Cell Line to Search for Osteosarcoma Agents

Osteosarcoma, a bone tumor of childhood, is lethal when unresponsive to chemotherapy. ER stress response, a cell survival mechanism that is triggered upon exposure to stressors such as chemotherapy, is one of the ways a tumor becomes resistant to chemotherapy. This project explores novel methods of producing a noncytotoxic EGFP-ATF6 construct to signal Endoplasmic Reticulum stress response.

Summary of Research

Osteosarcoma, a bone tumor of childhood, is lethal when unresponsive to chemotherapy. ER stress response, a cell survival mechanism that is triggered upon exposure to stressors such as chemotherapy, is one of the ways a tumor becomes resistant to chemotherapy. The transcription factor ATF6, the most reliable biomarker of ER stress, translocates from ER to
Golgi to nucleus to induce the unfolded protein response, resulting in chemoresistance. The development of future medications relies on the ability to assess movement and expression of
ATF6.

Methods of tracking ATF6 utilize fluorescent markers such as GFP, but the current EGFP-ATF6 plasmid is overexpressed and cytotoxic. This experiment seeks to modify the plasmid so that it can be effectively incorporated into an osteosarcoma cell line.

A novel EGFP-ATF6 plasmid was created in this experiment by removing the enhancer region using the restriction enzymes SnaBI and PciI, thereby preventing overexpression. XL1-Blue competent bacterial cells were used to produce the modified plasmid DNA. Cells from the osteosarcoma cell line 143B were transfected with the plasmid, and DTT treatments of various lengths showed that the modified EGFP-ATF6 molecule was not overexpressed, and was able to translocate from ER to Golgi to nucleus and thus mimic natural ATF6 movement. This movement was visualized using fluorescent microscopy. The modified EGFP-ATF6 plasmid created in this experiment can be used to create a new stable cell line, to be used in high throughput drug screening to search for agents that prevent ATF6 movement and UPR activation.

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Cell Line to Search for Osteosarcoma Agents

Osteosarcoma, a bone tumor of childhood, is lethal when unresponsive to chemotherapy. ER stress response, a cell survival mechanism that is triggered upon exposure to stressors such as chemotherapy, is one of the ways a tumor becomes resistant to chemotherapy. This project explores novel methods of producing a noncytotoxic EGFP-ATF6 construct to signal Endoplasmic Reticulum stress response.

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