Search PubMed⌕ Search

Biomedical subjects

H Rubin

Publications and source records attributed to H Rubin.

At least 163 records · Page 9Linked to original sources
PubMed1986

Development and inheritance of osmotic tolerance in a line of spontaneously transformed BALB/3T3 cells.

An in vitro line of transformed BALB/3T3 mouse fibroblasts was exposed to a 100 mM increase in the NaCl concentration of its growth medium. The rate of growth, as measured by the incorporation of tritiated thymidine, decreased almost 100-fold during the first 24 h of exposure to the hyperosmotic medium and then increased approximately 10-fold during the second 24 h of exposure. Cell counts of cultures passaged in medium with excess NaCl revealed a gradual increase in growth rate over a period of several weeks. The early kinetics by which salt tolerance developed in this cell line indicated a process of physiological adaptation rather than selection of preexisting variants in the control population. Clonally derived populations exposed to excess NaCl all showed a similar response. Cultures which tolerated a 100 mM increase in NaCl also grew well in medium containing 200 mM sucrose, indicating that their tolerance was not specific for NaCl. Although the initial response of cultures exposed to excess NaCl appeared to be one of physiological adaptation, tolerance for salt became hereditary after continued passage in hyperosmotic medium. Cultures that were returned to control conditions after prolonged exposure to excess NaCl inherited a high level of tolerance for salt which persisted for several hundred generations without selection.

Animals↗
PubMed1986

Dynamics of tumor growth and cellular adaptation after inoculation into nude mice of varying numbers of transformed 3T3 cells and of readaptation to culture of the tumor cells.

Decimal dilutions containing 5 X 10(5) to 5 X 10(1) cells were inoculated s.c. into nude mice and the course of tumor development was recorded. The highest concentration of cells produced rapidly growing poorly differentiated sarcomas within 2-3 weeks of their inoculation. Upon explantation the resultant tumors yielded cells which multiplied on plastic almost as rapidly as did their progenitors used to initiate the tumors, and had as high a colony forming efficiency in agar as long as tryptose phosphate broth was omitted from the agar medium. Tumors initiated by the lower concentrations of cells were disproportionately delayed in their appearance and tended to increase in size at a low rate. At least one tumor regressed and one which apparently regressed appeared again at a later time. These changes are characteristically described under the rubric of tumor regression. Host reactive cells such as neutrophils, eosinophils, macrophages, and fibroblasts were observed in some tumors. One of the tumors was a low grade hemangiosarcoma, another a well-differentiated fibrosarcoma, and the rest poorly differentiated sarcomas. Cells from two tumors initiated by 500 and 5000 cells multiplied slowly in early passages in culture, particularly when seeded at low densities at which they appeared to sustain cumulative damage even when multiplying. In later passages, the "low dose" tumor cells gained the capacity to multiply in culture after seeding at low densities, but it took up to 50 cell generations to reach this capacity. The loss of growth capacity on plastic of cells from the low dose tumors and its subsequent restoration by passaging in culture may provide a quantitative method for analyzing the type of cellular change which underlies tumor progression.

Adaptation, Physiological↗
PubMed1985

Evidence that intracellular magnesium is present in cells at a regulatory concentration for protein synthesis.

When extracellular magnesium is reduced by a factor of 50 (from 1.0 to 0.02 mM), the total intracellular magnesium of a spontaneously transformed clone of 3T3 cells decreases by 30-50%. Protein synthesis rates in these cells were measured as the intracellular magnesium decreased. Protein synthesis rates and magnesium content were found to decrease in parallel with each other. At 3 hr, a decrease to 84% of control values of magnesium content was accompanied by a decrease to 85% of control values of leucine incorporation rates. A larger inhibition had occurred by 12 hr, when the magnesium had decreased to 67% and leucine incorporation rates had decreased to 57%. When magnesium was restored to magnesium-deprived cells, both magnesium content and leucine incorporation increased about 2-fold by 1 hr. In the experiments reported here, initial small changes in magnesium content are associated with changes in protein synthesis rates. This strongly suggests that magnesium is present at a regulatory rather than excess concentration for protein synthesis. The results are consistent with a role for intracellular magnesium in the regulation of protein synthesis and support the hypothesis that magnesium has a central role in the regulation of metabolism and growth.

Adenosine Triphosphate↗
PubMed1985

Short-term fluctuations and long-term trends in anchorage-independent multiplication among cryopreserved subpopulations of a spontaneously transformed Balb/3T3 clone.

