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Expression of an IFN-Inducible Cellular Senescence Gene, IFI16, Is Up-Regulated by p53
Abstract and Figures
IFN-inducible IFI16 protein (encoded by IFI16 gene at 1q23.1) is the human member of the IFN-inducible structurally related p200 family proteins. Increased expression of the IFI16 protein, a positive modulator of p53-mediated transcription, in normal old human diploid fibroblasts (HDF) is associated with cellular senescence-mediated cell growth arrest. However, the underlying mechanisms that contribute to transcriptional activation of the IFI16 gene in old HDFs remain to be elucidated. Here, we reported that functional activation of p53 in normal young HDFs and p53-null Saos2 cell line resulted in transcriptional activation of the IFI16 gene. We identified a potential p53 DNA-binding site (indicated as IFI16-p53-BS) in the 5'-regulatory region of the IFI16 gene. Importantly, p53 bound to IFI16-p53-BS in a sequence-specific manner in gel-mobility shift assays. Furthermore, p53 associated with the 5'-regulatory region of the IFI16 gene in chromatin immunoprecipitation assays. Interestingly, p53 associated with the regulatory region of the IFI16 gene only on treatment of cells with DNA-damaging agents or in the old, but not in the young, HDFs. Importantly, our promoter-reporter assays, which were coupled with site-directed mutagenesis of IFI16-p53-BS, showed that p53 activates transcription of the IFI16 gene in HDFs through the p53 DNA-binding site. Together, our observations provide support for the idea that up-regulation of IFI16 expression by p53 and functional interactions between IFI16 protein and p53 contribute to cellular senescence.
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... PYCARD, binds AIM2 or IFI16 via its PYRIN domain and binds pro-caspase 1 via its CARD domain (reviewed in [14]). Interestingly, the gene for IFI16 has been shown to be a p53-regulated gene [15]. Hence, we explored possibility that the molecules forming at least one inflammasome type were expressed in A549 cells upon treatment with A + N or CPT, results are shown in Fig. 3C. ...
... Some genes coding for innate immunity proteins are regulated by p53. In addition to the aforementioned CASP1 [12] and IFI16 [15], p53 stimulates transcription of IRF5 [19], IRF7 [20], and ISG15 [21] among others. Thus, we hypothesized that p53 activated by A + N or CPT could stimulate the genes coding for proteins of innate immunity, which have not been so far identified as p53-regulated genes. ...
... Because the genes for IFI16 and IRF7 proteins are both regulated by p53 [15,20], we decided to explore if the STING protein, located in the signaling pathway between IFI16 and IRF7, can also be synergistically induced by actinomycin D and nutlin-3a or by CPT. Western blot analysis showed that this was indeed the case (Fig. 4A). ...
Actinomycin D and nutlin-3a (A + N) activate p53, partly through induction of phosphorylation on Ser392. The death of A549 cells induced by A + N morphologically resembles inflammation-inducing pyroptosis - cell destruction triggered by activated caspase-1. The treatment with A + N (or camptothecin) strongly upregulated caspase-1 and its two activators: IFI16 and NLRP1, however, caspase-1 activation was not detected. A549 cells may have been primed for pyroptosis, with the absence of a crucial trigger. The investigation of additional innate immunity elements revealed that A + N (or camptothecin) stimulated the expression of NLRX1, STING (stimulator of interferon genes) and two antiviral proteins, IFIT1 and IFIT3. IFI16 and caspase-1 are coded by p53-regulated genes which led us to investigate regulation of NLRP1, NLRX1, STING, IFIT1 and IFIT3 in p53-dependent mode. The upregulation of NLRP1, NLRX1 and STING was attenuated in p53 knockdown cells. The upsurge of the examined genes, and activation of p53, was inhibited by C16, an inhibitor of PKR kinase. PKR was tested due to its ability to phosphorylate p53 on Ser392. Surprisingly, C16 was active even in PKR knockdown cells. The ability of C16 to prevent activation of p53 and expression of innate immunity genes may be the source of its strong anti-inflammatory action. Moreover, cells exposed to A + N can influence neighboring cells in paracrine fashion, for instance, they shed ectodomain of COL17A1 protein and induce, in p53-dependent mode, the expression of gene for interleukin-7. Further, the activation of p53 also spurred the expression of SOCS1, an inhibitor of interferon triggered STAT1-dependent signaling. We conclude that, stimulation of p53 primes cells for the production of interferons (through upregulation of STING), and may activate negative-feedback within this signaling system by enhancing the production of SOCS1.
