Loss of Cyclin-Dependent Kinase Inhibitor Alters Oncolytic Adenovirus Replication and Promotes More Efficient Virus Production
<p>HCT-116 WT (wildtype) and p21<sup>−/−</sup> cells were infected with 1 MOI (multiplicity of infection) of CN702 virus and infected cells were harvested for total DNA at indicated time points. Panel shows adenoviral DNA amplified by qPCR primers to fiber gene. Data normalized to β-Actin. Error bars S.E (<b>A</b>). HCT-116 WT and p21<sup>−/−</sup> Cells were infected with 1 MOI of CN702 virus and infected cells were harvested for total DNA at indicated time points. Panel shows adenoviral DNA amplified by qPCR primers to fiber gene. Data normalized to β-Actin. Error bars represent ± S.E (<b>B</b>). Adenovirus entry by viral DNA qPCR. HCT-116 WT and p21<sup>−/−</sup> cells were infected with 1 MOI of CN702 virus and infected cells were harvested for total DNA at indicated time points. Panel shows adenoviral DNA amplified by qPCR primers to fiber gene. Data normalized to β-Actin. Error bars S.E (<b>C</b>). HCT-116 WT and p21<sup>−/−</sup> cells were infected with 5 or 10 MOI of CN702. At 24 h p.i. wells were imaged for GFP (green fluorescence protein) and fields were counted for GFP infected cells. No statistical difference was found in WT-infected cells vs. p21<sup>−/−</sup> infected cells (<b>D</b>). Statistical significance was defined as * <span class="html-italic">p</span> value ≤ 0.05.</p> "> Figure 2
<p>DBP foci formation in infected cells. HCT-116 WT and p21<sup>−/−</sup> were plated in chamber slides and cells were infected with 5 MOI of CN702 virus. Infected cells were stopped at indicated time points and immunofluoresence microscopy was performed (<b>A</b>) 6 h p.i. 60× representatives of DBP IF at the same time point indicative of centers of viral DNA replication. 6 h p.i. 40× Field representatives of DBP (green) and E1A (red) IF at the same time point (<b>B</b>). Note in HCT-116 WT has approximately 3-fold lower number of cells that are both E1A and DBP positive for the same time point than HCT-116 p21<sup>−/−</sup> cells. 6 h p.i. DBP (green) and E1A (red) IF at the same time point. Note in HCT-116 WT fewer cells are both E1A and DBP positive than HCT-116 p21<sup>−/−</sup> cells (<b>C</b>). 12 h p.i. representative images. Note similar amount of dual stained cells shows no statistical significant difference in infectivity at later time point (<span class="html-italic">p</span> value > 0.05) (<b>D</b>).</p> "> Figure 2 Cont.
<p>DBP foci formation in infected cells. HCT-116 WT and p21<sup>−/−</sup> were plated in chamber slides and cells were infected with 5 MOI of CN702 virus. Infected cells were stopped at indicated time points and immunofluoresence microscopy was performed (<b>A</b>) 6 h p.i. 60× representatives of DBP IF at the same time point indicative of centers of viral DNA replication. 6 h p.i. 40× Field representatives of DBP (green) and E1A (red) IF at the same time point (<b>B</b>). Note in HCT-116 WT has approximately 3-fold lower number of cells that are both E1A and DBP positive for the same time point than HCT-116 p21<sup>−/−</sup> cells. 6 h p.i. DBP (green) and E1A (red) IF at the same time point. Note in HCT-116 WT fewer cells are both E1A and DBP positive than HCT-116 p21<sup>−/−</sup> cells (<b>C</b>). 12 h p.i. representative images. Note similar amount of dual stained cells shows no statistical significant difference in infectivity at later time point (<span class="html-italic">p</span> value > 0.05) (<b>D</b>).</p> "> Figure 3
