1. Decreased Camptothecin Sensitivity of Cancer Stem-Like cell Population Correlates with Phosphorylation state of
DNA Topoisomerase I
6th World Congress on Biotechnology
New Delhi
Dr. Amit Roy
Assistant Professor Department of Biotechnology,
National Institute of Pharmaceutical Education and Research (NIPER),
Hajipur, India

3. DNA Topoisomerase I
DNA Topoisomerase I is an essential and ubiquitous enzyme that alters the topological changes of DNA by breaking and rejoining of DNA stands.
This ubiquitous enzyme play a pivotal role in vital cellular processes, like DNA replication, transcription, recombination, and chromosomal segregation during mitotic cell division.
Topoisomerase I induces a transient single-stranded break of DNA duplex and results in a reversible topoisomerase I-DNA covalent complex.
Type IA (Prokaryotic): Enzyme links with 5' phosphate of the DNA strand
Type IB (Eukaryotic): Enzyme links with 3' phosphate of the DNA strand

4. Human Topoisomerase IB
Most eukaryotic type IB topoisomerases are monomeric enzymes, including human Topo I, which comprises 765 amino acids (91 kDa).

5. Catalytic Mechanism of Topoisomerase IB Agarose Gel Elctrophoresis
Stand Rotation
Cleavage Religation
Electron Micrograph
Relaxed DNA
Supercoiled DNA

6. Camptothecin (CPT) Camptothecin (CPT) is a plant alkaloid.
CPT is most established inhibitor of DNA topoisomerase I.
Camptothecin is pharmacologically unique :
Topoisomerase I is the only target of this drug.
CPT binds neither to Top1 alone nor to DNA alone, but only to the TopI-DNA cleavable complex.

7. Rolling-circle Enhanced Enzyme Activity Detection (REEAD) System
Rolling Circle Amplification
Biosensor Microarray
Circularization
Hybridization of detection probe
Hybridization and Rolling circle

8. Rolling-circle Enhanced Enzyme Activity Detection
(REEAD) System
Control circle – Green
hTopI - Red

9. Models: HT-29 and Caco2
Colorectal cancer is one of the most common causes of cancer death worldwide.
Human epithelial colon carcinoma cell lines have been widely studied for their ability to differentiate into stem-like cells.
Topo I-DNA cleavable complex formation induced by CPT is 4 to 7-fold reduced in fully differentiated cells compared with proliferative cells in human intestinal cells.
Cleavable complex formation and cytotoxicity induced by CPT correlate with Topo I level and activity in cells at different stages in their differentiation.
Thus, high target levels correspond closely with drug sensitivity, since proliferating cells contain larger amounts of Topo I.

10. REEAD assay is highly sensitive to detect drug responsiveness
in cancer cells
uM CPT
HT Caco2
HT29 Caco2
hTopI
Loading Control
RT-PCR
NE of HT29
NE of Caco2
CPT Sensitivity
hTopoI activty

11. Cancer stem cells
Cancer stem cells (CSCs) are a small sub-population of cancer cells that have stem cell-like properties such as self-renewal and the ability to differentiate into multiple cell types.
CSCs are referred to as tumor-initiating cells (TICs) having the ability to maintain the malignant population.
Recent research suggested that CSCs are particularly resistant to conventional chemoradiotherapy compared with the non-CSCs.
CD44 and CD133 are cell-surface transmembrane glycoprotein, which has been used in the identification of putative CSCs from several solid tumors.
The CD44 and CD133 markers may be the best to identify tumor initiating cells of human colon cancer.

12. Differentiation of Cells

13. NaBt-treated Caco2 cells are hypersensitive to CPT

14. hTopI Activity and CPT Sensitivity of NE from NaBt-Treated Caco2 cells

15. Relaxation assay of endogenous Ni-NTA column purified hTopI from NaBt+/- Caco2 cells
Control hTopI
NaBt-Treated hTopI
Lane1: DNA control, Lanes 2-11: Eluted fractions (1 to 10) respectively, Lane 12: Rest of the eluted collection.

16. CPT Sensitivity of Ni-NTA Column purified
endogenous hTopI from NaBt+/- Caco2 cells

17. Dephosphorylation of Ni-NTA Column purified
endogenous hTopI and CPT Sensitivity

18. Isolation of CD44+ and CD44- cells by Cells Sorting

19. hTopI Activity and CPT Sensitivity of
Caco2 cells (CD44+/-) from Cells Extract

20. Phosphorylation of hTopI and CPT Sensitivity

21. Phosphorylation and Dephosphorylation
of cells extract and CPT Sensitivity

22. CONCLUSIONS
CD44 may be a good stem cell marker for colon cancer cell lines like Caco2.
Phosphorylation of TopI increases the enzyme activity and drug sensitivity.
Sodium butyrate (NaBt) treated cells are more drug sensitive because of overexpression and hyper-phosphorylation of TopI.
CD44+ cells are supposed to be cancer stem cells and are chemo-resistant. Because CD44+ cells having low TopI activity due to hypo-phosphorylation of enzyme.
These studies are expected to provide important insights into the cellular mechanisms behind TopI-targeted chemotherapy and actual reason of chemo-resistant of cancer stem-like cells.
It will provide the better means for optimizing current clinical use for the individual cancer patient.

23. PLoS ONE, 2014; 9(6): e99628

24. ACKNOWLEDGEMENTS Dr. Hemanta K. Majumder, IICB, Kolkata
Dr. Birgitta R. Knudsen, Aarhus University, Denmark
Félicie, Rikke, Magnus, Jørn,
Sissel, David, Christine, Pia
Gerda, Noriko  
Funding Agency:
Council of Scientific and Industrial Research (CSIR), Govt. of India
Danish Cancer Society, Denmark
Carlsberg Foundation, Denmark

25. THANK YOU