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PubMed1975

Transformation of Bacillus subtilis: transforming ability of deoxyribonucleic acid in lysates of L-forms or protoplasts.

The transformation of Bacillus subtilis by homologous deoxyribonucleic acid (DNA) made available by gently lysing a stable L-form or protoplast suspension was 3 to 10-fold more efficient than DNA isolated by conventional procedures. This increased transformation was not influenced by digestion with pronase, trypsin, or ribonuclease. Preincubation of isolated DNA with L-form lysates did not increase the transformation efficiency above that achieved with untreated, isolated DNA. In addition to displaying a higher efficiency of transformation, the DNA found in these gently prepared lysates was also able to co-transform heretofore unlinked markers at frequencies in excess of those found by congression. Comparison of the frequency of multiple marker transformations to single marker events as a function of DNA dilution conclusively proves that these markers originated from the same continuous strand of DNA.

Bacillus subtilis↗
PubMed1969

In vitro transformation by the adenovirus-simian virus 40 hybrid viruses. V. Virus-specific ribonucleic acid in cell lines transformed by the adenovirus 2-simian virus 40 and adenovirus 12-simian virus 40 transcapsidant hybrid viruses.

The ribonucleic acid-deoxyribonucleic acid hybridization technique was utilized to determine the presence of adenovirus (ad) and SV40 genetic information and to determine which ad genomes were present in clones of hamster cells transformed with the ad 2-SV40 and ad 12-SV40 transcapsidant hybrid virus populations. The results were correlated with the morphology of the transformed cells and colonies. It was found that cells transformed by either transcapsidant virus which had an SV40 morphology contained the ad 7 and SV40 genomes, whereas cells with a typical ad morphology contained only ad genetic information. Cells and colonies with morphological features of both ad- and SV40-transformed cells contained either the ad 2, or ad 12 genomes, depending on the transcapsidant used, together with the ad 7 and SV40 genomes. The results indicate the following: at least three different events occurred during transformation of hamster cells by the transcapsidant virus populations; the morphology of the resulting clones is determined by the viral genome(s) present; the linkage of the ad 7-SV40 genomes is confirmed since the ad 7- SV40 genomes were never found to be dissociated; the defective ad 7-SV40 genomes are capable of causing transformation; and the transcapsidant particle is probably composed of only ad 7 and SV40 genetic information.

Adenoviridae↗
PubMed1989

p53-plus-ras-transformed rat embryo fibroblasts express tumor-specific transplantation antigen activity which is shared by independently transformed cells.

p53-plus-ras-transformed rat cell lines express a tumor-specific transplantation antigen that is common to a number (85%) of independently derived p53-plus-ras-transformed cell lines. This has been shown by immunizing rats with irradiated p53-plus-ras-transformed cells and demonstrating protection of these animals by subsequent live-cell tumor challenge. Several c-myc-plus-ras-transformed cell lines (54% of the lines tested) and one adenovirus E1a-plus-ras-transformed cell line (9% of those tested) were shown to share a common tumor-specific transplantation antigen by their ability to immunize a rat against a p53-plus-ras cell line challenge. Several experimental approaches have been used to fractionate and identify the antigen common to these cell lines. The experimental results reported here make it clear that the p53 protein common to most of these transformed cell lines is not likely to be the tumor-specific transplantation antigen.

Animals↗
PubMed1999

The residues between the two transformation effector sites of Epstein-Barr virus latent membrane protein 1 are not critical for B-lymphocyte growth transformation.

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for EBV-mediated transformation of primary B lymphocytes. LMP1 spontaneously aggregates in the plasma membrane and enables two transformation effector sites (TES1 and TES2) within the 200-amino-acid cytoplasmic carboxyl terminus to constitutively engage the tumor necrosis factor receptor (TNFR)-associated factors TRAF1, TRAF2, TRAF3, and TRAF5 and the TNFR-associated death domain proteins TRADD and RIP, thereby activating NF-kappaB and c-Jun N-terminal kinase (JNK). To investigate the importance of the 60% of the LMP1 carboxyl terminus that lies between the TES1-TRAF and TES2-TRADD and -RIP binding sites, an EBV recombinant was made that contains a specific deletion of LMP1 codons 232 to 351. Surprisingly, the deletion mutant was similar to wild-type (wt) LMP1 EBV recombinants in its efficiency in transforming primary B lymphocytes into lymphoblastoid cell lines (LCLs). Mutant and wt EBV-transformed LCLs were similarly efficient in long-term outgrowth and in regrowth after endpoint dilution. Mutant and wt LMP1 proteins were also similar in their constitutive association with TRAF1, TRAF2, TRAF3, TRADD, and RIP. Mutant and wt EBV-transformed LCLs were similar in steady-state levels of Bcl2, JNK, and activated JNK proteins. The wt phenotype of recombinants with LMP1 codons 232 to 351 deleted further demarcates TES1 and TES2, underscores their central importance in B-lymphocyte growth transformation, and provides a new perspective on LMP1 sequence variation between TES1 and TES2.

