Paul J. Higgins

Tissue culture of human epithelial cells from benign colonic tumors

SummaryHuman colonic epithelial cells from three classes of benign tumors have been reproducibly cultured free of fibroblasts for 8 wk using a supplemented Medium 199 (M 199S). The cultured colonic cells were identified as epithelial by the presence of junctional complexes (tight junctions, gap junctions, and desmosomes), a brush border on the apical surface, keratin fibrils, and by both a close-packed columnar or cuboidal morphology and the capability to transport water and ions to form hemicysts. Colony formation was initiated by groups of epithelial cells, not by single cells, and was inhibited by cocultivation with either lethally irradiated 3T3 cells or human diploid fibroblasts. Enhancement of epithelial colony formation was observed following culture on nonadherent, “floating” substrates compared with substrates attached directly to the bottom of the culture dish.Replication of epithelial cells in M 199S from the class of benign colonic tumors least prone to malignancy, the tubular, was significantly enhanced by epidermal growth factor (EGF). In contrast, EGF did not stimulate the growth of cells in M 199S from the other classes of benign tumors, the villotubular and the villous, which exhibit more malignant potential. These data imply that premalignant colonic epithelial cells lose responsiveness to growth modulation by EGF as they progress toward frank carcinoma.

Enhanced albumin production by malignantly transformed hepatocytes during in vitro exposure to dimethylsulfoxide

The murine BW 1 and rat 32III 6/d tumor cell lines, derived from a spontaneous mouse hepatoma and a carcinogen-induced rat hepatocellular carcinoma, were used to investigate the effect of dimethylsulfoxide (DMSO) on liver cells in vitro. After a 4-day exposure to DMSO in final concentrations of 0.5 and 1.0%, BW 1 cell-associated albumin increased by 41.6 and 94.2%; extracellular albumin levels in these same cultures rose by 131.4 and 214.2%. Exposure of 32III 6/d cells to 2% DMSO produced increases in cell-associated and extra-cellular albumin concentrations of 67.8 and 188.7%, respectively. The lack of inducible gamma-glutamyl transpeptidase in BW 1 cells and its decrease in 32III 6/d cultures following DMSO treatment suggests that the DMSO-mediated enhancement of albumin production is not reflective of a random increase in the expression of cellular genes.

Information Transfer and Sperm Uptake by Mammalian Somatic Cells

Publisher Summary This chapter describes that sperm are endowed by their specific differentiation to interact with and to deliver their genetic components into ova for subsequent activation and function. Whether analogous genetic information can be transferred by sperm to mammalian somatic cells is unknown. The chapter describes that mammalian sperm interact with mammalian somatic cells as evidenced by actual penetration, the appearance of changes in morphology and growth characteristics, and the production of new gene products. These changes may be consequences of the direct acquisition and genetic expression of either the sperm chromatin itself or the reverse transcriptase containing complex within the sperm heads. The interaction of sperm with somatic cells that leads to fetal antigen expression and to changes resembling oncogenic transformation may involve a recapitulation, at least in part, of the program of postfertilization gene expression. The normal function of sperm is fertilization; the presence of its various components in somatic cells might very well induce changes analogous to those that occur in embryological and fetal development. It has been estimated that the amount of unique-sequence DNA that is transcribed in the rabbit blastocyst but no longer in the midgestation embryo is enough to specify some 23,000 different proteins; the corresponding difference between normal and virus-transformed mouse cells is regarded as sufficient to specify some 200,000 different proteins. To sort out the many steps involved in embryogenesis, fetal development, and oncogenesis in the face of such complexity is indeed a prodigious task.

