@article{mishra_gaddameedhi_2023, title={A new role of TRPM8 in circadian rhythm and molecular clock}, volume={1}, ISSN={["1748-1716"]}, url={https://doi.org/10.1111/apha.13934}, DOI={10.1111/apha.13934}, abstractNote={Over the past decades, studies of the TRPM8 channel, a non-selective cation channel, have provided much insight into the fundamental mechanisms of sensory neuron function that lead to the detection of cold sensors. In the current issue of Acta Physiologica, Reimundez et al1 provide evidence of a novel role of TRPM8 in circadian function in mice. The TRPM (transient receptor potential ion channels, where “m” stands for melastatin) is a family of eight different channels, TRPM1-TRPM8.2 The TRPM8 is a cold sensor and is also activated by chemical ligands such as menthol and icilin, but recently, their role in maintaining core body temperature has also been recognized.3, 4 TRPM8 is expressed in sensory neurons whose axonal afferents innervate peripheral tissues such as skin and oral cavity. Still, it has not been detected in the brain and spinal cord. Besides nerve tissues, TRPM8 is expressed in tissues such as the prostate, bladder, lungs, and urogenital tract. However, other than the sensory nervous system, the functional role of TRPM8 channels is not well understood. In this issue of Acta Physiologica, Reimundez et al1 provide solid experimental evidence for such a novel mechanism—where they demonstrate that TRPM8 might regulate Period 2 (Per2) mRNA levels in central clock (SCN) and peripheral clock (liver and white adipose tissue) and the circadian regulation of core body temperature. Their publication is among the most significant advances in the field of circadian regulation using cold sensors—it will probably set up the stage for several future research activities in which Reimundez and colleagues' ideas will be rigorously dissected and probed in circadian biology research. Previously, Ordás et al5 demonstrated the presence of TRPM8 fibers in the suprachiasmatic nucleus of the hypothalamus (SCN) of the brain, which is the principal circadian pacemaker in mammals and host a range of other physiological processes such as circadian oscillation, autonomic/peripheral and central nervous system function, regulating core body temperature and sleep–wake cycle. Since SCN receives axonal projections mainly from intrinsically photosensitive retinal ganglion cells (ipRGCs) that are responsible for resetting the circadian clock through SCN neuron activity with light as a primary zeitgeber.6 Here, authors used conventional PCR and mouse reporter lines to show the expression of TRPM8 in the inner retina, specifically in the ganglion cell layer (GC) and in the inner nuclear layer (INL). Next, they used the colocalization technique to demonstrate that melanopsin, a marker of ipRGCs7 and using mouse reporter lines, that TRPM8 expressing cells are indeed expressed in a subset of ipRGCs cells. Furthermore, they showed that TRPM8 is also expressed in cholinergic amacrine cells, characterized by releasing two neurotransmitters, GABA and acetylcholine. Injection of the fluorescent anterograde tracer cholera toxin subunit B (CTB-594) into both eyes in two TRPM8 reporter lines indicates the existence of ipRGCs expressing TRPM8 and projecting to the SCN (Figure 1). The choroid is an essential high blood flow vascular structure in the eye and is known to regulate ocular and retinal temperature8 and is richly innervated by sensory trigeminal nerve fibers; therefore, the authors next examined the role of TRPM8 in regulation of eye temperature (Teye) using infrared (IR) thermography. Interestingly, they discovered mice lacking TRPM8 started to decline at temperatures below 25°C, and Trpm8−/− mice displayed a significantly lower Teye than WT littermates when the temperature plate was around 15°C. This suggests a function of TRPM8 and ambient temperature in regulating choroid and ciliary body blood flow and, therefore, in controlling internal ocular temperature. The ability of mice to regulate homeostatic temperature control and the expression of TRPM8 in the ipRGCs is indicative but does not directly link that to central clock regulation. The authors performed expression and functional studies to investigate a direct link between TRPM8 and circadian clockwork at the SCN. First, they determined the expression of the Per2 gene, which is one of the essential components of core circadian clocks in the SCN.9 Authors found in the TRPM8 deficient mouse SCN a significant increase of ~40% in Per2 mRNA content in relation to WT littermates during the daytime (ZT4). Next, they examined one of the most relevant clock-controlled neuropeptide Arginine Vasopressin (AVP) in the SCN. AVP-expressing neurons in the SCN constitute the main output of this nucleus and are known to be involved in core temperature (Tc) circadian regulation.10 Like Per2, Trpm8−/− mice displayed greater AVP expression than control mice during the lights-on phase (~50% higher) but conserved the oscillation between day and nighttime. Briefly, these results suggest that the absence of TRPM8 does not prevent oscillatory expression of Per2 and AVP but involves modifying clockwork and neuropeptide levels in this hypothalamic nucleus, contributing to the regulation of the circadian oscillation of Tc. Second, Reimundez et al1 found that TRPM8 plays a significant role in regulating core body temperature where TRPM8 deficient mice show a significantly reduced temperature and increased amplitude both in light/dark (LD) and dark/dark (DD) conditions compared with its wild-type controls. Future studies investigating TRPM8 regulation on circadian rhythm properties such as period, acrophase, and mesor would be interesting. Third, authors extended the TRPM8 role in peripheral clocks liver and gonadal WAT by measuring Per2 mRNA levels during the day (ZT4) and night (ZT16). TRPM8 deficient mice liver has a significantly elevated Per2 levels during day and dampened levels during night compared to WT control. Interestingly, in gonadal WAT tissue, Per2 levels were significantly lower both in day and night compared to WT control. Future studies, investigating with more circadian timepoints (every 3 hours in a 24-hour cycle) on whether Per2 levels were significantly rhythmic would be interesting. The authors provide strong evidence of the TRPM8 channel as a connecting link between temperature and central and peripheral clocks, regulating the circadian oscillations of body temperature. However, the presented evidence is scant and indirect, especially using the global TRPM8 deficient mice and knowing that TRPM8 is also a peripheral sensor in the skin, which might play an indirect, yet a meaningful role in mediating this effect. Most importantly, extending the TRPM8 role to other core clock genes such as Cry1, Cry2, Per1, Bmal1, Clock, and Npas2 oscillation and circadian rhythmicity will be interesting. No conflict of interest to declare.}, journal={ACTA PHYSIOLOGICA}, author={Mishra, Santosh K. and Gaddameedhi, Shobhan}, year={2023}, month={Jan} } @article{koritala_dakup_porter_gaddameedhi_2023, title={The impact of shift-work light conditions on tissue-specific circadian rhythms of canonical clock genes: insights from a mouse model study}, url={https://doi.org/10.12688/f1000research.136998.1}, DOI={10.12688/f1000research.136998.1}, abstractNote={Background: The natural day-night cycle synchronizes our circadian rhythms, but modern work practices like night shifts disrupt this pattern, leading to increased exposure to nighttime light. This exposure is linked to various health issues. While some studies have explored the effects of night shifts on human circadian rhythms, there is limited research on the consequences of long-term exposure to shift-work light conditions. Rodents can provide valuable insights into these effects. This study aimed to examine how short- or long-term exposure to rotating shifts and chronic jetlag affects the core circadian oscillators in the liver and skin of mammals. Methods: C57BL/6J male mice were subjected to simulated shift-work light conditions, including short-term or long-term rotating shifts and chronic jet-lag conditions. Liver and skin samples were collected every four hours over a 24-hour period on the second day of constant darkness. RNA was extracted and qRT-PCR analysis was conducted to measure the circadian gene expression in liver and skin tissues. Circadian rhythm analysis using CircaCompare compared the control group to mice exposed to shift-work light conditions. Results: The liver's circadian clock is significantly altered in mice under long-term rotating shift conditions, with a lesser but still noticeable impact in mice experiencing chronic jetlag. However, short-term rotating shift conditions do not significantly affect the liver's circadian clock. Conversely, all three simulated shift conditions affect the skin's circadian clock, indicating that the skin clock is more sensitive to shift-work light conditions than the liver clock. Compared to the liver, the skin's circadian clock is greatly affected by long-term rotating shift conditions. Conclusions: The study findings indicate more pronounced disturbances in the canonical clock genes of the skin compared to the liver under simulated shift-work light conditions. These results suggest that the skin clock is more vulnerable to the effects of shift-work.}, journal={F1000Research}, author={Koritala, Bala S. C. and Dakup, Panshak P. and Porter, Kenneth I. and Gaddameedhi, Shobhan}, year={2023}, month={Jun} } @article{koritala_dakup_porter_gaddameedhi_2023, title={The impact of shift-work light conditions on tissue-specific circadian rhythms of canonical clock genes: insights from a mouse model study}, url={https://doi.org/10.12688/f1000research.136998.3}, DOI={10.12688/f1000research.136998.3}, abstractNote={Background: The natural day-night cycle synchronizes our circadian rhythms, but modern work practices like night shifts disrupt this pattern, leading to increased exposure to nighttime light. This exposure is linked to various health issues. While some studies have explored the effects of night shifts on human circadian rhythms, there is limited research on the consequences of long-term exposure to shift-work light conditions. Rodents can provide valuable insights into these effects. This study aimed to examine how short- or long-term exposure to rotating shifts and chronic jetlag affects the core circadian oscillators in the liver and skin of mammals. Methods: C57BL/6J male mice were subjected to simulated shift-work light conditions, including short-term or long-term rotating shifts and chronic jet-lag conditions. Liver and skin samples were collected every four hours over a 24-hour period on the second day of constant darkness. RNA was extracted and qRT-PCR analysis was conducted to measure the circadian gene expression in liver and skin tissues. Circadian rhythm analysis using CircaCompare compared the control group to mice exposed to shift-work light conditions. Results: The liver's circadian clock is significantly altered in mice under long-term rotating shift conditions, with a lesser but still noticeable impact in mice experiencing chronic jetlag. However, short-term rotating shift conditions do not significantly affect the liver's circadian clock. Conversely, all three simulated shift conditions affect the skin's circadian clock, indicating that the skin clock is more sensitive to shift-work light conditions than the liver clock. Compared to the liver, the skin's circadian clock is greatly affected by long-term rotating shift conditions. Conclusions: The study findings indicate more pronounced disturbances in the canonical clock genes of the skin compared to the liver under simulated shift-work light conditions. These results suggest that the skin clock is more vulnerable to the effects of shift-work.}, journal={F1000Research}, author={Koritala, Bala S. C. and Dakup, Panshak P. and Porter, Kenneth I. and Gaddameedhi, Shobhan}, year={2023}, month={Aug} } @article{koritala_dakup_porter_gaddameedhi_p._koritala_2023, title={The impact of shift-work light conditions on tissue-specific circadian rhythms of canonical clock genes: insights from a mouse model study [version 1; peer review: 1 approved, 1 approved with reservations]}, url={https://f1000research.com/articles/12-762/v1}, DOI={10.12688/f1000research.136998.2}, abstractNote={Background: The natural day-night cycle synchronizes our circadian rhythms, but modern work practices like night shifts disrupt this pattern, leading to increased exposure to nighttime light. This exposure is linked to various health issues. While some studies have explored the effects of night shifts on human circadian rhythms, there is limited research on the consequences of long-term exposure to shift-work light conditions. Rodents can provide valuable insights into these effects. This study aimed to examine how short- or long-term exposure to rotating shifts and chronic jetlag affects the core circadian oscillators in the liver and skin of mammals. Methods: C57BL/6J male mice were subjected to simulated shift-work light conditions, including short-term or long-term rotating shifts and chronic jet-lag conditions. Liver and skin samples were collected every four hours over a 24-hour period on the second day of constant darkness. RNA was extracted and qRT-PCR analysis was conducted to measure the circadian gene expression in liver and skin tissues. Circadian rhythm analysis using CircaCompare compared the control group to mice exposed to shift-work light conditions. Results: The liver's circadian clock is significantly altered in mice under long-term rotating shift conditions, with a lesser but still noticeable impact in mice experiencing chronic jetlag. However, short-term rotating shift conditions do not significantly affect the liver's circadian clock. Conversely, all three simulated shift conditions affect the skin's circadian clock, indicating that the skin clock is more sensitive to shift-work light conditions than the liver clock. Compared to the liver, the skin's circadian clock is greatly affected by long-term rotating shift conditions. Conclusions: The study findings indicate more pronounced disturbances in the canonical clock genes of the skin compared to the liver under simulated shift-work light conditions. These results suggest that the skin clock is more vulnerable to the effects of shift-work.}, note={peer review: 1 approved, 1 approved with reservations] F1000Research 2023, 12:762 (https://doi.org/10.12688/f1000research.136998.1}, journal={F1000Research}, author={Koritala, Bala S. C. and Dakup, Panshak P. and Porter, Kenneth I. and Gaddameedhi, Shobhan and P., Dakup Panshak and Koritala, Bala S. C.}, editor={P., Dakup Panshak and Shobhan, Gaddameedhi and Koritala, Bala S. C. and Porter, Kenneth I.Editors}, year={2023}, month={Jun} } @article{circadian dysregulation of human dna repair genes and elevated dna damage in simulated night shift schedule_2022, url={https://publons.com/wos-op/publon/54906417/}, journal={Sleep}, year={2022} } @article{koritala_porter_sarkar_gaddameedhi_2022, title={Circadian disruption and cisplatin chronotherapy for mammary carcinoma}, volume={436}, ISSN={["1096-0333"]}, url={https://publons.com/wos-op/publon/50851286/}, DOI={10.1016/j.taap.2022.115863}, abstractNote={Solid tumors are commonly treated with cisplatin, which can cause off-target side effects in cancer patients. Chronotherapy is a potential strategy to reduce drug toxicity. To determine the effectiveness of timed-cisplatin treatment in mammals, we compared two conditions: clock disrupted jet-lag and control conditions. Under normal and disrupted clock conditions, triple-negative mammary carcinoma cells were injected subcutaneously into eight-week-old NOD.Cg-Prkdcscid/J female mice. Tumor volumes and body weights were measured in these mice before and after treatment with cisplatin. We observed an increase in tumor volumes in mice housed under disrupted clock compared to the normal clock conditions. After treatment with cisplatin, we observed a reduced tumor growth rate in mice treated at ZT10 compared to ZT22 and untreated cohorts under normal clock conditions. However, these changes were not seen with the jet-lag protocol. We also observed greater body weight loss in mice treated with ZT10 compared to ZT22 or untreated mice in the jet-lag protocol. Our observations suggest that the effectiveness of cisplatin in mammary carcinoma treatment is time-dependent in the presence of the circadian clock.