1. Post-­‐transcrip,onal regula,on of
gene expression in UVB exposed
kera,nocytes
Newlyn Joseph
7/27/2015

2. UVB on Skin
• Damages skin cells and contributes
strongly to skin tumor genera,on.
• Pigmented spots and wrinkles
• Photoaging
• DNA damage (pyrimidine dimers,
CPDs)

3. Kera,nocyte Response
• The role of p53 (tumor
suppressor protein), with regards
to kera,nocytes, involves three
main courses of ac,on:
• Growth arrest
• DNA repair
• Apoptosis

4. Long-­‐term Goals
• Understanding the development of skin cancer requires understanding
of molecular mechanisms that control transla,onal ac,vity.
• Analyzing gene expression on a transla,onal level can shed light on
various cellular responses to UV, including ac,va,on of apopto,c
pathways, par,cularly in kera,nocytes.
• Polysomic profiling offers a robust way to assess the differen,al
transla,on of various transcripts.

5. Cell Stem Cell
Article
Sampath, et al., Cell Stem Cell 2:448, 2008.

6. Kera,nocyte Markers
• Extracellular Ca++ concentra,on is noted to induce differen,a,on
in HaCaT cells.
• Involucrin is expressed in differen,ated kera,nocytes (68 kDa)
• CK5 is expressed in undifferen,ated kera,nocytes (58 kDa)

7. Cultures
• HaCaT line is first permanent
epithelial cell line from adult
human skin that normally
differen,ates.
• Media (DMEM)
• FBS (was chelated)
• Calcium Chloride
• GlutaMAX
HaCaT High Ca++
(2.8 mM)
HaCaT Low Ca++
(0.03 mM)

8. 0729-Low Ca++ 10x 0729-Low Ca++ 4x
0731-Low Ca++ 10x 0731-Low Ca++ 4x
0729-High Ca++ 10x 0729-High Ca++ 4x
0731-High Ca++ 10x 0731-High Ca++ 4x

9. CK5 Expression
• Expected
to
be
expressed
in
undifferen1ated
HaCaT
cells
• Perhaps
expression
in
HEK293
• α-­‐CK5
at
1:2500
• 293
cells
do
not
express
CK5.
• Both
HaCaT
High
and
Low
express
CK5
(rela>ve
values
yet
to
be
normalized)
GAPDH 40

10. Involucrin Expression
• Marker
for
cell
differen1a1on
• Expected
in
High
Ca++
media
cells
• α-­‐Involucrin
at
1:200
and
1:2000
• 293
cells
express
involucrin
• Both
HaCaT
cells
fail
to
express
detectable
involucrin
(assay
will
be
repeated)
GAPDH 40
1:200 1:2000

11. UVB-­‐R on HaCaT
• ~9 million cell count in all samples
• P3 cells
• Harvested with 1 mL SDS lysis buffer
• Probe phosphorylated p53 as marker of
DNA repair mechanism ac,vity
• Probe γ-­‐H2AX as a marker of dsDNA
breaks.
• Normalize with total protein content via
Mini-­‐PROTEAN TGX Stain-­‐free
HaCaT
Cells
High Ca++
0 J/m2 6h
400 J/m2
6h
24h
Low Ca++
0 J/m2 6h
400 J/m2
6h
24h
Media
Conditions
UV
Conditions
Harvest
Time

12. Total Protein
• Total protein was detected via Mini-­‐
PROTEAN TGX stain-­‐free gel
• Total protein is rela,vely the same
amongst low Ca++ and high Ca++ HaCaT
cells.

13. UVB-­‐R on Low Ca++
• UV irradiated cells showed increased
levels of γ-­‐H2AX, indica,ng DNA damage.
• pp53 was present in the irradiated cells as
well, indica,ng repair mechanism ac,vity.
0J/m26h
400J/m26h
400J/m224h
pp53γ-H2AX

14. UVB-­‐R on High Ca++
• UV irradiated cells showed increased
levels of γ-­‐H2AX, indica,ng DNA damage.
• pp53 was present in the irradiated cells as
well, indica,ng repair mechanism ac,vity.
0J/m26h
400J/m26h
400J/m224h
pp53γ-H2AX

15. Future
• Assessment of the total levels of mRNA versus polysome-­‐complexed
mRNA in for various proteins (p53) via qPCR
• RNA sequencing of polysomes
• Insight into the differen,al transla,on of various transcripts
• Possible elucida,on of sequence mo,fs corresponding to regulatory binding
proteins.
• Affect of UVB on en,re transla,on profile during different ,me points acer
exposure.
• Preliminary insight into the ,me dependency of DNA repair mechanisms.

16. Thank You
• Sean Christesen MD, PhD
• Cate Muenker
• All of Lin Lab J