p53 mutant mice that display early ageing-associated phenotypes.

The p53 tumour suppressor is activated by numerous stressors to induce apoptosis, cell cycle arrest, or senescence. To study the biological effects of altered p53 function, we generated mice with a deletion mutation in the first six exons of the p53 gene that express a truncated RNA capable of encoding a carboxy-terminal p53 fragment. This mutation confers phenotypes consistent with activated p53 rather than inactivated p53. Mutant (p53+/m) mice exhibit enhanced resistance to spontaneous tumours compared with wild-type (p53+/+) littermates. As p53+/m mice age, they display an early onset of phenotypes associated with ageing. These include reduced longevity, osteoporosis, generalized organ atrophy and a diminished stress tolerance. A second line of transgenic mice containing a temperature-sensitive mutant allele of p53 also exhibits early ageing phenotypes. These data suggest that p53 has a role in regulating organismal ageing.     

Participation of p53 cellular tumour antigen in transformation of normal embryonic cells

The cellular tumour antigen p53 is found at elevated levels in a wide variety of transformed cells (for reviews see refs 1, 2). Very little is yet known about the precise relationship of p53 to malignant transformation. Although the increase in p53 levels could be a secondary by-product of the transformed state, it is equally possible that p53 is actively involved in altering cellular growth properties, especially as it has been implicated in the regulation of normal cell proliferation. We sought to test whether p53 could behave in a manner similar to known genes in a biological test system, and we demonstrate here that p53 can cooperate with the activated Ha-ras oncogene to transform normal embryonic cells. The resultant foci contain cells of a markedly altered morphology which produce high levels of p53. Cell lines established from such foci elicit tumours in syngeneic animals.     

Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas

Although cancer arises from a combination of mutations in oncogenes and tumour suppressor genes, the extent to which tumour suppressor gene loss is required for maintaining established tumours is poorly understood. p53 is an important tumour suppressor that acts to restrict proliferation in response to DNA damage or deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence. Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies. To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma. We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions. The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. Our study indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth.     

Growth regulation of a cellular tumour antigen, p53, in nontransformed cells

Many transformed cells in culture have been found to express elevated levels of a cellular tumour antigen, termed p53. This protein has also been implicated in the regulation of cellular growth. For these reasons experiments were designed to examine the expression of p53 as quiescent cultures of nontransformed 3T3 fibroblasts were stimulated to reenter the cell cycle. Synchronous populations of cells were obtained by releasing a culture from density-dependent inhibition of growth with the addition of fresh serum. Steady-state levels of p53 protein and mRNA were measured as a function of time after addition of serum to quiescent cultures and the rate of synthesis of p53 protein was analysed at a number of time points. The results, reported here, demonstrate an increase in the synthesis and steady-state levels of p53 protein and mRNA prior to DNA synthesis in late G1, and suggest a role for p53 in the progression of cells from a growth-arrested state to an actively dividing state.     

Fbxw7/Cdc4 is a p53-dependent, haploinsufficient tumour suppressor gene

The FBXW7/hCDC4 gene encodes a ubiquitin ligase implicated in the control of chromosome stability. Here we identify the mouse Fbxw7 gene as a p53-dependent tumour suppressor gene by using a mammalian genetic screen for p53-dependent genes involved in tumorigenesis. Radiation-induced lymphomas from p53+/- mice, but not those from p53-/- mice, show frequent loss of heterozygosity and a 10% mutation rate of the Fbxw7 gene. Fbxw7+/- mice have greater susceptibility to radiation-induced tumorigenesis, but most tumours retain and express the wild-type allele, indicating that Fbxw7 is a haploinsufficient tumour suppressor gene. Loss of Fbxw7 alters the spectrum of tumours that develop in p53 deficient mice to include a range of tumours in epithelial tissues such as the lung, liver and ovary. Mouse embryo fibroblasts from Fbxw7-deficient mice, or wild-type mouse cells expressing Fbxw7 small interfering RNA, have higher levels of Aurora-A kinase, c-Jun and Notch4, but not of cyclin E. We propose that p53-dependent loss of Fbxw7 leads to genetic instability by mechanisms that might involve the activation of Aurora-A, providing a rationale for the early occurrence of these mutations in human cancers.     

Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6.

Wild-type p53 protein has many properties consistent with its being the product of a tumour suppressor gene. Although the normal roles of tumour suppressor genes are still largely unknown, it seems that they could be involved in promoting cell differentiation as well as in mediating growth arrest by growth-inhibitory cytokines. Hence, the abrogation of wild-type p53 expression, which is a common feature of many tumours, could eliminate these activities. We have now tested this notion by restoring the expression of p53 in a murine myeloid leukaemic cell line that normally lacks p53. The use of a temperature-sensitive p53 mutant allowed us to analyse cells in which the introduced p53 had either wild-type or mutant properties. Although there seemed to be no effect on differentiation, the introduction of wild-type p53 resulted in rapid loss of cell viability in a way characteristic of apoptosis (programmed cell death). The effect of wild-type p53 was counteracted by interleukin-6. Thus products of tumour suppressor genes could be involved in restricting precursor cell populations by mediating apoptosis.     

Protein synthesis required to anchor a mutant p53 protein which is temperature-sensitive for nuclear transport

The p53 protein is rendered temperature-sensitive by a point mutation. Rat cells transformed by this mutant p53 and an activated ras oncogene grow well at 37 degrees C but cease DNA synthesis and cell division when shifted to 32 degrees C. Immunostaining demonstrates that the mutant p53 protein is in the nucleus of the arrested cells at 32 degrees C but in the cytoplasm of the growing cells at 37 degrees C. This is the first example of a protein which is temperature-sensitive for nuclear transport. The translocation from cytoplasm to nucleus and vice versa occurs 6 h after temperature shift and is coincident with the inhibition of DNA synthesis; transport from cytoplasm to nucleus does not require protein synthesis. Remarkably, inhibition of protein synthesis at 37 degrees C also results in the rapid appearance of mutant p53 in the cell nucleus. These results suggest the presence of a short-lived protein responsible for holding p53 in the cytoplasm at 37 degrees C but not at 32 degrees C. Analysis of a non-temperature-sensitive mutant p53 protein shows that its cytoplasmic location is sensitive to protein synthesis inhibitors but not to temperature.     

Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization

The p53 tumour suppressor is a short-lived protein that is maintained at low levels in normal cells by Mdm2-mediated ubiquitination and subsequent proteolysis. Stabilization of p53 is crucial for its tumour suppressor function. However, the precise mechanism by which ubiquitinated p53 levels are regulated in vivo is not completely understood. By mass spectrometry of affinity-purified p53-associated factors, we have identified herpesvirus-associated ubiquitin-specific protease (HAUSP) as a novel p53-interacting protein. HAUSP strongly stabilizes p53 even in the presence of excess Mdm2, and also induces p53-dependent cell growth repression and apoptosis. Significantly, HAUSP has an intrinsic enzymatic activity that specifically deubiquitinates p53 both in vitro and in vivo. In contrast, expression of a catalytically inactive point mutant of HAUSP in cells increases the levels of p53 ubiquitination and destabilizes p53. These findings reveal an important mechanism by which p53 can be stabilized by direct deubiquitination and also imply that HAUSP might function as a tumour suppressor in vivo through the stabilization of p53.     

Localization of gene for human p53 tumour antigen to band 17p13

Recently the gene for the cellular tumour antigen p53, a phosphoprotein found in increased concentration in a variety of human cells, had been mapped to region 17q22 by in situ hybridization techniques and has been shown to translocate to the chromosome carrying the translocation [t(15; 17)] associated with acute promyelocytic leukaemia (APL). Based on this finding it has been postulated that this gene has a role in the pathogenesis of APL. Here we present evidence that the gene for p53 is not located on the long arm of chromosome 17, but maps to band 17p13. We therefore suggest that this gene is not directly involved in the chromosome translocation observed in APL.     

PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression

Functional impairment of T cells is characteristic of many chronic mouse and human viral infections. The inhibitory receptor programmed death 1 (PD-1; also known as PDCD1), a negative regulator of activated T cells, is markedly upregulated on the surface of exhausted virus-specific CD8 T cells in mice. Blockade of this pathway using antibodies against the PD ligand 1 (PD-L1, also known as CD274) restores CD8 T-cell function and reduces viral load. To investigate the role of PD-1 in a chronic human viral infection, we examined PD-1 expression on human immunodeficiency virus (HIV)-specific CD8 T cells in 71 clade-C-infected people who were naive to anti-HIV treatments, using ten major histocompatibility complex (MHC) class I tetramers specific for frequently targeted epitopes. Here we report that PD-1 is significantly upregulated on these cells, and expression correlates with impaired HIV-specific CD8 T-cell function as well as predictors of disease progression: positively with plasma viral load and inversely with CD4 T-cell count. PD-1 expression on CD4 T cells likewise showed a positive correlation with viral load and an inverse correlation with CD4 T-cell count, and blockade of the pathway augmented HIV-specific CD4 and CD8 T-cell function. These data indicate that the immunoregulatory PD-1/PD-L1 pathway is operative during a persistent viral infection in humans, and define a reversible defect in HIV-specific T-cell function. Moreover, this pathway of reversible T-cell impairment provides a potential target for enhancing the function of exhausted T cells in chronic HIV infection.     

Restoration of p53 function leads to tumour regression in vivo

Tumorigenesis is a multi-step process that requires activation of oncogenes and inactivation of tumour suppressor genes. Mouse models of human cancers have recently demonstrated that continuous expression of a dominantly acting oncogene (for example, Hras, Kras and Myc) is often required for tumour maintenance; this phenotype is referred to as oncogene addiction. This concept has received clinical validation by the development of active anticancer drugs that specifically inhibit the function of oncoproteins such as BCR-ABL, c-KIT and EGFR. Identifying additional gene mutations that are required for tumour maintenance may therefore yield clinically useful targets for new cancer therapies. Although loss of p53 function is a common feature of human cancers, it is not known whether sustained inactivation of this or other tumour suppressor pathways is required for tumour maintenance. To explore this issue, we developed a Cre-loxP-based strategy to temporally control tumour suppressor gene expression in vivo. Here we show that restoring endogenous p53 expression leads to regression of autochthonous lymphomas and sarcomas in mice without affecting normal tissues. The mechanism responsible for tumour regression is dependent on the tumour type, with the main consequence of p53 restoration being apoptosis in lymphomas and suppression of cell growth with features of cellular senescence in sarcomas. These results support efforts to treat human cancers by way of pharmacological reactivation of p53.     

Selective activation of p53-mediated tumour suppression in high-grade tumours

Non-small cell lung carcinoma (NSCLC) is the leading cause of cancer-related death worldwide, with an overall 5-year survival rate of only 10-15%. Deregulation of the Ras pathway is a frequent hallmark of NSCLC, often through mutations that directly activate Kras. p53 is also frequently inactivated in NSCLC and, because oncogenic Ras can be a potent trigger of p53 (ref. 3), it seems likely that oncogenic Ras signalling has a major and persistent role in driving the selection against p53. Hence, pharmacological restoration of p53 is an appealing therapeutic strategy for treating this disease. Here we model the probable therapeutic impact of p53 restoration in a spontaneously evolving mouse model of NSCLC initiated by sporadic oncogenic activation of endogenous Kras. Surprisingly, p53 restoration failed to induce significant regression of established tumours, although it did result in a significant decrease in the relative proportion of high-grade tumours. This is due to selective activation of p53 only in the more aggressive tumour cells within each tumour. Such selective activation of p53 correlates with marked upregulation in Ras signal intensity and induction of the oncogenic signalling sensor p19(ARF)( )(ref. 6). Our data indicate that p53-mediated tumour suppression is triggered only when oncogenic Ras signal flux exceeds a critical threshold. Importantly, the failure of low-level oncogenic Kras to engage p53 reveals inherent limits in the capacity of p53 to restrain early tumour evolution and in the efficacy of therapeutic p53 restoration to eradicate cancers.     

