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Nonproducer (NP) clones of chicken and quail cells transformed by avian myelocytomatosis virus MC29-A were isolated in focus and agar colony assays. Several quail MC29 NP clones were developed into long-term cultures. They have been kept in continuous culture for 6 months and about 35 passages. They do not release virus particles detectable by [3H]uridine incorporation or reverse transcriptase assay. Rescue of transforming virus is possible at any time by superinfection of the NP cell clones with avian leukosis viruses such as Rous associated virus type 1 or ring-necked pheasant virus (RPV). [35S]Methionine pulse-labeled protein extracts of NP and of superinfected MC29 transformed cell cultures were analyzed by immune precipitation and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In NP cells the common precursor polypeptide for the structural proteins of avian RNA tumor viruses (pr76) is not synthesized. Instead, a major polypeptide with a molecular weight of 110,000–120,000 was observed (MC29–110K). In superinfected cells, both MC29–110K and pr76 are synthesized. The MC29–110K polypeptide was precipitated by an antiserum against whole virus (PR RSV-B) as well as by a monospecific anti-p27 serum. It was not precipitated by an anti-glycoprotein serum. Pulse-chase experiments showed that the MC29–110K polypeptide turned over at a rate comparable to that of pr76. However, none of the major structural proteins (p27, p19, p15) could be detected after the chase. Competition radioimmune assays demonstrated that protein extracts of NP MC29 cells contain inhibitory activity for precipitation of p19 and p27, but not for p15.
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... In QEF transfected with pRCAS-CDT1 or pRCAS-MC29, the ectopic p74 Cdt1 and p110 Gag-Myc proteins, as well as increased levels of endogenous p74 Cdt1 in cells transformed by pRCAS-MC29 were detected (Figure 5b). Cells transfected with the MC29 construct showed the typical morphology and capacity for anchorage-independent growth of fully v-myc-transformed cells 4 . QEF transfected with pRCAS-CDT1 were able to grow in semi-solid medium at relatively high numbers, although the colonies were significantly smaller than those induced by transformation with pRCAS-MC29 (Figure 5c). ...
... Cells and retroviruses. Cell culture, DNA transfection, and transformation of quail or chicken embryo fibroblasts (QEF, CEF) were performed as described 4,15 . The established quail cell lines Q8, QEF/MC29, QEF/Rc-Myc, QT6, VJ, VCD, R(-)3, and the cell lines Q/tM8 and Q/tM ON conditionally transformed by v-myc were used 4,15,32,33 . ...
... Cell culture, DNA transfection, and transformation of quail or chicken embryo fibroblasts (QEF, CEF) were performed as described 4,15 . The established quail cell lines Q8, QEF/MC29, QEF/Rc-Myc, QT6, VJ, VCD, R(-)3, and the cell lines Q/tM8 and Q/tM ON conditionally transformed by v-myc were used 4,15,32,33 . To generate CEF freshly transformed by the MC29 retroviruses, primary cells were transfected with pRCAS-MC29 as described 15 . ...
The c-myc protooncogene encodes the Myc transcription factor, a global regulator of fundamental cellular processes. Deregulation of c-myc leads to tumorigenesis, and c-myc is an important driver in human cancer. Myc and its dimerization partner Max are bHLH-Zip DNA binding proteins involved in transcriptional regulation of target genes. Non-transcriptional functions have also been attributed to the Myc protein, notably direct interaction with the pre-replicative complex (pre-RC) controlling the initiation of DNA replication. A key component of the pre-RC is the Cdt1 protein, an essential factor in origin licensing. Here we present data suggesting that the CDT1 gene is a transcriptional target of the Myc-Max complex. Expression of the CDT1 gene in v-myc-transformed cells directly correlates with myc expression. Also, human tumor cells with elevated c-myc expression display increased CDT1 expression. Occupation of the CDT1 promoter by Myc-Max is demonstrated by chromatin immunoprecipitation, and transactivation by Myc-Max is shown in reporter assays. Ectopic expression of CDT1 leads to cell transformation. Our results provide a possible direct mechanistic link of Myc's canonical function as a transcription factor to DNA replication. Furthermore, we suggest that aberrant transcriptional activation of CDT1 by deregulated myc alleles contributes to the genomic instabilities observed in tumor cells.
... The oncogenic potential of this virus appears to be due to a transformation-specific RNA sequence, v-myc, that is closely related to a genetic locus in chromosomal DNA of normal chicken cells, termed c-myc (2-4, 13, 24). Together with a partial complement of the gag gene, present in the RNA of replication-defective MC29, v-myc is expressed as a gag-related phosphoprotein of 110,000 daltons, termed pllO (5,6). Genetic evidence for the involvement of v-myc in oncogenesis rests on the following findings. ...
... The virus released by the producing clone was termed HBI-MC29/H (RPVH), and the virus rescued from the nonproducer clone by superinfection with a different stock of RPV was termed HBI-MC29/K (RPVK). Q10-MC29 (Q10-MCAV), released from the producer line Q10 of MC29-transformed quail fibroblasts (5), and MC29 (RPV), released from the quail nonproducer line Q8 (5) superinfected with RPVK, were used as sources of wtMC29. ...
region, v-myc,whichare deleted intdlOHRNA,arepresent inHBIRNA.Moreover, hybridization ofHBI RNA tomolecularly cloned subgenomic fragments oftitMC29 proviral DNA, followed byfingerprint analysis ofhybridized RNA,showed that theentire v-myc- specific RNA sequences defined previously arepresent. Hybridization tocloned DNA ofthenormal chicken locus c-mycshowsaclose relationship between HBI v-mycRNA andc-mycDNA,especially inthesequences whichweredeleted fromtdlOH-MC29. T,oligonucleotide mapsofHBIandtdlOHRNAs were prepared andcompared. Totalconservation oftheoligonucleotide pattern is observed intheoverlapping v-mycregions, while thepartial structural genesgag andenvshowsomevariations, mostofwhichcanbedirectly proven tobedueto point mutations orrecombination withhelper viral RNAsthatwereanalyzed in parallel. Recombination oftdlOH-MC29 withc-myc, followed byrecombinational andmutational changes inthestructural genesduring passage withhelper virus, could beapossible explanation fortheorigin ofHBI.
... The analysis revealed that high level expression of v-Jun led to strong activation of the TOJ3 Fig. 2. Specific activation of the TOJ3 promoter mediated by nonconsensus AP-1 binding sites. (A) 5.0 μg aliquots of DNA from the CAT gene constructs pCAT-qTOJ3 or pCAT-cTOJ3 containing the quail or chicken TOJ3 promoter regions, respectively, were transfected into normal quail embryo fibroblasts (QEF) or into QEF transformed by the ASV17 or MC29 retroviruses encoding Gag-Jun (Maki et al., 1987) or Gag-Myc (Bister et al., 1977) hybrid proteins, respectively. Equal amounts of DNA from the empty pCAT-Basic vector (Promega) and from the pCAT-qBKJ promoter construct of the direct AP-1 target gene BKJ Bister, 1995, 1998) containing two consensus AP-1 binding sites (5′-TGACTCA-3′) were used as controls. ...
... Preparation, culture, and transformation of chicken or quail embryo fibroblasts (CEF, QEF) derived from fertile Japanese quail (Coturnix japonica) or chicken (Gallus gallus) eggs have been described (Hartl and Bister, 1998;Bader et al., 2001;Hartl et al., 2001Hartl et al., , 2006. Infection of QEF with the avian retroviruses ASV17 (Maki et al., 1987) or MC29 (Bister et al., 1977) encoding Gag-Jun or Gag-Myc hybrid proteins, respectively, and of CEF with ASV17 was performed as described previously (Hartl and Bister, 1998;Hartl et al., 2001). ...
The TOJ3 gene was originally identified on the basis of its specific activation in avian fibroblasts transformed by the v-jun oncogene of avian sarcoma virus 17 (ASV17). Overexpression of TOJ3 induces cellular transformation of embryonic avian fibroblasts, revealing an intrinsic oncogenic potential. Transforming activity has also been demonstrated for MSP58, the human homolog of TOJ3, and oncogenic cell transformation by MSP58 is specifically inhibited by the tumor suppressor PTEN. To investigate the mechanism of aberrant TOJ3 gene activation in jun-transformed fibroblasts, the entire quail TOJ3 gene including 13 exons and the 5′ regulatory region was isolated. Functional analyses of the promoter by transcriptional transactivation assays revealed that the specific induction of TOJ3 is mediated by a cluster of three noncanonical AP-1 binding motifs (5′-CAGCTCA-3′ or 5′-CACCTCA-3′) which share the 3′ half-site with the consensus motif (5′-TGAC/GTCA-3′). Electrophoretic mobility shift assays and chromatin immunoprecipitation analyses showed that Jun binds to these motifs with an affinity similar to that observed for binding to an AP-1 consensus site. Noncanonical binding sites are also present in the chicken and human TOJ3/MSP58 promoter regions. These results confirm and extend the previous observation that TOJ3 represents an immediate effector gene of Jun and may point to an essential role of TOJ3/MSP58 in carcinogenesis involving aberrant AP-1 expression.
... Cell culture, DNA transfection, and transformation of CEFs and QEFs were performed as described (26). The avian retroviruses ASV17, NK24, MC29, MH2, and RSV, and the established quail cell lines Q8, MH2A10, VJ, and Q/tM ON were used (18,19,21,30). The retroviral constructs pRCAS-MC29 and pRCAS-MC29-IRES-v-Mil have been described (21). ...
... To construct pRCAS-BASP1, pRCAS-BASP1 derivatives, and pRCAS-BRAK, ClaI fragments from pA-BASP1, pA-BASP1 derivatives, or pA-BRAK were inserted into the pRCAS-BP vector as described (26). Colony assays and focus assays were done as described (26,30). To monitor focus formation, cells were fixed with ethanol and incubated for 30 min in Giemsas eosin methylene blue solution diluted 1:10. ...
Cell transformation by the Myc oncoprotein involves transcriptional activation or suppression of specific target genes with intrinsic oncogenic or tumor-suppressive potential, respectively. We have identified the BASP1 (CAP-23, NAP-22) gene as a novel target suppressed by Myc. The acidic 25-kDa BASP1 protein was originally isolated as a cortical cytoskeleton-associated protein from rat and chicken brain, but has also been found in other tissues and subcellular locations. BASP1 mRNA and protein expression is specifically suppressed in fibroblasts transformed by the v-myc oncogene, but not in cells transformed by other oncogenic agents. The BASP1 gene encompasses 2 exons separated by a 58-kbp intron and a Myc-responsive regulatory region at the 5' boundary of untranslated exon 1. Bicistronic expression of BASP1 and v-myc from a retroviral vector blocks v-myc-induced cell transformation. Furthermore, ectopic expression of BASP1 renders fibroblasts resistant to subsequent cell transformation by v-myc, and exogenous delivery of the BASP1 gene into v-myc-transformed cells leads to significant attenuation of the transformed phenotype. The inhibition of v-myc-induced cell transformation by BASP1 also prevents the transcriptional activation or repression of known Myc target genes. Mutational analysis showed that the basic N-terminal domain containing a myristoylation site, a calmodulin binding domain, and a putative nuclear localization signal is essential for the inhibitory function of BASP1. Our results suggest that down-regulation of the BASP1 gene is a necessary event in myc-induced oncogenesis and define the BASP1 protein as a potential tumor suppressor.
