Double edged sword: pediatric leukemia and the biology of adaptive immunity.
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Transcript of Double edged sword: pediatric leukemia and the biology of adaptive immunity.
double edged sword:pediatric leukemia and the biology of
adaptive immunity
Outline
pediatric leukemia and V(D)J Recombination
an in vitro assay that assesses aberrant V(D)J recombination in response to environmental exposure
mechanisms of genotoxin-induced recombination
A ROUGH AND RUDE IMMUNOLOGY PRIMER
Pediatric Leukemia
malignancies that arise from the uncontrolled proliferation of lymphoid or myeloid precursor cells
general types of pediatric leukemia include:
T- ALL & B-ALL
AML
PML
alterations of genomic DNA have been demonstrated to occur during fetal development.
Age specificity of pediatric leukemia
What is HPRT and why might we care?
X chromosome
Distribution of Aberrant V(D)J Recombinase Mediated Deletions and ALL in Children
Finette et al, 1996. Cancer Research
Incidence of ALL in Children and Young Adults
Over 50 publications linking V(D)J recombinase mediated genomic rearrangements with the development of lymphoid malignancies
% T
ota
l D
ele
tions
generating immune cell diversity
http://2012.igem.org/Team:Marburg_SYNMIKRO/Project
Normal
Abnormal
Cancer
Diversified Immune System
V(D)J Recombinase
consequences of a diverse immune system
Mechanism of V(D)J Recombination I
5’-CACAGTG-12/23-ACAAAAACC-3’
7-mer 9-mer
Recombination Signal Sequence (RSS)
Rag1
Rag2
I
II
DNA Damage & Repair
Mechanism of V(D)J Recombination II
Adapted from S.P. Jackson 2002 Carcinogenesis 25 (5):687
ArtemisXRCC4Ligase 4TdT
70 80
70 80
NHEJ proteins recognize RAG generated DSB and aid in the completion of V(D)J recombination
DNA-PK
DNA-PK
Ptrp
CAT
OOP
X
Rag1Rag2
OOP
OOPNHEJ
V(D)J Recombination of pGG51
AMP
CAT
Coding Joint
Ampr CamrPtrp
AMP
In vitro V(D)J recombination assay
Reh pre-B cells
X hours
pGG51
Amp/Cam colonies
Amp coloniesX 100 = % recombination frequency (Rf)
1 hour
amp amp/camamp amp/cam
Assay re-developed based on Gauss and Lieber 1993
48 hours
E. coli
Analysis of V(D)J signatures in rearranged plasmid substrates
pGG51
150 bp
5'...GCTTGG CTGCAGGTCGAC GGATCCCCGGGGATCAGCTTGGCGA...3'
Assessing DNArepair proteins
Gamma-radiation and Recombination frequency
Mutation Research/Fundamental and Molecular Mechanisms of MutagenesisVolume 686, Issues 1–2, 1 April 2010, Pages 30–38
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