a comparison of c myc regulated gene networks involved in tumourigenesis of two distinct tissues
Download
Skip this Video
Download Presentation
A Comparison of c-Myc Regulated Gene Networks Involved in Tumourigenesis of Two Distinct Tissues

Loading in 2 Seconds...

play fullscreen
1 / 19

A Comparison of c-Myc Regulated Gene Networks Involved in Tumourigenesis of Two Distinct Tissues - PowerPoint PPT Presentation


  • 59 Views
  • Uploaded on

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. Cancer. Normal Mitosis. Abnormal Mitosis. = Healthy Cell. = Healthy Cell. = Cancer Cell.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' A Comparison of c-Myc Regulated Gene Networks Involved in Tumourigenesis of Two Distinct Tissues' - kyros


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
a comparison of c myc regulated gene networks involved in tumourigenesis of two distinct tissues

A Comparison of c-MycRegulated Gene Networks Involved in Tumourigenesis of Two Distinct Tissues

Sam Robson

MOAC DTC, Senate House, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL

cancer
Cancer

Normal Mitosis

Abnormal Mitosis

= Healthy Cell

= Healthy Cell

= Cancer Cell

= Cancer Cell

= Apoptosis

= Apoptosis

Cell duplicates to form two identical daughter cells. Errors in DNA replication result in cell suicide (apoptosis) to avoid passing aberrant DNA to progeny.

Genetic defects prevent apoptotic pathways from activating, allowing abnormal cells to proliferate. With no proliferative control, tumours can form.

c myc
C-Myc

Legend

Myc Box I

Helix-Loop-Helix

Leucine Zipper

Myc Box II

Basic

Amino terminal

Carboxyl terminal

1

45

63

129

355

368

410

439

143

C-Myc Protein

Max Protein

Transactivation domain

Image adapted from Pelengaris et al. (2002).

transgenic model
Transgenic Model

Legend

Myc Box I

Helix-Loop-Helix

Leucine Zipper

Myc Box II

Basic

Estrogen Receptor

Max

CACGTG

TRRAP

Myc-Max complex binds E-box sequence of target gene

Transformation-Transcription domain Associated Protein (TRRAP) binds to MBII with help from MBI

Inactive MycERTAM

Active MycERTAM

TRRAP recruits a histone acetyltransferase (HAT). This acetylates nucleosomal histones resulting in chromatin remodelling, allowing access by RNA Polymerase for gene transcription

4-Hydroxytamoxifen

Myc

HAT

Max binds Myc at leucine helix-loop-helix zipper region

RNA Polymerase

4-OHT binds estrogen receptor opening up bHLHz domain.

Bound Heat Shock Protein 90

HSP90

ERTAM

slide5
Skin
  • 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.

Image taken from http://kidshealth.org/kid/body/skin_noSW.html

pancreas
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.

CACGTG

CACGTG

slide7

c-MycERTAMActivation in Pancreas – Apoptosis

CACGTG

CACGTG

INACTIVE

ACTIVE

+4OHT

Apoptosis outweighs proliferation

Islet involution

Pancreatic Islet β-Cells

Proliferation and Apoptosis

c-MycERTAM Activation in Skin – Proliferation

INACTIVE

ACTIVE

Cell Migration

+4OHT

Proliferation Increase in basal cell proliferation. More cells migrate through to squamous layer

Skin epidermisConstant renewal of cells from basal layer. Keratinised ‘squame’ cells lost from surface

Unchecked proliferation leads to many hallmarks of carcinoma. Tumour is localised with no metastasis seen.

microarray
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 hybridization

AAAA

B

AAAA

B

AAAA

B

AAAA

Microarray Hybridization

Biotin-labelled cRNA

Total RNA

cDNA

Reverse Transcription

In Vitro Transcription

Fragmentation, biotin labelling and hybridization.

Analysis in Genespring

laser capture microscopy
Laser Capture Microscopy

LCM Ependorf Tube

Membrane Slide

Tissue

Glass Slide Support

Laser

  • 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 microscopy1
Laser Capture Microscopy

1:

2:

Sweat gland

Path of laser

3:

4:

Sweat gland removed

Laser captured sweat gland

problems with pancreatic rna
Problems with pancreatic RNA
  • 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 rna1

Exocrine RNA

A

Fresh Sample

B

Islet RNA

C

18S and 28S peaks different heights

18S and 28S peaks different heights

Large unknown peak

Problems with pancreatic RNA
  • 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
Network Analysis
  • 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.

Image from Schäfer et al., 2005.

generalised linear models
Generalised Linear Models
  • Unsupervised linear regression technique.
  • Models data as a linear combination of variables:
  • 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
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
Acknowledgements

Project Supervisors:

Mike Khan

David Epstein

Stella Pelengaris

Group members:

Sylvie Abouna

Linda Cheung

Vicky Ifandi

Göran Mattson

Special thanks:

Helen Bird, Sue Davis, Lesley Ward, David Pritlove, Sean James, Paul Anderson

ad