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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The authors have nothing to declare.

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 ...

Overexpressing genes in cells is a important way to study gene function. Stable transfection with the retrovirus allows endogenous genes to be integrated into the host genome and be expressed continuously. We picked up single colonies of the retroviral infection and generated stable cell lines overexpressing FLAG-tagged DR3.The cell lines compensated for the unavailability of good DR3 antibody and provided excellent tools for DR3 function study post in vitro and in vivo. We generated the DR3 overexpression cell lines by picking up a single colony of the retroviral infection. Cell lines from single colony could maintain the homogeneity and purity.In addition, the protocol is easy and simple to handle. To begin this procedure, grow plat-A cells overnight in 60-millimeter dishes with four milliliters of DMEM. When cells reach 80 to 90%confluence, use transfection reagent to transfect them with two micrograms of the constructed plasmid as outlined in the manufacturer's manual.After 72 hours, collect the supernatant. Using a 0.45 micrometer sterile filter, filter the media, and keep the viral suspension in the dark at four degrees Celsius. Next, grow HT29 cells overnight in a 60-millimeter dish with DMEM, incubating the cells at 37 degrees Celsius with 5%carbon dioxide.When the cells reach 30 to 50%confluence, infect them with two milliliters of viral suspension in the presence of eight micrograms of polybrene per milliliter of viral suspension. Incubate the infected cells at 37 degrees Celsius for four to six hours. Then, aspirate the viral suspension.Add four milliliters of fresh DMEM and return the dish to the incubator. At 24 hours post infection, remove the media from the dishes and carefully wash the cells with two milliliters of pre-warmed PBS. Add one milliliter of trypsin EDTA, and incubate the cells at 37 degrees Celsius for three minutes.Using a microscope at 10 times magnification, observe the cells to ensure that most of them have detached. After this, add two milliliters of DMEM containing 10%FBS to stop the trypsinization, and collect the cell suspension in a 15-milliliter tube. Centrifuge at 200 times g for five minutes at room temperature, and remove the supernatant.Re-suspend the cell pellet in 10 milliliters of DMEM and gently pipette up and down to mix. Next, dilute the cells by factors of 30, 100, and 300. Seed the cells is 150-millimeter dishes that each contain 20 milliliters of DMEM containing blasticidin at a concentration of one microgram per milliliter.Incubate at 37 degrees Celsius with 5%carbon dioxide for approximately one to two weeks. During this period, use an inverted microscope at 10 times magnification to observe the colonies each day. Mark the well-isolated colonies on the bottom of the dish when the colonies'diameter is approximately one to two millimeters.When the incubation period is complete, aspirate the medium and wash the cells with three milliliters of pre-warmed PBS. Add two milliliters of trypsin EDTA in a 60-millimeter dish. Use sterile forceps to pick up autoclaved sterile cloning cylinders and place them in the dish.Then, pick up the cylinders which will now each contain approximately 30 microliters of trypsin EDTA, and gently place them over the marked colonies. Return the dish to the incubator for three minutes. After this, check the cells under a microscope to ensure that they've detached.When the cells have lifted up, at 70 microliters of culture medium to each cylinder to inactivate the trypsin. Use a 200-microliter pipette to gently mix the cell suspension, making sure not to move the cylinder while mixing. Set out two 24-well plates, and label them plate A and plate B.Add one milliliter of DMEM to each well.Add 30 microliters of cell suspension to each well of plate A, and add 70 microliters to the corresponding wells of plate B.Place the plates back into the incubator. When the cells in plate B are at 90%confluence, remove the media and carefully wash the cells with one milliliter of PBS. Removed the PBS completely, and add 50 microliters of 1X SDS-PAGE loading buffer to lyse the cells.After five minutes, transfer the cell lysate from the 24-well plate to 1.5-milliliter centrifuge tubes. Boil the samples at 100 degrees Celsius for 10 minutes. Run the samples on 10%SDS gel at a constant voltage of 80 volts for 15 minutes and then at 120 volts for one