Differentiation of New Metastatic Variants of B16 Melanoma Under Different Culture Conditions

The purpose of this study was to examine the differentiation of variant tumors of the B16 metastatic melanoma when tumors were grown serially under different culture conditions and transplanted into C57BL/6J black mice, lethal yellow A^y/a, albino c/c, and C⁺/c mutant mice. Morphological and biochemical markers of melanogenesis were examined in cells in culture and in the corresponding tumors. Cellular pigmentation was assessed in terms of the levels of DOPA and 5-S-CD and in terms of tyrosinase activity in the various cell lines and tumors. The observed change from high to low metastatic capacity, which was dependent on culture conditions, appeared to be unrelated to melanogenesis even though changes were observed in the biochemical melanotic phenotype. Overall, tumor cells from spontaneous pulmonary metastases appear to differentiate in ways that are unrelated to the instability of experimental metastatic capacity. The melanotic phenotype in albino c/c and C⁺/c mice was dependent on the phenotype of the parental tumors. A marked difference was observed between two pigmentation compartments, one of which was stable in the B16 control, while the other was unstable in YB16 and MB16 variant cells and in the tumors derived from them. It appears, therefore, that the metastatic capacity of B16 metastatic variants is changeable and is independent of the unstable melanogenic behavior. The production of metastases and the differentiation of tumors in the present experiments appeared to be related to the genetic background of the mice and the epigenetic metabolic environment of tumors and cells.     

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

Summary Twenty 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 10⁻⁸ M 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 10⁻⁶ M 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 10⁻⁸ M 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. This investigation was supported by PHS Grant CA 30220 awarded by the National Cancer Institute, DHHS, Grant 1203M awarded by the Council for Tobacco Research, Inc., and Grant RD-65 (for equipment) awarded by the American Cancer Society. Nude mice were provided by Dr. Wendall M. Farrow of Life Sciences, Inc., Resource Laboratory N01, CP6-1005 of the National Cancer Institute.     

Primary cultures and the levels of cytochromeP450 in hepatocytes from mouse,rat, hamster,and rabbit liver

Summary Hepatocyte primary cultures (HPC) derived from rat, mouse, hamster, and rabbit liver were characterized for a variety of parameters. The conditions that maximized recovery, attachment, and survival varied between species. Hepatocytes from all four species were capable of attaching in serum-free Williams’ medium E (WME), but optimal attachment as monolayer cultures was achieved for mouse and hamster HPC in medium receiving 1% calf serum supplementation. Hamster hepatocytes required additional cations, whereas rabbit and rat hepatocytes displayed maximal attachment in medium supplemented with 10% calf serum. Survival of mouse and rabbit hepatocytes after 24 h in serum supplemented media was in the order of 90%. Rat and hamster hepatocyte 24 h survival was approximately 70 and 60%, respectively, and was not significantly affected by serum supplementation. Hepatocytes from each species varied in their content of cytochromeP450 at the time of isolation and in the rate of reduction during culture. Mouse and rat hepatocytes demonstrated the most rapid decline in content during the initial 24 h in culture, whereas concentrations in rabbit hepatocytes were virtually unchanged. The rate of decline inP450 concentrations in hamster hepatocytes was intermediate between those displayed by rat and rabbit hepatocytes. These studies have delineated conditions useful for the culture of hepatocytes from four species and have documented the status of an important parameter of their functional capability. This study was supported by EPA contract 68-01-6179. C. J. Maslansky was a recipient of a Monsanto Fund Fellowship in Toxicology.     

Characterization of isolated Fe-loaded rat hepatocytes prepared by collagenase perfusion

Summary Hepatocytes from livers of rats loaded by Fe-dextran treatment were isolated by an in situ collagenase perfusion technique and evaluated for their biochemical, cytochemical, and morphological characteristics in cell culture. Iron loads 15 times higher than in normal rat liver cells isolated in the same way were retained in the preparations with 40% present as hemosiderin. A simple centrifugation-mathematical approach is described for the calculation of Fe content in the hepatocyte (95%) and reticuloendothelial (5%) fractions in the isolates. The cells were cultured for 22 h without loss of protein synthesis capability or significant changes in cell count, viability, endogenous glutamate-oxaloacetate transaminase (GOT) or Fe and were morphologically similar in most respects to unloaded (normal) hepatocytes similarly cultured. Studies are in progress to assess the utility of these preparations as a model for Fe mobilization from Fe-loaded animals. This work is supported by National Institutes of Health Grants AM 25647-03 (M. Dawson, Principal Investigator) and GM 28158-01 (C. Tyson, Principal Investigator). The technical assistance of Mr. Jack E. Dabbs, Mr. Charles Hart, and Mr. Randy Douglas is acknowledged.     

Rapid Cellular Regulation of D-Glucose Transport in Cultured Neural Cells

Previous studies have revealed two different kinds of regulation of glucose utilization in cell lines derived from the nervous system (Keller et al., 1981). We found glucose metabolism of C-6 glioma cells to be limited and regulated by membrane transport. In contrast, glucose utilization of C-1300 neuroblastoma (N2A) cells was limited by the known regulatory enzymes of the Embden-Meyerhof pathway. Under the given experimental conditions the "membrane-limited" C-6 glioma cells were characterized by periodically changing glucose transport rates and very low intracellular glucose concentrations, which remained constant in spite of widely differing transport rates. These findings suggest the close functional coupling between transport and phosphorylation required for the regulation of glucose transport by cellular metabolic needs.     

