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Hormonal regulation of the transformation phenotype in simian virus 40-transformed rat embryonic preadipose cell lines.

The regulation of transformed phenotypes was studied in newly isolated preadipose cell lines which were established after infection with simian virus 40 tsA58 dl2009. The clonal cell lines isolated exhibited most of the characteristics typical of transformed cells. The transformants, however, were able to differentiate into adipocytes in the presence of low calf serum (0.5%) and a combination of several hormones, including hydrocortisone and insulin. Treatment with insulin alone stimulated the growth of these cells but did not induce lipid accumulation without added hydrocortisone. The effect of hydrocortisone was accompanied by a restoration of growth control in the transformants after they reached high cell density. The blot hybridization analysis of cellular DNAs digested by restriction enzymes revealed that simian virus 40 genomes were integrated at multiple separate sites at which a head-to-tail oligomeric insertion took place. Large T antigen was synthesized in growing cells but was regulated at high cell density when cells were committed to differentiate by glucocorticoids. These results suggest that the glucocorticoid hydrocortisone is capable of restoring growth regulation at high cell densities to simian virus 40-transformed preadipose cell lines.

Adipose Tissue↗

Transformation-defective mutant of avian myeloblastosis virus that is temperature sensitive for production of transforming protein p45v-myb.

We have characterized a mutant of avian myeloblastosis virus (strain GA907/7) that shows a reduced capacity to transform myelomonocytic cells at the nonpermissive temperature. Myeloblasts transformed by this mutant suffer a substantial decrease in the amount of the transforming protein p45v-myb when shifted from the permissive to the nonpermissive temperature. We presume that the 5- to 10-fold decrease in the amount of p45v-myb causes the loss of the transformed phenotype. The decrease is due to a reduction in the level of v-myb mRNA. Mutant GA907/7 thus provides genetic evidence that p45v-myb is the transforming protein of avian myeloblastosis virus and apparently represents an unusual defect in the production or stability of mRNA.

Animals↗

Karyotype analysis and quantitation of viral transforming genes in Rous sarcoma virus transformed, revertant, and retransformed field vole cells.

Comparative studies of the number of cellular chromosomes and viral genes, including the gene required for malignant transformation, were performed on several clones of Rous sarcoma virus-transformed, revertant, and spontaneously retransformed field vole cells. The results of these studies indicate that no appreciable differences in either total viral gene equivalents or transforming gene sequences can be detected between transformed and revertant cell types, even though considerable differences in the number of certain chromosomes exist among the clones tested. Furthermore, no increase in the amount of total genes or transforming gene sequences accompanies retransformation of revertant clones, including clones that exhibited significant increases in chromosome number following retransformation.

Animals↗

The shape of the dose-response curve for radiation-induced neoplastic transformation in vitro: evidence for an adaptive response against neoplastic transformation at low doses of low-LET radiation.

A dose-response curve for gamma-radiation-induced neoplastic transformation of HeLa x skin fibroblast human hybrid cells over the dose range 0.1 cGy to 1 Gy is presented. In the experimental protocol used, the spontaneous (background) frequency of neoplastic transformation of sham-irradiated cultures was compared to that of cultures which had been irradiated with (137)Cs gamma radiation and either plated immediately or held for 24 h at 37 degrees C prior to plating, for assay for neoplastic transformation. The pooled data from a minimum of three repeat large-scale experiments at each dose demonstrated a reduced transformation frequency for the irradiated compared to the sham-irradiated cells for doses of 0.1, 0.5, 1, 5 and 10 cGy for the delayed-plating arm. The probability of this happening by chance is given by 1/2(n), where n is the number of observations (5); i.e., 1/32 congruent with 0.031. This is indicative of an adaptive response against spontaneous neoplastic transformation at least up to a dose of 10 cGy of gamma radiation. The high-dose data obtained at 30 and 50 cGy and 1 Gy showed a good fit to a linear extrapolation through the sham-irradiated, zero-dose control. The delayed-plating data at 10 cGy and below showed a statistically significant divergence from this linear extrapolation.

