Modal Chromosome Number Research Articles

Establishment of a spleen cell line from large yellow croaker Pseudosciaena crocea and its primitive application in foreign gene transfection

A large yellow croaker, Pseudosciaena crocea, spleen (LYCS) cell line was established and the feasibility of using it for foreign gene transfection was evaluaed in this study. Primary culture of LYCS cells was initiated from spleen tissue pieces, which were cultured at 25°C in Dulbecco’s modiced Eagle medium/F12 medium (DMEM/F12, 1:1) (pH7.2), supplemented with 20% fetal bovine serum, carboxymethyl chitosan, chondroitin sulfate, basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I). The cultured LYCS cells, in fibroblast shape, proliferated to 100% confluency 20 days later. Chromosome analyses indicated that the LYCS cells exhibited chromosomal aneuploidy with a modal chromosome number of 48 which displayed the normal diploid karyotype of P. crocea (6m+6sm+36t, NF=60). A LYCS cell line, with a population doubling time of 48.7 h at passage 60, has been established and subcultured to passage 70. Transgenic feasibility test demonstrated that positive green fluorescence protein (GFP) expression was observed in LYCS cells after pcDNA3.1-GFP plasmid transfection. In conclusion, a continuous foreign gene transfection feasible LYCS cell line has been established successfully. The cell line might serve as a valuable tool for studies of transgenic breeding and has potential applications for different kinds of cytotechnological studies.

Characterization of hTERT‐Immortalized Caprine Mammary Epithelial Cells

The aim of this article is to demonstrate and characterize caprine mammary epithelial cells (CMC) immortalized with human telomerase reverse transcriptase (hTERT) gene. Five immortalized CMCs were assigned to either myoepithelial or luminal epithelial groups based on their morphology and expression of cell lineage-specific intermediate filaments. Telomeric repeat amplification protocol revealed various telomerase activities in CMCs associated with their distinct proliferation potential. Karyotypic analysis showed three CMCs retained their modal Capra hircus chromosome number (2n = 60), whereas the remaining two CMCs were abnormal at 2n = 19 and 2n = 36. CMCs with abnormal karyotypes lost p53 protein after chemical-induced DNA damage and showed anchorage-independent growth in soft agar assay. In terms of functional differentiation, luminal CMCs organized into alveolus-like structures when grown in Matrigel. Furthermore, αs1- and β-casein gene was induced in luminal CMCs in response to lacto-hormones stimulation. Together these results showed that hTERT-immortalized CMCs retained major characteristics of mammary epithelial cells, and stability of the genome is required for maintaining normal mammary epithelium function. Application of CMCs can provide valuable models to study alveologenesis and lactogenesis of mammary epithelium and test the feasibility of recombinant constructs designed for the generation of transgenic livestock.

Establishment of a mammary carcinoma cell line from Syrian hamsters treated with N-methyl-N-nitrosourea

Clearly new breast cancer models are necessary in developing novel therapies. To address this challenge, we examined mammary tumor formation in the Syrian hamster using the chemical carcinogen N-methyl-N-nitrosourea (MNU). A single 50 mg/kg intraperitoneal dose of MNU resulted in a 60% incidence of premalignant mammary lesions, and a 20% incidence of mammary adenocarcinomas. Two cell lines, HMAM4A and HMAM4B, were derived from one of the primary mammary tumors induced by MNU. The morphology of the primary tumor was similar to a high-grade poorly differentiated adenocarcinoma in human breast cancer. The primary tumor stained positively for both HER-2/neu and pancytokeratin, and negatively for both cytokeratin 5/6 and p63. When the HMAM4B cell line was implanted subcutaneously into syngeneic female hamsters, tumors grew at a take rate of 50%. A tumor derived from HMAM4B cells implanted into a syngeneic hamster was further propagated in vitro as a stable cell line HMAM5. The HMAM5 cells grew in female syngeneic hamsters with a 70% take rate of tumor formation. These cells proliferate in vitro, form colonies in soft agar, and are aneuploid with a modal chromosomal number of 74 (the normal chromosome number for Syrian hamster is 44). To determine responsiveness to the estrogen receptor (ER), a cell proliferation assay was examined using increasing concentrations of tamoxifen. Both HMAM5 and human MCF-7 (ER positive) cells showed a similar decrease at 24 h. However, MDA-MB-231 (ER negative) cells were relatively insensitive to any decrease in proliferation from tamoxifen treatment. These results suggest that the HMAM5 cell line was likely derived from a luminal B subtype of mammary tumor. These results also represent characterization of the first mammary tumor cell line available from the Syrian hamster. The HMAM5 cell line is likely to be useful as an immunocompetent model for human breast cancer in developing novel therapies.