}, journal={TOXICOLOGY AND APPLIED PHARMACOLOGY}, author={Koritala, Bala S. C. and Porter, Kenneth I. and Sarkar, Soumyadeep and Gaddameedhi, Shobhan}, year={2022}, month={Feb} } @article{goodenow_greer_cone_gaddameedhi_2022, title={Circadian effects on UV-induced damage and mutations}, volume={789}, ISSN={["1388-2139"]}, url={https://publons.com/wos-op/publon/54906418/}, DOI={10.1016/j.mrrev.2022.108413}, abstractNote={Skin cancer is the most diagnosed type of cancer in the United States, and while most of these malignancies are highly treatable, treatment costs still exceed $8 billion annually. Over the last 50 years, the annual incidence of skin cancer has steadily grown; therefore, understanding the environmental factors driving these types of cancer is a prominent research-focus. A causality between ultraviolet radiation (UVR) exposure and skin cancer is well-established, but exposure to UVR alone is not necessarily sufficient to induce carcinogenesis. The emerging field of circadian biology intersects strongly with the physiological systems of the mammalian body and introduces a unique opportunity for analyzing mechanisms of homeostatic disruption. The circadian clock refers to the approximate 24-hour cycle, in which protein levels of specific clock-controlled genes (CCGs) fluctuate based on the time of day. Though these CCGs are tissue specific, the skin has been observed to have a robust circadian clock that plays a role in its response to UVR exposure. This in-depth review will detail the mechanisms of the circadian clock and its role in cellular homeostasis. Next, the skin's response to UVR exposure and its induction of DNA damage and mutations will be covered - with an additional focus placed on how the circadian clock influences this response through nucleotide excision repair. Lastly, this review will discuss current models for studying UVR-induced skin lesions and perturbations of the circadian clock, as well as the impact of these factors on human health.}, journal={MUTATION RESEARCH-REVIEWS IN MUTATION RESEARCH}, author={Goodenow, Donna and Greer, Adam J. and Cone, Sean J. and Gaddameedhi, Shobhan}, year={2022} } @article{kelly_yuen_satterfield_auchus_gaddameedhi_van dongen_liu_2022, title={Endogenous Diurnal Patterns of Adrenal and Gonadal Hormones During a 24-Hour Constant Routine After Simulated Shift Work}, volume={6}, ISSN={["2472-1972"]}, DOI={10.1210/jendso/bvac153}, abstractNote={Abstract Context Night-shift work causes circadian misalignment, predicts the development of metabolic diseases, and complicates the interpretation of hormone measurements. Objective To investigate endogenous circadian rhythms, dissociated from behavioral and environmental confounds, in adrenal and gonadal steroids after simulated shift work. Methods Fourteen healthy adults (ages 25.8 ± 3.2 years) were randomized to 3 days of night or day (control) shift work followed by a constant routine protocol designed to experimentally unveil rhythms driven endogenously by the central circadian pacemaker. Blood was sampled every 3 hours for 24 hours during the constant routine to concurrently obtain 16 Δ4 steroid profiles by mass spectrometry. Cosinor analyses of these profiles provided mesor (mean abundance), amplitude (oscillation magnitude), and acrophase (peak timing). Results Night-shift work marginally increased cortisol by 1 μg/dL (P = 0.039), and inactive/weak derivatives cortisone (P = 0.003) and 18-hydroxycortisol (P < 0.001), but did not alter the mesor of potent androgens testosterone and 11-ketotestosterone. Adrenal-derived steroids, including 11-ketotestosterone (P < 0.01), showed robust circadian rhythmicity after either day- or night-shift work. In contrast, testosterone and progesterone showed no circadian pattern after both shift work conditions. Night-shift work did not alter the amplitude or acrophase of any of the steroid profiles. Conclusion Experimental circadian misalignment had minimal effects on steroidogenesis. Adrenal steroids, but not gonadal hormones, showed endogenous circadian regulation robust to prior shift schedule. This dichotomy may predispose night-shift workers to metabolic ill health. Furthermore, adrenal steroids, including cortisol and the main adrenal androgen 11-ketostosterone, should always be evaluated during the biological morning whereas assessment of gonadal steroids, particularly testosterone, is dependent on the shift-work schedule.}, number={12}, journal={JOURNAL OF THE ENDOCRINE SOCIETY}, author={Kelly, Monica R. and Yuen, Fiona and Satterfield, Brieann C. and Auchus, Richard J. and Gaddameedhi, Shobhan and Van Dongen, Hans P. A. and Liu, Peter Y.}, year={2022}, month={Oct} } @article{dakup_greer_gaddameedhi_2022, title={Let's talk about sex: A biological variable in immune response against melanoma}, volume={35}, ISSN={["1755-148X"]}, url={https://doi.org/10.1111/pcmr.13028}, DOI={10.1111/pcmr.13028}, abstractNote={Abstract As science culture gravitates toward a more holistic inclusion of both males and females in research design, the outlining of sex differences and their respective intersections with disease physiology and pathophysiology should see reciprocal expansion. Melanoma skin cancer, for example, has observed a female advantage in incidence, mortality, and overall survival since the early 1970s. The exact biological mechanism of this trend, however, is unclear and further complicated by a layering of clinical variables such as skin phototype, age, and body mass index. In this perspective, we highlight epidemiological evidence of sex differences in melanoma and summarize the landscape of their potential origin. Among several biological hallmarks, we make a note of sex‐specific immune profiles—along with divergent hormonal regulation, social practices, DNA damage and oxidative stress responses, body composition, genetic variants, and X‐chromosome expression—as probable drivers of disparity in melanoma initiation and progression. This review further focuses the conversation of sex as an influencing factor in melanoma development and its potential implication for disease management and treatment strategies.}, number={2}, journal={PIGMENT CELL & MELANOMA RESEARCH}, publisher={Wiley}, author={Dakup, Panshak P. and Greer, Adam J. and Gaddameedhi, Shobhan}, year={2022}, month={Feb} } @article{kyle_bramer_claborne_stratton_bloodsworth_teeguarden_gaddameedhi_metz_van dongen_2022, title={Simulated Night- Shift Schedule Disrupts the Plasma Lipidome and Reveals Early Markers of Cardiovascular Disease Risk}, volume={14}, ISSN={["1179-1608"]}, url={https://publons.com/wos-op/publon/53707085/}, DOI={10.2147/NSS.S363437}, abstractNote={The circadian system coordinates daily rhythms in lipid metabolism, storage and utilization. Disruptions of internal circadian rhythms due to altered sleep/wake schedules, such as in night-shift work, have been implicated in increased risk of cardiovascular disease and metabolic disorders. To determine the impact of a night-shift schedule on the human blood plasma lipidome, an in-laboratory simulated shift work study was conducted.Fourteen healthy young adults were assigned to 3 days of either a simulated day or night-shift schedule, followed by a 24-h constant routine protocol with fixed environmental conditions, hourly isocaloric snacks, and constant wakefulness to investigate endogenous circadian rhythms. Blood plasma samples collected at 3-h intervals were subjected to untargeted lipidomics analysis.More than 400 lipids were identified and quantified across 21 subclasses. Focusing on lipids with low between-subject variation per shift condition, alterations in the circulating plasma lipidome revealed generally increased mean triglyceride levels and decreased mean phospholipid levels after night-shift relative to day-shift. The circadian rhythms of triglycerides containing odd chain fatty acids peaked earlier during constant routine after night-shift. Regardless of shift condition, triglycerides tended to either peak or be depleted at 16:30 h, with chain-specific differences associated with the direction of change.The simulated night-shift schedule was associated with altered temporal patterns in the lipidome. This may be premorbid to the elevated cardiovascular risk that has been found epidemiologically in night-shift workers.}, journal={NATURE AND SCIENCE OF SLEEP}, author={Kyle, Jennifer E. and Bramer, Lisa M. and Claborne, Daniel and Stratton, Kelly G. and Bloodsworth, Kent J. and Teeguarden, Justin G. and Gaddameedhi, Shobhan and Metz, Thomas O. and Van Dongen, Hans P. A.}, year={2022}, pages={981–994} } @article{muck_hudson_honn_gaddameedhi_van dongen_2022, title={Working around the Clock: Is a Person's Endogenous Circadian Timing for Optimal Neurobehavioral Functioning Inherently Task-Dependent?}, volume={4}, ISSN={["2624-5175"]}, url={https://publons.com/wos-op/publon/53707084/}, DOI={10.3390/clockssleep4010005}, abstractNote={Neurobehavioral task performance is modulated by the circadian and homeostatic processes of sleep/wake regulation. Biomathematical modeling of the temporal dynamics of these processes and their interaction allows for prospective prediction of performance impairment in shift-workers and provides a basis for fatigue risk management in 24/7 operations. It has been reported, however, that the impact of the circadian rhythm-and in particular its timing-is inherently task-dependent, which would have profound implications for our understanding of the temporal dynamics of neurobehavioral functioning and the accuracy of biomathematical model predictions. We investigated this issue in a laboratory study designed to unambiguously dissociate the influences of the circadian and homeostatic processes on neurobehavioral performance, as measured during a constant routine protocol preceded by three days on either a simulated night shift or a simulated day shift schedule. Neurobehavioral functions were measured every 2 h using three functionally distinct assays: a digit symbol substitution test, a psychomotor vigilance test, and the Karolinska Sleepiness Scale. After dissociating the circadian and homeostatic influences and accounting for inter-individual variability, peak circadian performance occurred in the late biological afternoon (in the "wake maintenance zone") for all three neurobehavioral assays. Our results are incongruent with the idea of inherent task-dependent differences in the endogenous circadian impact on performance. Rather, our results suggest that neurobehavioral functions are under top-down circadian control, consistent with the way they are accounted for in extant biomathematical models.}, number={1}, journal={CLOCKS & SLEEP}, author={Muck, Rachael A. and Hudson, Amanda N. and Honn, Kimberly A. and Gaddameedhi, Shobhan and Van Dongen, Hans P. A.}, year={2022}, month={Mar}, pages={23–36} } @article{sarkar_porter_dakup_gajula_koritala_hylton_kemp_wakamatsu_gaddameedhi_2021, title={Circadian clock protein BMAL1 regulates melanogenesis through MITF in melanoma cells}, volume={34}, ISSN={["1755-148X"]}, url={https://doi.org/10.1111/pcmr.12998}, DOI={10.1111/pcmr.12998}, abstractNote={Solar ultraviolet B radiation (UVB) is one of the leading causes of various skin conditions, including photoaging, sunburn erythema, and melanoma. As a protective response, the skin has inbuilt defense mechanisms, including DNA repair, cell cycle, apoptosis, and melanin synthesis. Though DNA repair, cell cycle, and apoptosis are clock controlled, the circadian mechanisms associated with melanin synthesis are not well understood. Using human melanocytes and melanoma cells under synchronized clock conditions, we observed that the microphthalmia-associated transcription factor (MITF), a rate-limiting protein in melanin synthesis, is expressed rhythmically with 24-hr periodicity in the presence of circadian clock protein, BMAL1. Furthermore, we demonstrated that BMAL1 binds to the promoter region of MITF and transcriptionally regulates its expression, which positively influences melanin synthesis. Finally, we report that an increase in melanin levels due to BMAL1 overexpression protects human melanoma cells from UVB. In conclusion, our studies provide novel insights into the mechanistic role of the circadian clock in melanin synthesis and protection against UVB-mediated DNA damage and genomic instability.}, number={5}, journal={PIGMENT CELL & MELANOMA RESEARCH}, publisher={Wiley}, author={Sarkar, Soumyadeep and Porter, Kenneth I and Dakup, Panshak P. and Gajula, Rajendra P. and Koritala, Bala S. C. and Hylton, Ryan and Kemp, Michael G. and Wakamatsu, Kazumasa and Gaddameedhi, Shobhan}, year={2021}, month={Jul} } @article{hayter_wehrens_van dongen_stangherlin_gaddameedhi_crooks_barron_venetucci_o' neill_brown_et al._2021, title={Distinct circadian mechanisms govern cardiac rhythms and susceptibility to arrhythmia}, volume={12}, ISSN={["2041-1723"]}, url={https://publons.com/wos-op/publon/45758593/}, DOI={10.1038/s41467-021-22788-8}, abstractNote={Electrical activity in the heart exhibits 24-hour rhythmicity, and potentially fatal arrhythmias are more likely to occur at specific times of day. Here, we demonstrate that circadian clocks within the brain and heart set daily rhythms in sinoatrial (SA) and atrioventricular (AV) node activity, and impose a time-of-day dependent susceptibility to ventricular arrhythmia. Critically, the balance of circadian inputs from the autonomic nervous system and cardiomyocyte clock to the SA and AV nodes differ, and this renders the cardiac conduction system sensitive to decoupling during abrupt shifts in behavioural routine and sleep-wake timing. Our findings reveal a functional segregation of circadian control across the heart's conduction system and inherent susceptibility to arrhythmia.}, number={1}, journal={NATURE COMMUNICATIONS}, author={Hayter, Edward A. and Wehrens, Sophie M. T. and Van Dongen, Hans P. A. and Stangherlin, Alessandra and Gaddameedhi, Shobhan and Crooks, Elena and Barron, Nichola J. and Venetucci, Luigi A. and O' Neill, John S. and Brown, Timothy M. and et al.}, year={2021}, month={Apr} } @article{liu_irwin_krueger_gaddameedhi_dongen_2021, title={Night shift schedule alters endogenous regulation of circulating cytokines}, volume={10}, url={https://doi.org/10.1016/j.nbscr.2021.100063}, DOI={10.1016/j.nbscr.2021.100063}, abstractNote={Night shift work is a risk factor for viral infection, suggesting that night shift schedules compromise host defense mechanisms. Prior studies have investigated changes in the temporal profiles of circulating cytokines important for priming and restraining the immune response to infectious challenges from night shift work, but not by way of a 24-h constant routine of continuous wakefulness devoid of behavioral or environmental influences. Hence the true endogenous pattern of cytokines, and the combined effect of sleep loss and circadian misalignment on these cytokines remains unknown. Here, 14 healthy young men and women underwent three days of either a simulated night shift or a simulated day shift schedule under dim light in a controlled in-laboratory environment. This was followed by a 24-h constant routine protocol during which venous blood was collected at 3-h intervals. Those who had been in the night shift schedule showed lower mean circulating TNF-α (t13 = -6.03, p < 0.001), without any significant differences in IL-1β, IL-8 and IL-10, compared with those who had been in the day shift (i.e., control) schedule. Furthermore, circulating IL-6 increased with time awake in both shift work conditions (t13 = 6.03, p < 0.001), such that temporal changes in IL-6 were markedly shifted relative to circadian clock time in the night shift condition. These results indicate that night shift work compromises host defense by creating cytokine conditions that initially impede anti-viral immunity (lower TNF-α) and may eventually promote autoimmunity (mistimed rise in IL-6).}, journal={Neurobiology of Sleep and Circadian Rhythms}, publisher={Elsevier BV}, author={Liu, Peter Y. and Irwin, Michael R. and Krueger, James M. and Gaddameedhi, Shobhan and Dongen, Hans P.A. Van}, year={2021}, month={May}, pages={100063} } @article{koritala_porter_arshad_gajula_mitchell_arman_manjanatha_teeguarden_van dongen_mcdermott_et al._2021, title={Night shift schedule causes circadian dysregulation of DNA repair genes and elevated DNA damage in humans}, volume={70}, ISSN={["1600-079X"]}, url={https://publons.com/wos-op/publon/35185561/}, DOI={10.1111/jpi.12726}, abstractNote={Circadian disruption has been identified as a risk factor for health disorders such as obesity, cardiovascular disease, and cancer. Although epidemiological studies suggest an increased risk of various cancers associated with circadian misalignment due to night shift work, the underlying mechanisms have yet to be elucidated. We sought to investigate the potential mechanistic role that circadian disruption of cancer hallmark pathway genes may play in the increased cancer risk in shift workers. In a controlled laboratory study, we investigated the circadian transcriptome of cancer hallmark pathway genes and associated biological pathways in circulating leukocytes obtained from healthy young adults during a 24-hour constant routine protocol following 3 days of simulated day shift or night shift. The simulated night shift schedule significantly altered the normal circadian rhythmicity of genes involved in cancer hallmark pathways. A DNA repair pathway showed significant enrichment of rhythmic genes following the simulated day shift schedule, but not following the simulated night shift schedule. In functional assessments, we demonstrated that there was an increased sensitivity to both endogenous and exogenous sources of DNA damage after exposure to simulated night shift. Our results suggest that circadian dysregulation of DNA repair may increase DNA damage and potentiate elevated cancer risk in night shift workers.