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Updated: Jul 2, 2025
Functional Characterization of Endogenously Expressed Human RYR1 Variants
Published on: June 9, 2021
Molecular characterization of ANKRD1 in rhabdomyosarcoma cell lines: expression, localization, and proteasomal
Emilija Milosevic1, Mirjana Novkovic1, Vittoria Cenni2,3
1Laboratory for Molecular Biology, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, 11042, Belgrade, Serbia.
Abstract:
Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children and adolescents. Respecting the age of the patients and the tumor aggressiveness, investigation of the molecular mechanisms of RMS tumorigenesis is directed toward the identification of novel therapeutic targets. To contribute to a better understanding of the molecular pathology of RMS, we investigated ankyrin repeat domain 1 (ANKRD1), designated as a potential marker for differential diagnostics. In this study, we used three RMS cell lines (SJRH30, RD, and HS-729) to assess its expression profile, intracellular localization, and turnover. They express wild-type ANKRD1, as judged by the sequencing of the open reading frame. Each cell line expressed a different amount of ANKRD1 protein, although the transcript level was similar. According to western blot analysis, ANKRD1 protein was expressed at detectable levels in the SJRH30 and RD cells (SJRH30 > RD), but not in the HS-729, even after immunoprecipitation. Immunocytochemistry revealed nuclear and cytoplasmic localization of ANKRD1 in all examined cell lines. Moreover, the punctate pattern of ANKRD1 staining in the nuclei of RD and HS-729 cells overlapped with coilin, indicating its association with Cajal bodies. We have shown that RMS cells are not able to overexpress ANKRD1 protein, which can be attributed to its proteasomal degradation. The unsuccessful attempt to overexpress ANKRD1 in RMS cells indicates the possibility that its overexpression may have detrimental effects for RMS cells and opens a window for further research into its role in RMS pathogenesis and for potential therapeutic targeting.
Insights
Ankyrin repeat domain 1 (ANKRD1) protein expression varies in pediatric Rhabdomyosarcoma (RMS) cells, with degradation limiting overexpression. This suggests ANKRD1 may be a therapeutic target for this childhood cancer.
Area of Science:
- Pediatric Oncology
- Molecular Pathology
- Cancer Biology
Background:
- Rhabdomyosarcoma (RMS) is a prevalent pediatric soft tissue cancer.
- Understanding RMS molecular mechanisms is key for identifying new therapeutic targets.
- Ankyrin repeat domain 1 (ANKRD1) is explored as a potential diagnostic marker.
Purpose of the Study:
- Investigate the expression, localization, and turnover of ANKRD1 in RMS.
- Determine if ANKRD1 can be overexpressed in RMS cells.
- Assess ANKRD1's potential as a therapeutic target.
Main Methods:
- Utilized three RMS cell lines (SJRH30, RD, HS-729).
- Performed gene sequencing, western blot, and immunocytochemistry.
- Analyzed ANKRD1 transcript and protein levels, localization, and degradation.
Main Results:
- All cell lines expressed wild-type ANKRD1 mRNA, but protein levels varied.
- ANKRD1 protein was detected in SJRH30 and RD cells, but not HS-729.
- ANKRD1 localized to the nucleus and cytoplasm, associating with Cajal bodies.
- RMS cells could not overexpress ANKRD1 due to proteasomal degradation.
Conclusions:
- ANKRD1 expression is tightly regulated in RMS cells, likely via proteasomal degradation.
- The inability to overexpress ANKRD1 suggests it may be detrimental to RMS cells.
- ANKRD1 warrants further investigation for its role in RMS pathogenesis and as a therapeutic target.
