Properties of the H-4-II-E tumor cell system. I. Growth and cell proliferation kinetics of an experimental hepatoma.

Growth and cell proliferation kinetics of hepatoma H-4-II-E and its tissue culture derivative have been studied to establish the characteristics of an in vivo--in vitro solid tumor model. The H-4-II-E line, originating from the Reuber H-35 hepatoma, can be maintained and studied either in cell culture or as a transplantable solid tumor in ACI male rats. In addition it allows for the in vitro assay of cell survival following treatment of animal tumors in situ. In vivo, hepatoma H-4-II-E is rapidly growing tumor with a mean doubling time of 49-2 hr. The cell cyle time is 39-1 hr with a cell loss factor of 0-32. Retrospective examination of tumor specimens obtained during the establishment of the H-4-II-E tumor system demonstrates that both structural as well as cell population changes have occurred. The biological characteristics of the primary tumor (H-35) and an early intermediate stage (H-35tc2) are compared with H-4-II-E and the histopathological, growth and cell kinetic changes are discussed.     

Properties of a murine monocytic tumor cell line J-774 in vitro. II. Enzyme activities.

Lysosomal enzyme activities, collagen degrading activity and sensitivity to bacterial infection were tested in a murine monocytic cell line, J-774, during cultivation with or without fetal calf serum (FCS) or endotoxin, and compared with the same parameters in normal murine peritoneal macrophages. The basic intracellular level of two out of three lysosomal enzyme activities tested (acid phosphatase and β-glucuronidase) and their extracellular release were higher in the J-774 cells than in normal macrophages, indicating that the tumor cells were more “activated”. This was further supported by the moderate increase in intracellular enzyme activities after FCS and endotoxin stimulation of the J-774 cells. Normal macrophages showed a much more impressive rise in these parameters after stimulation. Collagen-degrading activity was found at the same magnitude, or lower, in tumor cell cultures, compared to normal macrophage cultures. However, the activity in the tumor cultures was enhanced by endotoxin stimulation. The J-774 cells showed a higher sensitivity to bacterial contamination, tested after E. coli addition to the cultures, than normal macrophages. This high sensitivity could be prevented by pretreatment of the tumor cells with endotoxin.     

Activation of polycyclic hydrocarbons in Reuber H4-II-E hepatoma cells an in vitro system for the induction of SCEs

Activation of polycyclic aromatic hydrocarbons (PAHs) was examined in the Reuber H4-II-E established cell line without the use of exogenous enzyme preparations. Metabolism of PAHs to genotoxic products was determined by the induction of sister-chromatid exchanges (SCEs). The induction of SCEs followed a dose-response pattern with plateaus at high doses of PAH. The effects of metabolic enzyme inducers (3-methylcholanthrene, phenobarbital, Aroclor 1254) and the epoxide hydrase inhibitor 1,1,1-trichloropropylene oxide were assessed as changes in SCE induction and enhanced production of water-soluble metabolites. Results indicate that Reuber H4-II-E cells can be employed in the testing of carcinogens activated by the P₁-450 monooxygenase system and would be a useful in vitro system for the study of mechanisms of metabolic induction and their effect on genetic toxicity.     

A 60 kDa plasma membrane protein changes its localization to autophagosome and autolysosome membranes during induction of autophagy in rat hepatoma cell line, H-4-II-E cells.

We previously reported the preparation and characterization of an antibody against membrane fraction of autolysosomes from rat liver (J. Histochem. Cytochem. 38, 1571-1581, 1990). Immunoblot analyses of total membrane fraction of a rat hepatoma cell line, H-4-II-E cells by this antibody suggested that H-4-II-E cells expressed several autolysosomal proteins, including a protein with apparent molecular weight of 60 kDa. It was suggested that this 60 kDa protein was a peripheral membrane protein, because it was eluted from the membrane by sodium carbonate treatment. We prepared an antibody against this 60 kDa protein by affinity purification method, and examined its behavior during induction of autophagy. Autophagy was induced by transferring the cells from Dulbecco's modified Eagle medium (DMEM) containing 12% fetal calf serum into Hanks' balance salt solution. In DMEM, the 60 kDa protein showed diffused immunofluorescence pattern, and immunoelectron microscopy suggested that this protein was located on the extracellular side of the plasma membrane. After inducing autophagy, the immunofluorescence configuration of the 60 kDa protein changed from the diffused pattern to a granulous one. Immunoelectron microscopy showed that the 60 kDa protein was localized on the luminal side of the limiting membrane of autolysosomes and endosomes. In the presence of bafilomycin A1 which prevents fusion between autophagosomes and lysosomes, the 60 kDa protein was localized on the limiting membrane of the autophagosomes and endosomes. These results suggest that the 60 kDa protein is transported from the plasma membrane to the autophagosome membrane through the endosomes.     

