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PubMed1986

Establishment of two rabbit mammary epithelial cell lines with distinct oncogenic potential and differentiated phenotype after microinjection of transforming genes.

The goal of this work was to establish an assay for transformation of epithelial cells. Two epithelial cell lines were obtained after microinjecting transforming genes into primary rabbit mammary secretory cells. The cell lines were analyzed for their oncogenic potential and for the maintenance of a differentiated phenotype. A fully transformed cell line, which retained epithelial cell organization, was obtained by coinjecting simian virus 40 DNA and the activated human c-Ha-ras gene. The proliferation rate of these cells was high, with a doubling time of 16 h. Their growth was anchorage independent, and they had lost contact inhibition. The cells were tumorigenic in nude mice, but had no metastatic potential. Both microinjected DNAs were efficiently transcribed and translated, in contrast to the casein genes, which were expressed in primary cells but not in the transformed cell line. An immortalized cell line established after injection with simian virus 40 DNA alone was characterized by a moderate rate of proliferation with a doubling time of approximately 30 h. The growth of these cells was contact inhibited and anchorage dependent. The cells were not tumorigenic in nude mice. The viral DNA was expressed during early passages, as shown by the presence of the large T antigen in cell nuclei, but not at later passages. A high number of lactogenic hormone receptors were found associated with the cell surface. Despite the presence of these receptors, no induction of genes coding for milk proteins was observed after addition of prolactin. These data demonstrate that this assay system can be used to assess the immortalizing and transforming potential of candidate oncogenes in epithelial cells.

Animals↗
PubMed2002

Decreased efficiency of gamma-ray-induced DNA double-strand break rejoining in malignant transformants of human bronchial epithelial cells generated by alpha-particle exposure.

PURPOSE: To investigate the cytogenetic changes and DNA double-strand break (DSB) rejoining of transformed cell lines generated from human bronchial epithelial cells by alpha-particle exposure. MATERIALS AND METHODS: Transformed cell lines were derived from the HPV 18-immortalized human bronchial epithelial cell line BEP2D generated by 1.5 Gy of alpha-particles emitted by a 238Pu source. Two cell lines, BERP35T1 and BERP35T4, were investigated. Karyotypes were analyzed by trypsin/Giemsa banding. Cell survival was estimated by colony assay. PFGE was used to detect the DNA DSB. mRNA expression was analyzed by RT-PCR. RESULTS: Abnormal chromosomes 2 and 12 with elongated long arm and deletions of chromosomes 2, 12, 13 and 17 were observed in the transformed cell lines. BERP35T4 showed a much higher proportion of polyploid cells (40.5%) compared with parental BEP2D cells and the BERP35TI cell line (5%). BERP35T1 and BERP35T4 showed a markedly lower capacity for rejoining of gamma-ray-induced DNA DSB and increased radiosensitivity compared with parental BEP2D cells. The analysis of mRNA levels revealed a 2.5- to 6.5-fold down-regulated expression of the DNA repair genes XRCC-2, XRCC-3 and Ku80 in BERP35T1 and BERP35T4 cells. CONCLUSION: The karyotypic changes of chromosomes 2, 12, 13 and 17 and the deficiency of DSB rejoining could be related to the malignant transformation processing of BEP2D cells initiated by alpha-particle exposure.

Alpha Particles↗
PubMed2001

Primary tumor tissue lysates are enriched in heat shock proteins and induce the maturation of human dendritic cells.

Upon exposure to lysates or supernatants of necrotic transformed cell lines, human dendritic cells (DCs) undergo maturation. In contrast, DCs exposed to apoptotic transformed cell lines or necrotic lysates of primary cells remain immature. Analysis of supernatants of necrotic transformed cell lines showed them to be enriched in the heat shock proteins (hsp)70 and gp96, in contrast to supernatants of primary cells. Likewise, cells from a variety of primary human tumors contained considerably higher levels of hsp than their normal autologous tissue counterparts. Of the majority of human tumors enriched in hsps (hsp70 and/or gp96), their corresponding lysates matured DCs. The maturation effect of tumor cell lysates was abrogated by treatment with boiling, proteinase K, and geldanamycin, an inhibitor of hsps, suggesting that hsps rather than endotoxin or DNA were the responsible factors. Supporting this idea, highly purified, endotoxin-depleted hsp70, induced DC maturation similar to that seen with standard maturation stimuli LPS and monocyte conditioned medium. These results suggest that the maturation activity inherent within tumor cells and lines is mediated at least in part by hsps. The release of hsps in vivo as a result of cell injury should promote immunity through the maturation of resident DCs.

