"Laser Capture Microdissection" Mikroskop
Zusammenfassung der Projektergebnisse
The microscope is used for all tissue histology work in the groups of the Department of Biochemistry and Molecular Biology. For routine work, a technician is trained on the instrument and performs histology for all experiments in the Department. She also supervises external users and checks that they are competent for use. Some specific projects are listed below. Daniel Murphy/Martin Eilers: The central aim of the work using the laser capture microscope was to identify and functionally validate genes that are required for progression of lung tumours from the benign to the invasive stage, using a temporally tractable mouse model of lung cancer. The project involved laser-capture micro-dissection of morphologically distinct populations of tumour cells from within individual tumours, followed by gene expression analysis to identify genes whose expression increases as tumour progress to invasive disease. A number of other research groups have successfully analysed gene expression from large, welldeveloped late stage tumours, however, the novelty of this project was to push the limits of this technology in order to analyse the transition to more aggressive cancer at a very early stage of tumour development and with much less starting material (ie. much smaller populations of cells). Thus, the project encountered a number of technical challenges and although these slowed our progress somewhat, all such challenges were successfully overcome, laying a solid foundation for future investigation both within and out-with of the Murphy lab. The genes that were identified clearly define functional groups of progression genes and are currently being pursued in the Murphy Laboratory (which is not at the University of Glasgow/Beatson Institute of Cancer Research). Thomas Haaf: The AG Haaf at the Institute of Human Genetics is studying the role of epigenetic changes for the evolution of human cognitive functions, in particular language. Methylated DNA immunoprecipitation and ultra-high resolution NimbleGen tiling arrays identified differentially methylated regions between human and chimpanzee cortices in CNTNAP2, one of the largest genes in the human genome and bonafide language gene. Microarray analyses were performed with whole brain tissue, which represents a mixture of different cell types, most importantly neurons and glia cells. It is well known that methylation patterns can vary considerably between cell types. To exclude that the between-species differences are not merely caused by differences in the cell composition of human and chimpanzee cortices, but represent true evolutionary epigenetic changes, it was necessary to analyze the methylation patterns in pure cell fractions. A master student, Larissa Härtle, used the dissection microscope to isolate neurons from paraffin-embedded tissue sections by laser micro dissection. Neurons were identified by cresyl violet staining. Pools of 100 to 1000 micro-dissected neurons were collected in reaction tubes. Protocols were established for bisulfite-conversion and methylation analysis (by bisulfite pyrosequencing) of small amounts of DNA from 100-1000 micro-dissected cells. Manfred Gessler: We use the microscope as a histology microscope in two major projects: First, we analyze the role of Hey and Hes genes in the cardiovascular system and we have concentrated on postnatal angiogenesis in the retina of the newborn mouse as a model system. Here we rely on high-resolution fluorescence microscopy of so called „flat-mount“ retinas to characterize sprouting of individual endothelial cells. The conditional deletion of Hes1 in endothelia leads to a clear reduction of these sprouts and a subsequent delay in vascularization of the retina. Multicolor analysis with endothelial and pericyte markers revealed that the physiological delay in coverage of new sprouts by desmin positive pericytes is no longer seen in the mutants. Instead, all cells are covered by desmin. This suggests that there is a lack of immature cells at the front with premature differentiation and a concomitant reduction in proliferating cells in this critical area. These data are currently finalized for publication. Second, there is a lack of cell culture models for preclinical research into Wilms tumor biology that would allow analysis of tumor-specific gene defects and deregulated signaling pathways. Over the last years we have been able to establish cultures of the stromal and epithelial components of these. More recently we have even been able to culture the recalcitrant blastemal cells as spheroids in vitro and in xenografts. Especially for the characterization of these 3D spheroid cultures we need access to high quality microscopy equipment. We have been able to show that our tumor cultures react strongly upon treatment with retinoids and these findings have already made their way into first clinical trials. With our new and unpublished spheroid models we hope to get closer to the in vivo situation of these tumors that are typically made up of the three cell types blastema, stroma and epithelia. The cells are amenable to genetic modification opening up the possibility to engineer these to carry additional patient-derived mutations and to study their effects on cell morphology and behavior. AG Gaubatz: The aim of our work is to investigate whether DREAM and B-MYB contribute to tumorigenesis. To address this question we use a preclinical in vivo mouse model of non-small cell lung cancer (NSCLC) in the lab. In this model, lung tumorigenesis is driven by a conditional allele of oncogenic K-RasG12D and a conditional allele of p53. Activation of oncogenic K-Ras and deletion of p53 in the lung is achieved by intranasal delivery of adenoviral Cre-recombinase. To test whether DREAM contributes to tumor progression in this model, we use conditional alleles of B-MYB and LIN9, a core subunit of the DREAM complex. Compound mutant mice that harbor the conditional K-RasG12D allele in combination with conditional p53 and the conditional allele of LIN9 or B-MYB were created. Infection of these mice with Ade-Cre results in expression of oncogenic K-RasG12D, loss of p53 and loss of LIN9 or B-MYB. 13 weeks after tumor initiation, lung adenocarcinomas were analyzed by H&E staining. Microscopic pictures of stained lung sections were acquired with the microscope and tumor area was quantified.