A spontaneously transformed clone of Balb/3T3 cells which produced primitive pleomorphic sarcomas in nude mice was maintained in serial passage without cryopreservation. It was also cryopreserved at an early passage and thawed out at four successive intervals. The capacity of these 5 subpopulations to form colonies while suspended in agar (CFEag) was assayed repeatedly over a period of almost 3 years. The initial CFEag and the subsequent pattern of change were unique for each of the subpopulations. When later passages of the nonfrozen subpopulation were cryopreserved and then thawed, there was only a slight change in CFEag and colony size in agar. There were differences in multiplication rates of 3 of the subpopulations on plastic surfaces in the same rank order as their CFEag, but the differences were smaller. Short-term fluctuations in CFEag, however, were not paralleled by changes in multiplication rate on plastic. The nonfrozen subpopulation assumed the appearance of nontransformed fibroblasts when crowded for several days on plastic, and these "normalized" cells had a greatly reduced CFEag when assayed in agar. Neither the change in appearance nor the decrease in CFEag with prolonged incubation of crowded cells occurred with one of the cryopreserved subpopulations which had the same CFEag and growth rate on plastic as the nonfrozen subpopulation under optimal conditions. The evidence favors an epigenetic rather than a genetic origin of the variations in appearance and behavior of the subpopulations.

Animals↗
PubMed1985

Variation in agar growth of transformed 3T3 cells after tumor formation in nude mice.

Cells from a cloned line of spontaneously transformed 3T3 cells had a colony-forming efficiency in agar (CFEag) of about 10-15% and induced poorly differentiated sarcomas when injected into nude mice. These tumor cells were recultured in vitro and tested for their ability to grow in suspension. Initially, the tumor cells had a CFEag which was a hundredfold to a thousandfold lower than the cells that had been grown only in vitro. After 5-8 further weekly passages, however, the tumor lines recovered their original ability to grow in agar. For determination as to whether this increased CFEag was due to selection of cells with a higher CFEag from the tumor cell population or to adaptation of many of the tumor cells to agar growth, clones were isolated directly from a primary tumor, and each was tested weekly for agar colony formation. All of the tumor clones, as well as the uncloned tumor line, were able to recover their original ability to grow in agar. However, one tumor clone had a relatively high CFEag in the first assay, so the selection hypothesis could not be totally excluded. The initial low CFEag of the recultured cells was not due to the presence of normal nude mouse cells in the population. Before in vivo growth no clones could be isolated from the sublines that had as low a CFEag as the tumor cells isolated after in vivo growth. Tumor cells that had been repeatedly passaged in vivo still had a much lower CFEag than the input cells upon explantation into culture. The results suggest that phenotypic alterations observed during tumor growth and subsequent cultivation have an epigenetic basis.

Agar↗
PubMed1985

Inheritance of acquired changes in growth capacity of spontaneously transformed BALB/3T3 cells propagated in mice and in culture.

Five subclones were derived from a spontaneously transformed BALB/3T3 clone soon after its isolation. Despite their common clonal origin, the subclones were different from each other in appearance, colony-forming efficiency in agar (CFEag), and rate of tumor formation in mice. A comparison of growth properties during repeated passages in culture was made between the cells derived from the tumors and the cells used to initiate the tumors. In most cases, the tumor-derived cells had a much lower CFEag than did their parental in vitro-propagated cells, and the CFEag was restored slowly to the original level or remained at a reduced level during the period of study. In a few cases, the tumor-derived cells had almost as high a CFEag as their parental cells or were quickly restored to this level during cultivation. It was shown by karyotypic and clonal analysis that the reduced CFEag of the tumor-derived cells arose from a change in the transformed cells; i.e., it was not due to the presence of normal host cells in the explanted tumor. Clones of the tumor-derived cells tended to show the same patterns of change in CFEag as the uncloned tumor cell populations, but there were cases of individual variation in pattern among the tumor cell clones. Tumor cells with greatly reduced CFEag also grew a little more slowly on plastic than did the parental nontumor cells, and their growth rate tended to increase along with CFEag in long-term culture. Clonal analysis of one of the five original subclones failed to reveal cells which had the CFEag properties of its progeny tumor cells. This suggests that the reduction of CFEag during tumor formation arose by adaptation rather than selection of preexisting variants. A similar conclusion was drawn about the restoration of CFEag during cultivation of the tumor-derived cells. Although the decrease in CFEag which accompanied tumor formation varied in magnitude and stability, some tumor cell populations retained their reduced capacity through months of weekly passaging in culture, involving up to 100 cell divisions. The results are therefore consistent with the heretical notion of inheritance of acquired characteristics. In addition, the wide variation of in vitro growth capacity among tumors initiated by different subclones, and even among tumors initiated by the same subclone, raises the possibility that the complete chain of causality underlying the variation is intrinsically indeterminate.