Apoptosis↗
PubMed2006

A role for intercellular antigen transfer in the recognition of EBV-transformed B cell lines by EBV nuclear antigen-specific CD4+ T cells.

The CD4+ T cell response to EBV may have an important role in controlling virus-driven B lymphoproliferation because CD4+ T cell clones to a subset of EBV nuclear Ag (EBNA) epitopes can directly recognize virus-transformed lymphoblastoid cell lines (LCLs) in vitro and inhibit their growth. In this study, we used a panel of EBNA1, 2, 3A, and 3C-specific CD4+ T cell clones to study the route whereby endogenously expressed EBNAs access the HLA class II-presentation pathway. Two sets of results spoke against a direct route of intracellular access. First, none of the clones recognized cognate Ag overexpressed in cells from vaccinia vectors but did recognize Ag fused to an endo/lysosomal targeting sequence. Second, focusing on clones with the strongest LCL recognition that were specific for EBNA2- and EBNA3C-derived epitopes LCL recognition was unaffected by inhibiting autophagy, a postulated route for intracellular Ag delivery into the HLA class II pathway in LCL cells. Subsequently, using these same epitope-specific clones, we found that Ag-negative cells with the appropriate HLA-restricting allele could be efficiently sensitized to CD4+ T cell recognition by cocultivation with Ag-positive donor lines or by exposure to donor line-conditioned culture medium. Sensitization was mediated by a high m.w. antigenic species and required active Ag processing by recipient cells. We infer that intercellular Ag transfer plays a major role in the presentation of EBNA-derived CD4 epitopes by latently infected target cells.

Antigen Presentation↗
PubMed1988

Sequence of centromere separation: generation of unstable multicentric chromosomes in a rat cell line.

A transformed cell line, B1, of cerebral endothelial origin from the Wistar-Kyoto male rat has chromatid and chromosome type bridges in virtually every cell. It exhibits various dicentric and polycentric chromosomes. Most dicentrics are symmetric isochromosomes. Certain isodicentrics are present in a fair segment of the cell population; however, almost all cells have some newly arising isodicentrics. The live cells show a lengthened prometaphase. Anaphase is also retarded possibly due to the occurrence of bridges. At anaphase some multicentrics split at only one centromere. When pulled to the two poles the unsplit centromeres and the distal chromosome segment form a side arm bridge. Another mechanism appears to be a total lack of separation of daughter centromeres at meta-anaphase ('meiotic-like' behavior of mitotic chromosomes). This is realized by the pulling of each of the two unsplit centromeres to opposite poles and results in bridges with both sister chromatids running parallel to each other. A break at corresponding weak points in the two sister chromatids followed by rejoining can form a dicentric isochromosome. A third mechanism, the breakage-fusion-bridge cycle, is also operative but would not produce isodicentrics. In the case of the first two mechanisms some or all centromeres apparently split between telophase and onset of the following DNA synthesis rather than at the usual time at late metaphase. These observations may suggest some previously unknown behavior of multicentric chromosomes during mitosis.

Anaphase↗
PubMed1995

Determinants of cisplatin sensitivity in normal versus spontaneously transformed rat ovarian surface epithelial cells.

The chemosensitivity of spontaneously transformed rat ovarian surface epithelial (ROSE) cell lines was compared to that of the parental cells from which they were derived. Cisplatin cytotoxicity was determined in three nontransformed (early passage) and three transformed (late passage) ROSE cell lines. Transformed cells were uniformly more sensitive to cisplatin than parental cells (1.5- to 2.6-fold) and grew more rapidly (doubling time range 15-22 hr) than parental cells (28-37 hr). Increased doubling time correlated with decreased cisplatin sensitivity (rho = 0.771). Cisplatin accumulation did not correlate with cisplatin sensitivity. Glutathione (GSH) levels were higher in two of three early passage cell lines, but the correlation between GSH and decreased cisplatin sensitivity in the overall panel of cell lines was modest (rho = 0.549). No statistically significant differences in DNA-platinum binding were observed between early and late passage cell lines. However, initial levels of DNA-bound platinum correlated with cisplatin sensitivity (rho = 0.812) in the six-cell-line panel. GSH levels were inversely correlated with cisplatin accumulation (rho = -0.829) and DNA platination (rho = -0.786). Increased cisplatin sensitivity in spontaneously transformed ROSE cell lines showed a weak, inverse relationship with GSH levels, but more strongly correlated with their increased growth kinetics and to DNA platination.

