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Point mutations in c-Myc uncouple neoplastic transformation from multiple other phenotypes in rat fibroblasts.

Graves JA, Rothermund K, Wang T, Qian W, Van Houten B, Prochownik EV - PLoS ONE (2010)

Bottom Line: In addition to transforming various cell types, Myc also influences additional transformation-associated cellular phenotypes including proliferation, survival, genomic instability, reactive oxygen species production, and metabolism.Specifically, the higher rate of glucose metabolism known to be associated with wtMyc deregulation was found to be independent of transformation.These findings indicate that, while the Myc phenotypes examined here make additive contributions to transformation, none, with the possible exception of increased reliance on extracellular glutamine for survival, are necessary for achieving this state.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Department of Pediatrics, Children's Hospital of Pittsburgh of The University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States of America. gravja@chp.edu

ABSTRACT
Deregulation of c-Myc (Myc) occurs in many cancers. In addition to transforming various cell types, Myc also influences additional transformation-associated cellular phenotypes including proliferation, survival, genomic instability, reactive oxygen species production, and metabolism. Although Myc is wild type in most cancers (wtMyc), it occasionally acquires point mutations in certain lymphomas. Some of these mutations confer a survival advantage despite partially attenuating proliferation and transformation. Here, we have evaluated four naturally-occurring or synthetic point mutations of Myc for their ability to affect these phenotypes, as well as to promote genomic instability, to generate reactive oxygen species and to up-regulate aerobic glycolysis and oxidative phosphorylation. Our findings indicate that many of these phenotypes are genetically and functionally independent of one another and are not necessary for transformation. Specifically, the higher rate of glucose metabolism known to be associated with wtMyc deregulation was found to be independent of transformation. One mutation (Q131R) was greatly impaired for nearly all of the studied Myc phenotypes, yet was able to retain some ability to transform. These findings indicate that, while the Myc phenotypes examined here make additive contributions to transformation, none, with the possible exception of increased reliance on extracellular glutamine for survival, are necessary for achieving this state.

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Expression of Myc mutants in Rat1a cells.(a) Diagram of the Myc protein. The approximately 150 residue TRD is shaded, with an expanded MBII domain and relevant amino acids substitutions depicted below the diagram. The basic-helix-loop-helix dimerization domain at the extreme C-terminus of the protein is indicated by the checkered box. (b) Each of the indicated mutations, along with wtMyc was expressed in Rat1a fibroblasts following lentiviral-mediated transduction. An empty lentiviral vector infection served as a negative control. Pooled, blasticidin-resistant colonies were subjected to western analysis for either Myc or β-tubulin, which served as a loading control. The monoclonal antibody used to detect human Myc proteins had some cross reactivity with endogenous rat Myc.
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pone-0013717-g001: Expression of Myc mutants in Rat1a cells.(a) Diagram of the Myc protein. The approximately 150 residue TRD is shaded, with an expanded MBII domain and relevant amino acids substitutions depicted below the diagram. The basic-helix-loop-helix dimerization domain at the extreme C-terminus of the protein is indicated by the checkered box. (b) Each of the indicated mutations, along with wtMyc was expressed in Rat1a fibroblasts following lentiviral-mediated transduction. An empty lentiviral vector infection served as a negative control. Pooled, blasticidin-resistant colonies were subjected to western analysis for either Myc or β-tubulin, which served as a loading control. The monoclonal antibody used to detect human Myc proteins had some cross reactivity with endogenous rat Myc.

Mentions: Previous studies have shown that several naturally-occurring and synthetic MBII point mutations affect the ability of Myc to transform and/or promote proliferation and apoptosis. However, some of these proteins contained multiple TRD mutations, were assessed only for a limited number of phenotypes, or were evaluated in different cell types that complicated or precluded cross-study comparisons [10], [14], [15], [28]. We therefore re-examined these properties, as well as several additional ones, in the same cell line for four individual point mutants (Figure 1a). The Q131R and C133S constructs alter residues that have been targeted in previous studies [15]. The F138C mutant has been found to occur naturally in Burkitt's lymphoma, although it typically appears in combination with other mutations [5], [28]. The W135E mutant has been identified as having a reduced transformation capacity [15] and has been speculated to be deficient it its ability to interact with other proteins such as TRAAP [29]. These human proteins were expressed in Rat1a fibroblasts, because this cell background has been routinely used to evaluate numerous Myc phenotypes, including anchorage-independent transformation and in vivo tumorigenesis [14], [15], [30], [31]. Following high-efficiency lentiviral-mediated transduction, surviving clones were pooled and immediately examined for the expression of their respective proteins. As seen in Figure 1b, all four mutants were expressed at similar levels and were comparable to the expression of the wild-type Myc (wtMyc) protein.