... The v-myc allele is derived from the chicken cellular protooncogene c-myc by retroviral transduction [2,3]. The Myc protein product, initially identified as a viral Gag-Myc hybrid protein encoded by MC29 genomic RNA , is a transcriptional regulator of the basic/helix-loop-helix/leucine zipper (bHLH-LZ) protein family, forms heterodimers with the bHLH-LZ protein Max, binds to specific DNA sequence elements (E-boxes, preferentially CACGTG), and is the central node of a universal transcription factor network . In human cells, Myc transcription factor circuits control thousands of genes involved in essential cellular processes like growth, proliferation, differentiation, biosynthesis, energy metabolism, and apoptosis [7,8]. ...
... Selection was performed by adding 350 µg/ml of each antibiotic to the cell culture medium. Normal quail embryo fibroblasts (QEF) were prepared from 9-day-old embryos of Coturnix japonica as described [4,35]. The quail cell line QT6 transformed by the chemical carcinogen methylcholanthrene and the cell line Q/tM8 conditionally transformed by v-myc have been described before [32,33]. ...
The oncogenic bHLH-LZ transcription factor Myc forms binary complexes with its binding partner Max. These and other bHLH-LZ-based protein-protein interactions (PPI) in the Myc-Max network are essential for the physiological and oncogenic activities of Myc. We have generated a genetically determined and highly specific protein-fragment complementation assay based on Renilla luciferase to analyze the dynamic interplay of bHLH-LZ transcription factors Myc, Max, and Mxd1 in vivo. We also applied this PPI reporter to quantify alterations of nuclear Myc-Max complexes in response to mutational events, competitive binding by the transcriptional repressor Mxd1, or perturbations by small-molecule Myc inhibitors, including recently identified potent PPI inhibitors from a Kröhnke pyridine library. We show that the specificity of Myc-Max PPI reduction by the pyridine inhibitors directly correlates with their efficient and highly specific potential to interfere with the proliferation of human and avian tumor cells displaying deregulated Myc expression. In a direct comparison with known Myc inhibitors using human and avian cell systems, the pyridine compounds reveal a unique inhibitory potential even at sub-micromolar concentrations combined with remarkable specificity for the inhibition of Myc-driven tumor cell proliferation. Furthermore, we show in direct comparisons using defined avian cell systems that different Max PPI profiles for the variant members of the Myc protein family (c-Myc, v-Myc, N-Myc, L-Myc) correlate with their diverse oncogenic potential and their variable sensitivity to the novel pyridine inhibitors.
... In 1977, an avian cell line transformed by the v-myc-containing retrovirus MC29 was found to produce an unusual viral-related protein of 110,000 kDa (Bister et al. 1977). This protein represented the fusion of a truncated retrovirus core protein precursor with the MYC protein (Mellon et al. 1978;Rettenmier et al. 1979). ...
This review is intended to provide a broad outline of the biological and molecular functions of MYC as well as of the larger protein network within which MYC operates. We present a view of MYC as a sensor that integrates multiple cellular signals to mediate a broad transcriptional response controlling many aspects of cell behavior. We also describe the larger transcriptional network linked to MYC with emphasis on the MXD family of MYC antagonists. Last, we discuss evidence that the network has evolved for millions of years, dating back to the emergence of animals.
... Efficiencies of transformation for multiple experiments are averaged and summarized inTable 2. The inhibition of Myc-mediated transformation by compound NSC13728 is specific, whereas transformation by Src, Jun, or phosphoinositide 3-kinase was not significantly affected by the compound . In cell culture, NSC13728 inhibited the Myc-induced formation of transformed cell foci with an IC 50 of 3 M. Growth curves were determined with the Q8, an established QEF cell line transformed by the v-myc oncogene of avian acute leukemia virus MC29 (Bister et al., 1977 ). Compared with nontransformed QEF, Q8 cells reach a much higher density at each time point (SupplementalFig. ...
Many human cancers show constitutive or amplified expression of the transcriptional regulator and oncoprotein Myc, making Myc a potential target for therapeutic intervention. Here we report the down-regulation of Myc activity by reducing the availability of Max, the essential dimerization partner of Myc. Max is expressed constitutively and can form unstable homodimers. We have isolated stabilizers of the Max homodimer by applying virtual ligand screening (VLS) to identify specific binding pockets for small molecule interactors. Candidate compounds found by VLS were screened by fluorescence resonance energy transfer, and from these screens emerged a potent, specific stabilizer of the Max homodimer. In vitro binding assays demonstrated that the stabilizer enhances the formation of the Max-Max homodimer and interferes with the heterodimerization of Myc and Max in a dose-dependent manner. Furthermore, this compound interferes with Myc-induced oncogenic transformation, Myc-dependent cell growth, and Myc-mediated transcriptional activation. The Max-Max stabilizer can be considered a lead compound for the development of inhibitors of the Myc network.
... This is supported by the ability of such isolates, designated as mink cell focus forming (MCF) viruses, to induce morphologic alteration of mink cells in tissue culture (29). The more recent isolation of transforming variants from thymus cell-derived MCF virus stocks, one of which has been shown to be replication-defective (20) and to encode, as its major translation product, a 1 I0,000 Mr polyprotein (21), suggested a model analogous to that described for other mammalian RNA transforming viruses such as avian acute leukemia virus (4,7,10), several independent isolates of FeSV (5,6), and AbLV (8,9). ...
Mink cells nonproductively-infected with the weakly-transforming T-8 isolate of murine leukemia virus (MuLV) express a 110,000 mol wt polyprotein designated T-8 P110. By immunoprecipitation analysis, T-8 P110 is shown to contain AKR-MuLV amino terminal gag gene-specific components (p15, p12) but to lack p30, p10, gp70, and p15(E) antigenic determinants. These observations are further substantiated by tryptic peptide analysis indicating T-8 P110 to share approximately six lysine-containing tryptic peptides with AKR-MuLV Pr65gag, and none with AKr-MuLV Pr82env. Furthermore, of seven methionine-containing T-8 P110 tryptic peptides, at least four can be conclusively shown not to be present in either AKr-MuLV Pr180gag/pol or Pr82env. A clonal mink cell line nonproductively infected by T-8, and expressing high levels of P110, although not morphologically transformed, is shown to lack elevated levels of tyrosine-specific protein kinase activity and reduction of epidermal growth factor binding sites characteristic of cells transformed by many other RNA-transforming viruses. These findings argue either that the T-8 viral genome contains acquired cellular sequences encoding a portion of P110, or that T-8 P110 represents an inphase deletion of AKR-MuLV Pr180gag/pol with extensive posttranlational modification and that an as yet unidentified protein is responsible for T-8 associated transformation.
... Cells and Retroviruses. Cell culture, DNA transfection, and transformation assays of quail embryo fibroblasts (QEF) were performed as described (6,40,43). The construction of retroviral and Bluescript vectors carrying Hydra myc and max genes or Hydra-viral hybrid genes is described in SI Text. ...
The c-myc protooncogene encodes a transcription factor (Myc) with oncogenic potential. Myc and its dimerization partner Max are bHLH-Zip DNA binding proteins controlling fundamental cellular processes. Deregulation of c-myc leads to tumorigenesis and is a hallmark of many human cancers. We have identified and extensively characterized ancestral forms of myc and max genes from the early diploblastic cnidarian Hydra, the most primitive metazoan organism employed so far for the structural, functional, and evolutionary analysis of these genes. Hydra myc is specifically activated in all stem cells and nematoblast nests which represent the rapidly proliferating cell types of the interstitial stem cell system and in proliferating gland cells. In terminally differentiated nerve cells, nematocytes, or epithelial cells, myc expression is not detectable by in situ hybridization. Hydra max exhibits a similar expression pattern in interstitial cell clusters. The ancestral Hydra Myc and Max proteins display the principal design of their vertebrate derivatives, with the highest degree of sequence identities confined to the bHLH-Zip domains. Furthermore, the 314-amino acid Hydra Myc protein contains basic forms of the essential Myc boxes I through III. A recombinant Hydra Myc/Max complex binds to the consensus DNA sequence CACGTG with high affinity. Hybrid proteins composed of segments from the retroviral v-Myc oncoprotein and the Hydra Myc protein display oncogenic potential in cell transformation assays. Our results suggest that the principal functions of the Myc master regulator arose very early in metazoan evolution, allowing their dissection in a simple model organism showing regenerative ability but no senescence.
... The v-myc protein produced by the avian acute leukemia virus MC29 was first identified as a fusion protein containing a segment of the retroviral structural protein precursor covalently linked to the v-myc-polypeptide region (6). This protein and other myc-related fusion proteins were immunoprecipitated with antibodies against the viral structural polypeptide regions (7). Recently avian c-myc and v-myc proteins that are not linked to retroviral structural regions have been identified by means of antibodies raised against synthetic peptides corresponding to defined regions of the putative myc proteins (22, 22a) and against portions of myc-encoded proteins produced in bacterial expression vectors (2). ...
To examine myc protein products in the wide variety of human tumor cells having alterations of the c-myc locus, we have prepared an antiserum against a synthetic peptide corresponding to the predicted C-terminal sequence of the human c-myc protein. This antiserum (anti-hu-myc 12C) specifically precipitated two proteins of 64 and 67 kilodaltons in quantities ranging from low levels in normal fibroblasts to 10-fold-higher levels in Epstein-Barr virus-immortalized and Burkitt's lymphoma cell lines, to 20- to 60-fold-higher levels in cell lines having amplified c-myc. The p64 and p67 proteins were found to be highly related by partial V8 proteolytic mapping, and both were demonstrated to be encoded by the c-myc oncogene, using hybrid-selected translation of myc-specific RNA. In addition, the p64 protein was specifically precipitated from cells transfected with a translocated c-myc gene. Both p64 and p67 were found to be nuclear phosphoproteins with extremely short half-lives. In tumor cell lines having alterations at the c-myc locus due to amplification or translocation, we observed a significant change in the expression of p64 relative to p67 when compared with normal or Epstein-Bar virus-immortalized cells.
... The myc oncogene was originally identified as the transforming principle (v-myc) in the genome of avian acute leukemia virus MC29 encoding a single hybrid protein composed of partial structural (Gag) and Myc sequences (1,2). The highly oncogenic v-myc allele is derived from the chicken c-myc protooncogene by retroviral transduction (3,4). ...
The myc oncogene was originally identified as a transduced allele (v-myc) in the genome of a highly oncogenic avian retrovirus. The protein product (Myc) of the cellular c-myc proto-oncogene represents the key component of a transcription factor network controlling the expression of a large fraction of all human genes. Myc regulates fundamental cellular processes like growth, metabolism, proliferation, differentiation, and apoptosis. Mutational deregulation of c-myc leading to increased levels of the Myc protein is a frequent event in the etiology of human cancers. In this chapter, we describe cell systems and experimental strategies to monitor and quantify the oncogenic potential of myc alleles and to isolate and characterize transcriptional targets of Myc that are relevant for the cell transformation process. We also describe experimental procedures to study the evolutionary origin of myc and to analyze structure and function of the ancestral myc proto-oncogenes.
... MYC has been originally identified as the transforming determinant (v-myc) of avian acute leukemia virus MC29 in chicken (myelocytomatosis virus 29) (6). MYC was also isolated from the avian leukemia-and carcinoma-inducing MH2 virus, which carries in addition the v-mil(RAF) allele encoding a serine/threonine protein kinase (7). ...
