Characteristics of a new rapidly proliferating rabbit cell line

Summary A new tissue culture cell line (Calg-ARLC) has been established from an explant culture of adult rabbit lung. The Calg-ARLC line has been characterized with respect to morphology, chromosome constitution, tissue culture requirements, proliferative capacity, cell cycle, attainable synchrony, radioisotopically labeled precursor incorporation into nucleic acids and protein, and radioisotope sensitivity. The cells are fibroblast-like in appearance with a stabilized heteroploid chromosomal modal number of 35. They grow exponentially from high split ratios in several commercially available defined media with a generation time of 12 hr and are easily synchronized. Although sensitive to some isotopically labeled precursors, high specific activity nucleic acids have been isolated. The ARLC line is especially useful for the isolation of high specific activity nucleic acids and proteins of rabbit origin. The Calg-ARLC line should be invaluable in the fractionation of reiterated DNA sequences since no very rapidly reassociating DNA sequences such as those found in mouse are evident. This work was supported by operating grants from the National Cancer Institute of Canada and the National Research Council of Canada.     

Characterization of porcine stable kidney cell line adapted to hyperthermic temperature

The optimum temperature for the growth of porcine stable (PS) kidney cell line is 37 degrees C. We have adapted the cell line to grow at 40 degrees C. The original cell line grown at 37 degrees C has been denoted as PS-37, and the adapted new strain has been denoted as PS-40. Both the cell lines were screened for mycoplasma by Hoechst staining and tritiated uridine-uracil uptake and were found to be negative. Comparative characterization of PS-40 and its progenitor PS-37 cell line was done by using various parameters. The antigenic studies indicated that the new cell strain was not cross-contaminated with any other cell lines. It was observed that PS-40 cells were more fibroblastic with clean cytoplasm and appeared healthy. The growth of PS-40 cells was faster than the original cell line. The karyological study showed heteroploid chromosome number in PS-40 cells. The modal chromosome number of PS-40 cells was 58, whereas that of PS-37 cell line was 38. The lactic dehydrogenase isoenzyme pattern showed a cathodal shift of bands. The PS-40 cell strain could be cryopreserved and revived. The viability of PS-37 as well as PS-40 cell lines is in the range of 90-95%, and the growth characteristics of thawed cells showed six- to eightfold multiplications within 5 d. The virus susceptibility study revealed that the cytopathic effect was more profound and observed 1 d earlier in PS-40 cell line. Increased yields of Japanese encephalitis, Sindbis, and Semliki forest viruses were obtained by 1.8, 1.75, and 1.5 log plaque-forming units/ml, respectively. The yield of West Nile virus was, however, comparable to that in PS-37 cell line. Both the cell lines were refractory to Dengue viruses.     

Molecular cytogenetic analysis of repeated sequences in a long term wheat suspension culture

A rapidly growingTriticum aestivum L. (wheat) derived long term suspension culture (named TaKB1), that is probably not regenerable, was analysed for karyotype rearrangements, stability and changes in repetitive DNA. The cell line has an average chromosome number of 21 and the DNA amount of unreplicated cells of TaKB1 measured by flow cytometry is about 30% lower than an unreplicated (1C) bread wheat genome.In situ hybridization of a repetitive DNA sequence (pSc119.2), which occurs as tandemly repeated blocks (heterochromatin) in wheat, shows that chromosomes from the TakB1 line have fewer and weaker subtelomeric locations of the sequence than wheat, suggesting deletions of distal chromosome segments and a reduction in the sites and copy number of the sequence. Thein situ hybridization pattern and chromosome morphology allowed 27 chromosome types to be identified in the cell line. No two analysed cells contained the same chromosome complement, although some chromosome types were present in every cell. Using Southern hybridization the structure and copy number of a retroelement (Wis-2) and its flanking sequence was shown to be the same in the TaKB1 cell line and wheat. Anin situ analysis of rDNA in the TaKB1 cell line (using the probe pTa71) showed a reduction in number of sites and rRNA genes in each cell from that in wheat. Interphase cells of the cell line showed dispersed signal throughout the nucleolus with no evidence for clusters of condensed and inactive rRNA genes.     