Animals↗
PubMed1984

Solute concentration effects on the expression of cellular heterogeneity of anchorage-independent growth among spontaneously transformed BALB/c3T3 cells.

Clones were derived in culture from a tumor initiated by spontaneously transformed 3T3 cells and tested for their colony-forming efficiency in agar (CFEag). Incubation of petri dish cultures was done in subsaturation humidity to minimize mold contamination. There was great variation in CFEag between clones but also, under certain conditions, within clones. The most prominent condition that generated phenotypic diversity in CFEag was partial evaporation of the medium, which may occur during the protracted development of a mass population from a single cell. Evaporation was disproportionately great in 35-mm dishes and peripheral wells of multiwell plates. If the supraphysiological solute concentration resulting from evaporation was greater than 133% of normal, there was progressive suppression of cell growth in the succeeding transfer in agar or on plastic, even if isotonic medium was substituted 1 d before transfer. The effect of supraphysiological concentrations of all the solutes of the medium could be reproduced by simply increasing the NaCl concentration. Damaged cells were restored to their full growth potential after 3 d in isotonic medium. When nontransformed cells were chronically exposed to increased salt, irreversible increases in 2-deoxyglucose uptake were produced. With continued exposure of these cells to high salt, they became morphologically transformed, produced colonies in agar with high efficiency, and formed sarcomas when inoculated into nude mice.

Animals↗
PubMed1984

Early origin and pervasiveness of cellular heterogeneity in some malignant transformations.

Nontransformed BALB/3T3 cells were passaged weekly in monolayer culture on plastic dishes and aliquots were regularly assayed for colony production when suspended in agar. During several months of passaging, a single large colony arose once in the agar assay, and its constituent cells were isolated to form a subline of transformed cells. These cells had the fusiform and rounded morphologies characteristic of transformed cells and had a colony-forming efficiency in agar (CFEag) of approximately 10%. Five of the agar colonies were isolated at random and the cell populations of these primary subclones were further analyzed. Four of the five subclonal populations differed from each other in appearance slightly, but consistently, while the fifth had a markedly different colonial morphology. They also differed from one another in CFEag as well as in average diameter of the agar colonies. These general differences among the five subclones remained, although the CFEag and colony sizes changed recognizably in repeated weekly transfers. All of the subclones produced sarcomas in nude mice but did so at different rates. A secondary generation of five subclones was derived from each of three of the original subclonal populations. All the secondary subclones had the same morphology as the primary subclones from which they were derived. The averages of the CFEag and colony sizes of the secondary subclones from each of the subclones differed as a group from the other groups, but they also differed, to a lesser extent, among themselves. Despite the differences noted among the primary subclones in morphology, growth in agar, and tumor production in mice, they were virtually identical in their rapid growth rate on a plastic surface and all shared a high rate of glucose consumption. A second transformed clone arose among the continuously passaged nontransformed cells 5 months after the first one had appeared. Subclones of this clone did not differ recognizably from one another in morphology or in CFEag. The results indicate that recognizable heterogeneity can arise in some tumors during the earliest stages of their development and involve a high proportion of their constituent cells, while it may not become evident in other tumors until much later.

Animals↗
PubMed1984

Glycosylated fetal hemoglobin. Correlation with hyperglycemia and birth weight in infants of diabetic mothers.

Glycosylated fetal hemoglobin levels were measured in umbilical cord blood of normal infants and infants of diabetic mothers. The glycosylated fraction proved to be a stable compound; its level remained unchanged over a 19-day period. Exposure of fetal and adult hemoglobins to the same concentrations of glucose in vitro resulted in similar levels of glycosylated hemoglobins, suggesting that both types of hemoglobin are about equally reactive with glucose. Levels of glycosylated hemoglobin were significantly increased above normal in umbilical cord blood of infants of both Class A and Class B diabetic mothers. A significant relationship was found between macrosomia, reflected in birth weight ratios, and glycosylated hemoglobin from fetal erythrocytes in infants of diabetic mothers. While these data are consistent with the conclusion that glycosylated fetal hemoglobin levels are a function of fetal blood glucose concentrations in utero during the 2 mo before delivery, it is not known whether the glycosylated hemoglobin contributes to the abnormalities other than macrosomia found in infants of diabetic mothers.