hour.Then transfer the protein to a polyvinylidene fluoride membrane by wet transfer at a constant current of 400 milliamps for two hours. Dilute the primary antibody by a factor of 5, 000 in 5%nonfat milk that is dissolved in TBST. Add the FLAG antibody to the membrane, and incubate at four degrees Celsius overnight.Using a decoloring shaker set to 200 RPM, wash the membrane with TBST for 15 minutes, refreshing the TBST every five minutes. Then, dilute an anti-mouse secondary antibody by a factor of 10, 000 in 5%nonfat milk. Incubate the membrane with diluted secondary antibody at four degrees Celsius for five to six hours.Wash the membrane on the decoloring shaker again as previously described. After this, use Western enhanced chemiluminescence and a gel documentation system to image the membrane and detect the FLAG expression. First, collect both HT29 and HT29-DR3 cells by trypsinization, and use an automatic cell counter to measure the cell density.Seed the cells into the wells of 12-well plates with DMEM at a density of 30, 000 cells per well, and incubate overnight at 37 degrees Celsius with 5%carbon dioxide. The next day, add 10 nanomolar of diazonamide to each well that contains cells using 1%DMSO as a negative control, and transfer the plates to an incubator at 37 degrees Celsius with 5%carbon dioxide. After 48 hours of treatment, image the cells under a microscope at 10 times magnification.Next, seed both HT29 and HT29-DR3 cells into the wells of 96-well plate with DMEM at a density of 3, 000 cells per well, and allow them to grow at 37 degrees Celsius overnight. The next day, add dose-escalating diazonamide concentrations as outlined in the text protocol. After 48 hours of treatment, use a luminescence-based cell viability assay kit to measure the cell viability.Remove the 96-well plate from the incubator, and let it stand at room temperature for approximately 30 minutes. Then, add 50 microliters of assay reagents to each well, and shake the plate for two minutes at room temperature to lyse the cells. Incubate the plate at room temperature for 10 minutes, and then use a microplate reader to determine the luminescence of each well.Characterization of HT29 cells stably expressing DR3 reveals that the clones express varying levels of DR3 while the wild-type cells do not show exogenous gene expression. Genes one and five are seen to express the highest levels of DR3, and so are chosen for further experiments. The morphology of HT29 and HT29-DR3 cells are observed after being treated for 48 hours with diazonamide.HT29-DR3 cells display obvious apoptosis with a broken cell membrane and cell debris. However, HT29 cells show only mitotic arrest with intact cells that exhibit a round-up cell shape. The cell viability of these cell types is then determined after being treated for 48 hours with three nanomolar of diazonamide.HT29-DR3 cells are seen to have a cell death of over 80%while the parental HT29 cells demonstrate only a slight response in the form of mitotic arrest. This indicates that the overexpression of DR3 reconstituted the diazonamide-induced apoptotic pathway in these cells. Proper serial dilutions are important to get optimum density of single clones.It is also important to make sure every clone cylinder only contains one colony and to avoid contaminating nearby colonies. The DR3 overexpressing cell lines facilitated the biochemical study of the molecular mechanisms in vitro. We generated HT29-DR3 xenografts through subcutaneous injection of HT29-DR3 cells, and it helped elaborate the mechanisms of antimitotic agents-induced apoptosis in vivo.This protocol can be used to study genes of interest in other cell lines. In addition, gene knockout is another way for functional study, and our protocol can be adapted to the gene knockdown systems. Thus, it is generally applicable to elucidate gene functions.Remember to drop the virus relay to the garbage to specific containers for safety disposal.

establishing-cell-lines-overexpressing-dr3-to-assess-apoptotic

Research

JoVE Journal

Cancer Research

7.2K Görüntüleme.  Tsinghua University. Hücrelerdeki genlerin aşırı ifade etmesi gen fonksiyonlarını incelemenin önemli bir yoludur. Retrovirüs ile stabil transfeksiyon endojen genlerin konak genomuna entegre olmasını ve sürekli olarak ifade edilmesine olanak sağlar. Retroviral enfeksiyonun tek kolonilerini tespit ettik ve FLAG etiketli DR3'ü aşırı ifade eden sabit hücre hatları oluşturduk.Hücre hatları iyi DR3 antikor yetersizliği telafi ve DR3 fonksiyon çalışma sonrası in vitro ve in vivo için m?...