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Characterization of ET1ep-/- mouse line
By Steven MaMentor: Dr. Arup Indra
Pharmaceutical SciencesSep 25, 2009
Why ET1 (endothelin-1)?
• Known growth factor – hyperproliferative pathologies: psoriasis and cancer?
• Endothelin “axis” has been shown to promote
cancer progression (Bagnato et al. 2008)
o Melanomao Breast, prostate, brain, lung, bladder etc.
• Malignant melanoma is the leading cause of death (75%) in skin cancers - no curative measures yet (Jerant et al. 2000)
EndothelinsWhat we know:• 21 amino acid
peptides• Three isoforms, ET-
1, ET-2, ET-3• Produced by
keratinocytes at a basal level
http://upload.wikimedia.org/wikipedia/commons/a/ac/1EDP_human_endothelin1_03.png
What we don’t know:
• Autocrine functions in keratinocytes
Overview of Skin
• Many Layers95% are keratinocytes
• Melanocytes
o Secrete melanin – responsible for pigmentation (and tanning)
o Derived from neural crest cells (same as neurons)
Diferentiation
Proliferation
Hypothesis
ET1 has an autocrine effecton epidermal keratinocyte homeostasis, altering cellproliferation and differentiation.
ET1 has a paracrine effect on melanocytes.
Getting ET-1 out of epidermis
• Bacteriophage P1 site-specific Cre-lox recombination
• Flank target genes with loxP sites
• Cre – Cyclic Recombinase
• Human – mice homology http://upload.wikimedia.org/wikipedia/commons/5/58/CreLoxP_experiment.png
x
Techniques
• PCR: to confirm ET-1 ablation in epidermis
• Paraffin sections of skin treated with 4% PFA (paraformaldehyde) used for:
Hematoxylin and eosin staining
ImmunohistochemistryFontana Masson staining
Hematoxylin & eosin staining (HE)
• Stains nucleus blue and cytoplasm pink
• Popular method in histology, widely used in medical diagnosis, e.g. tumor biopsies
• Used to measure epidermal thickness
HFHFD
E{
DHF HF
E{
Data: epidermal thickness
ET1L2/L2 (wildtype)
ET1ep-/- (knockout)
ET1L2/L2 (wildtype) ET1ep-/- Epid
erm
al th
ickn
ess
(µ
m)
No significance
Immunohistochemistry (IHC - with paraffin sections)
• Use antibodies to detect specific proteins in skin sections
• Purpose: analyze cell differentiation and proliferation
• Used Cy3 2ndary antibody
Epidermal Proteins
Loricrin
Keratin 10 (K10)
Keratin 14(K14)
Late differentiating cells
Late differentiating cells
Early proliferating cells
Ki 67 Proliferation marker; present in all active cell phases (but not G0)
Red – Cy3Blue – Dapi (all cells)ET1L2/L2 (Wildtype) ET1ep-/-
Immunohistochemistry – differentiation
DE
K14
E
D
K10
D
E Loricrin
Red – Cy3Blue – Dapi (all cells)ET1L2/L2 (Wildtype) ET1ep-/-
Immunohistochemistry – proliferation
E
D
Ki 67
Counts (per 100 cells):
ET1L2/L2
(Wildtype)ET1ep-/-
(knockout)
7.68 5.57
Fontana-Masson Staining (FM)
Stains all nuclei red, but melanocytes black
• “Melanin stain”
• Used to analyze paracrine effect of keratinocyte ET-1 on neighboring melanocytes
Results thus far
• No significant autocrine effect on keratinocytes:– Proliferation– differentiation
• Significant effect on melanocyte proliferation
• Adult mice
Ongoing experiments
• UV treatment to new born mice–Skin samples at 0h, 24h,
48h time points–Fontana-Masson staining
analysis
• qPCR for melanogensis factors in UV treated mice
Special thanks to:
Dr. Kevin Ahern Dr. Arup IndraSteven Hyter Dr. Gitali IndraXiaobo Liang Gaurav BajajDan Coleman Zhixing Wang
References
• Jerant, A; Johnson, J; Sheridan, C; Cafferey, T. “Early Detection and Treatment of Skin Cancer.” American Family Physician. July 15, 2000
• Bagnato, A; Spinella, F; Rosano, L. “The endothelin axis in cancer: the promise and the challenges of molecularly targeted therapy. Can J Physiol Pharmacol. August, 2008. p473-84
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