Establishment of a rat hepatoma cell line which has ornithine carbamoyltransferase activity and grows continuously in arginine-deprived medium

The epithelial cell line, H4-II-E derived from Reuber hepatoma H35 has no significant activity of ornithine carbamoyltransferase (OCT, EC 2.1.3.3) and is not able to grow in arginine-deprived medium. A multi-step selection procedure is described which selects from H4-II-E populations, cells with OCT activity which can grow in arginine-deficient, ornithine-supplemented media.     

Genome structure of mink cell focus-forming murine leukemia virus in epithelial mink lung cells transformed vitro by iododeoxyuridine-induced C3H/MuLV cells

We characterized mink cell focus-forming murine leukemia viruses that were isolated from C3H/MCA-5 cells after induction with 5-iododeoxyuridine in culture. Mink lung epithelial cells malignantly transformed in vitro by induced virus were the source of four molecular clones of mink cell focus-forming virus. CI-1, CI-2, CI-3, and CI-4. Three clones, CI-1, CI-2, and CI-3, had full-length mink cell focus-forming viral genomes, one of which (CI-3) was infectious. In addition, we obtained a defective viral genome (CI-4) which had a deletion in the envelope gene. A comparison between the envelope genes of CI-4 and those of spleen focus-forming virus by heteroduplex mapping showed close homology in the substitution region and defined the deletion as being identical to the p15E deletion of spleen focus-forming virus. The recombinant mink cell focus-forming genomes are not endogenous in C3H/MCA-5 cells and therefore must have been formed in culture after induction by 5-iododeoxyuridine. CI-3, the infectious clone of mink cell focus-forming murine leukemia virus, was dualtropic, and mink cells infected with CI-3 were altered in their response to epidermal growth factor. In the presence of epidermal growth factor at 10 ng/ml, uninfected mink cells retained their epithelial morphology in monolayer culture and did not form colonies in soft agar. In contrast, CI-3 virus-infected mink cells grew with fibroblastic morphology in monolayer culture and showed an increased growth rate in soft agar in the presence of epidermal growth factor.     

Alterations in electrophoretic mobility, diaphorase activity, and terminal differentiation induced in murine erythroleukemia lines by differentiating agents

The electrophoretic mobilities (EPMs) and semiquinone reductase activities of two clones of Friend murine erythroleukemia (MEL) cells were investigated as a function of treatment with the inducing agents dimethylsulfoxide (DMSO) and hexamethylene bisacetamide (HMBA). As reported previously by others, the inducible clone DS19 lost its ability to grow in soft agar and expressed hemoglobin as judged by benzidine/H2O2 staining after 96 hours of treatment with 1% DMSO or 4 mM HMBA. In addition, its EPM fell by 14%, its semiquinone reductase activity by 40%, and its mean diameter by 10%. The second clone, R1, retained its ability to grow in soft agar and lacked hemoglobin expression after treatment with HMBA and DMSO, characterizing it as noninducible. However, R1 did demonstrate alterations in EPM, semiquinone reductase activity, and cell diameter that closely paralleled those found in DS19. Such responses were not seen in three non-MEL cell lines exposed to HMBA or DMSO, suggesting that clone R1 responded to these inducing agents in a cell-line specific manner but that its ability to complete the sequences necessary for differentiation may be blocked at an unknown point distal to the block characteristic of untreated cells. The data show that while a reduction in EPM, semiquinone reductase activity, and cell diameter accompany induced differentiation in MEL cells, such changes can occur in the absence of a commitment to terminal differentiation.     

