20), P. scalare (2n=20), P. vescum (2n=20) e P. rectum (2n=20). O nível diplóide (2n=20) é reportado pela primeira vez para P. malacophyllum. Os dados confirmam a predominância de acessos tetraplóides (43,6 por cento) no gênero e mostram um número incomumente elevado de espécies (44 por cento) e acessos diplóides (35,7 por cento). Estes resultados trazem novas perspectivas para programas de melhoramento, para estudos filogenéticos e para pesquisa orientada ao controle da apomixia, já que em Paspalum as plantas diplóides são tipicamente sexuais.

DNA Methylation of polyploid complex of Pinellia ternata by MSAP analysis / 中草药

Objective To analyze the DNA methylation status of polyploid complex of Pinellia ternata. Methods Methylation sensitive amplified polymorphism(MSAP) technique was carried out to analyze the methylation status of polyploid complex of P.ternata. Thirty-four selective primer amplifications were used to check the status of cytosine methylation DNA samples. Results A total of 7 708 bands were obtained.Among them 5 636 bands,each representing a recognition site cleaved by one or both of the isoschizomers(Hpa Ⅱ and Msp Ⅰ),were amplified.Furthermore,methylation patterns varied among the four polyploids:heptaploid,octoploid,nonuploid,and decaploid.Total and full methylation levels in P.ternata were 54%-58% and 24.1%-24.3%.All types of MSAP patterns detected in the study belonged to two classes,type Ⅰ and Ⅱ. 52.5% of detected bands belonged to Type Ⅰ; Another 47.5% were type Ⅱ,which showed the methylation differences among the four polyploids. Conclusion The results demonstrate DNA methylation events occur in P.ternata and the general methylation levels are higher.

Determination of Ciprofloxacin(CIPRO)and Metronidazole(MNZ)in Compound Ciprofloxacin Ear Drops by the Method of Nodical Polyploid UV-Spectrophotometry / 中国药房

OBJECTIVE:To simultaneously determine the contents of CIPRO and MNZ in compound ciprofloxacin ear dro_ ps by nodical polyploid UV-spectrophotometry.METHODS:CIPRO and MNZ were nodical absorption at wavelength of350.0nm and this was just the sum of isoabsorptive point of two components while DXM did not absorb UV ray at this point.Therefore350.0nm was adopted as the detecting wavelength for these two components.RESULTS:The average recovery and relative standard deviation of CIPRO were99.7%and1.4%respectively.The average recovery and relative standard de?viation of MNZ were100.7%and1.6%respectively.CONCLUSION:The method is accurate,rapid,simple and sensitive.This method can be used to control the quality of this preparation.