}, number={3}, journal={JOURNAL OF PINEAL RESEARCH}, author={Koritala, Bala S. C. and Porter, Kenneth I. and Arshad, Osama A. and Gajula, Rajendra P. and Mitchell, Hugh D. and Arman, Tarana and Manjanatha, Mugimane G. and Teeguarden, Justin and Van Dongen, Hans P. A. and McDermott, Jason E. and et al.}, year={2021}, month={Apr} } @article{xu_cheng_zhao_zhang_zhu_zhang_chen_wang_yan_robertson_et al._2021, title={Polymorphic tandem DNA repeats activate the human telomerase reverse transcriptase gene}, volume={118}, ISSN={["0027-8424"]}, url={https://publons.com/wos-op/publon/47740196/}, DOI={10.1073/pnas.2019043118}, abstractNote={Multiple independent sequence variants of the hTERT locus have been associated with telomere length and cancer risks in genome-wide association studies. Here, we identified an intronic variable number tandem repeat, VNTR2-1, as an enhancer-like element, which activated hTERT transcription in a cell in a chromatin-dependent manner. VNTR2-1, consisting of 42-bp repeats with an array of enhancer boxes, cooperated with the proximal promoter in the regulation of hTERT transcription by basic helix-loop-helix transcription factors and maintained hTERT expression during embryonic stem-cell differentiation. Genomic deletion of VNTR2-1 in MelJuSo melanoma cells markedly reduced hTERT transcription, leading to telomere shortening, cellular senescence, and impairment of xenograft tumor growth. Interestingly, VNTR2-1 lengths varied widely in human populations; hTERT alleles with shorter VNTR2-1 were underrepresented in African American centenarians, indicating its role in human aging. Therefore, this polymorphic element is likely a missing link in the telomerase regulatory network and a molecular basis for genetic diversities of telomere homeostasis and age-related disease susceptibilities.}, number={26}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Xu, Tao and Cheng, De and Zhao, Yuanjun and Zhang, Jinglong and Zhu, Xiaolu and Zhang, Fan and Chen, Gang and Wang, Yang and Yan, Xiufeng and Robertson, Gavin P. and et al.}, year={2021}, month={Jun} } @article{khalyfa_gaddameedhi_crooks_zhang_li_qiao_trzepizur_kay_andrade_satterfield_et al._2020, title={Circulating Exosomal miRNAs Signal Circadian Misalignment to Peripheral Metabolic Tissues}, volume={21}, ISSN={["1422-0067"]}, url={https://publons.com/wos-op/publon/33435120/}, DOI={10.3390/ijms21176396}, abstractNote={Night shift work increases risk of metabolic disorders, particularly obesity and insulin resistance. While the underlying mechanisms are unknown, evidence points to misalignment of peripheral oscillators causing metabolic disturbances. A pathway conveying such misalignment may involve exosome-based intercellular communication. Fourteen volunteers were assigned to a simulated day shift (DS) or night shift (NS) condition. After 3 days on the simulated shift schedule, blood samples were collected during a 24-h constant routine protocol. Exosomes were isolated from the plasma samples from each of the blood draws. Exosomes were added to naïve differentiated adipocytes, and insulin-induced pAkt/Akt expression changes were assessed. ChIP-Seq analyses for BMAL1 protein, mRNA microarrays and exosomal miRNA arrays combined with bioinformatics and functional effects of agomirs and antagomirs targeting miRNAs in NS and DS exosomal cargo were examined. Human adipocytes treated with exosomes from the NS condition showed altered Akt phosphorylation responses to insulin in comparison to those treated with exosomes from the DS condition. BMAL1 ChIP-Seq of exosome-treated adipocytes showed 42,037 binding sites in the DS condition and 5538 sites in the NS condition, with a large proportion of BMAL1 targets including genes encoding for metabolic regulators. A significant and restricted miRNA exosomal signature emerged after exposure to the NS condition. Among the exosomal miRNAs regulated differentially after 3 days of simulated NS versus DS, proof-of-concept validation of circadian misalignment signaling was demonstrated with hsa-mir-3614-5p. Exosomes from the NS condition markedly altered expression of key genes related to circadian rhythm in several cultured cell types, including adipocytes, myocytes, and hepatocytes, along with significant changes in 29 genes and downstream gene network interactions. Our results indicate that a simulated NS schedule leads to changes in exosomal cargo in the circulation. These changes promote reduction of insulin sensitivity of adipocytes in vitro and alter the expression of core clock genes in peripheral tissues. Circulating exosomal miRNAs may play an important role in metabolic dysfunction in NS workers by serving as messengers of circadian misalignment to peripheral tissues.}, number={17}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Khalyfa, Abdelnaby and Gaddameedhi, Shobhan and Crooks, Elena and Zhang, Chunling and Li, Yan and Qiao, Zhuanhong and Trzepizur, Wojciech and Kay, Steve A. and Andrade, Jorge and Satterfield, Brieann C. and et al.}, year={2020}, month={Sep} } @article{dakup_porter_little_zhang_gaddameedhi_2020, title={Sex differences in the association between tumor growth and T cell response in a melanoma mouse model}, volume={69}, url={https://doi.org/10.1007/s00262-020-02643-3}, DOI={10.1007/s00262-020-02643-3}, abstractNote={Epidemiological evidence suggests that females have an advantage over males in cases of melanoma incidence, progression, and survival. However, the biological mechanisms underlying these sex differences remain unclear. With the knowledge that females generally have a more robust immune system than males, we investigated sex differences in melanoma progression in a B16-F10/BL6 syngeneic mouse model. We observed significantly less tumor volume and growth rate over 14 days in female mice compared to male mice. Furthermore, higher populations of CD4+ and CD8+ T cells, which indicate adaptive immune responses, were found in the circulating blood and tumors of females and corresponded with less tumor growth, and vice versa in males. Our results highlight a mouse model that represents melanoma progression in the human population and displays a higher immune response to melanoma in females compared to males. These findings suggest that the immune system may be one of the mechanisms responsible for sex differences in melanoma.}, number={10}, journal={Cancer Immunology, Immunotherapy}, publisher={Springer Science and Business Media LLC}, author={Dakup, Panshak P. and Porter, Kenneth I. and Little, Alexander A. and Zhang, Hui and Gaddameedhi, Shobhan}, year={2020}, month={Oct}, pages={2157–2162} } @article{solar ultraviolet-induced dna damage response: melanocytes story in transformation to environmental melanomagenesis._2020, url={https://doi.org/10.1002/em.22370}, DOI={10.1002/em.22370}, abstractNote={Exposure to sunlight is both beneficial, as it heats the planet to a comfortable temperature, and potentially harmful, since sunlight contains ultraviolet radiation (UVR), which is deemed detrimental for living organisms. Earth's ozone layer plays a vital role in blocking most of the extremely dangerous UVC; however, low frequency/energy UVR (i.e., UVB and UVA) seeps through in minute amount and reaches the Earth's surface. Both UVB and UVA are physiologically responsible for a plethora of skin ailments, including skin cancers. The UVR is readily absorbed by the genomic DNA of skin cells, causing DNA bond distortion and UV-induced DNA damage. As a defense mechanism, the DNA damage response (DDR) signaling in skin cells activates nucleotide excision repair (NER), which is responsible for the removal of UVR-induced DNA photolesions and helps maintain the genomic integrity of the cells. Failure of proper NER function leads to mutagenesis and development of skin cancers. One of the deadliest form of skin cancers is melanoma which originates upon the genetic transformation of melanocytes, melanin producing skin cells. NER is a well-studied DNA repair system in the whole skin, as a tissue, but not much is known about it in melanocytes. Therefore, this review encapsulates NER in melanocytes, with a specific focus on its functional regulators and their cross talks due to skin heterogeneity and divulging the potential knowledge gap in the field.}, journal={Environmental and molecular mutagenesis}, year={2020}, month={May} } @article{dakup_porter_gaddameedhi_2020, title={The circadian clock protects against acute radiation-induced dermatitis}, volume={399}, url={https://doi.org/10.1016/j.taap.2020.115040}, DOI={10.1016/j.taap.2020.115040}, abstractNote={Radiation-induced dermatitis is a common occurrence in cancer patients undergoing radiation therapy (RT) and is caused when ionizing radiation (IR) induces DNA strand breaks in skin cells. The wide use of RT in cancer treatments makes it important to minimize RT-induced toxicities including radiodermatitis. This study sought to determine if the circadian clock plays a protective role in minimizing radiodermatitis. We treated mice in control (Day Shift), environmentally-disrupted (Rotating Shift) and genetically-disrupted (Per 1/2-/-) circadian conditions with 6 Gy of IR to the whole body. There was a significantly increased number of radiodermatitis spots on mice with circadian clock disruption compared to control mice. Additionally, circadian clock disrupted mice exhibited reduced protein levels of Bmal1, a phenomenon that sensitized circadian synchronized keratinocytes to IR-induced DNA damage. Furthermore, the skin phenotype results corresponded with significantly reduced body weights and increased genomic DNA damage in blood cells of mice with clock disruption compared to control mice. These findings suggest that the circadian clock plays a protective role in IR-induced DNA damage and skin toxicity, possibly through BMAL1-dependent mechanisms, and potentially impacts RT-associated radiodermatitis in cancer patients.}, journal={Toxicology and Applied Pharmacology}, publisher={Elsevier BV}, author={Dakup, Panshak P. and Porter, Kenneth I. and Gaddameedhi, Shobhan}, year={2020}, month={Jul}, pages={115040} } @article{the circadian clock protects against ionizing radiation-induced cardiotoxicity._2020, url={https://doi.org/10.1096/fj.201901850RR}, DOI={10.1096/fj.201901850rr}, abstractNote={Radiation therapy (RT) is commonly used to treat solid tumors of the breast, lung, and esophagus; however, the heart is an unintentional target of ionizing radiation (IR). IR exposure to the heart results in chronic toxicities including heart failure. We hypothesize that the circadian system plays regulatory roles in minimizing the IR-induced cardiotoxicity. We treated mice in control (Day Shift), environmentally disrupted (Rotating Shift), and genetically disrupted (Per 1/2 mutant) circadian conditions with 18 Gy of IR to the heart. Compared to control mice, circadian clock disruption significantly exacerbated post-IR systolic dysfunction (by ultrasound echocardiography) and increased fibrosis in mice. At the cellular level, Bmal1 protein bound to Atm, Brca1, and Brca2 promoter regions and its expression level was inversely correlated with the DNA damage levels based on the state of the clock. Further studies with circadian synchronized cardiomyocytes revealed that Bmal1 depletion increased the IR-induced DNA damage and apoptosis. Collectively, these findings suggest that the circadian clock protects from IR-induced toxicity and potentially impacts RT treatment outcome in cancer patients through IR-induced DNA damage responses.}, journal={FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, year={2020}, month={Jan} } @article{cardiac autonomic activity during simulated shift work_2019, url={https://publons.com/wos-op/publon/32745708/}, DOI={10.2486/INDHEALTH.2018-0044}, abstractNote={Shift work leads to adverse health outcomes including increased risk of cardiovascular disease. Heart rate (HR) and heart rate variability (HRV) are measures of cardiac autonomic activity and markers of cardiovascular disease and mortality. To investigate the effects of shift work on cardiac autonomic activity, we assessed the influence of simulated night work on HR and HRV, and dissociated the direct effects of circadian misalignment from those of sleep displacement and altered physical activity patterns. A total of 29 subjects each participated in one of two in-laboratory, simulated shift work studies. In both studies, EKG was continuously monitored via Holter monitors to measure HR and the high frequency (HF) component of HRV (HF-HRV). We found endogenous circadian rhythmicity in HR and HF-HRV. Sleep and waking physical activity, both displaced during simulated night work, had more substantial, and opposite, effects on HR and HF-HRV. Our findings show systematic but complex, interacting effects of time of day, sleep/wake state, and physical activity on cardiac autonomic activity. These effects need to be taken into account when evaluating HR and HRV in shift work settings and when interpreting these measures of cardiac autonomic activity as markers of cardiovascular disease.}, journal={Industrial Health}, year={2019} } @article{it's about time: advances in understanding the circadian regulation of dna damage and repair in carcinogenesis and cancer treatment outcomes_2019, url={https://publons.com/wos-op/publon/32413204/}, journal={Yale Journal of Biology and Medicine}, year={2019} } @article{it's about time: advances in understanding the circadian regulation of dna damage and repair in carcinogenesis and cancer treatment outcomes._2019, url={https://europepmc.org/articles/PMC6585512}, journal={The Yale journal of biology and medicine}, year={2019}, month={Jun} } @article{circadian clock protects against radiation-induced dermatitis and cardiomyopathy in mice_2018, url={https://publons.com/wos-op/publon/32413206/}, DOI={10.1158/1538-7445.AM2018-4159}, abstractNote={Abstract Radiation therapy (RT) is commonly used in cancer treatment. Despite technological advances to improve precision external-beam RT in treating breast, lung, and esophageal tumors, a substantial number of patients receive radiation to their hearts and the external skin region. This radiation insult results in acute and chronic toxicities including dermatitis and cardiomyopathy, affecting the patient's quality of life and survival. Hence, reducing toxicities associated with RT are desirable outcomes. With the knowledge that the natural circadian clock, present in almost every cells of the body including the skin and heart, regulates DNA repair, cell death and proliferation, and cardiac function processes, we hypothesize that molecular defensive mechanisms of the circadian system play regulatory roles to minimizing RT-induced skin and heart toxicity in cancer patients. To investigate acute toxicity of RT, studies were performed using SKH-1 hairless mice with healthy clock (wild-type day shift (DS)) and environmentally disrupted clock (wild-type rotating shift (RS) and genetically disrupted clock (Per1/2 knockout (Per1/2-/-)) conditions. We treated all groups (n=9 mice per group) with a single dose RT (6 grays of ionizing radiation) to the whole body. We used body weight as an indicator of overall toxicity, red spots as a measure of dermatitis, and left ventricular ejection fraction (LVEF) by ultrasound echocardiography as a readout for cardiomyopathy, relative to the measurements pre-RT as controls. After sacrifice, genotoxicity was detected using alkaline comet assay in PBMCs. Worse toxicities were observed until 2 weeks post-RT in mice with circadian clock disruption compared to mice with healthy clocks. Body weights of the disrupted clock groups were significantly reduced by up to 10% on day 2, unlike the DS group which reduced by only 2%. However, there was body weight loss recovery from day 6 in clock disrupted mice but this recovery was not sustained in Per1/2-/- mice. Dermatitis was counted where visible and there was 2.4- and 1.7- fold increases in RS and Per1/2-/- mice compared to DS mice. In the heart, LVEF was significantly decreased in RS and Per1/2-/- mice by days 1 and 4, and began to recover by day 8 in RS. Comparatively, the LVEF in DS mice was not significantly reduced. On a cellular level, genotoxicity in blood cells were significantly increased in RS and Per1/2-/- mice relative to DS mice. Taken together, these findings strongly suggest that the healthy circadian clock protects the whole body, skin, and heart from RT-induced toxicity. Future work includes mechanistic understanding of how the natural circadian clock system can be harnessed to minimize toxicity against RT. The successful completion of this project will provide a novel mechanistic understanding into the protective role of the circadian clock against acute and chronic skin and heart toxicities for cancer patients undergoing RT. Citation Format: Panshak P. Dakup, Kenneth I. Porter, Zhaokang Cheng, Shobhan Gaddameedhi. Circadian clock protects against radiation-induced dermatitis and cardiomyopathy in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4159.