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John A. Mkpa Claflin University/University of South Carolina Cancer Research Training Program

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. Cervical Cancer. Approximately 500,000 cases worldwide (Globocan, 2000)

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John A. Mkpa Claflin University/University of South Carolina Cancer Research Training Program

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

  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)

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

  7. TGF- TGF- TGF- PO4 R-Smad TGF- RI PO4 TGF- RII PO4 R-Smad Co-Smad PO4 R-Sma Co-Smad TF’s Growth inhibition of epithelial cells TF’s R-Sma Co-Smad Target gene Signal Transduction Pathway of TGF-β Cell membrane Cytoplasm Nucleus

  8. d1 d2 d4 d5 Standard Deviation of cycles for 5 normal HKc: TGF-b RI 0.5, TGF-b RII 0.53 TGF-b RI TGF-b RII TGF- Receptor Type I, but not Type II, mRNA Levels Decrease as HKc/HPV16 Progress

  9. What is the Cause of a Decrease in TGF- Type I ReceptorExpression 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-b receptor type I promoter region does not appear to be responsible for decreased expression of the TGF-b 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.

  12. Scriptaid • Histone Deacetylase Inhibitor (BIOMOL) • 6-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxyhexanamide • MF C18H18N2O4 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+[3H]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 Colleagues NCI

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