Effect of laminin on karyotypic variability in cell line of Indian muntjac skin fibroblasts

The numerical and structural karyotypic variability has been investigated in the Indian muntjac skin fibroblasts cell line M and karyotypic variant of this line M' on cultivation on a laminin 2/4 coated surface. In cell line M, cultivated on the laminin-coated surface for 4 and 14 days, and in karyotypic variant M', cultivated for 2, 4 and 14 days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in frequency of cells with model numbers of chromosomes, and an increase in frequency of cells with lower chromosome numbers. As a result, new modal chromosome numbers form. The frequency of cells with 4 chromosomes increases significantly; as a rule, such cells are absent in the control cell variants. Many new additional structural variants of the karyotype (SVK) appear. Detachment of cells M' from the laminin-coated surface followed by a 2 day cultivation on a hydrophilic surface, commonly used for routine cell cultivation, does not restore the control cell distribution for chromosomal number. The frequency of chromosomal aberrations on cultivation of the laminin-coated surface does not change relatively to controls. The observed alterations seem to be due to both disturbances of mitotic apparatus and selection of SVK, which are more advantageous to changed culture conditions of the cell population.     

Myoblast senescence in muscular dystrophy

The limited proliferative capacity of normal diploid cells predicts that the utilization of cell divisions in vivo should reduce the lifespan of cells in culture. Because of the continuing demands for muscle regeneration in muscular dystrophy, myoblasts isolated from affected muscles should thus show a decrease in the number of cell divisions they are capable of expressing in culture. This hypothesis was tested by examining the proliferative capacity of myoblasts from different muscles for normal line 412 and dystrophic line 413 chickens of various ages. Prior to approx. 2 months of age, dystrophic myoblasts exhibited a relatively normal proliferative lifespan. By 5 months of age, myoblasts from the severely affected pectoralis major showed a 40% reduction in their proliferative potential, while myoblasts from the less affected posterior latissimus dorsi muscle showed a 25% decrease in their cultured lifespan. The time course of the appearance of a decreased proliferative capacity only after the disease has been clinically manifested strongly supports it representing a secondary response rather than it being an intrinsic property of dystrophic myoblasts. A hypothesis for manipulating the pattern of stem cell division in order to increase the mass of muscle produced from a constant number of cell divisions is presented. If myoblast senescence and the consequent failure of muscle regeneration is a contributing factor in the progressive deterioration of muscle function in the disease, then this hypothesis might provide an important therapeutic strategy for ameliorating the course of muscular dystrophy.     

In vitro culture of hybridoma cells in agarose beads producing antibody secretion for two weeks

A new process for embedding cells in agarose is described. Beads were obtained by extruding an ultralow gelling temperature agarose solution in a capillary containing a hydrophobic medium flowthrough. The toxicity of the procedure has been evaluated by monitoring the energy status of agarose-embedded C(6) glioma cells with (31)P nuclear magnetic resonance (NMR). Suspension and microbead cultures of hybridoma cell line were compared. In suspension culture the number of cells and the antibody concentrations increased for 5 days before the stationary phase began, when the cultures were stopped. In agarose bead cultures, the gel provided an enormous support surface area (50 m(2)/ mL of gel). It was possible to seed 20-fold more cells. The gel pressure modified the proliferative process and antibody pattern secretion. In particular, the antibodies could be harvested for two weeks.     