MYC represents a transcription factor with oncogenic potential converting multiple cellular signals into a broad transcriptional response, thereby controlling the expression of numerous protein-coding and non-coding RNAs important for cell proliferation, metabolism, differentiation, and apoptosis. Constitutive activation of MYC leads to neoplastic cell transformation, and deregulated MYC alleles are frequently observed in many human cancer cell types. Multiple approaches have been performed to isolate genes differentially expressed in cells containing aberrantly activated MYC proteins leading to the identification of thousands of putative targets. Functional analyses of genes differentially expressed in MYC-transformed cells had revealed that so far more than forty upregulated or downregulated MYC targets are actively involved in cell transformation or tumorigenesis. However, for determination which of the known, or yet unidentified targets are responsible for processing the oncogenic MYC program, further systematic and selective approaches are required. The search for critical targets in MYC-dependent tumor cells is exacerbated by the fact that during tumor development, cancer cells progressively evolve in a multistep process thereby acquiring their characteristic features in an additive manner. Functional expression cloning, combinatorial gene expression and appropriate in vivo tests could represent adequate tools for dissecting the complex scenario of MYC-specified cell transformation. In this context, the central goal is to identify a minimal set of targets that suffices to phenocopy oncogenic MYC. Recently developed genomic editing tools could be employed to confirm the requirement of crucial transformation-associated targets. Knowledge about essential MYC regulated genes is beneficial to expedite the development of specific inhibitors to interfere with growth and viability of human tumor cells in which MYC is aberrantly activated. Approaches based on the principle of synthetic lethality using MYC-overexpressing cancer cells and chemical or RNAi libraries have been employed to search for novel anticancer drugs, also leading to the identification of several druggable targets. Targeting oncogenic MYC effector genes instead of MYC may lead to compounds with higher specificities and less side effects. This class of drugs could also display a wider pharmaceutical window because physiological functions of MYC, which are important for normal cell growth, proliferation, and differentiation would be less impaired.
... These GST-fusion proteins were used in pull-down assays performed with whole cell extracts from quail embryo fibroblasts (QEF) transformed by MC29. As expected, the MC29-encoded 110-kDa Gag-Myc hybrid protein  coprecipitated with GST-Max, but with similar efficiency also with GST-CaM ( Figure 1B). No complex formation of p110 was observed with GST alone, or with GST-BASP1, confirming previous results that Myc and BASP1 do not interact directly . ...
The bHLH-LZ (basic region/helix-loop-helix/leucine zipper) oncoprotein Myc and the bHLH-LZ protein Max form a binary transcription factor complex controlling fundamental cellular processes. Deregulated Myc expression leads to neoplastic transformation and is a hallmark of most human cancers. The dynamics of Myc transcription factor activity are post-translationally coordinated by defined protein-protein interactions. Here, we present evidence for a second messenger controlled physical interaction between the Ca2+ sensor calmodulin (CaM) and all Myc variants (v-Myc, c-Myc, N-Myc, and L-Myc). The predominantly cytoplasmic Myc:CaM interaction is Ca2+-dependent, and the binding site maps to the conserved bHLH domain of Myc. Ca2+-loaded CaM binds the monomeric and intrinsically disordered Myc protein with high affinity, whereas Myc:Max heterodimers show less, and Max homodimers no affinity for CaM. NMR spectroscopic analyses using alternating mixtures of 15N-labeled and unlabeled preparations of CaM and a monomeric Myc fragment containing the bHLH-LZ domain corroborate the biochemical results on the Myc:CaM interaction and confirm the interaction site mapping. In electrophoretic mobility shift assays, addition of CaM does not affect high-affinity DNA-binding of Myc:Max heterodimers. However, cell-based reporter analyses and cell transformation assays suggest that increasing CaM levels enhance Myc transcriptional and oncogenic activities. Our results point to a possible involvement of Ca2+ sensing CaM in the fine-tuning of Myc function.
... Mammalian cells are refractory to the action of most single oncogenes; in contrast, avian cells are highly sensitive indicators of the oncogenic potential of single agents. They can be readily transformed by single oncoproteins, for example, Myc, Jun, Qin, P3K or Akt, all of which are either inactive or extremely inefficient as single oncogenic agents in mammalian cell systems (Aoki et al., 1998; Bister et al., 1977; Chang et al., 1997; Himly et al., 1998). Rheb is an essential component of the Akt-TOR signaling chain. ...
Rheb (Ras-homolog enriched in brain) is a component of the phosphatidylinositol 3-kinase (PI3K) target of rapamycin (TOR) signaling pathway, functioning as a positive regulator of TOR. Constitutively active mutants of Rheb induce oncogenic transformation in cell culture. The transformed cells are larger and contain more protein than their normal counterparts. They show constitutive phosphorylation of the ribosomal protein S6 kinase and the eukaryotic initiation factor 4E-binding protein 1, two downstream targets of TOR. The TOR-specific inhibitor rapamycin strongly interferes with transformation induced by constitutively active Rheb, suggesting that TOR activity is essential for the oncogenic effects of mutant Rheb. Rheb-induced transformation is also dependent on a C-terminal farnesylation signal that mediates localization to a cellular membrane. An engineered N-terminal myristylation signal can substitute for the farnesylation. Immunofluorescence localizes wild-type and mutant Rheb to vesicular structures in the cytoplasm, overlapping with the endoplasmic reticulum.
We examined expression of the c-myc locus in four cell lines established from bursal lymphomas induced by avian leukosis virus. In all four lines the level of myc-related RNA was elevated. In three lines a majority of the myc-containing RNAs lacked viral-LTR-related sequences, in contrast to results obtained with primary tumors. This suggests that LTR sequences are not required for maintenance of high level c-myc expression. One line, RP9, has a complex pattern of myc RNAs containing LTR sequences, and one of these RNAs is packaged into virions. Using hybrid selection of RNAs with myc DNA, followed by in vitro translation, we detected translation of myc-related proteins from RNA of all four cell lines. The sizes of these proteins differ among the cell lines. The major polypeptides detected were 64, 57, and 54 kilodaltons. Events leading to elevation of c-myc transcription may be accompanied by alterations in mRNA initiation or processing that generate different protein products.
Clones of rat cells transformed by avian myelocytomatosis virus strain MC29 and avian erythroblastosis virus have been isolated. These cells display an altered morphology and are able to form colonies in soft agar. Focus-forming virus can be rescued from the transformed rat cells by fusion with chick cells.
The screening of a >9000 compound library of synthetic DNA binding molecules for selective binding to the consensus sequence of the transcription factor LEF-1 followed by assessment of the candidate compounds in a series of assays that characterized functional activity (disruption of DNA-LEF-1 binding) at the intended target and site (inhibition of intracellular LEF-1-mediated gene transcription) resulting in a desired phenotypic cellular change (inhibit LEF-1-driven cell transformation) provided two lead compounds: lefmycin-1 and lefmycin-2. The sequence of screens defining the approach assures that activity in the final functional assay may be directly related to the inhibition of gene transcription and DNA binding properties of the identified molecules. Central to the implementation of this generalized approach to the discovery of DNA binding small molecule inhibitors of gene transcription was (1) the use of a technically nondemanding fluorescent intercalator displacement (FID) assay for initial assessment of the DNA binding affinity and selectivity of a library of compounds for any sequence of interest, and (2) the technology used to prepare a sufficiently large library of DNA binding compounds.
The src gene product of Harvey murine sarcoma virus is a 21,000-dalton guanine nucleotide-binding protein. We have recently shown that a wide variety of vertebrate cell strains and cell lines express much lower levels of an endogenous p21 immunologically related to the Harvey murine sarcoma virus-coded p21. In this report, we have examined the levels of endogenous p21 in a unique hemopoietic precursor cell line, 416B, which was originally described as a continuous cell line of a hemopoietic stem cell, CFU-S. The currently available 416B cells express markedly elevated levels of endogenous p21. The level of endogenous p21 in the 416B cells is 5- to 10-fold higher than the level of p21 in Harvey murine sarcoma virus-infected cells and more than 100 times higher than the level of endogenous p21 that we have observed in a variety of other fresh or cultured cells. The results indicate that marked regulation of the levels of an endogenous sarc gene product can occur, and speculation about a possible role for endogenous p21 in normal hemopoietic stem cells is discussed.
A cell line derived from a transplantable chicken hepatoma induced by virus MC29 was established. This cell line grew permanently over 2 years and could be easily recovered from frozen state. Morphology of cells did not change during the period of cultivation. Karyological analysis revealed the hypodiploid stemline with reduced numbers of microchromosomes. The cell line was not transplantable in 1-day-old chicks, but induced multiple tumour nodules in the mesenterium and liver about 8 weeks postinoculation. The cell line is virus productive. The analysis of viral proteins with the gag specific determinants showed the presence of pr76 and p110.
Nonproducer clines of chicken bone marrow cells and quail embryo cells transformed by avian myelocytomatosis virus strain CMII were isolated. Analysis of [35S]methionine-labeled cell extracts of the nonproducer clones by immune precipitation showed that none of the three viral structural protein precursors, Pr76gag, gPr95env, and Pr180gag-pol were synthesized, but instead a 90,000 molecular weight protein (CMII-90K) were isolated. By using specific antisera this protein was shown to be related to the gag gene product, but not to the products of the pol or env genes. Competition radioimmunoassays showed that non-producer cells expressed inhibitory activity for p19 but not for p27 or p15. Tryptic peptide analysis of CMII-90K, Pr76gag, gPr95env, and the β-subunit of the viral reverse transcriptase confirmed the immunological data that the CMII-90K protein was shown to contain the p19 truptic peptides plus peptide which were specific for CMII and not related to env or pol gene products. The possible role of the CMII-90K protein in cell transformation is discussed.
The patterns of oncovirus protein biosynthesis are essentially similar for avian and mammalian viruses. In each case the four major internal structural proteins are synthesized as a precursor polypeptide of about 75 000 daltons, the product of the gag gene. Translation occurs on genome-sized mRNA. This polyprotein is cleaved in a series of steps to give the mature proteins. The mechanism and localization of cleavage have not yet been clarified. Viral reverse transcriptase, the product of the pol gene, also is translated on genome-sized mRNA as a precursor, which is a "read-through" product of the neighbouring gag gene. The two major envelope proteins are translated as a glycosylated precursor of apparent molecular weight about 90 000 from the env gene located on a sub-genomic RNA species. The precursor is transported to the plasma membrane where it may mark the site of virus budding. It is cleaved in transport or on the membrane, but the resulting two mature envelope proteins remain tied by disulfide bonds. Sarc, the protein product of the src gene that is responsible for transformation, is translated from a different viral mRNA than the structural proteins. Sarc has not been definitively characterized in any system.
We have identified an avian oncovirus-specific polypeptide of 120,000 dalton molecular weight (P120) in extracts of uninfected chick embryo fibroblast cells (CEF). P120 can be detected in CEF positive for the expression of avian oncovirus group-specific antigen (gs+), but its presence cannot be correlated with phenotypic expression of endogenous oncovirus envelope glycoprotein (chf). Immune precipitation experiments indicate that P120 contains antigenic determinants of the virion core proteins (products of the gag gene) and reverse transcriptase, but not of the envelope glycoprotein gp85. Double-label ion-exchange chromatography of tryptic digests of P120 reveals that this polypeptide contains amino acid sequences related to the virion core proteins p270, p19 and p12 of the chicken endogenous virus RAV-0, but lacks the tryptic peptides of p15. In addition, P120 contains three tryptic peptides which appear to be present in both the α and β subunits of reverse transcriptase. The order of the tryptic peptides on P120 was determined using the pactamycin procedure. The data demonstrate that the P120 tryptic peptides which are presumed to be related to reverse transcriptase lie between the gag region and the carboxy terminus of the protein. Our data are consistent with the idea that P120 is equivalent to a gag-pol polyprotein that is missing both p15 and significant portions of reverse transcriptase. In pulse-chase experiments, P120 does not appear to be specifically cleaved to generate virion structural proteins. Furthermore, we have been unable to detect P120 in RAV-0 producing line 7 and line 15 CEF. Instead, only Pr76gag (the direct precusor to virion core proteins in productively infected cells) is observed in these cells. Thus P120 is unlikely to act as a functional intermediate in virus maturation. We have also observed that infection of gs+ CEF with exogenous avian sarcoma and leukemia viruses results in a significant but variable decrease in the level of P120.