... Moreover, in human lung cancer H1299 (p53 null) cells engineered to inducibly express wild-type p53, we observed increased TBK1, STING, and IRF3 phosphorylation in response to p53 expression (Figure S1E). The phosphorylation of these proteins likely reflects the p53-dependent induction of IFI16, which cooperates with cGAS to activate TBK1-STING-IRF3 ( Figure S1E) (Almine et al., 2017;Jonsson et al., 2017;Song et al., 2008). In contrast, IFI16 levels were not affected by mtp53 knockdown or overexpression in different cell lines (Figures S1F and S1G). ...
... Our analysis of human triple-negative breast cancer data from TCGA found several cGAS/STING-regulated chemokines and cytokines, including IFNB1, to be more positively correlated with wild-type p53 than with mtp53. The precise mechanism underlying this remains to be determined, but it is potentially medi-ated by the wild-type p53 transcriptional target IFI16 (Almine et al., 2017;Jonsson et al., 2017;Munoz-Fontela et al., 2008;Song et al., 2008;Takaoka et al., 2003). Loss of wild-type p53 combined with an ATR inhibitor and DNA damage has been shown to induce a STAT1-dependent inflammatory response that does not require cGAS (Chen et al., 2020). ...
Certain mutant p53 variants suppressed cGAS–STING signaling to enable cancer cell immune escape.
... Moreover, in human lung cancer H1299 (p53 null) cells engineered to inducibly express wild-type p53, we observed increased TBK1, STING, and IRF3 phosphorylation in response to p53 expression (Figure S1E). The phosphorylation of these proteins likely reflects the p53-dependent induction of IFI16, which cooperates with cGAS to activate TBK1-STING-IRF3 ( Figure S1E) (Almine et al., 2017;Jonsson et al., 2017;Song et al., 2008). In contrast, IFI16 levels were not affected by mtp53 knockdown or overexpression in different cell lines (Figures S1F and S1G). ...
... Our analysis of human triple-negative breast cancer data from TCGA found several cGAS/STING-regulated chemokines and cytokines, including IFNB1, to be more positively correlated with wild-type p53 than with mtp53. The precise mechanism underlying this remains to be determined, but it is potentially medi-ated by the wild-type p53 transcriptional target IFI16 (Almine et al., 2017;Jonsson et al., 2017;Munoz-Fontela et al., 2008;Song et al., 2008;Takaoka et al., 2003). Loss of wild-type p53 combined with an ATR inhibitor and DNA damage has been shown to induce a STAT1-dependent inflammatory response that does not require cGAS (Chen et al., 2020). ...
Mutant p53 (mtp53) proteins can exert cancer-promoting gain-of-function activities. We report a mechanism by which mtp53 suppresses both cell-autonomous and non-cell-autonomous signaling to promote cancer cell survival and evasion of tumor immune surveillance. Mtp53 interferes with the function of the cytoplasmic DNA sensing machinery, cGAS-STING-TBK1-IRF3, that activates the innate immune response. Mtp53, but not wild-type p53, binds to TANK-binding protein kinase 1 (TBK1) and prevents the formation of a trimeric complex between TBK1, STING, and IRF3, which is required for activation, nuclear translocation, and transcriptional activity of IRF3. Inactivation of innate immune signaling by mtp53 alters cytokine production, resulting in immune evasion. Restoring TBK1 signaling is sufficient to bypass mtp53 and lead to restored immune cell function and cancer cell eradication. This work is of translational interest because therapeutic approaches that restore TBK1 function could potentially reactivate immune surveillance and eliminate mtp53 tumors.