Adaptation, Physiological↗

[Agrobacterium rhizogenes-mediated transformation and the regeneration of transformants in Alhagi pseudalhagi].

The regenerated shoot segments of Alhagi pseudalhagi were sliced and infected with Agrobacterium rhizogenes strain A4. The hairy roots and transformed calli were obtained through selection on hormone free MS medium. The transformants were cultured on MS medium with 2 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5-1 mg/L 6-benzylaminopurine (6-BA) to induce calli. 3 mg/L 6-BA and 0.5 mg/L naphthalene acetic acid (NAA) were applied for shoot differentiation. Shoots were planted on MS medium with 2 mg/L indole-3-butyric acid (IBA) and produced roots. Opine analysis proved the integration and expression of T-DNA in over 95% hairy roots, 75% transformed calli and transformed plantlets respectively. The 81% hairy root cells had normal chromosome numbers (2n = 18). The alterations of chromosome number were observed. After one year of subculturing, the regeneration ability of transformants was maintained.

DNA, Bacterial↗

Differential tyrosine-specific phosphorylation of integrin in Rous sarcoma virus transformed cells with differing transformed phenotypes.

We have investigated the localization and phosphorylation of the fibronectin receptor in chick embryo fibroblasts transformed either by wild-type Prague C strain Rous sarcoma virus, which induces a rounded, less adhesive phenotype with a loss of surface fibronectin and gross cytoskeletal changes, or transformed by rASV2234.3, a variant which induces a flat, adhesive morphology with the retention of surface fibronectin and more normal cytoskeleton. Immunofluorescence showed co-distribution of pp60v-src with integrin and fibronectin fibrils in rASV2234.3-transformed cells. Total levels and surface expression of integrin were unchanged in both transformed cell types compared with untransformed cells. However, whereas integrin band 3 (beta 1 subunit) in Prague C-transformed cells was hyperphosphorylated on tyrosine, this was reduced virtually to normal in rASV2234.3-transformed cells. A similar differential phosphorylation of integrin band 3 could be found in membranes phosphorylated in vitro. rASV2234.3 pp60v-src was less efficient in phosphorylating a synthetic peptide containing the putative integrin tyrosine phosphorylation site, indicating that this variant pp60v-src has an altered substrate specificity compared to Prague C pp60v-src. The correlation between tyrosine-specific phosphorylation of integrin and loss of surface fibronectin suggests that this could play an important role in the induction of the transformed phenotype.

Amino Acids↗

Altered microfilament structure in cells transformed with a temperature-sensitive transformation mutant of murine sarcoma virus.

The structure and distribution of microfilaments were examined by electron microscopy in uninfected normal rat kidney (NRK) cells, murine sarcoma virus (MSV)-transformed NRK cells, and NRK cells infected with a cold-sensitive transformation mutant of MSV, i.e., NRK (MSV-1b) cells, grown at both permissive (39 degrees) and nonpermissive (33 degrees) temperature. The uninfected cells contained numerous microfilaments which were especially prominent at sites of intercellular adherens junctions. In contrast, the MSV-transformed cells contained few microfilaments and did not form adherens junctions. At 33 degrees, the NRK (MSV-1b) cells appeared normal but formed an altered form of adherens junction with disorganized microfilaments. At 39 degrees, these cells resembled NRK cells transformed by wild-type MSV but still formed a few of the altered type of adherens junctions. Disorganized adherens junction microfilaments were also found in cells newly infected with wild-type MSV. These results suggest that the perturbed assembly of microfilaments at adherens junctions may be an intermediate stage in the loss of adherens junctions during viral transformation.

Cell Line↗

Transformation by basic fibroblast growth factor requires high levels of expression: comparison with transformation by hst/K-fgf.