SV40 T基因转化的山羊乳腺上皮细胞系及其生物学特性

目的建立能用于乳腺特异表达基因构件质量检验的山羊乳腺上皮细胞系.方法根据已发表的SV40病毒T基因序列设计引物,以整合有SV40 DNA早期基因区的COS-1细胞基因组DNA为模板,用高保真PCR扩增SV40 T基因.将获得的SV40 T基因克隆入真核表达载体,并用获得的重组表达质粒转染山羊原代乳腺上皮细胞.经有限稀释和反复传代后获得转化细胞克隆,对其生物学特性进行研究.结果扩增出序列正确的SV40T基因,重组质粒转染获得的转化细胞的对数生长期为接种后第4天,细胞群体倍增时间为23.5*!h,克隆形成率为26.7%.DNA斑点杂交试验证明转化细胞的基因组中整合有SV40 T基因,染色体核型分析试验表明转化细胞的核型无明显异常,裸鼠接种试验证明转化细胞不能形成肿瘤,软琼脂集落形成试验表明转化细胞在软琼脂中不能生长.部分细胞克隆已在体外传30代以上,保持正常乳腺上皮细胞的形态特征,在胶原基质上能形成腺泡样结构.结论本研究获得的SV40 T基因转化的山羊乳腺上皮细胞具有转化细胞系的生物学特性.     

Suppression of cell proliferation and deoxyribonucleic acid synthesis in the cloned rat hepatoma H4-II-E cells overexpressing regucalcin.

The role of endogenous regucalcin (RC) in the regulation of cell proliferation was investigated in the cloned rat hepatoma H4-II-E cells overexpressing RC stably. H4-II-E cells were transfected with RC/pCXN2 vector and the multiple neomycin-resistant clones which overexpress stably RC were selected. The RC content of RC/pCXN2-transfected cells used in this study was 19.7-fold as compared with that of the parental wild type H4-II-E cells. Wild type H4-II-E cells, pCXN2 vector-transfected cells (mock type), and RC/pCXN2-transfected cells (transfectants) were cultured for 24, 48, and 72 h in the presence of fetal bovine serum (10% FBS). Cell numbers of wild and mock type were significantly increased with the time course of culture. Cell numbers of transfectants was significantly suppressed as compared with that of wild and mock type. Deoxyribonucleic acid (DNA) synthesis activity in the nuclear fraction of H4-II-E cells was significantly suppressed in transfectants with culture for 12-48 h. The presence of anti-RC monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant increase in DNA synthesis activity in the nuclei of wild type and transfectants; this increase was remarkable in transfectants. The effect of anti-RC monoclonal antibody (50 ng/ml) in increasing DNA synthesis activity in transfectants was completely prevented by the addition of regucalcin (1 microM). This study demonstrates that cell proliferation is suppressed in the cloned rat hepatoma H4-II-E overexpressing RC stably.     

Expression of v-src and chicken c-src in rat cells demonstrates qualitative differences between pp60v-src and pp60c-src

The retroviral oncogene v-src arose by transduction of the cellular gene c-src. The similarity between these genes raised the possibility that c-src might be able to elicit neoplastic growth. We explored this by constructing a chimeric plasmid that allows the expression of chicken c-src. A rat cell line containing ten times the normal intracellular level of pp60c -src was isolated after transfecting rat-2 cells with the chimeric DNA. These cells produce the protein encoded by c-src ( pp60c -src) in quantities at least three times greater than required to achieve transformation by the product of v-src ( pp60v -src). The cells remain phenotypically normal, contain actin cables, and do not grow in soft agar. However, transfection of the cell line containing elevated cells of pp60c -src or Rat-2 cells with a molecular clone of v-src produces cells that exhibit properties of biologically transformed cells: round morphology, disrupted actin cables, and ability to grow in soft agar.     

Alterations in cellular phenotype induced by the type 5 adenovirus E1A and the beta 1 protein kinase C genes in cloned rat embryo fibroblast cells.

