Abstract
In mammals, circadian rhythms in physiological function are generated by a molecular oscillator driven by transcriptional-translational feedback loop consisting of negative and positive regulators. Disruption of this circadian clock machinery is thought to increase the risk of cancer development, but the potential contributions of each component of circadian clock to oncogenesis have been little explored. Here we reported that negative and positive transcriptional regulators of circadian feedback loop had different roles in oncogene-induced neoplastic transformation. Mouse embryonic fibroblasts prepared from animals deficient in negative circadian clock regulators, Period2 (Per2) or Cryptochrome1/2 (Cry1/2), were prone to transformation induced by co-expression of H-ras(V12) and SV40 large T antigen (SV40LT). In contrast, mouse embryonic fibroblasts prepared from mice deficient in positive circadian clock regulators, Bmal1 or Clock, showed resistance to oncogene-induced transformation. In Per2 mutant and Cry1/2-null cells, the introduction of oncogenes induced expression of ATF4, a potent repressor of cell senescence-associated proteins p16INK4a and p19ARF. Elevated levels of ATF4 were sufficient to suppress expression of these proteins and drive oncogenic transformation. Conversely, in Bmal1-null and Clock mutant cells, the expression of ATF4 was not induced by oncogene introduction, which allowed constitutive expression of p16INK4a and p19ARF triggering cellular senescence. Although genetic ablation of either negative or positive transcriptional regulators of the circadian clock leads to disrupted rhythms in physiological functions, our findings define their different contributions to neoplastic cellular transformation.
Highlights
The circadian clock is a timekeeping system that allows organisms to adapt their physiological and behavioral functions to anticipatory changes in their environment
Mouse embryonic fibroblasts (MEFs)3 prepared from Per2 mutant (Per2m/m) or Cry1/2-null (Cry1/2Ϫ/Ϫ) mice were prone to oncogenic transformation induced by coexpression of H-rasV12 and SV40 large T antigen (SV40LT); these oncogene induced expression of cellular senescence-associated proteins in Bmal1-null (Bmal1Ϫ/Ϫ) or Clock mutant (Clk/Clk) cells, resulting in failure of their neoplastic transformation
By contrast, when the circadian rhythms are disrupted due to a dysfunction in the transcriptional activator BMAL1 or CLOCK, activating transcription factor 4 (ATF4) is unresponsive to oncogenic stimuli, and elevations in the expression of p16INK4a and p19ARF causes cellular senescence
Summary
Treatments of Animals and Cells—Per2m/m, Bmal1Ϫ/Ϫ, and Clock mutant (Clk/Clk) mice with an ICR background and wild type mice of the same strain were housed in a temperaturecontrolled (24 Ϯ 1 °C) room under a 12-h light:12-h dark cycle. MEFs were infected with 1 ϫ 106 colony-forming units/ml retroviral vectors expressing H-rasV12 and SV40LT [14]. Plasmid vectors (pcDNA3.1; Invitrogen) expressing Per, Bmal, or Atf were transfected into oncogene-introduced MEFs. Transgeneexpressing cells were selected with G418 (Wako, Osaka, Japan), and individual colonies were expanded and maintained in media containing 4 g/ml G418. Male NOD-SCID mice were purchased from Charles River They were inoculated with oncogenic-transformed MEFs (5 ϫ 106 cells/mouse). Expression vectors for mouse Per, Cry, Bmal, Clock, and ATF4 were constructed using complementary DNAs (cDNAs) obtained by RT-PCR derived from mouse hepatic RNA. To confirm the expression of H-rasV12 and SV40LT, diluted cDNA samples were subjected to PCR as described above, and PCR products were run on agarose gel after staining with ethidium bromide.
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