}, journal={Cancer Research}, year={2018} } @article{circulating plasma exosomes may signal circadian clock misalignment to peripheral tissues_2018, url={https://publons.com/wos-op/publon/35726971/}, journal={American Journal of Respiratory and Critical Care Medicine}, year={2018} } @article{skene_skornyakov_chowdhury_gajula_middleton_satterfield_porter_dongen_gaddameedhi_2018, title={Separation of circadian- and behavior-driven metabolite rhythms in humans provides a window on peripheral oscillators and metabolism}, volume={115}, url={https://doi.org/10.1073/pnas.1801183115}, DOI={10.1073/pnas.1801183115}, abstractNote={Significance Shift workers, whose schedules are misaligned relative to their suprachiasmatic nuclei (SCN) circadian pacemaker, are at elevated risk of metabolic disorders. In a study of simulated day- versus night-shift work followed by a constant routine, we separated plasma-circulating metabolites according to whether their 24-h rhythms aligned with the central SCN pacemaker or instead reflected externally imposed behavioral schedules. We found that rhythms in many metabolites implicated in food metabolism dissociated from the SCN pacemaker rhythm, with the vast majority aligning with the preceding sleep/wake and feeding/fasting cycles. Our metabolomics study yields insight into the link between prolonged exposure to shift work and the spectrum of associated metabolic disorders by providing a window into peripheral oscillators and the biobehavioral factors that orchestrate them.}, number={30}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Skene, Debra J. and Skornyakov, Elena and Chowdhury, Namrata R. and Gajula, Rajendra P. and Middleton, Benita and Satterfield, Brieann C. and Porter, Kenneth I. and Dongen, Hans P. A. Van and Gaddameedhi, Shobhan}, year={2018}, month={Jul}, pages={7825–7830} } @article{the circadian clock regulates cisplatin-induced toxicity and tumor regression in melanoma mouse and human models._2018, url={https://europepmc.org/articles/PMC5865687}, DOI={10.18632/oncotarget.24539}, abstractNote={// Panshak P. Dakup 1 , Kenneth I. Porter 1 , Alexander A. Little 1 , Rajendra P. Gajula 1 , Hui Zhang 1 , Elena Skornyakov 2, 3 , Michael G. Kemp 4 , Hans P.A. Van Dongen 2, 5 and Shobhan Gaddameedhi 1, 2 1 Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Spokane, WA, USA 2 Sleep and Performance Research Center, Washington State University, Spokane, WA, USA 3 Department of Physical Therapy, Eastern Washington University, Spokane, WA, USA 4 Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, OH, USA 5 Elson S. Floyd College of Medicine, Washington State University, Spokane, WA, USA Correspondence to: Shobhan Gaddameedhi, email: shobhan.gaddameedhi@wsu.edu Keywords: circadian rhythm; DNA repair; cisplatin; toxicity; melanoma Received: December 19, 2017     Accepted: February 10, 2018     Epub: February 20, 2018     Published: March 06, 2018 ABSTRACT Cisplatin is one of the most commonly used chemotherapeutic drugs; however, toxicity and tumor resistance limit its use. Studies using murine models and human subjects have shown that the time of day of cisplatin treatment influences renal and blood toxicities. We hypothesized that the mechanisms responsible for these outcomes are driven by the circadian clock. We conducted experiments using wild-type and circadian disrupted Per1/2 -/- mice treated with cisplatin at selected morning (AM) and evening (PM) times. Wild-type mice treated in the evening showed an enhanced rate of removal of cisplatin-DNA adducts and less toxicity than the morning-treated mice. This temporal variation in toxicity was lost in the Per1/2 -/- clock-disrupted mice, suggesting that the time-of-day effect is linked to the circadian clock. Observations in blood cells from humans subjected to simulated day and night shift schedules corroborated this view. Per1/2 -/- mice also exhibited a more robust immune response and slower tumor growth rate, indicating that the circadian clock also influences the immune response to melanoma tumors. Our findings indicate that cisplatin chronopharmacology involves the circadian clock control of DNA repair as well as immune responses, and thus affects both cisplatin toxicity and tumor growth. This has important implications for chronochemotherapy in cancer patients, and also suggests that influencing the circadian clock (e.g., through bright light treatment) may be explored as a tool to improve patient outcomes.}, journal={Oncotarget}, year={2018}, month={Feb} } @article{uv-b-induced erythema in human skin: the circadian clock is ticking._2018, url={https://doi.org/10.1016/j.jid.2017.09.002}, DOI={10.1016/j.jid.2017.09.002}, abstractNote={Acute exposure of skin to UV-B causes DNA damage and sunburn erythema in both mice and humans. Previous studies documented time-of-day-related differences in sunburn responses after UV-B exposure in mice. Because humans are diurnal and mice are nocturnal, the circadian rhythm in human skin was hypothesized to be in opposite phase to the rhythm in mice. A study by Nikkola et al. demonstrates that humans are more prone to sunburn erythema after evening exposure to solar UV-B radiation as compared with morning exposure. Acute exposure of skin to UV-B causes DNA damage and sunburn erythema in both mice and humans. Previous studies documented time-of-day-related differences in sunburn responses after UV-B exposure in mice. Because humans are diurnal and mice are nocturnal, the circadian rhythm in human skin was hypothesized to be in opposite phase to the rhythm in mice. A study by Nikkola et al. demonstrates that humans are more prone to sunburn erythema after evening exposure to solar UV-B radiation as compared with morning exposure. Because mice are nocturnal and humans are diurnal, their circadian clock outputs are out of phase. Based on this argument, and previous data relating time-of-day UV exposure to severity of UV effects in mouse skin, it was proposed that humans would be more prone to sunburn after late afternoon UV exposure rather than morning exposure.The majority of living organisms on the Earth are photosensitive and are influenced by the solar day and night cycle created by the axial rotation of the Earth. Consequently, cells have developed an amazing time-keeping mechanism known as the circadian clock that is self-sustained and oscillates with a periodic cycle of approximately 24 hours. In mammals, the molecular clock is entrained daily by the morning sunlight entering the eyes to reset the master clock located in the suprachiasmatic nucleus of the brain. The suprachiasmatic nucleus, via various hormonal and neuronal signals, synchronizes peripheral tissues including the heart, lung, liver, and skin. At the molecular level, the circadian clock is made up of primary as well as secondary feedback loops that are primarily responsible for approximately 24-hour rhythmicity of clock-controlled genes. Proteins including brain and muscle ARNT-like protein 1 (BMAL1), circadian locomotor output kaput (CLOCK), cryptochromes (CRY 1, 2), and periods (PER 1, 2, 3) make up a transcriptional-translational feedback loop that is initiated by dimerization of BMAL1 and CLOCK and the subsequent binding of this complex to the E-Box sequence (CACGTG or CACGTT) found in the promoter region of target clock-controlled genes (Lowrey and Takahashi, 2011Lowrey P.L. Takahashi J.S. Genetics of circadian rhythms in Mammalian model organisms.Adv Genet. 2011; 74: 175-230Crossref PubMed Scopus (409) Google Scholar). Tissue-specific circadian expression patterns have been observed in as many as 10% of mammalian genes, and many of these genes regulate cell cycle, metabolism, cell death, and DNA repair processes (Sancar et al., 2015Sancar A. Lindsey-Boltz L.A. Gaddameedhi S. Selby C.P. Ye R. Chiou Y.Y. et al.Circadian clock, cancer, and chemotherapy.Biochemistry. 2015; 54: 110-123Crossref PubMed Scopus (109) Google Scholar). Because mice are nocturnal and humans are diurnal, their circadian clock outputs are out of phase. Based on this argument, and previous data relating time-of-day UV exposure to severity of UV effects in mouse skin, it was proposed that humans would be more prone to sunburn after late afternoon UV exposure rather than morning exposure. Apart from its life-giving quality, the Sun emits harmful UVR that induces DNA damage in skin. Exposure to UVR causes a plethora of skin-related issues including photoaging, sunburn erythema, and skin cancers including melanomas, the deadliest skin cancers. In recent years, the skin’s circadian clock has been shown to regulate various cellular responses after UVR exposure, including nucleotide excision repair, cell cycle checkpoints, oxidative stress, and apoptosis (Dakup and Gaddameedhi, 2017Dakup P. Gaddameedhi S. Impact of the circadian clock on UV-induced DNA damage response and photocarcinogenesis.Photochem Photobiol. 2017; 93: 296-303Crossref PubMed Scopus (37) Google Scholar, Gaddameedhi et al., 2011Gaddameedhi S. Selby C.P. Kaufmann W.K. Smart R.C. Sancar A. Control of skin cancer by the circadian rhythm.Proc Natl Acad Sci USA. 2011; 108: 18790-18795Crossref PubMed Scopus (163) Google Scholar, Geyfman et al., 2012Geyfman M. Kumar V. Liu Q. Ruiz R. Gordon W. Espitia F. et al.Brain and muscle Arnt-like protein-1 (BMAL1) controls circadian cell proliferation and susceptibility to UVB-induced DNA damage in the epidermis.Proc Natl Acad Sci USA. 2012; 109: 11758-11763Crossref PubMed Scopus (162) Google Scholar, Plikus et al., 2015Plikus M.V. Van Spyk E.N. Pham K. Geyfman M. Kumar V. Takahashi J.S. et al.The circadian clock in skin: implications for adult stem cells, tissue regeneration, cancer, aging, and immunity.J Biol Rhythms. 2015; 30: 163-182Crossref PubMed Scopus (103) Google Scholar, Sancar et al., 2015Sancar A. Lindsey-Boltz L.A. Gaddameedhi S. Selby C.P. Ye R. Chiou Y.Y. et al.Circadian clock, cancer, and chemotherapy.Biochemistry. 2015; 54: 110-123Crossref PubMed Scopus (109) Google Scholar, Wang et al., 2017Wang H. van Spyk E. Liu Q. Geyfman M. Salmans M.L. Kumar V. et al.Time-restricted feeding shifts the skin circadian clock and alters UVB-induced DNA damage.Cell Rep. 2017; 20: 1061-1072Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). These robust protective mechanisms attenuate unwanted consequences of UVR-mediated DNA damage. Skin clock regulation of nucleotide excision repair has been well characterized using the SKH-1 hairless mouse model, where it was observed that an elevated rate of UV photoproduct removal correlated with higher expression of the core nucleotide excision repair factor Xpa in the evening as compared with the morning (Gaddameedhi et al., 2011Gaddameedhi S. Selby C.P. Kaufmann W.K. Smart R.C. Sancar A. Control of skin cancer by the circadian rhythm.Proc Natl Acad Sci USA. 2011; 108: 18790-18795Crossref PubMed Scopus (163) Google Scholar). However, acute exposure to UVR also causes a UV-induced inflammatory reaction that is characterized by vasodilation and increased blood flow to the dermis, resulting in sunburn erythema in the affected skin. Using SKH-1 hairless mice, we have shown that more sunburn erythema occurs in morning solar UV-B-treated mice than in evening-treated animals (Gaddameedhi et al., 2015Gaddameedhi S. Selby C.P. Kemp M.G. Ye R. Sancar A. The circadian clock controls sunburn apoptosis and erythema in mouse skin.J Invest Dermatol. 2015; 135: 1119-1127Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). We determined that during morning time, when DNA repair is lowest and DNA synthesis is highest, the DNA damage caused by UVR leads to stronger activation of the Atr protein kinase, which is known to be activated in response to replication stress and to regulate the stability of the tumor suppressor p53. This results in increased numbers of sunburn apoptotic cells and enhanced inflammation in mouse skin. Because mice are nocturnal and humans are diurnal, their circadian clock outputs are out of phase. Based on this argument, and previous data relating time-of-day UV exposure to severity of UV effects in mouse skin, it was proposed that humans would be more prone to sunburn after late afternoon UV exposure rather than morning exposure. Nikkola et al. evaluated the circadian time effect of narrow band-UV-B exposure on sunburn erythema in human skin. They selected 19 subjects (16 females and 3 males) having skin types II and III as defined by Fitzpatrick sun-reactive skin type scale (Fitzpatrick, 1988Fitzpatrick T.B. The validity and practicality of sun-reactive skin types I through VI.Arch Dermatol. 1988; 124: 869-871Crossref PubMed Scopus (3021) Google Scholar). Subjects were exposed to increasing doses of narrow band-UV-B up to 4 suberythemal dose, once in the morning between 7 am and 9 am on one buttock and once in the evening between 7 pm and 9 pm on the other buttock of the same subject. Twenty-four hours after UV-B treatment, the erythema of the skin patches was quantified and skin biopsies were irradiated with the highest dose of UV-B (i.e., 4 suberythemal dose = 40 mJ/cm2 CIE (Commission Internationale de l'Eclairage, Vienna, Austria)) were collected. Protein levels of the tumor suppressor proteins p53 and the clock proteins CRY1 and CRY2 were measured in biopsies. Interestingly, the authors found that human subjects treated with UV-B in the evening had a significantly higher erythema index scores in comparison with the subjects exposed in the morning. In addition, different levels of CRY1 and CRY2 were detected in irradiated skin using immunohistochemistry, and human subjects with a negligible amount of CRY2 showed more erythema relative to individuals with high levels of CRY2. This is a novel observation. The authors speculated that CRY2 protects against UV-B-mediated skin damage. In a different study, the Lamia group reported that CRY2 regulated c-MYC proteasomal degradation by acting as a cofactor for the SCF substrate adaptor FBXL3 (an E3 ligase protein) (Huber et al., 2016Huber A.L. Papp S.J. Chan A.B. Henriksson E. Jordan S.D. Kriebs A. et al.CRY2 and FBXL3 cooperatively degrade c-MYC.Mol Cell. 2016; 64: 774-789Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Because c-MYC plays important roles in cell proliferation, cell growth, and apoptosis, high levels of CRY2 could enhance c-MYC degradation, which in turn might reduce sunburn-induced apoptosis and other characteristic features of sunburn erythema. It would be interesting to evaluate levels of core clock proteins including CRY1, 2, PER1, 2, 3, BMAL1, and CLOCK in a larger sample set. Also, knowing the chronotype (behavioral manifestation of the underlying biological clock) of the patients with negligible amount of CRY2 levels would facilitate understanding the effects of circadian clock disruption on sunburn erythema. Collectively, the above additional information would define mechanistic similarities and circadian phase output differences between human and mouse models as shown in Figure 1. In addition to CRY protein levels, Nikkola et al. also measured p53 concentrations in skin biopsies and observed increased p53 levels in the morning samples compared with the evening group. This observation appears to contradict the predicted human skin sunburn model, but the authors suggested that the possible reasons for this observation were either delayed (24 hours after UV-B irradiation) biopsy collection in comparison with the mouse study previously conducted by us or a dissociation of human p53 elevation and erythema as previously suggested (Healy et al., 1994Healy E. Reynolds N.J. Smith M.D. Campbell C. Farr P.M. Rees J.L. Dissociation of erythema and p53 protein expression in human skin following UVB irradiation, and induction of p53 protein and mRNA following application of skin irritants.J Invest Dermatol. 1994; 103: 493-499Abstract Full Text PDF PubMed Google Scholar). However, the paper by Healy et al. did not consider the circadian aspect of p53 and erythema, nor were subjects selected according to chronotype. Thus, the observations by Healy et al. may not be directly relatable to the current study. In contrast, strong evidence of p53 involvement in sunburn erythema was shown by Brash’s group using p53 knockout mouse models, where they demonstrated that deleting p53 gene in mouse skin decreased the number of sunburn-mediated apoptotic cells after exposure to UV-B (Ziegler et al., 1994Ziegler A. Jonason A.S. Leffell D.J. Simon J.A. Sharma H.W. Kimmelman J. et al.Sunburn and p53 in the onset of skin cancer.Nature. 1994; 372: 773-776Crossref PubMed Scopus (1348) Google Scholar). In another recent paper (Guan et al., 2016Guan L. Suggs A. Ahsanuddin S. Tarrillion M. Selph J. Lam M. et al.2016 Arte poster competition first place winner: circadian rhythm and UV-induced skin damage: an in vivo study.J Drugs Dermatol. 