The E1A gene of adenovirus type 5 (Ad5) induces morphological transformation and anchorage-independent growth in cloned rat embryo fibroblast (CREF) cells. In contrast, CREF cells transfected with a beta 1 protein kinase C (PKC) gene and expressing low-levels of beta 1 PKC display a CREF-like morphology and do not form colonies when grown in agar. The combination of Ad5 E1A and low-level beta 1 PKC expression in the same CREF cell line results in an enhanced ability to grow when suspended in agar. In Ad5 E1A and Ad5 E1A + low-level beta 1 PKC expressing CREF clones, the tumor promoting agent 12-0-tetradecanoyl-phorbol-13-acetate (TPA) further enhances anchorage-independence. In contrast, TPA does not induce CREF cells or transfected CREF cells expressing low-levels of beta 1 PKC to grow in agar. Low-level beta 1 PKC expression in transfected CREF cells is associated with a modest 1.2 to 1.6-fold increase in binding of [3H]-phorbol-12,13-dibutyrate (PDBu) and only a 2.3-fold increase in PKC enzymatic activity. In contrast, specific beta 1 PKC-retroviral vector transformed CREF clones (CREF-RV-PKC) display higher levels of PKC mRNA, PDBu binding and PKC enzymatic activity. A majority of CREF-RV-PKC clones exhibit a transformed morphology and grow more rapidly in monolayer culture, form macroscopic colonies in agar in the absence of TPA and in many independent clones TPA further enhances anchorage-independent growth. This effect is not directly related to the level of enhanced [3H]-PDBu binding. The present study indicates that the effect of beta 1 PKC on cellular phenotype in immortal rat embryo cells is complex and is affected by its mode of insertion into CREF cells, i.e. transfection versus retroviral insertion. In addition, the combination of a transfected Ad5 E1A and a beta 1 PKC gene in the same CREF clone results in an enhanced expression of the transformed phenotype in both the absence and presence of TPA.     

Culture of adult rat lung cells: Benzo(a)pyrene metabolism and mutagenesis

Summary A method is described for obtaining and culturing large numbers of lung cells from normal adult male rats. The lungs were perfused in situ to remove blood cells and then perfused via the trachea with a trypsin-collagenase solution to initiate tissue digestion. The tissue was further digested in the enzyme solution and approximately 2×10⁸ viable lung cells were obtained per animal. Primary cultures contained a mixed cell population. Through eight subcultures about 70% of the cell population possessed an epithelial-like morphology, whereas the remaining 30% was fibroblast-like. Three clones of epithelial-like cells were isolated at the fourth subculture. The mass culture lung cells and the epithelial-like clone that was studied retained a normal karyotype and did not grow in soft agar. Both the mass culture cells and the epithelial clone metabolized the lung carcinogen benzo(a)pyrene (BP) to water-soluble products. Furthermore, the mass culture lung cells metabolized BP to intermediate(s) which mutated Chinese hamster V79 cells from ouabain sensitivity to ouabain resistance. These lung cell cultures have potential use in cell transformation, mutation and carcinogen metabolism studies. Visiting scientist from Hungary. This research was supported by Grant 5 R01 CA20022 and Public Health Service Contract N01 CP33278 from the Division of Cancer Cause and Prevention, National Cancer Institute, National Institutes of Health.     

Normal mouse serum contains peptides which induce fibroblasts to grow in soft agar

The untransformed mouse fibroblast cells NIH/3T3, C3H/10T1/2, and rat NRK cells do not grow in soft agar in medium supplemented with 10% fetal calf serum. When fetal calf serum in the growth medium was supplemented with less than 1% of sera from mice or other vertebrates, however, these cells responded, forming large colonies. The morphology of soft agar colonies was a function of the treated cell type. In the presence of 10% serum from C57BL/6 mice, NRK cells grew to smooth-surfaced spherical colonies, while NIH/3T3 colonies showed individual round cells on their surface and C3H/10T1/2 cells grew as extended cells forming columns of end to end connected fibroblasts. Mus Musculus Castaneus-Epithelial (MMC- E) cells were not stimulated to grow in soft agar under these conditions. The major fibroblast colony-inducing factor (F-CIF) was partially purified from mouse serum by acid/ethanol-extraction, gel permeation chromatography, and reverse-phase high-pressure liquid chromatography. F-CIF is a polypeptide, which does not compete for binding to epidermal growth factor (EGF) receptors, but stimulates normal fibroblasts to form small colonies in semisolid medium and very large colonies in the presence of added EGF (2 ng/ml). In contrast to unfractionated mouse serum, purified F-CIF did not induce C3H/10T1/2 cells to grow in soft agar, suggesting that serum contains additional cell type-specific agar growth-stimulating activities.     