B-Lymphocytes↗
PubMed1980

The preparation of transforming DNA from Mycoplasma hominis strain Sprott tetr and quantitative studies of the factors affecting the genetic transformation of Mycoplasma salivarium strain S9 tets to tetracycline resistance.

DNA extracted by a standard method from Mycoplasma hominis Sprott, resistant to 100 micrograms tetracycline, permitted the quantitative genetic transformation of tetracycline-sensitive Mycoplasma salivarium to resistance. The yield was 1 microgram DNA/10(9) cells. This DNA enabled determination of the optimum conditions for making M. Salivarium competent with CaCl2 and for studying some factors affecting transformation. Mycoplasma salivarium was transformed to resistance to 10, 20, and 30 micrograms tetracycline but not to 40 micrograms. The optimum DNA concentration for transforming resistance to 10, 20, and 30 micrograms tetracycline was the same, i.e., 50 micrograms DNA/10(8) viable cells. Treatment with DNase indicated that DNA uptake took 30 min. Competition between transforming DNA and DNA from calf thymus and M. salivarium tets inhibited transformation.

Calcium Chloride↗
PubMed1986

Transforming activity of DNA extracted from BKV-transformed hamster cells after passages in culture.

Primary fetal rat fibroblasts (FRF cells) were transfected with DNA extracted from BKV-transformed hamster kidney cells at low passage after transformation (HKBKlp cells) and at high passage after transformation (HKBKhp cells). Two transformed rat cell lines were obtained, FRF-DNA-HKBKlp cells and FRF-DNA-HKBKhp cells, transformed by DNA extracted from HKBKlp cells and HKBKhp cells, respectively. They were characterized by a number of biological properties and by tumorigenicity and metastatic ability in newborn hamsters. The two transformed FRF cell lines exhibited exactly inverted properties in comparison with HKBK cells, in particular FRF-DNA-HKBKlp cells showed a low level of nuclear T antigen and a diffuse appearance of TSTA at the cell surface, whereas FRF-DNA-HKBKhp cells demonstrated a high level of T antigen and the capping of TSTA in the cell membrane. The tumorigencity and the metastatic ability were greater with FRF-DNA-HKBKlp cells, while FRF-DNA-HKBKhp cells appeared to be less tumorigenic and metastatic in newborn hamsters.

Animals↗
PubMed1987

Marmoset lymphoblastoid cells transformed by NPC-KT-derived Epstein-Barr virus which possesses transforming and superinfecting characteristics.

New world monkey (cotton-top marmoset; CTM) lymphocytes were transformed by two different strains of Epstein-Barr virus (EBV) derived from human nasopharyngeal epithelial/hybrid cells (NPC-KT and A2L/AH). M-KT cells were a CTM lymphoblastoid cell line which was transformed by EBV derived from NPC-KT cells (NPC-KT EBV). M-BA2L cells were a CTM lymphoblastoid cell line which was transformed by EBV derived from A2L/AH cells (A2L/AH EBV). EBV obtained from M-KT cells, like parental NPC-KT EBV, could transform human cord blood lymphocytes (HCBLs) and superinfect Raji cells. EBV obtained from M-BA2L cells, like parental A2L/AH EBV, could transform HCBLs but could not superinfect Raji cells. We have compared the EBV-DNA associated with M-KT cells to NPC-KT EBV-DNA. The results obtained using a DNA restriction enzyme (Hind III) showed that virus DNA prepared from NPC-KT cells is different from EBV-DNA prepared from CTM lymphocytes transformed by EBV derived from infectious mononucleosis (B95-8), but that virus DNA prepared from M-KT cells is identical with parental NPC-KT EBV. These results indicate the possibility that a single NPC-KT EBV is associated with two biological activities.

