dna repair and intracellular communication
TRANSCRIPT
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DNA Repair and Intracellular Communication
Daniel B. Yarosh, Ph.D.SVP, Basic Science Research
Estee Lauder Co.
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1953: Birth of Molecular Genetics Watson and Crick
Heredity / Control of Cell FunctionDNA is a sensor for cell damage
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UVINDIRECT
Free Radicals
DIRECT
DNA-AGC^T-
DNA-AoGCT-
CellularOxidationProducts
ANTIOXIDANTS
UV DAMAGE TO DNA
Sunscreens
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Thymine CytosineCyclobutane pyrimidine dimer
Direct DNA Damage
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Can Antioxidants Prevent CPD?
Murray et al. (Pinnell) JAAD 59:418, 2008
15% Vitamin C, 1% Vitamin E and 0.5% Ferulic Acid (CEFer)
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Indirect DNA Damage
8-oxo-Guanine
ROS
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UV DAMAGE TO DNAType of Damage DNA Repair Enzyme
Direct:Cyclobutane pyrimidine T4 endonuclease V
dimer UV EndonucleasePhotolyase
Indirect:ROS → 8-oxo-Gua OGG1
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T4 Endonuclease V
Bacteriophage T4 recombinant protein
Recognition and incision in one polypeptide
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Liposome Delivery
DNA repair enzymes
LIPOSOME
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DNA Repair in Human Skin
Untreated
Single UVB dose (1,500J/m2)6h treatment with the DNA Repair Lotion (2mg/cm2)
DNA Repair Lotion
Zastrow and Olivier, 2004 ICP, Jeju Korea
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DNA Repair in Human Skin
UV
UV+Photosomes
Human skin: UVB
CPD (green)
Photosomes (1h) reduce CPD by 45%
(Stege et al., PNAS 97:1790, 2000)
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OGG1 Repair of 8-oxo-Gua
SKH‐1 mice: UV followed by OGG1 liposomes
Wulff et al., Photochem. Photobiol. 84:317, 2008
8oxoGua
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Organ Transplant Patientsand Skin Cancer
180,000 living transplant patients
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Nickel sulfate:wheal and flareimmune response
UV erythema and suppressionof immune response
Photosomes prevent UV-B erythemaand restore immune response
Stege et al., PNAS 97:1790, 2000
DNA Damage and Immunity
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Local Contact Hypersensitivity
FITC
FITCDNA
RepairUV-B
3 days 5 days 1 day
measureearswelling
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DNA Repair Prevents Immunosuppression at Remote Sites
FITC +UV +Placebo +T4N55
6
7
8
9
10
CH
S R
espo
nse
Mea
n Ea
r Sw
ellin
g
p<0.05
Kripke et al. Exp Derm 5:173, 1996
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Langerhans Cells Flee Skin
Kuchel et al. PPS 4:577, 2005
Dendritic cells in human skin (red)After 4 days of solar simulating light
Treated with Placebo liposomes (Empty)
orT4N5 Liposome Lotion
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T-cells in Lymph Node
Phase contrast FITC (antigen) DiI (Liposome marker)
Yarosh et al, JID 103:461, 1994
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Cell to Cell Communication
2 MED (UVA,B) 6 hours
anti-IL-10 antibody staining
T4N5 LiposomeLotion
Placebo
Wolf et al. JID 114:149-156, 2000
DNA Damage induces IL-10
DNA repair reduces expression of IL-10
Human Skin
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Wound Healing Protease: MMP-1
Direct UV damageto collagen
UV to fibroblast
UV to keratinocytereleases cytokine
MMP-1 release
collagen
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Induction of MMP-1 in Skin
Modified from: Fisher et al., New England Journal of Medicine, 337:1419, 1997
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Cell to Cell Communication
UVB 500 J/m2
Keratinocytes+ DNA Repair
24h media
Fibroblasts
48 h media
MMP-1Collagen
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MMP-1 Gene Expression
Normal human fibroblasts treated with media from NHEKNHEK: 500 J/m2 UV-B + T4N5 Liposomes (1 µg/ml) for 1 hr post-UV.MMP-1 mRNA expression measured by Real-Time PCR (RT-PCR)
1
1.5
2
2.5
3
3.5
4
0 10 20 30
UV-BUV-B+T4N5None
Hours Media Exposure
Rel
ativ
e In
crea
se M
MP-
1Human Fibroblasts Receiving NHEK Media
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50
100
MM
P-1
/GA
PD
H (%
Con
trol)
Significance: * p<0.05
0.0 0.1 1.0 0.1 1.0
150
200
(%)
- UVB
+ UVB
0.0 0.1 1.0
MMP-1
GAPDH
0.1 1.0Blank
LiposomeUV
Endonuclease(liposomal M. luteus
extract)
(%)
- UVB
+ UVB BlankLiposome
UVEndonuclease
(liposomal M. luteus extract)
UV endonuclease significantly suppressed the Kc-induced MMP-1 expression in Fb
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OGG1 Inhibition of MMP-1MMP1 in Fb
48h after UV-Kc media
0.0 1.0 2.50.00
0.25
0.50
0.75
1.00
1.25 Empty LiposomesOGG1 Liposomes
Liposomes (µg/ml)
MM
P-1
(ng/
ml)
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DNA Damage and Cell Communication
Kinase CascadeSelective transcript activation
CYTOKINES:IMMUNE SUPPRESSIONWOUND HEALINGVIRAL ACTIVATION
DNA PK (PI3K family)ATM, ATR, mTOR
RNA-Pol
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The New Synthesis• DNA damage injures a cell• Injured cells communicate
– Cytokines release– Immune suppression– Wound healing response– Viral activation
• Undamaged cells respond to DNA damage• Extracellular signals contribute to skin
cancer
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Acknowledgements
AGI Dermatics Cosmos ResearchKen Smiles, PhD Hitoshi Masaki, PhDMatt Canning Yasunobu OchiaiKelly Dong Kuri ObayashiStephanie Nay Yuri OkanoNoah DamaghiStephaney Picart THE XP STUDY GROUPNelli Markova, PhD Margaret Kripke labNeveena Juruvoska, PhD Jean Krutmann lab