Michael E. Geusz

Circadian rhythms in mouse suprachiasmatic nucleus explants on multimicroelectrode plates

The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus functions as a circadian pacemaker. This study used multimicroelectrode plates to measure extracellular action potential activity simultaneously from multiple sites within the cultured mouse SCN. Neurons within the isolated mouse SCN expressed a circadian rhythm in spontaneous firing rate for weeks in culture.

Long-term monitoring of circadian rhythms in c-fos gene expression from suprachiasmatic nucleus cultures

BACKGROUND The AP-1 family of transcription factors has been implicated in the control of the expression of many genes in response to environmental signals. Previous studies have provided temporal profiles for c-fos expression by taking measurements from many animals at several points in time, but these studies provide limited information about dynamic changes in expression. Here, we have devised a method of continuously measuring c-fos expression. RESULTS A transgenic mouse line expressing the human c-fos promoter linked to the firefly luciferase reporter gene (fos/luc) was generated to continuously monitor c-fos gene expression. A second transgenic mouse line expressing luciferase under the control of the cytomegalovirus promoter (CMV/luc) served as a control. Luminescence originating from identifiable brain regions was imaged from fos/luc brain slice cultures. Expression of the fos/luc transgene accurately reflected transcriptional responses of the endogenous c-fos gene. Dynamic changes in fos/luc expression in suprachiasmatic nuclei (SCN) explant cultures were monitored continuously, and luminescence showed almost 24 hour rhythms lasting up to five circadian cycles. In contrast, bioluminescence monitored from CMV/luc SCN explant cultures was not rhythmic. CONCLUSION The fos/luc transgenic mouse will be useful for long-term, non-invasive monitoring of c-fos transcriptional responses to the changing cellular environment. Circadian rhythms in c-fos expression can be monitored non-invasively in real time from the SCN, clearly demonstrating that c-fos transcription is regulated by the circadian clock.

Effects of curcumin on stem-like cells in human esophageal squamous carcinoma cell lines

BackgroundMany cancers contain cell subpopulations that display characteristics of stem cells. Because these cancer stem cells (CSCs) appear to provide resistance to chemo-radiation therapy, development of therapeutic agents that target CSCs is essential. Curcumin is a phytochemical agent that is currently used in clinical trials to test its effectiveness against cancer. However, the effect of curcumin on CSCs is not well established. The current study evaluated curcumin-induced cell death in six cancer cell lines derived from human esophageal squamous cell carcinomas. Moreover, these cell lines and the ones established from cells that survived curcumin treatments were characterized.MethodsCell loss was assayed after TE-1, TE-8, KY-5, KY-10, YES-1, and YES-2 cells were exposed to 20–80 μM curcumin for 30 hrs. Cell lines surviving 40 or 60 μM curcumin were established from these six original lines. The stem cell markers aldehyde dehydrogenase-1A1 (ALDH1A1) and CD44 as well as NF-κB were used to compare CSC-like subpopulations within and among the original lines as well as the curcumin-surviving lines. YES-2 was tested for tumorsphere-forming capabilities. Finally, the surviving lines were treated with 40 and 60 μM curcumin to determine whether their sensitivity was different from the original lines.ResultsThe cell loss after curcumin treatment increased in a dose-dependent manner in all cell lines. The percentage of cells remaining after 60 μM curcumin treatment varied from 10.9% to 36.3% across the six lines. The cell lines were heterogeneous with respect to ALDH1A1, NF-κB and CD44 expression. KY-5 and YES-1 were the least sensitive and had the highest number of stem-like cells whereas TE-1 had the lowest. The curcumin-surviving lines showed a significant loss in the high staining ALDH1A1 and CD44 cell populations. Tumorspheres formed from YES-2 but were small and rare in the YES-2 surviving line. The curcumin-surviving lines showed a small but significant decrease in sensitivity to curcumin when compared with the original lines.ConclusionOur results suggest that curcumin not only eliminates cancer cells but also targets CSCs. Therefore, curcumin may be an effective compound for treating esophageal and possibly other cancers in which CSCs can cause tumor recurrence.

