Expression of the Reverse Transcriptase Domain of Telomerase Reverse Transcriptase Induces Lytic Cellular Response in DNA-Immunized Mice and Limits Tumorigenic and Metastatic Potential of Murine Adenocarcinoma 4T1 Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plasmids
2.2. Peptides and Recombinant Proteins Used for Immunoassays
2.3. Transient Expression of TERT in Mammalian Cells
2.4. Generation of 4T1luc2 Derivatives Expressing rtTERT by Lentiviral Transduction
2.5. Extraction of Nucleic Acids and Analysis of Genomic rtTERT Inserts in 4T1luc2 Daughter Clones
2.6. Reverse Transcription and Analysis of rtTERT mRNA Expression by Semiquantitative PCR
2.7. Analysis of Expression of Endogenous TERT in 4T1luc2 Clones by Immunofluorescent Microscopy
2.8. Assessment of Genetic Stability of 4T1luc2 Clones
2.9. Cell Cycle Analysis of 4T1luc2 Clones
2.10. Animals and Animal Experiments
2.11. DNA Immunization of Mice
2.12. In Vivo Bioluminescent Imaging
2.13. End Point of DNA Immunization Experiment
2.14. Assessment of Cellular Immune Response
2.15. Assessment of Anti-TERT Antibody Response
2.16. Assessment of In Vivo Tumorigenicity of 4T1luc2 Clones Expressing rtTERT
2.17. End Point of Tumor Challenge Experiment, Collection of Mouse Organs, and Rapid Ex Vivo Assessment of Metastases
2.18. Evaluation of Cellular Response against TERT in Mice Implanted with 4T1luc2 Clones
2.19. Tumor Histology and Ex Vivo Assessment of the Metastases
2.20. Statistical Analysis
3. Results
3.1. Design and Expression of Synthetic Rat TERT Gene
3.2. TERT Epitope Map and Selection of TERT-Derived Peptides for the Analysis of Anti-TERT Immune Response
3.3. Immunization and In Vivo Assessment of the Development of Immune Response by Bioluminescent Imaging
3.4. Assessment of Cellular Immune Response
3.5. Antibody Response
3.6. Generation of 4T1luc2 Clones Expressing RT Domain of Rat TERT
3.7. Tumorigenic Potential of 4T1luc2 Clones Expressing RT-TERT
3.8. Metastatic Potential of 4T1luc2 Clones Expressing rtTERT
3.9. Cellular Immune Response against Epitopes of TERT in Mice Implanted with rtTERT-Expressing 4T1luc2 Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Data Availability
References
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Name | Amino Acid (aa) Sequence | Position in Rat TERT, 1st and Last aa Residue | Identity to Mouse TERT, Common aa/Total aa (%) | Identity to Human TERT, Unique aa/Total aa (%) | Class I Score for H2 Db Mice (IEDB) | Class I Score for H2 Dd Mice (IEDB) | The Highest Class II Scores (IEDB) | T Cell Epitopes Localized in the Region |
---|---|---|---|---|---|---|---|---|
TERT 1 | PPSLTGARRLVEIIFLGSRPRTSGPFC | 356–382 | 24/27 (89) | 19/27 (70) | 0.47 | 0.58 | 1.28–1.33 | [30,31] |
TERT 2 | ILAMFLFWLMDTYVVQLLRSFFYITETT | 530–558 | 26/28 (93) | 22/28 (79) | 0.3727 | 0.349 | 6.48–9.01 | [30,32,33,34,35,36] |
TERT 3 | QKNRLFFYRKSVWSKLQSIGIRQQL | 559–584 | 24/25 (96) | 24/25 (96) | −0.185 | −0.268 | 2.65–7.13 | [30,34,35,36,37,38] |
TERT 4 | DTWLAMPICRLRFIPK | 600–616 | 16/16 (100) | 7/16 (44) | 0.31 | 0.19 | 9.44 | [32,39] |
