Replication Study: Transcriptional amplification in tumor cells with elevated c-Myc

Abstract

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Blum et al., 2015), that described how we intended to replicate selected experiments from the paper 'Transcriptional amplification in tumor cells with elevated c-Myc' (Lin et al., 2012). Here we report the results. We found overexpression of c-Myc increased total levels of RNA in P493-6 Burkitt's lymphoma cells; however, while the effect was in the same direction as the original study (Figure 3E; Lin et al., 2012), statistical significance and the size of the effect varied between the original study and the two different lots of serum tested in this replication. Digital gene expression analysis for a set of genes was also performed on P493-6 cells before and after c-Myc overexpression. Transcripts from genes that were active before c-Myc induction increased in expression following c-Myc overexpression, similar to the original study (Figure 3F; Lin et al., 2012). Transcripts from genes that were silent before c-Myc induction also increased in expression following c-Myc overexpression, while the original study concluded elevated c-Myc had no effect on silent genes (Figure 3F; Lin et al., 2012). Treating the data as paired, we found a statistically significant increase in gene expression for both active and silent genes upon c-Myc induction, with the change in gene expression greater for active genes compared to silent genes. Finally, we report meta-analyses for each result.

Keywords: Reproducibility Project: Cancer Biology; biochemistry; c-Myc; cancer biology; gene expression; human; metascience; replication; reproducibility.

Figure 1.. Induction of c-Myc in P493-6 cells and impact on total RNA levels.

P493-6 cells were grown in the presence of tetracycline (Tet) for 72 hr and switched into Tet-free growth medium to induce c-Myc expression. Cells were cultured in two separate lots of serum. (A) Representative Western blot using an anti-c-Myc antibody (top panels) or an anti-ß-Actin antibody (bottom panel). Two exposures of the anti-c-Myc antibody are presented to facilitate detection of c-Myc. (B) Quantification of total RNA levels (ng of total RNA per 1,000 cells) for cells at 0, 1, and 24 hr after release from Tet. Means reported and error bars represent s.e.m. from three independent biological repeats. For serum lot one, one-way ANOVA on total RNA levels of all groups; F(2, 6)=1.25, p=0.353. Planned contrast between 0 hr and 24 hr; t(6) = 1.02, p=0.347 with a priori alpha level = 0.05. For serum lot two, one-way ANOVA on total RNA levels of all groups; F(2, 6)=21.87, p=0.00176. Planned contrast between 0 hr and 24 hr; t(6) = 5.03, p=0.0024 with a priori alpha level = 0.05. Additional details for this experiment can be found at https://osf.io/tfd57/.

Figure 2.. Digital gene expression analysis.

P493-6 cells grown in the presence of tetracycline (Tet) for 72 hr for repression of the conditional pmyc-tet construct, were switched into Tet-free growth medium to induce c-Myc expression. Cells were cultured in two separate lots of serum. Transcripts/cell estimates from NanoString nCounter gene expression assays (1369 genes assay) for active (left) and silent (right) genes at 0, 1, and 24 hr after release from Tet. Active genes expressed greater than one transcript/cell. Silent genes expressed less than 0.5 transcript/cell. Box and whisker plots with median represented as the line through the box and whiskers representing values within 1.5 IQR of the first and third quartile. Cells grown in serum lot one: active genes = 708, silent genes = 580. Cells grown in serum lot two: active genes = 719, silent genes = 573. Confirmatory analysis is reported in Table 1 and exploratory statistical analysis is reported in Table 2 and Table 3. Additional details for this experiment can be found at https://osf.io/fn2y4/.

Figure 2—figure supplement 1.. Logarithmic expression of genes.

This is the same experiment as in Figure 2. (A–B, E–F) Gene expression data plotted on a log₂ transformed scale for active (A, E) and silent (B, F) genes at 0, 1, and 24 hr after release from Tet for both lots of serum. (C–D, G–H) Box and whisker plots showing gene expression changes (log₂ ratio) between the indicated times for active (C, G) and silent (D, H) genes. Median represented as the line through the box and whiskers representing values within 1.5 IQR of the first and third quartile. Additional details for this experiment can be found at https://osf.io/fn2y4/.

Figure 2—figure supplement 2.. Comparison of gene expression data as continuous.

This is the same experiment as in Figure 2. (A–C, E–G) Scatter plots of log₂ transformed gene expression data for all genes analyzed at the indicated times on the y and x axes for both lots of serum. Active genes are blue, silent genes are red, and genes that are neither active or silent (expression was more than 0.5 transcript/cell and less than one transcript/cell at time 0 hr) are white. (D, H) Box and whisker plots showing gene expression changes (log₂ ratio) between the indicated times for all genes analyzed for both lots of serum. Median represented as the line through the box and whiskers representing values within 1.5 IQR of the first and third quartile. Additional details for this experiment can be found at https://osf.io/fn2y4/.

Figure 3.. Meta-analyses of each effect.

Effect size and 95% confidence interval are presented for Lin et al., 2012, this replication study (RP:CB), and a random effects meta-analysis of those two effects. Cohen’s d is the standardized difference between the two measurements, with a larger positive value indicating total RNA levels are increased at 24 hr compared to 0 hr. The effect size r is a standardized measure of the correlation (strength and direction) of the association between gene expression and c-Myc induction, with a larger positive value indicating gene expression increased during the course of c-Myc induction. Sample sizes used in Lin et al., 2012 and this replication attempt are reported under the study name. (A) Total RNA levels in P493-6 cells 0 hr compared to 24 hr after release from tetracycline (meta-analysis p = 0.0488). (B) Gene expression of active or silent genes are shown for all comparisons. Active genes: 0 hr compared to 1 hr (meta-analysis p = 1.12x10^-7), 0 hr compared to 24 hr (meta-analysis p = 7.01x10^-4), 1 hr compared to 24 hr (meta-analysis p = 0.0129). Silent genes: 0 hr compared to 1 hr (meta-analysis p = 0.203), 0 hr compared to 24 hr (meta-analysis p = 7.10x10^-17), 1 hr compared to 24 hr (meta-analysis p = 0.0571). Additional details for these meta-analyses can be found at https://osf.io/5yscz/.