Presented by Johann Campbell

1. Genetic reprogramming in pathways of colonic cell maturation induced by short chain fatty acids: Comparison with Trichostatin A, Sulindac, and Curcumin and cmplications for chemoprevention of colon cancer. Presented by Johann Campbell Mariadason, J., G., Corner, and L. Augenlicht. 2000. Genetic reprogramming in pathways of colonic cell maturation induced by short chain fatty acids: comparison with trichostatin A, sulindac, and curcumin and implications for chemoprevention of colon cancer.Cancer Res. 60:4561-4572.

2. Background • Butyrate produced in intestine by microbial fermentation • Butyrate is an inhibitor of HDAC activity • TSA also inhibits HDAC activity • Sulindac induces G0-G1 cell cycle arrest and apoptosis • Curcumin induces G2-M cell cycle arrest and apoptosis

3. Cell Cycle

4. Investigating genomic activity • Cell lines used were SW60 and Caco-2 • Microarray technology • Probe labels included Cy3 and Cy5 • 8063 arrays used out of a total of 18,000 available • Western Blot (Western Immunoblot)

5. Microarray Technology • Micro slide coated with DNA sequences from a database of about 18,000 • mRNA from desired genes are labeled fluorescent dye • 2 groups of labeled mRNA used : Control and Test • Labeled mRNA allowed to hybridize on microslide • Microslide washed to remove unbound mRNA • Fluorescence measured for ratios of bound Control : Test

6. Courtesy National Human Genome Institute

7. Microarray Results • In the control group the distribution of sequences remained tightly around the mean • In the treated test groups, there was a spread of ratios • The spread indicates gene activity • The width of the spread is co-relational to the activity

9. Gene activity • A Treeview and Cluster program was used to visualize genetic reprogramming • Red indicates an increase in gene expression • Green indicates a decrease in gene expression • Activity among gene clusters are compared

11. Response to Butyrate • After 48 hours about 256 sequences were increased in expression by butyrate • About 333 were repressed after 48 hours • Of the 589 altered sequence, only 345 are named

12. Response to all reagents • Some reagents displayed similar activity patterns at similar times • Some reagents displayed similar activity at different times • Time similarities/differences were graphed using the N-Euclidean distances

14. Histone Acetylation • Histone acetylation peaks at different times • TSA peaks at 2 hours • Butyrate peaks at 16 hours

16. Butyrate and Sulindac activity • Both butyrate and sulindac stimulate similar cell cycle arrest • Both are similar in inducing apoptosis • Both displayed progressive gene activity with time

18. Further analysis of Butyrate and Sulindac • Sulindac and butyrate displayed some similarity in the type of gene activity • About 145 were common • About 53 showed of the 143 showed opposite reactions

20. Analysis of gene recruitment of butyrate and sulindac • Class of genes active in signal transduction and transcription alteration • 2 groups defined: group 1 signaling (33 increased expression) and group 2 signaling (45 repressed expression)

23. Cell cycle response • 61 genes involved in cell cycle regulation were chosen • Euclidean distances calculated for each response maximally with time • Butyrate was similar to TSA in affecting these genes • Butyrate was less like curcumin in affecting genes responsible for cell cycle regulation • The same analysis was done on cells from Caco-2 lines • Butyrate response was similar to arrested Caco-2 cells

25. Conclusion • Butyrate was found to play a role in gene activity in colonic cells • Butyrate is also able to induce a cell cycle arrest by its activities on cell cycle regulating genes • Butyrate shows similar activities on genes similar to TSA, opposite to curcumin • Butyrate had limited similar effects compared to sulindac

26. Questions