2016; 15: 1124-1130PubMed Google Scholar) in which erythema induction was examined as a function of time-of-day of UV exposure, the authors reported enhanced erythema in human subjects after morning treatment in comparison with evening exposure. However, this study used solar simulated radiation, which is primarily composed of UV-A radiation that is much less efficient at inducing canonical UV bipyrimidine dimers in DNA, rather than narrow band-UV-B as in the Nikkola et al. study. Guan et al. also did not evaluate core clock gene expression levels in skin biopsies of the subjects, which is important to understand the circadian phase of the observations. In future studies, it will be interesting to determine how different experimental parameters and variables in the Guan et al. and Nikkola et al. studies contribute to the unique time-of-day erythemal responses in humans. It should be noted that there are additional factors that may contribute to circadian effects on UV responses in human skin, such as skin type, sleep cycles, and feeding behaviors of the participating human volunteers. These issues are more difficult to control for in human studies than in mice. In a recent elegant study, the Andersen group showed that restricting feeding to the inactive phase (during day time) in mice shifts the skin’s circadian clock and its response to solar UV-B irradiation (Wang et al., 2017Wang H. van Spyk E. Liu Q. Geyfman M. Salmans M.L. Kumar V. et al.Time-restricted feeding shifts the skin circadian clock and alters UVB-induced DNA damage.Cell Rep. 2017; 20: 1061-1072Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The group concluded that daytime feeding reverses diurnal sensitivity to UVB-induced DNA damage specifically by influencing the oscillatory expression pattern of the Xpa gene. Feeding and social behaviors of human subjects may also affect UV responses and should therefore be considered when designing future studies involving humans. A few possible solutions to this problem include evaluating participating human subjects in sleep research labs, where their sleep cycles, physical activity, and feeding patterns can be closely monitored, or formulating detailed questionnaires for the volunteers at the time of enrollment. Collectively, the Nikkola et al. and Guan et al. papers show involvement of circadian timing in UVR-induced sunburn erythema in humans, but report different observations relating the intensity of sunburn erythema to time-of-day UVR exposure. Observations of core clock gene expression pattern in the −/+UV-B-treated skin samples bolster the involvement of circadian clock regulation in UV-B-induced sunburn erythema in humans. However, it would have been fascinating to perform additional mechanistic studies to understand the signaling events occurring through the skin’s circadian clock on UVR exposure and correlate the results with the observed phenotype in humans. Our environment is constantly changing, challenging us both socially and biologically, and pushing us to adopt erratic time schedules. Irregular and unhealthy sleep cycles and feeding patterns are disrupting our natural circadian rhythm and making us vulnerable to environmental stresses that vary depending on the time-of-day. Despite the strong evidence that the circadian clock impacts on sunburn erythema in mice, there are still active debate about the vulnerability of human skin to UVR-induced erythema as a function of the time-of-day of exposure. One reason for continued controversy could be huge variations in the natural rhythm among humans, which make such studies much more difficult and time consuming. Hence, special efforts should be made to select appropriate human volunteers. Identification of the optimal time-of-day to minimize sunburn erythema in humans may soon be possible, and may be consistent with the model proposed by Gaddameedhi et al., 2015Gaddameedhi S. Selby C.P. Kemp M.G. Ye R. Sancar A. The circadian clock controls sunburn apoptosis and erythema in mouse skin.J Invest Dermatol. 2015; 135: 1119-1127Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar. Until then, minimization of sun exposure and photoprotection should continue to be recommended. The authors state no conflict of interest. The authors thank Dr Michael G. Kemp at Wright State University and Panshak Dakup at WSU for critical reading of this commentary. SG was supported by National Institutes of Health grant 4R00ES022640 and WSU College of Pharmacy. Circadian Time Effects on NB-UVB–Induced Erythema in Human Skin In VivoJournal of Investigative DermatologyVol. 138Issue 2PreviewUVR-induced skin erythema occurs after DNA damage, a consequence of the cell signaling cascades and synthesis of various cytokines and inflammatory mediators detected as vasodilation, inflammation, and apoptosis (Sklar et al., 2013). UVB-induced carcinogenesis has been linked to circadian time in mice, because those subjected to chronic UVB radiation in the morning had a 5-fold higher number of invasive squamous cell carcinomas compared with the evening-treated group (Gaddameedhi et al., 2011). Full-Text PDF Open Archive}, journal={The Journal of investigative dermatology}, year={2018}, month={Feb} } @article{chronopharmacology of cisplatin: role of the circadian rhythm in modulating cisplatin-induced toxicity in melanoma mouse model_2017, url={https://publons.com/wos-op/publon/32413210/}, DOI={10.1158/1538-7445.AM2017-1120}, abstractNote={Purpose: The goal of this research is to highlight novel and divergent molecular mechanisms of the circadian system in modulating the response of cisplatin therapy against melanoma tumors. Cisplatin is one of the most commonly used chemotherapeutic drugs in treating a variety of tumors including cancers of ovaries, testis, lungs, blood and solid tumors of the head and neck, and is more recently under clinical trials for potential application in melanoma tumors. However, the major limitation of cisplatin as a chemotherapeutic drug is its tumor resistance and nephrotoxicity. Hence, improving the effectiveness and reducing the toxicities associated with cisplatin therapy are desirable outcomes. Studies on human models have shown decreased renal and blood toxicity by time-of-the-day. Consequently, our project seeks to study the chronopharmacological effects and understand the mechanisms that have been successfully demonstrated in human models. We hypothesize a mechanistic, circadian rhythm-based cause for these outcomes. Experimental Design: Studies were done on B16F10 melanoma mouse models of wild-type and circadian disrupted Per1/2 -/- animals treated with 3 doses of 5 mg/kg cisplatin in the morning (7 AM) and evening (5 PM). Animal weights and tumor sizes were measured regularly post-treatment. Upon sacrifice, tissues (skin, kidney, and tumor) were harvested and analyzed for cisplatin-induced toxicities and DNA damage responses using H & E staining, western blot, and KIM-1 and immuno-slot blot assays. Results: Weight measurements show a clock-regulated response to cisplatin toxicity. AM-treated wild-type animals showed significant weight loss compared to PM-treated wild-type animals. This treatment time differential is lost in the Per1/2 -/-. On a molecular level, kidney tissue DNA samples showed clock-controlled cisplatin-DNA repair activity in wild-type animals compared to Per1/2 -/-. These initial findings strongly suggest that nephrotoxicity and DNA damage response function might be regulated by the circadian rhythm. Conclusions: These findings indicate a possible mechanism for the chronopharmacology of cisplatin in minimizing the toxicity associated with it and reveal a target for future study of additional mechanistic causes of circadian dosing changes in cisplatin and other genotoxic stress-mediated anti-cancer agents. Citation Format: Panshak Dakup, Kenneth Porter, Rajendra Gajula, Shobhan Gaddameedhi. Chronopharmacology of cisplatin: role of the circadian rhythm in modulating cisplatin-induced toxicity in melanoma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1120. doi:10.1158/1538-7445.AM2017-1120}, journal={Cancer Research}, year={2017} } @article{impact of the circadian clock on uv-induced dna damage response and photocarcinogenesis_2017, url={https://publons.com/wos-op/publon/32413211/}, DOI={10.1111/PHP.12662}, abstractNote={The skin is in constant exposure to various external environmental stressors, including solar ultraviolet (UV) radiation. Various wavelengths of UV light are absorbed by the DNA and other molecules in the skin to cause DNA damage and induce oxidative stress. The exposure to excessive ultraviolet (UV) radiation and/or accumulation of damage over time can lead to photocarcinogenesis and photoaging. The nucleotide excision repair (NER) system is the sole mechanism for removing UV photoproduct damage from DNA, and genetic disruption of this repair pathway leads to the photosensitive disorder xeroderma pigmentosum (XP). Interestingly, recent work has shown that NER is controlled by the circadian clock, the body's natural time-keeping mechanism, through regulation of the rate-limiting repair factor xeroderma pigmentosum group A (XPA). Studies have shown reduced UV-induced skin cancer after UV exposure in the evening compared to the morning, which corresponds with times of high and low repair capacities, respectively. However, most studies of the circadian clock–NER connection have utilized murine models, and it is therefore important to translate these findings to humans to improve skin cancer prevention and chronotherapy.}, journal={Photochemistry & Photobiology}, year={2017} } @article{shift work: disrupted circadian rhythms and sleep-implications for health and well-being._2017, url={https://europepmc.org/articles/PMC5647832}, DOI={10.1007/s40675-017-0071-6}, abstractNote={Our 24/7 society is dependent on shift work, despite mounting evidence for negative health outcomes from sleep displacement due to shift work. This paper reviews short- and long-term health consequences of sleep displacement and circadian misalignment due to shift work. We focus on four broad health domains: metabolic health, risk of cancer, cardiovascular health, and mental health. Circadian misalignment affects these domains by inducing sleep deficiency, sympathovagal and hormonal imbalance, inflammation, impaired glucose metabolism, and dysregulated cell cycles. This leads to a range of medical conditions, including obesity, metabolic syndrome, type II diabetes, gastrointestinal dysfunction, compromised immune function, cardiovascular disease, excessive sleepiness, mood and social disorders, and increased cancer risk. Interactions of biological disturbances with behavioral and societal factors shape the effects of shift work on health and well-being. Research is needed to better understand the underlying mechanisms and drive the development of countermeasures.}, journal={Current sleep medicine reports}, year={2017}, month={Apr} } @article{chronopharmacology of cisplatin: role of the circadian rhythm in modulating the cisplatin transporters levels_2016, url={https://publons.com/wos-op/publon/32413213/}, journal={The FASEB Journal}, year={2016} } @article{circadian clock, cancer, and chemotherapy_2015, url={https://publons.com/wos-op/publon/6089121/}, DOI={10.1021/BI5007354}, abstractNote={The circadian clock is a global regulatory system that interfaces with most other regulatory systems and pathways in mammalian organisms. Investigations of the circadian clock-DNA damage response connections have revealed that nucleotide excision repair, DNA damage checkpoints, and apoptosis are appreciably influenced by the clock. Although several epidemiological studies in humans and a limited number of genetic studies in mouse model systems have indicated that clock disruption may predispose mammals to cancer, well-controlled genetic studies in mice have not supported the commonly held view that circadian clock disruption is a cancer risk factor. In fact, in the appropriate genetic background, clock disruption may instead aid in cancer regression by promoting intrinsic and extrinsic apoptosis. Finally, the clock may affect the efficacy of cancer treatment (chronochemotherapy) by modulating the pharmacokinetics and pharmacodynamics of chemotherapeutic drugs as well as the activity of the DNA repair enzymes that repair the DNA damage caused by anticancer drugs.}, journal={Biochemistry}, year={2015} } @article{commentary: chemiexcitation of melanin derivatives induces dna photoproducts long after uv exposure._2015, url={https://europepmc.org/articles/PMC4594339}, DOI={10.3389/fphys.2015.00276}, abstractNote={GENERAL COMMENTARY article Front. Physiol., 06 October 2015Sec. Redox Physiology Volume 6 - 2015 | https://doi.org/10.3389/fphys.2015.00276}, journal={Frontiers in physiology}, year={2015}, month={Oct} } @article{development and validation of a new transgenic hairless albino mouse as a mutational model for potential assessment of photocarcinogenicity._2015, url={https://doi.org/10.1016/j.mrgentox.2015.08.001}, DOI={10.1016/j.mrgentox.2015.08.001}, abstractNote={Short-term phototoxicity testing is useful in selecting test agents for the longer and more expensive photocarcinogenesis safety tests; however, no validated short-term tests have been proven reliable in predicting the outcome of a photocarcinogenesis safety test. A transgenic, hairless, albino (THA) mouse model was developed that carries the gpt and red/gam [Spi(-)] genes from the gpt delta mouse background and the phenotypes from the SKH-1 mouse background to use as a short-term test in lieu of photocarcinogenesis safety tests. Validation of the THA mouse model was confirmed by exposing groups of male mice to sub-erythemal doses of ultraviolet B (UVB) irradiation for three consecutive days emitted from calibrated overhead, Kodacel-filtered fluorescent lamps and measuring the mutant frequencies (MFs) in the gpt and red/gam (Spi(-)) genes and types of mutations in the gpt gene. The doses or irradiation were monitored with broad-spectrum dosimeters that were calibrated to a NIST-traceable standard and cumulative CIE-weighted doses were 20.55 and 41.0mJ/cm(2) (effective). Mice were sacrificed 14 days after the final UVB exposure and MFs in both the gpt and red/gam genes were evaluated in the epidermis. The exposure of mice to UVB induced significant ten- to twelve-fold increases in the gpt MF and three- to five-fold increases in the Spi(-) MF over their respective background MF, 26±3×10(-6) and 9±1×10(-6). The gpt mutation spectra were significantly different between that of the UVB-irradiated and that of non-irradiated mice although the mutation spectra of both groups were dominated by C→T transitions (84% and 66%). In mice exposed to UVB, the C→T transitions occurred almost exclusively at dipyrimidine sites (92%), whereas in non-irradiated control mice, the C→T transitions occurred at CpG sites (86%). These results suggest that the newly developed THA mice are a useful and reliable model for testing UVB-induced mutagenicity in skin tissue. The application of this model for short-term prediction of solar-induced skin carcinogenicity is presently under investigation.}, journal={Mutation research. Genetic toxicology and environmental mutagenesis}, year={2015}, month={Aug} } @article{oncogenic braf(v600e) induces clastogenesis and uvb hypersensitivity._2015, url={https://europepmc.org/articles/PMC4491700}, DOI={10.3390/cancers7020825}, abstractNote={The oncogenic BRAF(V600E) mutation is common in melanomas as well as moles. The roles that this mutation plays in the early events in the development of melanoma are poorly understood. This study demonstrates that expression of BRAF(V600E) is not only clastogenic, but synergizes for clastogenesis caused by exposure to ultraviolet radiation in the 300 to 320 nM (UVB) range. Expression of BRAF(V600E) was associated with induction of Chk1 pS280 and a reduction in chromatin remodeling factors BRG1 and BAF180. These alterations in the Chk1 signaling pathway and SWI/SNF chromatin remodeling pathway may contribute to the clastogenesis and UVB sensitivity. These results emphasize the importance of preventing sunburns in children with developing moles.