New model systems provide insights into Myc-induced transformation

Abstract

The ability of Myc to promote cellular transformation is well established; however, a better understanding of the mechanisms through which Myc mediates tumorigenesis is essential for the development of therapeutic approaches to target this potent oncoprotein. Structure-function studies in rodent fibroblast cells have provided the basis for much of our current understanding of these mechanisms. To build on these approaches, we have characterized three novel human cell line models of Myc-dependent transformation: MCF10A, SH-EP Tet21/N-Myc, and LF1/TERT/LT/ST cells. We have also evaluated Myc family proteins (c-Myc and L-Myc), a naturally occurring isoform of Myc (MycS), and a set of N-terminal domain mutants (ΔMBII, W135E, T58A) for their ability to promote anchorage-independent growth in these models. Taken together, these results provide the field with three new human cell-based models to study Myc activity, highlight the importance of cellular context, and challenge the paradigm that the ability of Myc to promote tumorigenesis is exclusively MBII-dependent.

Figure 1. Human Myc promotes cellular transformation in MCF10A, SH-EP and LF1/3T human cell lines

Control green fluorescent protein (GFP) or human Myc cDNA were introduced by infection with ecotropic, replication-incompetent retrovirus into human cell lines, MCF10A, SH-EP and LF1/3T, as described previously (Wu et al., 2004) and stable pools were isolated by fluorescence-activated cell sorting (FACS) for green fluorescent protein (GFP) expression. A) MCF10A cells (a kind gift from Senthil Muthuswamy, Ontatio Cancer Institute) were cultured as described previously (Debnath et al., 2003). Growth factor withdrawal was achieved by culturing cells in media containing 0.05% horse serum and supplemented with only 10 μL/mL insulin for one hour. Ectopic Myc expression was confirmed by immunoblotting of lysates isolated from asynchronously growing cells in full growth media and from cells subjected to 1 hour of serum and growth factor withdrawal. B) Transformation was evaluated by anchorage-independent colony growth in soft agar. Soft agar experiments were completed as previously described with the following modifications, 5 000 cells were seeded per 35 mm petri dish in triplicate, and colonies (greater than 6 cells) were counted at the end of a 2–3 week period (Oster et al., 2003). Transformation data is presented as a relative number of colonies compared to cells expressing wild-type Myc. Data represents the mean ± standard deviation from three independent experiments, **p<0.01, ***p<0.001, paired t-test. C) SH-EP Tet21/N-Myc cells (a kind gift from Manfred Schwab, German Cancer Research Center) were cultured in RPMI 1640 with 10% FBS (Breit and Schwab, 1989). Ectopic Myc expression was confirmed by immunoblotting. This experiment was conducted both in the presence and absence of N-Myc expression. 1 μg/mL tetracycline (Sigma, St. Louis, MO) was added to growth media 48 hours prior to experiments to inactivate N-Myc expression. D) Soft agar transformation experiments were conducted as above, both in the absence (N-Myc ON) and presence (N-Myc OFF) of tetracycline. Data represents the mean ± standard deviation from two independent experiments. E) LF1/TERT/LT/ST cells (a kind gift from John Sedivy, Brown University) were grown in HAM F10 media and supplemented with 15% fetal bovine serum (FBS) (Wei et al., 2003). Ectopic Myc expression was confirmed by immunoblotting. F) Soft agar transformation experiments were conducted as above and data represents the mean ± standard deviation from three independent experiments, **p<0.01, paired t-test.

Figure 2. Transformation in MCF10A and SH-EP cells is MBII-dependent

The panel of Myc cDNAs were introduced by infection with ecotropic, replication-incompetent retrovirus into MCF10A and SH-EP cells as described previously (Wu et al., 2004). For all SH-EP experiments, 1 μg/mL tetracycline (Sigma, St. Louis, MO) was added to the media 48 hours prior to experiments to inactivate N-Myc expression. A,D) Protein expression was evaluated by immunoblotting. MCF10A cells were harvested under both asynchronously growing and 1 hour serum and growth factor withdrawl conditions. * indicates non-specific bands. B,E) Cell proliferation was assessed by subconfluently seeding 4000 cells/well in a 24-well dish in triplicate. Cells were counted daily over a 5 day period using a Coulter Counter, or haemocytometer. Population doubling times were calculated using GraphPad Prism software (v2.0b) and are presented as mean ± standard deviation for 3–5 independent experiments. C,F) Soft agar experiments were completed as described in Figure 1. Transformation data is presented as a relative number of colonies compared to cells expressing wild-type Myc, with mean ± standard deviation for 3–6 independent experiments. *p<0.05, **p<0.01, ***p<0.001, paired t-test.

Figure 3. Transformation in LF1/3T cells is MBII-independent

The panel of Myc cDNAs were introduced by infection with ecotropic, replication-incompetent retrovirus into LF1/3T cells as described previously (Wu et al., 2004). A) Protein expression was confirmed by immunoblotting. * indicates non-specific bands. B) Doubling times were determined as described in Figure 2 and are presented as mean ± standard deviation from three independent experiments. C) Soft agar colony formation experiments were performed as described in Figure 1. Transformation data is presented as a relative number of colonies compared to cells expressing wild-type Myc, with mean ± standard deviation for three independent experiments. **p<0.01 paired t-test. D) Rat-1A cells were cultured in DMEM H21 with 10% FBS. Control GFP or Myc cDNAs were introduced by infection with ecotropic, replication-incompetent retrovirus. Protein expression was confirmed by immunoblotting. E,F) Soft agar colony formation experiments were completed as described in Figure 1 with the following modifications, 250 Rat-1A cells were seeded per 35 mm petri dish in triplicate. Data represents the mean ± standard deviation for two independent experiments.