Animals↗
PubMed1995

The Syrian hamster embryo (SHE) cell transformation system: a biologically relevant in vitro model--with carcinogen predicting capabilities--of in vivo multistage neoplastic transformation.

Neoplastic transformation is a multistep process that can be modeled in vitro using Syrian hamster embryo (SHE) cells. SHE cells multistage transformation involves several intermediate stages, including morphological transformation, immortality, acquisition of tumorigenicity, and malignant progression. Analysis of the molecular alterations that occur at each stage indicated that morphological transformation results from both carcinogen-induced irreversible chromosomal/genetic mutations and reversible genetic events, including altered DNA methylation. Morphological transformation results from a block in the cellular differentiation of progenitor and determined stem-like cells in the SHE cell population via alternation in the expression of the H19 tumor suppressor gene and other genes. Immortality results from genetic mutations in growth factor responsiveness, including loss of growth suppression by TGF beta and autocrine growth factor production, and genomic stability, resulting in genomic instability and an increased mutation rate. Acquisition of tumorigenicity involves loss of tumor suppressor gene function, altered mitogenic signal transduction, mutation of oncogenes, acquisition of anchorage independent growth, and chromosomal aberrations. Malignant progression is associated with alterations in extracellular matrix growth characteristics, alterations in cytoskeleton structure, elevated fibrinolytic activity, secretion of proteases, and changes in extracellular matrix protein secretion. Together, these changes model the alterations observed during in vivo neoplastic transformation and possibly explain why the SHE assay, as a carcinogen screening tool, is able to identify carcinogens with a 80 to 85% accuracy.

Animals↗
PubMed2001

[Mechanism of transformation in Pichia methanolica yeast: transforming and nontransforming genes].

Two types of genes were found in the study of transformation in yeast Pichia methanolica: transforming (Trg) and nontransforming (Ntg) genes. Transforming genes (P-ADE7,4 and S-LEU2), as linear DNA molecules, can transform competent cells with high efficiency inversely proportional to the molecule size. Nontransforming genes (P-ADE5 and H-LEU2) transform P. methanolica cells at an extremely low rate even when they are combined with transforming genes. The analysis showed that linear DNA molecules with Trg and Ntg can be either rearranged and integrated in random sites of the recipient genome or form circular plasmids, which are capable of autonomous replication irrespective of the presence of specific replicative elements.

Gene Deletion↗
PubMedDate not supplied

Selective and non-selective apoptosis induction in transformed and non-transformed fibroblasts by exogenous reactive oxygen and nitrogen species.

This paper defines selective and nonselective apoptosis induction in transformed and nontransformed fibroblasts by extracellular reactive oxygen and nitrogen species (ROS and RNS). Superoxide anions do not exhibit direct apoptosis inducing potential, whereas their dismutation product hydrogen peroxide induces apoptosis nonselectively in transformed and nontransformed fibroblasts. Myeloperoxidase converts hydrogen peroxide into the selective apoptosis mediator HOCl, which interacts with transformed target cell-derived superoxide anions and generates apoptosis-inducing hydroxyl radicals. Nitric oxide does not induce apoptosis directly in fibroblasts. However, interaction of nitric oxide with transformed cell-derived superoxide anions leads to the generation of the ultimate apoptosis inducer, peroxynitrite. Peroxynitrite by itself does not discriminate between transformed and nontransformed cells. The direction of generation of apoptosis inducing ROS and RNS to the site of superoxide anion production has relevance for the selectivity of ROS and RNS-based natural antitumor systems, as extracellular superoxide anion generation represents a hallmark of the transformed state.

Animals↗
PubMed2006

[Combination of growing culture transformation and resting cells transformation of Pseudomonas putida NA-1 for production of 6-hydroxynicotinic acid].