<p>HCT-116 WT and p21<sup>−/−</sup> cells were infected with 1 MOI of CN702. At indicated time points, infected cells were harvested for total RNA. 100 ng of RNA was used to run Nanostring nCounter Assay using custom Ad5 code set. Ad5 mRNA counts were normalized to internal controls and panel of housekeeping genes and quantitation of Adeno Early (<b>A</b>), Late (<b>B</b>), and VA RNA mRNA expression. The yellow, orange, and green colors indicated statistical significance with different <span class="html-italic">p</span> value. Significance was defined as * <span class="html-italic">p</span> ≤ 0.05 (<b>C</b>). HCT-116 WT cells were transfected with plasmid expressing p21 shRNA or control vector. 24 h post transfection cells were infected with 2 MOI of CN702 and 10 MOI of FFIG (Fiber-IRES-GFP) reporter virus and GFP readings were taken at indicated time points (<b>D</b>). CN702 infection time course protein expression by Westerns blot. HCT-116 WT and p21<sup>−/−</sup> cells were infected with 1 MOI of CN702. At indicated time points, cells were harvested and adenovirus proteins were analyzed by Western blot (<b>E</b>).</p> "> Figure 4
<p>Viral burst size is significantly larger in HCT-116 p21<sup>−/−</sup> cells. HCT-116 WT and p21<sup>−/−</sup> cells were plated in 10-cm dishes and infected with 250 or 500 PFU of AdTrack-HisE1A-E1B virus. After 1 h, virus was removed and overlaid with 0.1% Agarose Media mixture. GFP fluorescence was imaged at indicated time points to follow viral burst (<b>A</b>). HCT-116 WT and p21<sup>−/−</sup> cells were plated in 10-cm dishes and infected with 250 or 500 PFU of CN702 virus. After 1 h, virus was removed and overlaid with 0.1% Agarose Media mixture. 26 days p.i. overlays were stained with neutral red overnight (<b>B</b>). HCT-116 WT and p21<sup>−/−</sup> cells were infected with various MOI of CN702. At 72 h, p.i. cell monolayers were stained with 0.5% crystal violet in methanol to visualize viral CPE (<b>C</b>). Assessing infectious particle titer over time. HCT-116 WT and p21<sup>−/−</sup> cells were plated in 6 well plates and infected with 10 MOI of CN702. Cells and media were harvested at indicated time points and viral titer was done on 293 HEK cells (<b>D</b>). Statistical significance was defined as * <span class="html-italic">p</span> ≤ 0.05.</p> ">
Abstract
:1. Introduction
2. Results
2.1. DNA Replication Is Significantly Increased in the Absence of p21
2.2. Oncolytic Viral DNA Replication Foci Form Earlier in p21−/− Cells
2.3. Viral Transcription and Late Gene Translation Is Higher in p21−/− Cells
2.4. HCT-116 p21 WT Infected Cells Produce Smaller Plaques Which Develop Slower
2.5. Lower Amount of Oncolytic Virus in p21 Intact Cells Is Independent of Infection Time
3. Materials and Methods
3.1. Cell Culture and Antibodies
3.2. Indirect Immunofluorescence
3.3. Western Blot Analysis
3.4. Viral Stock Preparation and Real-Time PCR
Fiber Sense | CCCATTGGGGGATACAAAGGGAGGA |
Fiber Antisense | GCAGATGAAGCGCGCGCAAGACCGT |
E4 Sense | GTAATTCACCACCTCCCGGTA |
E4 Antisense | GGCTCTCCACTGTCATTGTTC |
Actin Sense | GTACCACTGGCATCGTGATGGACT |
Actin Antisense | CCGCTCATTGCCAATGGTGAT |
3.5. Plaque Assay
3.6. Output to Input Assay
3.7. Nanostring nCounter Assay
3.8. Crystal Violet Staining
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Höti, N.; Johnson, T.J.; Chowdhury, W.H.; Rodriguez, R. Loss of Cyclin-Dependent Kinase Inhibitor Alters Oncolytic Adenovirus Replication and Promotes More Efficient Virus Production. Cancers 2018, 10, 202. https://doi.org/10.3390/cancers10060202
Höti N, Johnson TJ, Chowdhury WH, Rodriguez R. Loss of Cyclin-Dependent Kinase Inhibitor Alters Oncolytic Adenovirus Replication and Promotes More Efficient Virus Production. Cancers. 2018; 10(6):202. https://doi.org/10.3390/cancers10060202
Chicago/Turabian StyleHöti, Naseruddin, Tamara Jane Johnson, Wasim H. Chowdhury, and Ronald Rodriguez. 2018. "Loss of Cyclin-Dependent Kinase Inhibitor Alters Oncolytic Adenovirus Replication and Promotes More Efficient Virus Production" Cancers 10, no. 6: 202. https://doi.org/10.3390/cancers10060202