Warren I. Schaeffer

Terminology Associated with Cell, Tissue and Organ Culture, Molecular Biology and Molecular Genetics

When areas of research become interdisciplinary their jargon, techniques and bodies of information are utilized widely in diverse disciplines. At such times, reciprocal use of terminology by individuals who had not previously used it is often misused and, therefore, confusion occurs. This confusion is especially acute in the field of vertebrate, invertebrate and plant cell culture. There is hardly a field of biological investigation in which culturing of such cells is not employed. Similarly, molecular biology and molecular genetics are lending their technology to an everwidening group of researchers who are communicating in a more global sense via scientific presentations, publications and research proposals. Unfortunately often the writer and reader represent different areas of specialization who have been brought together by the common technology used in their work. As such, misuse of terminology can prove unfortunate indeed; anything from inability to repeat a piece of research to problems in publishing a paper or obtaining funding of a research proposal. The following glossary, approved by the Tissue Culture Association Terminology Committee is published in an effort to increase communication between scientists and between scientists and the lay community.When areas of research become interdisciplinary their jargon, techniques and bodies of information are utilized widely in diverse disciplines. At such times, reciprocal use of terminology by individuals who had not previously used it is often misused and, therefore, confusion occurs. This confusion is especially acute in the field of vertebrate, invertebrate and plant cell culture. There is hardly a field of biological investigation in which culturing of such cells is not employed. Similarly, molecular biology and molecular genetics are lending their technology to an everwidening group of researchers who are communicating in a more global sense via scientific presentations, publications and research proposals. Unfortunately often the writer and reader represent different areas of specialization who have been brought together by the common technology used in their work. As such, misuse of terminology can prove unfortunate indeed; anything from inability to repeat a piece of research to problems in publishing a paper or obtaining funding of a research proposal. The following glossary, approved by the Tissue Culture Association Terminology Committee is published in an effort to increase communication between scientists and between scientists and the lay community.

Establishment and characterization of a bovine mammary epithelial cell line with unique properties.

SummaryClonal cell lines (BME-UV) were established from primary epithelial cells by stable transfection with a plasmid, carrying the sequence of the simian virus 40 early region mutant tsA58, encoding the thermolabile large T antigen. The BME-UV cells have undergone more than 300 population doublings and produce intranuclear large T antigen. At low confluency, growing islands of cells are apparent exhibiting the characteristic cobblestone morphology of epithelial cells. The BME-UV cells expressed functional markers such as microvilli and desmosomes and biochemical markers of mammary epithelial cells such as a repertoire of cytokeratins. The BME-UV cells are capable of synthesizing low levels of α-lactalbumin and α8l (50 ng/ml of medium/24 h). One of the cell lines, BME-UV1 showed enhanced proliferation in the presence of epidermal growth factor (EGF) and insulinlike growth factor I (IGF-I). The BME-UV1 cell line is the only known bovine mammary epithelial cell line responsive to EGF. The BME-UV cells grown on collagen at low confluency are capable of developing very long projections that most likely allow for communication between cells at a distance from each other. The BME-UV cells may become a valid model system to examine bovine mammary epithelial proliferation and differentiation and cell-to-cell communication.

The identity and nuclear uptake of a cytosolic binding protein for 3-methylcholanthrene

Abstract The ability of 3-methylcholanthrene to interact noncovalently with rat liver cytosolic proteins was studied using Sephadex G200 chromatography. A specific 3-methylcholanthrene binding fraction from Sephadex G200 chromatography, termed peak B, when incubated with rat liver nuclei was able to translocate 3-methylcholanthrene into the nucleus. This translocation occurred faster and was quantitatively greater than the binding of 3-methylcholanthrene in buffer to nuclei. In addition, the nuclear uptake of peak B was increased by prewarming, suggesting that a heat-sensitive activation step may occur prior to the translocation process. However, no evidence was found on sucrose gradients for any conformational change in the protein fraction studied here. The translocation to the nucleus was temperature and time dependent. An examination of the characteristics of this 3-methylcholanthrene binding protein using Sephacryl S200 column chromatography showed a small number of high-affinity, saturable, binding sites to be present. These had an apparent dissociation constant, Kd, of 2.8 n m and a binding capacity of 770 fmol/mg of cytosolic protein. The selectivity of this protein was examined by competition studies and, in general, polycyclic hydrocarbons competed for the binding site, except for anthracene and phenanthrene. Of the inducers studied, 5,6-benzoflavone was a strong competitor. No competition was found with 12-O-tetradecanoyl phorbol-13-acetate, 2,6-ditertbutyl-p-cresol, β-retinyl acetate, or a number of steroids, except for 17β-estradiol which exhibited moderate binding. Peak B had a sedimentation coefficient of 4.2 S when analyzed on a linear sucrose gradient. Chromatography of peak B on a calibrated Sephacryl S200 column gave a molecular weight corresponding to 44,600 ± 4000.

A diploid rat liver cell culture

SummaryA method for culturing and cloning a cell line derived from rat hepatic tissue is described. The cloned cells in culture were diploid, epithelioid in morphology, and synthesized tyrosine aminotransferase. The cytotoxic effects of the hepatocarcinogen aflatoxin B1 on these cells were compared to the effects on an established cell culture. The diploid culture was found to be more resistant to the toxin as measured by cell counts and residual protein as well as by [14C]uridine incorporation.