Birth Weight↗
PubMed1984

Mg2+ mitigates Ca2+-dependent cell killing by ionophore A23187.

The Ca2+, Mg2+ ionophore A23187 kills cultured cells in a manner which is dependent upon millimolar concentrations of Ca2+ (Schanne et al., 1979). The killing is thought to be caused by an increase in the cellular Ca2+ content following exposure of cells to the ionophore. We have found that the Ca2+-dependent killing of Balb/c 3T3 cells by ionophore A23187 is reversed by raising, and potentiated by lowering, the extracellular Mg2+ concentration. Ionophore treatment (5 micrograms/ml) causes a decrease in Ca2+ content within minutes, possibly by raising the intracellular concentration of free Ca2+ and thus stimulating its efflux. The response of Ca2+ content to ionophore concentration is biphasic, with low doses causing a decrease and high doses an increase. The sparing effect of Mg2+ on Ca2+-dependent killing of cells by the ionophore suggests that the ionophore kills cells by increasing the cytoplasmic concentration of free Ca2+, thereby creating a competition between Ca2+ and Mg2+ for certain Mg2+-requiring reactions needed for cell survival.

Animals↗
PubMed1984

High-frequency variation and population drift in a newly transformed clone of BALB/3T3 cells.

During repeated passage of BALB/3T3 cells and testing for anchorage-independent growth, a single transformed clone was isolated from agar, and five subclones were derived from it. These subclones differed from one another in morphology on a solid substratum, efficiency and size of colony formation in agar, and rate of tumor formation in nude mice. With weekly passage over a period of 6 months, the differences in morphology and growth in agar gradually decreased. The subclone which produced the fastest-growing tumors in nude mice after 4 weeks of culture produced the slowest-growing tumors after 18 weeks, and a change in the opposite direction was made by another subclone. There was no difference among the subclones in growth rate on plastic. The distribution of chromosome numbers was heterogeneous but overlapping in all the primary subclones at 16 and 24 weeks, with no statistically significant difference in the mean number of chromosomes per subclone. An extremely high degree of variation must have occurred to produce the multiple differences between the subclones, and the same type of variation could have been responsible for the subsequent changes with repeated passage. The high frequency and graded nature of the changes and the concurrent involvement of several traits suggest an epigenetic basis for the variation.

Animals↗
PubMed1984

Variation in capacity for anchorage-independent growth among agar-derived clones of spontaneously transformed BALB/3T3 cells.

A subline of cloned spontaneously transformed BALB/3T3 cells had a colony-forming efficiency (CFE) in agar of 5 to 20%. Individual agar colonies isolated and reseeded into agar were not significantly more efficient at initiating colonies than the original unselected subline. Four successive cycles of agar growth and selection also failed to increase the mean CFE in agar. Randomly selected clones isolated on a plastic surface all had the capacity to grow in agar. These results suggest that the failure of the majority of the cells to grow in agar is not the result of an intrinsic or heritable inability to do so. The ability to initiate a colony in agar seems to vary phenotypically from cell to cell. In contrast, agar colonies isolated from some tumor cell lines (originating from related spontaneously transformed 3T3 cells) and reseeded in agar had a higher CFE than the unselected tumor cell lines. In one case, this increased CFE in agar was lost when the cells were passaged on plastic without further selection for agar growth. Thus, expression of the anchorage-independent phenotype may vary, even among related cloned populations of transformed cells.

Agar↗
PubMed1984

Adaptive changes in spontaneously transformed Balb/3T3 cells during tumor formation and subsequent cultivation.

Cells from a clone of Balb/3T3 cells, which underwent spontaneous morphologic transformation and consistently had a high colony-forming efficiency when suspended in agar [colony-forming efficiency in agar (CFEag)], also formed primitive, pleomorphic sarcomas when inoculated into N:NIH(S)II-nu/nu mice. Cells cultured from 1 of the tumors had only 10% of the CFEag of those used to initiate the tumor but drifted back to the original high level during the first 2 months in culture. Twelve clones of the tumor cells differed widely from one another in CFEag in each of seven successive assays over a 10-week period, although the difference was most pronounced in the first assay. The CFEag for some of the clones fluctuated greatly in successive assays. Two of the clones usually scored lower than the others, and their average CFEag for the seven assays was significantly lower than that of the others. The ability of cells from a second tumor to form colonies in agar also increased with successive passaging. All clones from the second tumor increased in CFEag to a similar extent as the parental population in the same time period, suggesting the increase was the result of adaptation rather than selection of preexisting variants. A tumor produced from a different subpopulation of transformed cells yielded cells with the same CFEag as the input cells. Thus some populations of transformed cells change some of their characteristics when passaged in animals and again when passaged in culture.