TERT 5 | SLLHFFLRFVRHSVVKIDGRFYVQ | 791–815 | 16/24 (67) | 12/24 (50) | 0.379 | 0.4973 | 9.4 | [30,36] |
TERT 6 | QQDGLLLRFVDDFLLVTPHL | 845–865 | 19/20 (95) | 17/20 (85) | 0.32 | 0.438 | >10 | [33,34,35] |
TERT 7 | KTVVNFPVETGALGGAAPHQLPAHCLFPW | 888–917 | 26/29 (90) | 22/29 (76) | 0.2196 | 0.5080 | 7.75–10.73 | [30,31,37] |
TERT 8 | LGGAAPHQLPAHCLFPWCGLLLDTRTLE | 901–929 | 26/28 (93) | 23/28 (82) | 0.355 | 0.333 | 11.25 | [30,31,33,37] |
TERT 9 | FLDLQVNSLQTVCINIYKIFLLQAYRFHACVI | 973–1001 | 32/32 (100) | 29/32 (90) | 0.176 | 0.1579 | 7.81–10.77 | [30,31,33,34,35,36,38] |
Clones of 4T1luc2 Cells with rtTERT Inserts, Full Name | Abbreviated Name | Nn of Genomic Copies of rtTERT by Actb | Nn of Genomic Copies of rtTERT by Mstn | rtTERT mRNA Expression Relative to HPRT1 |
---|---|---|---|---|
4T1luc2_rtTERT_B5 | B5 | 0.57 ± 0.08 | 0.45 ± 0.02 | 1.00 ± 0.00 |
4T1luc2_rtTERT_C6 | C6 | 1.33 ± 0.14 | 1.05 ± 0.09 | 6.56 ± 1.63 |
4T1luc2_rtTERT_H9 * | H9 | 2.96 ± 0.24 | 1.66 ± 0.19 | 15.28 ± 1.72 |
4T1luc2_rtTERT_F1 | F1 | 0.70 ± 0.06 | 0.70 ± 0.06 | 5.95 ± 0.70 |
4T1luc2 | 4T1luc2 | N/A | N/A | N/A |
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Jansons, J.; Bayurova, E.; Skrastina, D.; Kurlanda, A.; Fridrihsone, I.; Kostyushev, D.; Kostyusheva, A.; Artyuhov, A.; Dashinimaev, E.; Avdoshina, D.; et al. Expression of the Reverse Transcriptase Domain of Telomerase Reverse Transcriptase Induces Lytic Cellular Response in DNA-Immunized Mice and Limits Tumorigenic and Metastatic Potential of Murine Adenocarcinoma 4T1 Cells. Vaccines 2020, 8, 318. https://doi.org/10.3390/vaccines8020318
Jansons J, Bayurova E, Skrastina D, Kurlanda A, Fridrihsone I, Kostyushev D, Kostyusheva A, Artyuhov A, Dashinimaev E, Avdoshina D, et al. Expression of the Reverse Transcriptase Domain of Telomerase Reverse Transcriptase Induces Lytic Cellular Response in DNA-Immunized Mice and Limits Tumorigenic and Metastatic Potential of Murine Adenocarcinoma 4T1 Cells. Vaccines. 2020; 8(2):318. https://doi.org/10.3390/vaccines8020318
Chicago/Turabian StyleJansons, Juris, Ekaterina Bayurova, Dace Skrastina, Alisa Kurlanda, Ilze Fridrihsone, Dmitry Kostyushev, Anastasia Kostyusheva, Alexander Artyuhov, Erdem Dashinimaev, Darya Avdoshina, and et al. 2020. "Expression of the Reverse Transcriptase Domain of Telomerase Reverse Transcriptase Induces Lytic Cellular Response in DNA-Immunized Mice and Limits Tumorigenic and Metastatic Potential of Murine Adenocarcinoma 4T1 Cells" Vaccines 8, no. 2: 318. https://doi.org/10.3390/vaccines8020318
APA StyleJansons, J., Bayurova, E., Skrastina, D., Kurlanda, A., Fridrihsone, I., Kostyushev, D., Kostyusheva, A., Artyuhov, A., Dashinimaev, E., Avdoshina, D., Kondrashova, A., Valuev-Elliston, V., Latyshev, O., Eliseeva, O., Petkov, S., Abakumov, M., Hippe, L., Kholodnyuk, I., Starodubova, E., ... Isaguliants, M. (2020). Expression of the Reverse Transcriptase Domain of Telomerase Reverse Transcriptase Induces Lytic Cellular Response in DNA-Immunized Mice and Limits Tumorigenic and Metastatic Potential of Murine Adenocarcinoma 4T1 Cells. Vaccines, 8(2), 318. https://doi.org/10.3390/vaccines8020318