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Assays to measure p53-dependent and -independent apoptosis
Darren C Phillips1, Sean P Garrison, John R Jeffers
1Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA.
Abstract:
Paramount to the maintenance of normal tissue homeostasis is the induction of programmed cell death, otherwise known as apoptosis. Several disease states, including cancer, are characterized by an inability to remove unwanted cells due to a failure to commit to apoptosis. What is more, apoptosis is the central functional response behind many agents utilized in the treatment of cancer. Many of these antitumorigenic agents rely on the activation of the tumor suppressor p53. As the physiological "guardian of the genome," p53's normal function is to sense stressed or damaged cells and arrest proliferation, allowing time for cellular repair. However, if the damage is excessive, cells are removed prior to the onset of malignancy through apoptosis. Current chemotherapeutic strategies manipulate this property by damaging cells and turning on p53's transcriptional function, which consequently upregulates the expression of proapoptotic proteins such as Puma. We have also demonstrated that Puma is capable of inducing apoptosis independent of p53. In this regard, defects in the apoptotic machinery or in p53 function itself lead to a resistant phenotype that in cancer results in chemotherapeutic failure, and more often than not, poor prognosis. This chapter describes protocols for the determination of p53-dependent and -independent apoptosis utilizing primary cells from genetically altered mice.
Insights
Programmed cell death, or apoptosis, is crucial for tissue health and cancer treatment. This study details methods to measure apoptosis, essential for understanding cancer therapy resistance.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Apoptosis, or programmed cell death, is vital for tissue homeostasis and is a key mechanism targeted by cancer therapies.
- Dysfunctional apoptosis contributes to cancer development and resistance to chemotherapy.
- The tumor suppressor p53 plays a critical role in initiating apoptosis in response to cellular damage.
Purpose of the Study:
- To describe protocols for assessing apoptosis.
- To investigate both p53-dependent and p53-independent apoptosis.
- To utilize primary cells from genetically modified mice for apoptosis studies.
Main Methods:
- Utilizing primary cells derived from genetically altered mouse models.
- Implementing protocols to determine p53-dependent apoptosis.
- Implementing protocols to determine p53-independent apoptosis.
Main Results:
- Apoptosis is essential for removing damaged cells and preventing cancer.
- Chemotherapeutic agents often function by activating p53 to induce apoptosis.
- Puma can induce apoptosis independently of p53, highlighting alternative apoptotic pathways.
- Defects in apoptosis or p53 function lead to chemoresistance and poor cancer prognosis.
Conclusions:
- Understanding apoptosis pathways is critical for improving cancer treatment efficacy.
- Protocols for assessing p53-dependent and -independent apoptosis are valuable tools in cancer research.
- Defects in apoptosis machinery contribute to therapeutic failure in cancer patients.
