Apr 142010
 
ResearchBlogging.orgAlthough we are most familiar with the circadian rhythm from its effects on our physiological state, the roots of the phenomenon lie in the molecular biology of individual cells. The circadian rhythm is the result of a transcriptional control system that regulates the levels of many different proteins in the cell with the passing of time. Not all of the proteins subject to this control have yet been catalogued, and as a result some surprising effects are still being discovered. A recent article in Proceedings of the National Academy of Sciences from the Sancar lab at UNC suggests that circadian control of a DNA repair factor may be a way to enhance the effectiveness of a chemotherapeutic agent. The article is open access, so I encourage you to open it up and read along.

Previously, the Sancar group has shown that the circadian rhythm affects DNA repair in brain cells. In that case, DNA had been damaged by UV irradiation, a lesion that had to be replaced by the excision repair mechanism. Because one of the critical factors for this kind of repair, the Xeroderma Pigmentosum A protein (XPA), undergoes a circadian oscillation, the efficiency of repair depends on the time of day at which the damage occurred. Kang et al. hypothesized that this circadian dependence could also be true for other forms of DNA damage that undergo excision repair.

This led them to cisplatin, a drug used as primary chemotherapy or part of a combinatorial regimen for several kinds of cancer. Cisplatin creates covalent bonds crosslinking DNA bases in an intra-strand or inter-strand manner. These covalent linkages make replication (and therefore mitosis) impossible, and elicit a DNA repair response. If the cell cannot clear the crosslink, it will either die by apoptosis, or if the apoptotic response is suppressed, fail to produce viable daughter cells. Either way, the growth of the tumor is suppressed. The excision-repair pathway is the only mechanism of clearing this kind of DNA damage, so Kang et al. thought that there might be a robust circadian dependence. In order to test this idea, they carried out experiments using extracts from mouse liver and testis.

Figure 1 shows the results of their experiment using liver extract. Panel C summarizes the key results (data shown in panels A and B) that XPA mRNA, XPA protein, and excision repair efficiency are correlated with the dark/light cycle the mice are experiencing, with the highest levels of protein and repair occurring in the late afternoon, and the lowest levels in the very early morning (around 5 AM). Panel E shows a comparison of excision repair between normal mice and ones that have been genetically modified to lack cryptochrome, a critical circadian clock protein. In these CryDKO mice the time of day has no effect on the efficiency of repair, and as panel F shows, they also do not have the daily fluctuation in XPA mRNA and protein levels. These results suggest that circadian control of XPA expression levels dictates repair efficiency in liver tissues — as panel D shows, addition of exogenous XPA protein can recover the excision repair activity. However, there was no detectable circadian dependence for excision repair activity in testis, as Figure 2 shows using similar experiments.

Circadian control of XPA activity is possible because the protein doesn’t last very long in the cell. Figure 3 shows experiments using two inhibitors: cycloheximide (CHX), which prevents protein synthesis, and MG132, which prevents protein degradation by the proteasome. The gel in panel A shows that in two different types of cells, CHX treatment caused XPA protein to disappear over a period of three hours, in contrast to the control protein actin, which was unaffected. Addition of MG132 caused XPA to accumulate with time, although actin levels were again constant. The fairly rapid degradation of XPA protein means that the overall quantity of that protein in a cell will be highly dependent on the concentration of its mRNA transcript. That is, you can only have high XPA levels if there’s a lot of mRNA so ribosomes can continue to produce it. Circadian control of transcription is therefore able to regulate protein concentration. The authors hypothesize that this tight circadian control may have developed to prevent deleterious effects of non-specific DNA repair activity during times when there is no chance of UV insult, although there is no specific evidence to support this conjecture presently.

The MG132 experiment indicates that XPA is degraded by ubiquitin ligation and subsequent destruction in the proteasome. Figures 4 and 5 show a variety of evidence indicating that this process is mediated by the ubiquitin ligase HERC2. The experiments in figure 4 establish that HERC2 binds to XPA (panels A and B) and colocalizes with it in the cell (panels C and D). The gels in Figure 5 show that when HERC2 levels in the cell are knocked down by RNA inhibition using siRNA specific for that protein, CHX treatment does not cause XPA to degrade. These facts indicate that HERC2 is the ubiquitin ligase for XPA. The direct effect of HERC2 activity on the repair of cisplatin DNA damage is shown by figure 6. Here, cells were treated with cisplatin and HERC2 siRNA. The left side of panel A shows the clearance of cisplatin adducts from A549 cells over time (the right side shows the total DNA). As you can see, addition of HERC2 siRNA allows for more rapid clearance of the adduct, with a particularly dramatic effect at low dosage.

Unfortunately, due to its extreme toxicity, treating cancer cells with CHX is not a viable strategy for chemotherapy. However, knowing that the level of XPA protein in some target cells varies in a predictable way with the time of day can help doctors optimize treatments for maximum effectiveness. In particular, for cancers originating in tissues that have a strong circadian rhythm and intact XPA, early-morning treatment with cisplatin may be more effective than treatment at other times of the day. More experiments are needed before this can be formally recommended — in particular, whole-animal studies and human trials will be necessary to definitively establish the effect. If these results hold up in whole organisms, however, the circadian effect on DNA repair may become a valuable tool for optimizing some chemotherapy regimens.

Kang, T., Lindsey-Boltz, L., Reardon, J., & Sancar, A. (2010). “Circadian control of XPA and excision repair of cisplatin-DNA damage by cryptochrome and HERC2 ubiquitin ligase”. Proceedings of the National Academy of Sciences, 107 (11), 4890-4895 DOI: 10.1073/pnas.0915085107

Full Disclosure: I have previously collaborated with Aziz and his group on a research project on eukaryotic cryptochromes.