... Subsequently, Lin et al. show that IFI16 functions as a tumor suppressor in hepatocellular carcinoma (HCC) by activating the p53 signaling pathway and inflammasome (40). In turn, functional activation of p53 stimulates the transcription of IFI16 through associating with the regulatory region of the IFI16 gene in the cells treated with DNA-damaging agents, suggesting a positive feedback loop between p53 and IFI16 (41). A recent research indicates that IFI16 positively regulates programmed cell death 1 ligand 1 (PD-L1) in cervical cancer cells by activating the STING-TBK1-NF-kB pathway, which can interact with the proximal region of the PD-L1 promoter to facilitate PD-L1 expression, and promoting the progression of cervical cancer (42). ...
... For instance, as mentioned above, IFI16 directly binds to the C-terminal region of p53 and enhances p53mediated transcriptional activation (37,38). Moreover, p53 also facilitates IFI16 transcription by directly binding to the promoter region of IFI16 and thus provides positive feedback regulation of p53 signaling (41). The IL-6/JAK/STAT3 pathway plays a key role in the growth and development of many human cancers (63). ...
IFI16, hnRNPA2B1, and nuclear cGAS are nuclear-located DNA sensors that play important roles in initiating host antiviral immunity and modulating tumorigenesis. IFI16 triggers innate antiviral immunity, inflammasome, and suppresses tumorigenesis by recognizing double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), damaged nuclear DNA, or cooperatively interacting with multiple tumor suppressors such as p53 and BRCA1. hnRNPA2B1 initiates interferon (IFN)-α/β production and enhances STING-dependent cytosolic antiviral signaling by directly binding viral dsDNA from invaded viruses and facilitating N ⁶ -methyladenosine (m ⁶ A) modification of cGAS, IFI16, and STING mRNAs. Nuclear cGAS is recruited to double-stranded breaks (DSBs), suppresses DNA repair, and promotes tumorigenesis. This review briefly describes the nuclear functions of IFI16, hnRNPA2B1, and cGAS, and summarizes the transcriptional, post-transcriptional, and post-translational regulation of these nuclear DNA sensors.
... p53 binds to the p53 DNA-binding site (BS) (known as IFI16-p53-BS) in the 5′-regulatory region of the IFI16 gene. 24 It has been shown that IFI16 is associated with transcription factor E2F1 and retinoblastoma tumor-suppressor protein Rb. This interaction could inhibit Rb-E2F1mediated transcriptional repression. ...
... 62-64 AIM2 resembles IFI16 and is activated through p53-and NF-кB-dependent pathways. 24,65,66 V. MNDA MNDA was identified in human leukemia cell line 60 as a 55-kDa protein. MNDA is predominantly expressed in myeloid and granulocyte-monocyte lineages. ...
The innate immune system is the first line of defense against microbial pathogens. The response of innate
immunity is initiated by molecules known as pattern recognition receptors (PRRs). Such responses are often triggered by
nucleic acids that are delivered to the cytoplasm or nucleus of cells. The ability to recognize foreign nucleic acids in these
two locations is an important defense mechanism of the human innate immune system. Several PRRs are located in the
cytosol or nucleus and detect foreign DNAs. The pyrin and hematopoietic interferon-inducible nuclear (PYHIN) domain
protein is a family of PRRs that includes interferon-inducible protein 16, absent in melanoma 2, PYHIN 1 (or interferon-inducible protein X, as it is also known), myeloid cell nuclear differentiation antigen, and pyrin domain only protein 3.
These nuclear and cytosolic sensors play an essential part in host defense of intracellular pathogens. In addition, members
of the PYHIN family are critical regulators of immune response, apoptosis, cell growth, differentiation, and transcription.
In this review, we summarize important characteristics of these innate immune sensors and their roles in several diseases.
A better understanding of the role of DNA sensors in the nucleus and cytoplasm will lead to the development of novel
therapeutic approaches to control infections and associated diseases.
... IFI16, a member of the interferoninducible p200-protein family (PYHIN200 or HIN-200 protein family), is characterized by a 200-amino-acid motif containing a DNA binding domain at the C-terminus and a PYRIN domain at the N-terminus [11]. IFI16 may regulate a variety of biological processes, including cell differentiation, proliferation, senescence, apoptosis, and inflammasome assembly, according to researchers in the field of cellular biology [12][13][14][15]. It was reported that IFI16 could be regarded as a DNA sensor that is implicated in the innate immune response, particularly in defense against viral infections and activation of the interferon gene pathway [15][16][17]. ...
