Differences between the Ribonucleic Acids of Transforming and Nontransforming Avian Tumor Viruses

Duesberg, Peter H.; Vogt, Peter K.

doi:10.1073/pnas.67.4.1673

Cited by 256 publications

(234 citation statements)

References 27 publications

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“…After the discovery of the 'cancercausing' sarcoma virus by Rous in 1909 3,4 research into genetics continued and the hypothetical gene thought to be responsible for a cell becoming cancerous was named an oncogene 5 . In the 1960s the src (sarcoma) oncogene was discovered in Rous's sarcoma virus (RSV) 6 and in 1975 this oncogene was found in not only cancerous cells 7 , but also in normal healthy cells 8 . This led researchers to hypothesize that the src oncogene was triggered to become cancerous by the introduction of the virus into the cells.…”

Section: Cancermentioning

confidence: 99%

Role of the pro-survival molecule Bfl-1 in melanoma

Hind

Carter

Harris

et al. 2015

The International Journal of Biochemistry & Cell Biology

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Melanoma, the cancer derived from the melanocytes of the skin, is becoming an ever more common cancer in the ageing population of the developed world and displays an intrinsic resistance to current therapies. This chemo resistance makes metastatic melanoma an extremely difficult cancer to treat leading to a 5 year survival rate of just 20%. As such, it is important to unearth new potential drug targets to increase the prospects for melanoma patients.Bfl-1 is a pro-survival protein of the Bcl-2 family of apoptosis regulating proteins. It has been found to be over-expressed in a small subset of chemo resistant tumours, including melanoma. Accordingly, I characterised the molecule in melanoma cells and determined its role in protecting these cells from chemotherapy-induced apoptosis. I elucidated the expression profile and half-lives of the protein and mRNA across a panel of melanoma cell-lines and healthy melanocytes. I also confirmed, using pathway specific inhibitors, that Bfl-1 was transcriptionally regulated by the NFB pathway and concluded through pulsechase experiments that Bfl-1 protein was degraded, at least in part, by the proteasome. Subcellular fractionation and indirect immunofluorescence techniques elucidated that Bfl-1 was located mostly at the mitochondria in both resting and apoptotic cells, with a diffuse level present throughout the cytoplasm. As well as characterising the protein in both melanoma cells and healthy primary melanocytes, I determined its role in the resistance of these cells to apoptosis. Over-expressed Bfl-1 was found to protect melanoma cells from apoptosis caused by a range of chemotherapeutic agents in A375 melanoma cells, which naturally expressed very low levels of Bfl-1. Further, knock down of Bfl-1 using siRNA technology in melanoma cells revealed the dependence of these cells on Bfl-1 for their resistance to certain chemotherapeutic agents currently used in melanoma treatment, including the MEK inhibitor PD901. All together, this research contributes to our understanding of Bfl-1 and highlights it as a potentially important therapeutic target in metastatic melanoma.

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Section: Cancermentioning

confidence: 99%

Role of the pro-survival molecule Bfl-1 in melanoma

Hind

Carter

Harris

et al. 2015

The International Journal of Biochemistry & Cell Biology

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“…A maximum of 10-13% of the viral RNA formed RNase-resistant duplexes with cDNAc0; this value was achieved when the complementary sequences of DNA and RNA were annealed at a ratio of 1-3 (Table 1). These findings indicate that cDNAsarc is a reasonably uniform representation of 10-13% of the ASV genome (about 1300 nucleotides), which represents about 60% of the deletion in the strain of td virus used for the selection (15)(16)(17)(18). Second, we isolated the viral RNA that hybridized to cDNAsrc and analyzed the two-dimensional chromatogram of oligonucleotides released from this RNA by hydrolysis with Ti RNase (Sankyo) in low salt according to published procedures (19,20); homochromatography was carried out with Homomix B (20).…”

mentioning

confidence: 99%

Nucleotide sequences related to the transforming gene of avian sarcoma virus are present in DNA of uninfected vertebrates.

Spector¹,

Varmus²,

Bishop³

1978

Proc. Natl. Acad. Sci. U.S.A.