Establishment and characterization of a new cell line derived from kidney of grouper, Epinephelus akaara (Temminck & Schlegel), susceptible to Singapore grouper iridovirus (SGIV)

A marine fish cell line derived from the kidney of red-spotted grouper, Epinephelus akaara, designated as EAGK was established and characterized. The EAGK cells multiplied well in Leibovitz's L-15 medium containing 10% foetal bovine serum at 25 °C and have been subcultured for more than 90 passages. Karyotyping, chromosomal typing and ribosomal RNA (rRNA) genotyping analysis revealed that EAGK had a modal diploid chromosome number of 82 and was a fibroblast cell line originated from grouper. A severe cytopathic effect was observed in EAGK cells incubated with Singapore grouper iridovirus (SGIV), but not with soft-shelled turtle iridovirus, viral nervous necrosis virus or spring viraemia of carp virus. SGIV replication was further confirmed by immunofluorescence, electron microscopy and virus titre determination. Bright fluorescence was observed after transfection with fluorescent protein reporter plasmids, indicating that EAGK cells can be used to identify gene functions in vitro. In addition, the cell organelles including mitochondria and endoplasm reticulum changed and aggregated around virus factories after SGIV infection, suggested that the EAGK cell line could be an important tool for investigation of iridovirus-host interactions.

Cytogenetic Studies of Human Esophageal Squamous Cell Carcinoma and Gastric Adenocarcinoma Cell Lines

Esophagus and gastric cancer are one of the most common in Asian countries, but molecular event in the carcinogenesis remains as largely unidentified associated oncogenes. Detailed karyotypic analyses were performed on 4 cell lines derived from malignant cells of a patient with esophagus carcinoma of both diagnosis and metastasis stages and 2 cell lines from 2 independent patients with gastric cancer of metastasis stages. Most of these cells examined were near triploid with modal chromosome number of 59-73. These cells had a complex karyotype with more than 10 rearrangements. The karyotypic pattern was relatively stable; consistent alterations involved long arm of chromosomes 2, 9 and 12 and short arm of chromosomes 3 in both diagnosis and metastasis stages in esophagus cell lines. Those alterations of gastric cancer involved chromosomes 7 and 11. Higher rearrangements associated with translocations and deletions were observed at 2q23 of chromosome2, 3p14 of chromosome 3, 4q11 of chromosome 4 and 11q25 of chromosome 11 in esophagus cancer; while 7p13 of chromosome 7 and 11p15 of chromosome 11 in gastric cancer. The overall chromosome pattern was not similar between these cancers, although these tumors arise from anatomically or biologically related site. Unknown oncogenes localizing on these chromosome breakpoints for translocation or deletion region might be associated with the pathogenesis of esophagus or gastric cancer. These newly established cell lines will be useful tools in the study of the molecular pathogenesis and for testing new therapeutic reagents for these cancers in the future.

Cytogenetic studies of human esophageal squamous cell carcinoma and gastric adenocarcinoma cell lines

Esophagus and gastric cancer are one of the most common in Asian countries, but molecular event in the carcinogenesis remains as largely unidentified associated oncogenes. Detailed karyotypic analyses were performed on 4 cell lines derived from malignant cells of a patient with esophagus carcinoma of both diagnosis and metastasis stages and 2 cell lines from 2 independent patients with gastric cancer of metastasis stages. Most of these cells examined were near triploid with modal chromosome number of 59-73. These cells had a complex karyotype with more than 10 rearrangements. The karyotypic pattern was relatively stable; consistent alterations involved long arm of chromosomes 2, 9 and 12 and short arm of chromosomes 3 in both diagnosis and metastasis stages in esophagus cell lines. Those alterations of gastric cancer involved chromosomes 7 and 11. Higher rearrangements associated with translocations and deletions were observed at 2q23 of chromosome2, 3p14 of chromosome 3, 4q11 of chromosome 4 and 11q25 of chromosome 11 in esophagus cancer; while 7p13 of chromosome 7 and 11p15 of chromosome 11 in gastric cancer. The overall chromosome pattern was not similar between these cancers, although these tumors arise from anatomically or biologically related site. Unknown oncogenes localizing on these chromosome breakpoints for translocation or deletion region might be associated with the pathogenesis of esophagus or gastric cancer. These newly established cell lines will be useful tools in the study of the molecular pathogenesis and for testing new therapeutic reagents for these cancers in the future.