Background
Lupus nephritis (LN) is one of the most severe complications of systemic lupus erythematosus (SLE). However, the current management of LN remains unsatisfactory due to sneaky symptoms during early stages and lack of reliable predictors of disease progression.
Methods
Bioinformatics and machine learning algorithms were initially used to explore the potential biomarkers for LN development. Identified biomarker expression was evaluated by immunohistochemistry (IHC) and multiplex immunofluorescence (IF) in 104 LN patients, 12 diabetic kidney disease (DKD) patients, 12 minimal change disease (MCD) patients, 12 IgA nephropathy (IgAN) patients and 14 normal controls (NC). The association of biomarker expression with clinicopathologic indices and prognosis was analyzed. Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) were utilized to explore potential mechanisms.
Results
Interferon-inducible protein 16 (IFI16) was identified as a potential biomarker for LN. IFI16 was highly expressed in the kidneys of LN patients compared to those with MCD, DKD, IgAN or NC. IFI16 co-localized with certain renal and inflammatory cells. Glomerular IFI16 expression was correlated with pathological activity indices of LN, while tubulointerstitial IFI16 expression was correlated with pathological chronicity indices. Renal IFI16 expression was positively associated with systemic lupus erythematosus disease activity index (SLEDAI) and serum creatinine while negatively related to baseline eGFR and serum complement C3. Additionally, higher IFI16 expression was closely related to poorer prognosis of LN patients. GSEA and GSVA suggested that IFI16 expression was involved in adaptive immune-related processes of LN.
Conclusion
Renal IFI16 expression is a potential biomarker for disease activity and clinical prognosis in LN patients. Renal IFI16 levels may be used to shed light on predicting the renal response and develop precise therapy for LN.
... The p53 protein stimulates the expression of other components of inflammasomes -IFI16 [62]. The expression thereof may also be stimulated by interferon alpha [53]. ...
The p53 tumor suppressor protein is best known as an inhibitor of the cell cycle and an inducer of apoptosis. Unexpectedly, these functions of p53 are not required for its tumor suppressive activity in animal models. High-throughput transcriptomic investigations as well as individual studies have demonstrated that p53 stimulates expression of many genes involved in immunity. Probably to interfere with its immunostimulatory role, many viruses code for proteins that inactivate p53. Judging by the activities of immunity-related p53-regulated genes it can be concluded that p53 is involved in detection of danger signals, inflammasome formation and activation, antigen presentation, activation of natural killer cells and other effectors of immunity, stimulation of interferon production, direct inhibition of virus replication, secretion of extracellular signaling molecules, production of antibacterial proteins, negative feedback loops in immunity-related signaling pathways, and immunologic tolerance. Many of these p53 functions have barely been studied and require further, more detailed investigations. Some of them appear to be cell-type specific. The results of transcriptomic studies have generated many new hypotheses on the mechanisms utilized by p53 to impact on the immune system. In the future, these mechanisms may be harnessed to fight cancer and infectious diseases.
... Since IFI16 is not a drug resistance protein itself but is involved in different cellular processes we focused on genes "related to" expression and physiological role of IFI16. As IFI16 expression is regulated by interferons [24,29,64,65], steroid hormones [37,66,67] and p53 protein [34,40,68,69] we compared expression of these genes, but we did not observe any differences in their expression between cell lines. On the other hand, IFI16 is involved in cellular senescence-associated cell growth arrest by interaction with p53/p21 pathway and positive regulation of p53 dependent genes [33,34,40,47,50] and Rb/E2F pathways [29] and negative regulation of pRb dependent genes [70,71]. ...
Background
Inherent or developed during treatment drug resistance is the main reason for the low effectiveness of chemotherapy in ovarian cancer. IFI16 is a cytoplasmic/nuclear protein involved in response to virus’s infection and cell cycle arrest associated with the cellular senescence.