Properties of a murine monocytic tumor cell line J-774 in vitro. I. Morphology and endocytosis.

A murine monocytic tumor cell line J-774 was maintained in culture in the presence or absence of endotoxin, in an attempt to induce differentiation similar to that found in activated peritoneal macrophages. The morphology and Fc and C₃ receptor functions of attachment and ingestion were compared in the treated and untreated cultures. J-774 cells maintained in culture for 72 h seemed to resemble endotoxin-activated macrophages rather than normal peritoneal macrophages. A striking amount of ruffling was observed on the surface of the cells cultured for 3–4 days both in the presence and in the absence of endotoxin. As compared to the peritoneal macrophage where particles attached to the C₃ receptors are not ingested unless the cells are activated, J-744 cells attached and ingested particles via the C₃ receptor even without stimulation. The presence of endotoxin in the culture medium of these cells gave rise to more efficient phagocytosis but did not effect the temperature sensitivity of the phagocytic receptors. Both in treated and untreated cultures attachment to the Fc receptor was less dependent on the temperature than that of the C₃ receptor while ingestion was more sensitive in the Fc receptor as compared with the C₃ receptor.     

In vitro cultivation of parthenogenesis-inducing Wolbachia in an Aedes albopictus cell line

Parthenogenesis‐inducing (PI) Wolbachia, infecting the parasitoid wasp Trichogramma kaykai Pinto and Stouthamer (Hymenoptera: Trichogrammatidae), were successfully maintained and cultivated in a mosquito, Aedes albopictus (Skuse) (Diptera: Culicidae), cell line, NIAS‐AeAl‐2. A parthenogenesis‐inducing (PI) Wolbachia strains with wasp ovaries were introduced into flasks with cultures of the cell line. The PI Wolbachia proliferated in NIAS‐AeAl‐2 as confirmed using a quantitative polymerase chain reaction method. Our final goal is to induce parthenogenesis in other hymenopteran species of commercial interest through the artificial transfer of PI Wolbachia. As a step towards this goal, we microinjected pupae of Trichogramma with PI Wolbachia culture in NIAS‐AeAl‐2 cells and PI Wolbachia was detected from recipient by PCR with Wolbachia specific primers (wsp gene).     

In vitro mitogenic and steroidogenic effects of ACTH analogues on an adrenal tumor cell line (Y-1)

Native porcine adrenocorticotropin (ACTH_1–39) as well as synthetic adrenocorticotropin (ACTH_1–24) increase cAMP and steroid production and inhibit DNA synthesis in an adrenal cell line. The COOH terminal sequence of both peptides as well as β-endorphin have no effects, while the NH₂ terminal sequence of ACTH as well as α-MSH which have very low stimulatory effect on cAMP production, have a mitogenic effect. These results suggest that ACTH might have in vitro some mitogenic action on adrenal cell, but this effect is blunted by cAMP accumulation during hormonal stimulation. The results can also explain the in vivo and in vitro contradictory effects of the hormone on adrenal cell replication.     

Phosphorylation of DNA topoisomerase II in a human tumor cell line.

The phosphorylation of the nuclear enzyme DNA topoisomerase II was characterized from HeLa human cervical carcinoma cells labeled with 32Pi. Analysis of topoisomerase II immunoprecipitates from 32P-labeled HeLa cells indicated that phosphorylation of the enzyme occurred at serine residues. Incorporation of 32P into topoisomerase II was not due to other types of phosphomodifications such as poly(ADP-ribosylation) or covalent interactions of the enzyme with nucleic acids. The stability of topoisomerase II protein and topoisomerase II phosphorylation was also investigated in HeLa cells. Topoisomerase II protein was relatively stable, having a half-life of approximately 27 h. Phosphorylation of HeLa topoisomerase II was also remarkably stable with a T1/2 of 17 h.     

In vitro inhibition of hemopoietic cell line growth by hepatitis B virus.

The effects of hepatitis B virus (HBV) on established human cell lines of various tissue origins were evaluated by clonal or colorimetric assays in methylcellulose culture. HBV exposure inhibited the growth of six hemopoietic cell lines, while similar incubation did not affect the growth of seven nonhemopoietic carcinoma cell lines of breast, colon, liver, and stomach origin. The inhibition of hemopoietic cell line colony formation was dependent on the presence of intact viral (Dane) particles and the ratio of exposure of virions to cells and was reversible with antibodies to pre-S1, pre-S2, and S envelope protein epitopes. Purified HBV DNA, surface antigen pre-S antigens, and core antigen did not inhibit cell line growth. These results further demonstrate the tropism of HBV for cells of hemopoietic origin, confirming our previous findings on the effects of HBV on the growth of normal bone marrow progenitor cells in vitro. Established human tissue culture cell lines may be used to study the interactions of hemopoietic cells with HBV.     