Development and characterization of a fibroblastic-like cell line from caudal fin of the red-line torpedo, Puntius denisonii (Day) (Teleostei: Cyprinidae)

A fibroblastic-like cell line was established from the ornamental ¢sh, red-line torpedo (Puntius denisonii). The red-line torpedo fin (RTF) cell line is being maintained in Leibovitz’s L-15 mediumsupplementedwith 10% fetal bovine serum (FBS) for over 1year at 28 1C on a continuous basis in normal atmosphere. The growth rate of RTF cells increased as the FBS proportion increased from 5% to 20% at 28 1C with optimum growth at the concentrations of 10% FBS. The morphology of RTF cell was predominantly fibroblastic like. Propagation of these cell lines was serum dependent, with a low plating efficiency (o15%). Karyotyping analysis of RTF cells at the 25th passage indicated that the modal chromosome number was 2n550. The cell line was cryopreserved in liquid nitrogen at �196 1C and could be recovered from storage after 6 months with good cell viability. Polymerase chain reaction amplification of a fragment of two mitochondrial genes, 16S rRNA and CO1, con¢rmed the identity of these cell lines with those reported from this animal species, confirming that the cell lines originated from P. denisonii. The bacterial extracellular products from Vibrio cholerae MTCC3904 and Aeromonas hydrophila were found to be toxic to RTF. The cell lines were not susceptible to viral nervous necrosis virus, a marine ¢sh virus.

Chromosomal study of a lamprey (Lampetra zanandreai Vladykov, 1955) (Petromyzonida: Petromyzontiformes): conventional and FISH analysis

Karyotype and other chromosomal characteristics in the Adriatic brook lamprey Lampetra zanandreai, representative of one of the most ancestral group of vertebrates, were examined using conventional (Ag-staining, C-banding as well as CMA(3) and DAPI fluorescence) and molecular (FISH with 18/28S rDNA and EcoRI satDNA as probes) protocols with metaphase chromosomes derived from whole blood cultures. The chromosome complement had a modal diploid chromosome number of 2n = 164, as in other petromyzontid lamprey species. Ag-staining and CMA(3) fluorescence, as well as FISH with 18/28S rDNA probes, detected nucleolar organizer regions (NORs) close to the centromeres of the biarmed chromosomes of pairs 1 and 2, the largest chromosome pairs of the complement. In addition to NORs, CMA(3) fluorescence revealed positive signals in approximately 40 other chromosomes. DAPI stained mostly centromeric regions of many chromosomes as well as conspicuously massive blocks overlapping NOR sites. C-banding evidenced a large amount of constitutive heterochromatin in somatic chromosomes, with approximately 40 C-positive acrocentric elements completely heterochromatic, corresponding with the 40 CMA(3)+ chromosomes and positive heterochromatic blocks in pericentromeric regions of chromosome pairs 1 and 2. Polymerase chain reaction (PCR)-based cloning of satellite DNA with primers derived from Petromyzon marinus centromeric sequences was successful for L. zanandreai genomic DNA. The sequence was AT-rich (59%) and characterized by short consensus motifs similar to other centromeric satellite motifs. FISH using satDNA clones as a probe produced a fluorescent signal on a single pair of small chromosomes. This sequence was PCR-amplified also in L. planeri and P. marinus genomic DNA, and the evolution of this repetitive element in the above species was analysed.

