Showing papers by "Paul B. Fisher published in 1983"
TL;DR: DNA and RNA filter transfer hybridization analysis substantiated the conclusions that those cell lines capable of complementing viral host range mutants contained the appropriate viral DNA sequences and cytoplasmic polyadenylated RNA species.
Abstract: The recombinant plasmid pSV2-gpt, which contains the Escherichia coli XGPRT gene under the control of a simian virus 40 early promoter, was modified to contain the type 2 adenovirus (Ad2) XhoI-C (0 to 15.5 map units) restriction endonuclease fragment. Plasmid (pLB206) DNA was introduced into human KB cells by Ca2+-mediated DNA transfection, and transformants were selected in medium containing xanthine, aminopterin, and mycophenolic acid, as a consequence of expression of the dominant, selectable XGPRT gene. A series of 13 gpt+ cell lines were isolated and tested for their ability to complement Ad5 deletion mutants in E1a (H5dl312) and E1b (H5dl315). Four classes of gpt+ KB cell lines were identified, including clones constitutively expressing both E1a and E1b, only E1a, or only E1b or not expressing either E1a or E1b. DNA and RNA filter transfer hybridization analysis substantiated the conclusions that those cell lines capable of complementing viral host range mutants contained the appropriate viral DNA sequences and cytoplasmic polyadenylated RNA species. DNA filter transfer hybridization studies also revealed that the transfected vector DNA was stably integrated into chromosomal DNA in the KB transformants and the number of integrated sites ranged from 1 to 3. The gpt+ KB cell line that only expressed E1b gene functions only contained viral E1b gene sequences; those cell lines that expressed neither E1a nor E1b gene function contained only small or no regions of Ad2 DNA. When weaned off the selective medium, transformed KB cell lines stably maintained their inserted DNA in the absence of selective pressure and could easily be adapted to growth in suspension culture.
66 citations
TL;DR: These studies suggest that some of the antitumor effects of interferon may relate to its capacity to modulate cellular differentiation.
Abstract: We have studied the effects of human leukocyte interferon produced in bacteria and diterpene phorbol ester tumor promoters on differentiation of normal human myoblast cultures derived from mature skeletal muscle. Interferon (100-5,000 units/ml) induced an acceleration of myotube formation and creatine kinase (CK; EC 2.7.3.2) isoenzyme transition from CK-BB to CK-MM. Heat-inactivated or trypsin-treated interferon did not affect the differentiation process. In contrast, the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), but not its inactive structural analogues phorbol and 4 alpha-phorbol 12,13-didecanoate, caused a dose-dependent (0.01-100 ng/ml) inhibition of myotube formation and CK isoenzyme transition. Neither interferon nor TPA had a significant effect on myoblast proliferation prior to fusion, and the cloning efficiencies were similar as well. Opposing effects of interferon and TPA were also demonstrated by simultaneous application of these agents to the cultures. These studies suggest that some of the antitumor effects of interferon may relate to its capacity to modulate cellular differentiation.
56 citations
TL;DR: Findings indicate that the gene product affected by the hr1 mutation is cold sensitive and is essential for the expression of the characteristics that identify the transformed cell.
Abstract: hr1, an E1a (0-4.5 map units) host range mutant of type 5 adenovirus (Ad5), transformed a cloned rat embryo fibroblast (CREF) cell line at about a 5-fold higher frequency than wild-type (wt) Ad5 when cells were cultured at 37 degrees C. However, if the cells were infected with hr1 and maintained at 32 degrees C morphological transformation did not occur. When infected cells were shifted from 32 degrees C to 37 degrees C 2 wk postinfection, the frequency of transformation by 6 wk was identical to that of cells grown continuously at 37 degrees C, whereas cultures shifted from 37 degrees C to 32 degrees C 2 wk postinfection displayed a greater than 96% reduction in morphological transformation. hr1-transformed cells had a fibroblastic morphology as contrasted with the typical epithelioid morphology of wt Ad5-transformed cells, but hr1- and wt Ad5-transformed cells had similar saturation densities, growth rates, and agar cloning efficiencies when assayed at 37 degrees C. However, when cells transformed by hr1 at 37 degrees C were grown at 32 degrees C, they had a saturation density close to that of normal CREF cells and grew at a lower efficiency in agar than wt-transformed cells. DNA transfer/hybridization analysis of two hr1-transformed cloned cell lines, A2 and B3, indicated that A2 cells contained a complete integrated copy of the Ad5 genome, whereas in B3 cells only part of the Ad5 genome was integrated. RNA transfer and RNA/DNA filter hybridization analyses indicated that the types of viral messenger RNAs and the relative amounts of RNA transcribed were similar in the A2 and B3 cell lines when they were grown at 32 degrees C and 37 degrees C. Indirect immunofluorescence, with antisera from hamsters bearing Ad-induced tumors, indicated a temperature dependence in staining--i.e., cells grown at 37 degrees C or shifted from 32 degrees C to 37 degrees C contained intense, particulate staining in the nuclear region, whereas the staining was decreased significantly in cells cultured at 32 degrees C and in cells shifted from 37 degrees C to 32 degrees C. These findings indicate that the gene product affected by the hr1 mutation is cold sensitive and is essential for the expression of the characteristics that identify the transformed cell.