In vitro photosensitisation of a murine mammary adenocarcinoma cell line with Verteporfin.

Benzoporphyrin derivative monoacid ring A (BPD-MA) is a second generation hydrophobic photosensitiser for PDT that has been approved for ocular disease treatment. In the present paper we report the results of in vitro studies on the photosensitising activity of Verteporfin (liposomally formulated BPD-MA) using an adenocarcinoma derived cell line. Our findings show a quick and efficient uptake of Verteporfin by LM3 cells, reaching maxima concentrations after 5 hr exposure to 18 microg Verteporfin/ml. Independently on the concentration, plateau levels are attained 5 hr after exposure to Verteporfin. Exposure of the cells to the photosensitiser appears to be safe in the darkness within a broad range of concentrations. The hydroxyl radical scavenger mannitol afforded the highest protection against PDT, while L-tryptophan, a well known and efficient singlet oxygen quencher was not an effective protector at all, showing scavenging activity only when it was supplemented at concentration as high as 10 mM and when 50% of the cells were affected, showing that in addition to singlet oxygen, which is considered the primary cytotoxic agent in PDT, other interconvertible reactive oxygen specie (ROS), in particular HO are also generated. Verteporfin-PDT also induced morphological features typical of apoptotic cells. Results of the present work show that the LM3 adenocarcinoma cell can be effectively sensitised with Verteporfin-PDT.     

Introduction of the H-2Dk gene into a class I-negative tumor cell line confers interferon-gamma inducibility upon the silent endogenous H-2Kk gene.

Kgv cells do not constitutively express class I mRNA or protein. Interferon (IFN)-gamma, but not IFN-alpha/beta, induces H-2Dk expression. IFN does not induce H-2Kk expression. We examined constitutive and IFN-inducible class I expression on Kgv cells stably transfected with genomic clones of H-2Kk or H-2Dk and on somatic cell hybrid lines constructed between Kgv cells and constitutively class I-positive cells of a distinguishable H-2 haplotype. Our results suggest that both the lack of constitutive class I expression and the inability of IFN-alpha/beta to induce class I expression on Kgv cells are primarily due to cis-regulatory mechanisms. However, stable introduction of the H-2Dk gene into Kgv cells conferred IFN-gamma inducibility upon the silent endogenous H-2Kk gene. Therefore, the failure of IFN-gamma to induce H-2Kk expression on Kgv cells is due, at least in part, to a trans-regulatory mechanism.     

In vitro development and characterization of a manatee bronchial cell line

In the present study, culture conditions that promote the growth and differentiation of manatee respiratory tract epithelial cells toward a mucociliary phenotype were determined. Characterization of a manatee-specific cell line enables investigators to conduct in vitro testing where live-animal experimentation is not possible. Cell cultures were established from both explants and enzymatically dissociated cells that were isolated from manatee bronchial tissue. To modulate their differentiation, bronchial epithelial cells were grown on Transwell collagen membranes either submerged or at an air-liquid interface. Growth on a collagen membrane at an air-liquid interface and medium supplemented with retinoic acid was required to promote a mucociliary phenotype. When cells were grown in submerged cultures without retinoic acid, they appeared more squamous and were not ciliated. Intracellular keratin proteins were detected in both submerged and interface cultures. Cultured manatee bronchial epithelial cells will facilitate future studies to investigate their potential role in pulmonary disease associated with brevetoxicosis after exposure to the red-tide organism, Karenia brevis.     

45T-1, an established cell line with characteristics of Sertoli cells, forms organized aggregates in vitro after exposure to tumor necrosis factor alpha.

