The p53 gene is highly crucial in cell biology, and thus the realm of cell replacement therapy. The function of this gene is to control the cell cycle and stop tumor formation, which resulted in its nickname the “tumor suppressor gene.”
Author Photos (left to right): Sushil K. Jaiswal, Ph.D., John J. Oh, post-baccalaureate trainee, and Melvin L. DePamphilis, Ph.D. Image Credit: AlphaMed Press.
But earlier attempts to determine whether the p53 gene is responsible for programmed cellular death (apoptosis)—caused by DNA damage in pluripotent embryonic stem cells (ESCs)—generated contradictory results. Preliminary studies claimed that it was not, and subsequent studies concluded that it was.
Published recently in the Stem Cells journal, a new study sifts through this maze of contradictions to eventually establish that the numerous roles of the p53 gene in the regulation and apoptosis of cell cycle are acquired during the differentiation of pluripotent stem cells.
Melvin L. DePamphilis, Ph.D., Section Chief of Eukaryotic DNA Replication at the National Institute of Child Health & Human Development, Bethesda, Maryland, performed the study together with his Institute collaborators Sushil K. Jaiswal, Ph.D., and John J. Oh, post-baccalaureate trainee.
If ESCs are to be used to form the basis of cell replacement therapies, then identifying the role or roles of p53 in pluripotent stem cells is essential, Our goal was to finally resolve this question of whether p53 is essential for inducing cell cycle arrest and/or apoptosis prior to ESC differentiation.”
Melvin L. DePamphilis PhD, Section Chief of Eukaryotic DNA Replication, National Institute of Child Health & Human Development
Most of the previous studies employed a chemotherapy drug, known as Adriamycin/doxorubicin (ADR), to cause apoptosis. The effects of ADR lead to DNA replication and mitosis, resulting in the build-up of double-stranded DNA breaks in proliferating cells. The p53 gene can be triggered by damaged DNA to begin apoptosis and inhibit the duplication of damaged chromosomes.
As the p53 levels increase, they trigger the production of a protein known as p21, which stops the cycle governed by the p53 gene. It does this by attaching to and preventing the activity of the Cdk/cyclin complexes, the function of which is to turn off the cell cycle and permit time to repair the damaged DNA.
Using a 40-fold range of ADR concentrations and also other chemotherapy drugs (WX8 and staurosporine), the research team from the Institute similarly induced apoptosis as part of the current study. These drugs were all tested across a 50-fold range of cell seeding densities.
To prevent the possibility that conclusions relied on either the source or derivation of p53-/- ESCs, wild-type as well as p53-/- ESCs—extracted directly from mouse blastocysts—were defined in parallel with ESCs where the p53 genes were removed in vitro from ESCs harboring conditional p53 gene knockouts.
According to Dr. Jaiswal, “ESCs isolated from p53-/- blastocysts presented a chronic phenotype, whereas p53-/- ESCs engineered in vitro presented an acute phenotype that could be compared directly with their parental ESCs.”
The outcomes revealed that irrespective of derivation, the ESCs did not need p21, p53, or PUMA (another important protein implicated in apoptosis induced by the p53 gene) either to stimulate the G2-checkpoint, which makes sure that cells do not trigger mitosis until the damaged DNA is repaired, or to go through apoptosis quickly and efficiently.
The effects of ADR concentration and cell confluency were marginal, but the effects of cell differentiation were dramatic; p53 dependent regulation of cell division and apoptosis were acquired during p53-dependent cell differentiation.”
John J. Oh, Post-Baccalaureate Trainee, National Institute of Child Health & Human Development
These outcomes made the scientists conclude that the latest study “unequivocally” proves that the numerous roles of p53 in the regulation and apoptosis of cell cycle are initially acquired during the differentiation of pluripotent stem cells.
The new report by Jaiswal et al. is extremely important in reconciling contradictory results concerning the role of p53 in the stem cell field.”
Dr Jan Nolta, Editor-in-Chief, Stem Cells
“The authors have now definitely shown that the importance of p53 in cell cycle arrest and initiation of apoptosis is first acquired during the differentiation of pluripotent stem cells,” Dr Nolta concluded.
Jaiswal, S. K., et al. (2020) Cell cycle arrest and apoptosis are not dependent on p53 prior to p53-dependent embryonic stem cell differentiation. Stem Cells. doi.org/10.1002/stem.3199.