a comparison of c-myc regulated gene networks involved in tumourigenesis of two distinct tissues
Post on 16-Jan-2016
23 Views
Preview:
DESCRIPTION
TRANSCRIPT
A Comparison of c-Myc Regulated Gene Networks Involved in
Tumourigenesis of Two Distinct Tissues
Sam Robson
MOAC DTC, Senate House, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL
CancerNormal Mitosis Abnormal Mitosis
Genetic defects prevent apoptotic pathways from activating, allowing abnormal cells to proliferate. With no proliferative control, tumours can form.
Cell duplicates to form two identical daughter cells. Errors in DNA replication result in cell suicide (apoptosis) to avoid passing aberrant DNA to progeny.
= Healthy Cell
= Cancer Cell
= Apoptosis
= Healthy Cell
= Cancer Cell
= Apoptosis
C-Myc
C-Myc Protein
45 63 129 143 355 368 410 4391
Carboxyl terminal
Amino terminal
Transactivation domain
Myc Box IMyc Box IIBasic
Helix-Loop-HelixLeucine Zipper
Legend
Max Protein
Image adapted from Pelengaris et al. (2002).
Transgenic Model
Myc Box IMyc Box IIBasic
Helix-Loop-HelixLeucine ZipperEstrogen Receptor
Legend
ERTAM HSP90
MycInactive MycERTAM
Bound Heat Shock Protein 90
4-Hydroxytamoxifen
4-OHT binds estrogen receptor opening up bHLHz domain.
Max Max binds Myc at leucine helix-loop-helix zipper region
Active MycERTAM
Myc-Max complex binds E-box sequence of target gene
TR
RA
P
Transformation-Transcription domain Associated Protein (TRRAP) binds to MBII with help from MBI
HA
T
RNA Polymerase
TRRAP recruits a histone acetyltransferase (HAT). This acetylates nucleosomal histones resulting in chromatin remodelling, allowing access by RNA Polymerase for gene transcription
CACGTG
Skin
Image taken from http://kidshealth.org/kid/body/skin_noSW.html
• Cells proliferate from epidermal stem cells in basal layer.
• Migration towards surface – cells become keratinized.
• Stratum corneus layer made up of highly keratinized nuclei-free cells – squames.
• Squames constantly shed from epidermal surface.
• Homeostasis within the skin very important.
Pancreas
• Homogenous groupings of cells within the exocrine – Islets of Langerhans.
• Islets contain predominately β-cells – sole source of insulin
• Insulin responsible for glucose metabolism.
• Loss of insulin leads to Type I diabetes.
• Pancreatic ducts transport pancreatic enzymes.
c-MycERTAM Activation in Pancreas – Apoptosis
c-MycERTAM Activation in Skin – Proliferation
INACTIVE ACTIVE
Pancreatic Islet β-Cells
Proliferation and Apoptosis
Apoptosis outweighs
proliferation
Islet involution
Skin epidermis Constant renewal of
cells from basal layer. Keratinised ‘squame’ cells lost from surface
Proliferation Increase in basal cell
proliferation. More cells migrate through to squamous layer
INACTIVE ACTIVE
+4OHT
+4OHT
Unchecked proliferation leads to many hallmarks of carcinoma.
Tumour is localised with no metastasis seen.
Cell Migration
Microarray
•Features measure one nucleotide sequence (25mers).
•Hundreds of identical 25mers per feature.
•11-20 features per gene.
•25mer sequence specifically binds biotin labelled cDNA.
•Fluorescence readings give relative RNA concentration – equivalent to gene expression.
Images courtesy of Affymetrix - www.affymetrix.com
Microarray HybridizationTotal RNA
Reverse TranscriptionAAAA
AAAA
AAAA
AAAA
cDNA
In Vitro Transcription
B
B
B
Biotin-labelled cRNA
Fragmentation, biotin labelling
and hybridization.
Analysis in Genespring
Cell cycle – Cancer reversal
Cell Death – Cancer reversal
Laser Capture Microscopy
Laser Glass Slide Support
Membrane Slide
LCM Ependorf
Tube
Tissue
• Tissue section bound to membrane of LCM slide. Glass slide used as support on LCM platform.
• “Sticky” ependorf tube lid lowered onto membrane. Laser cuts designated area for dissection. Raising lid lifts cut material from LCM platform for RNA extraction.
Laser Capture Microscopy
Sweat gland
Path of laser
1:
4:3:
2:
Sweat gland
removed
Laser captured
sweat gland
Problems with pancreatic RNA
Freshsection
Airdriedsection
Fixed section
Stained section
Control sample
18s and 28s peaks similar heights
18s and 28s peaks different heights
18s and 28s peaks non-existant
18s and 28s peaks non-existant
18s and 28s peaks non-existant
A EDCBFreshsection
Airdriedsection
Fixed section
Stained section
Freshsection
Airdriedsection
Fixed section
Stained section
Control sample
18s and 28s peaks similar heights
18s and 28s peaks different heights
18s and 28s peaks non-existant
18s and 28s peaks non-existant
18s and 28s peaks non-existant
A EDCB
• RNA degraded naturally in cells by the enzyme RNase.
• Pancreas rife with RNase activity.
• Integrity of RNA gradually decreases throughout LCM procedure.
• RNA fully degraded by the time of tissue collection.
Problems with pancreatic RNA
A Fresh Sample
Islet RNA
Exocrine RNA
B C
18S and 28S peaks different heights
18S and 28S peaks different heights
Large unknown peak
• RNA integrity in islets is good compared to RNA integrity in exocrine tissue.
• Implies that islet RNA is not subject to same degradation as that in the exocrine.
• Possible that structure of islets protects islet cell RNA from ductal RNases.
Network Analysis
Image from Schäfer et al., 2005.
•Empirical Bayesian approach estimates gene network structure from microarray data.
•Problems – Sample size small for number of nodes (genes).
•Allows estimation of gene interactions in complex system.
Generalised Linear Models• Unsupervised linear regression technique.• Models data as a linear combination of variables:
...321
332211 nnn xbxbxbay
• Gives the most statistically relevant variables.
• Implementation in Genespring for public use.
• Makes no assumptions of data and works with unbalanced experiments – Useful for clinical data.
Conclusion• c-Myc known to be very important in cancer
formation.• c-Myc function in cancer onset still not fully
understood.• In vivo analysis of early c-Myc activity will help to
disentangle the web of c-Myc functionality.• Understanding of the route of tumourigenesis
will hopefully aid in development of gene specific cancer therapies.
Acknowledgements
Project Supervisors:
Mike Khan
David Epstein
Stella Pelengaris
Group members:
Sylvie Abouna
Linda Cheung
Vicky Ifandi
Göran MattsonSpecial thanks:
Helen Bird, Sue Davis, Lesley Ward, David Pritlove, Sean James, Paul Anderson
top related