1. Characterization of ET1ep-/- mouse line
By Steven Ma
Mentor: Dr. Arup Indra
Pharmaceutical Sciences
Sep 25, 2009

2. Why 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)
Melanoma
Breast, 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)

3. Endothelins What we don’t know: Autocrine functions in keratinocytes
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

4. Overview 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 healing
Inflammation response due to sun damage (red skin)
Temperature regulation through sweat secretion
Diferentiation
Proliferation

5. Hypothesis on epidermal keratinocyte homeostasis, altering cell
ET1 has an autocrine effect
on epidermal keratinocyte
homeostasis, altering cell
proliferation and differentiation.
ET1 has a paracrine effect on melanocytes.

6. 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 x
Mice and humans share 99% of their genes
90 percent of genes associated with disease are identical in the human and the mouse, supporting the use of mice as model organisms.

7. 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

8. 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

9. Data: epidermal thicknessHF E{
Epidermal thickness (µm)
No significance
ET1L2/L2 (wildtype) HF D E{
ET1L2/L2 (wildtype)
ET1ep-/-
ET1ep-/- (knockout)

10. Immunohistochemistry (IHC - with paraffin sections)
Use antibodies to detect specific proteins in skin sections
Purpose: analyze cell differentiation and proliferation
Used Cy3 2ndary antibody

11. 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)

12. Immunohistochemistry – differentiation
Red – Cy3
Blue – Dapi (all cells)
ET1L2/L2 (Wildtype)
ET1ep-/- D E
Loricrin E D
K10 D E
K14

13. Immunohistochemistry – proliferation
Red – Cy3
Blue – Dapi (all cells)
ET1L2/L2 (Wildtype)
ET1ep-/- E D
Ki 67
Counts (per 100 cells):
Arup: please check the comment (the yellow thing)
ET1L2/L2 (Wildtype)
ET1ep-/- (knockout)
7.68
5.57

14. 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

15. Fontana-Masson Stain
ET1L2/L2 (Wildtype)
ET1ep-/-

16. Fontana-Masson Stain: melanocyte count compared

17. Results thus far No significant autocrine effect on keratinocytes:
Proliferation
differentiation
Significant effect on melanocyte proliferation
Adult mice

18. 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
Do IHC with TRP-1 (melanocyte protein marker) and compare between adult and neonatal skin
FM stain count in epidermis and in dermis separately
Why UV treatment on new born and not adults?

19. Fontana-Masson Stain:
UV treatment – 0 hour time point
Count melanocytes in epidermis and dermis separately

20. Special thanks to:
Dr. Kevin Ahern Dr. Arup Indra Steven Hyter Dr. Gitali Indra Xiaobo Liang Gaurav Bajaj Dan Coleman Zhixing Wang

21. 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, p473-84

22. Questions?