Polyoma viral middle T-antigen is required for transformation.

To determine whether small or middle T-antigen (or both) of polyoma virus is required for transformation, we constructed mutants of recombinant plasmids which bear the viral oncogene and measured the capacity of these mutants to transform rat cells in culture. Insertion and deletion mutations in sequences encoding small and middle T-antigens (79.7, 81.3, and 82.9 map units) rendered the DNA incapable of causing transformation by the focus assay. Similar mutations in sequences that encoded middle but not small T-antigen (89.7, 92.1, and 96.5 map units) generally abolished the transforming activity of the DNA. However, two mutants (pPdl1-4 and PPd12-7) that carried deletions at 92.1 map units retained the capacity to transform cells; pPdl1-4 did so at frequencies equal to those of the parental plasmid, whereas pPdl2-7 transformed at 10% the frequency of its antecedent. From these studies we conclude that small T-antigen alone is insufficient to cause transformation and that middle T-antigen is required for transformation, either in combination with small T-antigen or by itself.     

T-antigen interactions with chromatin and p53 during the cell cycle in extracts from xenopus eggs.

The role of SV40 large tumor T-antigen in replication of viral DNA is well established, but it is still unclear how T-antigen triggers cellular replication and cell transformation in non-permissive cells. Here, we used Xenopus egg extracts which reproduce most nuclear events linked to the cell cycle in vitro to analyze its interaction with genomic chromatin during the cell cycle. We show that T-antigen associates with chromatin before the nuclear membrane formation, and further demonstrate that the nuclear membrane is not necessary for its import into the nucleus. We show that the interaction of T-antigen with the endogenous chromatin does not occur at replication foci nor at RPA pre-replication centers. Immunoprecipitations as well as sucrose gradient experiments, indicate that the endogenous pool of p53 interacts with T-antigen. In addition, a transient association of both proteins with the nuclear matrix is observed during the ongoing DNA synthesis. These data are discussed in view of the T-antigen and p53 activity during the cell cycle.     

Comparative study of rabies virus persistence in human and hamster cell lines.

Persistent infections by rabies virus in BHK-21/13S and HEp-2 cells were studied comparatively. No evidence of interferon production, selection of virus-resistant cells, or integration of the viral genome could be found. Persisting viruses replicated efficiently at 34, 36, and 40 degrees C. Both persistently infected cultures released defective interfering virus particles. A cyclical pattern of infection, which was not characteristic of the persistently infected HEp-2 system, was observed in persistently infected BHK cultures. The virus from persistently infected BHK cultures lost its virulence for mice, whereas the virus from persistently infected HEp-2 cultures retained mouse-killing capacity for more than 3 years.     

Hepatocyte cell lines established from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene.

To establish cell lines exhibiting differentiation phenotypes, the immortalized cell lines were rapidly established from the primary culture of different tissues of transgenic mice harboring SV40 temperature-sensitive large T-antigen gene. The established cell lines grew at permissive temperature (33 degrees C), but not at nonpermissive temperature (39 degrees C). Several different cell types could be rapidly immortalized and cloned from the adult transgenic mice tissues. Among those cell lines, the established hepatocyte cell lines (TLR cell lines) exhibited liver-specific morphological and biochemical properties, but their properties were not coupled with the growth condition modified by temperature. The hepatocyte cell lines showed an inducibility of P450IA1 by 3-methylcholanthrene as observed in rat livers and this liver-specific function was stable even after 6 months of culture by continuous passages.     

SV-40 transformation: effect on GM2 ganglioside in cultured cell lines.

In previous work, we observed the presence of substantially elevated levels of GM2 after Simian Virus 40 (SV-40) transformation of human fetal brain cells. This elevated level of GM2 contrasted with the reports of many other investigators who had often observed decreased levels of GM2 and a simplification of ganglioside pattern in various non-neural rodent cell lines. In order to determine if the increase in GM2 in the transformed human brain cells would also be found in transformed rodent brain cells, we analyzed ganglioside changes after transformation in mouse brain cell lines and observed the increase in GM3 and low levels or lack of GM2 usually noted in rodent SV-40 transformed cell lines. In addition, we analyzed changes after SV-40 transformation in three human fibroblast lines and found that all three lines contained substantially elevated levels of GM2 after SV-40 transformation. As a result of this study, our earlier work on SV-40 transformed human brain cells, and occasional other reports of high levels of GM2 in human SV-40 transformed cell lines, elevated levels of GM2 may be considered a marker for SV-40 transformed human cells of both fibroblastic and neural origin.     