In the testis TNF is produced by germinal cells. The putative role of tumor necrosis factor alpha (TNF) in development and differentiation was investigated in 45T-1 mouse cell cultures, a cell line with characteristic markers of Sertoli cells, established from transgenic mouse families expressing the polyoma large T antigen in their testes. Exposure to TNF elicited a gradual assembly of the cells of the monolayer into highly organized spheroids. The first morphological sign of the changes was detected one week after TNF treatment by anti-desmin immunostaining which showed the formation of foci in the culture consisting of several hundred cells connected by an increasing number of cell contacts. Between days 10-20 the cells formed large ovoid or vermiform aggregates covered by several layers of flat, elongated cells. These cells extended septae into the inner mass of the spheroids consisting of loosely arranged, large polygonal or palisadic cells. The spheroids were surrounded by radially arranged elongated cells covered by small blebs. TNF treatment upregulated laminin expression in 45T-1 cell cultures, which is known to induce formation of cord-like structures by Sertoli cells in vitro. Coculturing 45T-1 cells with immortalized germinal cells or TNF-producing HeLa cells also lead to the formation of spheroids. These observations suggest that TNF production of germinal cells might contribute to the organization/differentiation of Sertoli cells.     

T98G: an anchorage-independent human tumor cell line that exhibits stationary phase G1 arrest in vitro.

T98 and T98G are two related cell lines that were derived from a human glioblastoma multiforma tumor. T98G has almost twice as many chromosomes as T98, suggesting that it is a polyploid variant of T98. Three aspects of control of cellular proliferation were studied in T98 and T98G cells in comparison to WI-38 normal human diploid cells. WI-38 cells have the following properties: (1) they can undergo only a limited number of population doublings in vitro; (2) they cannot proliferate without anchorage; and (3) they become arrested in G1 phase under stationary phase conditions. T98 cells differ from normal cells in all three of these properties, as do many other transformed cell lines. However, the derivative of T98, namely T98G, expresses an unique combination of normal and transformed aspects of the control of cellular proliferation. T98G cells are like normal cells in that they become arrested in G1 phase under stationary phase conditions, yet they also exhibit the transformed characteristics of anchorage independence and immortality. Thus, T98G cells demonstrate that transformation to immortality and anchorage independence can exist without concomitant loss of the normal mechanism for G1 arrest in response to stationary phase conditions. This result supports the hypothesis that each of these three aspects of control of cellular proliferation can be altered independently. Partially transformed cell lines, such as T98G, should be useful for sorting out the biochemical changes associated with transformation in each of these aspects.     

In vitro selection of a cell line for resistance to lysis by tumor necrosis factor-alpha selects for reduced tumorigenicity

Experimentally, TNF-alpha can mediate the hemorrhagic necrosis of certain tumors. Furthermore, evidence indicates that natural cytotoxic (NC) activity, a cell-mediated cytolytic activity that utilizes TNF-alpha in the lysis of target cells, is involved in preventing the outgrowth of certain NC/TNF-alpha-sensitive tumor cells. These observations raise the issue of whether soluble TNF-alpha normally serves as a tumor surveillance mechanism preventing the outgrowth of some tumors. To address this issue, we have used TNF-alpha to select TNF-alpha-resistant variants from the NC/TNF-alpha-sensitive mouse fibroblast cell line 10ME. Previously, we have demonstrated that 10ME is tumorigenic in immune-deficient mice but fails to form tumors in normal mice. Moreover, selection of NC-resistant variants from 10ME selects for both TNF-alpha resistance and tumorigenicity in normal mice. As cells that have been selected for NC resistance form tumors in normal mice, whereas the NC-sensitive parental cell line does not, it seems that escape from NC activity is sufficient to significantly increase the tumorigenic potential of the cell line. We show that the selection with TNF-alpha, although associated with NC resistance, does not increase the tumorigenic potential of 10ME cells but reduces it. Thus, NC activity appears to function as a mechanism to prevent tumor formation, and escape from NC activity allows for tumor formation; TNF-alpha does not have similar activity. Moreover, this suggests that NC activity is not equivalent to soluble TNF-alpha activity, but utilizes TNF-alpha more efficiently than soluble TNF-alpha, or NC activity involves both TNF-alpha and other effector mechanisms.     

In vitro induction of cecropin genes--an immune response in a Drosophila blood cell line.

The Drosophila melanogaster cell line mbn-2 was explored as a model system to study insect immune responses in vitro. This cell line is of blood cell origin, derived from larval hemocytes of the mutant lethal (2) malignant blood neoplasm (1(2)mbn). The mbn-2 cells respond to microbial substances by the activation of cecropin genes, coding for bactericidal peptides. The response is stronger than that previously described for SL2 cells, and four other tested Drosophila cell lines were totally unresponsive. Bacterial lipopolysaccharide, algal laminarin (a beta-1,3-glucan), and bacterial flagellin were strong inducers, bacterial peptidoglycan fragments gave a weaker response, whereas a formyl-methionine-containing peptide had no effect. Experiments with different drugs indicate that the response may be mediated by a G protein, but not by protein kinase C or eicosanoids, and that it requires a protein factor with a high rate of turnover.