The current technology for microbial hydroxylation of nicotinic acid is a procedure of resting cells transformation. Here it was reported that non-degradation of 6-hydroxynicotinic acid during hydroxylation of nicotinic acid by resting cells was due to existing of nicotinic acid in the transformation mixture, not the death of cells. Therefore it was attempted that the induced cultivation of Pseudomonas putida NA-1 for hydroxylation activity by nicotinic acid was used for accumulation of 6-hydroxynicotinic acid to establish a new technology, a combined technology of growing culture transformation and resting cells transformation. In the combination technology, when 1% of nicotinic acid was kept in cultivation medium by supplement of nicotinic acid at intervals during cultivation of Pseudomonas putida NA-1, the growing culture could consistently hydroxylate nicotinic acid without degradation of its product which was accumulated linearly in the cultivation medium with progress of cultivation; the resting cells obtained from cultivation broth after growing culture transformation still processed a very high hydroxylation activity and could be used to next resting transformation of nicotinic acid. By using of the combined technology, the inducer was saved and the production yield of 6-hydroxynicotinic acid increased above 65%.

Nicotinic Acids↗
PubMed1985

[Analysis of Epstein-Barr virus transformation of human lymphocytes: susceptibility of B lymphocyte subpopulations and differentiation stage of the transformed cells].

The subpopulations of B lymphocytes in human adult peripheral blood which are susceptible to infection and transformation by Epstein-Barr virus (EBV), and also the stage of EBV-transformed cells in B cell differentiation lineage were identified. The EBV susceptibility were analyzed by two-color immunofluorescence and colony formation in semisolid agar. Surface Ig-bearing lymphocytes of 3 major Ig classes, IgM, IgG, and IgA, in peripheral blood expressed viral nuclear antigen (EBNA) after exposure to the virus, indicating that all these subpopulations are susceptible to EBV infection. In contrast, cytoplasmic Ig-positive lymphocytes were totally resistant to infection. The Ig-positive cells of either Ig class proliferated equally after EBV infection and formed colonies in semisolid agar, thereby showing that these subpopulations are also susceptible to transformation by EBV. Analysis of clonally transformed cells originating from each target cell by using a series of newly developed monoclonal antibodies to B cell differentiation antigens showed that EBV-transformed cells corresponded to the immunoblast stage of B cell differentiation. These data suggest that EBV infects and transforms mature B lymphocytes, and makes them differentiated to the more advanced immunoblast stage within the terminal B cell differentiation process.

Antibodies, Monoclonal↗
PubMed1986

The set of growth factors stimulatory for a transformed rat cell of line NRK-49F depends on the identity of the transforming virus.

Re-cloned rat fibroblast line NRK-49F was transformed by human adenovirus type 5 which had been grown in human cells of the KB carcinoma line. Cell subclones were isolated from seven independent transformation events, and seven untransformed cell subclones were isolated in parallel from control cultures which had been inoculated with lysate of uninfected KB cells. The adenovirus-transformed cells differed from the control untransformed fibroblasts by being typically small and cuboidal, showing multilayered growth, producing colonies in soft-agar medium, and growing to higher saturation density. Adenovirus-transformed subclones showed no infective virus and no consistent difference from control subclones in karyotype (mode = 41). Like the original re-cloned NRK-49F cells, all fourteen subclones required epidermal growth factor, fibronectin, insulin, and retinoic acid for optimal growth in serum-free culture. Thus, transformation by HA5 did not alter the required set, in contrast to the earlier finding that transformation of this same re-cloned line by polyoma virus eliminated the requirements for insulin and retinoic acid.

Adenoviridae↗
PubMed1987

A transforming gene, hst, found in NIH 3T3 cells transformed with DNA from three stomach cancers and a colon cancer.

DNA samples from 5 out of 15 stomach cancers and from 5 out of 15 colon cancers showed transforming activity in NIH 3T3 cells upon transfection. A transforming gene from an NIH 3T3 transformant induced by transfection of DNA from a stomach cancer was cloned, and showed transforming activity amounting to 120 focus-forming units/micrograms. The transforming gene was identified as hst. The hst gene was also responsible for acquisition of the transforming activity in DNA samples from 2 other stomach cancers and one colon cancer.

Animals↗
PubMed1984

Generation of new transforming viruses from SIRC cells "phenotypically transformed" in the medium with low concentrations of serum or calcium ions.