}, journal={Cancers}, year={2015}, month={Jun} } @article{the circadian clock controls sunburn apoptosis and erythema in mouse skin_2015, url={https://publons.com/wos-op/publon/48039280/}, DOI={10.1038/JID.2014.508}, abstractNote={Epidemiological studies of humans and experimental studies with mouse models suggest that sunburn resulting from exposure to excessive UV light and damage to DNA confers an increased risk for melanoma and non-melanoma skin cancer. Previous reports have shown that both nucleotide excision repair, which is the sole pathway in humans for removing UV photoproducts, and DNA replication are regulated by the circadian clock in mouse skin. Furthermore, the timing of UV exposure during the circadian cycle has been shown to affect skin carcinogenesis in mice. Because sunburn and skin cancer are causally related, we investigated UV-induced sunburn apoptosis and erythema in mouse skin as a function of circadian time. Interestingly, we observed that sunburn apoptosis, inflammatory cytokine induction, and erythema were maximal following an acute early-morning exposure to UV and minimal following an afternoon exposure. Early-morning exposure to UV also produced maximal activation of ataxia telangiectasia mutated and Rad3-related (Atr)-mediated DNA damage checkpoint signaling, including activation of the tumor suppressor p53, which is known to control the process of sunburn apoptosis. These data provide early evidence that the circadian clock has an important role in the erythemal response in UV-irradiated skin. The early morning is when DNA repair is at a minimum, and thus the acute responses likely are associated with unrepaired DNA damage. The prior report that mice are more susceptible to skin cancer induction following chronic irradiation in the AM, when p53 levels are maximally induced, is discussed in terms of the mutational inactivation of p53 during chronic irradiation. Epidemiological studies of humans and experimental studies with mouse models suggest that sunburn resulting from exposure to excessive UV light and damage to DNA confers an increased risk for melanoma and non-melanoma skin cancer. Previous reports have shown that both nucleotide excision repair, which is the sole pathway in humans for removing UV photoproducts, and DNA replication are regulated by the circadian clock in mouse skin. Furthermore, the timing of UV exposure during the circadian cycle has been shown to affect skin carcinogenesis in mice. Because sunburn and skin cancer are causally related, we investigated UV-induced sunburn apoptosis and erythema in mouse skin as a function of circadian time. Interestingly, we observed that sunburn apoptosis, inflammatory cytokine induction, and erythema were maximal following an acute early-morning exposure to UV and minimal following an afternoon exposure. Early-morning exposure to UV also produced maximal activation of ataxia telangiectasia mutated and Rad3-related (Atr)-mediated DNA damage checkpoint signaling, including activation of the tumor suppressor p53, which is known to control the process of sunburn apoptosis. These data provide early evidence that the circadian clock has an important role in the erythemal response in UV-irradiated skin. The early morning is when DNA repair is at a minimum, and thus the acute responses likely are associated with unrepaired DNA damage. The prior report that mice are more susceptible to skin cancer induction following chronic irradiation in the AM, when p53 levels are maximally induced, is discussed in terms of the mutational inactivation of p53 during chronic irradiation. zeitgeber time Excessive exposure to solar UVR has a variety of adverse effects on the skin, such as aging, sunburn, and the induction of melanoma and non-melanoma skin cancers (Ziegler et al., 1994Ziegler A. Jonason A.S. Leffell D.J. et al.Sunburn and p53 in the onset of skin cancer.Nature. 1994; 372: 773-776Crossref PubMed Scopus (1348) Google Scholar; Geller et al., 2012Geller A.C. Balk S.J. Fisher D.E. Stemming the tanning bed epidemic: time for action.J Natl Compr Canc Netw. 2012; 10: 1311-1314Crossref PubMed Scopus (6) Google Scholar; Sorrentino et al., 2014Sorrentino J.A. Krishnamurthy J. Tilley S. et al.p16INK4a reporter mice reveal age-promoting effects of environmental toxicants.J Clin Invest. 2014; 124: 169-173Crossref PubMed Scopus (52) Google Scholar). The sunburn process is an early inflammatory response to UV exposure that is regulated by the tumor suppressor protein p53 and follows molecular and cellular responses that include DNA repair, DNA damage checkpoint signaling, and apoptosis. Evidence from both epidemiological studies on humans (Whiteman et al., 2001Whiteman D.C. Whiteman C.A. Green A.C. Childhood sun exposure as a risk factor for melanoma: a systematic review of epidemiologic studies.Cancer Causes Control. 2001; 12: 69-82Crossref PubMed Scopus (506) Google Scholar) and experimental studies with mouse melanoma models (Noonan et al., 2001Noonan F.P. Recio J.A. Takayama H. et al.Neonatal sunburn and melanoma in mice.Nature. 2001; 413: 271-272Crossref PubMed Scopus (311) Google Scholar; Kannan et al., 2003Kannan K. Sharpless N.E. Xu J. et al.Components of the Rb pathway are critical targets of UV mutagenesis in a murine melanoma model.Proc Natl Acad Sci USA. 2003; 100: 1221-1225Crossref PubMed Scopus (104) Google Scholar) suggest that sunburn poses a significant risk of developing malignant melanoma, which is the deadliest form of skin cancer. Moreover, owing to changes in lifestyle and the environment, the incidence of sunburn-associated malignant melanoma has been steadily increasing (Fisher and James, 2010Fisher D.E. James W.D. Indoor tanning–science, behavior, and policy.N Engl J Med. 2010; 363: 901-903Crossref PubMed Scopus (81) Google Scholar), and novel approaches are needed to address this problem. Organisms from bacteria to humans possess circadian clocks, which are molecular time-keeping systems that maintain daily rhythms in physiological and biochemical processes with a periodicity of ∼24 hours (Reppert and Weaver, 2001Reppert S.M. Weaver D.R. Molecular analysis of mammalian circadian rhythms.Annu Rev Physiol. 2001; 63: 647-676Crossref PubMed Scopus (1210) Google Scholar; Lowrey and Takahashi, 2004Lowrey P.L. Takahashi J.S. Mammalian circadian biology: elucidating genome-wide levels of temporal organization.Annu Rev Genomics Hum Genet. 2004; 5: 407-441Crossref PubMed Scopus (762) Google Scholar; Partch et al., 2014Partch C.L. Green C.B. Takahashi J.S. Molecular architecture of the mammalian circadian clock.Trends Cell Biol. 2014; 24: 90-99Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar). At the molecular level, circadian oscillation in higher organisms is generated by a transcription–translation feedback loop in which the heterodimeric transcription factor CLOCK/BMAL1 drives the expression of many clock-controlled target genes, including the Cryptochrome (Cry1/2) and Period (Per1/2) genes, by binding to E-box elements in their promoters. A negative feedback loop is formed when CRY/PER protein complexes shuttle into the nucleus, inhibit CLOCK/BMAL1-mediated transactivation, and thereby inhibit their own transcription (Vitaterna et al., 1999Vitaterna M.H. Selby C.P. Todo T. et al.Differential regulation of mammalian period genes and circadian rhythmicity by cryptochromes 1 and 2.Proc Natl Acad Sci USA. 1999; 96: 12114-12119Crossref PubMed Scopus (563) Google Scholar; Reppert and Weaver, 2001Reppert S.M. Weaver D.R. Molecular analysis of mammalian circadian rhythms.Annu Rev Physiol. 2001; 63: 647-676Crossref PubMed Scopus (1210) Google Scholar; Sahar and Sassone-Corsi, 2009Sahar S. Sassone-Corsi P. Metabolism and cancer: the circadian clock connection.Nat Rev Cancer. 2009; 9: 886-896Crossref PubMed Scopus (410) Google Scholar). Loss of CRY/PER by proteolytic degradation re-initiates transactivation by CLOCK/BMAL1 (Lowrey and Takahashi, 2004Lowrey P.L. Takahashi J.S. Mammalian circadian biology: elucidating genome-wide levels of temporal organization.Annu Rev Genomics Hum Genet. 2004; 5: 407-441Crossref PubMed Scopus (762) Google Scholar; Antoch and Kondratov, 2010Antoch M.P. Kondratov R.V. Circadian proteins and genotoxic stress response.Cir Res. 2010; 106: 68-78Crossref PubMed Scopus (42) Google Scholar). Consequently, the genes and pathways that are controlled by the molecular clock have a periodicity of ∼24 hours. The expression of as many as 10% of mammalian genes is regulated by the circadian clock (Akhtar et al., 2002Akhtar R.A. Reddy A.B. Maywood E.S. et al.Circadian cycling of the mouse liver transcriptome, as revealed by cDNA microarray, is driven by the suprachiasmatic nucleus.Curr Biol. 2002; 12: 540-550Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar; Hughes et al., 2009Hughes M.E. DiTacchio L. Hayes K.R. et al.Harmonics of circadian gene transcription in mammals.PLoS Genet. 2009; 5: e1000442Crossref PubMed Scopus (492) Google Scholar), and most of the circadian gene expression patterns are tissue specific (Partch et al., 2014Partch C.L. Green C.B. Takahashi J.S. Molecular architecture of the mammalian circadian clock.Trends Cell Biol. 2014; 24: 90-99Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar). Among the proteins that are regulated by the clock is XPA (xeroderma pigmentosum complementation group A), which is one of the six core factors that are required for removing UV photoproducts from DNA. Patients with the disease xeroderma pigmentosum exhibit solar sensitivity and a greatly increased risk of skin cancer (Kraemer et al., 1987Kraemer K.H. Lee M.M. Scotto J. Xeroderma pigmentosum. Cutaneous, ocular, and neurologic abnormalities in 830 published cases.Arch Dermatol. 1987; 123: 241-250Crossref PubMed Scopus (964) Google Scholar). In mice, the levels of XPA RNA and protein have been found to be rhythmic in brain (Kang et al., 2009Kang T.H. Reardon J.T. Kemp M. et al.Circadian oscillation of nucleotide excision repair in mammalian brain.Proc Natl Acad Sci USA. 2009; 106: 2864-2867Crossref PubMed Scopus (159) Google Scholar), liver (Kang et al., 2010Kang T.H. Lindsey-Boltz L.A. Reardon J.T. et al.Circadian control of XPA and excision repair of cisplatin-DNA damage by cryptochrome and HERC2 ubiquitin ligase.Proc Natl Acad Sci USA. 2010; 107: 4890-4895Crossref PubMed Scopus (174) Google Scholar), and, more recently, skin (Gaddameedhi et al., 2011Gaddameedhi S. Selby C.P. Kaufmann W.K. et al.Control of skin cancer by the circadian rhythm.Proc Natl Acad Sci USA. 2011; 108: 18790-18795Crossref PubMed Scopus (164) Google Scholar), which is known to harbor a functional circadian clock that regulates gene expression (Tanioka et al., 2009Tanioka M. Yamada H. Doi M. et al.Molecular clocks in mouse skin.J Invest Dermatol. 2009; 129: 1225-1231Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar; Sporl et al., 2012Sporl F. Korge S. Jurchott K. et al.Kruppel-like factor 9 is a circadian transcription factor in human epidermis that controls proliferation of keratinocytes.Proc Natl Acad Sci USA. 2012; 109: 10903-10908Crossref PubMed Scopus (99) Google Scholar). Oscillation of XPA levels was found to parallel the oscillation of repair activity in these tissues (Kang et al., 2009Kang T.H. Reardon J.T. Kemp M. et al.Circadian oscillation of nucleotide excision repair in mammalian brain.Proc Natl Acad Sci USA. 2009; 106: 2864-2867Crossref PubMed Scopus (159) Google Scholar; Gaddameedhi et al., 2011Gaddameedhi S. Selby C.P. Kaufmann W.K. et al.Control of skin cancer by the circadian rhythm.Proc Natl Acad Sci USA. 2011; 108: 18790-18795Crossref PubMed Scopus (164) Google Scholar; Kang et al., 2011Kang T.H. Reardon J.T. Sancar A. Regulation of nucleotide excision repair activity by transcriptional and post-transcriptional control of the XPA protein.Nucleic Acids Res. 2011; 39: 3176-3187Crossref PubMed Scopus (92) Google Scholar). In addition to excision repair oscillation, using a circadian clock–deficient genetic mouse model, we and others have shown that DNA replication activity is circadian in nature in proliferating tissues such as the intestine and the epidermis (Gaddameedhi et al., 2011Gaddameedhi S. Selby C.P. Kaufmann W.K. et al.Control of skin cancer by the circadian rhythm.Proc Natl Acad Sci USA. 2011; 108: 18790-18795Crossref PubMed Scopus (164) Google Scholar; Geyfman et al., 2012Geyfman M. Kumar V. Liu Q. et al.Brain and muscle Arnt-like protein-1 (BMAL1) controls circadian cell proliferation and susceptibility to UVB-induced DNA damage in the epidermis.Proc Natl Acad Sci USA. 2012; 109: 11758-11763Crossref PubMed Scopus (162) Google Scholar). This circadian rhythmicity of DNA replication is antiphase to that of DNA repair. Interestingly, mice were found to be more sensitive to the development of skin cancer when chronically irradiated in the early morning (when repair activity was low and DNA synthesis was high) than when chronically irradiated in the late afternoon (when repair activity was high and DNA synthesis was low). Melanomas and basal cell carcinomas in humans are commonly associated with episodes of sunburn early in life. Sunburn is initiated by excessive acute UVR–induced DNA damage and is followed by p53-dependent apoptosis and inflammation (Ziegler et al., 1994Ziegler A. Jonason A.S. Leffell D.J. et al.Sunburn and p53 in the onset of skin cancer.Nature. 1994; 372: 773-776Crossref PubMed Scopus (1348) Google Scholar). The importance of DNA damage and repair to the induction of sunburn is indicated by the greatly enhanced apoptotic and inflammatory responses to UVR seen in XPA-knockout mice (Miyauchi-Hashimoto et al., 1996Miyauchi-Hashimoto H. Tanaka K. Horio T. Enhanced inflammation and immunosuppression by ultraviolet radiation in xeroderma pigmentosum group A (XPA) model mice.J Invest Dermatol. 1996; 107: 343-348Abstract Full Text PDF PubMed Scopus (85) Google Scholar; Okamoto et al., 1999Okamoto H. Mizuno K. Itoh T. et al.Evaluation of apoptotic cells induced by ultraviolet light B radiation in epidermal sheets stained by the TUNEL technique.J Invest Dermatol. 1999; 113: 802-807Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar; Brash et al., 2001Brash D.E. Wikonkal N.M. Remenyik E. et al.The DNA damage signal for Mdm2 regulation, Trp53 induction, and sunburn cell formation in vivo originates from actively transcribed genes.J Invest Dermatol. 2001; 117: 1234-1240Abstract Full Text Full Text PDF PubMed Google Scholar; Katiyar et al., 2011Katiyar S.K. Mantena S.K. Meeran S.M. Silymarin protects epidermal keratinocytes from ultraviolet radiation-induced apoptosis and DNA damage by nucleotide excision repair mechanism.PLoS One. 2011; 6: e21410Crossref PubMed Scopus (84) Google Scholar). In fact, apoptosis and sunburn may be considered to be readouts of the level of DNA damage that is produced by UVR. Sunburn-associated apoptosis is mediated by the tumor suppressor p53 (Ziegler et al., 1994Ziegler A. Jonason A.S. Leffell D.J. et al.Sunburn and p53 in the onset of skin cancer.Nature. 1994; 372: 773-776Crossref PubMed Scopus (1348) Google Scholar) and is considered to be a protective mechanism by which potentially neoplastic cells are removed from the skin. Although sunburn apoptosis and skin cancer are both produced by DNA damage and involve p53, there are important distinctions that can be made regarding how p53 affects these processes. Apoptosis and sunburn are acute responses mediated by p53 regulation of gene expression, whereas carcinogenesis results from chronic exposures during which p53 and its protective proapoptotic functions are commonly lost by mutational inactivation. Because the levels of repair and replication are under the control of the circadian clock in the skin and likely contribute to many biological responses to UVR, we examined how the circadian clock influences the erythemal response to UVR in mouse skin. We found elevated erythemal responses following UV exposures in the early morning in comparison with exposures in the late afternoon. This enhanced response appears to be not only owing to circadian control of repair and replication but also to circadian clock regulation of p53. Evidence that skin carcinogenesis is influenced by the timing of UVR exposure during the circadian cycle (Gaddameedhi et al., 2011Gaddameedhi S. Selby C.P. Kaufmann W.K. et al.Control of skin cancer by the circadian rhythm.Proc Natl Acad Sci USA. 2011; 108: 18790-18795Crossref PubMed Scopus (164) Google Scholar), combined with the well-known association between sunburn and skin cancer (Noonan et al., 2001Noonan F.P. Recio J.A. Takayama H. et al.Neonatal sunburn and melanoma in mice.Nature. 2001; 413: 271-272Crossref PubMed Scopus (311) Google Scholar; Whiteman et al., 2001Whiteman D.C. Whiteman C.A. Green A.C. Childhood sun exposure as a risk factor for melanoma: a systematic review of epidemiologic studies.Cancer Causes Control. 2001; 12: 69-82Crossref PubMed Scopus (506) Google Scholar; Geller et al., 2012Geller A.C. Balk S.J. Fisher D.E. Stemming the tanning bed epidemic: time for action.J Natl Compr Canc Netw. 2012; 10: 1311-1314Crossref PubMed Scopus (6) Google Scholar), indicated that the development of sunburn may also be controlled by the circadian clock. To investigate this hypothesis, SKH-1 hairless mice were immobilized in a restraining tube containing four holes (0.5-cm diameter) in the top, and each mouse was irradiated with four different doses of UVR on the dorsal aspect. This arrangement resulted in the production of discreet circular areas of erythema, depending on the dose. An “AM group” of mice (n=6) was irradiated at ZT21 (zeitgeber time 21), and a “PM group” (n=6) was irradiated at ZT09. In conventional terms, with lights on at 7 AM, ZT21 corresponds to 4 AM and ZT9 corresponds to 4 PM. Each mouse was scored for erythema daily for 6 days following UVR. The results are shown graphically in Figure 1a. The extent of the erythemal response was greater in the AM group than in the PM group, particularly following doses of 400 and 500 J m−2 of UVR. The experiment was repeated with a second set of mice, and the same trend was observed (Supplementary Figure 1 online). Thus, the sensitivity of mice toward sunburn is controlled by the time of day of UV exposure, and this sensitivity as a function of circadian time parallels the sensitivity of mice toward the development of UVR-induced skin cancer (Gaddameedhi et al., 2011Gaddameedhi S. Selby C.P. Kaufmann W.K. et al.Control of skin cancer by the circadian rhythm.Proc Natl Acad Sci USA. 2011; 108: 18790-18795Crossref PubMed Scopus (164) Google Scholar), such that the greatest responses occur following early AM exposures. Download .pdf (.5 MB) Help with pdf files Supplementary Material The erythemal response proceeds through the production of proinflammatory cytokines by neutrophils and macrophages in the skin as a result of UV damage (Faustin and Reed, 2008Faustin B. Reed J.C. Sunburned skin activates inflammasomes.Trends Cell Biol. 2008; 18: 4-8Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). We used a mouse cytokine antibody array to detect cytokines in extracts of either unirradiated whole mouse skin or skin collected 12 hours following irradiation with 500 J m−2 of UVR, which elicits erythema. This array contains 40 different antibodies, and protein/cytokine expression patterns can be determined in a single assay at the protein level. Many of the proteins detected with this array (Supplementary Figure 2A and B online) were clearly induced by UV and tended to be more abundant following morning exposure compared with evening exposure. Seven proteins were induced to a significantly (P<0.05) greater extent following a morning exposure compared with an evening exposure (Figure 1b). Among these, IFN-γ, TNF-α, IL-12p70, and MIP-1α are known to be proinflammatory cytokines, whereas IP-10 and KC are chemokines that have essential roles in the initiation and/or promotion of inflammation. Most interestingly, both TNF-α and IFN-γ are known to be involved in UVR-induced erythema and melanoma promotion in mice (Murakawa et al., 2006Murakawa M. Yamaoka K. Tanaka Y. et al.Involvement of tumor necrosis factor (TNF)-alpha in phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin edema in mice.Biochem Pharmacol. 