RNA tumor viruses, commonly called “oncornavirus,” are essentially categorized as type-A, type-B, type-C, and type-D, primarily on the basis of morphologic criteria. Type-C RNA tumor viruses are regarded as the most extensively studied class of oncornaviruses. This group of viruses is characterized by their centrally located nucleoid and a pattern of virion assembly that occurs as a budding process at the plasma membrane. This chapter focuses on type-C viruses and their translational products. It also presents RNA tumor viruses, essentially known as “RNA sarcoma viruses.” Type-C oncornaviruses can be distinguished from either type-B or type-D viruses on the basis of both morphologic criteria and the divalent cation preference of their RNA-dependent DNA-polymerase. A number of structural proteins of all type-C oncornavirus isolates essentially share cross-reactive interspecies antigenic determinants. The type-C virus genome consists of two identical 35 S RNA subunits joined near their 5′ terminus in a hydrogen-bonded dimer linkage structure. The genome is terminally redundant containing short identical segments at its 5′ and 3′ ends that provide a means of forming circular structures during replication. The mammalian type-C viral genome appears to be arranged in the order 5′ gag-pol-env 3′. In the case of replication-defective mammalian sarcoma viruses, src sequences acquired from the host cell genome by genetic recombination are located at varying positions, frequently extending into the 3′terminus of the gag gene.
We have analyzed normal rat kidney cells nonproductively infected with the Friend strain of spleen focus-forming virus (SFFV) for the presence of murine leukemia virus-specific type C viral proteins. SFFV was found to code for the p15 and p12 proteins of Friend-murine leukemia virus as determined by immunological typing of their antigenic determinants. Molecular-size analysis of p15 and p12 proteins in SFFV nonproductively infected normal rat kidney cells indicated that these proteins are translated as parts of polyprotein molecules. The apparent molecular weights of the polypeptides bearing p12 antigenic determinants revealed the presence of translational products of the SFFV genome that could not be accounted for by know type C virus helper structural proteins.
This chapter describes the models for the structure and morphogenesis of avian and murine type-C retroviruses. These are based on the molecular arrangement of virion structural components in the particle as well as on available data relating to the biosynthesis of these products. Since the avian and murine retroviruses are the most thoroughly characterized, the chapter focuses mainly on these systems. The visualization of the morphology of type-C viruses by electron microscopy has been generally impaired because of their labile structure and artifacts generated by various staining methods. However, gentle purification procedures combined with improved sample preparation and staining techniques have permitted a visualization of the fine structure of type-C viruses from a variety of species. Recent electron microscopic investigations with murine and avian type-C viruses revealed the presence of a thin layer, the inner coat (IC), located just beneath the viral envelope. The IC is apparently attached to the envelope since it remains in its normal configuration even in instances where the inner components of the virus are well separated from the envelope. The viral envelope and IC surround the virus core that consists of an outer core shell (CS) of hexagonally arranged subunits, enclosing the internal electron-dense ribonucleoprotein complex (RNP). The RNP appears as a filamental strand in the form of a spiral. The localization of the analogous avian and murine structural components in the virion is also summarized.
The virion RNA of avian myelocytoma virus MC29 was hybridized to full genome length DNA of the Prague strain of Rous sarcoma virus and analyzed by heteroduplex mapping in the electron microscopy. The results show that MC29 specific sequences for which there are no homologous counterparts in the Rous sarcoma virus genome make up a contiguous stretch of RNA about 1.8 kilobases long. These sequences are located approximately in the middle of the genome, replacing the 3' half of the gag gene, the entire pol gene, and the 5' portion of the env gene, which are absent from MC29. This MC29 specific genetic substitution may contain information for the leukemogenic transformation of the host cell.
Avian myeloblastosis virus contains a proteolytic activity that can cleave in vitro the viral precursor polypeptide Pr76gag. This substrate was prepared by radioactive labeling in vivo followed by immune precipitation, polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate, and elution from the gel. The major products of this reaction include the mature virion proteins p27 and p15, as well as an unstable fragment containing both of these proteins. Several other fragments are also formed, but mature p12 and the major p19 species are not. The cleavage of undenatured Pr76 bound to antibodies and formallin-fixed Staphylococcus yields similar fragments. The viral proteolytic enzyme is indistinguishable from the structural protein p15. Cleavage of Pr76 by p15 is optimal in the pH range 4–7 and is stimulated by salt. The activity of the enzyme is not inhibited by reagents specific for proteases with serine at their active sites, but is partially inhibited by reagents specific for thiols. Proteolysis is highly specific. Under the conditions used for Pr76 cleavage, p15 does not introduce breaks into mixtures of cellular proteins eluted in parallel to Pr76 from SDS-containing gels. However, it does fragment proteins that contain all or parts of the amino acid sequence of Pr76. These proteins include the precursor polypeptide for viral reverse transcriptase (Pr180gag-pol), a virus-related protein found in uninfected gs+ chick cells (P120), viral proteins from cells infected with avian erythroblastosis virus (P75) or with avian myelocytomatosis virus MC29 (P110).
Nonproducer clones of chicken fibroblasts or erythroblasts transformed by avian eryth-roblastosis virus (AEV) of strains R and ES4 were isolated. They do not release virus particles detectable by [3H]uridine incorporation or reverse transcriptase assays. Transforming virus can be rescued from these clones by superinfection with avian leukosis viruses of sub-groups A, B, C, and D. Analysis of [35S]methionine-labeled cell extracts of the nonproducer clones by immune precipitation showed that none of the three viral structural protein precursor polyproteins, Pr76gag, gPr95env, and Pr180gag-pol were synthesized, instead a 75,000 molecular weight protein (AEV-75K) was isolated. By using specific antisera, this protein was shown to be antigenically related to the gag gene, but not to the pol or env genes. Pulse-chase experiments showed that the AEV-75K protein was turned over but none of the major structural proteins (p27, p19, or p15) could be detected after the chase. Competition radioimmunoassays showed that nonproducer cells expressed inhibitory activity only for p19; no inhibitory activity related to p27 or p15 could be demonstrated. Tryptic peptide analysis of the AEV-75K protein confirmed the immunological data in that the fingerprint of the AEV-75K protein was distinct from those of Pr76gag, gPr95env, and the β-subunit of the reverse transcriptase. The possible role of the AEV-75K protein in transformation is discussed.
The myelocytomatosis viruses are a family of replication-defective avian retroviruses that cause a variety of tumours in chickens and transform both fibroblasts and macrophages in culture through the activity of their oncogene v-myc
1. A closely related gene (c-myc)2,3 is found in vertebrate animals and is thought to be the progenitor of v-myc
4,5. Changes in the expression and perhaps the structure of c-myc have been implicated in the genesis of avian6,7, murine8–10 and human11–14 tumours (for a review, see ref. 15). Elucidation of the mechanisms by which v-myc and c-myc might elicit tumorigenesis requires identification of the proteins encoded by these genes. To this end, we have expressed a portion of v-myc in a bacterial host and used the resulting protein to raise antisera that react with myc proteins. We report here that v-myc and c-myc encode closely related proteins with molecular weights (MWs) of ~58,000. Integration of retroviral DNA near or within c-myc in avian lymphomas6,7 apparently enhances expression of the gene6,7. Here we have used cells from one such tumour to identify the protein encoded by c-myc and find that the coding domain for the gene is probably intact.
A plasmid, pJL6, was constructed that contains a unique ClaI site twelve codons beyond the bacteriophage λcII gene initiation codon. This site allowed us to fuse the carboxy-terminal sequences of the avian myelocytomatosis virus (MC29) v-myc gene to the amino-terminal portion of the cII gene. Transcription of the hybrid gene is controlled from the phage λ pL promoter. When this promoter is derepressed. Escherichia coli cells harboring the chimeric plasmid produce a level of cII-myc fusion protein greater than 5% of total cellular protein. Antibodies raised by this protein immunoprecipitate the MC29 gag-myc gene product, P110gag-myc
Infection of chicken embryo fibroblasts by Rous sarcoma virus induces a variety of alterations in cellular growth and morphology. We have used two-dimensional polyacrylamide gel electrophoresis to examine the effects of viral transformation on the pattern of synthesis and phosphorylation of cellular polypeptides. Infection by Rous sarcoma virus does not appear to induce the de novo synthesis, or the complete suppression, of any of the [35S]methionine-labeled cellular polypeptides that can be resolved with this technique; however, there are quantitative changes in a minor fraction (approximately 4%) of the [35S]methionine-labeled polypeptides. When cells labeled with [32P]orthophosphate were examined, a phosphorylated polypeptide, Mr 36,000, was detected in transformed cells; this polypeptide appears within 20 min when cells infected by a temperature-sensitive mutant of Rous sarcoma virus are shifted from the nonpermissive to the permissive temperature. Phosphorylation of the 36,000 Mr polypeptide thus represents an early event in the process of transformation, and it is possible that this polypeptide is a target for the kinase activity associated with pp60src.
The MYC oncogene was originally identified as a transduced allele (v-myc) in the genome of the highly oncogenic avian retrovirus MC29. The protein product (MYC) of the cellular MYC (c-myc) protooncogene represents the key component of a transcription factor network controlling the expression of a large fraction of all human genes. MYC regulates fundamental cellular processes like growth control, metabolism, proliferation, differentiation, and apoptosis. Mutational deregulation of MYC, leading to increased levels of the MYC protein, is a frequent event in the etiology of human cancers. In this chapter, we describe cell systems and experimental strategies to quantify the oncogenic potential of MYC alleles, to test MYC inhibitors, and to monitor MYC-specific protein-protein interactions that are relevant for the cell transformation process. We also describe experimental procedures to study the evolutionary origin of MYC and to analyze structure, function, and regulation of the ancestral MYC proto-oncogenes.
The prototypes of the human MYC and RAF gene families are orthologs of animal proto-oncogenes that were originally identified as transduced alleles in the genomes of highly oncogenic retroviruses. MYC and RAF genes are now established as key regulatory elements in normal cellular physiology, but also as major cancer driver genes. Although the predominantly nuclear MYC proteins and the cytoplasmic RAF proteins have different biochemical functions, they are functionally linked in pivotal signaling cascades and circuits. The MYC protein is a transcription factor and together with its dimerization partner MAX holds a central position in a regulatory network of bHLH-LZ proteins. MYC regulates transcription conducted by all RNA polymerases and controls virtually the entire transcriptome. Fundamental cellular processes including distinct catabolic and anabolic branches of metabolism, cell cycle regulation, cell growth and proliferation, differentiation, stem cell regulation, and apoptosis are under MYC control. Deregulation of MYC expression by rearrangement or amplification of the MYC locus or by defects in kinase-mediated upstream signaling, accompanied by loss of apoptotic checkpoints, leads to tumorigenesis and is a hallmark of most human cancers. The critically controlled serine/threonine RAF kinases are central nodes of the cytoplasmic MAPK signaling cascade transducing converted extracellular signals to the nucleus for reshaping transcription factor controlled gene expression profiles. Specific mutations of RAF kinases, such as the prevalent BRAF(V600E) mutation in melanoma, or defects in upstream signaling or feedback loops cause decoupled kinase activities which lead to tumorigenesis. Different strategies for pharmacological interference with MYC- or RAF-induced tumorigenesis are being developed and several RAF kinase inhibitors are already in clinical use.