Basic fibroblast growth factor is a potent mitogen for a variety of cell types and has been suggested to have transforming activity. To test this hypothesis, we have introduced a human bFGF cDNA into NIH 3T3 cells either by DNA transfection or by retrovirus infection. We have compared the properties of cell lines obtained with cells prepared similarly but expressing the hst/K-fgf growth factor. While bFGF does not contain an amino terminal signal sequence and is not secreted from cells in which it is synthesized, hst/K-fgf does contain a signal sequence and is secreted from cells. Our results show that the transformed phenotype correlates directly with the level of bFGF expression, since all transformed clones expressed high levels of bFGF, while nontransformed clones expressed comparatively low levels of bFGF. In contrast, even low levels of hst/K-fgf expression resulted in a transformed phenotype. These results suggest that bFGF is an inefficient transforming protein and that this may relate to its lack of secretion.

Animals↗

Multistep nature of X-ray-induced neoplastic transformation in golden hamster embryo cells: expression of transformed phenotypes and stepwise changes in karyotypes.

We have examined the expression of transformed phenotypes and genetic changes associated with the expression of each transformed phenotype after X-ray irradiation. Unirradiated cells grown at a constant growth rate until 8 passages (population doubling number, 15) exhibited little morphological change and ceased to divide thereafter. X-irradiated cells escaped from senescence and showed morphological alteration and anchorage independence after a population doubling number of 20. The acquisition of tumorigenicity in nude mice was observed much later (35 population doublings after irradiation). From cytogenetic analysis, all anchorage-independent clones were consistently found to have trisomy of chromosome 7. Furthermore, cells derived from tumors contained three copies of chromosome 9q in addition to the trisomy of chromosome 7. We have not detected any augmented expression of v-Ha-ras- and v-myc-related oncogenes with RNA dot-blot analysis and could not find activation of any type of oncogenes by NIH3T3 transfection experiments. Our studies demonstrated that X-ray-induced neoplastic transformation is a multistep phenomenon and that the numerical change of specific chromosomes may play an important role in the expression of each transformed phenotype. The results suggest that different endogenous oncogenes, other than the ras gene family and myc oncogene, could be responsible for the progressive nature of neoplastic transformation.

Animals↗

Transforming activity of human c-Ha-ras-1 proto-oncogene generated by the binding of 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole and 4-nitroquinoline N-oxide: direct evidence of cellular transformation by chemically modified DNA.

An activity that transforms NIH 3T3 cells was generated by the in vitro modification of plasmids containing the human c-Ha-ras-1 proto-oncogene with the synthesized ultimate carcinogen, 2-acetoxyamino-6-methyldipyrido[1,2-a:3',2'-d]-imidazole (N-OAc-Glu-P-1). DNAs isolated from the transformed cells were analyzed by restriction fragment length polymorphism (RFLP) assay using the restriction enzyme Msp I. Of fourteen transformants studied, six contained a mutation in the region of the CCGG sequence of the eleventh and the twelfth codons, in which GG corresponds to the first two nucleotides of the twelfth codon. Transforming activity was also generated by the chemical modification of the plasmids with 4-acetoxyaminoquinoline N-oxide (N-OAc-4AQO). The results clearly indicate that formation of DNA adducts with N-OAc-Glu-P-1 or N-OAc-4AQO causes the induction of transformation of mammalian cells.

4-Nitroquinoline-1-oxide↗

The intracellular distribution of the transformation-associated protein p53 in adenovirus-transformed rodent cells.

The intracellular distribution of the transformation-associated cellular protein p53 was studied by indirect immunofluorescence in a series of adenovirus-transformed rodent cells. In most adenovirus 2 or 5 (group C) transformed cell lines p53 was detected in discrete areas in nuclei and in all cell lines p53 was also present in a perinuclear structure. The adenovirus 2 or 5 E1B-58 kD protein, previously found to form molecular complexes with p53 in group C transformed cells, colocalized with p53 in both intracellular locations. Further studies on the region of the cell corresponding to the perinuclear body containing p53 showed that it frequently included the centrosomes of the transformed cell. The intranuclear p53 was released by mild DNAase I digestion.

