PROJECT 1 : In vitro genetics and imaging of molecular signaling pathways

The regulation of gene expression is one of the most critical cellular functions in defining not only cell type specificity, but also phenotypic variation among individuals. Therefore, it is a key factor in shaping the complex pathophysiology of disease. Moreover, it is likely that significant portions of gene expression differences among humans are genetic. Understanding the molecular basis of these differences could hold significant promise for personalized medicine.

I. Our laboratory became interested in the genetic basis of individual differences via research into the human circadian clock. In our previous studies, a lentiviral cell-based system has been used to monitor real-time circadian rhythms directly in human skin fibroblasts (Figure 1A). Utilizing this powerful system, we discovered that the circadian period length obtained from the human skin fibroblasts widely varies among human individuals [1], and correlates directly with human physiological circadian period length [2]. Such period length measured in vitro is in turn directly predictive of human diurnal behavior [3].
   

Figure 1:  Circadian bioluminescence recording from primary fibroblasts (Brown et al., 2005)

We are currently using these technologies to investigate circadian function during ageing and in disease.

II. The circadian molecular pathway represents only one of many signaling cascades responsible for the regulation of human diurnal physiology and behavior, and similar technologies could equally be applied to these other cascades. Therefore, we have extended our focus to other major signal transduction pathways, including MAP- Kinase pathways, the inflammatory responses (IFN-γ), immune system responses (NFAT and NFkB), as well as apoptosis (p53). For this purpose, we developed a novel auto-normalized lentivector-based system, which allows us to detect real-time drug-induced activation of various signaling pathways directly in human primary cell lines (Figure 2). .

Figure 2: Bioluminescence profiles of human fibroblasts transduced with pathway specific reporters. Drugs used for activation: CREB-Forskolin (5μM); Elk1-phorbol 12-myristate 13-acetate (PMA, 100ng/ml)); c-JUN-Dexamethasone (5μM); CHOP-methyl methanesulfonate (MMS, 10ng/ml); NFAT-phorbol 12-myristate 13-acetate (PMA)/calcimycin (100ng/ml); NFkB-human tumor necrosis factor α (hTNFα, 50ng/ml), SRE-Fetal bovine serum (FBS, 20%); p53-Actinomycin D(5nM).

The profiling techniques developed for the analysis of cellular signal transduction pathways could have a significant impact not only for basic research but as well for its application to medicine. In the area of basic research, identification of genetic modifier loci that might correlate with a variety of difficult-to-access behavioral phenotypes (daily behavior, memory consolidation, or mood) will bring more insights into the knowledge of natural human variability regulated by the complex operation of these pathways. Furthermore, the same technologies that we have developed for the determination of natural inter-individual variation in the human cellular signaling could be equally suited to trace and study the behavior of signaling pathways within other healthy and pathological tissues, such as developing and progressing tumors (Figure 3).

Day   5        7          9a          9b          9c

Figure 3: Monitoring the behavior of signaling pathways within the growing tumors. Left panel: In vivo bioluminescence imaging of Elk1 signaling measured on a fifth, seventh and ninth day post injection. Three different time points were measured during the last ninth day. The 9.0. point refers to the time of the measurement before the injection of a specific pathway activator; the point 9.3. refers to the time of the measurement 3 hours after the injection of the activators and the point 9.7 refer to the time of the measurement 7 hours after the injection of the activators directly into the mouse tumors. Activators used for injection: Elk1 – PMA (2.5µg/mouse. Right panels: Charts representing bioluminescence measurement of both A) Elk1 and B)CHOP signaling. Red coloured points on the chart correspond to the time point measurements 9.0, 9.3 and9.7. Error bars represent the standard deviation (N=4 animals per pathway).

1. Steven A Brown, F.F.-O., Emi Nagoshi, Conrad Hauser,Cristiana Juge, Christophe A Meier, Rachel Chicheportiche, Jean-Michel Dayer, Urs Albrecht and Ueli Schibler, The Period Length of Fibroblast Circadian Gene Expression Varies Widely among Human Individuals. PLoS Biol., 2005. 3(10): p. e338

2. Pagani, L., et al., The Physiological Period Length of the Human Circadian Clock In Vivo Is Directly Proportional to Period in Human Fibroblasts. PLoS ONE. 5(10): p. e13376.

3. Brown, S.A., et al., Molecular insights into human daily behavior. Proceedings of the National Academy of Sciences, 2008. 105(5): p. 1602-1607.

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