Acute High-Dose X-Radiation-Induced Genomic Changes in A549 Cells

Accidents with ionizing radiation often involve single, acute high-dose exposures that can lead to acute radiation syndrome and late effects such as carcinogenesis. To study such effects at the cellular level, we investigated acute ionizing radiation-induced chromosomal aberrations in A549 adenocarcinoma cells at the genome-wide level by exposing the cells to an acute dose of 6 Gy 240 kV X rays. One sham-irradiated clone and four surviving irradiated clones were recovered by minimal dilution and further expanded and analyzed by chromosome painting and tiling-path array CGH, with the nonirradiated clone 0 serving as the control. Acute X-ray exposure induced specific translocations and changes in modal chromosome number in the four irradiated clones. Array CGH disclosed unique and recurrent genomic changes, predominantly losses, and revealed that the fragile sites FRA3B and FRA16D were preferential regions of genomic alterations in all irradiated clones, which is likely related to radioresistant S-phase progression and genomic stress. Furthermore, clone 4 displayed an increased radiosensitivity at doses >5 Gy. Pairwise comparisons of the gene expression patterns of all irradiated clones to the sham-irradiated clone 0 revealed an enrichment of the Gene Ontology term "M Phase" (P = 6.2 × 10(-7)) in the set of differentially expressed genes of clone 4 but not in those of clones 1-3. Ionizing radiation-induced genomic changes and fragile site expression highlight the capacity of a single acute radiation exposure to affect the genome of exposed cells by inflicting genomic stress.

Establishment of human infancy hemangioma-derived endothelial cell line XPTS-1 and animal model of human infancy hemangioma

To establish an immortalized human infancy hemangioma-derived endothelial cell line (HemEC) and animal model of human infancy hemangioma. Hemangioma-derived endothelial cells from specimen of human infancy hemangioma were cultured in vitro and monocloed, and then its growth curve was made, karyomorphism of chromosome analyzed, morphologic characteristics observe, factor VIII related antigen identified by immunohistochemical method.Vascular endothelial growth factor receptor 2 (VEGFR-2) was detected by flow cytometry. HemEC were inoculated subcutaneously in athymic mouse to establish animal model of infancy hemangioma. The animal model was observed closely and its pathological characteristic was also studied. The cultural cells grew active, and immortalized spontaneously when they were subcultured on sixteenth generation. This cell line was cultivated for more than 70 times within one year and in good condition after freezing and resuscitating once and again, and had the morphologic character of HemEC. The cell population doubling time was 22 h. Factor VIII and VEGFR-2 were expressed positively. Karyo type analysis of the cell line showed abnormal diploid with the modal chromosomal number varying between diploid and triploid. The cell line was then named XPTS-1. The animal model of infancy hemangioma was successfully established and its character of histopathology was similar with that of infancy hemangioma. The cell line of HemEC was successfully established and immortalized spontaneously, and had the morphologic and biological character of HemEC. The animal model of infancy hemangioma was successfully established and showed the character of histopathology similar with that of infancy hemangioma.

Effect of Mycoplasma salivarium with and without L-arginine on karyotypic variability in cell line of Indian muntjac skin fibroblasts at long-term cultivation

The effect of Mycoplasma salivarium on the numerical and structural karyotypic variability was studied in the markerless cell line of Indian muntjac skin fibroblasts (line M) during long-term cultivation with and without L-arginine. The cultivation of mycoplasma-contaminated cells for 15 and 30 days did not change the character of cell distribution for the number of chromosomes. In contaminated cells cultivated for 60 and 75 days, the character of cell distribution for the chromosome number changed. These changes involve bimodal distribution for the chromosome number due to a significant decrease in frequency of the cells with the modal number of chromosomes with the main structural variant of karyotype (SVK) 2 + 2 + 1 + 1 + 1 and an increase in the frequency of cells with the submodal number of chromosomes with a main SVK of 2 + 2 + 1 + 1. Furthermore, a significant increase in the frequency of cells with lower numbers of chromosomes was observed after 60 days compared to that after 75 days of cultivation. After the cultivation of the contaminated and control cells in the medium with an elevated concentration of L-arginine for 60 days, the numerical parameters were unchanged relative to the control. The cultivation of contaminated cells for 60 days followed by the addition of L-arginine for 15 days restored the numerical parameters to the control level. In the contaminated cells, the frequency of chromosomal aberrations for 30, 60, and 75 days increased significantly compared to the control variant. After 30 days of cultivation, a small but statistically significant increase took place due to a uniform slight increase in the frequency of chromosomal aberrations of all types. After 60 and 75 days, a greater increase occurred due to a statistically significant increase in the frequency of chromosomal and chromatid breaks. Moreover, after 60 days, the level of dicentrics (telomeric associations) mainly produced by chromosomes 1 and 2 increased significantly. The role of dicentrics as for a means of adaptation for markerless cell lines to the condition of cultivation and the role of L-arginine in the restoration of the normal karyotypic structure of the line M cell population at mycoplasmal contamination are discussed.