Constitutive phosphorylation of eps8 in tumor cell lines: relevance to malignant transformation.

eps8, a recently identified tyrosine kinase substrate, has been shown to augment epidermal growth factor (EGF) responsiveness, implicating it in EGF receptor (EGFR)-mediated mitogenic signaling. We investigated the status of eps8 phosphorylation in normal and transformed cells and the role of eps8 in transformation. In NIH 3T3 cells overexpressing EGFR (NIH-EGFR), eps8 becomes rapidly phosphorylated upon EGF stimulation. At receptor-saturating doses of EGF, approximately 30% of the eps8 pool is tyrosine phosphorylated. Under physiological conditions of activation (i.e., at low receptor occupancy), corresponding to the 50% effective dose of EGF for mitogenesis, approximately 3 to 4% of the eps8 contains phosphotyrosine. In human tumor cell lines, we detected constitutive tyrosine phosphorylation of eps8, with a stoichiometry (approximately 5%) similar to that associated with potent mitogenic response in NIH-EGFR cells. Overexpression of eps8 was able to transform NIH 3T3 cells under limiting conditions of activation of the EGFR pathway. Concomitant tyrosine phosphorylation of eps8 and shc, but not of rasGAP, phospholipase C-gamma, and eps15, was frequently detected in tumor cells. This suggested that eps8 and shc might be part of a pathway which is preferentially selected in some tumors. Cooperation between these two transducers was further indicated by the finding of their in vivo association. This association was, at least in part, dependent on recognition of shc by the SH3 domain of eps8. Our results indicate that eps8 is physiologically part of the EGFR-activated signaling and that its alterations can contribute to the malignant phenotype.     

Antigen-specific. I region-restricted interactions in vitro between tumor cell lines and T cell hybridomas

A series of H-2d B cell tumor lines and one monocytic tumor cell line were shown to be capable of I region-restricted antigen presentation to I-A-d- and I-Ed- restricted, antigen-specific cloned T cell hybridomas. For the most part, antigen presentation correlated with the present of Ia antigens on the presenting cells, although in a few interesting cases Ia-expression lines failed to present antigen. These T cell hybridomas, together with the B cell and to monocyte tumor cell lines, offer a unique set of tools to study the phenomenon of I region-restricted antigen presentation.     

Stimulation with autologous lymphoblastoid cell lines: lysis of Epstein-Barr virus-positive and -negative cell lines by two phenotypically distinguishable effector cell populations

Human lymphocytes, stimulated in vitro for 6 days with x-irradiated or glutaraldehyde-treated autologous Epstein-Barr (EB) virus-transformed lymphoblastoid cell lines (LCL), are cytotoxic for autologous and allogeneic EB+ LCLs as well as for several EB- cell lines that are also susceptible to lysis by interferon-activated natural killer (NK) cells. To determine whether the apparent nonspecific lysis mediated by LCL-stimulated cells is due to a mixture of effector cells directed against different target cells, advantage was taken of our recent finding that monoclonal antibody OKT8 reacts with human cytotoxic T lymphocytes but not with NK cells or NK-like cells generated in mixed leukocyte cultures. The depletion of OKT8+ cells from LCL-stimulated cultures by treatment with OKT8 and complement abolished or markedly depleted cytotoxicity against all EB+ target cells tested, whereas cytotoxicity against EB-, NK-sensitive cell lines including K562, MOLT-4 and HSB-2 was not or only minimally reduced. These results indicate that stimulation with autologous LCL results in the generation of OKT8+ cytotoxic T lymphocytes that lyse EB virus-transformed LCL and OKT8- NK-like cells that lyse EB-, NK-sensitive cells.     

