Name: establishing cell lines overexpressing dr3 to assess the apoptotic response to anti-mitotic therapeutics
Uploaded: 2019-01-11T15:00:00
Description: Watch this Scientific Journal Video about Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics at JoVE.com

This work was supported by grants 53110000117 (to G.W.) and 043222019 (to X.W.) from Tsinghua University.

Please note that all the mouse experiments described here were approved and performed in accordance with the Institutional Animal Care and Use Committee (IACUC) at Tsinghua University.
1. Generation of DR3 Overexpression Cell Lines
<ol>
	<li>Construct the plasmid for DR3 expression (pMXs-IRES-DR3-FLAG) by inserting full-length DR3 cDNA with 3x FLAG at the C-terminus into retroviral vector pMXs-IRES-Blasticidin at the restriction sites of BamHI/XhoI.</li>
	<li>Grow Plat-A cells in 60 mm dishe...

<ol>
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In this manuscript, we describe a method to generate HT29-DR3 stable cell lines. HT29-DR3 cells provide a model for studying the molecular mechanisms by which DR3 contributes to apoptosis induced by antimitotic agents. This approach is versatile and repeatable. To ensure the success of the procedure, four key steps of the protocol need to be considered. First, in order to produce a high enough virus titer, it is recommended to perform DNA transfection when the Plat-A cells are at 80% - 90% confluence. Second, the viral s...

Heterologous gene expression is often employed to study the function of genes of interest. Two methods, transient and stable transfection, are prevalently used in cells and molecular biology to insert a segment of DNA or RNA into a host cell1,2. Transiently-transfected DNA or RNA cannot be passed on to daughter cells, so the genetic alteration can only be retained for a short period of time. On the other hand, in stably transfected cells, exogenous genes are integrated into the host cell genome, sustaining its expression in the cell line. Thus, stable transfection is a method that is usually reserved for research into long-term genetic regulation. The stable cell line can also be transplanted as xenografts into mouse models for in vivo study3. Stable transfection can be divided into two categories: viral and nonviral. Compared to nonviral transfection, viral infection can transfer genes into a wider variety of human cells with a higher efficiency4,5,6,7. Furthermore, a stable cell line from a single clone offers the advantage of homogeneity and clonal purity.
Uncontrolled cell growth and division are the most distinguishing features of cancer. In clinical settings, antimitotic agents are the primary treatments for many types of tumors8. However, some significant limitations of antimitotic agents cannot be ignored. First, these chemotherapies can also kill normal cells along with the undesired cancerous cells and, thus, can result in severe side effects8. Second, antitubulin drugs are not effective against all types of tumors, despite tubulin&#39;s ubiquitous expression in a wide variety of different tissues as a cytoskeletal protein9,10. It is unclear why antitubulin agents showed promising efficacy against ovarian, lung, and hematological cancers in clinical treatment, but not in kidney, colon, or pancreatic cancers11.&#160;Finally, even patients with the same type of tumor can respond&#160;to the antimitotics differently in an unpredictable manner. There may be a key effector molecule that affects the sensitivity of different patients to antimitotic agents, resulting in the diverse outcomes seen in clinical treatment12. Thus, the potential differential sensitivities of patients to antimitotic treatment should be taken into consideration in order to optimize therapeutic interventions13.
A high-throughput whole-genome siRNA library screen demonstrated that DR3 knockdown could render sensitive cells resistant to antimitotic drugs, implicating a role for DR3 in chemotherapy-induced apoptosis14. As a member of the tumor necrosis factor receptor (TNFR) superfamily, DR3 has been reported to mediate cell apoptosis in various systems15,16,17,18. Thus, we set out to establish stable cell lines overexpressing DR3 to study the molecular mechanisms by which antimitotic drugs induce apoptosis. An appropriate cell model for studying DR3 overexpression can be provided by the human colon cancer cell line HT29, which was found to be DR3-deficient. Additionally, in the clinic, colon cancers are insensitive to antimitotic drugs19.
In this article, we describe a method which allows the generation of an HT29-DR3 stable cell line through retroviral infection. We further show how to validate the DR3 expression in these cells using western blotting and how to assess the sensitivity to antimitotic agents by using a cell viability assay and morphological observation. The cells are amplified from single clones and, thus, have the advantage of a homogeneous genetic background. In addition, the FLAG tag on DR3 allowed for the visualization of cellular DR3 by microscopy and analysis of gene expression and protein interaction by biochemical approaches. Furthermore, the cells can be xenografted in mice to further analyze tumor progression in response to antimitotics in vivo14.
The HT29-DR3 cell system presented here offered an effective tool for studying the molecular mechanisms of how antimitotics kill cancer cells14. Since overexpression and knockdown cell lines are common tools for studying gene function, this protocol can be adapted easily to other genes of interest or other cell lines and, thus, turned into an extensively used approach.

