Optimization of oncogene expression through intra‐population competition

Abstract

Although functional roles have been assigned to many genes, e.g. those involved in cell-cycle regulation, growth signaling, or cancer, considerably less is known about the quantitative relationship between gene expression levels and outcome. We devised an intra-population competition to study oncogene dosage. Cell populations were engineered to express a range of H-Ras oncogene levels. Cells with different levels of H-Ras then "competed" for an increased share of the total cell population. Using flow cytometry to track the population composition over time, we determined the relationship between the different H-Ras oncogene expression levels and the net proliferation rate. Under culture conditions in which wild-type Ras activation was suppressed, we found that increased and maximal net proliferation occurred when the H-Ras G12V oncogene was expressed at a level 1.2-fold that of wild-type Ras. As the H-Ras G12V expression levels increased above this optimal level, proliferation rates decreased. Our findings suggest that the tumor evolution process may optimize gene expression levels for maximal cell proliferation. In principle, engineered intra-population competitions can be used to determine proliferation rates associated with the level of any ectopically expressed gene. The approach also may be used to determine proliferation rates associated with different cell species in a heterogeneous population or to improve the proliferation rate of a cell line. We also envision that the tracking of intra-population competitions could be utilized to investigate the evolution of tumors in the body.

Quantitative analysis of mammalian translation initiation sites by FACS‐seq

“…HCT‐116 cells were cultured in McCoy's 5A medium (HyClone Laboratories, Logan, UT) with 10% FBS. Where indicated, 1.75 μM imatinib (Novartis, Basel, Switzerland) was added (Muljo & Schlissel, ; Ferreira & Wang, ). All cells were cultured with 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C with 5% CO 2 .…”

Engineering ribosomal leaky scanning and upstream open reading frames for precise control of protein translation

“…Alternatively, researchers may use our vectors that already contain the engineered RNA leaders (available through Addgene). To date, our lab has utilized our RNA leader sequences to tune the expression levels of GFP, mCherry, mTagBFP, mKate, c-Myc, p53, p21, H-Ras, 20 FKBP, and DHFR. For applications where the protein of interest can be toxic or applications where conditional or dynamic control of expression is desired, we have found it desirable to specify the magnitude of expression by controlling translation and to conditionally turn on expression using small-molecule inducible control of protein localization (estrogen-receptor fusion with nuclear localization induced by addition of 4-hydroxytamoxifen) and stability (DHFR-fusion stabilized by trimethoprim, Fig.…”

Editorial: Biomolecular engineering – latest advances and applications

“…The final session of the second day, “Biomolecular Engineering for Cell and Tissue Engineering,” was co‐chaired by Prof. Clifford Wang from Stanford University. In this issue, Ferreira and Wang [8] report a novel approach for quantifying the relationship between gene expression levels and the net proliferation rate. The outcomes of this study can be potentially used for investigating the evolution of tumors in the body.…”