Following the introductory discussion in Section I of the general characteristics of tumor viruses, this section endeavors to provide a systematic overview of the molecular biology of viral carcinogenesis. However, before coming to specific examples of RNA or DNA tumor viruses we will review some of the important methodological and conceptual tools to provide a framework for understanding many of the aspects discussed in the subsections on the tumor viruses. Thus, the section begins with a discussion of the assay systems for cell transformation and oncogenesis and of the properties of transformed cells (in subsection I). This is followed (in subsection II) by an illustrative, systematic review of the vast area of the signal transduction network which functions principally in the cytoplasm in eukaryotic cells. Although in many cases the oncogenic property of a virally-transduced gene may have provided the first insight into, and experimental handle on, the functions of various components of this highly complex network, we have chosen to outline normal function first. In subsection III we discuss and exemplify the tumor-suppressor genes, a number of which play key roles in the control of normal cell cycling and the regulation of cell proliferation.
The relationship between two types of retroviral onc genes and cellular structural homologs termed proto-onc genes was studied. The type I Rous sarcoma virus (RSV) src gene, which is unrelated to essential virion genes, was found to have a complete structural homolog in cloned chicken DNA based on fingerprinting RNA-DNA hybrids. By the same techniques only the specific part (mcv) of the type II MC29 virus onc gene, which is a hybrid that also includes part (Δ) of the structural gag gene of retroviruses (Δ gag-mcv), was found to have a structural homolog in the cell. Hence, the onc gene of MC29 does not have a complete homolog in the cell. Both onc-related cellular loci are not linked to any other virion sequences. Presumed host markers of certain viral src genes, said to be experimentally transduced from the cell, were not detected in the proto src-locus. The cellular mcv-locus was found to be interrupted by one sequence of non-homology relative to the viral counterpart; the src-locus is known to be interrupted by six. We deduce that there is a close qualitative sequence-homology between the virion gene-unrelated sequences of viral onc genes and cellular proto-onc genes. However, functional homology between viral one genes and proto-onc loci cannot be deduced due to the different arrangements of onc-related sequences in viruses and cells and to scattered single nucleotide differences in their primary structures and due to the lack of Δ gag in cellular prototypes of hybrid onc genes, such as Δ gag mcv. Considering the genetic structures of RSV and MC29 and those of the corresponding cellular DNA loci, it follows that the generation of viruses like RSV and MC29 by transduction of cellular sequences into the genome of a retrovirus must have involved rare, illegitimate recombinations and specific deletions.
The transformation-specific oncogenes (or v-onc genes) of highly oncogenic retroviruses represent transduced mutant alleles of cellular oncogenes whose normal nonmutated alleles are termed protooncogenes (or c-onc genes). Protooncogenes are obviously nononcogenic in their normal cellular environment, and most of them presumably fulfil essential physiological functions in cell growth and development. The transition from a normal chromosomal allele with physiological function to a transduced mutant allele with oncogenic function is always accompanied by changes in gene structure and control of gene expression. However, it is largely unknown which of the qualitative or quantitative changes in gene structure and expression are necessary or even sufficient for oncogenic activation. Furthermore, there is as yet no unequivocal identification of the relevant biochemical function(s) of the protein products of both the mutant (transduced) and the normal (chromosomal) allele of an oncogene. Hence, the molecular mechanisms of cell transformation by v-onc genes are still obscure.
RNA tumour viruses, or oncoviruses as they are now called, belong to the family Retroviridae, comprising the retroviruses which also include foamy virus (Spumavirinae) and Maedi/Visna virus (Lentivirinae). Oncoviruses are divided according to a morphological classification into Type B, C and D viruses (1). Type C viruses have been isolated from or identified in numerous vertebrate hosts ranging from fish to mammals, and also from mosquitos. Oncoviruses are known to cause a variety of neoplasms in their natural host species. Thus the lymphomatous leukoses of cattle, cats, mice and chickens are typically caused by Type C oncoviruses, whereas Type B and D oncoviruses are associated with mammary carcinomas. Rare, acute neoplasms, such as sarcomas, and erythroid and myeloid leukaemias,are also recognised to result from retrovirus infection, as well as non-malignant diseases, such as osteopetrosis in chickens, anaemia in cats, and possibly autoimmune and paralytic diseases in mice. The problem of identifying retroviruses with neoplastic potential in humans remains equivocal, although tantalising items of evidence continue to be thrown up, as exemplified by Thiry’s contribution to this volume.
MC29 avian myelocytomatosis virus is an acute leukemia virus and the prototype of the MC29-subgroup of the avian leukosis-sarcoma genus of type C retroviruses. It causes myelocytomatosis, endotheliomas, liver and kidney carcinomas, and sarcomas in chickens. Biochemical and genetic analyses of the RNA genome of MC29 led to the discovery of the myc oncogene. The MC29 genome contains partial complements of the essential virion genes gag and env, and v-myc as the transforming principle. The single protein product of MC29, p110gag-myc, is a hybrid translational product of the partial gag gene and the v-myc sequences. The retroviral v-myc oncogene is a transduced mutant allele of the cellular protooncogene c-myc. The protein product of c-myc (Myc) is a bHLH-Zip protein, forms heterodimers with the bHLH-Zip protein Max, binds to specific DNA sequence elements (E-boxes) and is the central part of a transcription factor network controlling fundamental cellular processes like growth, metabolism, proliferation, differentiation, and apoptosis. Mutation and deregulation of c-myc is a frequent event in human tumorigenesis, occuring in about 30% of all human cancers. Myc and Max proteins were identified in early diploblastic organisms, suggesting that the principal functions of the Myc master regulator arose very early in metazoan evolution.
Avian oncogenic retrovirus MH2 carries two cell-derived oncogenes, v-mil and v-myc. From an infectious stock of MH2 a spontaneous deletion mutant, MH2D12, that has lost most of the v-mil gene but has retained a complete and functional v-myc gene, has been isolated. Nonproducer quail embryo cells transformed by MH2D12 in the absence of helper virus contain two virus-specific proteins: a gag-related protein of 53,000 Da (p53gag), and a v-myc gene product of 59,000/61,000 Da (p59/61v-myc) indistinguishable from the v-myc protein encoded by MH2. MH2D12 viral RNA contains all T1-oligonucleotides specific for the MH2 v-myc gene but none of those characteristic for the v-mil gene. The genetic structure of molecularly cloned proviral DNA of MH2D12 was revealed by restriction mapping, blot hybridization, heteroduplex analysis, and nucleotide sequencing. The MH2D12 provirus is homologous to the MH2 genome but has suffered a deletion of 1271 nucleotides from the central region encompassing the 3′ end of Δgag and all of v-mil except the very 3′ 31 nucleotides directly adjacent to the v-myc gene. A nine-nucleotide overlap of homology to gag or mil at the Δgag/Δmil junction suggests that recombination between homologous sequence elements of the Δgag and v-mil domains of MH2 was involved in the genesis of MH2D12. The nucleotide sequence analysis predicts that the carboxyterminal 17 amino acids of p539gag are encoded by the residual v-mil sequences and by intron-derived v-myc sequences. Transformation of quail embryo cells by MH2D12 can be assayed by focus and colony formation of transformed cells. This indicates that the v-mil gene is not essential for these activities. However, size and morphology of foci and colonies, and cellular morphology of cultured MH2D12-transformed cell lines can easily be distinguished from those observed in cell transformation by MH2 and resemble more those seen in cell transformation by viruses containing the myc oncogene only.
The LSCC-SF-MC29 cell culture model system was further characterized by studies on the provirus content of the cells, the host range and the subgroup specificity of the produced virus. The transforming potential of the Mc29 virus was evaluated by the focus-forming and colony-forming assays on primary cell cultures and continuous cell lines of avian and mammalian origin. The in ovo effects of the myelocytomatosis virus Mc29 on 15I line White Leghorn chicken embryos were studied by routine histopathological methods. Six avian leucosis virus-specific proviral sequences were detected by PCR analysis in the genome of LSCC-SF-MC29 cells. The presence of a Mc29 provirus-specific sequence located in the gag-myc region was confirmed. Using primers designed to dif-ferentiate ALV subgroups, amplification product was obtained with subgroup B/D-specific PCR primers. As it was expected, the subgroup E-specific PCR primers amplified the endogenous ALV sequences. In vitro studies on the host range of Mc29 virus showed that the primary cultures of chicken and hamster cells and a continuous hamster cell line were susceptible, while the cultures of primary quail cells and of a permanent line of duck embryo cells were resistant to the transforming effect of the virus. In ovo, the inoculated Mc29 virus in-duced hyperplasic and preneoplastic lesions in the embryonal liver and pancreas and myxomas of the neck.
This chapter discusses the adventures in mycology. Many of the now familiar concepts concerning the molecular biology of cancer are derived from the study of viral oncology. This includes a major contribution to the list of the dominantly acting transforming genes called “oncogenes.” Although the study of the viral oncogenes currently is not the dominant theme in the molecular biology of cancer, an understanding of the contributions from viral research is a necessary element in understanding any of the oncogenes. The myc gene is discovered in the avian acute transforming retroviruses, CMII, MC29, MH2, and OK I0. The viruses in this group cause a broad spectrum of malignancies in vivo, including sarcomas, carcinomas, and myelocytomas, and also possess the ability to transform fibroblasts, epithelial cells, and bone marrow cells in culture. As for all of the other retroviral oncogenes, the viral myc gene (v-myc) is derived from the host cell genome by a recombination event between a replication competent retrovirus and a preexisting host cell gene, in this case the cellular myc gene (c-myc).
To identify and characterize the proteins encoded by the erbA proto-oncogene, we expressed the C-terminal region of v-erbA in a bacterial trpE expression vector system and used the fusion protein to prepare antiserum. The anti-trp-erbA serum recognized the P75gag-erbA protein encoded by avian erythroblastosis virus and specifically precipitated six highly related proteins ranging in size from 27 to 46 kilodaltons from chicken embryonic erythroid cells. In vitro translation of a chicken erbA cDNA produced essentially the same pattern of proteins. Partial proteolytic maps and antigenicity and kinetic analyses of the in vivo and in vitro proteins indicated that they are related and that the multiple bands are likely to arise from internal initiations within c-erbA to generate a nested set of proteins. All of the c-erbA proteins are predominantly associated with chicken erythroblast nuclei. However, Nonidet P-40 treatment resulted in extraction of the three smaller proteins, whereas the larger proteins were retained. During differentiation of erythroid cells in chicken embryos, we found maximal levels of c-erbA protein synthesis at days 7 to 8 of embryogenesis. By contrast, c-erbA mRNA levels remained essentially constant from days 5 to 12. Together, our results indicate that posttranscriptional or translational mechanisms are involved in regulation of c-erbA expression and in the complexity of its protein products.