Fibrinolytic activity in a human fibrosarcoma cell line and evidence for the induction of plasminogen activator secretion during tumor formation.

Seven clones were isolated from the HT1080 human fibrosarcoma cell line using a fibrinagarose overlay technique. Three of these clones induced lysis of the fibrin overlay, whereas four did not. The extracellular and intracellular levels of protease were then measured using 125I-fibrin plates incubated with acid-treated human serum. The extracellular protease can be directly assayed in the medium from cells incubated with 10% fetal calf serum. Although there were large differences in the amounts of protease secreted by these two sets of clones, the intracellular levels of protease were similar. No significant differences were found between the abilities of the cells to grow in soft agar or as tumors in immunosuppressed hamsters. However, cells grown from tumors derived from all the low secretors of protease showed an increase in the amount of protease secreted. It appeared, therefore, that the secretion of protease might be selected for or induced during tumor growth. Further detailed studies with one of the low secreting clones (clone E) suggested an inductive rather than a selective mechanism for this increase in extracellular plasminogen activator.     

Rapid determination of DNA synthesis in adherent cells grown in microtiter plates

A specific, rapid, and economical method for measuring the extent of DNA synthesis in adherent rat hepatoma H4-II-E cells grown in 96-well microtiter plates is described. The adherent cells were pulsed for 1 h with [methyl-3H]thymidine, released from the substratum by trypsinization, and collected on fiberglass filters with a MASH II cell harvester. The amount of radioactivity incorporated was directly proportional to the number of cells per well. Growth curves generated by measuring [methyl-3H]thymidine incorporation and counting the number of cells per well were identical. Experiments with inhibitors of DNA, protein, and RNA synthesis demonstrated that this method selectively measured DNA synthesis. In addition, [3H]thymidine uptake showed excellent correlation with autoradiographic assessment of DNA synthesis. This specific and sensitive method for determining DNA synthesis in microtiter cultures should facilitate studies of effects of various growth-controlling agents on epithelial, fibroblastic, and other cells which grow as adherent cells in culture.     

Progression of the phenotype of transformed cells after growth stimulation of cells by a human papillomavirus type 16 gene function.

Alteration of the growth properties of the established murine fibroblast cell lines NIH 3T3 and 3Y1 was studied in monolayer cultures and in cells suspended in semisolid medium after introduction of a cloned human papillomavirus type 16 (HPV16) DNA. HPV 16 DNA stimulated both cell lines to grow beyond their saturation densities in monolayer cultures without any apparent morphological changes or tendency to pile up. These cells were also stimulated to grow in soft agar. Since essentially all the cells that received the viral gene were stimulated to grow, the growth-stimulatory activity of HPV16 appeared to be due to the direct effect of a viral gene function. The NIH 3T3 cells showed an additional change in growth properties upon prolonged incubation of dense monolayers of cells containing the HPV16 DNA; morphologically recognizable dense foci appeared at a frequency of about 10(-3). These cells, when cloned from the foci, grew more rapidly in soft agar than the parental cells and were morphologically transformed. In other words, there were two sequential steps in cell transformation induced by HPV16. Practically all the viral DNAs were present in the cells as large rearranged multimers and were integrated into host chromosomal DNA. There was no obvious difference in the state of viral DNA in the cells of the original clone or the three subclones derived from it as dense foci. There was no difference in the amount or the number of viral RNA species expressed in the cells at these two stages. The secondary changes in the growth properties of NIH 3T3 cells appear to be due to some cellular alterations.     

