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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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Cell death mediated by Myc proteins.The indicated Rat1a cell lines were seeded into 12 well pates at 105 cells/well and allowed to achieve approximately 80% confluency. At the indicated times, viable cell numbers in triplicate plates were determined by flow cytometry using an Annexin V-Propidium Iodide staining protocol as described in Materials and Methods. The results represent the average amount of staining for the triplicate samples +/− 1 SE. The experiment was repeated on at least three occasions with similar results. (a) Serum withdrawal. The cells were washed in PBS and incubated in serum-free medium for the remainder of the study. (b) Glutamine withdrawal. The cells were washed in PBS and incubated in glutamine-free medium for the remainder of the study.
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pone-0013717-g003: Cell death mediated by Myc proteins.The indicated Rat1a cell lines were seeded into 12 well pates at 105 cells/well and allowed to achieve approximately 80% confluency. At the indicated times, viable cell numbers in triplicate plates were determined by flow cytometry using an Annexin V-Propidium Iodide staining protocol as described in Materials and Methods. The results represent the average amount of staining for the triplicate samples +/− 1 SE. The experiment was repeated on at least three occasions with similar results. (a) Serum withdrawal. The cells were washed in PBS and incubated in serum-free medium for the remainder of the study. (b) Glutamine withdrawal. The cells were washed in PBS and incubated in glutamine-free medium for the remainder of the study.

Mentions: Myc confers a proliferative advantage when over-expressed in various cell backgrounds including Rat1a cells [32]. We therefore compared the growth rates of the six Rat1a cell lines depicted in Figure 1 under non-limiting growth conditions (Figure 2). When viable, adherent cells were enumerated, it was observed that all MBII mutants and wtMyc cells grew modestly faster than did the vector control line. C133S and W135E cells also attained approximately a 1.5-2-fold higher final density and remained viable longer. We next asked whether any of the mutations altered the well-known ability of Myc over-expression to sensitize cells to pro-apoptotic stimuli [15], [33], [34], [35]. We therefore grew each of the cell lines to approximately 80% confluency under non-limiting conditions, then changed to media lacking either serum (Figure 3a) or glutamine (Figure 3b), and subsequently assessed viability. As seen in Figure 3a, wtMyc cells showed the expected high sensitivity to serum deprivation as manifested by an approximately 9-fold higher fraction of apoptotic cells at 72 hours when compared to vector cells. W135E and C133S cells were almost as sensitive as wtMyc cells to serum withdrawal, whereas Q131R cells and F138C cells, like vector cells, were resistant.


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)

Cell death mediated by Myc proteins.The indicated Rat1a cell lines were seeded into 12 well pates at 105 cells/well and allowed to achieve approximately 80% confluency. At the indicated times, viable cell numbers in triplicate plates were determined by flow cytometry using an Annexin V-Propidium Iodide staining protocol as described in Materials and Methods. The results represent the average amount of staining for the triplicate samples +/− 1 SE. The experiment was repeated on at least three occasions with similar results. (a) Serum withdrawal. The cells were washed in PBS and incubated in serum-free medium for the remainder of the study. (b) Glutamine withdrawal. The cells were washed in PBS and incubated in glutamine-free medium for the remainder of the study.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965668&req=5

pone-0013717-g003: Cell death mediated by Myc proteins.The indicated Rat1a cell lines were seeded into 12 well pates at 105 cells/well and allowed to achieve approximately 80% confluency. At the indicated times, viable cell numbers in triplicate plates were determined by flow cytometry using an Annexin V-Propidium Iodide staining protocol as described in Materials and Methods. The results represent the average amount of staining for the triplicate samples +/− 1 SE. The experiment was repeated on at least three occasions with similar results. (a) Serum withdrawal. The cells were washed in PBS and incubated in serum-free medium for the remainder of the study. (b) Glutamine withdrawal. The cells were washed in PBS and incubated in glutamine-free medium for the remainder of the study.
Mentions: Myc confers a proliferative advantage when over-expressed in various cell backgrounds including Rat1a cells [32]. We therefore compared the growth rates of the six Rat1a cell lines depicted in Figure 1 under non-limiting growth conditions (Figure 2). When viable, adherent cells were enumerated, it was observed that all MBII mutants and wtMyc cells grew modestly faster than did the vector control line. C133S and W135E cells also attained approximately a 1.5-2-fold higher final density and remained viable longer. We next asked whether any of the mutations altered the well-known ability of Myc over-expression to sensitize cells to pro-apoptotic stimuli [15], [33], [34], [35]. We therefore grew each of the cell lines to approximately 80% confluency under non-limiting conditions, then changed to media lacking either serum (Figure 3a) or glutamine (Figure 3b), and subsequently assessed viability. As seen in Figure 3a, wtMyc cells showed the expected high sensitivity to serum deprivation as manifested by an approximately 9-fold higher fraction of apoptotic cells at 72 hours when compared to vector cells. W135E and C133S cells were almost as sensitive as wtMyc cells to serum withdrawal, whereas Q131R cells and F138C cells, like vector cells, were resistant.

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