Usage of vertebrate, invertebrate and plant cell, tissue and organ culture terminology

The multidisciplinary use of cell, tissue and organ culture techniques, and the attendant use of terms in an ever widening sense, has made it necessary to bring together those terms most often used by scientists employing these techniques. Thus, the Tissue Culture Association Terminology Committee publishes the following integrated listing in an attempt to avoid the confusion which arises when scientists of different disciplines give more than one definition to a particular term or use more than one term to describe the same phenomenon.

Efficient detection of soft agar grown colonies using a tetrazolium salt

The use of 2-(p-iodophenyl)-3-(p-nitrophenul)-5-phenyl tetrazolium chloride at concentrations of from 0.25-1.0 mg/ml, when added to plates containing soft agar grown colonies, colors the cell clusters a deep brick red against a colorless background. This greatly facilitates the quantitation of such colonies since clusters of cells containing as few as 16-32 cells are visible macroscopically. Incubation for 20 h is optimal although coloration takes place as early as 6-8 h.

A diploid rat liver cell culture. III. Characterization of the heteroploid morphological variants which develop with time in culture.

Abstract A substrain was developed from one of three morphological variants which were isolated from a late passage cloned epithelial rat liver cell strain. The substrain was studied at sequential passages, over time, with respect to clonal morphology, growth, plating efficiency, packed cell volume, saturation density, chromosome number, karyology, ability to grow in soft agar, and degree of malignant transformation. The substrain was determined to be the progenitor of the other two morphological variants and the progression in morphological variance follows the stemline hypothesis. A marker chromosome was found to be a stable component of the genetic constitution of all cells in the substrain. What is observed appears to be population growth dynamics concomitant with a cellular aging phenomenon.

Cytoplasmic suppression of malignancy

SummaryUsing both normal and transformed rat liver epithelial cells to prepare cytoplasmic hybrids (cybrids) we have found evidence to support the theory that the cytoplasm from a normal cell can suppress tumorigenicity. A unique aspect of this study is that all of the cells utilized, both normal and malignantly transformed, were derived from an original cloned cell. We found that fusing cytoplasts from normal cells to malignantly transformed whole cells resulted in cybrid clones which, when injected into newborn rat pups, isogenic with those from which the cell culture was initiated, yielted tumors in 51% of the animals injected compared to 92% of the animals injected with the tumorigenic parent. Those animals that did develop tumors from the cybrid cells survived longer than those injected with cells from the tumorigenic parent. Thus, the cybrid, formed of cytoplasm from both parents, was less tumorigenic than the malignantly transformed parent cell. When reconstituted cells were prepared by fusing cytoplasts from normal cells with karyoplasts from malignantly transformed cells, a situation in which essentially all of the cytoplasm of the reconstituted cell is derived from normal cells, the tumorigenic phenotype was extinguished.

A diploid rat liver cell culture. IV. Malignant transformation by aflatoxin B1.

SummaryChronic exposure of a cloned rat hepatocyte culture (RL-PR-C) to a subtoxic, sublethal dose of aflatoxin B1 resulted in malignant transformation. Continuous exposure to aflatoxin B1 caused increasing tumorigenic potential as tested by back injection into isogenic animals. Control cultures exhibited spontaneous transformation, although approximately 20 passages beyond the chemically induced event. Neither aflatoxin-treated nor control cultures exhibited cytopathological morphology, formation of cell foci, growth in soft agar, or irregular fibroblast-like growth patterns that could be specifically related to the onset of tumorigenic potential. In general, those parameters commonly used to monitor fibroblast cultures for transformation in vitro were not applicable for assessing the tumorigenic potential of these epithelial cells. Karyotypic analyses revealed no specific chromosomal aberrations associated with aflatoxin treatment; however, chromosomal instability was a property of the tumorigenic cell populations. Injection of both aflatoxin-treated and control cultures at passage 56 resulted in tumors indicative of both carcinoma and sarcoma indicating to us the multipotency of these epithelial cells transformed in vitro.

ESTABLISHMENT AND CHARACTERIZATION OF A BOVINE MAMMARY MYOEPITHELIAL CELL LINE

SummaryThe thermolabile large T-antigen, encoded by the simian virus 40 early region mutant tsA58, was used to establish clonal cell lines (BMM-UV) from primary bovine myoepithelial cells. The BMM-UV cells have undergone more than 300 population doublings without any signs of senescence, and they contain the intranuclear large T antigen. At low confluency, they grow in a spindlelike manner and develop very long projections that most likely allow for communication of cells at a distance from each other. Establishment results in a decrease in the number of cells that contract in response to oxytocin compared with the parental nontransfected cells (20% versus 45%). Oxytocin responsiveness of BMM-UV cells increases when the cells are cultured in a medium supplemented with staphylococcal proteases. Proliferation of BMM-UV cells increases when they are cultured in the presence of epidermal growth factor (10 ng/ml) or insulinlike growth factor I (50 ng/ml). The BMM-UV cells may become a useful model to study growth properties, cell-to-cell communication, and the function of bovine mammary myoepithelial cells.