Methods
Here we performed a detailed IFI16 expression analysis in ovarian cancer cell lines sensitive (A2780) and resistant to doxorubicin (DOX) (A2780DR1 and A2780DR2) and paclitaxel (PAC) (A2780PR1). IFI16 mRNA level, protein level in the nuclear and cytoplasmic fraction (Western blot analysis), the protein expression in cancer cells and nuclei (immunofluorescence analysis) and cancer patient lesions (immunohistochemistry) were performed in this study.
Results
We observed upregulation of IFI16 expression in drug resistant cell lines with dominant cytoplasmic localization in DOX-resistant cell lines and nuclear one in the PAC-resistant cell line. The most abundantly overexpressed isoforms of IFI16 were IFI16A and IFI16C. Finally, an analysis of a histological type of ovarian cancer (immunohistochemistry) showed expression in serous ovarian cancer.
Conclusions
Expression of IFI16 in drug-resistant cell lines suggests its role in drug resistance development in ovarian cancer. Expression in serous ovarian cancer suggests its role in the pathogenesis of this histological type.
Cellular changes that are linked to aging in humans include genomic instability, telomere attrition, epigenetic alterations, mitochondrial dysfunction, cellular senescence, and altered intercellular communications. The extent of the changes in these aging hallmarks and their interactions with each other are part of the human aging. However, the molecular mechanisms through which the aging hallmarks interact with each other remain unclear. Studies have indicated a potential role for the type I interferon (IFN) and p53-inducible IFI16 proteins in interactions with the aging hallmarks. The IFI16 proteins are members of the PYHIN protein family. Proteins in the family share a DNA-binding domain (the HIN domain) and a protein-protein interaction pyrin domain (PYD). IFI16 proteins are needed for cytosolic DNA-induced activation of the cGAS-STING pathway for type I IFN (IFN-β) expression. The pathway plays an important role in aging-related inflammation (inflammaging). Further, increased levels of the IFI16 proteins potentiate the cell growth inhibitory functions of the p53 and pRb tumor suppressors proteins. Moreover, IFI16 proteins are needed for most aging hallmarks. Therefore, here we discuss how an improved understanding of the role of the IFI16 proteins in integration of the aging hallmarks has potential to improve the human health and lifespan.
The coevolution of vertebrate and mammalian hosts with DNA viruses has driven the ability of host cells to distinguish viral from cellular DNA in the nucleus to induce intrinsic immune responses. Concomitant viral mechanisms have arisen to inhibit DNA sensing. At this virus–host interface, emerging evidence links cytokine responses and cellular homeostasis pathways, particularly the DNA damage response (DDR). Nuclear DNA sensors, such as the interferon (IFN)-γ inducible protein 16 (IFI16), functionally intersect with the DDR regulators ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK). Here, we discuss accumulating knowledge for the DDR-innate immunity signaling axis. Through the lens of this infection-driven signaling axis, we present host and viral molecular strategies acquired to regulate autoinflammation and antiviral responses.
Cellular response to DNA damage is multifacted in nature and involves a complex signaling network in which p53 functions as a "molecular node" for converging signals. p53 has been implicated in a variety of cellular processes primarily functioning as a transcription factor and also in a transcription-independent manner. It is rapidly activated following DNA damage with phosphorylation as one of the initial signals. Cellular context as well as the type and severity of DNA damage determine p53 activation code, and its activities are regulated predominantly through protein degradation, post-translational modification and interactions with various cellular co-factors. These events are crucial in decision making by p53 as it has the ability to receive, assess and integrate different signals and route them accordingly to induce cell death or promote cell survival. In this decision making process, its transcriptional role to activate a specific subset of target genes linked to inducing cell cycle arrest or apoptosis is critical that is further fine-tuned by its transcription-independent function. This article reviews the current state of knowledge about the role of p53 in determining the fate of cells that have incurred DNA damage.