2006; 71: 1331-1336Crossref PubMed Scopus (135) Google Scholar; Zaidi et al., 2011Zaidi M.R. Davis S. Noonan F.P. et al.Interferon-gamma links ultraviolet radiation to melanomagenesis in mice.Nature. 2011; 469: 548-553Crossref PubMed Scopus (213) Google Scholar). In addition, it was recently reported that TNF-α levels are influenced by the circadian rhythm in mouse macrophages (Keller et al., 2009Keller M. Mazuch J. Abraham U. et al.A circadian clock in macrophages controls inflammatory immune responses.Proc Natl Acad Sci USA. 2009; 106: 21407-21412Crossref PubMed Scopus (556) Google Scholar). TREM-1, which is also more highly induced by UV in the morning, is a receptor that is found on immune cells. TREM-1 is involved in antigen detection, secretion of inflammatory mediators, and increased acute inflammatory response, and it is upregulated during the inflammatory response (Bouchon et al., 2001Bouchon A. Facchetti F. Weigand M.A. et al.TREM-1 amplifies inflammation and is a crucial mediator of septic shock.Nature. 2001; 410: 1103-1107Crossref PubMed Scopus (841) Google Scholar). Several additional cytokines appeared to be more highly induced in the morning (Supplementary Figure 2C online), but were of borderline statistical significance. These included IL-2, IL-4, IL-7, IL-13, and MIG. Overall, we find that the inflammatory cytokine response parallels the erythemal response and supports the physiological data that show a role of the time of day of UV exposure in the erythemal response. At the cellular level, erythemogenic doses of UVR are associated with apoptosis (also called “sunburn apoptosis”) (Ziegler et al., 1994Ziegler A. Jonason A.S. Leffell D.J. et al.Sunburn and p53 in the onset of skin cancer.Nature. 1994; 372: 773-776Crossref PubMed Scopus (1348) Google Scholar). To determine the effect of the circadian clock on sunburn apoptosis, we irradiated C57BL/6 mice with UVR either in the early morning or the late afternoon and then collected mouse skin at 0, 6, and 12 hours after UVR. Apoptosis was measured using the fluorometric TUNEL assay in which fragmented DNA from apoptotic cells is end-labeled with fluorophore. Figure 2 shows a greater apoptotic response in the AM group compared with the PM group. To determine whether sunburn apoptosis is controlled by the circadian clock, we used mice in which the clock was disrupted by mutating essential clock genes. In both Cry1, Cry2 double knockout mice (Cry1/2-/- in Figure 2a and b), and Per1, Per2 double knockout mice (Per1/2-/- in Figure 2c and d), the effect of the time of the day on sunburn apoptosis was abolished. The elevated apoptosis in wild-type mice irradiated in the AM was found to be correlated with reduced repair of UV-induced DNA photoproducts (Gaddameedhi et al., 2011Gaddameedhi S. Selby C.P. Kaufmann W.K. et al.Control of skin cancer by the circadian rhythm.Proc Natl Acad Sci USA. 2011; 108: 18790-18795Crossref PubMed Scopus (164) Google Scholar). Although it has been reported that p53 contributes to excision repair and UV survival in human cells (Ford and Hanawalt, 1997Ford J.M. Hanawalt P.C. Expression of wild-type p53 is required for efficient global genomic nucleotide excision repair in UV-irradiated human fibroblasts.J Biol Chem. 1997; 272: 28073-28080Crossref PubMed Scopus (334) Google Scholar; Ferguson and Oh, 2005Ferguson B.E. Oh D.H. Proficient global nucleotide excision repair in human keratinocytes but not in fibroblasts deficient in p53.Cancer Res. 2005; 65: 8723-8729Crossref PubMed Scopus (26) Google Scholar), mouse p53 protein does not seem to have a role in excision repair or UV survival (Ishizaki et al., 1994Ishizaki K. Ejima Y. Matsunaga T. et al.Increased UV-induced SCEs but normal repair of DNA damage in p53-deficient mouse cells.Int J Cancer. 1994; 58: 254-257Crossref PubMed Scopus (52) Google Scholar), which could be owing to the fact that the mouse XPE gene, which encodes the DDB2 protein, does not have a p53 recognition domain (Tan and Chu, 2002Tan T. Chu G. p53 Binds and activates the xeroderma pigmentosum DDB2 gene in humans but not mice.Mol Cell Biol. 2002; 22: 3247-3254Crossref PubMed Scopus (141) Google Scholar). Therefore, circadian oscillation of p53 in mouse skin may not have a significant role in regulating excision repair capacity. In addition to the C57BL/6 mouse strain, we also observed a circadian effect of sunburn apoptosis in SKH-1 hairless mice by classical hematoxylin and eosin staining (Supplementary Figure 3 online). Thus, enhanced apoptosis parallels the enhanced sunburn and reduced repair that is observed following exposure in the AM. Both sunburn and the apoptotic response are known to be controlled by the tumor suppressor p53 (Ziegler et al., 1994Ziegler A. Jonason A.S. Leffell D.J. et al.Sunburn and p53 in the onset of skin cancer.Nature. 1994; 372: 773-776Crossref PubMed Scopus (1348) Google Scholar), and the absolute protein level, phosphorylation status, and activity of p53 are known to be induced by DNA damage. We therefore first investigated p53 protein levels following UVR as a function of circadian time. Skin was collected from SKH-1 mice at 0, 1, and 2 hours following irradiation and analyzed for p53 content by immunoblotting. The results (Figure 3a and b) show that p53 accumulated to a greater extent in mice irradiated in the AM in comparison with mice irradiated in the PM. The cell cycle regulatory protein P21, which is directly controlled at the level of transcription by p53, also showed a stronger induction following exposure in the AM than following exposure in the PM (Figure 3a and c). Together, these results demonstrate that both p53 protein levels and activity following UVR are controlled by the circadian clock. The p53 protein is regulated by Mdm2, which ubiquitinates p53 to promote its degradation by the proteosome. We noticed that higher Mdm2 protein levels in the PM samples (Figure 3a and d) were correlated with a lower induction of p53 protein by UV (Figure 3a and b), which indicated that circadian control of Mdm2 protein may contribute to the p53 response. In an effort to better understand the circadian regulation of p53, we examined the expression of p53 and Mdm2 in unirradiated mouse skin as a function of circadian time. We found that both proteins were rhythmically expressed in both C57BL/6 and SKH-1 mice (Figure 3e and f and Supplementary Figure 4 online). In contrast, p53 and Mdm2 mRNA levels appeared to be nonrhythmic (Supplementary Figure 5 online). Furthermore, the mRNA levels of p53 were not induced by UVR (Supplementary Figure 5C online). The results in Figure 3e and f show that p53 and Mdm2 protein oscillations are in phase with one another. When the levels of both proteins are low in the early AM, interactions between the two would be expected to be minimal, which could result in less ubiquitination and degradation of p53 and hence higher levels of p53 and a more robust p53-dependent response, including apoptosis. The diminished capacity of Mdm2 to target p53 for degradation in the AM is therefore seen as a likely contributor to the extreme elevation in p53 levels following UVR in the AM (Figure 3a and b). UV induction in the AM propels the level of p53 from its nadir of circadian expression to greatly exceed its peak circadian expression level, which is near the detection limit at the zero irradiation time point in Figure 3a. Although additional factors, including p19ARF and HAUSP, have been reported to affect Mdm2 and p53 function (Zhang et al., 1998Zhang Y. Xiong Y. Yarbrough W.G. ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways.Cell. 1998; 92: 725-734Abstract Full Text Full Text PDF PubMed Scopus (1397) Google Scholar; Li et al., 2004Li M. Brooks C.L. Kon N. et al.A dynamic role of HAUSP in the p53-Mdm2 pathway.Mol Cell. 2004; 13: 879-886Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar; Khoronenkova et al., 2012Khoronenkova S.V. Dianova I.I. Ternette N. et al.ATM-dependent downregulation of USP7/HAUSP by PPM1G activates p53 response to DNA damage.Mol Cell. 2012; 45: 801-813Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), neither of these proteins displayed a circadian pattern of expression (data not shown). We therefore conclude that low levels of Mdm2 in the morning and high levels in the afternoon may contribute to the different effects of UVR on p53 protein induction and activity at these time points.Figure 5Proposed model for the role of the circadian clock in sunburn inflammation. Low levels of repair and high levels of DNA replication in the AM lead to unrepaired UV photoproducts in DNA that cause replication stress and to enhanced DNA damage checkpoint signaling compared with responses in the PM. The enhanced Atr-mediated checkpoint signaling in the AM coupled with reduced levels of Mdm2 lead to greater phosphorylation, stabilization, and activity of p53, which leads to more apoptosis and sunburn following UV exposure in the AM. Atr, ataxia telangiectasia mutated and Rad3-related.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The lower levels of UV photoproduct repair and the higher levels of DNA synthesis that are observed in the AM (Gaddameedhi et al., 2011Gaddameedhi S. Selby C.P. Kaufman}, journal={Journal of Investigative Dermatology}, year={2015} } @article{dna repair synthesis and ligation affect the processing of excised oligonucleotides generated by human nucleotide excision repair._2014, url={https://europepmc.org/articles/PMC4176238}, DOI={10.1074/jbc.m114.597088}, abstractNote={Ultraviolet (UV) photoproducts are removed from genomic DNA by dual incisions in humans in the form of 24- to 32-nucleotide-long oligomers (canonical 30-mers) by the nucleotide excision repair system. How the small, excised, damage-containing DNA oligonucleotides (sedDNAs) are processed in cells following the dual incision event is not known. Here, we demonstrate that sedDNAs are localized to the nucleus in two biochemically distinct forms, which include chromatin-associated, transcription factor II H-bound complexes and more readily solubilized, RPA-bound complexes. Because the nuclear mobility and repair functions of transcription factor II H and RPA are influenced by post-incision gap-filling events, we examined how DNA repair synthesis and DNA ligation affect sedDNA processing. We found that although these gap filling activities are not essential for the dual incision/sedDNA generation event per se, the inhibition of DNA repair synthesis and ligation is associated with a decrease in UV photoproduct removal rate and an accumulation of RPA-sedDNA complexes in the cell. These findings indicate that sedDNA processing and association with repair proteins following the dual incisions may be tightly coordinated with gap filling during nucleotide excision repair in vivo.}, journal={The Journal of biological chemistry}, year={2014}, month={Aug} } @article{dual modes of clock:bmal1 inhibition mediated by cryptochrome and period proteins in the mammalian circadian clock._2014, url={https://europepmc.org/articles/PMC4173159}, DOI={10.1101/gad.249417.114}, abstractNote={The mammalian circadian clock is based on a transcription–translation feedback loop (TTFL) in which CLOCK and BMAL1 proteins act as transcriptional activators of Cryptochrome and Period genes, which encode proteins that repress CLOCK–BMAL1 with a periodicity of ∼24 h. In this model, the mechanistic roles of CRY and PER are unclear. Here, we used a controlled targeting system to introduce CRY1 or PER2 into the nuclei of mouse cells with defined circadian genotypes to characterize the functions of CRY and PER. Our data show that CRY is the primary repressor in the TTFL: It binds to CLOCK–BMAL1 at the promoter and inhibits CLOCK–BMAL1-dependent transcription without dissociating the complex (“blocking”-type repression). PER alone has no effect on CLOCK–BMAL1-activated transcription. However, in the presence of CRY, nuclear entry of PER inhibits transcription by displacing CLOCK–BMAL1 from the promoter (“displacement”-type repression). In light of these findings, we propose a new model for the mammalian circadian clock in which the negative arm of the TTFL proceeds by two different mechanisms during the circadian cycle.}, journal={Genes & development}, year={2014}, month={Sep} } @article{highly specific and sensitive method for measuring nucleotide excision repair kinetics of ultraviolet photoproducts in human cells_2014, url={https://publons.com/wos-op/publon/9035191/}, DOI={10.1093/NAR/GKT1179}, abstractNote={Abstract The nucleotide excision repair pathway removes ultraviolet (UV) photoproducts from the human genome in the form of short oligonucleotides ∼30 nt in length. Because there are limitations to many of the currently available methods for investigating UV photoproduct repair in vivo, we developed a convenient non-radioisotopic method to directly detect DNA excision repair events in human cells. The approach involves extraction of oligonucleotides from UV-irradiated cells, DNA end-labeling with biotin and streptavidin-mediated chemiluminescent detection of the excised UV photoproduct-containing oligonucleotides that are released from the genome during excision repair. Our novel approach is robust, with essentially no signal in the absence of UV or a functional excision repair system. Furthermore, our non-radioisotopic methodology allows for the sensitive detection of excision products within minutes following UV irradiation and does not require additional enrichment steps such as immunoprecipitation. Finally, this technique allows for quantitative measurements of excision repair in human cells. We suggest that the new techniques presented here will be a useful and powerful approach for studying the mechanism of human nucleotide excision repair in vivo.}, journal={Nucleic Acids Research}, year={2014} } @article{dna damage-specific control of cell death by cryptochrome in p53-mutant ras-transformed cells_2013, url={https://publons.com/wos-op/publon/9037698/}, DOI={10.1158/0008-5472.CAN-12-1994}, abstractNote={Abstract The main feedback loop driving circadian rhythm in mice is controlled, in part, by the genes encoding the cryptochromes Cry1 and Cry2. Targeted mutation of both Cry1 and Cry2 delay the early onset of tumor formation in p53-null mutant mice. Furthermore, Ras-transformed p53- and Cry-null mouse skin fibroblasts are more sensitive than p53 mutants to apoptotic cell death initiated by agents that activate either the intrinsic or the extrinsic apoptosis pathways. Here, we investigated the effect of Cry1 and Cry2 mutations on cell death by other genotoxic agents that generate alkylated bases, interstrand crosslinks, DNA–protein crosslinks, and double-strand breaks. Both ultraviolet (UV) and the UV mimetic compound oxaliplatin and the radiomimetic compound doxorubicin promoted apoptosis by upregulating the tumor suppressor p73. However, only the UV and oxaliplatin-induced upregulation of p73 mediated by the transcription factor Egr1, but not the doxorubicin-induced upregulation mediated by the transcription factor E2F1, was enhanced by Cry1/Cry2 double mutation. Accordingly, Egr1 downregulation reduced oxaliplatin-induced apoptosis, whereas E2F1 downregulation reduced doxorubicin-induced apoptosis. Our findings establish distinct roles for cryptochromes in intrinsic apoptosis induced by UV mimetic and radiomimetic agents. Cancer Res; 73(2); 785–91. ©2012 AACR.