Animals↗
PubMed1983

Understanding cancer.

Explore the source record for details and available documents.

Cell Transformation, Neoplastic↗
PubMed1983

Growth stimulatory precipitates of Ca2+ and pyrophosphate.

Inorganic pyrophosphate (PPi) forms an insoluble precipitate with calcium in growth medium when its concentration exceeds about 0.1 mM. This PPi precipitate can reproduce the effects of 10% calf serum on all cell processes examined in Balb/c 3T3 cells, including hexose uptake and metabolism to lactate, 3H-uridine, and 3H-choline uptake, and the incorporation of 3H-leucine and 3H-thymidine into trichloroacetic acid (TCA)-insoluble material. Concentrations of PPi insufficient to form a precipitate are without effect on cell metabolism. The precipitates are most effective when prepared with concentrations of PPi just sufficient to result in precipitate formation and become considerably less effective as the PPi concentration increases, even though the quantity of precipitate formed continues to increase with PPi concentration up to 1 mM PPi. Precipitates formed at low PPi concentrations consist largely of Ca2+ (81% of cations), PPi (77% of anions), and Pi (23% of anions). Precipitates formed with higher concentrations of PPi contain proportionately less Ca2+ and Pi and more monovalent cations and PPi. We have distinguished cell surface-bound PPi from intracellular PPi by differential extraction. The quantity of surface-bound PPi increases sharply when the PPi concentration reaches the point of precipitate formation. If the precipitate is prevented from binding to the cell surface by inverting monolayer cultures in precipitate-containing medium, the cells are not stimulated. These findings suggest that the binding of PPi precipitate to the cell surface is involved in the stimulation of cell metabolism by PPi. PPi precipitates do not absorb serum mitogens or inhibitors from the culture medium, nor do they affect the binding of 125I-platelet-derived growth factor to its specific cell-surface receptor, suggesting that PPi precipitates do not act directly through either of these mitogen-receptor systems. In analogy to cell stimulation by epidermal growth factor and by antigens, we suggest that PPi may be active only in the form of a precipitate because multivalent binding of receptors with formation of clusters is required for stimulation. The inhibitory effects of high concentrations of PPi may be due to interference by free PPi with formation of active receptor clusters.

Animals↗
PubMed1983

Colony morphology and heritability of anchorage-independent growth among spontaneously transformed Balb/3T3 cells.

A clone of spontaneously transformed Balb/3T3 cells produced four different types of colonies in agar. Flat colonies arose at the interface between bottom and top layers of agar, two types of somewhat flattened compact colonies arose within the top layer, and mixed colonies developed when the compact colonies contacted the interface. The proportions of different colony types could be altered by varying the volume, concentration, and gelling rate of the agar. Spherical colonies only appeared in ungelled medium. None of the colony types bred true, showing that their morphologies depended on local conditions. When cells from a subpopulation with low colony-forming efficiency in agar (CFE) were cloned on plastic, all clones formed colonies in agar and did so with about the same CFE as the parental population. Colonies isolated from agar had no higher CFE than the parental population. The results indicate a great phenotypic heterogeneity in anchorage-independent growth, even in cloned populations of transformed cells.

Agar↗
PubMed1983

Heritable variations in growth potential and morphology within a clone of Balb/3T3 cells and their relation to tumor formation.

A nontransformed clone and a spontaneously transformed clone were isolated from a twice-recloned line of Balb/3T3 cells. At different times two sublines were initiated from the nontransformed clone, and three were initiated from the transformed clone. The sublines were maintained in parallel passages under the same conditions. Each subline was distinctive in appearance and fell into the same rank order in a variety of growth parameters in vitro. Colony formation in agar and tumor formation in mice occurred only in the morphologically transformed sublines, but there was no quantitative correlation between the two properties or with the rate of glucose utilization. Two of the cell populations derived from noninbred NIH nude mouse tumors of the 3 transformed sublines differed in agar colony formation from the parental sublines. The results indicate that there is an immense capacity for variation in cultured animal cells involving many unrelated characteristics expressed in a way that is difficult, if not impossible, to explain by conventional genetic models.

Agar↗