Retroviruses are the original source of oncogenes. The discovery and characterization of these genes was made possible by the introduction of quantitative cell biological and molecular techniques for the study of tumour viruses. Key features of all retroviral oncogenes were first identified in src, the oncogene of Rous sarcoma virus. These include non-involvement in viral replication, coding for a single protein and cellular origin. The MYC, RAS and ERBB oncogenes quickly followed SRC, and these together with PI3K are now recognized as crucial driving forces in human cancer.
A major concern of cell biologists, virologists, and immunologists alike, has been the attempt to better understand the molecular mechanisms which underlie malignant transformation. The type C RNA tumor viruses are of particular interest in this regard since they are natural etiologic agents of neoplastic disease in a wide variety of animal species. While it has been appreciated that these viruses are able to cause leukemias and sarcomas in susceptible species, it is also clear that some type C isolates are able to produce plasmacytomas, carcinomas of the liver and kidney, osteopetrosis, and infectious anemias as well. From the viewpoint of the molecular biologist, the type C viruses, like the small DNA tumor viruses, offer an attractive model system for studies of cellular transformation since the genetic complexity of these organisms is relatively small. Moreover, recent technologic advances in nucleic acid and protein chemistry have facilitated an extensive description of their molecular structure. The aim of this review is to provide the reader with a contemporary view of type C RNA tumor viruses, emphasizing their origins from normal cellular genes. It is this latter concept which distinguishes the RNA tumor viruses from other extrinsic etiologic agents of cancer, and raises the possibility that a better understanding of these agents will facilitate the eventual recognition of subsets of cellular genes involved in malignant disease. An attempt has been made to focus on recent data obtained in selected model systems, rather than to provide a comprehensive, historical review. As such, many unanswered questions are raised concerning the molecular basis of viral-cell interactions, both in vitro and in vivo. It is hoped, however, that this approach will point out some issues that are of prime concern to those working directly in this field.
Using labeled cDNA specific for the detection of the src gene of avian sarcoma viruses, we find that avian myelocytomatosis virus strain MC29 and avian erythroblastosis virus strain ES4 lack nucleotide sequences related to the src gene. Furthermore, chicken fibroblasts as well as hematopoietic cells, infected and transformed with these viruses, show no enhanced level of transcription of the cellular nucleotide sequences related to the src gene of avian sarcoma viruses. These two viruses may thus contain their own transforming gene(s) or induce cellular genes unrelated to the src-like cellular sequences.
Cell lines transformed by woolly monkey sarcoma virus (WSV) in the absence of infectious virus production were analyzed for the expression of woolly monkey helper viral p30, p12, and gp70 antigens. Several lines produced high levels of both p30 and p12, whereas gp70 was not detectable. One transformed clone expressed only p12, and in another cell line, none of the helper viral antigens were detected. The properties of each sarcoma virus bred true upon transmission, indicating that each variant represents a distinct genotype. The different cell lines were examined with respect to properties characteristic of the transformed state. The in vitro growth properties and oncogenicity of each WSV-transformed clone were indistinguishable, indicating that transformation by WSV occurs independently of the expression of at least three helper viral polypeptides.
Forty proteins with polypeptide chains of well characterized molecular weights have been studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate following the procedure of Shapiro, Viñuela, and Maizel (Biochem. Biophys. Res. Commun., 28, 815 (1967)). When the electrophoretic mobilities were plotted against the logarithm of the known polypeptide chain molecular weights, a smooth curve was obtained. The results show that the method can be used with great confidence to determine the molecular weights of polypeptide chains for a wide variety of proteins.
The genetic analysis of RNA tumor viruses has two main objectives: (1) to provide an understanding of virus replication and (2) to explain virus-induced transformation of the host cell. Virus replication results from a complex interaction of viral and cellular genomes. Viral genetics, however, considers only the virus side of this interaction; the numerous and specific cellular functions which are required for the synthesis of infectious virus will have to be defined by a genetic analysis of the host cell. In virus-induced transformation, viral genetic information presumably interferes with the genetic regulatory apparatus of the host cell, and here again it is important to realize that focusing on the viral information will reveal only part of a very complex interaction between two organisms. Despite these obvious limitations, the viral genome is at the moment that partner in this interaction which is more amenable to experimental study and offers realistic opportunities for an increase of our insight into virus replication and cellular transformation.
Infection of chick embryo cell monolayers in high multiplicity with strain MC29 leukosis virus in conventional tissue culture induces rapid, massive morphological conversion within 4 to 7 days. Treatment of the cells in appropriately lower inoculation multiplicities results in the occurrence of infectious centers distinguished as foci of converted cells. When the infected cells are overlaid with agar, the number of foci occurring in 7 days is closely proportional to virus dose. This relationship is applicable to bioassay of strain MC29 leukosis virus with reproducibility and precision of results compatible with practical quantitative studies on the agent. The bioassay procedure and the rapid massive culture conversion make available a unique avian virus-tumor system for studies on cell-virus interrelationships in the induction of neoplasia.
In order to determine whether or not every Rous-infected cell produces infectious progeny in the absence of helper virus, a number of cloned lines of L-R cells (leukosis virus negative Rous cells, formerly called NP cells) were tested for spontaneous virus production. It was found that there were two classes of L-R lines, one producing RSV infectious for Japanese quail embryo or type chick embryo cells and another producing virus particles that were not detectably infectious for any cell line tested.
A selective technique based on infective centers is described for isolating host range recombinants of avian RNA tumor viruses. This method was exploited to obtain recombinants between the genome of chick helper factor (chf) and nondefective strains of Rous sarcoma virus (RSV), including temperature-sensitive mutants. Recombinants of chf with the defective Bryan strain RSV could not be isolated. A majority of nondefective RSV particles selected as carrying the chf host range also carried the parental RSV host range; i.e., were of dual host range, but the progeny segregated into parental or recombinant genotypes. These observations suggested that the particles were at least partially diploid or polyploid and represented unstable “heterozygotes.” Genotypic mixing was not evident in chf-negative cells which contain viral DNA but not viral RNA, suggesting that genetic reassortment occurs among RNA molecules. A model is proposed in which reassortment of independent genome segments may be converted into stable recombinants following provirus formation in the next replicative cycle.
As is usual for Cold Spring Harbor meetings, this was the right meeting at the right time. There were enough half-completed experiments to leave something to think about and enough completed experiments to generate a sense of satisfaction. The major focus of the meeting was on the molecular biology of tumor viruses, while the problem of how tumor viruses can cause tumors was hardly mentioned at all. It is clear that within a very few years the molecular biology of tumor viruses will be understood in great detail. Hopefully, then, the groundwork will exist from which to consider how these viruses can cause tumors.
After an exhausting but exhilarating week in which 146 papers were presented, even a cursory summary would be longer than anyone would care to read. This will be, therefore, a selected review of aspects of the meeting, most of which fall into a set of simple...
Two nonglycosylated structural proteins of avian RNA tumor viruses, p15 and p19, were examined for the presence of subgroup-specific antigenic determinants by using competition radioimmunoassays. A comparison of viruses from subgroups A, B, and C revealed that subgroup B and C virus proteins were equally efficient in inhibiting the homologous radioimmunoassays which used antiserum against B77 and iodinated p15 and p19 from B77. On the other hand, subgroup A virus proteins were less efficient than subgroup C viruses in causing inhibition in these assays. That these differences in inhibition were due to true immunological differences was confirmed by using heterologous competition radioimmunoassays. Since it was possible to distinguish the p19 or p15 of subgroup A viruses from the corresponding proteins of either subgroup B or subgroup C virus, recombinant viruses from crosses between leukosis viruses of subgroup A and sarcoma viruses of subgroups B or C were examined. The recombinant viruses have the envelope glycoprotein gene (env) of the subgroup A virus and the sarcoma gene (src) of the subgroup B or C virus. The results show that 14 clones out of 17 examined had p19 from the sarcoma virus. RNA fingerprinting has shown that the src gene is located close to the 3′ end of the genome and is closely followed towards the 5′ end by the env gene. The observed linkage between src and p19 can be explained by postulating that the gene for p19 is located close to the 5′ end of the genome and that recombination takes place between circular forms of the virus genome.
The translation product of the gag gene of mammalian type-c- RNA viruses is a 65,000-68,000 molecular weight precursor polypeptide (Pr65) whose cleavage leads to the formation of four virion proteins, p30, p15, p12 and p10. An immunological approach has been used to establish the arrangement of the sequences coding for these proteins within the viral genome as (5') p15-p12-p30-p10 (3').
Chicken embryo fibroblasts infected by avian RNA tumor viruses have been analyzed by immune precipitation for the synthesis of virus-specific proteins. Three virus-specific precursor polyproteins with molecular weights of 180,000, 95,000, and 76,000 were detected following pulse-labeling of the cells. The 76,000-dalton polyprotein is the precursor to the nonglycosylated gag proteins of the virion as shown earlier by Vogt and Eisenman (1973, Proc. Nat. Acad. Sci. USA70, 1734–1738). By using monospecific antisera it was possible to identify the 180,000-dalton polyprotein as having sequences in common with the virion polymerase and the gag proteins and the 95,000-dalton polyprotein as being related to the virion glycoproteins gp85 and gp37. Although the 180,000-dalton protein contains sequences for the gag proteins, pulse-chase experiments indicated that it did not represent a precursor to the major 76,000 dalton gag precursor polyprotein and is most likely synthesized by an occasional read-through of the messenger RNA for the gag precursor polyprotein.
The authors review and describe experiments directed towards 1) outling the cleavage scheme for the formation of the virion structural proteins from the precursor, 2) determining the order of the virion proteins on the precursor, and 3) investigating the synthesis of viral specific proteins in several transformed nonproducing heterologous cell lines.
The four major internal structural proteins (the group-specific antigens) of avian myeloblastosis virus are formed by sequential cleavage of a precursor polypeptide with Mr = 76,000 (Pr76). The evidence for this conclusion is based on analysis of immune precipitates from lysates of AMV§-infected cells treated with a multivalent antiserum directed against these proteins. Sodium dodecyl sulfate gel electrophoresis of such immune precipitates from cells pulse-labeled with [35S]-methionine reveals five metabolically unstable radioactive polypeptides. These polypeptides behave kinetically as precursors to virion proteins. Double-label ion-exchange chromatography of tryptic digests of the unstable polypeptides demonstrates that the largest precursor, Pr76, contains the amino acid sequences of all four virion proteins. It appears not to contain the sequence of the fifth and smallest internal virion protein. The four smaller precursors are intermediate cleavage products of Pr76.The arrangement of the virion proteins in Pr76 was determined by labeling cells shortly after inhibiting polypeptide chain initiation. The relative amounts of radioactivity both in completed virion proteins and in the tryptic peptides of Pr76 implies the same order for three of the four proteins. The exact position of one protein remains uncertain.On the basis of these experiments, we propose a cleavage pathway for the generation of the structural proteins of AMV. We also demonstrate that cleavage of precursors can proceed in crude extracts of AMV-infected cells. This proteolysis, while resistant to several protease inhibitors, is completely blocked by addition of agents that disrupt membranes.
The use of yolk sac macrophages from an inbred chicken line (line 6) has led to an improved and more reliable assay for avian myeloblastosis virus (AMV) transforming activity. The assay has been instrumental in defining some of the biological characteristics of AMV, namely, envelope antigenicity, host range, and sensitivity to viral interference. The assay also demonstrated the defectiveness of AMV and the need for a helper virus for its full replicative expression.