A Novel Cell Line from Liver of Stone Flounder, Kareius bicoloratus, and Its Susceptibility to Different Iridoviruses

A novel cell line (SFL) was established from the liver of stone flounder, Kareius bicoloratus, and its susceptibility to different iridoviruses was evaluated. The SFL cells grew well in Dulbecco's Modified Eagle Medium supplemented with fetal bovine serum, basic fibroblast growth factor, and insulin-like growth factor-II, and have been subcultured over 82 passages. The optimal growth temperature was 22 C. The SFL cells were fibroblastic in morphology and grew at a steady rate, with a population doubling time of 38.8 h. Karyotype analysis showed that SFL cells exhibited chromosomal aneuploidy with a modal chromosome number of 48. The susceptibility evaluation of SFL cells revealed that cytopathic effects (CPE) appeared after infection by different iridoviruses, lymphocystis disease virus (LCDV) and turbot reddish body iridovirus (TRBIV). In addition, a large number of TRBIV and LCDV particles were observed in the infected SFL cells by electron microscope examination. It is suggested that the SFL cell line could be used as a valuable tool for isolation and propagation of different iridoviruses.

The prognostic significance of cytogenetics and molecular profiling in multiple myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by very complex cytogenetic and molecular genetic aberrations. In newly diagnosed symptomatic patients, the modal chromosome number is usually either hyperdiploid with multiple trisomies or hypodiploid with one of several types of immunoglobulin heavy chain (Ig) translocations. The chromosome ploidy status and Ig rearrangements are two genetic criteria that are used to help stratify patients into prognostic groups based on the findings of conventional cytogenetics and fluorescence in situ hybridization (FISH). In general, the hypodiploid group with t(4;14)(p16;q32) or t(14;16)(q32;q23) is considered a high-risk group, while the hyperdiploid patients with t(11;14)(q13;q32) are considered a better prognostic group. As the disease progresses, it becomes more proliferative and develops a number of secondary chromosome aberrations. These secondary aberrations commonly involve MYC rearrangements, del(13q), del(17p), and the deletion of 1p and/or amplification of 1q. Of the secondary aberrations, del(17p) is consistently associated with poor prognosis. All of these cytogenetic aberrations and many additional ones are now identified by means of high resolution molecular profiling. Gene expression profiling (GEP), array comparative genomic hybridization (aCGH), and single-nucleotide polymorphism (SNP) arrays have been able to identify novel genetic aberration patterns that have previously gone unrecognized. With the integration of data from these profiling techniques, new subclassifications of MM have been proposed which define distinct molecular genetic subgroups. In this review, the findings from conventional cytogenetics, interphase FISH, GEP, aCGH, and SNP profiles are described to provide the conceptual framework for defining the emerging molecular genetic subgroups with prognostic significance.

Phylogeny of Sphaerium solidum (Bivalvia) based on karyotype and sequences of 16S and ITS1 rDNA

Abstract The present work represents the first karyological and molecular characterisation of Sphaerium solidum, a rare European clam. Specimens of S. solidum were collected in Lithuania and Hungary. The modal diploid chromosome number found in both populations was 2n=30. Small, biarmed B chromosomes were found in 42.3% of cells studied in clams from Lithuania and in 11.8% of cells in clams from Hungary. Comparative analysis revealed no significant (P<0.05) interspecific differences in chromosome morphology of S. solidum and that of previously studied S. corneum. DNA sequence analyses of S. solidum showed no interpopulation differences in ITS1; moreover, only one site was different from ITS1 of S. corneum. However, differences in mitochondrial 16S sequence of S. solidum were revealed: two haplotypes in Lithuania and three in Hungary were identified. The genetic characteristics revealed in this study do not support ascription of S. solidum and S. corneum to different subgenus, Cyrenastrum and Sphaerium s. str., respectively. Comparative cytogenetic analysis disclosed that the chromosome morphology could be conserved in some sphaeriid species during speciation despite the fact that most other species in this family undergo radical karyotypic differentiation.