Animals↗
PubMed1993

Clonal expansion of cells with trisomy of chromosomes 12 and X in an EBV-transformed lymphoblastoid cell line and establishment of a tumorigenic monoclonal cell line (48,XX,+X,+12).

Clonal expansion of cells with a certain abnormal chromosome constitution has been demonstrated in EBV-transformed lymphoblastoid cell lines, and a new monoclonal tumorigenic cell line (48,XX,+X,+12) has been established. Based on these data and our previous hypothesis that predisposition to tumor development could be associated with inherited susceptibility of certain chromosomes and chromosome regions to breakage, we emphasize that cytogenetic analysis of immortalized cell populations established from normal individuals and cancer patients might be of great importance for predicting development of different primary and secondary malignancies.

Animals↗
PubMed1987

Malignant progression of a transformed rat cell line by transfer of the v-fos oncogene.

Transfer of the v-fos oncogene into a rat cell line transformed by Rous sarcoma virus increased both the spontaneous and experimental lung-metastasis. Metastatic ability of each v-fos transferred cell line was dependent on both the manner of integration and transcriptional amount of the v-fos oncogene, but did not correlate with the growth rate in vivo. Expression of the src, myc or ras genes were not altered by transfer of the v-fos gene, except that the myc expression was enhanced in the cell line, which acquired augmentation of growth rate in vivo but not metastatic potential to the lung. Cells of the metastatic lung nodules of each cell line also possessed exogenous fos DNA and the transcripts. These results suggest that v-fos oncogene functions in the transfected cells and causes malignant progression.

Animals↗
PubMed1999

Apoptosis and catastrophic cell death in benzo[a]pyrene-transformed human breast epithelial cells.

Apoptosis and mitotic death, bi- and multinucleation, giant cells and micronucleation were investigated in human breast epithelial cell lines transformed by benzo[a]pyrene (BP) (BP1, BP1-E and BP1-E1 cells) and in BP1 cells transfected with the c-Ha-ras oncogene (BP1-Tras cells). Since BP induces apoptosis and the abnormal expression of ras genes elicits catastrophic mitosis, both cell death phenomena were expected to occur in this system, especially in BP1-Tras cells. Regardless of the cell line considered, single-nucleate cells were found to be eliminated preferentially through apoptosis, while bi- and multinucleate cells were eliminated through catastrophic mitosis. Apoptosis and catastrophic mitosis were observed in all cell lines but were significantly more frequent in BP1-Tras cells. The abnormal expression of Ha-ras in the latter cells may enhance in this system the effects of the BP apoptosis path reported for BP-transformed Hepa 1c1c7 hepatoma cells. Transfection with the ras oncogene also enhanced the mitotic disturbances, which produced multi- and micronucleation and mitotic death, possibly because of the genomic instability promoted by this oncogene in the BP-transformed cell line.

Apoptosis↗
PubMed1969

Gangliosides in DNA virus-transformed and spontaneously transformed tumorigenic mouse cell lines.

In mouse cell lines transformed by SV40 virus, a decrease was observed in the higher ganglioside homologues disialo-ceramidetetrahexoside and monosialo-ceramidetetrahexoside. Such change was observed only in cells which carry the virus genome, and it correlated with increased saturation density in tissue culture and with rejection in immunologically competent syngeneic host. This indicates that the change is one of the virus-regulated functions, and it is postulated that it relates to the rejection of the virus-transformed cells.

Animals↗
PubMed1981

NADH cytochrome b5 reductase activity in lymphoid cell lines. Expression of the defect in epstein Barr virus transformed lymphoblastoid cell lines from patients with recessive congenital methemoglobinemia.

Recessive congenital methemoglobinemia (RCM) is due to the homozygous deficiency of NADH-cytochrome b5 reductase (EC 1.6.2.2.). In type I disease, in which the patients are only methemoglobinemic, the enzyme defect is fully expressed in the erythrocytes, whereas the leukocytes are much less affected. In type II disease, in which the patients are, in addition, mentally retarded, the defect is generalized to all the tissues including cultured fibroblasts. In the present study we have investigated Epstein-Barr virus (EBV) transformed lymphoid cell lines (LCL) derived from patients with both types of cytochrome b5 reductase deficiency and from nondeficient individuals. The total cytochrome b5 reductase activity of the control LCL was found to be similar whatever the LCL origin, except for one lymphoma line (Daudi). The enzyme from the control LCL (c 252/B 95) was found to be immunologically related to the human soluble erythrocyte cytochrome b5 reductase, indicating that it is the product of the same gene: the DIA1 (diaphorase) locus. The LCL derived from one patient with the type I disease and two patients with the type II disease were investigated.l In the former the defect was expressed to a lesser degree than in the cases with mental retardation in which the defect was much pronounced, and involved both the mitochondrial and the microsomal fraction. This indicated that all the subcellular forms of the cytochrome b5 reductase are under the same genetic control. Altogether, these data show that the LCL are a favorable material for studying both types of cytochrome b5 reductase deficiency and for investigating in depth the molecular aspects of this metabolic disease.