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>C11965500BT</td>
			<td>Supplemented with 10% FBS and 1% Penicilin/Streptomycin</td>
		</tr>
		<tr>
			<td>Luminescent Cell Viability Assay</td>
			<td>Promega</td>
			<td>G7571</td>
			<td></td>
		</tr>
		<tr>
			<td>Paclitaxel</td>
			<td>Selleck</td>
			<td>S1150</td>
			<td></td>
		</tr>
		<tr>
			<td>pMXs-IRES-Blasicidin</td>
			<td>Cell Biolabs</td>
			<td>RTV-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>C14190500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Invitrogen</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>1,5-Dimethyl-1,5-diazaundecamethylene polymethobromide</td>
			<td>Santa Cruz</td>
			<td>sc-134220</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Transfection Reagent</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse secondary antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Flag antibody</td>
			<td>Sigma</td>
			<td>F-3165</td>
			<td></td>
		</tr>
		<tr>
			<td>HT29 cell line</td>
			<td>ATCC</td>
			<td>HTB-38</td>
			<td></td>
		</tr>
		<tr>
			<td>plat-A cell line</td>
			<td>Cell Biolabs</td>
			<td>RV-102</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF Immobilon-P</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cloning Cylinder</td>
			<td>Sigma</td>
			<td>C1059</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Imaging Multi-Mode Reader</td>
			<td>Biotek</td>
			<td>BTCYT3MV</td>
			<td></td>
		</tr>
		<tr>
			<td>CKX53 Inverted Microscope</td>
			<td>Olympus</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well cell culture plates</td>
			<td>Nest</td>
			<td>712001</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture plates</td>
			<td>Nest</td>
			<td>705001</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Nest</td>
			<td>601052</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m steril filter</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810 R</td>
			<td>Eppendorf</td>
			<td>5810000327</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well White Polystyrene Microplate&#160;</td>
			<td>Corning</td>
			<td>3903</td>
			<td></td>
		</tr>
		<tr>
			<td>Western ECL Substrate</td>
			<td>BIO-RAD</td>
			<td>1705060</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageQuant LAS 4000</td>
			<td>GE Healthcare</td>
			<td>ImageQuant LAS 4000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Decoloring Shaker&#160;</td>
			<td>HINOTECH</td>
			<td>TS-2000A</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>InvivoGen</td>
			<td>ant-bl-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated cell counter</td>
			<td>BIO-RAD</td>
			<td>TC 20</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher</td>
			<td>31985070</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishing cell lines overexpressing dr3 to assess the apoptotic response to anti-mitotic therapeutics

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell lines or increasing its expression with overexpression cell lines. Transient and stable transfection are two methods that are often used for exogenous gene expression. Transient transfection is only useful for short-term expression, whereas stable transfection allows exogenous genes to be integrated into the host cell genome where it will be continuously expressed. As a result, stable transfection is usually employed for research into long-term genetic regulation. Here we describe a simple protocol to generate a stable cell line overexpressing tagged death receptor 3 (DR3) to explore DR3 function. We picked single clones after a retroviral infection in order to maintain the homogeneity and purity of the stable cell lines. The stable cell lines generated using this protocol render DR3-deficient HT29 cells sensitive to antimitotic drugs, thus reconstituting the apoptotic response in HT29 cells. Moreover, the FLAG tag on DR3 compensates for the unavailability of good DR3 antibody and facilitates the biochemical study of the molecular mechanism by which antimitotic agents induce apoptosis.

A characterization of HT29 cells stably expressing DR3 is shown in Figure 1. The clones express varying levels of DR3, while the wild-type cells, which serve as a negative control, do not show exogenous gene expression. Here we only show five clones, among which clone 1 and 5 express the highest level of DR3. We chose clone 1 and 5 for the following experiments.
The morphology of HT29 and HT29-DR3 c...

Watch this Scientific Journal Video about Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics at JoVE.com

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics

Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell ...

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