Effect of immobilized laminin on karyotypic variability in two karyotypically different variants of the indian muntjac skin fibroblast cell line

The numerical and structural karyotypic variability has been investigated in MTs of the markerless cell line of Indian muntjac skin fibroblasts, as well as in its karyotypic variant MT^D cultivated on a laminin 2/4 coated surface. In the MT cell line preincubated in serum-free medium for 2.5 and 1.0 h, then cultivated on a laminin-coated surface in serum-containing medium for one, two, and three days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in the frequency of cells with modal numbers of chromosomes and an increase in frequency of cells with lower chromosome numbers. Some new additional structural variants of the karyotype (SVK) appeared. The observed alterations seem to be due to disturbances of the chromosome segregation and the establishment of a new advantageous balanced karyotypic structure. In the karyotypic variant MT^D differing from MT by an increased number of dicentrics (telomeric associations) cultivated under the same conditions, the character of cell distribution for the chromosome number did not change. In the MT cell line, the frequency of chromosomal aberrations did not change relative to control variants. In the karyotypic variant MT^D under the same conditions, the frequency of chromosomal aberrations significantly increased after three days mainly due to the formation of dicentrics. These results confirm the conclusion that, like aneuploidy, the formation of dicentrics in markerless cell lines appears to be the way in which the cell population adapts to unfavorable environmental factors. Possible reasons for differences in the character of the numerical and structural karyotypic variability between the MT cell line and its karyotypic variant MT^D are discussed.     

The influence of a substrate including extracellular matrix proteins on karyotypic variability in two cell lines of Indian muntjac skin fibroblasts

The influence of cell-culture conditions on numerical and structural karyotypic variability has been investigated in two Indian muntjac skin fibroblast “markerless” cell lines, M and MT. The cells were cultivated on a substrate consisting of extracellular matrix proteins (ECMs) synthesized by human mesenchymal stem cells (SC5-MSC). The character of cell distribution for the chromosome number of the cell line M changed after cultivation for 1 and 4 days as compared to control cells, which were cultured on a hydrophilic surface without ECM coating. These changes involve a significant decrease in frequency of cells with the modal numbers of chromosomes and an increase in frequency of cells with lower chromosome numbers. Many new types of additional structural variants of the karyotype (SVK) appear. The MT cell line, differing from M line in the number of homologous chromosomes, exhibited a character of cell distribution similar to that of the M line for the chromosome number for only 1 day after cultivation on the ECM substrate, but not after 4 days under the same culture conditions, when no difference from the control cells was observed. Further cultivation of MT cells for 8 days did not change the character of cell distribution for the chromosome number relative to the control variant. The observed alterations seem to be due to disturbances in the correct chromosome-segregation process, which were caused by an abrupt shift in the cell-culture conditions. Analysis of the structural karyotypic variability revealed a significant increase in frequency of chromosomal aberrations in the M cell line for 1 and 4 days in culture on the ECM substrate as compared to the control cells. The frequency of dicentric chromosomes (telomeric associations) was increased and constituted more than 50% of all chromosome aberrations. No increase in frequency of chromosome aberrations was observed for MT cells cultured under the same conditions. It can be suggested that the differing by the karyotypic structure, but the genetically identical cell lines have different response to the substrate. In contrast to the M line, in the MT line, a fast normalization of numerical karyotypic characteristics and no enhancement of structural karyotypic variability takes place. This provides a possibility to cultivate an MT cell on the given protein substrate while maintaining a balanced karyotypic structure characteristic of MT cell line.     

Characterization of an HPV-negative cell line (FR-car) derived from a cervical squamous intraepithelial lesion

Summary A new cell line, FR-car, has been established from a biopsy of a low-grade human cervical squamous intraepithelial lesion (SIL). We confirmed the epithelial origin of the cells by keratin staining using polykeratin, AE1/AE3 and CAM 5.2 antibodies. Sixty percent to 80% of the cultured cells stained positive for proliferative cell nuclear antigen (PCNA) and Ki-67. There was no overexpression of p53. Karyotyping revealed that the cell line was hypodiploid with clonal abnormalities on chromosome 6 and 16. Sections of a biopsy adjacent to the lesion from which the culture was initiated tested positive for human papillomavirus (HPV) 18 DNA by the polymerase chain reaction, but cultured cells tested at several passages were HPV-negative by either type-specific or consensus PCRs. This HPV-negative SIL line may be useful in studies into the cell biology of dysplastic epithelium.     

