Graduation Date

Fall 12-18-2015

Document Type

Thesis

Degree Name

Master of Science (MS)

Programs

Medical Sciences Interdepartmental Area

First Advisor

Aimin Peng, PhD

Abstract


Purpose: This thesis attempted to quantitatively analyze the individual cell fate choice in resistant head and neck UM-SCC38 cells exposed to cisplatin using the most current techniques available.

Methods: UM-SCC-38 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). They were treated with cisplatin and ATM/ATR inhibitors of known dosages. Using live cell imaging, one hundred cells were tracked in each experiment and their behaviors were analyzed and entered into Microsoft Excel Spreadsheet to generate cell profile graphs. HaCaT cells, non-tumorigenic keratinocyte cell line, were also analyzed using live cell imaging and their cell fate profiles generated to better understand the resistance of SCC-38 to cisplatin.

Results: Our study revealed a highly heterogeneous pattern of cell fate choices in SCC-38, in comparison to that of the control, HaCaT, cells. In both SCC-38 and HaCaT cell lines, the majority of cell death occurred in the immediate interphase without mitotic entry, whereas significant portions of SCC-38 cells survived the treatment via either checkpoint arrest or checkpoint slippage. Cells that exhibited checkpoint slippage were primarily treated or exposed to cisplatin at late-S and G2 phases. Our study also revealed cells in M-phase were hypersensitive to cisplatin. Moreover, although the cisplatin resistant progression of mitosis exhibited no delay in general, greatly prolonged mitosis correlated with the induction of cell death in mitosis. This finding suggested a combinatorial treatment using cisplatin and an agent that blocks mitotic exit, Mg-132. Consistently, we showed a strong synergy between cisplatin and the proteasome inhibitor Mg-132. Finally, targeting DNA damage checkpoint using ATR inhibitor effectively sensitized SCC-38 to cisplatin treatment. To our surprise, targeting checkpoint eliminated both checkpoint arrest and checkpoint slippage, and augmented the induction of cell death in interphase without mitotic entry.

Conclusion: The diverse cell fate choices of SCC-38 and HaCaT cells were confirmed using live cell imaging. Our results showed the majority of cell death occurred in interphase without mitotic entry and a significantly smaller portion of SCC-38 cells died after the cisplatin treatment when compared to HaCaT. On the other hand, analysis of the surviving SCC-38 cells revealed the co-existence of checkpoint arrest and checkpoint slippage. However, caffeine was shown to abolish these surviving mechanisms in cisplatin treated cells. Moreover, our combination therapy of cisplatin plus MG-132 showed strong synergistic effect on SCC-38 cell death. Overall, our study revealed new insights into chemoresistance and suggested combinatorial strategies that potentially overcome cancer resistance.

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