Manifestation of malignancy in somatic hybrids obtained by the cell fusion of 2 tumor lines

Malignancy of 6 independent hybrid clones derived from fusion of two tumor cell lines of Djungarian hamster, which had been transformed with SV40 virus, was studied. In most of the hybrid clones, suppression of the ability to grow progressively in vivo and the increase in the latent period of tumor occurrence were observed. These data bear witness to suggestion about the existence of different genetic alterations in these tumor cells. Suppression of malignancy does not depend on the genome mutations leading to cell polyploidization, since no decrease in tumorigenicity was found in polyploid cell clones of the high tumor cell line. These polyploid cells can actively form colonies in the soft agar medium.     

Characterization of extended primary and secondary cultures of hamster tracheal epithelial cells

Summary Studies on the regulation of differentiation in airway epithelial cells have been hampered by the lack of cell culture systems that differentiate in vitro. One such system that does exhibit differentiation is hamster tracheal epithelial cells (HTE). A major problem with this system, however, is that at the time cells differentiate, they lyze the collagen gel upon which they grow, resulting in termination of the culture. Here we report that by growing the HTE cells at 32° instead of 37°C we can totally prevent lysis of the collagen gel. Cells grown at this lower temperature maintain their differentiated phenotype as evidenced by abundant mucus granules and the secretion of authentic mucus glycoproteins into the culture media. We have also developed a method for subculturing the primary cells which allows growth and differentiation in secondary culture. The HTE cells were capable of being passaged at least three times and did not become transformed as judged by their inability to grow in soft agar and to produce tumors in syngeneic animals. This improved HTE cell culture system will allow detailed studies on the mechanisms which regulate growth, differentiation, and mucus secretion in surface airway epithelial cells. This work was supported in part by grants HL-19717 and HL-36854 from the National Institutes of Health, Bethesda, MD.     

Anchorage-independence as an index of proliferative potential and maturational age: a comparison of the growth of normal, primary explanted bovine granulosa cells in semisolid agar and in liquid culture

When seeded at low-density, normal primary explanted granulosa cells will grow to form clones of functionally differentiated cells in both semisolid agar and in liquid culture. The anchorage-independent clonogenic granulosa cell differs from the anchorage-dependent granulosa cells detected in clonal liquid culture in a number of properties. Basal cloning efficiency in liquid culture is up to 50-fold higher than in agar culture. In serum supplemented medium (20% fetal calf serum) cloning efficiency in liquid culture is unaltered in the presence of added epidermal growth factor (EGF), whereas, agar cloning efficiency is augmented six-fold when cells are incubated under identical conditions. Cells derived from primary anchorage-independent clones, when dispersed and replated, will generate secondary anchorage-independent clones and anchorage-dependent liquid clones. On the other hand, although cells derived from parallel primary anchorage-dependent clones will also generate secondary anchorage-dependent clones, generation of secondary anchorage-independent clones is not detectable. These findings suggest that the anchorage-independent clonal agar assay may be detecting a developmentally earlier granulosa cell subpopulation than is detectable in the liquid culture assay.     

Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts.

Fibroblast cell lines, designated R- and W cells, were generated, respectively, from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 insulin-like growth factor receptor, and from their wild-type littermates. W cells grow normally in serum-free medium supplemented with various combinations of purified growth factors, while pre- and postcrisis R- cells cannot grow, as they are arrested before entering the S phase. R- cells are able to grow in 10% serum, albeit more slowly than W cells, and with all phases of the cell cycle being elongated. An activated Ha-ras expressed from a stably transfected plasmid is unable to overcome the inability of R- cells to grow in serum-free medium supplemented with purified clones. Nevertheless, even in the presence of serum, R- cells stably transfected with Ha-ras, alone or in combination with simian virus 40 large T antigen, fail to form colonies in soft agar. Reintroduction into R- cells (or their derivatives) of a plasmid expressing the human insulin-like growth factor I receptor RNA and protein restores their ability to grow with purified growth factors or in soft agar. The signaling pathways participating in cell growth and transformation are discussed on the basis of these results.     

