Aaron E. Freeman

I. In vitrol transformation of rat embryo cells: correlations with the known tumorigenic activities of chemicals in rodents.

SummarySusceptibility to chemically induced transformation changed as a rat embryo cell culture was passaged. For the first 35 to 60 passages, the cultures were diploid and resistant to transformation by chemical carcinogens. However, cultures infected with a murine leukemia virus were transformed by chemicals. For the next 60 passages, the cultures were heteroploid, but retained contact inhibition and were not tumorigenic. Even without addition of heterotypic viruses, these heteroploid cultures could be transformed by chemicals, but the endogenous rat C-type virus could be demonstrated in the transformed cultures. At higher passages, the rates of spontaneous transformation gradually increased so that the cultures could not be used for transformation studies.Chemically induced transformation of the stable heteroploid cell line (F1706) was manifested by an easy to read focal alteration. Initial observations based on these foci were confirmed by inoculating the morphologically altered cells into isogeneic newborn rats.A number of carcinogenic and noncarcinogenic chemical analogues were tested for their ability to transform F1706 cultures. The compounds tested included 4 azo dyes, 12 polycyclic hydrocarbons, 12 aromatic amines, and 7 miscellaneous compounds. Based on the known activities of the same chemicals in rodents, certain active compounds failed to induce transformation in any test, and others induced transformation in only some tests, but these in vitro tests, if used as a screening assay, would have been correct in 82% of all individual tests, and over-all, would have correctly predicted the carcinogenic activity of 33 of the 35 agents tested.Susceptibility to chemically induced transformation changed as a rat embryo cell culture was passaged. For the first 35 to 60 passages, the cultures were diploid and resistant to transformation by chemical carcinogens. However, cultures infected with a murine leukemia virus were transformed by chemicals. For the next 60 passages, the cultures were heteroploid, but retained contact inhibition and were not tumorigenic. Even without addition of heterotypic viruses, these heteroploid cultures could be transformed by chemicals, but the endogenous rat C-type virus could be demonstrated in the transformed cultures. At higher passages, the rates of spontaneous transformation gradually increased so that the cultures could not be used for transformation studies. Chemically induced transformation of the stable heteroploid cell line (F1706) was manifested by an easy to read focal alteration. Initial observations based on these foci were confirmed by inoculating the morphologically altered cells into isogeneic newborn rats. A number of carcinogenic and noncarcinogenic chemical analogues were tested for their ability to transform F1706 cultures. The compounds tested included 4 azo dyes, 12 polycyclic hydrocarbons, 12 aromatic amines, and 7 miscellaneous compounds. Based on the known activities of the same chemicals in rodents, certain active compounds failed to induce transformation in any test, and others induced transformation in only some tests, but these in vitro tests, if used as a screening assay, would have been correct in 82% of all individual tests, and over-all, would have correctly predicated the carcinogenic activity of 33 of the 35 agents tested.

Calcium sensitivity of cell cultures derived from adenovirus-induced tumors.

Summary Cell lines derived from adeno-virus-induced tumors or adenovirus-trans-formed cell lines clumped or retracted in 7.5 mM calcium or less, a characteristic generally not shown by cells derived from other virus-induced tumors or cells transformed by other viruses. Similarly, standard primary, diploid, and heteroploid cell cultures were not sensitive to 7.5 mM calcium. In support of these observations, the use of a low calcium medium facilitated the passaging of adenovirus-trans-formed cells in tissue culture; however, the use of a completely calcium-free medium resulted in a deficiency which caused detachment of the culture. The calcium effect may be useful as a marker to substantiate other evidence that a tumor was induced by adeno-virus or that a cell line was transformed by adenovirus.

Effects of Laminin, Fibronectin and Type IV Collagen on Liver Cell Cultures

The effects on mouse liver cells of laminin, fibronectin and type IV collagen, all of which are the main matrix of the basement membrane, were studied. Laminin, a glycoprotein isolated from cultures of rat yolk sac carcinoma cells, promoted the attachment of mouse fetal liver cells to laminin-coated dishes, but did not have a strong influence upon the attachment of normal adult liver cells. On the other hand, fibronectin which was purified from mouse plasma promoted the attachment of adult liver cells but not that of fetal liver cells. The number of neonatal liver cells attached to the surfaces coated was intermediate between those of fetal and adult liver cells in each matrix. DNA synthesis and cell proliferation during the culture of full-term fetal liver cells in laminin-coated dishes were higher than those in fibronectin- or type IV collagen-coated dishes. The amount of alpha-fetoprotein secreted in the laminin-coated dishes was more than in other groups. No differences in secretion of albumin into media, however, were observed in either group. These results suggest that laminin may be necessary for cell growth, tissue organization and cell differentiation during the normal development of liver in vivo.