Glycosphingolipids of human urothelial cell lines with different grades of transformation

Neutral glycolipids and gangliosides from seven human urothelial cell lines, differing in grades of transformation (TGr), were characterized by fast atom bombardment mass spectrometry, exoglycosidase treatment and an immunostaining procedure. The major neutral glycolipids identified in all cell lines studied included CMH, CDH, CTH, globoside and paragloboside, the gangliosides were G_M3, G_M2, sialosylparagloboside and G_D1a. The following observations were made: 1. G_M2 was the major ganglioside in the TGrll cell lines (non-tumorigenic, non-invasive), but a minor component in the TGrIII cell lines (tumorigenic, invasive). 2. All components showed C16:0 and C24:0 as major fatty acids, but in the TGrIII cell lines the fatty acid composition of CMH and some of the gangliosides were more complex showing unsaturated and hydroxy-fatty, acids as well.Abbreviations CMH Monohexosylceramide - CDH Lactosylceramide (Galß1-4GlcCer) - CTH Globotriaosylceramide (Gal1-4Galß1-4GlcCer) - Globoside (GalNAcß1-3Gal1-4Galß1-4GlcCer) - Paragloboside (Galß1-4GlcNacß1-3Galß1-4GlcCer) - 3L_M1 Slalosylparagloboside (Neu5Ac2-3Galß1-4GlcNacß1-3Galß1-4GlcCer) - Aslalo-G_M2 (GalNAcß1-4Galß1-4GlcCer) - AsialoG_M1 (Galß1-3GalNAcß1-4Galß1-4GlcCer) - Hex Hexosyl - HexNAc 2-acetamido-2-deoxyhexosyl - HPTLC high performance thin layer chromatography - FAB-MS fastatom bombardment mass spectrometry - TGr transformation grade Ganglios are named according to Svennerholm [1]     

Dipeptidyl peptidase IV in two human glioma cell lines.

There is growing evidence that dipeptidyl peptidase IV [DPP-IV, EC 3.4.14.5] takes part in the metabolism of biologically active peptides participating in the regulation of growth and transformation of glial cells. However, the knowledge on the DPP-IV expression in human glial and glioma cells is still very limited. In this study, using histochemical and biochemical techniques, the DPP-IV activity was demonstrated in two commercially available human glioma cell lines of different transformation degree, as represented by U373 astrocytoma (Grade III) and U87 glioblastoma multiforme (Grade IV) lines. Higher total activity of the enzyme, as well as its preferential localisation in the plasma membrane, was observed in U87 cells. Compared to U373 population, U87 cells were morphologically more pleiomorphic, they were cycling at lower rate and expressing less Glial Fibrillary Acidic Protein. The data revealed positive correlation between the degree of transformation of cells and activity of DPP-IV. Great difference in expression of this enzyme, together with the phenotypic differences of cells, makes these lines a suitable standard model for further studies of function of this enzyme in human glioma cells.     

Competitive interactions and persistence of two nematode species that parasitize Drosophila recens

Drosophila recens is parasitized in the wild by two nematodes, Howardula aoronymphium , a host generalist, and Parasitylenchus nearcticus , a host specialist known only from D .  recens . In order to understand how these two parasite species coexist, we compared their ability to infect and grow in D .  recens , their effects on host fecundity and survival, and whether one parasite species was competitively superior in double infections. The specialist nematode P. nearcticus had greater rates of infection and reproduction than the generalist H. aoronymphium , and completely sterilized females in single and mixed infections. The specialist was competitively superior in mixed infections, as generalist motherworms were significantly smaller than in single infections. These results suggest that P. nearcticus might competitively exclude H. aoronymphium if D. recens were the only host available. It is likely that H. aoronymphium persists in D. recens by transmission from other, more suitable host species.     

Propagation of arthropod-borne Rickettsia spp. in two mosquito cell lines

Rickettsiae are obligate intracellular alphaproteobacteria that include pathogenic species in the spotted fever, typhus, and transitional groups. The development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences among and between pathogenic and nonpathogenic species of rickettsiae. Although several rickettsial species have been grown in tick cells, tick cells are more difficult to maintain and they grow more slowly than insect cells. Rickettsia-permissive arthropod cell lines that can be passaged rapidly are highly desirable for studies on arthropod-Rickettsia interactions. We used two cell lines (Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B) that have not been used previously for the purpose of rickettsial propagation. We optimized the culture conditions to propagate one transitional-group rickettsial species (Rickettsia felis) and two spotted-fever-group rickettsial species (R. montanensis and R. peacockii) in each cell line. Both cell lines allowed the stable propagation of rickettsiae by weekly passaging regimens. Stable infections were confirmed by PCR, restriction digestion of rompA, sequencing, and the direct observation of bacteria by fluorescence in situ hybridization. These cell lines not only supported rickettsial growth but were also permissive toward the most fastidious species of the three, R. peacockii. The permissive nature of these cell lines suggests that they may potentially be used to isolate novel rickettsiae or other intracellular bacteria. Our results have important implications for the in vitro maintenance of uncultured rickettsiae, as well as providing insights into Rickettsia-arthropod interactions.     

