Phenotypic effects of the circadian gene Cryptochrome 2 on cancer-related pathways

Abstract

Background: Circadian genes continue to gain attention as important transcriptional regulators with the potential to influence a variety of biological pathways, including many cancer-related processes. The core circadian gene cryptochrome 2 (CRY2) is essential for proper circadian timing, and is a key component of the negative arm of the circadian feedback loop. As such, aberrant expression of CRY2 may influence carcinogenic processes and thereby impact cancer susceptibility.

Methods: We silenced CRY2 in breast cancer cell lines (MCF-7) using small-interfering oligos (siRNA) and measured the impact of CRY2 knockdown on a number of cancer-relevant parameters. Cell cycle distribution, cell viability, and apoptotic response were measured in CRY2 knockdown (CRY2-) and normal (CRY2+) cell populations using flow cytometry in cells with and without exposure to a mutagen challenge. DNA damage accumulation was measured using the single cell gel electrophoresis (comet) assay, and damage was quantified using the Olive tail moment, which considers the amount and distance of DNA migration away from the nucleus, indicative of DNA strand breaks. Expression changes in cancer-relevant transcripts were measured by whole genome microarray. The Student's t-test was used for statistical comparisons, and P-values obtained from the microarray were adjusted for multiple comparisons using the false discovery rate correction, in order to obtain an adjusted Q-value for each observation.

Results: The comet assay results indicated that upon exposure to the same dose of chemical mutagen, CRY2- cells accumulate significantly more unrepaired DNA damage than CRY2+ cells (P = 0.040), suggesting that CRY2 may be important for DNA repair. In addition, a number of transcripts with relevance for DNA damage repair displayed altered expression following CRY2 silencing. These included BCCIP (Q = 0.002), BCL2 (Q = 0.049), CCND1 (Q = 0.009), CDKN1A (Q < 0.001), GADD45A (Q = 0.002), HERC5 (Q < 0.001), MCM5 (Q = 0.042), PPP1R15A (Q < 0.001), SUMO1 (Q < 0.001), and UBA1 (Q = 0.023). However, no significant influence of CRY2 knockdown on cell cycle distributions, cell cycle checkpoints in response to mutagen challenge, or apoptotic response was detected.

Conclusions: In total, these data suggest a limited, but potentially important role for CRY2 in the regulation of DNA damage repair and the maintenance of genomic stability. Future investigations may focus on identifying the mechanisms by which CRY2 may regulate the expression of transcripts with known relevance for carcinogenesis.

Figure 1

Cell cycle distributions in CRY2- and CRY2+ cells in mock and mutagen treated cell populations. Both in the absence and presence of mutagen, cells with reduced CRY2 had a similar cell cycle distribution as cells with normal CRY2 levels, suggesting no direct impact of CRY2 on cell cycle progression or cell cycle checkpoints in response to DNA damage. All results are based on the average of triplicate experiments, and error bars are for SEM.

Figure 2

Cell death and apoptosis in CRY2+ and CRY2- cell populations. No differences were observed in CRY2 knockdown cells in terms of necrosis or apoptosis, with or without mutagen exposure. All results are based on the average of triplicate experiments, and error bars are for SEM.

Figure 3

Comet assay results for cells with reduced and normal CRY2. DNA damage was measured by the Olive tail moment, which considers the amount and distance of DNA migration away from the nucleus, indicative of DNA strand breaks. Higher values correspond to increased damage. Upon exposure to the same concentration of mutagen, CRY2- cells accumulated significantly more DNA damage compared to normal cells (P = 0.040). Comet assay results are from duplicate experiments performed on 50 cells each. All data are aggregated (i.e. 100 cells per treatment), and error bars are for SEM.