Growth effects and metabolism of arginine in stationary and rapidly-dividing sugarcane cell suspensions

The effect of 300 μ M arginine on growth of sugarcane cell suspensions was studied. Cells transferred to defined media in the stationary growth stage showed a greater requirement for exogenous arginine than cells similarly transferred in the rapidly dividing stage. Cell arginine levels, rates of arginine synthesis, and enzymes of arginine synthesis all decreased in cells entering the stationary stage. It is concluded that stationary stage cells are deficient in their ability to synthesize arginine and are therefore dependent upon an exogenous supply to resume growth in fresh media.     

Quinine and quinidine production by cinchona leaf,root and unorganized cultures

Serially propagated Cinchona ledgeriana and C. succirubra (Rubiaceae) leaf, root and unorganized suspension cultures established from germinated seeds were studied for quinine and quinidine production. Leaf organ cultures were grown and subcultured in Murashige and Skoog's Revised Tobacco Medium supplemented with benzyladenine; root organ cultures were grown on the same medium supplemented with indolebutyric acid; and unorganized suspension cultures were grown on the same medium supplemented with 2,4-dichlorophenoxyacetic acid and benzyladenine. On a dry weight basis, leaf organ cultures of C. ledgeriana contained 0.06 % quinine and 0.05 % quinidine and of C. succirubra contained 0.04 % quinine and 0.04 % quinidine. No quinine and quinidine were detected in either root organ or unorganized suspension cultures.     

Differentiation of embryogenic pollen in cold-treated buds ofNicotiana tabacum var. Badischer burley and nutritional requirements of the isolated pollen to form embryos

Summary Embryogenic pollen were selectively isolated from buds after cold treatment at 10 °C for 10 days; it was immaterial whether the buds were taken from short day and low temperature (SD and LT; 8 hours light, 18 °C) or long day and high temperature (LD and HT; 16 hours light, 24 °C) plants. However, in buds from SD and LT plants the differentiation of embryogenic pollen could be detected as early as 7 days after the cold treatment, and pollen from these plants formed embryos at higher frequency (up to 4% of cultured pollen) than those from LD and HT plants (up to 1% only).The embryogenic pollen, in isolated buds, differentiated by way of pollen dimorphism. During cold treatment a fraction of pollen remained small, retained clear cytoplasm and was capable of embryogenesis in comparison to gametophytic pollen which enlarged and acquired granular cytoplasm. In our experiments cold treatment was a key factor in the induction of pollen dimorphism. This aspect of cold treatment in pollen embryogenesis is reported for the first time and was possible on the basis of selection of embryogenic pollen by density gradient centrifugation. The ratio of embryogenic pollen was about one fifth of the total population.The nutritional requirements of isolated pollen for embryogenesis were rather simple. These pollen formed embryos which readily developed into plantlets on a mineral medium supplemented with sucrose provided the pH was 6.8.     

Growth characteristics of human epidermal kerationcytes from newborn foreskin in primary and serial cultures

Summary Using gels of acid-soluble, collagen as a culture surface, trypsin-released keartinocytes from 0.1-mm, split-thickness sections of newborn foreskin may be plated with high efficiency and subcultured at a 1∶5 split a 2- to 3-week intervals for three subpassages. When plated at a density of 3.2×10⁴ cells per cm², keratinocytes attach to the gel with an efficiency of over 70%; after a lag phase of 3 days, the cells multiply exponentially with a doubling time of 60 hr. Cultures reach a growth-plateau phase at a density of 47.7×10⁴ cells per cm². Both hydrocortisone and epidermal growth factor (EGF) stimulate slightly the growth of primary cultures; both factors are required for proliferation of the 2nd and further passage of keratinocytes. As the cultures reach, confluence multilayers, of stratified cells are formed and cells of squamous morphology are spontaneously released from the surface. When the released cells and the attached cells are pulsed with [³H]-histidine and [¹⁴C]-leucine, a higher ratio of histidine to leucine is observed in the released cells indicating the biochemical onset of maturation. Orange G-Aniline Blue staining of the released cells show some of the cells to be completely keratinized. Fibrous proteins extracted from the cultured cells and analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis display the characteristic stratum corneum proteins of 60,000 and 66,000 daltons. Supported in part by Grants AM 14121 and AM 19595 of the U.S. Public Health Service.     

Improved basal medium for Y-1 mouse adrenal cortex tumor cells in culture

Summary An improved basal medium is presented that requires only minimal supplementation with dialyzed fetal bovine serum or bovine serum albumin and fetuin to be comparable to Ham's F-10, which requires 15% horse serum (HS) and 2.5% fetal bovine serum (FBS) for the growth and function of Y-1, mouse adrenal cortex tumor, cells. Cell monolayers maintained for up to 2 weeks without any protein supplementation have retained their steroid response to ACTH. The medium differs from Ham's F-10 in its buffer composition and higher calcium-ion concentration. This medium should be a useful adjunct to studies pertaining to steroid and lipid intermediary metabolism, the retention of a specialized physiological function in a chemically defined medium, and the mechanism of hormonal response. Supported by the Medical Research Service of the Veterans Administration.