A New Method for Identifying Stem-Like Cells in Esophageal Cancer Cell Lines

Cancer stem cells (CSCs) appear to resist chemo-radiotherapy and initiate tumor recurrence in patients. Isolation and further characterization of this subpopulation is important for targeting CSCs. Flow cytometry using Aldefluor, a fluorescent substrate of aldehyde dehydrogenase, has been used to isolate CSCs from various cancer cell lines. However, new techniques are needed to locate and identify CSCs in culture for live-cell analyses such as fluorescence microscopy without introducing artifacts during cell sorting and to observe CSC and non-CSC interactions. Previously, we characterized a distinct CSC subpopulation within human esophageal cancer cell lines (ESCC). In this study we introduce the attached-cell Aldefluor method (ACAM) to detect CSCs in ESCC cell lines (KY-5, KY-10, TE-1, TE-8, YES-1, YES-2). To validate this technique, we isolated CSCs from the YES-2 parental line using standard Aldefluor flow cytometry to create a cell line enriched in CSCs (YES-2CSC). This line showed significantly greater ACAM staining and higher CD44 levels than YES-2. ACAM also showed significantly higher ALDH activity in YES-2CSC than in YES-2S, a cell line that has a diminished CSC subpopulation after having survived treatment with curcumin. ACAM stained cells within tumorspheres made from the CSC-enriched line but not differentiating cells from the tumorspheres. This study also demonstrates a new method for generating and growing tumorspheres without the growth factor supplements normally used in medium to form tumorspheres. ACAM should be evaluated using other cancer cell lines to further substantiate its effectiveness and to characterize CSCs in culture through various imaging techniques.

Monitoring immediate-early gene expression through firefly luciferase imaging of HRS/J hairless mice

BackgroundGene promoters fused to the firefly luciferase gene (luc) are useful for examining gene regulation in live transgenic mice and they provide unique views of functioning organs. The dynamics of gene expression in cells and tissues expressing luciferase can be observed by imaging this enzymes bioluminescent oxidation of luciferin. Neural pathways involved in specific behaviors have been identified by localizing expression of immediate-early genes such as c-fos. A transgenic mouse line with luc controlled by the human c-fos promoter (fos::luc) has enabled gene expression imaging in brain slice cultures. To optimize imaging of immediate-early gene expression throughout intact mice, the present study examined fos::luc mice and a second transgenic mouse containing luc controlled by the human cytomegalovirus immediate-early gene 1 promoter and enhancer (CMV::luc). Because skin pigments and hair can significantly scatter light from underlying structures, the two transgenic lines were crossed with a hairless albino mouse (HRS/J) to explore which deep structures could be imaged. Furthermore, live anesthetized mice were compared with overdosed mice.ResultsBioluminescence imaging of anesthetized mice over several weeks corresponded with expression patterns in mice imaged rapidly after a lethal overdose. Both fos::luc and CMV::luc mice showed quantifiable bright bioluminescence in ear, nose, paws, and tail whether they were anesthetized or overdosed. CMV::luc and fos::luc neonates had bioluminescence patterns similar to those of adults, although intensity was significantly higher in neonates. CMV::luc mice crossed with HRS/J mice had high expression in bone, claws, head, pancreas, and skeletal muscle, but less in extremities than haired CMV::luc mice. Imaging of brain bioluminescence through the neonatal skull was also practical. By imaging luciferin autofluorescence it was clear that substrate distribution did not restrict bioluminescence imaging to capillaries after injection. Luciferin treatment and anesthesia during imaging did not adversely affect circadian rhythms in locomotor activity.ConclusionsImaging of gene expression patterns with luciferase can be extended from studies of live animals to rapid imaging of mice following a pentobarbital overdose before significant effects from postmortem changes occurs. Bioluminescent transgenic mice crossed with HRS/J mice are valuable for examining gene expression in deep tissues.

Calcium imaging in organotypic cultures of the rat suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) of the hypothalamus contains a circadian pacemaker responsible for several circadian rhythms. Retinal cell projections to the SCN carry light information that phase shifts the pacemaker through the release of excitatory amino acids. To study this pathway, the Ca(2+)-sensitive dyes Fluo-3 and Fura-2 were used in organotypic slice cultures of rat SCN to visualize changes in intracellular Ca2+ of individual cells. After at least two weeks of culture, Ca2+ responses were measured in response to agonists of glutamate receptors in the presence of tetrodotoxin (TTX). Cells that showed a Ca2+ increase in response to N-methyl-D-aspartate (NMDA) and non-NMDA agonists also showed immunoreactivity towards vasoactive intestinal peptide (VIP), providing further evidence that VIP-containing neurons receive direct retinal input. The cells differed in their responses to the NMDA and non-NMDA agonists, suggesting that the cells contain differing densities of glutamate receptor subtypes.

