Small molecule-induced pancreatic exocrine transdifferentiation: Assay development

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Small molecule-induced pancreatic exocrine
transdifferentiation Assay development

• Kristin Rose
• Bridget Wagner, Ph.D.
• Broad Chemical Biology, Broad Institute of MIT
  Harvard
• Summer 2008

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Pancreas

• Endocrine
• Islets of Langerhans large spherical cellular
  clusters
• Secrete hormones
• a glucagon
• b insulin
• d somatostatin
• PP pancreatic polypeptide
• Exocrine
• Acini small berry-like clusters
• Secrete digestive enzymes to intestine via ducts
  (trypsin, chymotrypsin)

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Type 1 Diabetes

• Autoimmune permanent destruction of
• insulin-producing ß cells
• No cure, lethal without insulin injections
• Islet transplantation last resort,
• but risk of rejection, requires lifelong
• immunosuppresant use
• So what can we do?

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Transdifferentiation

• Process of switching between
• differentiated states
• Goal Increase ß cell mass
• Exocrine ? insulin-secreting?

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Project Cell Lines
AR42J ARIP Commercially available rat
pancreatic exocrine tumor cell lines AR42J
acinar ARIP ductal
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Project Cell Lines

• Why?
• Research literature shows these cell lines being
  induced to express insulin by various treatments,
  including conophylline
• Natural product-derived small molecule!
• Encouragement to develop an assay

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Assay Development 1 qPCR
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Preliminary Results

• Establish basal levels of expression
• Quantitative comparison of untreated AR42J cells
  to INS-1 cells
• INS-1 is a ß cell-derived rat tumor cell line
  (insulinoma)

Gene Fold difference of INS-1 to AR42J
Insulin 7786
Gcg --
PP 0.38
Pdx1 2771.91
Glut2 250.73
MafA 68.12
Nkx2.2 47.95
Pax4 15.69
Beta2 11.52
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Testing to identify a positive control

• Hepatocyte Growth Factor (HGF)
• Literature-based positive control
• Activin A
• Peptide (TGF-b family) with role in endocrine
  function
• Glucagon-Like Peptide-1 (GLP-1)
• Promotes b cell function
• Exendin-4
• Peptide analog of GLP-1, isolated from saliva of
  the Gila monster, a poisonous lizard
• Trichostatin A (TSA)
• Small-molecule histone deacetylase (HDAC)
  inhibitor

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Treatment effects on insulin gene expression in
AR42J cells
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Assay Development 2 Insulin protein expression
(1)
(2)
MIN6 cells (mouse beta)
nuclei insulin
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Assay Development 2 Insulin protein expression
Untreated AR42J
Untreated ARIP
nuclei insulin
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Summary and future directions

• Plan Screen for compounds to induce insulin
  expression in exocrine cells
• First step Assay development and identification
  of positive controls
• Approach 1 gene expression
• HGFactivin and exendin-4 increase ins mRNA in
  AR42J
• Need to optimize TSA concentration
• Need to optimize GLP-1 treatment
• Approach 2 protein expression
• Immunofluorescence-based approach
• Ideal for high-throughput screening
• But more stringent screening condition (all the
  way to protein)
• Working on positive control conditions

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Acknowledgements
Broad Chemical Biology Deepika Walpita Stefan
Kubicek Tammy Gilbert Dina Fomina Yuan
Yuan Danny Chou Alex Gitlin Yiying Xu Bridget
Wagner Stuart Schreiber
Broad/Summer Research Program in Genomics Maura
Silverstein Lucia Vielma Shawna Young Bruce
Birren Eric Lander