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. 2010 Oct 28;5(10):e13717.
doi: 10.1371/journal.pone.0013717.

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

J Anthony Graves  1 , Kristi Rothermund, Tao Wang, Wei Qian, Bennett Van Houten, Edward V Prochownik
Affiliations

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

J Anthony Graves et al. PLoS One. .

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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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. 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.
Figure 2
Figure 2. Effect of Myc point mutations on proliferation.
The indicated Rat1a cell lines were seeded at 104 cells/well in 12 well plates and allowed to attach in medium containing 10% FBS. At the indicated times, the total cell number in triplicate wells was determined. The points shown represent the average number of cells/well +/− 1 standard error (SE). The experiment was repeated on at least three occasions with similar results. Only adherent cells were counted. The number of cells at the latter time points may be affected by contact inhibition or cell loss due to overgrowth.
Figure 3
Figure 3. 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.
Figure 4
Figure 4. GI induced by Myc proteins.
(a) Induction of tetraploidy. Each of the depicted cell lines showed indistinguishable cell cycle parameters during logarithmic growth in the absence of colcemid (top row). Treatment of vector cells with colcemid resulted in the expected accumulation of G2/M-arrest cells containing a 4N DNA content, whereas a significant fraction of wtMyc cells accumulated tetraploid DNA content (8N bottom row) , . Note the varying degrees of tetraploidy induction in all mutant cell lines except Q131R. (b) Induction of ROS. Each of the cell lines was exposed to CM-H2-DCFDA and was then assessed by flow cytometry. Note the >3-fold higher levels of ROS in wtMyc cells compared to vector control cells as previously described , and that only C133S cells were able to reproduce this effect. p-values were calculated by a two-tailed t-test in Microsoft Excel when compared to vector cells: (*) p≤0.05; (**) p≤0.005.
Figure 5
Figure 5. OCR and ECAR in Rat1a cells expressing human Myc point mutants.
(a) A Seahorse Bioscience XF24 Extracellular Flux Analyzer was used for the real-time determination of metabolism. OCR (OXPHOS) was expressed as a function of time. Each inhibitor as described in Results was injected at the times indicated by the vertical lines (injections 1–4). A typical experiment, performed in triplicate wells is shown. The experiment was repeated on at least three occasions with similar results. (b) Areas under the curve (AUC) were calculated with the software provided by the manufacturer and used to compare the various cell lines' total reserve respiratory capacity relative to that of vector control cells, which are arbitrarily set at 1. The average of three to five individual experiments, where each cell line was measured at least in triplicate is displayed +/− 1 SE. p-values of the comparisons with vector control cells were calculated by a two-tailed t-test in Microsoft Excel: (*) p≤0.05; (**) 0.01. This graph represents the AUCs for the time point just prior to injection 2 until the time point just before injection 4. This represents the total reserve respiratory capacity of each cell line. (c) ECARs (glycolysis) were expressed as a function of time. The vertical lines represent the same injections as previously described. The experiment was repeated on at least three occasions with similar results. (d) AUCs for the time point just prior to injection 1 to the time point just prior to injection 2 were calculated. This is the best representation of the glycolytic potential of these strains. p-values were calculated and represented as described for (b).
Figure 6
Figure 6. Effect of Myc point mutations on transformation.
(a) In vitro transformation. Anchorage-independent clonogenic growth in soft agar was assessed as previously described , . 12–14 days after plating, the total number of macroscopically visible colonies on each plate was quantified. Numbers shown represent the average number of colonies seen in triplicate cultures +/− 1 SE. p-values of the comparisons with vector control cells were calculated by a two-tailed t-test in Microsoft Excel: (**) p≤0.01; (***) ≤0.005. (b) In vivo transformation. 5×106 of the indicated Rat1a cell lines were inoculated subcutaneously into the flanks of nude mice. Each inoculum was performed in triplicate and tumor sizes were evaluated weekly. The data shown represents two individually repeated experiments. Averages tumor sizes +/− 1 SE are depicted as a function of time.
Figure 7
Figure 7. 18F-2DG imaging by PET.
Duplicate tumor-bearing mice inoculated with wtMyc, Q131R, or F138C cells were imaged by PET following injection of 18F-labeled 2DG. Dotted lines indicate tumor boundaries. Arrows indicate the urinary bladder, where the isotope is expectedly the most concentrated. The average relative SUV is indicated with the value for wtMyc set at 1.
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