The temperature-sensitive defect in replication of LA334, a double mutant of Rous sarcoma virus, has been characterized both biologically and biochemically. This mutant is complemented for replication at the nonpermissive temperature by both leukosis virus and by RSV(−). Both experiments indicate that LA334 synthesizes functional glycoproteins at the restrictive temperature. This conclusion was confirmed by interference experiments which showed that LA334 induced specific cellular resistance at 41° against superinfection with viruses of subgroup C. Noninfectious virus particles are synthesized at 41° and possess a higher density in sucrose gradients (1.175 g/ml) when compared to those produced at 35° (1.15 g/ml). In addition to the major viral structural proteins these noninfectious virions contain four novel polypeptides, at least three of which appear to be viral in origin. An analysis of viral polypeptide processing indicates that the rate of cleavage of the polyprotein precursor to nonglycosylated structural polypeptides is reduced significantly at the restrictive temperature. Addition of cycloheximide to infected cultures does not prevent the rapid production of infectious virus, seen on shifting cells from the restrictive to the permissive temerature, which suggests that proteins synthesized at 41° can be processed correctly at 35°. Large budding structures that lack the characteristic morphology of C-type particles are visible in electron micrographs of cells infected with the mutant at the nonpermissive temperature. Large virions corresponding in size to some of the budding structures are detectable in negatively stained preparations of noninfectious virus.
We have prepared radioactive DNA (cDNAsarc) complementary to nucleotide sequences which represent at least a portion of the viral gene(s) required for neoplastic transformation of fibroblasts by an avian sarcoma virus. The genetic complexity of cDNAsarc (~1600 nucleotides) is sufficient to represent an entire cistron. The genomes of three independent isolates of avian sarcoma viruses share nucleotide sequences closely related to cDNAsarc, whereas the sequences are absent from transformation-defective mutants of avian sarcoma viruses, several avian leukosis viruses, a non-pathogenic endogenous virus of chickens (Rous-associated virus-O), sarcoma-leukosis viruses of mice and cats, and mouse mammary tumor virus. We conclude that the transforming gene(s) of all avian sarcoma viruses have closely related or common genetic lineages distinct from the transforming genes in sarcoma viruses of other species. Our results conform to previous reports that transformation-defective variants of avian sarcoma viruses are mutants with identical regions deleted from each subunit of a polyploid genome.
INFECTION of fibroblasts by avian sarcoma virus (ASV) leads to
neoplastic transformation of the host cell. Genetic analyses have
implicated specific viral genes in the transforming
process1-4, and recent results suggest that a single viral
gene is responsible4. Normal chicken cells contain DNA
homologous to part of the ASV genome5-8 moreover, embryonic
fibroblasts from certain strains of chickens can produce low titres of
infectious type C viruses either spontaneously9 or in
response to various inducing agents10. None of the viruses
obtained from normal chicken cells, however, can transform fibroblasts,
and results with molecular hybridisation indicate that the nucleotide
sequences responsible for transformation by ASV are not part of the
genetic complement of the normal cell11. We demonstrate here
that the DNA of normal chicken cells contains nucleotide sequences
closely related to at least a portion of the transforming gene(s) of
ASV; in addition, we have found that similar sequences are widely
distributed among DNA of avian species and that they have diverged
roughly according to phylogenetic distances among the species. Our data
are relevant to current hypotheses of the origin of the genomes of RNA
tumour viruses12 and the potential role of these genomes in
Treatment of avian RNA tumor virus-infected chicken fibroblasts with 20 mM glucosamine for 8 hr leads to the inhibition of virion synthesis (Hunter et al., 1974). In this report we have investigated the mechanism of inhibition. A kinetic analysis on the inhibition of virion production revealed that the effect of glucosamine was essentially complete 5 hr after its addition, by this time virion production has been inhibited 50-fold. An analysis of the effect of glucosamine on cellular and intracellular virus protein synthesis revealed that by 5 hr cellular protein synthesis was inhibited 60% and virus protein synthesis was specifically inhibited by a further 75%. The major nonglycosylated viral structural proteins (p27, p19, p15, and p12) are synthesized as a 76,000 dalton precursor (Pr76) which is subsequently cleaved to the virion proteins. The effects of glucosamine on this process were examined by pulse-chase experiments. After 5 hr of treatment with glucosamine only 5% of the precursor synthesized in a 15-min pulse was cleaved in an hour, compared with 54% in untreated cells. Chases in the absence of glucosamine demonstrated that the precursor synthesized in the presence of glucosamine could be cleaved and assembled into virions. SDS-PAGE analysis of these virions showed that they lacked radioactively labeled glycoproteins, gp85 and gp37. Thus, glucosamine inhibits the replication of avian RNA tumor viruses by a combination of an inhibition of cellular protein synthesis, viral structural protein synthesis, and Pr76 cleavage.
The effects of ultraviolet (uv) irradiation on transforming and replicating capacities of avian oncoviruses and on the synthesis of virus specific products after infection with irradiated virus were studied. Different strains of nondefective avian sarcoma viruses were inactivated at the same rate following single-hit kinetics. The 37% survival dose D37 (1/e) was 736 erg mm−2 on average. A comparison of the inactivation kinetics in a focus assay (transforming capacity) and an infectious center assay (replicating and transforming capacity) showed no partial inactivation of the virus genome; focus and infectious center formation were inactivated at the same rate. Similar results were obtained when the replicating capacity of the avian sarcoma virus was measured in a plaque assay; focus and plaque formation were inactivated at the same rate. No repair of the uv damage by either complementation or recombination with exogenous or endogenous avian leukosis virus could be demonstrated. The rates of inactivation of avian sarcoma virus assayed in focus and infectious center tests on chick embryo fibroblasts expressing or not expressing chicken helper factor, on chick embryo cells preinfected with RAV-1, and on Peking duck cells were identical. Nondefective avian sarcoma virus and deletion mutants of avian sarcoma virus defective for replication or transformation were inactivated at the same rate. Biochemical analysis of the DNA extracted from a Japanese quail tumor cell line (QT-6) 26 hr after infection with irradiated avian sarcoma virus strain B77 showed a decrease of total virus specific DNA and of full-length covalently closed circular (form I) viral DNA synthesis with increase of the uv dose. Virus-specific RNA synthesis, measured by hybridization of labeled RNA extracted from chicken embryo fibroblasts infected with irradiated virus to viral DNA, and particle production, assayed by uridine incorporation, were also inhibited with increasing uv dose. The inactivation rates for virus-specific DNA and RNA synthesis and for particle production were very similar, but lower than the rate for the loss of infectivity.
Three clones of morphologically altered cells (L(-)MC29) of singular properties were isolated from MC29 (subgroup A) leukosis virus-infected chick embryo cells. Supernatant fluids from cultures of the cloned cells produced no transforming or interfering activity on chick embryo cells susceptible to known avian leukosis-sarcoma viruses. No virus associated with the cells was demonstrable by fluorescent-antibody staining or by electron microscopy. All L(-)MC29 clone cells were activated, however, by four strains of Rous-associated viruses (RAV) representative of A, B, C, and D subgroup avian leukosis viruses and by two strains of MC29 virus. Virus L(-)MC29 cells activated by superinfection with RAV-1 and RAV-2 was characterized by helper-dependent and helper-independent properties. These findings suggest that the strain MC29 leukosis virus, or a component thereof, possesses properties of defectiveness similar to those of the Bryan high-titer Rous sarcoma virus.
Publisher Summary This chapter discusses on the term leukosis virus, which will be used to designate any avian tumor virus strain that causes neoplastic disorders originating predominantly in the hematopoietic system. The majority of all avian tumor viruses belong in this category. Most of the remaining strains cause primarily solid tumors of connective tissue origin and may be classed as avian sarcoma viruses. The experimental work during the past few years has focused on a few prototypes, notably, Rous sarcoma virus (RSV), Rous associated virus (RAV), avian myeloblastosis virus strain BAI A (AMV), strain R erythroblastosis virus, and strain RPL 12 avian leukosis virus. The basic anatomy of avian tumor viruses is well established, and to a certain degree morphology has become correlated with function, mainly by the differentiation of coat and core properties in defective RSV. The geometrical as well as chemical architecture of the nucleocapsid needs further study. An immediate question in the chapter concerns the size and uniformity of viral RNA molecules, and whether the nucleocapsid contains one or several of such molecules. The central and most fundamental problem of the avian tumor virus field is doubtlessly the mechanism of virus-induced malignant transformation. Although the past few years have seen impressive progress in the anatomy and multiplication of avian tumor viruses, advances in the understanding of viral carcinogenesis have been slow; perhaps the main finding in this respect is the lack of an absolute link between virus synthesis and cellular transformation. Malignant cellular behavior and unrestricted growth cannot be separated from differentiation and normal control.
A quantitative assay for a leukosis virus has been developed. Chick embryo yolk sac and bone marrow cultures were infected with serial dilutions of avian myeloblastosis virus and were overlayed with purified agar dissolved in nutrient medium. Under these conditions well defined foci of transformed cells developed. There was a linear relationship between the number of these foci and the concentration of virus.
Strain MC29 avian leukosis (myelocytomatosis) virus induced infection, elaboration of virus, and morphological alteration in chick embryo cells in vitro. Virus liberation began within 18 hr, morphological change was detectable at about 40 hr, and the cultures could be completely altered within 80 hr after infection. Altered cells were about half the volume and grew at approximately twice the rate of uninfected elements. The output of virus estimated by electron microscopy was about 140 particles per cell per hr. Deoxyribonucleic acid remained constant, but ribonucleic acid increased in both infected and control cells in adjustment to culture environment. The rates of uptake and incorporation of (3)H-uridine and the incorporation of (3)H-thymidine increased in the infected cells with onset of morphological change but were unaffected by processes of infection and virus elaboration per se. Incorporation of a (14)C-amino acid mixture was slightly greater in the infected than in control cells. The speed of continuity of infection and massive morphological alteration constitute a unique response to avian tumor viruses, and the system gives promise of singular value for detailed studies of the processes of infection and morphological change.
Polycations enhance the infectivity of avian sarcoma viruses for chick embryo fibroblast cultures up to 80-fold. The enhancement is restricted to members of avian tumor virus subgroups B, C, and D and to RSV(O). Subgroup A viruses are either unaffected or inhibited by polycations. The polycation-mediated enhancement is at least in part due to an increased adsorption rate of virus to cell, but may also involve penetration. Viral growth rates are not influenced by cationic polymers. Polyanions reduce the infectivity of avian sarcoma viruses and are also able to neutralize the virus-enhancing activity of polycations. An exception is dextran sulfate which causes virus inhibition only at low concentrations (less than 4 μg/ml), but enhances viral infectivity at higher concentrations. Extracts from normal or leukosis virus-infected chick embryo fibroblasts also enhance focus formation by certain avian sarcoma viruses. The characteristics of this enhancement are similar to that mediated by polycations.
A continuous line of embryo cells can be transformed by murine sarcoma virus (MSV). Of twenty foci selected at limiting MSV dilution, eighteen release both MSV and murine leukemia virus (MuLV). Two focus-derived lines, however, show no physical or biological evidence of virus production and contain no evidence of antigens of the murine sarcoma-leukemia complex. These lines have altered properties in tissue culture and in vivo and are morphologically indistinguishable from virus releasing MSV-transformed mouse lines. The addition of “helper” MuLV results in the rescue of the MSV genome with host range and neutralization characteristics of the MuLV used to rescue it. These findings show that virus production and release is not necessary for the maintenance of the transformed state in mouse cells and suggest that MSV is capable of initiating transformation without MuLV. In the nonproducer lines the sarcoma genome can be passed from cell to daughter cell for over 100 cell generations in the absence of any detectable virus expression. A preliminary report of some of these findings has been presented (Aaronson, 1970a).
