Canine Mammary Tumor Cell Lines Derived from Metastatic Foci Show Increased RAD51 Expression but Diminished Radioresistance via p21 Inhibition
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Cells and Cell Culture
2.2. Double Thymidine Block
2.3. Western Blot Analysis
2.4. Induction of DNA Damage
2.5. MTT Assay
2.6. Tumor Cell Migration Assay
2.7. Immunostaining of RAD51 Foci and Microscopy
2.8. cDNA Cloning, Sequencing, and Structure Analysis of Canine p21
2.9. Cell Cycle Analysis
2.10. Microsatellite Genotyping of CHMp and CHMm Using PCR and Amplified Fragment Length Polymorphism Analysis
2.11. p53 Tetramerization Reporter Assay
2.12. Statistical Analysis
3. Results
3.1. RAD51 Expression Was Different in Each Canine Mammary Tumor Cell Line
3.2. Cell Survival after Irradiation
3.3. Comparison of Migration Abilities after Radiation
3.4. CHMm Cells Had Reduced Ability to Form RAD51 Foci after Irradiation
3.4.1. Irradiation Does Not Lead to Cell Cycle Arrest in CHMm
3.4.2. Analysis of Cell Identity Using Microsatellite Analysis
3.4.3. Cloning and Structural Analysis of Canine PALB2
3.4.4. Confirmation of Cross-Reactivity of Anti-Human p21 Antibodies against Canine p21
3.4.5. p21 Expression Was Induced by DNA Damage in CHMp, but Not CHMm
3.4.6. The p21 Transcriptional Activation of CHMm Cells Was Significantly Reduced
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Shimakawa, K.; Ochiai, K.; Hirose, S.; Tanabe, E.; Michishita, M.; Sakaue, M.; Yoshikawa, Y.; Morimatsu, M.; Tajima, T.; Watanabe, M.; et al. Canine Mammary Tumor Cell Lines Derived from Metastatic Foci Show Increased RAD51 Expression but Diminished Radioresistance via p21 Inhibition. Vet. Sci. 2022, 9, 703. https://doi.org/10.3390/vetsci9120703
Shimakawa K, Ochiai K, Hirose S, Tanabe E, Michishita M, Sakaue M, Yoshikawa Y, Morimatsu M, Tajima T, Watanabe M, et al. Canine Mammary Tumor Cell Lines Derived from Metastatic Foci Show Increased RAD51 Expression but Diminished Radioresistance via p21 Inhibition. Veterinary Sciences. 2022; 9(12):703. https://doi.org/10.3390/vetsci9120703
Chicago/Turabian StyleShimakawa, Kei, Kazuhiko Ochiai, Sachi Hirose, Eri Tanabe, Masaki Michishita, Motoharu Sakaue, Yasunaga Yoshikawa, Masami Morimatsu, Tsuyoshi Tajima, Masami Watanabe, and et al. 2022. "Canine Mammary Tumor Cell Lines Derived from Metastatic Foci Show Increased RAD51 Expression but Diminished Radioresistance via p21 Inhibition" Veterinary Sciences 9, no. 12: 703. https://doi.org/10.3390/vetsci9120703