Projektbezogene Publikationen (Auswahl)
- (2011) The MK5/PRAK Kinase and Myc Form a Negative Feedback Loop that Is Disrupted during Colorectal Tumorigenesis. Molecular Cell, 41:445-57
Kress, T.R., Cannell, I.G., Brenkman, A.B., Gaestel, M., N'Burgering, B.M., Bushell, M., Rosenwald, A., and Eilers, M.
- (2011). Retinoic acid pathway activity in Wilms tumors and characterization of biological responses in vitro. Molecular cancer 10, 136
Wegert, J., Bausenwein, S., Kneitz, S., Roth, S., Graf, N., Geissinger, E., and Gessler, M.
- (2012) Deregulated MYC expression induces dependence upon AMPK-related kinase 5. Nature, 483:608-12
Liu, L., Ulbrich, J., Müller, J., Wüstefeld, T., Aeberhard, L., Kress, T.R., Muthalagu, N., Rudalska, R., Moll, R., Kempa, S., Zender, L., Eilers, M., and Murphy, D.J.
- (2012). Characterization of primary Wilms tumor cultures as an in vitro model. Genes, chromosomes & cancer 51, 92-104
Wegert, J., Bausenwein, S., Roth, S., Graf, N., Geissinger, E., and Gessler, M.
- (2013) Miz1 is required to maintain autophagic flux. Nature Communications, 4:2535
Wolf, E., Gebhardt, A., Kawauchi, D., Walz, S., Björn von Eyss, B., Wagner, N., Renninger, C., Krohne, G., Asan, E., Roussel, M., and Eilers, M.
- (2014) Bim is a critical mediator of Myc-induced apoptosis in multiple solid tissues. Cell Reports, doi: pii: S2211-1247(14)00659-7
Muthalagu, N., Junttila, M., Wiese, K.E., Bauer, G., Eilers, M., Evan, G.I., and Murphy, D.J.
(Siehe online unter https://doi.org/10.1016/j.celrep.2014.07.057) - (2014) Miz1 deficiency in the mammary gland causes a lactation defect by attenuated stat5 expression and phosphorylation. PLoS One 9: e89187
Sanz-Moreno A, Fuhrmann D, Wolf E, von Eyss B, Eilers M, Elsasser HP
(Siehe online unter https://doi.org/10.1371/journal.pone.0089187) - (2014) The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer. Journal of Clinical Investigation
Diefenbacher, M.E., Popov, N., Blake, S.M., Schülein-Völk, C., Nye,E., Spencer-Dene, B., Jaenicke, L.A., Eilers, M. and Axel Behrens
(Siehe online unter https://doi.org/10.1172/JCI73733) - (2014) Tumor cell-specific inhibition of MYC function using small molecule inhibitors of the HUWE1 ubiquitin ligase. EMBO Molecular Medicine, (2014) 6, 1525-1541
Peter, S., Bultinck, J., Myant, K., Jaenicke, L.A., Walz, S:, Müller, J., Gmachl, M., Treu, M., Boehmelt, G., Ade, C.P., Schmitz, W., Wiegering, A., Otto, C., Popov, N., Sansom, O., Kraut, N., and Eilers, M.
(Siehe online unter https://dx.doi.org/10.15252/emmm.201403927) - Widespread differences in cortex DNA methylation of the “language gene” CNTNAP2 between humans and chimpanzees. Epigenetics 2014;9:533-5
Schneider E., El Hajj N., Richter S., Roche-Santiago J., Nanda I., Schempp W., Riederer P., Navarro B., Bontrop R.E., Kondova I., Scholz C.J., Haaf T.
(Siehe online unter https://doi.org/10.4161/epi.27689)