Establishment of a cell line from the brain of grouper (Epinephelus akaara) for cytotoxicity testing and virus pathogenesis

We established and characterized a continuous cell line (EAGB) from the brain of grouper (Epinephelus akaara). EAGB cells multiplied well in MEM that was supplemented with 10% fetal bovine serum at 30°C. The EAGB cell line consisted predominantly of fibroblast-like cells from grouper, which was confirmed by the sequence of mitochondrial 18s rRNA. Karyotyping indicated that the modal chromosome number was 60. The transfection efficiency of pEGFP-N3 into EAGB cells reached 34%. Bacterial cytotoxicity studies showed that the extracellular products from Vibrio alginolyticus or Vibrio anguillarum were toxic to EAGB cells. In addition, EAGB cells were susceptible to Singapore grouper iridovirus (SGIV) and viral nervous necrosis virus (VNNV), which was demonstrated by the presence of a severe cytopathic effect (CPE), abundant virus particles, and increased viral titers. Although the CPE was observed in soft-shelled turtle iridovirus (STIV)-infected EAGB cells, the virus titer was not increased and virus particles were not observed by electron microscopy. Nuclear morphological observations showed that STIV infection induced typical apoptotic bodies, whereas SGIV infection evoked non-apoptotic death in EAGB cells. The newly established EAGB cell line provided a useful tool for genetic manipulation, cytotoxicity testing, and virus pathogenesis.

Characteristics and establishment of cell lines from human gastrointestinal stromal tumors.

To establish the cell line from specimens of resectable human gastrointestinal stromal tumors (GIST) and to verify the characteristics of cell biology in vitro. The tissues from biopsies of human GIST were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum. After growing to 90% confluence, the cells were detached for subculture and their characteristics, including morphology, growth kinetics, karyotype analysis, immunohistochemical analysis and tumorigenicity in nude mice were determined. GIST named GIST-H1 was successfully established. The cell line was passaged for more than 60 times 1 year. The characteristics demonstrated: The population doubling time calculated in the log phase of growth was 47.5 h. The cloning efficiency in the soft agar averaged 24.8%. Electronmicroscopically, there were rich ribosomes and mitochondrion in the cytoplasm. Immunohistochemical analysis showed CD117(+), SMA(+), dog-1(+), CD34(-), and S-100(-). Karyotype analysis illustrated aneuploidy with the modal chromosomal number 60-98. The GIST cells transplanted in nude mice had high tumorigenicity. The immortalized GIST cells are developed in vitro and have specific characteristics of GIST.

Prevalence of B chromosomes in Orthoptera is associated with shape and number of A chromosomes

We analyze the prevalence of B chromosomes in 1,601 species of orthopteran insects where chromosome number and shape are known. B chromosomes have been reported in 191 of these species. Bs are not uniformly distributed among orthopteran superfamilies, with evident hotspots in the Pyrgomorphoidea (32.3% of species carrying Bs), Grylloidea (14.9%), Acridoidea (14.6%) and Tetrigoidea (14.3%). As expected under the theory of centromeric drive, we found a correlation between B chromosome presence and A chromosome shape-Bs are more frequent in karyotypes with more acrocentric A chromosomes. We also found that Bs are less common in species with high chromosome numbers and appear to be most common at the modal chromosome number (2n = 24). Study effort, measured for each genus, was not associated with B prevalence, A chromosome shape or A chromosome number. Our results thus provide support for centromeric drive as an important and prevalent force in the karyotypic evolution of Orthoptera, just as it appears to be in mammals. We suggest that centromeric drive may provide a mechanistic explanation for White's principle of karyotypic orthoselection.

Cytogenetic characterization of low-passage cell line of follicular thyroid adenoma

