Sir Presentation

The detection of differences between normal and cancerous stem cells through analysis of morphology, gene expression, and effects of DCA

Justina Cho, Jimmy Liu Advisers: Ms. Marie Olszewski, Dr. Morris Kletzel

Acknowledgements We would like to thank Ms. Marie Olszewski, Dr. Morris Kletzel, Wei Huang, and Dr. Judith A. Scheppler for their continual support and guidance.

Introduction • • • • •

Dichloroacetate as a promising cancer therapy Mitochondrial action Versatility Non-toxic Previous studies

Methods • Culturing cells • Examining gene expression ▫ RT-PCR

• Testing cell viability ▫ Trypan-Blue

• Applying dichloroacetate (DCA)

Results

Figure 1. Bar graph of the cell concentration as time increases. K562 was cultured and the cell concentration of the controls (without DCA) were recorded at various times in a 14 day period.

Results

Figure 2. Protocol a: The effects of increasing DCA concentration on the cell concentration of K562. Cell concentration (K/uL) of K562 cell cultures with no DCA and cultures with 2.5 mM, 5mM, 10mM, and 20mM were observed after 168hrs after the addition of DCA.

Results

Figure 3. Protocol b: K562 cell concentrations on day 14 of the culture and 96 hours after adding DCA to certain cultures. 10mM and 20mM DCA concentrations were tested and compared to the day 14 control

Results

Figure 4. Protocol c: BM2, a normal stem cell sample, was treated with various concentrations of DCA. The resulting cell concentrations after 48 hrs of immersion in DCA were compared to the control, which had been cultured for a total of 14 days.

Results

Figure 5. WT1 expression in K562 controls, 10mM, 20 mM, and BM2. WT1 expression is relatively low for BM2, and K562 cells in 20mM DCA concentrations.

Results

Figure 6. WT1 expression in K562 controls, 10mM and 20mM DCA treated K562 samples, and BM2. The resulting data was examined through the LightCycler Data Analysis.

Discussion • Control group K562 proliferates normally • Protocol A: increased DCA concentration correlated with decreased cell concentration ▫ Decreased cell proliferation

• Protocol B: Confirming efficacy of 10mM and 20mM concentrations of DCA after 96 hours

Discussion • DCA tested on non-cancerous stem cells ▫ No effects detected

• DCA inhibits cell proliferation by restoring apoptosis (Bonnett et al.) • WT1 gene controls cell proliferation • WT1 expression in samples

Conclusion • Increased DCA concentration correlated with decreased K562 proliferation • WT1 gene inhibited in 20mM DCA-treated group • DCA does not affect non-cancerous cells • Viable cancer treatment

What the future has in store… • Further DCA tests to solidify data • Test cancerous stem cells • Test DCA with samples of healthy and cancerous cells combined

References Bonnet, S., Archer, S. L., Allalunis-Turner, J., & Haromy, A.(2007). A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth. Cancer Cell, 11, 37-51. Hernandez-Cabellero, E., Mayani, H., Monetsinos, J.J., Arenas, D., Salamanca, F., & Penaloza R.(2007). In vitro leukemic cell differentiation and WT1 gene expression. Leukemia Research. 31(3), 395-397. Jørgensen, H., Holyoake, T. (2007). Characterization of cancer stem cells in chronic myeloid leukaemia. Biochem Soc Trans.. 35, 134751.

References Lee, J. T., & Herlyn, M. (2007). Old disease, new culprit: Tumor stem cells in cancer [Electronic version]. Journal of Cellular Physiology. 213(3), 603-609. from PubMed. Young, N.S., & Hwang-Chen, S.P. (1981). Anti-K562 cell monoclonal antibodies recognize hematopoietic progenitors. Proc Natl Acad Sci U S A. 11, 7073-7077. Zou, G. (2007). Cancer stem cells in leukemia, recent advances. Journal of Cellular Physiology, 213(2), 440-444.