Inhibition of p53-Dependent Apoptosis in Cancer Therapy
Inhibition of p53-Dependent Apoptosis in Cancer Therapy
By Ryan Handzlik
The p53 tumor suppressor gene is inactivated in a variety of human cancers at a high frequency. When oncogenic mutations occur, the cell employs numerous protective mechanisms limiting the expansion of genomically unstable cells by suppressing proliferation or initiating a degradative response.
Expression of p53 in cells with metastatic potential stimulates growth arrest or an apoptotic response. Factors determining the type of response mechanism that occurs depends on cell type, cell environment, and factors such as oncogene expression.3 There is significant evidence linking p53 overexpression to side effects associated with cancer treatment in healthy irradiated cells. An understanding of p53 transcriptional activation, and its primary upstream and downstream effector genes, has led to numerous proposals for curtailing p53-associated side effects. Komarov and colleagues proposed that an effective p53 inhibitor can potentially reverse the side effects associated with cancer treatment.
Background
Transcription of the p53 gene is regulated by several upstream protein kinases. ATM and DNA-dependent protein kinase (DNA-PK) are two examples of upstream effector proteins that trigger a number of cell cycle checkpoints leading to growth arrest, DNA repair, and apoptosis. The kinases mediate a response to DNA damage by regulating the expression of the p53 gene through a predominantly unknown series of pathways.3 In contrast, the Mdm-2 protein inactivates p53 by binding directly to it, or stimulating its degradative pathway. There is evidence that Mdm-2 is amplified in a variety of tumors.3
DNA-PK binds to Mdm-2 and prevents inactivation of p53. Therefore, DNA-PK plays an important role in stabilizing p53 so that it can function as a cell cycle checkpoint in the mitotic cell. The p19 gene, a member of the INK4 tumor suppressor family, targets Mdm-2 and stabilizes functional p53. Recent evidence has linked its mutation or deletion to a variety of human cancers.3
Post-transcriptional regulators of p53 operate through direct binding and inhibition. E1B proteins synthesized by the adenovirus suppress p53 function by post-transcriptional inactivation following infection. E1B19K, a product of the adenovirus, is a functional homologue of Bcl-2 and will be discussed later.3 E6, a product of the papillomavirus, also has the capacity to bind and inactivate p53. In addition, the simian virus 40 large T antigen has also been associated with direct p53 inactivation. The products of these p53 inhibitory viral genomic sequences do not necessarily prevent apoptosis, but most likely delay p53-mediated responses in the infected cell.
The p53 gene mediates cell cycle arrest by activating downstream effector genes including GADD45 and Waf1/p21.2 Both Gadd45 and Waf1/p21 levels are overexpressed following exposure to radiation. It has been determined that a consensus binding sequence on p53 binds and regulates the expression of the human Gadd45 gene.2 The p53 gene also acts as a transcription factor for the Waf1/p21 gene. As p53 levels increase following radiation, p21 levels increase in a dose responsive manner.2 It has been proposed that ionizing radiation decreases cellular growth through cellular mechanisms involving p53 and p21. Expression of p53 and p21 are associated with cell cycle delay during G1-S stages of the cell cycle. The p21 gene inhibits the formation of the cyclinD/cdk4 complex. Without formation of the protein complex, no subsequent phosphorylation of pRB (retinoblastoma gene) occurs and cell cycle progression is inhibited. Therefore, the transcriptional activation of p21 mediated by p53 halts cell cycle progression and plays an integral role in promoting G1 arrest following cell irradiation. There is evidence that G1 arrest is accompanied by an increase in p53 following irradiation in cultured thyroid cells.2 These results suggest that radiation-induced G1 arrest is p53 dependent.2
p53 and Apoptosis
The p53 gene also initiates a series of cellular responses leading to apoptosis following DNA damage. It has been reported that cellular exposure to ionizing radiation and chemotherapeutic agents will result in the initiation of a signal transduction pathway that increases p53 levels in response to DNA damage.1 Transcriptional regulation of the bcl-2 and bax gene appear to be primary targets in the mediation of the cellular response. Mediation of the bcl-2/bax gene ratio by p53 appears to be a major factor in determining whether a cell enters apoptosis.1 Specifically, upregulation of bcl-2 results in growth arrest, while bax-induced expression promotes apoptosis.1 There is evidence that overexpression of functional bcl-2 and activated c-myc genes cooperate in transgenic mice to promote extended cell growth and tumorigenesis. Overexpression of bcl-2 and a variety of oncogenes have also been associated with tumor formation early in life. These results suggest that bcl-2 overexpression promotes cell survival and apoptotic inhibition. It is responsible for cellular quiescence and inhibits reentry into the cell cycle.3 In contrast, the p53-induced Bax subfamily (Bax, Bak, and Bok) promotes programmed cell death in response to apoptotic factors. It is hypothesized that the balance between apoptotic and anti-apoptotic factors determines whether or not a cell enters apoptosis.
