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Characterization of
ep-/ET1
mouse line
By Steven Ma
Mentor: Dr. Arup Indra
Pharmaceutical Sciences
Sep 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 Melanoma
o 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)
Endothelins
What we know:
• 21 amino acid peptides
• Three isoforms, ET-1,
ET-2, ET-3
• Produced by
keratinocytes at a
basal level
What we don’t know:
• Autocrine functions in
keratinocytes
http://upload.wikimedia.org/wikipedia/commons/a/ac/1EDP_human_endot
helin1_03.png
Overview of Skin
• Many Layers
95% 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 effect
on epidermal keratinocyte
homeostasis, altering cell
proliferation 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
x
• Cre – Cyclic
Recombinase
• Human – mice
homology
http://upload.wikimedia.org/wikipedia/commons/5/58/CreLoxP_experiment.png
Techniques
• PCR: to confirm ET-1
ablation in epidermis
• Paraffin sections of skin
treated with 4% PFA
(paraformaldehyde) used for:
Hematoxylin and eosin staining
Immunohistochemistry
Fontana 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
Data: epidermal thickness
D
HF
HF
ET1L2/L2 (wildtype)
E{
D
Epidermal thickness (µm)
E{
No significance
ET1L2/L2 (wildtype)
HF
HF
ET1ep-/- (knockout)
ET1ep-/-
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
Late differentiating cells
Keratin 10
(K10)
Late differentiating cells
Keratin 14
(K14)
Early proliferating cells
Ki 67
Proliferation marker;
present in all active cell phases
(but not G0)
Immunohistochemistry – differentiation
ET1L2/L2 (Wildtype)
E
Red – Cy3
Blue – Dapi (all cells)
Loricrin
D
E
K10
D
K14
E
D
ET1ep-/-
Immunohistochemistry – proliferation
ET1L2/L2 (Wildtype)
Red – Cy3
Blue – Dapi (all cells)
E
D
Ki 67
Counts (per 100 cells):
ET1L2/L2
(Wildtype)
ET1ep-/(knockout)
7.68
5.57
ET1ep-/-
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
Fontana-Masson Stain
ET1L2/L2 (Wildtype)
ET1ep-/-
Fontana-Masson Stain:
melanocyte count compared
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
Fontana-Masson Stain:
UV treatment – 0 hour time point
Special thanks to:
Dr. Kevin Ahern
Steven Hyter
Xiaobo Liang
Dan Coleman
Dr. Arup Indra
Dr. Gitali Indra
Gaurav Bajaj
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
Questions?