Alterations in growth rate and cell cycle kinetics of rat liver tumor cells cultured in ethanol-containing medium: In vitro model of proliferative restriction in response to ethanol exposure

Mechanisms related to the growth suppressive effect of acute ethanol exposure on liver cells were investigated using an established line of ethanol-sensitive rat hepatic tumor cells (32IIIA) and recently developed cytochemical methods for analysis of hepatocyte cell cycle kinetics. Exposure of exponentially growing 32IIIA cells to ethyl alcohol (range 10-100 mM in the growth medium) for a period of 3 days resulted in concentration-dependent decreases (4-25%) in final population density and increases (18-35%) in mean population doubling time compared to untreated cells. Viability was unaffected by ethanol exposure in the concentrations indicated and for the duration period utilized, approximating 94% under all experimental conditions. Multiparametric flow cytometric analysis revealed significant ethanol-associated differences in specific growth parameters and growth state compartments of 32IIIA hepatic tumor cell populations. Most prominent was an ethanol-associated and concentration-dependent (a) increase in the fraction of cells in the G1 phase of the cell cycle, (b) increase in the coefficient of variation in the G1 DNA content measurement, and (c) accumulation (in the G1 phase) of cells with a very low mean RNA content. Increases in each of these cytochemically-defined parameters reflected increasing levels of ethanol in the growth medium. This study indicates that the effects of ethanol on cultured cells of hepatic origin are quite complex. It is concluded that the inhibition of proliferation observed during acute ethanol exposure of liver-derived 32IIIA cells in vitro is due to an accumulation of cells in the G1 compartment.

Incubation of Rat Hepatic Tumor Cells with Ethanol and Acetaldehyde in vitro: Effects on Growth Rate, Albumin Secretion and Cellular Protein Content

The in vitro response (defined as changes in growth rate, cellular protein content, and albumin secretion) of liver epithelial cells to the putative hepatotoxins ethanol and acetaldehyde were evaluated using the well-characterized 32IIIA 6/2d rat liver tumor clonal cell line. Exposure of hepatic tumor cells to ethanol (50-100 mM) for a period of 3 days reduced final population density (apparently due to a drug-induced increase in mean cell cycle transit time), reduced secretion of albumin, and increased the mean cellular protein content. Since these ethanol-associated effects were also observed in cells cultured in growth medium containing acetaldehyde (0.1 mM), and were inhibited by simultaneous addition of pyrazole, the changes in the parameters measured in this study appear to be induced by products of ethanol metabolism. These data complement recent in vivo studies implicating acetaldehyde as an inhibitor of hepatocyte secretory function. The 32IIIA 6/2d liver cell system, thus, responds to certain hepatotoxic compounds in a manner analogous to the in vivo organ and may facilitate future analysis of molecular mechanisms underlying alcohol-induced liver disease under defined culture conditions.

Secretion of albumin and alpha-foetoprotein by dimethylsulphoxide-stimulated hepatocellular carcinoma cells.

Exposure of BW77-1 and BW77-2 mouse hepatic tumour cells to the polar solvent dimethylsulphoxide (DMSO) altered extracellular accumulation of albumin and alpha-foetoprotein (AFP) and perturbed their cell cycle kinetics. The amount of albumin secreted into the culture growth medium was dependent on the concentration of DMSO used. Hepatic tumour cells cultured in 1 and 2% DMSO accumulated 50% and 111% more albumin, respectively, than non-DMSO-stimulated cells during the final 24 h of a 4-day exposure to the polar solvent. Commitment of mouse hepatoma cells to increased albumin secretion was temporally dependent, requiring a minimum of 48 h in the presence of DMSO. The AFP level in 1% DMSO-treated cultures was also significantly increased, compared with control cells. Unlike albumin secretion, however, exposure of hepatic tumour cells to 2% DMSO did not further increase (but slightly decreased) extracellular AFP accumulation. Treatment of BW77-1 cells with DMSO resulted in a gradual decline in the percentage of 2C DNA content cells (diploid G1 population) and in a corresponding increase in the proportion of cells with a 4C DNA content (generation of either a G2 or tetraploid G1 population). The extent of this shift directly reflected the concentration of polar solvent in the medium and paralleled the DMSO-induced stimulation in albumin secretion. DMSO-stimulated hepatic tumour cells, therefore, may prove useful in the elucidation of specific regulatory events underlying control of gene expression during the hepatocyte cell cycle.