Molecular changes associated with cellular aging in a strain of human diploid fibroblasts, IMR-90, were addressed by analyzing the expression of the tumor suppressor protein, p53. In all studies, IMR-90 cultures were characterized as "young" or "near-senescent" based on morphology, rate of population doubling, capacity for DNA synthesis, and presence of established markers for senescence. When p53 was immunoprecipitated by monoclonal antibodies and detected by Western immunoblot analysis, more protein per cell was detected in the near-senescent cultures. A greater than 10-fold increase in p53 protein was measured with the PAb 1801 (N-terminal-specific) anti-p53 antibody, whereas PAb 122 (C-terminal-specific) measured a 5-fold increase. Although near-senescent cultures demonstrated a higher level of p53 than young cells, these cultures had similar charges and molecular weight p53 isoforms when analyzed by two-dimensional Western blots. When p53 RNA was compared to total RNA there was a decrease in p53 RNA with age, but on a per cell basis p53 RNA was elevated. These results provide evidence for transcriptional regulation of p53 during aging and support the hypothesis that elevated levels of p53 protein may play a role in cellular senescence.
The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA-damaging agents.
Activation of the p53 transcription factor in response to a variety of cellular stresses, including DNA damage and oncogene activation, initiates a program of gene expression that blocks the proliferative expansion of damaged cells. While the beneficial impact of the anticancer function of p53 is well established, several recent papers suggest that p53 activation may in some circumstances act in a manner detrimental to the long-term homeostasis of the organism. Here, we discuss the significant participation of p53 in three non-mutually exclusive theories of human aging involving DNA damage, telomere shortening, and oxidative stress. These “good cop/bad cop” functions of p53 appear to place it at the nexus of two opposing forces, cancer and aging. By extension, this relationship implies that therapies aimed to reduce cancer and postpone aging, and thereby increase longevity, will necessarily work either upstream or downstream, but not on the level of, p53.
The therapeutic responsiveness of genetically defined tumors expressing or devoid of the p53 tumor suppressor gene was compared
in immunocompromised mice. Tumors expressing the p53 gene contained a high proportion of apoptotic cells and typically regressed
after treatment with gamma radiation or adriamycin. In contrast, p53-deficient tumors treated with the same regimens continued
to enlarge and contained few apoptotic cells. Acquired mutations in p53 were associated with both treatment resistance and
relapse in p53-expressing tumors. These results establish that defects in apoptosis, here caused by the inactivation of p53,
can produce treatment-resistant tumors and suggest that p53 status may be an important determinant of tumor response to therapy.
The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA-damaging agents.
gamma-Irradiation of human diploid fibroblasts in the G1 interval caused arrest of the cell cycle prior to S phase. This cell cycle block was correlated with a lack of activation of both cyclin E-Cyclin-dependent kinase 2 (Cdk2) and cyclin A-Cdk2 kinases and depended on wild-type p53. Although the accumulation of cyclin A was strongly inhibited in gamma-irradiated cells, cyclin E accumulated and bound Cdk2 at normal levels but remained in an inactive state. We found that both whole-cell lysates and inactive cyclin E-Cdk2 complexes prepared from irradiated cells contained an activity capable of inactivating cyclin E-Cdk2 complexes. The protein responsible for this activity was shown to be p21CIP1/WAF1, recently described as a p53-inducible Cdk inhibitor. Our data suggest a model in which ionizing radiation confers G1 arrest via the p53-mediated induction of a Cdk inhibitor protein.
Normally growing cells promptly cease DNA synthesis when exposed to genotoxic stresses, such as radiation, and this cell-cycle arrest prevents the accumulation of mutations. The transcription factor interferon regulatory factor (IRF)-1 is essential for the regulation of the interferon system, inhibits cell growth, and manifests tumour-suppressor activities. Here we show that mouse embryonic fibroblasts (EFs) lacking IRF-1 are deficient in their ability to undergo DNA-damage-induced cell-cycle arrest. A similar phenotype has been observed in EFs lacking the tumour suppressor p53 (refs 8, 9), although the expression of IRF-1 and p53 are independent of one another. Furthermore, we show that transcriptional induction of the gene encoding p21 (WAF1, CIP1), a cell-cycle inhibitor, by gamma-irradiation is dependent on both p53 and IRF-1, and that the p21 promoter is activated, either directly or indirectly, by both in a transient cotransfection assay. These two tumour-suppressor transcription factors therefore converge functionally to regulate the cell cycle through the activation of a common target gene.