Animals↗

Anchorage-independent growth of v-myc-transformed Balb/c 3T3 cells is promoted by platelet-derived growth factor or co-transformation by other oncogenes.

Murine fibroblasts transformed by the myc oncogene have a reduced growth factor dependence for both anchorage-dependent and anchorage-independent proliferation. Here we show that v-myc-transformed Balb/c 3T3 cells require, in addition to insulin, only platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) for anchorage-dependent growth in serum-free media. PDGF, however, cannot efficiently be substituted by EGF for anchorage-independent growth. The results suggest that constitutive v-myc expression reduces cellular growth factor requirements by non-autocrine mechanisms for proliferation in monolayer cultures. In contrast, v-myc-transformed cells require plasma components and growth factors of the 'competence' type for anchorage-independent growth. We also demonstrate that the requirement for PDGF by the myc-transformed cells can be abrogated by v-K-ras, v-src and v-fos but not the v-raf oncogene. The results demonstrate that oncogenes can cooperate in the expression of the transformed phenotype by also drastically reducing cellular growth factor requirements.

Animals↗

Genetic transformation in Streptococcus sanguis. Effects on genetic transformation by culture filtrates of Streptococcus sanguis (serogroups H and W) and streptococcus mitis (mitior) with reference to identification.

Streptococcus sanguis (serogroups H and W) strains are frequently competent in genetic transformation. In a collection of 18 strains of S. sanguis isolated from blood cultures, 12 strains were spontaneously competent in transformation by streptomycin-resistant DNA, and three strains were induced to competence by the use of culture filtrates containing competence factor (CF). Culture filtrates from spontaneously competent S. sanguis strains only induced competence in transformation of strains of the same species. In addition to the three "constitutional" types of S. sanguis with regard to specificity of transformation previously reported (originally represented by the strain Challis, 13b and NCTC 7863), a possible fourth type, (represented by strain 21452) was found. Further studies are required to ascertain the role of these types. The "constitutional" types are characterized by the range of activity of their CF's. These specificities seem useful in identification of S. sanguis and its separation from S. mitis (mitior). Tests of spontaneously competent strains with culture filtrates from other competent strains showed variable effects on the transformation frequencies in different strains.

Bacterial Proteins↗

A rapid screening for the specific DNA sequence: analysis of transforming DNA segments in adenovirus-transformed cells.

The viral DNA sequences in cells transformed by adenovirus type-12 (Ad12) DNA fragments were investigated by spot hybridization, a detection by autoradiography of nucleic acid hybrids formed between cell DNAs spotted on a membrane filter and various nick-translated Ad12 DNA fragments. In CY1 cell line, a rat cell line transformed by the EcoRI-C fragment (left hand 16%), all of the HindIII fragments included in the EcoRI-C fragment are shown to be present. In GY1 cell line, a rat cell line transformed by the HindIII-G fragment (left hand 7%), both of the BpaI-H and a part of the BpaI-J, two components consisting of the HindIII-G fragment, were found. A dominant presence of the Ad12 BpaI-H fragment (left hand end 4.5%) of the Ad12 DNA molecule, approximately 60% of the Ad12 transforming DNA sequences (Ad12 HindIII-G), was also shown in GY1. Spot hybridization used in the experiment will be of general use for detecting viral nucleic acid sequences in cells and provides a simple and useful screening method for investigating viral etiology of tumors and transformed cells.

Adenoviruses, Human↗

[Malignant transformation of thyroid cancer; poorly differentiated cancer and anaplastic cancer transformation].

The prognosis of thyroid cancer depends largely on histological differentiation and clinical stage. The prognosis in anaplastic cancer is worst, in well differentiated cancer it is best; poorly differentiated cancer takes a middle position. The prognosis of patients with differentiated cancer is generally good, however, there are cases with malignant transformation such as poorly differentiated, anaplastic and squamous-cell cancer transformation. We studied the frequency and the inducement factors of malignant transformation in patients with thyroid cancer. The frequency of poorly differentiated, anaplastic and squamous-cell cancer transformation was 13.6%, 6.4% and 0.7%, respectively. In anaplastic cancer transformation, irradiation is an important factor.