Analysis of the state of differentiation of two rat colon carcinoma clones with distinct tumorigenic properties.

The presence of some characteristics of normal rat intestinal epithelial cells was studied on two clones originating from a single rat colon carcinoma. These clones differed by their tumorigenic properties in the syngeneic host. However, they grow similarly in vitro and in immuno-deprived animals. The PROb clone which had the ability to form progressive tumors in the syngeneic host appeared to possess more features of differentiated cells than the REGb clone which was immunologically rejected by syngeneic hosts. Indeed, the morphology of the cells was different, the REBb cells having a more fibroblastic appearance while the PROb cells had the capacity to form domes characterizing the functional polarization of the cell layer. The two clones could also be distinguished by their expression of proteins of intermediate filaments. Both expressed cytokeratins showing their epithelial origin, but only REGb cells displayed vimentin which is characteristic of mesenchymal or poorly differentiated epithelial cells. Furthermore, analysis of the expression of a series of glycoconjugate tissue antigens and an unknown protein (p120) showed that the PROb cells resembled more the normal adult digestive epithelium than the REGb cells did. In conclusion, it appears that in this model, the most aggressive cells, those resisting to the constraints imposed by the immune system, are also the more differentiated ones.     

Down-regulation of cytokeratin 14 mRNA in polyoma virus middle T-transformed rat liver epithelial cells.

We have recently shown that rat liver nonparenchymal epithelial cells, such as T51B cells, selectively express cytokeratin (CK) 14 as a partner of CK8 in their intermediate filaments, and we proposed CK14 as a unique cell lineage marker of the liver epithelial cell population (R. Blouin, M-J. Blouin, I. Royal, A. Grenier, A. Loranger, D. R. Roop, and N. Marceau, Differentiation, submitted for publication, 1992). In the present study, T51B-261A (spontaneously transformed) and T51B-261B (aflatoxin B1-treated) clones and clones derived from T51B cells transfected with SV40 large T (LT) and polyoma virus middle T (MT) were used to investigate CK gene expression in nontransformed and transformed liver epithelial cells. T51B-261A, T51B-261B, MT-T51B, and LT/MT-T51B clones all grew in calcium-deficient medium and formed colonies in soft agar, whereas LT-T51B clones did not grow at all in either one of these assays. T51B-261A and T51B-261B clones formed small, slow growing tumors when injected into newborn syngenic rats, whereas the MT-T51B and LT/MT-T51B clones produced rapidly forming, large tumors. There was no effect of cell transformation on CK expression, except in the clones expressing MT, where the CK intermediate filaments were completely lost. Analyses of [35S]methionine incorporation into the Triton-resistant cytoskeleton and of total proteins confirmed that CKs were absent. In contrast, vimentin intermediate filaments remained unaffected in all of the clones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Entamoeba histolytica: generation and characterization of hybrid clones

Transference of DNA to Entamoeba histolytica was carried out by polyethylene glycol fusion of two amebic clones with different phenotypes. Clone C9, strain HM1:IMSS, was the donor. It is emetine-resistant, highly phagocytic and virulent, and grows in soft agar. Clone L6, strain HM1:IMSS, the recipient, is emetine-sensitive, phagocytic, and virulence-deficient, and it does not grow in soft agar. Clones L6 and C9 have shown high stability in their virulence phenotypes since their isolation more than 5 years ago. Before fusion experiments, clone C9 was incubated in 20 micrograms/ml bromodeoxyuridine for 24 hr, and then, irradiated with 310 nm light to complete inactivation. Controls ensured that all irradiated trophozoites died after 24 hr of incubation at 37 degrees C. Irradiated C9 trophozoites were fused with L6 trophozoites, and hybrids were selected by their ability to grow in the presence of emetine. All hybrids, independently generated, grew poorly in soft agar and showed both an intermediate emetine-resistance and rate of phagocytosis. Some of them destroyed efficiently cell monolayers, but interestingly, they showed differences in their ability to produce hepatic abscesses in hamsters.