Cell Line↗
PubMed1982

Demonstration of the tumorigenicity of transformed rat kidney cell-lines by intravenous allotransplantation in the neonate.

The tumorigenic potential of allogeneic rat kidney cell-lines transformed in vivo by a carcinogenic dose of dimethylnitrosamine (DMN) was investigated by intravenous injection of single cell suspensions into neonatal outbred rats within 24 h of birth. The cell-lines tested included mesenchymal populations obtained from DMN-induced renal mesenchymal tumors (RRMT-2,-8,-9) and transformed mesenchymal (TRKM-5,-7,-8) and epithelial (TRKE-1) cell-lines derived from the kidneys of rats treated only hours previously with the carcinogen. Additionally, spontaneous nephroblastoma-derived embryonal cell-lines (REN-1,-2) were included in the study to extend the potential of the neonatal rat system over a range of differentiation lineages. The transplantation system proved to be a rapid and efficacious assay for demonstrating the malignancy of the mesenchymal and embryonal cell-lines. The major sites for tumor growth were the lungs, heart and eye, but differences in organ predilection were observed for individual cell-lines. The transformed epithelial cell-line (TRKE-1) proved refractory to the single intravenous inoculation but was transplantation-positive when a follow-up subcutaneous dose was administered several days later. The resultant growths produced by the diverse differentiation lineages were histologically characteristic for the tumor tissue affiliate of each cell-line. The results demonstrate the utility of this transplantation system for testing the malignant potential of morphologically transformed cells across the allogeneic barrier as well as proving that DMN is capable of inducing malignant transformation in both mesenchymal and epithelial cell types of the rat kidney after a very short period of exposure in vivo.

Animals↗
PubMed1983

Adenovirus type 12 specific cell surface antigen in transformed cells is a product of the E1b early region.

Six syngeneic rat cell lines transformed with isolated or cloned left end fragments of adenovirus type 12 (Ad12) DNA were used in a study of the Ad12-specific cell surface antigen. Cells transformed with EcoRI-C, SalI-C, and HindIII-G fragments of Ad12 DNA fragment-transformed express the E1a and a part of or a complete E1b early regions. Two AccI-H DNA fragment-transformed cell lines contain and express only the E1a region. All these cells contain nuclear Ad12 antigen. Cytotoxic antibodies raised against syngeneic EcoRI-C DNA fragment-transformed cells kill EcoRI-C, SalI-C and HindIII-G fragment-transformed cells, but fail to kill cells transformed with AccI-H DNA fragment. Immunofluorescence analysis shows that such antibodies, which stain the surface of cells expressing the E1b region, do not stain the surface of AccI-H DNA fragment-transformed cells. Cells transformed with AccI-H fragment are also not killed by secondary cytolytic T cells, raised and effective against cells transformed with EcoRI-C, SalI-C, and HindIII-G DNA fragments. Cells transformed with AccI-H fragment do not elicit cytolytic T cells against any of the studied cell lines. The only Ad12-specific product shared by all cell lines killed in cytolytic assays which is absent from AccI-H fragment-transformed cells is the E1b 18K protein. Since it has also been shown in other studies that this protein is associated with the cellular membrane, the simplest interpretation of these data is that the Ad12-specific cell surface antigen in transformed rat cells is a product of the left end of the E1b region.

Adenoviridae↗
PubMedDate not supplied

In vitro cytocidal effect of lytic peptides on several transformed mammalian cell lines.

Several types of transformed mammalian cells, derived from established cell lines, were found to be lysed in vitro by three novel lytic peptides (SB-37, SB-37*, and Shiva-1). This is in contrast with the behavior of normal cells, where the observed lytic activity of the peptides is greatly reduced. Based on experiments utilizing compounds which disrupt the cytoskeleton (colchicine and cytochalasin-D), it is surmised that alterations in the cytoskeleton of transformed cells increase their sensitivity to the cytolytic activity exerted by the peptides, primarily by causing a loss of osmotic integrity. Thus, a stable and regenerative cytoskeletal system, as that possessed by normal cells, would seem requisite to withstanding the lytic effects of the peptides.

