Presentation on theme: "Characterization of ET1ep-/- mouse line"— Presentation transcript:
1Characterization of ET1ep-/- mouse line By Steven MaMentor: Dr. Arup IndraPharmaceutical SciencesSep 25, 2009
2Why ET1 (endothelin-1)? Melanoma Known growth factor – hyperproliferative pathologies: psoriasis and cancer?Endothelin “axis” has been shown to promote cancer progression (Bagnato et al. 2008)MelanomaBreast, prostate, brain, lung, bladder etc.Cite these (Last name, et al year)Malignant melanoma is the leading cause of death (75%) in skin cancers - no curative measures yet (Jerant et al. 2000)
3Endothelins What we don’t know: Autocrine functions in keratinocytes What we know:21 amino acid peptidesThree isoforms, ET-1, ET-2, ET-3Produced by keratinocytes at a basal levelWhat we don’t know:Autocrine functions in keratinocytes
4Overview of Skin Many Layers Melanocytes 95% are keratinocytes Secrete melanin – responsible for pigmentation (and tanning)Derived from neural crest cells (same as neurons)Skin is an unappreciated homeostasis regulator (people usually first think of kidneys or the liver)Crucial to our evolution as a land species (dry air)Proliferation is crucial to wound healingInflammation response due to sun damage (red skin)Temperature regulation through sweat secretionDiferentiationProliferation
5Hypothesis on epidermal keratinocyte homeostasis, altering cell ET1 has an autocrine effecton epidermal keratinocytehomeostasis, altering cellproliferation and differentiation.ET1 has a paracrine effect on melanocytes.
6Getting ET-1 out of epidermis Bacteriophage P1 site-specific Cre-lox recombinationFlank target geneswith loxP sitesCre – CyclicRecombinaseHuman – micehomologyxMice and humans share 99% of their genes90 percent of genes associated with disease are identical in the human and the mouse, supporting the use of mice as model organisms.
7Techniques PCR: to confirm ET-1 ablation in epidermis Paraffin sections of skin treated with 4% PFA (paraformaldehyde) used for:Hematoxylin and eosin stainingImmunohistochemistryFontana Masson staining
8Hematoxylin & eosin staining (HE) Stains nucleus blue and cytoplasm pinkPopular method in histology, widely used in medical diagnosis, e.g. tumor biopsiesUsed to measure epidermal thickness
10Immunohistochemistry (IHC - with paraffin sections) Use antibodies to detect specific proteins in skin sectionsPurpose: analyze cell differentiation and proliferationUsed Cy3 2ndary antibody
11Epidermal Proteins Loricrin Late differentiating cells Keratin 10 (K10)Late differentiating cellsKeratin 14(K14)Early proliferating cellsKi 67Proliferation marker; present in all active cell phases (but not G0)
17Results thus far No significant autocrine effect on keratinocytes: ProliferationdifferentiationSignificant effect on melanocyte proliferationAdult mice
18Ongoing experiments UV treatment to new born mice Skin samples at 0h, 24h, 48h time pointsFontana-Masson staining analysisqPCR for melanogensis factors in UV treated miceDo IHC with TRP-1 (melanocyte protein marker) and compare between adult and neonatal skinFM stain count in epidermis and in dermis separatelyWhy UV treatment on new born and not adults?
19Fontana-Masson Stain: UV treatment – 0 hour time pointCount melanocytes in epidermis and dermis separately
20Special thanks to:Dr. Kevin Ahern Dr. Arup Indra Steven Hyter Dr. Gitali Indra Xiaobo Liang Gaurav Bajaj Dan Coleman Zhixing Wang
21ReferencesJerant, A; Johnson, J; Sheridan, C; Cafferey, T. “Early Detection and Treatment of Skin Cancer.” American Family Physician. July 15, 2000Bagnato, A; Spinella, F; Rosano, L. “The endothelin axis in cancer: the promise and the challenges of molecularly targeted therapy. Can J Physiol Pharmacol. August, p473-84