The effect of methadone and naloxone on cultured rat liver cells

Opioids modify hepatic function in vivo, however, a direct effect of this class of drugs on liver cells has not been demonstrated. The potential effect of opioids on liver parenchymal-type cells, therefore, was studied using cultured albumin-secreting rat hepatoma cells. Liver cells were incubated with methadone or naloxone individually or in combination. Dose-response analysis indicated that concentrations of either or both drugs in excess of 2.5 X 10(-5) M were clearly cytotoxic to cultured hepatoma cells. At a concentration (2.5 X 10(-7) M) at which no adverse effect on hepatoma cell viability or growth rate was observed, naloxone, methadone, or naloxone plus methadone produced a 26, 41, and 63% decrease in trichloroacetic acid-precipitable protein (per 10(6) cells), respectively, when compared to control. The inhibitory effect of naloxone, methadone, or naloxone plus methadone on albumin accumulation in the culture medium was greater than the drug-induced reduction of protein synthesis. These studies demonstrate that opioids have several direct effects on cultured hepatoma cells and, therefore, raise the question of whether clinically significant hepatic dysfunction may be produced by the direct action of opioids on the liver.

Differential association of fetal antigen with hepatoma tissue grown in vivo and in vitro.

Abstract Production of two distinct fetal antigens, alpha-fetoprotein (AFP) and γ-fetal antigen (γ-FA) was associated with growth of the BW7756 mouse hepatoma in vivo . Synthesis of AFP, but not γ-FA, continued during in vitro propagation of the tumor cells. After re-inoculation of these cultured hepatoma cells into inbred mice, both AFP and γ-FA could be detected again in the growing tumor tissue and in the sera of tumor-bearing mice. It is evident that different growth conditions effect synthesis of these two tumor-associated antigens.

Dimethylsulfoxide-induced alterations in the growth properties and protein composition of in vitro-propagated murine hepatoma cells

The effects of the differentiation-inducing agent dimethylsulfoxide (DMSO) on the growth properties and protein synthetic capacity of BW77-1 mouse hepatoma cells were compared at various media concentrations of the polar solvent. DMSO produced a dose-dependent reduction in final population density, reduced or eliminated cell piling and stimulated synthesis of albumin. The rising albumin content reflected dose-related DMSO-induced increases in total cellular protein and in the albumin contribution to total cellular protein. In order to determine whether viral gene expression was associated with DMSO-induced stimulation of albumin synthesis, BW77-1 cultures were examined for the production of ecotropic and xenotropic type C virus. The BW77-1 hepatic tumor cell line was determined to be a nonproducer of type C virus by assays designed to measure extracellular reverse transcriptase, viral env gene product and infectivity on mouse and mink indicator cells. Type C virus could not be induced in BW77-1 cultures by treatment with DMSO under conditions which lead to a reduced proliferative capacity and enhanced expression liver-specific genes.

Cell cycle compartments of adult mouse hepatocytes identified by flow cytometric analysis of total cellular and nuclear RNA content: Effect of aging on G1 substates

A model of the hepatocyte cell cycle was computer-constructed using two-parameter cytochemical data obtained by flow cytometric measurements of the RNA and DNA content of individual liver cells (or isolated liver cell nuclei). In young mice (under 10 weeks post-partum), the diploid (2C DNA) G1 phase was resolved into 3 distinct subpopulations of liver cells distinguishable on the basis of RNA content. These substates were conventionally designated G1Q (quiescent non-cycling cells; lowest RNA content), G1A (cells of the indeterminate phase; do not directly enter the DNA synthetic compartment; intermediate RNA content) and G1B (immediate pre-DNA synthetic cells; highest G1 RNA content). An early G1-associated event during postnatal aging of the mouse liver (pre-10 weeks post-partum) involved a shift from a predominant G1A/B substate to a predominant G1Q substate. Later stages in aging were characterized by a gradual decline in the percentage of G1A/B cells in the 2C G1 compartment. Greater than 80% of the total 2C G1 liver cell population of animals approaching 2 years of age consisted of GIQ hepatocytes. This increase in the quiescent cell population (G1Q) and decrease in the cycling G1 substates (G1A/B) within the mouse liver as a function of age may be involved in the aging-associated diminution of the hepatic compensatory regenerative response in mammals.