1. The Effects of Histone Deacetylase Inhibitor on the Expression of TGF-  Sensitivity in HKc/DR Cells John A. Mkpa Claflin University/University of South Carolina Cancer Research Training Program

2. Cervical Cancer Approximately 500,000 cases worldwide (Globocan, 2000) 3,900 deaths in the US in 2004 (ACS, 2004) 10,200 new cases in the US in 2004 (ACS, 2004) Susceptibility to cervical cancer 4 times more for women living in less developed countries (Globocan, 2000)

3. Why Human Papillomavirus? Double stranded DNA virus HPV is the primary etiologic agent for cervical carcinomas and precursor lesions HPV is the most common sexually transmitted viral infection There are over 100 genotypes of HPV High risk subtypes (HPV16, 18, etc.) are present in almost all cervical carcinomas

4. Model System (in Vitro) Normal Human Keratinocytes (HKc) HPV16-Immortalized HKc (HKc/HPV16) Transfection with HPV16 DNA Growth Factor Independent HKc (HKc/GFI) Medium Lacking EGF and BPE Differentiation-Resistant HKc (HKc/DR) Medium Containing Serum and Calcium Tumors Transfection with ras/HSV2, Injected into Nude Mice

5. Loss of TGF-β Sensitivity from HKc/HPV16 to HKc/DR Loss of sensitivity to TGF-  as HKc/HPV16 cells progress in vitro (Borger, et al., J. Virology, 2000) TGF-  receptor type I, but not the type II mRNA levels decrease as HKc/HPV16 progress in vitro determined by RPA (Mi, et al., J. Virology, 2000) Re-expression of the TGF-  receptor type I into HKc/DR allows growth inhibition by TGF-  (Mi, et al., J. Virology, 2000) (Mi, et al., J. Virology, 2000)

6. TGF-β Has an antiproliferative effect on epithelial and endothelial cells Three receptors have been identified

7. TGF-  Signal Transduction Pathway of TGF-β TGF-  RII TGF-  RI TGF-  PO 4 TGF-  R-SmaCo-Smad Target gene Growth inhibition of epithelial cells TF’s PO 4 R-Smad R-SmaCo-Smad Nucleus Cytoplasm Cell membrane

8. TGF-  Receptor Type I, but not Type II, mRNA Levels Decrease as HKc/HPV16 Progress TGF-  RI TGF-  RII Standard Deviation of cycles for 5 normal HKc : TGF-  RI 0.5, TGF-  RII 0.53 d1d2d4d5

9. What is the Cause of a Decrease in TGF-  Type I Receptor Expression in HKc/DR Cells?

10. What we know Decrease in mRNA levels of TGF-  receptor type I, not the TGF-  type II, in HKc/DR confirmed using Real Time qPCR No methylation apparent in normal HKc or HKc/HPV16 Mutations in and methylation of the TGF-  receptor type I promoter region does not appear to be responsible for decreased expression of the TGF-  receptor type I in HKc/DR

11. Hypothesis The HKc/DR DNA is tightly bound by histones thereby inhibiting its transcription. This could be directly connected to the resistance of the HKc/DR to regain TGF-  sensitivity. The HKc/DR DNA is tightly bound by histones thereby inhibiting its transcription. This could be directly connected to the resistance of the HKc/DR to regain TGF-  sensitivity.

12. Scriptaid Histone Deacetylase Inhibitor (BIOMOL) 6-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N- hydroxyhexanamide MF C 18 H 18 N 2 O 4 and MW 326.4 Soluble in DMSO (4mg/ml) and Ethanol (0.3mg/ml) Facilitated functional independence of ligand- stimulated transcriptional activation and histone acetylation states (Su, et al., C. Research,2000)

13. Experimentation Plate the cells appropriately Scriptaid treatment (2µg/ml) Scriptaid+TGF-β treatments Scriptaid+TGF-β+[ 3 H]Thymidine treatments Collect and count cells using a scintillation counter (Thymidine Uptake) Analyze data

14. Confirmation of TGF-  Sensitivity of D1++

15. D1++ vs. D1DR

16. TGF-  Sensitivity?

17. D1DR w/Scriptaid vs. Control

18. Observations As expected, the D1++ remain sensitive to TGF- . Even though the data we have is inconclusive, there seems to be a potential for the recovery of TGF-  sensitivity in the D1DR cells with Scriptaid treatments. The cell counts for the D1DR cells treated with Scriptaid were unusually very low. This could be an unfavorable reaction to the Scriptaid treatments.

19. Future Work Continued studies to determine whether the HKc/DR are really resistant to TGF- .

20. Acknowledgements GOD Omar Bagasra, M.D. Kim Creek, Ph.D. Lucia Pirisi-Creek M.D. Dr. Chandrashekhar Patel Twaina Harris Bertha Taylor Fang Wan Yi Chen Anjali Bheda Diego Altomare Chinmay Trivedi Diedre Irick ColleaguesNCI