Glycolipids and fatty acids of two dog kidney cell lines.

Glycolipid and fatty acid compositions were studied in whole cells and plasma membranes from two dog kidney cell lines (Madin-Darby and SV40-transformed cells) grown in monolayer and suspension cultures. Glycolipids, which account for 5% or less of the total lipids in dog kidney cells, were substantially increased in plasma membranes relative to whole cells. Sialoglycolipids more complex than a Tay-Sachs-like ganglioside were not found in any whole-cell or plasma-membrane preparation of this study. Dog kidney cells transformed by SV40 virus contained primarily a less complex sialoglycolipid, haematoside. Neutral glycolipids comprised 26-43% of the total glycolipid content in Madin-Darby preparations, whereas in transformed cells and membranes neutral glycolipids constituted only 1-22% of the total glycolipid content. Ceramide trihexoside was found in Madin-Darby cultures, but not in transformed cultures. The values for short-chain fatty acids from neutral glycolipids and for saturated fatty acids were generally higher than the values for these fatty acids in calf serum.     

Characteristics of alpha-adrenoceptors in two human colorectal cancer cell lines.

The DiFi and HT-29 human colorectal cancer cell lines were characterized and compared with respect to binding properties of alpha adrenoceptors present on the cell surface. Both cell lines possessed alpha-1 and alpha-2 adrenoceptors of high affinity; however, DiFi cells were rich in alpha-1 adrenoceptors, whereas HT-29 cells were rich in alpha-2 adrenoceptors. In each cell line, specificity of radioligand binding to alpha-1 or alpha-2 adrenoceptors was proved via competition studies using non-tritiated drugs. We believe this to be the first characterization of alpha-1 adrenoceptors in cell line HT-29 and of alpha-1 and alpha-2 adrenoceptors in DiFi cells. Differences between these cell lines in alpha adrenoceptor expression are discussed in relation to colon carcinogenesis. The high level of alpha-1 adrenoceptors seen in DiFi cells should make this cell line useful in studies of the function and regulation of this adrenoceptor subtype.     

Partial transformation of mouse fibroblastic and epithelial cell lines with the v-myc oncogene

To investigate the role of the myc gene in mammalian cell transformation, plasmid constructs containing the v-myc oncogene and a co-selectable G418 resistance marker were introduced into both mouse fibroblasts (NIH-3T3) and bladder epithelial cells (BBN3 and BBN7). After transfection or microinjection of DNA, no transformed foci could be detected on confluent monolayers but, when the cells were cultured under conditions in which individual cells were allowed to grow and form colonies, morphological transformation was observed. Unlike ras-transformed NIH-3T3 cells, v-myc-transformed cells were unable to grow in serum-free medium and therefore still required exogenous growth factors. v-myc-transformed NIH-3T3 cells were poor at forming foci when co-cultivated with untransformed cells; however, the efficiencies could be increased by addition of EGF to the medium. Both v-myc-transformed fibroblasts and epithelial cells acquired the ability to grow in soft agar, though at efficiencies lower than the corresponding ras transformants. Subcutaneous inoculation of v-myc-transformed NIH-3T3 cells into nude mice resulted in no tumours within 6 weeks. After protracted periods (2-3 months) a few tumours were detected, but at a frequency barely above that for spontaneous tumour formation. Epithelial cells transformed by v-myc were either non-tumorigenic or gave a very low incidence of tumours. We conclude that the v-myc oncogene induces morphological changes and anchorage independence in immortal mouse fibroblasts and epithelial cell lines but further events are required for the cells to become tumorigenic.     

Chromosome changes in human monocytic cell lines with in vitro spontaneous malignant transformation

Summary Serial chromosome studies were performed on four monocytic cell lines established from bone marrow samples of patients suffering from hematopoietic disorders other than leukemia. A spontaneous in vitro transformation towards a malignant phenotype has been found to be related to the karyotype evolution. The correlation between the chromosome changes of these cell lines and those described in human cancer and leukemia is discussed.     

