This document summarizes an experiment examining the effects of UVB exposure on keratinocyte gene expression and translation. Keratinocytes were cultured under low and high calcium conditions to induce different levels of differentiation. Cells were exposed to UVB radiation and harvested at various time points. Markers of DNA damage (γ-H2AX) and DNA repair (phosphorylated p53) increased after UVB exposure in both culture conditions. Future work will analyze mRNA and polysome-associated mRNA levels to understand differential gene translation in response to UVB exposure over time. This may provide insight into molecular mechanisms controlling translational activity during DNA damage responses.
CAP Trapper Technologies and Applications, CAP Analysis of Gene Expression (C...Laura Berry
Presented at the NGS Tech and Applications Congress: USA. To find out more, visit:
www.global-engage.com
Masayoshi Itoh is a Senior Scientist at the RIKEN Center for Life Science Technologies (CLST) and Coordinator of the RIKEN Preventive Medicine and Diagnostics Innovation Program (PMI). In this presentation Masayoshi introduces CAP trapper technologies and presents the findings of the FANTOM5 project.
DNA Replication stress is any hindrance to progression or movement of the replication fork. Recently, its been related to genomic instability and cancer.
The Forsythe Immune Protocol, outcome based InvestigationTahoe eLab
Top Ten Take Home Points:
Integrative cancer medicine combines conventional and alternative treatments
Hope in victory over cancer with integrative cancer therapies
Genomic Testing (CST) on whole blood isolates circulating tumor blood cells
Genomic testing offers a blue print for individual’s cancer treatments
Genomic testing defines top chemo agents most effective in the treatment of one’s cancer
Genomic testing isolates supplements, herbs and vitamins that are most effective in the treatment of one’s cancer
Insulin Potentiated Therapy (IPT) uses insulin as its target agent
CST + IPT + Lipoic –Acid-Palladium (LAPd) Compound produces higher survivorship rates
Forsythe Immune Protocol™
shows overall survivorship rate of 70% over a 96 month period in 1400 Stage
IV cancer patients
10. Freedom to choose alternative cancer treatments is your right
CAP Trapper Technologies and Applications, CAP Analysis of Gene Expression (C...Laura Berry
Presented at the NGS Tech and Applications Congress: USA. To find out more, visit:
www.global-engage.com
Masayoshi Itoh is a Senior Scientist at the RIKEN Center for Life Science Technologies (CLST) and Coordinator of the RIKEN Preventive Medicine and Diagnostics Innovation Program (PMI). In this presentation Masayoshi introduces CAP trapper technologies and presents the findings of the FANTOM5 project.
DNA Replication stress is any hindrance to progression or movement of the replication fork. Recently, its been related to genomic instability and cancer.
The Forsythe Immune Protocol, outcome based InvestigationTahoe eLab
Top Ten Take Home Points:
Integrative cancer medicine combines conventional and alternative treatments
Hope in victory over cancer with integrative cancer therapies
Genomic Testing (CST) on whole blood isolates circulating tumor blood cells
Genomic testing offers a blue print for individual’s cancer treatments
Genomic testing defines top chemo agents most effective in the treatment of one’s cancer
Genomic testing isolates supplements, herbs and vitamins that are most effective in the treatment of one’s cancer
Insulin Potentiated Therapy (IPT) uses insulin as its target agent
CST + IPT + Lipoic –Acid-Palladium (LAPd) Compound produces higher survivorship rates
Forsythe Immune Protocol™
shows overall survivorship rate of 70% over a 96 month period in 1400 Stage
IV cancer patients
10. Freedom to choose alternative cancer treatments is your right
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.
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)
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