Identification of a chromosome that controls malignancy in Chinese hamster cells.

A chromosome that controls malignancy in Chinese hamster cells has been identified by analysis of the Giemsa banding pattern of a malignant cell line transformed by simian virus 40 (SV40), non-malignant revertants from this line, segregants from the revertants that were again malignant and a cell line transformed by methylcholanthrene. The malignant cell line transformed by SV40 was near diploid and had gained additional material of chromosome 3. Revertants with a suppression of malignancy and malignant revertants from which they were derived. Malignancy of these cells was associated with the ability to form colonies in agar. Cells of a line transformed by methylcholanthrene were malignant, formed almost no colonies in agar and the only chromosome change from the normal diploid chromosome banding complement was the addition of a long arm of chromosome 3. The results indicate that chromosome 3 carriers gene(s) that control malignancy in Chinese hamster cells in cell lines transformed by a viral or a chemical carcinogen and that malignancy was induced in both cell types by an increase of these genes.     

Karyotype variation of CHO host cell lines over time in culture characterized by chromosome counting and chromosome painting

Genomic rearrangements are a common phenomenon in rapidly growing cell lines such as Chinese hamster ovary (CHO) cells, a feature that in the context of production of biologics may lead to cell line and product instability. Few methods exist to assess such genome wide instability. Here, we use the population distribution of chromosome numbers per cell as well as chromosome painting to quantify the karyotypic variation in several CHO host cell lines. CHO‐S, CHO‐K1 8 mM glutamine, and CHO‐K1 cells adapted to grow in media containing no glutamine were analyzed over up to 6 months in culture. All three cell lines were clearly distinguishable by their chromosome number distribution and by the specific chromosome rearrangements that were present in each population. Chromosome Painting revealed a predominant karyotype for each cell line at the start of the experiment, completed by a large number of variants present in each population. Over time in culture, the predominant karyotype changed for CHO‐S and CHO‐K1, with the diversity increasing and new variants appearing, while CHO‐K1 0 mM Gln preferred chromosome pattern increased in percent of the population over time. As control, Chinese hamster lung fibroblasts were shown to also contain an increasing number of variants over time in culture.     

Establishment and characterization of endometrial undifferentiated carcinoma cell line (TMCC-2.U)

We established new cell line designed TMCC-2.U, which suggested transformation to undifferentiated carcinoma, derived from endometrial clear cell carcinoma cell line (TMCC-2). The monolayer culture cell showed a pavement arrangement and spindle like shape. A rough-endoplasmic reticulum, mitochondria etc. are well developed. But cytoplasmic endocrine granulosa were so poorly, it suggests functional developments are poor. The TMCC-2.U cells were transplanted to nude mice which showed no typical pattern suggested undifferentiated carcinoma. Their chromosome number varied and the mode is 78. Marker chromosome were found frequency. Growth pattern and production of tumor marker are clearly differentiate from TMCC-2. As mensioned above, TMCC-2.U cell line will be very valuable in basic research on mechanism of transformation and effects of patient's serum on hystogenesity.     

Establishment and characterization of a cell line from the american opossum (Didelphys virginiana)

Summary A permanent tissue-cultured cell line (designated OK) has been established from kidney tissue of an adult American opossum. The OK line has been characterized with respect to morphology, chromosome constitution, tissue-culture requirements, and attainable mitotic arrest. The cells are epithelial-like with a stable nondiploid chromosomal modal number of 23. Cells grown in Eagle's minimal essential medium with 10% fetal bovine serum have a mean doubling time of 18 hr. The cell line OK is potentially useful for the isolation and purification of the mammalian X chromosome because of the size differential between the smaller X's and the larger autosomes. This work was supported by NIH grant HD-04420.     

A gene involved in control of human cellular senescence on human chromosome 1q.