Point mutations in c-Myc uncouple neoplastic transformation from multiple other phenotypes in rat fibroblasts.

Graves JA, Rothermund K, Wang T, Qian W, Van Houten B, Prochownik EV - PLoS ONE (2010)

Expression of Myc mutants in Rat1a cells.(a) Diagram of the Myc protein. The approximately 150 residue TRD is shaded, with an expanded MBII domain and relevant amino acids substitutions depicted below the diagram. The basic-helix-loop-helix dimerization domain at the extreme C-terminus of the protein is indicated by the checkered box. (b) Each of the indicated mutations, along with wtMyc was expressed in Rat1a fibroblasts following lentiviral-mediated transduction. An empty lentiviral vector infection served as a negative control. Pooled, blasticidin-resistant colonies were subjected to western analysis for either Myc or β-tubulin, which served as a loading control. The monoclonal antibody used to detect human Myc proteins had some cross reactivity with endogenous rat Myc.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965668&req=5

pone-0013717-g001: Expression of Myc mutants in Rat1a cells.(a) Diagram of the Myc protein. The approximately 150 residue TRD is shaded, with an expanded MBII domain and relevant amino acids substitutions depicted below the diagram. The basic-helix-loop-helix dimerization domain at the extreme C-terminus of the protein is indicated by the checkered box. (b) Each of the indicated mutations, along with wtMyc was expressed in Rat1a fibroblasts following lentiviral-mediated transduction. An empty lentiviral vector infection served as a negative control. Pooled, blasticidin-resistant colonies were subjected to western analysis for either Myc or β-tubulin, which served as a loading control. The monoclonal antibody used to detect human Myc proteins had some cross reactivity with endogenous rat Myc.
Mentions: Previous studies have shown that several naturally-occurring and synthetic MBII point mutations affect the ability of Myc to transform and/or promote proliferation and apoptosis. However, some of these proteins contained multiple TRD mutations, were assessed only for a limited number of phenotypes, or were evaluated in different cell types that complicated or precluded cross-study comparisons [10], [14], [15], [28]. We therefore re-examined these properties, as well as several additional ones, in the same cell line for four individual point mutants (Figure 1a). The Q131R and C133S constructs alter residues that have been targeted in previous studies [15]. The F138C mutant has been found to occur naturally in Burkitt's lymphoma, although it typically appears in combination with other mutations [5], [28]. The W135E mutant has been identified as having a reduced transformation capacity [15] and has been speculated to be deficient it its ability to interact with other proteins such as TRAAP [29]. These human proteins were expressed in Rat1a fibroblasts, because this cell background has been routinely used to evaluate numerous Myc phenotypes, including anchorage-independent transformation and in vivo tumorigenesis [14], [15], [30], [31]. Following high-efficiency lentiviral-mediated transduction, surviving clones were pooled and immediately examined for the expression of their respective proteins. As seen in Figure 1b, all four mutants were expressed at similar levels and were comparable to the expression of the wild-type Myc (wtMyc) protein.

Bottom Line: In addition to transforming various cell types, Myc also influences additional transformation-associated cellular phenotypes including proliferation, survival, genomic instability, reactive oxygen species production, and metabolism.Specifically, the higher rate of glucose metabolism known to be associated with wtMyc deregulation was found to be independent of transformation.These findings indicate that, while the Myc phenotypes examined here make additive contributions to transformation, none, with the possible exception of increased reliance on extracellular glutamine for survival, are necessary for achieving this state.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Department of Pediatrics, Children's Hospital of Pittsburgh of The University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States of America. gravja@chp.edu

ABSTRACT
Deregulation of c-Myc (Myc) occurs in many cancers. In addition to transforming various cell types, Myc also influences additional transformation-associated cellular phenotypes including proliferation, survival, genomic instability, reactive oxygen species production, and metabolism. Although Myc is wild type in most cancers (wtMyc), it occasionally acquires point mutations in certain lymphomas. Some of these mutations confer a survival advantage despite partially attenuating proliferation and transformation. Here, we have evaluated four naturally-occurring or synthetic point mutations of Myc for their ability to affect these phenotypes, as well as to promote genomic instability, to generate reactive oxygen species and to up-regulate aerobic glycolysis and oxidative phosphorylation. Our findings indicate that many of these phenotypes are genetically and functionally independent of one another and are not necessary for transformation. Specifically, the higher rate of glucose metabolism known to be associated with wtMyc deregulation was found to be independent of transformation. One mutation (Q131R) was greatly impaired for nearly all of the studied Myc phenotypes, yet was able to retain some ability to transform. These findings indicate that, while the Myc phenotypes examined here make additive contributions to transformation, none, with the possible exception of increased reliance on extracellular glutamine for survival, are necessary for achieving this state.

Show MeSH
Related in: MedlinePlus