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See related article by Brown and Wouters, Cancer Res 1999;59:1391–9.

In 1999, a review on apoptosis, p53, and tumor cell sensitivity to anticancer agents was published in Cancer Research (1). TP53 is one of the most commonly mutated tumor suppressor genes in human cancer. This review accurately encapsulates the body of data collected at the time with regard to cells lacking p53 and their sensitivity to anticancer agents using short-term assays. Importantly, the authors of this review, Brown and Wouters, stress the importance of using long-term clonogenic assays to reveal the true sensitivity of cells to anticancer agents. This review in part led to a flurry of further investigation on p53 and its role in cancer and sensitivity to radiation, chemotherapy, and other anticancer agents.

The major arguments laid out by the authors of this review are: (i) short-term assays can underestimate overall cell killing; (ii) p53 status did not seem to affect the sensitivity of cells of nonhematologic origin to genotoxic stress. The conclusions were contrary to the belief in the cancer field that tumor cells bearing p53 mutations were resistant to apoptosis and to cancer treatment (2–5). This review was written as a cautionary tale for investigators moving their studies from tissue culture–based work to in vivo mouse models. Many times, moving results from cell-based assays does not translate into effective use in vivo. Although the authors made very important points regarding the use of long-term clonogenic assays to assess the sensitivity of tumor cells to genotoxic agents, what was lacking at the time of publication were in vivo knockin mouse models with the p53 mutations that are commonly detected in human cancer patients. Over the last 17 years, several mouse models with p53 point mutations have been genetically engineered (6–9); two additional p53 family members, p63 and p73, were identified and found to contribute to p53 tumor suppression (10). Using these additional mouse models, novel insights and therapies to target the p53 pathway have been made.

Two landmark articles with p53 hotspot mutations corresponding to p53R175H and p53R273H provided evidence of p53-mutant gain of function in cancer (7, 9). These mice displayed a distinct tumor spectrum to mice lacking p53, and also, tumors from these mice with p53 mutations metastasized (7, 9). Generation of these models also led to the ability to reactivate p53 in p53 nullizygous mice (11) and in those with mutant p53 (12) as a means for therapy. These mouse models show distinct mechanisms for p53 tumor suppression in different tumor types. Upon reactivation of p53 in p53 nullizygous mice, induction of apoptosis was critical for regression of lymphomas, while induction of cellular senescence is the key mechanism for regression of sarcomas (11); however, in mice with a p53 missense mutation, reactivation of p53 kept tumors from progressing but did not completely cause tumor regression (12). These results clearly indicate the differences between loss of p53 and the gain-of-function and dominant-negative effects of point mutant p53. Other mouse models with p53 mutations in crucial sites of acetylation of the protein have revealed other biological functions, such as metabolism (8) and ferroptosis (6), and are important for p53 tumor-suppressive functions. In many ways, the results of these in vivo experiments could have been predicted on the basis of the long-term clonogenic assays described by Brown and Wouters, and the result that apoptosis did not play a significant role in genotoxic sensitivity.

In the late 1990s, the p53 family members, p63 and p73, were discovered (13). These genes were found to have overlapping functions with p53 (10, 13–15). Moreover, all three p53 family members were found to be necessary for p53 to execute the full apoptotic program (10), and mice deficient for combinations of p53 family members had increased tumor burden and metastasis (16). Further investigation on the p53 family members revealed the presence of the isoforms of p63 and p73 (13). Importantly, groups of isoforms with opposing functions existed. There were some isoforms, which more closely resemble p53, known as the TA isoforms of p63 and p73. They are named TA isoforms due to the N-terminal acidic transactivation (TA) domain. There also exist isoforms lacking this domain known as the ΔN isoforms of p63 and p73. In cell culture assays, the TA isoforms were found to have proapoptotic functions, whereas the ΔN isoforms had prosurvival functions (13–15). Recently, genetically engineered mouse models have unequivocally shown that TAp63 and TAp73 are tumor suppressor genes through their abilities to regulate miRNA biogenesis, senescence, and metabolism (17–21) and that ΔNp63 and ΔNp73 are oncogenes though their activities in metabolic reprogramming (20, 22). These findings are important in the context of one of the observations highlighted in the Brown and Wouters review, that is, that mutations in p53 have been shown to confer sensitivity to drugs, such as nitrogen mustard and cisplatin (23, 24). In the study with murine teratocarcinoma cells, p53 protected against cisplatin-induced apoptosis, yet sensitized to cell killing determined by colony formation (24). The authors provide an argument due to the levels of cisplatin used and that may have yielded no apoptosis followed by lower colony formation. One other explanation could be the activity of the p53 family members and their isoforms, which have not been tested in this system.

The p63 and p73 mouse models described above have led to an understanding of the interplay of the p53 family not only in apoptosis, but in other antitumorigenic functions, such as metabolism (20). This knowledge has allowed the design of potential novel therapies for the p53 pathway. These include the use of antidiabetic drugs, such as pramlintide (20), and miRNA therapies (25). The information detailed in the review by Brown and Wouters is still relevant today and is crucial to build a clear understanding of the long-term efficacy of these treatments using cell-based systems prior to testing using in vivo mouse models.

In summary, the review by Brown and Wouters provided the foundation for further investigation by the p53 field to use not only more sophisticated methods to measure tumor cell sensitivity (long-term assays instead of short-term ones) but also more sophisticated mouse models to unveil sensitivity to anticancer treatments. Furthermore, the discovery of the p53 family members, p63 and p73, around the time of the publication of the review by Brown and Wouters, led to further insights and explanations to some of the puzzling findings on p53 status and sensitivity to genotoxic stress.

No potential conflicts of interest were disclosed.

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