Normal cells in culture exhibit limited division potential and have been used as a model for cellular senescence. In contrast, tumor-derived or carcinogen- or virus-transformed cells are capable of indefinite division. Fusion of normal human diploid fibroblasts with immortal human cells yielded hybrids having limited life spans, indicating that cellular senescence was dominant. Fusions of various immortal human cell lines with each other led to the identification of four complementation groups for indefinite division. The purpose of this study was to determine whether human chromosome 1 could complement the recessive immortal defect of human cell lines assigned to one of the four complementation groups. Using microcell fusion, we introduced a single normal human chromosome 1 into immortal human cell lines representing the complementation groups and determined that it caused loss of proliferative potential of an osteosarcoma-derived cell line (TE85), a cytomegalovirus-transformed lung fibroblast cell line (CMV-Mj-HEL-1), and a Ki-ras(+)-transformed derivative of TE85 (143B TK-), all of which were assigned to complementation group C. This chromosome 1 caused no change in proliferative potential of cell lines representing the other complementation groups. A derivative of human chromosome 1 that had lost most of the q arm by spontaneous deletion was unable to induce senescence in any of the immortal cell lines. This finding indicates that the q arm of human chromosome 1 carries a gene or set of genes which is altered in the cell lines assigned to complementation group C and is involved in the control of cellular senescence.     

Establishment of a new cell line from lepidopteran epidermis and hormonal regulation on the genes

When an insect molts, old cuticle on the outside of the integument is shed by apolysis and a new cuticle is formed under the old one. This process is completed by the epidermal cells which are controlled by 20-hydroxyecdysone (20E) and juvenile hormone. To understand the molecular mechanisms of integument remolding and hormonal regulation on the gene expression, an epidermal cell line from the 5th instar larval integument of Helicoverpa armigera was established and named HaEpi. The cell line has been cultured continuously for 82 passages beginning on June 30, 2005 until now. Cell doubling time was 64 h. The chromosomes were granular and the chromosome mode was from 70 to 76. Collagenase I was used to detach the cells from the flask bottom. Non-self pathogen AcMNPV induced the cells to apoptosis. The cell line was proved to be an epidermal cell line based on its unique gene expression pattern. It responded to 20E and the non-steroidal ecdysone agonist RH-2485. Its gene expression could be knocked down using RNA interference. Various genes in the cell line were investigated based on their response to 20E. This new cell line represents a platform for investigating the 20E signaling transduction pathway, the immune response mechanism in lepidopteran epidermis and interactions of the genes.     

Establishment and characterization of a cell line from the American opossum (Didelphys virginiana)

A permanent tissue-cultured cell line (designated OK) has been established from kidney tissue of an adult American opossum. The OK line has been characterized with respect to morphology, chromosome constitution, tissue-culture requirements, and attainable mitotic arrest. The cells are epithelial-like with a stable nondiploid chromosomal modal number of 23. Cells grown in Eagle's minimal essential medium with 10% fetal bovine serum have a mean doubling time of 18 hr. The cell line OK is potentially useful for the isolation and purification of the mammalian X chromosome because of the size differential between the smaller X's and the larger autosomes.     

Spontaneous transformation of a cloned cell line of normal diploid bovine vascular endothelial cells

Summary We report here the spontaneous transformation of a normal diploid bovine fetal aortic endothelial cell line. This cell line showed a period of rapid proliferation, followed by a period of declining proliferative activity, as judged by both a decline in the number of population doublings achieved from seeding to subcultivation and a decrease in the fraction of cells incorporating [³H]thymidine. During the decline in proliferation, foci of small cells appeared amid a background of larger senescent-appearing cells. The cultures then regained proliferative activity and have been maintained in our laboratory for more than 22 months. The transformants are characterized by (a) an indefinite life span, (b) a morphology that is more spindle shaped as compared to pretransformants, and (c) heteroploidy with chromosome translocations. This work was supported by the U.S. National Institute of Health (NIH) Grant AG-00378. S. D. G. is a predoctoral trainee supported by U.S. Public Health Service Grant CA-09191-06, E. H. is supported under NIH Grant AG0-2100, and W. W. N. is the S. Emlen Stokes Professor of Genetics at the Institute for Medical Research.