Adult↗

Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection.

Freeze-thaw transformation provides a simple and rapid method to transform Agrobacterium tumefaciens directly with plasmid DNA. Competent A. tumefaciens cells of strains LBA4404, GV3850 and EHA101 were transformed with four to nine plasmids differing in size, size of insert and in some cases sensitivity to antibiotics. A threefold to fourfold increase in transformed colonies per microgram of DNA was obtained by freezing cells with liquid nitrogen vs. dry ice/ethanol. Freezing cells in liquid nitrogen followed by incubation of transformed cells in a low concentration of appropriate antibiotics prior to plating resulted in a ninefold increase in colonies obtained compared with the procedure of freezing cells in dry ice/ethanol without the incubation period in the low concentration of antibiotics prior to plating. Restriction fragments of the expected sizes from the plasmids indicated that the procedural modifications did not cause apparent recombinations in the region of the inserts.

Agrobacterium tumefaciens↗

Bcl-2 and CrmA have different effects on transformation, apoptosis and the stability of I kappa B-alpha in chicken spleen cells transformed by temperature-sensitive v-Rel oncoproteins.

The retroviral oncoprotein v-Rel is a member of the Rel/ NF-kappa B family of transcription factors. We have previously characterized two v-Rel mutants (v-G37E and v-R273H) that are temperature-sensitive (ts) for transformation and immortalization of chicken spleen cells in vitro. We have now constructed vectors for the co-expression of wild-type or ts mutant v-Rel proteins and the anti-apoptosis proteins Bcl-2 or CrmA. The formation of v-Rel-transformed colonies is enhanced in the presence of overexpressed Bcl-2. Moreover, co-expression of Bcl-2 suppresses apoptosis that is induced when ts v-Rel-transformed cells are shifted to the non-permissive temperature. However, co-expression of Bcl-2 in these cells does not affect ts functions of v-Rel, such as DNA binding and stabilization of I kappa B-alpha. In contrast, co-expression of CrmA does not suppress apoptosis, but does block an amino-terminal proteolysis of I kappa B-alpha that occurs in ts v-G37E-transformed cells shifted to the nonpermissive temperature, indicating that an ICE-like protease activity is not involved in apoptosis in these cells but is involved in proteolysis of I kappa B-alpha. In addition, CrmA can block cycloheximide-induced amino-terminal processing of I kappa B-alpha in spleen cells transformed by wild-type v-Rel. In summary, these results suggest that v-Rel immortalizes chicken spleen cells through a pathway that involves the Bcl-2 family of proteins, and suggest that one pathway of proteolysis of I kappa B-alpha involves an ICE-like protease.

Animals↗

Carbohydrate compositions of normal, spontaneously transformed, and virally transformed cells derived from BALB/c mice.

Carbohydrate compositions of chloroform: methanol-soluble and -insoluble complex polysaccharides have been studied in cell lines derived from BALB/c mice. The cells used in these studies include normal cells, spontaneous and viral transformants that cause tumors that regress, and spontaneous and viral transformants that cause progressively growing tumors that kill immunocompetent BALB/c mice. The carbohydrates were determined by gas-liquid chromatography. Some of the transformed cell lines compared with normal cells have altered carbohydrate compositions, including decreased sialic acid levels and decreased N-acetylgalactosamine in chloroform: methanol-soluble material. Significant decreases in total carbohydrates of chloroform: methanol-soluble material were also observed in some of the transformed cells. However, these changes alone do not predict cancer. Transformed cell lines that cause progressively growing tumors tend to have fewer alterations in carbohydrates of complex polysaccharides than lines causing tumors that regress.

Animals↗