So, what determines whether or not a cell enters p53-dependent apoptosis following genotoxic insult? Recent evidence tends to associate tissue specific initiating signals with programmed cell death response. It has been shown that the induced expression of p53 initiates a series of pathways leading to either apoptosis or cell cycle arrest. Current models suggest that upstream initiating signals may contribute to the regulation of p53, and apoptotic p53 induction is determined in a tissue specific manner. Midgley and colleagues reported two important conclusions concerning p53 induction in irradiated mice. They were able to confirm that some tissues show a dramatic increase in p53 in response to radiation while others do not.4 In addition, they confirmed that select tissues responded to p53 induction with apoptosis (spleen, thymus).4 This means that both the induction of p53 and the response to p53 are regulated in a tissue specific manner.4
Therapeutic Approaches to Cancer Treatment Modalities
The problems associated with chemotherapy and radiation therapy are demonstrated through numerous side effects linked to the p53-mediated apoptotic response. Radiation is used to treat tumors in tissues that possess deficient p53 genes. Side effects associated with radiation occur in p53-sensitive tissues immediately following radiation. Most therapies are designed to control these side effects by suppressing normal p53 activity during cancer treatment.
Radiation only has a therapeutic effect on those tissues that lack functional p53. For example, the healthy tissues of p53-deficient mice are known to suffer less damage than healthy tissues of wild type p53 mice following genotoxic insult.5 The results show a higher rate of survival among p53 deficient mice than among the wild-type.5 These experiments suggest that p53 contributes to the toxic side effects associated with cancer treatment. For this reason, potential therapeutic agents have been designed around the suppression of p53 function for the purpose of alleviating the toxic side effects associated with cancer treatment.
Several therapeutic approaches have been designed around the functional inhibition of p53. It was mentioned earlier that viral infection can result in the accumulation of E1B proteins. Proteins produced by the adenovirus have the capacity to bind and inactivate p53 resulting in apoptotic suppression. This method of inhibiting p53-dependent apoptosis has recently been exploited for its therapeutic potential in cancer treatment.3 Other approaches explored for their therapeutic potential include the manipulation of downstream effectors of p53 as opposed to the tumor suppressor gene itself. For example, inhibition or downregulation of bax is a potential mechanism for apoptotic suppression. These therapeutic approaches are designed with the intent of eliminating the apoptotic response mechanism resulting from genotoxic insult during cancer treatment.
Komarov and colleagues successfully designed an inhibitor of p53 that can potentially be used to alleviate many of the side effects associated with cancer treatment. They examined the effect of a p53 antagonist in an irradiated cell, and discovered that it prevented apoptosis. The p53 inhibitor, pifithrin (PFT), protected mice from the lethal genotoxic stress associated with cancer treatment without promotion of subsequent tumors in healthy cells. They found that PFT treatment resulted in an increase in the long-term survival of cells due to suppression of p53-dependent apoptosis.
If PFT therapy is evaluated on the basis of the programmed cell death response, treated cells have a higher survival rate than untreated cells that would normally undergo apoptosis. Mice pretreated with PFT lost less weight than untreated mice following exposure to radiation.5 These results suggest that the drug prevents the apoptotic response associated with radiation in p53 wild type cells. PFT did not rescue p53-null mice following irradiation.5 These results suggest that PFT acts through a p53-dependent mechanism. The response associated with temporary suppression of p53 by PFT in wild type mice appears to be different from the response in p53 deficient mice following radiation. It is also suggested that PFT reduces the p53-dependent suppression of DNA replication in rapidly proliferating tissues following gamma irradiation.5 There is also evidence that untreated cells posed an increased risk for new cancer development. In addition, PFT proved to be an effective therapeutic agent by diminishing the apoptotic response of intestinal cells following irradiation.5
It was observed that PFT-dependent cell recovery following irradiation was dependent on the time and duration of treatment. Komarov et al reported that PFT application prior to irradiation had no protective effect. In contrast, PFT treatment for a short three-hour time period immediately following irradiation had a significant effect on cell recovery, while application for a prolonged 24-hour time period proved to be most effective.5 No protection occurred if treatment began three hours following irradiation. Their conclusion was that the p53 dependent apoptotic response is reduced within several hours, and completely eliminated within 24 hours of irradiation.5
Conclusion
Radiation therapy and chemotherapy have been employed as a form of cancer treatment for the purpose of eliminating unwanted cells predisposed to tumorigenesis. Most cells that have lost functional p53 through deletion or mutation are unable to respond to DNA damage or general genomic instability. Healthy cells that have the capacity to detect DNA damage resulting from genotoxic insult can initiate growth arrest and apoptosis. When these cells are irradiated during cancer treatment, unwanted cell death is triggered by a p53-dependent apoptotic protective mechanism.
In order to compensate for the inability of current therapeutic modalities to select for tumor specific cells during cancer treatment, the possibilty of utilizing pifithrin or other compounds with similar activity may be beneficial for reducing the side effects associated with cancer treatment. PFT has the potential to become an effective therapeutic agent because its effects are p53 dependent, and therefore should not affect the sensitivity of p53 deficient cells associated with cancer. (Mr. Handzlik is a graduate student at Roswell Park Cancer Institute, Buffalo, New York.)
References
1. Saito Y, Milross CG, Hittelman WN, et al. Int J Radiat Oncol Biol Phys 1997;38:623-631.
2. Namba H, Hara T, Tukazaki T, et al. Can Res 1995;55:2075-2080.
3. Evan G, Littlewood T. Science 1998;281:1317-1322.
4. Midgley CA, Owens B, Briscoe CV, et al. J Cell Sci 1995;108:1843-1848.
5. Komarov PG, Komarova EA, Kondratov RV, et al. Science 1999;285:1733-1737.
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