Identification of human brain tumour initiating cells

The cancer stem cell (CSC) hypothesis suggests that neoplastic clones are maintained exclusively by a rare fraction of cells with stem cell properties. Although the existence of CSCs in human leukaemia is established, little evidence exists for CSCs in solid tumours, except for breast cancer. Recently, we prospectively isolated a CD133+ cell subpopulation from human brain tumours that exhibited stem cell properties in vitro. However, the true measures of CSCs are their capacity for self renewal and exact recapitulation of the original tumour. Here we report the development of a xenograft assay that identified human brain tumour initiating cells that initiate tumours in vivo. Only the CD133+ brain tumour fraction contains cells that are capable of tumour initiation in NOD-SCID (non-obese diabetic, severe combined immunodeficient) mouse brains. Injection of as few as 100 CD133+ cells produced a tumour that could be serially transplanted and was a phenocopy of the patient's original tumour, whereas injection of 10(5) CD133- cells engrafted but did not cause a tumour. Thus, the identification of brain tumour initiating cells provides insights into human brain tumour pathogenesis, giving strong support for the CSC hypothesis as the basis for many solid tumours, and establishes a previously unidentified cellular target for more effective cancer therapies.     

Clonal deletion of V beta 14-bearing T cells in mice transgenic for mammary tumour virus.

Autoreactive T lymphocytes are clonally deleted during maturation in the thymus. Deletion of T cells expressing particular receptor V beta elements is controlled by poorly defined autosomal dominant genes. A gene has now been identified by expression of transgenes in mice which causes deletion of V beta 14+ T cells. The gene lies in the open reading frame of the long terminal repeat of the mouse mammary tumour virus.     

Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants

The c-Myc oncoprotein promotes proliferation and apoptosis, such that mutations that disable apoptotic programmes often cooperate with MYC during tumorigenesis. Here we report that two common mutant MYC alleles derived from human Burkitt's lymphoma uncouple proliferation from apoptosis and, as a result, are more effective than wild-type MYC at promoting B cell lymphomagenesis in mice. Mutant MYC proteins retain their ability to stimulate proliferation and activate p53, but are defective at promoting apoptosis due to a failure to induce the BH3-only protein Bim (a member of the B cell lymphoma 2 (Bcl2) family) and effectively inhibit Bcl2. Disruption of apoptosis through enforced expression of Bcl2, or loss of either Bim or p53 function, enables wild-type MYC to produce lymphomas as efficiently as mutant MYC. These data show how parallel apoptotic pathways act together to suppress MYC-induced transformation, and how mutant MYC proteins, by selectively disabling a p53-independent pathway, enable tumour cells to evade p53 action during lymphomagenesis.     

Virus-mediated killing of cells that lack p53 activity

A major goal of molecular oncology is to identify means to kill cells lacking p53 function. Most current cancer therapy is based on damaging cellular DNA by irradiation or chemicals. Recent reports support the notion that, in the event of DNA damage, the p53 tumour-suppressor protein is able to prevent cell death by sustaining an arrest of the cell cycle at the G2 phase. We report here that adeno-associated virus (AAV) selectively induces apoptosis in cells that lack active p53. Cells with intact p53 activity are not killed but undergo arrest in the G2 phase of the cell cycle. This arrest is characterized by an increase in p53 activity and p21 levels and by the targeted destruction of CDC25C. Neither cell killing nor arrest depends upon AAV-encoded proteins. Rather, AAV DNA, which is single-stranded with hairpin structures at both ends, elicits in cells a DNA damage response that, in the absence of active p53, leads to cell death. AAV inhibits tumour growth in mice. Thus viruses can be used to deliver DNA of unusual structure into cells to trigger a DNA damage response without damaging cellular DNA and to selectively eliminate those cells lacking p53 activity.     

Overproduction of p53 antigen makes established cells highly tumorigenic

The p53 cellular tumour antigen, long known to be overproduced in a variety of neoplastically transformed cells, was recently shown to be directly involved in transformation. Thus, p53 can complement activated Ha-ras in transforming secondary rat embryo fibroblasts into grossly altered, tumorigenic cells. Moreover, p53 can also be shown to possess immortalizing activity. Our previous results indicated, however, that the contribution of p53 to the transformation was not synonymous with immortalization, suggesting that the two activities of the protein are probably separable. We demonstrate here that this is indeed the case, as overproduction of p53 in an established cell line, while not causing gross morphological changes, endows these cells with an overt tumorigenic potential. Furthermore, the tumorigenic efficiency of such cell lines may be correlated with the extent of p53 over-production.