Using Period knockout mice as tools for investigating circadian timekeeping
There are at least two distinct circadian timekeeping mechanisms in the central nervous system of mammals, the light-entrainable oscillator (LEO) in the suprachiasmatic nucleus (SCN) and the food-entrainable oscillator (FEO), which controls food anticipatory activity.
Another relatively understudied putative oscillator, whose output can only be observed in the presence of methamphetamine, is the methamphetamine-sensitive circadian oscillator (MASCO). We and others have demonstrated that the FEO and MASCO may use the same molecular timekeeping mechanism (which differs from the timekeeping of the SCN), or, alternatively, they may be the same oscillator.
Despite exhaustive efforts in searching for the location of the FEO, its anatomical locus has not been identified. We have shown that the FEO and MASCO are functional in LEO-disabled mice (in mice lacking all three paralogs of the Period gene: Per1/2/3 knockout mice). Thus, these mice are a novel and unique tool for revitalizing the search for the FEO.
Our studies of the FEO and MASCO in Per1/2/3 knockout mice have also revealed intriguing characteristics about their SCN and behavioral rhythms. Although Per1 and Per2 have been traditionally regarded as the components of the circadian timekeeping machinery that respond to environmental cues, such as light, temperature and locomotor activity; we have found that Per1/2/3 knockout mice retain responsiveness to the environment. They are rhythmic in the light-dark cycle with an advanced phase of activity onset and exhibit phase-dependent masking (acute activity suppression by light).
Moreover, Per1/2/3 knockout mice display transients when the light-dark is shifted. Our findings indicate that although the LEO is disabled in constant darkness, Per1/2/3 knockout mice are light-responsive and light drives oscillations in the Per1/2/3 knockout oscillator (most likely the SCN). Our lab will seek to understand the molecular mechanisms of entrainment using this unique model by employing in vivo multi-unit recording of neural activity, in situ hybridization, and immunocytochemistry.
Reconnecting the circadian and cell cycles as a cancer therapeutic
Two prominent timekeeping systems – the cell cycle which controls cell division, and the circadian system – are found in nearly all living organisms. Twenty-four-hour fluctuations in cell division have been observed in numerous species ranging from unicellular organisms to humans. This suggests that the circadian system is regulating cell cycle progression.
In addition, multiple lines of evidence suggest that circadian dysregulation accompanies or is a prerequisite for cancer development and progression. We developed a novel approach for monitoring real-time cell cycle gene expression and found that the cell cycle rhythm is not coupled to the circadian clock in immortalized fibroblasts and carcinoma cells, even though the rhythm of circadian gene expression is normal. This indicates that cell cycle dysregulation can occur even when the circadian rhythm is intact.
Recently, several laboratories have demonstrated that stem cells do not have circadian rhythms, indicating that circadian control of cell mitosis is not advantageous in rapidly-dividing cells. This led us to develop the novel hypothesis that cancer cells achieve rapid cell division by disconnecting the circadian system from control of the cell cycle (rather than suppressing the circadian rhythm as in stem cells because the circadian rhythm is already developed in cancerous cells).
Our lab will continue to investigate coupling between the circadian and cell cycles using the novel approach of simultaneously monitoring the circadian and cell cycle gene expression rhythms in tissues and single cells in real-time. The relationship between circadian and cell cycles will be examined in normal tissues and in cancer cells.
Furthermore, this novel dual luciferase system could be used for high-throughput screening of putative cancer drugs and for phenotyping the circadian and cell cycles in tumor cells explanted from patients and transfected with reporter constructs. Consequently, chrono-cancer treatment could be tailored to each individual.