}, journal={Cancer Research}, year={2013} } @article{nucleotide excision repair in human cells: fate of the excised oligonucleotide carrying dna damage in vivo._2013, url={https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23749995/?tool=EBI}, DOI={10.1074/jbc.m113.482257}, abstractNote={Nucleotide excision repair is the sole mechanism for removing the major UV photoproducts from genomic DNA in human cells. In vitro with human cell-free extract or purified excision repair factors, the damage is removed from naked DNA or nucleosomes in the form of 24- to 32-nucleotide-long oligomers (nominal 30-mer) by dual incisions. Whether the DNA damage is removed from chromatin in vivo in a similar manner and what the fate of the excised oligomer was has not been known previously. Here, we demonstrate that dual incisions occur in vivo identical to the in vitro reaction. Further, we show that transcription-coupled repair, which operates in the absence of the XPC protein, also generates the nominal 30-mer in UV-irradiated XP-C mutant cells. Finally, we report that the excised 30-mer is released from the chromatin in complex with the repair factors TFIIH and XPG. Taken together, our results show the congruence of in vivo and in vitro data on nucleotide excision repair in humans.}, journal={The Journal of biological chemistry}, year={2013}, month={Jun} } @article{effect of circadian clock mutations on dna damage response in mammalian cells._2012, url={https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22918252/?tool=EBI}, DOI={10.4161/cc.21771}, abstractNote={The circadian clock is a global regulatory mechanism that confers daily rhythmicity on many biochemical and physiological functions, including DNA excision repair in mammalian organisms. Here, we investigated the effect of the circadian clock on the major DNA damage response pathways by using mouse cell lines mutated in genes encoding proteins in the positive (Bmal1, CLOCK) or negative (Cry 1/2, Per 1/2) arms of the transcription-translation feedback loop that generates the circadian clock. We find that cells mutated in these genes are indistinguishable from wild-type in their response to UV, ionizing radiation and mitomycin C. We conclude that either the majority of DNA damage response reactions are not controlled by the circadian clock or that, even if such a control exists at the organism level, it is supplanted by homeostatic control mechanisms at the cellular level in tissue culture. We suggest that caution must be exercised in extrapolating from experiments in tissue culture to whole animals with respect to the effect of the circadian clock on cellular response to DNA damaging agents.}, journal={Cell cycle (Georgetown, Tex.)}, year={2012}, month={Aug} } @article{gaddameedhi_selby_kaufmann_smart_sancar_2011, title={Control of skin cancer by the circadian rhythm}, volume={108}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.1115249108}, DOI={10.1073/pnas.1115249108}, abstractNote={Skin cancer is the most common form of cancer in the United States. The main cause of this cancer is DNA damage induced by the UV component of sunlight. In humans and mice, UV damage is removed by the nucleotide excision repair system. Here, we report that a rate-limiting subunit of excision repair, the xeroderma pigmentosum group A (XPA) protein, and the excision repair rate exhibit daily rhythmicity in mouse skin, with a minimum in the morning and a maximum in the afternoon/evening. In parallel with the rhythmicity of repair rate, we find that mice exposed to UV radiation (UVR) at 4:00 AM display a decreased latency and about a fivefold increased multiplicity of skin cancer (invasive squamous cell carcinoma) than mice exposed to UVR at 4:00 PM. We conclude that time of day of exposure to UVR is a contributing factor to its carcinogenicity in mice, and possibly in humans.}, number={46}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Gaddameedhi, S. and Selby, C. P. and Kaufmann, W. K. and Smart, R. C. and Sancar, A.}, year={2011}, month={Oct}, pages={18790–18795} } @article{melanoma and dna damage from a distance (farstander effect)._2011, url={https://doi.org/10.1111/j.1755-148X.2010.00805.x}, DOI={10.1111/j.1755-148x.2010.00805.x}, abstractNote={It has been known for a long time that cancer, even in the absence of metastasis, has adverse systemic effects on the organism. The mechanism of these global effects is complex. A clue to the possible mechanism comes from what is known about the bystander effect (BE). Although BE has been used to describe the reaction in tissue adjacent to the irradiated area more generally the term is applied to the effect of cancer cells on the adjacent normal tissue. It is generally believed that cytokines such as TGF-β and reactive oxygen species (ROS) released from cancer cells mediate the BE. The paper by Redon et al. (2010) shows that the same or similar mechanisms that are responsible for BE may cause systemic effects as well (farstander effect, FE). These findings provide significant insight into the systemic effects of cancer as well as the potential link between chronic inflammation and cancer. The basic experimental design of Redon et al. (2010) was to subcutaneously implant B16 melanoma and M5076 reticulum sarcoma into syngeneic C57BL/6 mice and similarly COLON26 carcinoma into syngeneic BALB/C mice. After the tumors reached about 200 mg, the animals were sacrificed to look for systemic genotoxic stress, as determined by DNA damage checkpoint response (Sancar et al., 2004) and oxidative base damage. For DNA damage checkpoint response, the authors measured H2AX phosphorylation, which is a classical sign of the checkpoint response to DNA double-strand breaks (DSBs). As a readout for oxidative damage they measured thymine glycols, 8-oxoguanine, and apurinic/apyrimidinic (AP sites) in DNA, all known to be induced by ROS. The authors found that all three cancers induced systemic effects on distant organs, but only in those organs in which there is active cell proliferation, such as the gastrointestinal tract (GIT) and the skin. These results suggest that S-phase cells of the skin and GIT are more sensitive to DSB formation through FE, as seen previously for BE in an IR-induced cell culture system (Burdak-Rothkamm et al., 2007; Burdak-Rothkamm et al., 2008). Importantly, Redon et al. also show that at the hair bulb (where stem cells are located) of the hair follicles in the skin, as well as at base of the duodenum (where proliferating cells are located) of the tumor implanted mice have up to threefold more γH2AX foci than at the other parts of the same tissue. Even though the authors did not look for stem cell markers at the base of the hair bulb or at the duodenum, it is likely that the elevated γH2AX foci are from the stem cells which may be more sensitive to tumor-induced near and far signals. Figure 1 shows the schematic representation of the tumor-induced spontaneous systemic FE response in vivo. Model for the tumor-induced distant DNA damage and checkpoint response in vivo. Activated cytokines such as chemokine (CCL2) from the tumor microenvironment induce DNA damage such as double-strand breaks (DSBs) and oxidative clustered DNA lesions (OCDLs) in proliferating cells of the skin, hair follicles, and gastrointestinal tract (GIT) tissues either through DNA replication stress or through ATM/ATR signaling. In less/non-proliferating cells, there is only oxidative damage. Tissues like liver and kidney do not exhibit any DNA damage or checkpoint response, possibly because cells in these organs are non-proliferating and contain high levels of antioxidants or fewer inflammatory response mediators. Previous studies have indicated that the macrophage-mediated inflammatory response of the radiation-induced damaged cells may be responsible for bystander signal-mediated DNA damage in neighboring cells (Prise and O’Sullivan, 2009). Taking a clue from these studies, Redon et al. examined the serum levels of 59 cytokines, both from the tumor implanted mice and from control mice. Of these, four cytokines (CCL2, CCL4, CCL7, and CXCL10) were significantly elevated in the serum of tumor-bearing mice compared with the controls. This finding suggested that these cytokines may be the potential signal transducers for the DNA damage response observed in distant organs of the tumor-bearing mice. Since CCL-2 (also known as MCP-1, monocyte chemoattractant protein-1) is associated with various kinds of inflammation and cancer progression (Conti and Rollins, 2004), the authors focused on this cytokine to further investigate its role in systemic FE. Importantly, when the authors implanted the same tumors into CCL-2 deficient mice, the systemic effects of the tumors were eliminated as evidenced by lack of either DSBs or oxidative clustered DNA lesions in GIT. Even though skin was not analyzed in CCL-2 deficient mice, we assume that the same might be true for skin as well. Overall, these results indicate that although tumor cells are capable of inducing DNA damage in distant places, it appears that cytokines released by the immune cells activated by the tumor are responsible for the long-distance DNA damage and checkpoint activation. Apart from its value in shedding light on the mechanism of systemic effects of cancer in general and melanoma in particular, this study opens a new area of inquiry into how the inflammatory response initiated DNA damage response may lead to systemic pathological changes at the organism level. Skin is the major organ of the body exposed to all kinds of environmental insults, in particular to ultraviolet radiation from sun light on a daily basis. The connections among the skin, DNA damage, and melanoma are important subjects from a biomedical standpoint, as well as providing a model to understand the role of FE of melanoma on systemic response in distance organs. Further studies are needed to evaluate the generality of the findings reported in this paper. First, in the study by Redon et al., the tumors were implanted into healthy recipient mice. While this is the commonly used approach in cancer research, the findings cannot be generalized because it is conceivable that the tumor implantation process activated the host-immune system, resulting in a general inflammatory response. In addition, the authors used fairly large tumors to detect the systemic DNA damage and the checkpoint response. Assuming a stochastic process, it is expected that the FE would be present at all phases of the tumor progression. Nevertheless, this must be demonstrated experimentally for the findings in this paper to become relevant to understand the systemic effect of cancer and for cancer prevention and management.}, journal={Pigment cell & melanoma research}, year={2011}, month={Feb} } @article{similar nucleotide excision repair capacity in melanocytes and melanoma cells._2010, url={https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20501836/?tool=EBI}, DOI={10.1158/0008-5472.can-10-0095}, abstractNote={Sunlight UV exposure produces DNA photoproducts in skin that are repaired solely by nucleotide excision repair in humans. A significant fraction of melanomas are thought to result from UV-induced DNA damage that escapes repair; however, little evidence is available about the functional capacity of normal human melanocytes, malignant melanoma cells, and metastatic melanoma cells to repair UV-induced photoproducts in DNA. In this study, we measured nucleotide excision repair in both normal melanocytes and a panel of melanoma cell lines. Our results show that in 11 of 12 melanoma cell lines tested, UV photoproduct repair occurred as efficiently as in primary melanocytes. Importantly, repair capacity was not affected by mutation in the N-RAS or B-RAF oncogenes, nor was a difference observed between a highly metastatic melanoma cell line (A375SM) or its parental line (A375P). Lastly, we found that although p53 status contributed to photoproduct removal efficiency, its role did not seem to be mediated by enhanced expression or activity of DNA binding protein DDB2. We concluded that melanoma cells retain capacity for nucleotide excision repair, the loss of which probably does not commonly contribute to melanoma progression.}, journal={Cancer research}, year={2010}, month={May} } @article{association between the unfolded protein response, induced by 2-deoxyglucose, and hypersensitivity to cisplatin: a mechanistic study employing molecular genomics._2009, url={https://doi.org/10.4103/0973-1482.55146}, DOI={10.4103/0973-1482.55146}, abstractNote={Background: The specific signaling that occurs between the endoplasmic reticulum (ER) and the nucleus in response to ER stress is known as the unfolded protein response (UPR). Specific induction of GRP78 (glucose-regulated protein of Mr 78 kDa) is an integral component of ER stress and the UPR. We first discovered that the up-regulation of GRP78 is associated with augmented sensitivity/apoptosis of cancer cells to clinically used alkylating/platinating agents. Objectives: To decipher molecular mechanisms that associate induction of the UPR/GRP78 with augmented sensitivity/apoptosis to cisplatin. Materials and Methods: A549 cells were exposed to 2-deoxyglucose (2dG) to induce the UPR/GRP78, followed by cisplatin treatment. We used human cDNA microarray containing 42,000 ESTs as well as pathway-specific macroarrays for apoptosis, cell cycle, and MAP kinase signaling pathways containing 100-280 genes and subsequently examined the pertinent transcript levels. The results obtained from these studies were confirmed by examining relevant protein levels and the enzymatic activity. Results: We demonstrate that the induction of UPR/GRP78 alone causes a decrease in the transcript levels of DNA repair genes and DNA damage check point genes, and an increase in the transcript levels of apoptotic genes. Furthermore, we show that cisplatin treatment after the induction of UPR/GRP78 is facilitating the mitochondria-mediated apoptotic cascades through the initial activation of caspase-2 and down-regulation of genes involved in DNA repair. Conclusions: Our study will shed new insight as to the increased understanding of the mechanisms of the UPR/GRP78 modulation of molecular and cellular responses to cisplatin that will allow strategies for transferring bench side results to the bed.}, journal={Journal of cancer research and therapeutics}, year={2009}, month={Sep} } @article{loss of cryptochrome reduces cancer risk in p53 mutant mice._2009, url={https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/19188586/?tool=EBI}, DOI={10.1073/pnas.0813028106}, abstractNote={It is commonly thought that disruption of the circadian clock increases the cancer incidence in humans and mice. However, it was found that disruption of the clock by the Cryptochrome (Cry) mutation in mice did not increase cancer rate in the mutant mice even after exposing the animals to ionizing radiation. Therefore, in this study we tested the effect of the Cry mutation on carcinogenesis in a mouse strain prone to cancer because of a p53 mutation, with the expectation that clock disruption in this sensitized background would further increase cancer risk. Paradoxically, we find that the Cry mutation protects p53 mutant mice from the early onset of cancer and extends their median lifespan approximately 50%, in part by sensitizing p53 mutant cells to apoptosis in response to genotoxic stress. These results suggest alternative therapeutic approaches in management of cancers associated with a p53 mutation.}, journal={Proceedings of the National Academy of Sciences of the United States of America}, year={2009}, month={Feb} } @article{deciphering the association between up-regulation of glucose regulated protein of mr-78 kda (grp78) and hypersensitivity to cisplatin employing comparative genomics_2008, url={https://publons.com/wos-op/publon/54906423/}, journal={The FASEB Journal}, year={2008} } @article{up-regulation of glucose regulated protein of m-r-78kda (grp78) followed by cisplatin treatment leads to mitochondrial mediated apoptosis through caspase-2 activation_2006, url={https://publons.com/wos-op/publon/54906422/}, journal={Cancer Research}, year={2006} } @article{in-gel precipitation of enzymatically released phosphate._2004, url={https://doi.org/10.1016/j.ab.2004.07.010}, DOI={10.1016/j.ab.2004.07.010}, abstractNote={The phosphate precipitation reaction using ammonium molybdate and triethylamine under low pH has been applied to gel-based assays for detecting phosphate-releasing enzymes. The sensitivity of the assay is 10 pmol Pi/mm2 of 1.5-mm-thick gel. The assay is applicable to enzymes with a wide range of optimal pH, from acid (pH 4.5) to alkaline phosphatase (pH 9.7), and to enzymes that use acid-labile substrates such as apyrase and glutamine synthetase. Using a negative staining approach, maltose phosphorylase, a phosphate-consuming enzyme, can also be detected. The assay was used to detect glutamine synthetase isoforms, separated by nondenaturing polyacrylamide gel electrophoresis from crude maize extracts. For downstream applications such as staining gels for proteins, the gels with precipitate should be incubated in 10 mM dithiothreitol or beta-mercaptoethanol until the precipitate is dissolved and then thoroughly washed in water. In comparison to calcium phosphate precipitation or the phosphomolybdate-malachite green method, this method is more sensitive. It is a very simple, rapid, versatile, reproducible, and inexpensive method that could be a useful tool in enzymological studies.}, journal={Analytical biochemistry}, year={2004}, month={Nov} }