The work reported here demonstrates the possibility of rapid generation of highly transforming viruses from rabbit epithelial corneal cells (SIRC) originated from simian sarcoma associated virus (SSAV). The results of the experiments indicate a possible association of the phenomenon of "phenotypic transformation" with the expression of cellular genes with a potential transforming activity. SIRC/SSAV phenotypic transformation was induced by cultivation of these cells in the medium with a low concentration of serum or calcium ions. During subsequent infection of mink lung fibroblasts the authors succeeded in obtaining two new transforming SSAV isolates which induced transformation foci of various morphological types in cellular lines permissive for SSAV replication.

Animals↗
PubMed1997

Transformation of rabbit vascular smooth muscle cells by human cytomegalovirus morphological transforming region I.

The association of human cytomegalovirus with atherosclerosis and the monoclonal hypothesis of atherogenesis suggested that transformation of vascular smooth muscle cells may be an outcome of the virus-host cell interaction. To test this hypothesis, rabbit aorta smooth muscle cells were transfected with the morphological transforming region I (mtrI) of human cytomegalovirus (HCMV) linked to the neomycin resistance gene. Foci of neomycin-resistant and morphologically transformed cells were isolated and expanded into fourteen RCMV strains. Eight of these strains acquired immortalization, but only one strain (RCMV-21) retained recombined viral sequences integrated in the cellular DNA. RCMV strains were heterogeneous in their morphology, expression of smooth muscle alpha-actin, growth, and mitogenic response to serum and fibroblast growth factor (FGF)-2 and -4. All RCMV strains assayed except RCMV-3 showed DNA synthesis in low serum medium and, with the exception of RCMV-1 cells, all showed a significant mitogenic response to FGF-2 and FGF-4, Maintenance of the transformed phenotype appeared independent of the retention of the transforming viral sequences, which was suggestive of a "hit-and-run" mechanism. These results suggested that morphological transformation by HCMV DNA sequences could enhance the mitogenic response of vascular smooth muscle cells to fibroblast growth factors.

Animals↗
PubMed1983

Similar transforming growth factors (TGFs) produced by cells transformed by different isolates of feline sarcoma virus.

Fisher rat embryo cells transformed by each of three independent isolates of feline sarcoma virus (FeSV) are shown to release transforming growth factors (TGFs) into cell culture medium. These acid- and heat-stable peptides compete for binding to, and stimulate phosphorylation of, EGF membrane receptors and promote anchorage-independent cell growth. Cells transformed by the Gardner and Synder-Theilen strains of FeSV produce high titers of TGF (60-200 ng eq EGF/liter) while cells transformed by McDonough FeSV produce TGF at only low levels (less than 10 ng eq EGF/liter). Growth factors produced by cells transformed by each of the three FeSV isolates functionally and biochemically resemble each other, mouse sarcoma growth factor (SGF), and TGFs produced by human tumor cells.

Animals↗
PubMed1988

Positive selection of human cells lacking several transformation parameters from an SV40-transformed culture by means of parvovirus H-1.

The simian virus 40 (SV40)-transformed, newborn human kidney cell line NB-F was found to be heterogeneous with respect to its sensitivity to parvovirus H-1. The majority of the cells sustain a productive H-1 infection which eventually causes their lysis. Yet, a small fraction of the cells appears to be much less susceptible to H-1. Such a resistance to H-1 infection is a stable, transmissible property of this subpopulation of cells which was denoted NB-FR. The heterogeneity of NB-F cells is also apparent from the distribution of their karyotypes, which is bimodal and peaks at 114 and 46 chromosomes/cell. In contrast, the great majority of NB-FR cells contain 41-50 chromosomes. H-1-resistant and -sensitive cells appear to be related in several respects: they both contain morphologically human chromosomes as well as multiple SV40 DNA inserts, and could not be distinguished by isoenzyme typing. It was investigated whether the degree of sensitivity to H-1 infection correlated with other phenotypic properties of the human cell derivatives. NB-F cultures exhibit a series of transformation parameters, such as SV40 T-antigen expression, poor contact inhibition, clonogenicity in semi-solid medium and high lectin agglutinability, which are all much reduced or even undetectable in NB-FR cells. These observations suggest that cell susceptibility to H-1 segregates with marker(s) of in vitro malignant transformation. Moreover, the data indicate that parvoviruses can be used to preferentially remove transformants from a mixed culture of normal and transformed cells.

Antigens, Viral, Tumor↗