41 citations
TL;DR: It is demonstrated that human myoblasts can be immortalized by SV40, and this procedure may prove useful for generating large quantities of genetically deficient human cells for biochemical and molecular analysis.
Abstract: Molecular studies of the biochemical alterations involved in human myopathies have been restricted because of the finite life-span and slow growth rate of cultures derived from primary tissue. Because the tumor virus simian virus 40 (SV40) can alter both the growth properties and longevity of human cells, we have infected skeletal muscle cultures derived from four biopsies with a small-plaque variant of SV40 and analyzed the biological and biochemical properties of cloned myoblast derivatives. At early times after infection, myoblasts fused normally into multinucleated myotubes, and both unfused and fused cells contained SV40 tumor antigen (T antigen). After six to eight subcultures after infection, the ability of myoblasts to fuse diminished, and clonal cell lines were generated with increased growth rates and saturation densities. Transformed cultures also lost contact inhibition of growth and became anchorage independent. Unlike untransformed myoblasts, SV40-transformed clones did not undergo an increase in creatine kinase activity or a transition of creatine kinase isoenzymes from the BB form to the muscle-specific MM form. Analysis of the pattern of SV40 DNA integration by Southern blotting hybridization analysis in two cloned SV40-transformed myoblast cell lines (KJ-SV40 and PK-SV40) indicated that KJ-SV40 contained at least one site of SV40 DNA integration into chromosomal DNA and PK-SV40 contained at least three sites of SV40 DNA covalently linked to cellular DNA. Cell lysates and growth medium from PK-SV40 transformants contained infectious small-plaque variant SV40, whereas KJ-SV40 did not contain or produce detectable virus. These studies demonstrate that human myoblasts can be immortalized by SV40. This procedure may prove useful for generating large quantities of genetically deficient human cells for biochemical and molecular analysis.
35 citations
TL;DR: These results provide the first evidence that cells transformed by an adenovirus can produce a growth factor(s) that inhibits EGF‐receptor binding and induces anchorage‐independent growth of normal cells.
Abstract: Transformation of Sprague-Dawley rat embryo (RE) cells and a cloned Fischer rat embryo cell line (CREF) with wild-type (Ad5) or a temperature-sensitive DNA-minus mutant (H5ts125) of type 5 adenovirus results in a reduction in binding of epidermal growth factor (EGF) to cell surface receptors. A reduction in EGF binding is also seen in a Syrian hamster embryo cell line transformed by a hexon mutant of Ad5. In contrast, a human embryonic kidney cell line (293) transformed by sheared Ad5 DNA or transfected clones of KB cells expressing the E1 transforming region of Ad5 do not show a decrease in receptor binding. When cocultivated, the adenovirus transformed rat cells were able to induce the growth in agar of normal CREF cells. Medium from Ad5 transformed RE cells stimulated the growth in agar of CREF cells and also inhibited [125I]-EGF binding in CREF cells. When fractionated by gel filtration, two peaks of [125I]-EGF inhibiting activities were obtained with apparent molecular weights of 35,000 and 16,000. These results provide the first evidence that cells transformed by an adenovirus can produce a growth factor(s) that inhibits EGF-receptor binding and induces anchorage-independent growth of normal cells.
19 citations
TL;DR: The results indicate that thyroid hormone modulates an early stage involved in adenovirus transformation and that it also enhances the expression of the transformed state in previously transformed cells.
Abstract: We have examined the effect of triiodothyronine (T3) on de novo transformation of a cloned population of Fischer rat embryo fibroblasts (CREF) by a temperature-sensitive mutant (H5ts125) of type 5 adenovirus and on the expression of the transformed phenotype in these cells. When CREF cells were grown in medium lacking T3 before, during, and after infection with H5ts125, the yield of transformed foci was half that in the cultures supplemented with 1 nM T3. Selective addition or removal of T3 during various phases of the transformation process indicated that the hormone exerted its maximal effect within 72 hr after viral infection. T3 was also required for optimal growth in agar of two clones of CREF cells previously transformed by type 5 adenovirus, wt-3A and ts-7E. The tumor promoter 12-O-tetradecanoylphorbol 13-acetate could substitute for T3 in enhancing growth in agar of wt-3A but not of ts-7E, suggesting that the promoter and T3 modify anchorage-independent growth by different mechanisms. Normal CREF cells and both of the transformed CREF clones grew equally well in monolayer culture in medium containing or lacking T3. Both of the transformed CREF clones contained a lower number of nuclear T3 receptors than did CREF cells and they bound somewhat lower levels of phorbol dibutyrate. These results indicate that thyroid hormone modulates an early stage involved in adenovirus transformation and that it also enhances the expression of the transformed state in previously transformed cells.
15 citations