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A 60 kd MDM2 isoform is produced by caspase cleavage in non-apoptotic tumor cells
R Pochampally1, B Fodera, L Chen
1Louisiana State University Medical Center, Department of Microbiology, New Orleans, 70112, USA.
Abstract:
The MDM2 oncogene product is a regulator of the p53 tumor suppressor. MDM2 is cleaved by Caspase 3 (CPP32) during apoptosis after aspartic acid-361, generating a 60 kd fragment. Here we report that human tumor cell lines often express high levels of a 60 kd MDM2 isoform (p60) in the absence of apoptosis. We demonstrate that p60 is a product of caspase cleavage of full length MDM2 after residue 361. The protease that cleaves MDM2 in non-apoptotic cells appears to be distinct from the apoptosis-specific Caspase 3, since Caspase 3 substrate poly(ADP-ribose) polymerase (PARP) is not cleaved in cells producing p60. The p60 form of MDM2 is a significant fraction of the p53-bound MDM2 protein in certain tumor cells, suggesting that it functions in the regulation of p53. p60 is also detected in breast tumors overexpressing MDM2. These observations suggest that MDM2 is regulated by caspase processing in non-apoptotic cells, and may account for the MDM2 proteins of similar mobility seen in tumors and other cell lines.
Insights
A 60 kDa MDM2 protein fragment (p60) is generated in non-apoptotic tumor cells via caspase cleavage. This p60 isoform regulates the p53 tumor suppressor and is detected in breast tumors.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- MDM2 oncogene product regulates the p53 tumor suppressor.
- MDM2 is cleaved by Caspase 3 during apoptosis, yielding a 60 kDa fragment.
- High levels of a 60 kDa MDM2 isoform (p60) are observed in tumor cells without apoptosis.
Purpose of the Study:
- Investigate the generation and function of the p60 MDM2 isoform in non-apoptotic tumor cells.
- Determine if p60 is produced by Caspase 3 or a distinct protease.
- Assess the role of p60 in p53 regulation and its presence in human tumors.
Main Methods:
- Western blot analysis to detect MDM2 isoforms and cleaved PARP.
- Caspase activity assays.
- Immunoprecipitation to study p53-MDM2 interactions.
Main Results:
- Human tumor cell lines express a 60 kDa MDM2 isoform (p60) in the absence of apoptosis.
- p60 is generated by caspase cleavage of full-length MDM2 after residue 361.
- The protease cleaving MDM2 in non-apoptotic cells is distinct from Caspase 3, as PARP remains uncleaved.
- p60 is a significant fraction of p53-bound MDM2 in some tumor cells.
- p60 is detected in breast tumors overexpressing MDM2.
Conclusions:
- MDM2 is regulated by caspase processing in non-apoptotic cells, producing the p60 isoform.
- The p60 MDM2 isoform likely functions in p53 regulation within tumor cells.
- This non-apoptotic caspase processing may explain MDM2 protein variants observed in tumors.