Circadian Regulation of a Viral Gene Promoter in Live Transgenic Mice Expressing Firefly Luciferase

PurposeThis study was conducted to test for possible circadian control of viral infection in live animals using bioluminescence imaging of a firefly luciferase transgene.MethodsTransgenic mice expressing the firefly luciferase gene under the control of the promoter and enhancer of the human cytomegalovirus major immediate-early gene (CMV::luc) were examined through whole-animal imaging. Mice were crossed with HRS/J hairless albino mice to improve imaging of deep structures.ResultsTransgene expression in the extremities and head was elevated around dusk in mice maintained in cycles of light and dark. Signal was also elevated during the animals night in mice maintained in extended darkness. The viral promoter was induced during the active phase of the circadian locomotor rhythm in several tissues. Both the acinar cells and islets expressed the transgene in dissociated pancreas cultures.ConclusionsThese results suggest that viruses may exploit the circadian system for optimal timing of infection at particular phases in several tissue types.

An opsin-based photopigment mediates phase shifts of the Bulla circadian pacemaker

Summary1.The spectral response of the circadian pacemaker of the eye of the mollusk Bulla gouldiana was examined in two ways: by using the latency of the first light-evoked compound action potential (CAP) as an acute photoresponse of the putative pacemaker cells of the eye, the basal retinal neurons (BRNs), and by measuring the effectiveness of monochromatic light pulses at resetting the pacemaker.2.Through measurements of the spectral sensitivity of the acute response of the BRNs, a photopigment absorbing maximally near 490 nm (λmax) was described. Action spectra of the acute response following isolation of the BRNs, by surgical removal of the distal photoreceptor layer or the use of low Ca2+ media to block chemical synapses on the BRNs, further suggested that a 490 nm λmax photopigment is used in generating the acute light response. The spectral sensitivity of eyes adapted to a dim background illumination also agreed with the expected absorption of a 490 λmax rhodopsin.3.The effectiveness of monochromatic light pulses at shifting the phase of the circadian rhythm in CAP frequency suggested that the photopigment used in the entrainment of the pacemaker is the opsin based molecule identified through acute response measurements.

Elevated mPer1 gene expression in tumor stroma imaged through bioluminescence

The tumor stroma has significant effects on cancer cell growth and metastasis. Interactions between cancer and stromal cells shape tumor progression through poorly understood mechanisms. One factor regulating tumor growth is the circadian timing system that generates daily physiological rhythms throughout the body. Clock genes such as mPer1 serve in molecular timing events of circadian oscillators and when mutated can disrupt circadian rhythms and accelerate tumor growth. Stimulation of mPer1 by cytokines suggests that the timing of circadian oscillators may be altered by these tumor‐derived signals. To explore tumor and stromal interactions, the pattern of mPer1 expression was imaged in tumors generated through subcutaneous injection of Lewis lung carcinoma (LLC) cells. Several imaging studies have used bioluminescent cancer cell lines expressing firefly luciferase to image tumor growth in live mice. In contrast, this study used non‐bioluminescent cancer cells to produce tumors within transgenic mice expressing luciferase controlled by the mPer1 gene promoter. Bioluminescence originated only in host cells and was significantly elevated throughout the tumor stroma. It was detected through the skin of live mice or by imaging the tumor directly. No effects on the circadian timing system were detected during three weeks of tumor growth according to wheel‐running rhythms. Similarly, no effects on mPer1 expression outside the tumor were found. These results suggest that mPer1 activity may play a localized role in the interactions between cancer and stromal cells. The effects might be exploited clinically by targeting the circadian clock genes of stromal cells.

Opsin-like immunoreactivity in the circadian pacemaker neurons and photoreceptors of the eye of the opisthobranch mollusc Bulla gouldiana

Abstract.Circadian pacemaker cells in the eyes of the opisthobranch mollusc Bulla gouldiana generate a near 24-h rhythm in the frequency of optic nerve impulses. Previous electrophysiological studies suggest that these basal retinal neurons are intrinsically photosensitive and transduce light signals that shift the phase of their pacemaker mechanism. To test whether the pacemaker neurons contain opsin-like proteins, several polyclonal antibodies that recognize opsins of vertebrate photoreceptors have been tested on histological sections of the eye and on the neurons in primary cell culture. The antibodies label both the pacemaker cells and the large distal photoreceptors that surround the lens. Immunoblot analyses of the proteins of the eye have identified a single band at 62±4 kDa. These opsin antibodies may label the photopigment used in the entrainment of the circadian pacemaker.