A SIMPLIFIED METHOD FOR THE CLONING OF HETEROPLOID AND DIPLOID MAMMALIAN CELLS.

Summary and conclusions A method which appears a practical and easy way to produce cloned cell lines is presented, with some observations and problems to be considered when cloning. Although the method attempts to derive cell lines from single cells by taking many precautions, it cannot be definitely stated that this goal is actually accomplished. It has yet to be demonstrated satisfactorily that a single diploid cell can in fact be cloned. The method, however, definitely produces cell lines which are derived from such a reduced population that morphological differences cannot be demonstrated within a clone, but can be demonstrated between cells from different clones.

Duct, exocrine, and endocrine components of cultured fetal mouse pancreas

SummaryTwenty to twenty-two days postcoitum mouse fetal pancreas organ bits were cultured on the dermal surface of irradiated pigskin as a substrate. The medium used for long term culture consisted of Eagle’s Minimum Essential Medium with the addition of 10% bovine serum, 0.02 U/ml insulin, 0.025 μg/ml glucagon, 3.63 μg/ml hydrocortisone, 100 μg/ml soybean trypsin inhibitor or 10−8M atropine. When the medium lacked trypsin inhibitor or atropine but contained the three hormones, the pigskin support began to be destroyed after 2 to 4 wk in culture. Thereafter, the cultured cells could not grow and survive on the digested pigskin. When 10−6M atropine was added to the medium, amylase secretion from cultured cells and destruction of pigskin were inhibited completely but pancreas cells could not grow or survive. In contrast, 100 μg/ml soybean trypsin inhibitor or 10−8M atropine permitted cell growth, permitted amylase secretion from the cultured acinar cells, and prevented the destruction of pigskin. Under these conditions pancreas cells migrated or grew or both from the organ bits onto the surface of the pigskin dermis and organoid aggregations formed. Hydrocortisone was needed to permit growth for more than 2 wk. Glucagon and insulin had additive effects. Light and electron microscopic observations indicated the culture of at least five kinds of cells, i.e., duct, acinar, centroacinar, endocrine, and mesenchymal. The majority of cultured cells were duct cells and acinar cells. There were few mesenchymal cells. Mouse pancreas cells were cultured for at least 12 wk by this method.

Transformation of Fischer Rat Embryo Cells by the Combined Action of Murine Leukemia Virus and (-)-Trans- 9-Tetrahydrocannabinol

Summary The cannabinoid, Δ9THC [(-)-trans-Δ9-tetrahydrocannabinol], was found to transform high passage Fischer rat cells chronically infected with Rauscher leukemia virus, but the transformation could be demonstrated only after extended subculture (13 passages) of the cells. Under the same conditions, control cultures and cultures treated with Δ8THC [(-)-trans-Δ8-tetrahydrocannabinol], CBN [cannabinol], or CBD [transcannabinol], were not transformed (20 passages). In contrast, cultures treated with 3MC [3-methylcholanthrene] were transformed in three subpassages. Thus, Δ9THC is the most active of the major cannabinoids as a transforming agent, but this activity is weak as compared to 3MC.

A simple interferon assay as an adjunct for determining the genus of origin of cell cultures

SummaryA short, simple test involving interferon-mediated protection of cells in vitro from cytopathogenic effects produced by vesicular stomatitis virus or Sindbis virus has been developed to help in determining the genus of origin of cells. By using the observed pattern of protection of five cell types by five interferons, cells could be grouped as of primate, rabbit, rat, and mouse origins. The primate grouping resulted from bilateral cross-reactions between the human and monkey systems. An unexpected observation was that African green monkey interferon preparations protect both monkey and rat cells, but not the converse. Chromosomal analysis of the cell cultures confirmed the genus determined by the interferon test.