Neoplasias arising from follicular thyroid cells provide a model for multistep tumorigenesis (Sozzi et al., Cancer Genet Cytogenet 1992;64:38e41). Adenomas of the thyroid are rare examples of epithelial tumors frequently displaying specific chromosomal translocations. The most commonly reported translocations involve chromosomes 2, 3, and 5 (Rippe et al., Oncogene 2003; 22:6111e4; Roque et al., Genes Chromosomes Cancer 2003;36: 292e302; Belge et al., Cancer Genet Cytogenet 1998;101: 42e8). We report the isolation and characterization of a new low-passage follicular thyroid adenoma cell line, with similar chromosomal alterations as described previously. A fresh sample was obtained from Tumour Bank of Osuna Hospital, belonging to the Tumour Banks of the Andalusia Network, from a 52year-old women diagnosed with a follicular thyroid adenoma. The sample was processed and placed in culture with specific media until growth was achieved. The culture was analyzed to determine cellular morphology, and was characterized in various phases of generation for lineage specificity by immunohistochemistry. Moreover, telomerase activity was quantified by qPCR using a telomerase repeat amplification protocol, and the DNA profile of the cell line was compared to the original tumor sample using polymorphisms in STR loci. We performed cytogenetic analysis by G-banding and spectral karyotyping. After incubation for a few weeks, cells grew as a firm adherent monolayer with spindle-epithelial like morphology, and formed piled-up cell colonies. The epithelial nature of the cultured cells was confirmed by immunocytochemical analysis using an anti-cytokeratin cocktail. Telomerase activity was positive and the cells showed a single DNA profile, concordant with the tumor origin of the sample. Cytogenetic analysis, in middle passage, showed a modal chromosome number of 44e45 with three clonal rearrangements: an unbalanced translocation between chromosomes 2 and 3 [t(2;3) (q32;?)], between chromosomes 2 and 10 [(t(2;10)(q24;?)] and a derivative marker chromosome from 5 and 13 [t(5;13) (?;p11.2)]. Furthermore, we observed a deletion of chromosome 13 (13q21) and a monosomy of chromosomes 3 and X in all analyzed metaphases. These results indicate that the features of this cell line are similar to follicular thyroid adenomas described by others and, therefore, offer researchers a good tool for cancer investigation. RECURRENT COPY NUMBER ALTERATIONS IN ESOPHAGEAL SQUAMOUS CELL CARCINOMAS

Establishment and characterization of a new marine fish cell line derived from red‐spotted grouper Epinephelus akaara

A new marine fish cell line, EAGL, derived from the liver of red-spotted grouper Epinephelus akaara was established and characterized. The cells multiplied well in minimum essential medium (MEM) supplemented with 10% foetal bovine serum (FBS) at temperatures between 25 and 30° C. The growth rate of this cell line increased as the proportion of FBS increased from 5 to 20% at 25° C, with maximum growth at the concentration of 15 or 20% FBS. Morphologically, the cells were epithelial-like and the presence of pancytokeratin confirmed their epithelial origin. Chromosome analysis revealed that the modal chromosome number was 48. The susceptibility of the cell line to four fish viruses was tested. Significant cytopathic effect (CPE) was only observed in Singapore grouper iridovirus (SGIV)-infected cells, and the virus replication was further confirmed by immunofluorescence, electron microscopy and real-time reverse-transcription (RT)-PCR assay. When the cells were transfected with pEGFP-N3 plasmid, bright fluorescent signals were observed, suggesting that this cell line can be used for transgenic and genetic manipulation studies.

Establishment of a novel human esophageal squamous cell carcinoma cell line (ESC-410) and its partial biological characterization

Esophageal cancer exhibits an uneven geographical distribution strikingly, resulting in focal endemic high-incidence areas in several countries worldwide including China, which might be associated with the environmental and genetic risk factors in those areas. Permanent cancer cell lines are invaluable tools in understanding the biology of cancers and experimental therapeutics. To enrich cell line panel and animal models of human esophageal squamous cell carcinoma (ESCC) from different geographical areas and investigate the environmental and genetic risk factors in the carcinogenesis of ESCC, a novel human esophageal squamous cancer cell line (ESC-410) was established. The cell line grew adherent as a monolayer and maintained stable growth rate with a doubling time of 53 h and distinct epithelial morphological appearance; it was maintained in vitro for 18 months and subcultured for more than 50 passages. Ultrastructural examination revealed large irregular nuclei, desmosome, and tonofilaments; karyotype analysis showed a modal number of chromosomes that ranged from 35 to 73, with a median of 57, and 77% of analyzed cells were hyperdiploidy; reverse transcription polymerase chain reaction (RT-PCR) detected the mRNA expressions of CK8, CK18, and CK19 in the established cells; immunofluorescence assay identified the protein expressions of neurotrophin receptor p75 and integrin α6 (CD49f) in the ESC-410 cell line; xenotransplantation of ESC-410 cells into athymic nude mice subcutaneously induced the formation of solid tumor masses in about 2 weeks. By histopathological examination, heterogeneity of xenograft tumor was observed, as same as that of human primary ESCC. All findings and evidence in this experimental study suggested that this cell line might be a useful model in vitro and in vivo in cellular and molecular studies as well as in testing novel therapies for human ESCC.