Characterization and transplantation of two neuronal cell lines with dopaminergic properties

Immortalized rat mesencephalic cells (1RB₃AN₂₇) produced dopamine (DA) at a level that was higher than produced by undifferentiated or differentiated murine neuroblastoma cells (NBP₂) in culture. Treatment of 1RB₃AN₂₇ and NBP₂ cells with a cAMP stimulating agent increased tyrosine hydroxylase (TH) activity and the intensity of immunostaining for the DA transporter protein (DAT). 1RB₃AN₂₇ cells were labelled with primary antibodies to neuron specific enolase (NSE) and nestin and exhibited very little or no labeling with anti-glial fibrillary acidic protein (GFAP). 1RB₃AN₂₇ cells exhibited β- and α-adrenoreceptors, and prostaglandin E₁ receptors, all of which were linked to adenylate cyclase (AC). Dopamine receptor (D₁) and cholinergic muscarinic receptors linked to AC were not detectable. The levels of PKCα and PKCβ isoforms were higher than those of PKCγ and PKCδ in 1RB₃AN₂₇ cells. The 1RB₃AN₂₇ cells were more effective in reducing the rate of methamphetamine-induced turning in rats with unilateral 6-OHDA lesion of the nigrostriatal system than differentiated NBP₂ cells. The grafted 1RB₃AN₂₇ were viable as determined by DiI labelling, but they did not divide and did not produce T-antigen protein; however, when these grafted cells were cultured in vitro, they resumed production of T-antigen and proliferated after the primary glia cells and neurons of host brain died due to maturation and subsequent degeneration. Examination of H&E stained sections of the grafted sites revealed no evidence of infiltration of inflammatory cells in the grafted area suggesting that these cells were not immunogenic. They also did not form tumors.     

EBV transformation and cell culturing destabilizes DNA methylation in human lymphoblastoid cell lines

Recent research suggests that epigenetic alterations involving DNA methylation can be causative for neurodevelopmental, growth and metabolic disorders. Although lymphoblastoid cell lines have been an invaluable resource for the study of both genetic and epigenetic disorders, the impact of EBV transformation, cell culturing and freezing on epigenetic patterns is unknown. We compared genome-wide DNA methylation patterns of four white blood cell samples, four low-passage lymphoblastoid cell lines pre and post freezing and four high-passage lymphobastoid cell lines, using two microarray platforms: Illumina HumanMethylation27 platform containing 27,578 CpG sites and Agilent Human CpG island Array containing 27,800 CpG islands. Comparison of genome-wide methylation profiles between white blood cells and lymphoblastoid cell lines demonstrated methylation alterations in lymphoblastoid cell lines occurring at random genomic locations. These changes were more profound in high-passage cells. Freezing at low-passages did not have a significant effect on DNA methylation. Methylation changes were observed in several imprinted differentially methylated regions, including DIRAS3, NNAT, H19, MEG3, NDN and MKRN3, but not in known imprinting centers. Our results suggest that lymphoblastoid cell lines should be used with caution for the identification of disease-associated DNA methylation changes or for discovery of new imprinted genes, as the methylation patterns seen in these cell lines may not always be representative of DNA methylation present in the original B-lymphocytes of the patient.     

Differentiation of two mouse cell lines is associated with hypomethylation of their genomes.

Methyl-accepting assays and a sensitive method for labeling specific CpG sites have been used to show that the DNA of F9 embryonal carcinoma cells decreases in 5-methylcytosine content by ca. 9% during retinoic acid-induced differentiation, whereas the DNA of dimethyl sulfoxide-induced Friend murine erythroleukemia (MEL) cells loses ca. 3.8% of its methyl groups. These values correspond to the demethylation of 2.2 X 10(6) and 0.9 X 10(6) 5'-CpG-3' sites per haploid genome in differentiating F9 and MEL cells, respectively. Fluorography of DNA restriction fragments methylated in vitro and displayed on agarose gels showed that demethylation occurred throughout the genome. In uninduced F9 cells, the sequence TCGA tended to be more heavily methylated than did the sequence CCGG, whereas this tendency was reversed in MEL cells. The kinetics of in vitro DNA methylation reactions catalyzed by MEL cell DNA methyltransferase showed that substantial numbers of hemimethylated sites accumulate in the DNA of terminally differentiating F9 and MEL cells, implying that a partial loss of DNA-methylating activity may accompany terminal differentiation in these two cell types.