Amino Acid Sequence↗
PubMed2001

The effect of porphyrins on normal and transformed mouse cell lines in the presence of visible light.

Photodynamic therapy consists of the uptake of a photosensitizing dye, often a porphyrin, by tumor tissue and subsequent irradiation of the tumor with visible light of an appropriate wavelength matched to the absorption spectrum of the photosensitizing dye. This class of molecules produces reactive oxygen species when activated by light, resulting in a direct or indirect cytotoxic effect on the target cells. Photodynamic therapy has been used in the treatment of cancer but the technology has a potential for the treatment of several disease conditions mainly because of its selectivity. However, it is not clear why the porphyrins are retained preferentially by abnormal tissue. This paper describes a study of the effect of the association of porphyrin and visible light on two mouse fibroblast cell lines: A31, normal cells and B61, an EJ-ras transformed variant of A31. Two water-soluble porphyrins were used, a positively charged one, tetra(N-methyl-4-pyridyl)porphyrin chloride, and a negatively charged one, tetra(4-sulfonatophenyl)porphyrin-Na salt (TPPS4) in order to assess the effect on cell survival. The results suggest that the B61 cell line is more sensitive to incubation with the anionic porphyrin (TPPS4) followed by light irradiation and that the anionic porphyrin is more efficient in killing the cells than the cationic porphyrin.

Animals↗
PubMed1995

A transformed murine myocardial vascular endothelial cell clone: characterization of cells in vitro and of tumours derived from clone in situ.

In the course of maintaining a cloned murine myocardium-derived endothelial cell line (mouse heart endothelial cell clone 5; MHEC5) a spontaneously transformed variant has been identified (clone MHEC5-T). On injection into histocompatible mice, clone MHEC5-T uniformly generated epithelioid haemangioendotheliomas. Clone MHEC5-T underwent significant additional alterations in addition to the acquisition of tumour-forming potential in vivo along with the diagnostic correlate of loss of cellular contact inhibition in vitro. Whereas the transformed cells maintained lectin-binding properties characteristic of endothelial cells, they lost the cell surface receptor(s) for acetylated low density lipoprotein and no longer bound antibodies to either angiotensin converting enzyme or von Willebrand factor-associated antigen. Vascular cell adhesion molecule-1 (VCAM-1), expressed constitutively on the parent clone, was down-regulated in the transformed cell line. The transformed cells acquired immunoreactivity to antibodies directed against cytokeratin, and they showed a markedly increased response to migration-inducing factors in vitro. The cell line described in this report demonstrates that the in vitro transformation of myocardium-derived endothelial cells can lead through transitional stages of differentiation to a new stable phenotype characterized by endothelial--to--epithelioid transition. The study of MHEC5-T cells, in addition to providing insight into the biology of cardiac neoplasms, may help to elucidate regulatory mechanisms involved in endothelial cell activation, transition and transformation.

Animals↗
PubMed1975

Cell properties after repeated transplantation of spontaneously and of SV40 virus transformed mouse cell lines. I. Growth in culture.

"Spontaneously" or SV40 virus transformed AL/N mouse cell lines were passed repeatedly through syngeneic mice. Cell lines were re-established in culture from minced pieces of tumors in the presence of concentrated fetal calf serum or from tumor cells dispersed by trypsin. The aim of this study was to compare the two cell lines in regard to the selection processes which operate during such procedures by characterization of the resulting cell lines. Measurements of growth in tissue culture on substratum showed no significant difference between any of the transformed cell lines. The SV40 transformed cells and its derivative cells had a low anchorage requirement for growth. The greatest anchorage requirement for growth was in the normal untransformed cells and in the derivative cells from the "spontaneously" transformed cells which were established from minced tumors. The spontaneously transformed cells and all derivative cells had high tumorigenicity (TD50 is less than 10-2). The SV40 transformed cells had no observable tumorigenicity (TD50 is greater than 10-8), except when injected into irradiated mice (TD50 = 1-5 X 10-5 in the immunocompetent mice, 5 X 10-4 in the irradiated mice). The SV40 transformed derivative cells maintained their SV40 specific T antigen and their susceptibility to lysis by specific antiserum.

Animals↗