Deoxyribonucleic acid (DNA) polymerase activity can be elicited in purified preparations of avian myeloblastosis virus and Rous sarcoma virus (Schmidt-Ruppin strain) by treatment with nonionic detergent. The enzyme(s) and its synthetic products appear to be virion-associated. Enzymatic activity can be inhibited by pretreatment with either ribonuclease (8- to 10-fold inhibition) or actinomycin D (twofold inhibition). By contrast, rifampin has little, if any effect. The enzyme(s) synthesizes two primary products, a ribonucleic acid (RNA):DNA hybrid and DNA which is free of RNA. The results of both zonal and equilibrium centrifugation indicate that nascent chains of DNA are associated with the 70S viral RNA. It is concluded that at least two enzymatic activities are under study: transcription of DNA from viral RNA, and subsequent, additional synthesis of DNA, utilizing product of the initial reaction as template.
Gel electrophoresis of the dissociated 60–70 S RNA prepared from cloned avian sarcoma viruses showed a single major peak corresponding to size class a. Class b RNA was not detectable in these preparations. Class a RNA derived from the Schmidt-Ruppin strain of Rous sarcoma virus had a slightly but reproducibly lower electrophoretic mobility than class a RNA of Prague strain Rous sarcoma virus. The presence of avian tumor virus group specific (gs) antigens in chicken cells before infection did not detectably influence the electropherograms of the dissociated avian sarcoma virus RNA: cloned sarcoma virus from gs positive and gs negative cells showed identical gel patterns. However, two avian sarcoma virus clones derived from a clonal colony of transformed cells contained a new RNA component which migrated slower than class a RNA. This unusual component was found consistently in the original clonal cultures but was not seen if new cells were infected with the same virus, suggesting a cell mediated modification of viral RNA.Additional evidence was obtained for the absence of class a RNA from avian tumor viruses which cannot form foci in fibroblast cultures. Several leukosis and transformation defective viruses which had not been analyzed previously showed class b RNA only. No significant differences between the class b RNA preparations obtained from several independently isolated transformation defective viruses were detected.
Antibody to partially disrupted avian myeloblastosis virus was used to selectively precipitate newly synthesized intracellular viral polypeptides from extracts of infected chicken cells. When analyzed by sodium dodecyl sulfate-gel electrophoresis, immune precipitates from extracts of cells pulse-labeled for 10 min with [(35)S]methionine contain none of the major virion polypeptides. Instead they show prominent viral specific polypeptides of molecular weight 76,000 and 12,000, as well as minor quantities of other labeled polypeptides. From pulse-chase kinetics and two-dimensional tryptic finger-prints it appears that the large polypeptide is a precursor of at least the two major virion proteins of molecular weights 24,000 and 11,000, while the smaller is a precursor of the 11,000-dalton virion protein.
By clone purification of avian myelocytomatosis virus MC29, a virus strain, MCV-B, was isolated which was found to belong to subgroup B of avian leukosis-sarcoma viruses. It contained a nontransforming virus in excess of a transforming virus defective for reproduction. MCV-B retained the capacity to transform hematopoietic chicken tissues at a high efficiency. It was also capable of inducing two types of foci and agar suspension colonies in cell cultures derived from 11-day-old chicken embryos. These alterations were shown to be due to a morphological transformation of two different types of target cells designated s and l. Several lines of evidence indicate that a single agent is able to transform both types of target cells. Pure cultures of s-type cells were obtained by cloning of uninfected chicken cells. They were highly susceptible to transformation with Rous sarcoma virus (RSV) but resistant to transformation with avian myeloblastosis virus (AMV) and myeloblastosis associated virus (MAV). Efforts to clone-purify uninfected l-type cells have failed so far. It is suggested that this type of cell is derived from the hematopoietic system and is the in vitro target cell for transformation with AMV.
A colony of transformed cells was isolated from chick-embryo cells infected with a stock of nondefective Schmidt-Ruppin strain of Rous sarcoma virus. The virus recovered from this colony was a stable defective mutant very similar to the Bryan strain of Rous sarcoma virus in the following characteristics: (i) noninfectiousness of virus particles released from transformed cells that lack helper factor; (ii) formation of infectious pseudotypes by coinfection with avian leukosis virus or by interaction with endogenous-helper factor in chicken cells; (iii) ability of the noninfectious form of virus to transform chick-embryo cells in the presence of ultraviolet light-inactivated Sendai virus; (iv) absence of glycoprotein in the noninfectious form; (v) failure to produce nondefective virus by recombination with avian leukosis virus; and (vi) segregation of polymerase-negative virus.
The morphology of transformed cells is characteristic of those infected by the Schmidt-Ruppin strain. The demonstration of segregation of such a defective virus from nondefective sarcoma virus and failure to detect revertants of this mutant suggest that the deletion of some genes may be involved in this mutation.
SUMMARY A transplantable cell line was isolated from a liver tumor induced by the MC29 strain of avian leukosis virus. It was found that its ability to develop into a tumor when inoculated in recipient birds (both intramuscularly and in the wing web) was dependent on a morphological alteration which took place after the cells had been in culture for some time. The transplantable cell exhibited unusual properties, compared with other MC29 altered cells, and appears to represent a unique target cell for the MC29 virus.
Endogenous leukosis-like viruses of ring-necked pheasants (Phasianus colchicus) and golden pheasants (Chrysolophus pictus) have been isolated and characterized. The majority of the normal pheasant embryo cultures contain helper activity for the defective Bryan high titer strain of Rous sarcoma virus. The Rous sarcoma pseudotypes produced with endogenous helper activity from ring-necked pheasants belong to subgroup F. The pseudotypes from golden pheasant cells constitute subgroup G. Subgroup F and G pseudotypes can infect all known genetic types of chicken fibroblasts as well as pheasant and Japanese quail cells, but do not plate on goose cells. Duck cells are resistant to subgroup G but not to F.The subgroup F and G helper viruses isolated from Rous sarcoma viral pseudotypes show interference with their homologous subgroup. RAV-61, a standard of subgroup F, interferes with pseudotypes produced with endogenous helper activity from ring-necked pheasant cells but not with subgroup G pseudotypes.Subgroups F and G do not cross-react with subgroup A to E in neutralization tests. Some normal ring-necked pheasant sera have anti-F activity.Subgroup F and probably also G leukosis-like viruses can undergo genetic recombination with nondefective avian sarcoma viruses.
The RNAs and proteins specified by five early genes of bacteriophage T7 have been identified by electrophoresis on sodium dodecyl sulfate, polyacrylamide gels. Extracts of cells infected by different deletion strains and point mutants of T7 are analyzed on a slab gel system in which 25 samples can be run simultaneously and then dried for autoradiography. The high capacity of this system makes it possible to do many types of experiment that would be extremely tedious by other means.The five early genes are designated 0.3,0.7, 1, 1.1 and 1.3, in order from left to right on the T7 genetic map. The stop signal that prevents host RNA polymerase from transcribing into the late region of T7 DNA is located to the right of gene 1.3 (ligase). Most deletions that affect gene 1.3 also delete the stop signal, and some of them affect at least one late protein, the 1.7 protein. Several small, early RNAs can be resolved that are not affected by any of the deletions. These small RNAs could not come from between the five early genes or from the right end of the early region, and other work (Dunn & Studier, 1973) indicates that at least some of them come from the region to the left of gene 0.3.Deletions have been found that enter either end of the gene 1 RNA or the right ends of the 0.3 or 1.1 RNAs without seeming to affect the proteins specified by these RNAs. Perhaps all of the early messenger RNAs of T7 have untranslated regions at both ends. Some deletions that enter the left end of the gene 1 RNA reduce the amount of gene 1 protein that is synthesized, presumably by interfering with initiation of protein synthesis.
A simple method is described for detecting 3H in polyacrylamide gels by scintillation autography (fluorography) using X-ray film. The gel is dehydrated in dimethyl sulphoxide, soaked in a solution of 2,5-diphenyloxazole (PPO) in dimethylsulphoxide, dried and exposed to RP Royal “X-Omat” film at -70 °C. Optimal conditions for each step are described. β-particles from 3H interact with the 2,5-diphenyloxazole emitting light which causes local blackening of an X-ray film. The image produced resembles that obtained by conventional autoradiography of isotopes with higher emission energies such as 14C. 3000 dis. 3H/min in a band in a gel can be detected in a 24-h exposure. Similarly 500 dis./min can be detected in one week.
When applied to the detection of 35S and 14C in polyacrylamide gels, this method is ten times more sensitive than conventional autoradiography. 130 dis. 35S or 14C/min in a band in a gel can be detected in 24 h.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
There occurs a characteristic morphological conversion when certain cell cultures are infected with avian myeloblastosis virus (AMV). AMV induces the formation of at least two, new and stable, probably neoplastic types of cells—myeloblasts and osteoblasts. The converting effect of AMV upon cell cultures appears to be similar to the oncogenic effect of this virus in vivo. Conversion depends upon the presence of susceptible target cells. The presented evidence indicates that these target cells are some mesenchymal precursors of myeloblasts in the normal sequence of development. When cells, other than target cells, become infected by AMV they apparently retain their normal morphology but become virus producers; these cells multiply at least as well as noninfected cells.The formation of discrete foci of converted cells in monolayer cultures infected with AMV provides an in vitro assay system.
A simple and rapid method is presented for the preparation of I/sup 131/- labeled human growth hormone of high specific radioactivity (240-300 mu C/ mu g). Low amounts of carrierfree I/sup 131/ iodide (2 mC) are allowed to react, without prior treatment, with small quantities of protein (5 mu g) in a highyield reaction (approx. 70% transfer of I/sup 131/ to protein). The degree of chemical substitution is minimized (0.5- 1.0 atom of iodine/molecule of protein) by the use of carrier-free I/sup 131/ iodide. The I/sup 131/-labeled hormone (up to 300 mu C/ mu g) contains no detectable degradation products and is immunologically identical with the unlabeled hormone. The loss of immunological reactivity at high specific radioactivities or at high levels of chemical substitution with STAI/sup 127/!iodine is demonstrated. (auth)
This chapter focuses on avian virus growth and their etiologic agents. Despite great apparent differences in pathogenesis and etiology of the growths, numerous findings have consistently suggested a distinct continuity of inter associations, both in the natural occurrence and in the viral transmission of the various neoplastic states. A notable trend in the recent investigation of these avian tumors has been a shift in principle away from the earlier purely qualitative biologic experimentation and toward the application of methods designed to afford quantitation and definitive correlation of data of diverse sorts. The most recent work with the avian tumor viruses has been concerned with further characterization of various individual strains of the agents and with penetration into the still obscure province of cell–virus interactions at the ultrastructural and biochemical levels. Although the evidence of viral etiology has been well established, the concepts of the etiologic interrelationships of the diseases have represented, to this day, practically every conceivable variation of the potentialities. The factors that influence and regulate both quantitative and qualitative aspects of host response to the tumor agents do not differ from those concerned with the occurrence of other virus diseases. Among the most helpful advances in the study of the avian tumor viruses is the recent, very considerable increase in the knowledge of the nature of the agents themselves. Systematic utilization of tissue culture systems for study of the avian tumor viruses has been undertaken only in recent years. It has been the intent of this chapter to attempt some correlation of the many data obtained, primarily in the more recent years, in the study of the avian tumors and their filterable etiologic agents.