206

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We have detected nucleotide sequences related to the transforming gene of avian sarcoma virus (ASV) in the DNA of uninfected vertebrates. Purified 1 for review). The rate of evolution of such sequences has been estimated from the final extent of annealing and thermal stability of duplexes formed between cellular DNA and virus-specific reagents. In general, virusspecific DNA in cellular genomes appears to evolve at least as rapidly as cellular unique-sequence DNA; this pattern has been documented for viral DNA endogenous to primates (2), cats (3), rodents (4), and birds (5-10). In some instances, the apparently rapid evolution of virus-specific DNA could be explained by its relatively recent introduction into the germ line of a species by infection with a virus originating in another species (3).In the cases cited, the hybridization reagents were not specific for defined portions of the viral genome, and the endogenous viruses under study were, in most cases, not oncogenic. Stehelin et al. (11) isolated radioactive DNA (cDNAsarc) complementary to most or all of the viral gene(s) (src) required for transformation of fibroblasts by avian sarcoma virus (ASV) (11-13).cDNA,.rc annealed to DNA not only from normal chickens, the presumed natural host for ASV, but also to DNA from several other birds spanning 100 X 106 years of evolution (10). This apparent conservation of nucleotide sequences related to src (hereafter called "sarc sequences") during avian speciation contrasted sharply with the lack of conservation of sequences encoding a nontransforming virus endogenous to chickens and closely related to ASV. Moreover, the conservation of cellular sarc sequences suggested that they might have an important, but as yet unknown, function, and that they might be present, albeit in a diverged form, in vertebrates other than birds.In the previous study (10) (14). Homogenized tissue or cells were suspended in buffer containing 0.1 M NaCI, 10 mM Tris-HCI (pH 7.4), 1 mM EDTA, 0.5% sodium dodecyl sulfate (NaDodSO4), and 500 ,ug of Pronase per ml at 370 for 1-2 hr and extracted with phenol/chloroform/isoamyl alcohol (25:24:1 vol/vol). The high molecular weight DNA was then spooled out of solution after addition of ethanol. High molecular weight salmon sperm and calf thymus DNA (Worthington Biochemical Corp.) were treated as above.The DNA was reduced to 6-7 S and residual RNA was digested by boiling in 0.3 M NaOH for 20 min. The samples were then neutralized with HCl, adjusted to 0.5% NaDodSO4, treated with 500 ,ug of Pronase per ml for 45 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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“…Double-stranded RNAs have been reported (14) in extracts of cells infected with vaccinia virus (a DNA virus) and may play a role in the replication of RNA viruses. Moreover, in the intact interferon-treated, vaccinia virus-infected L-cell all protein synthesis is inhibited (15), as would be expected if small amounts of viral dsRNA were produced.…”

Section: Resultsmentioning

confidence: 99%

Interferon-induced inhibition of protein synthesis in L-cell extracts: an ATP-dependent step in the activation of an inhibitor by double-stranded RNA.

Roberts¹,

Clemens²,

Kerr³

1976

Proc. Natl. Acad. Sci. U.S.A.

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ABSIRACT The translation of encephalomyocarditis virionRNA in extracts from interferon-treated L-cells is inhibited by the addition of double-stranded RNA (dsRNA) at 400 ng/ml. A similar inhibition in response to dsRNA is seen in control cell extracts supplemented with small amounts of a postribosomal supernatant fraction from interferon-treated cels (interferon cell sap): Neither interferon cell sap nor dsRNA alone is inhibitory in control systems. The inhibition is much reduced if translation is carried out at low ATP concentrations. Conversely, the inhibitory capacity of the interferon cell sap is increased 100-fold if it is preincubated with dsRNA and ATP prior to its addition to the protein-synthesizing system. After this preincubation all detectable dsRNA can be removed without any diminution of the inhibitory activity of the ceU sap. These results are compatible with a two-step model for the inhibition in which a pre-inhibitor is activated by dsRNA, the activated inhibitor then interacting with the protein synthesis system to inhibit translation. Double-stranded RNA (dsRNA) inhibits protein synthesis in animal cells and cell-free systems (1-4). Interferon treatment of cells renders them more sensitive to the toxic effects of dsRNA (5); and with extracts from interferon-treated L-cells, the translation of encephalomyocarditis virion RNA (EMC RNA) in the cell-free system shows an enhanced sensitivity to inhibition by dsRNA (6). The latter inhibition is specific for dsRNA, and the interferon dose-response curve and heat-inactivation studies show a close correlation between the antiviral activity of interferon and the enhanced sensitivity to dsRNA observed in the cell-free system (6).Recently we have found that the addition of small amounts of a postribosomal supernatant fraction from interferon-treated cells (interferon cell sap) renders cell-free protein-synthesizing systems from control cells sensitive to inhibition by dsRNA*. This has permitted the quantitation of a putative dsRNAdependent inhibitor in the interferon cell sap. Here (10). Postmitochondrial supernatant fractions (S10) were prepared from L-cells (9) and preincubated and dialyzed as described (11). Interferon and control cell saps were prepared from S10 that had not been preincubated. These were centrifuged at 100,000 X g for 2 hr at 40, and the supernatant cell saps were dialyzed as above.Amino Acid Incorporation Assays. Preincubated and dialyzed S10 from control cells provided the ribosomes, enzymes, factors, and tRNA for the amino acid incorporation assays. Interferon and control cell saps and dsRNA were added with and without preincubation, as indicated in the individual experiments. The conditions used in the assay of the incorporation of a mixture of fourteen "4C-labeled amino acids (54 mCi/ milliatom of carbon, The Radiochemical Centre, Amersham, England) were as described, with the omission of the reticulocyte initiation factors (11). Incubations were for 120 min at 300 unless otherwise stated, and 10-,sl aliquots were assayed for ...

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Differences between the Ribonucleic Acids of Transforming and Nontransforming Avian Tumor Viruses

Cited by 256 publications

References 27 publications

Role of the pro-survival molecule Bfl-1 in melanoma

Role of the pro-survival molecule Bfl-1 in melanoma

Nucleotide sequences related to the transforming gene of avian sarcoma virus are present in DNA of uninfected vertebrates.

Interferon-induced inhibition of protein synthesis in L-cell extracts: an ATP-dependent step in the activation of an inhibitor by double-stranded RNA.

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