Human papillomavirus type 16 E6 and E7 oncogenes abrogate radiation-induced DNA damage responses in vivo through p53-dependent and p53-independent pathways

E6 and E7 oncoproteins from high risk human papillomaviruses (HPVs) transform cells in tissue culture and induce tumors in vivo. Both E6, which inhibits p53 functions, and E7, which inhibits pRb, can also abrogate growth arrest induced by DNA-damaging agents in cultured cells. In this study, we have used transgenic mice that express HPV-16 E6 or E7 in the epidermis to determine how these two proteins modulate DNA damage responses in vivo. Our results demonstrate that both E6 and E7 abrogate the inhibition of DNA synthesis in the epidermis after treatment with ionizing radiation. Increases in the levels of p53 and p21 proteins after irradiation were suppressed by E6 but not by E7. Through the study of p53-null mice, we found that radiation-induced growth arrest in the epidermis is mediated through both p53-dependent and p53-independent pathways. The abrogation of radiation responses in both E6 and E7 transgenic mice was more complete than was seen in the p53-null epidermis. We conclude that E6 and E7 each have the capacity to modulate p53-dependent as well as p53-independent cellular responses to radiation. Additionally, we found that the conserved region (CR) 1 and CR2 domains in E7 protein, which are involved in the inactivation of pRb function and required for E7’s transforming function, were also required for E7 to modulate DNA damage responses in vivo. Thus pRb and/or pRb-like proteins likely mediate both p53-dependent and p53-independent responses to radiation.     

Differential suppression by protease inhibitors and cytokines of apoptosis induced by wild-type p53 and cytotoxic agents.

Apoptosis induced in myeloid leukemic cells by wild-type p53 was suppressed by different cleavage-site directed protease inhibitors, which inhibit interleukin-1 beta-converting enzyme-like, granzyme B and cathepsins B and L proteases. Apoptosis was also suppressed by the serine and cysteine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone (TPCK) [corrected], but not by other serine or cysteine protease inhibitors including N alpha-p-tosyl-L-lysine chloromethylketone (TLCK), E64, pepstatin A, or chymostatin. Protease inhibitors suppressed induction of apoptosis by gamma-irradiation and cycloheximide but not by doxorubicin, vincristine, or withdrawal of interleukin 3 from interleukin 3-dependent 32D non-malignant myeloid cells. Induction of apoptosis in normal thymocytes by gamma-irradiation or dexamethasone was also suppressed by the cleavage-site directed protease inhibitors, but in contrast to the myeloid leukemic cells apoptosis in thymocytes was suppressed by TLCK but not by TPCK. The results indicate that (i) inhibitors of interleukin-1 beta-converting enzyme-like proteases and some other protease inhibitors suppressed induction of apoptosis by wild-type p53 and certain p53-independent pathways of apoptosis; (ii) the protease inhibitors together with the cytokines interleukin 6 and interferon-gamma or the antioxidant butylated hydroxyanisole gave a cooperative protection against apoptosis; (iii) these protease inhibitors did not suppress induction of apoptosis by some cytotoxic agents or by viability-factor withdrawal from 32D cells, whereas these pathways of apoptosis were suppressed by cytokines; (iv) there are cell type differences in the proteases involved in apoptosis; and (v) there are multiple pathways leading to apoptosis that can be selectively induced and suppressed by different agents.     

Thermodynamic stability of wild-type and mutant p53 core domain

Some 50% of human cancers are associated with mutations in the core domain of the tumor suppressor p53. Many mutations are thought just to destabilize the protein. To assess this and the possibility of rescue, we have set up a system to analyze the stability of the core domain and its mutants. The use of differential scanning calorimetry or spectroscopy to measure its melting temperature leads to irreversible denaturation and aggregation and so is useful as only a qualitative guide to stability. There are excellent two-state denaturation curves on the addition of urea that may be analyzed quantitatively. One Zn²⁺ ion remains tightly bound in the holo-form of p53 throughout the denaturation curve. The stability of wild type is 6.0 kcal (1 kcal = 4.18 kJ)/mol at 25°C and 9.8 kcal/mol at 10°C. The oncogenic mutants R175H, C242S, R248Q, R249S, and R273H are destabilized by 3.0, 2.9, 1.9, 1.9, and 0.4 kcal/mol, respectively. Under certain denaturing conditions, the wild-type domain forms an aggregate that is relatively highly fluorescent at 340 nm on excitation at 280 nm. The destabilized mutants give this fluorescence under milder denaturation conditions.     

p53-independent apoptosis induced by paclitaxel through an indirect mechanism

The efficacy of chemotherapeutic agents may be determined by a number of different factors, including the genotype of the tumor cell. The p53 tumor suppressor gene frequently is mutated in human tumors, and this may contribute to chemotherapeutic resistance. We tested the requirement for wild-type p53 in the response of tumor cells to treatment with paclitaxel (trade name Taxol), an antineoplastic agent that stabilizes cellular microtubules. Although paclitaxel is broadly effective against human tumor xenografts in mice, including some known to carry p53 mutations, we found that p53-containing mouse tumor cells were significantly more sensitive to direct treatment with this drug than were p53-deficient tumor cells. In an attempt to reconcile this apparent discrepancy, we examined the requirement for p53 in the cytotoxic effects of tumor necrosis factor α (TNF-α), a cytokine released from murine macrophages upon paclitaxel treatment. Conditioned medium from paclitaxel-treated macrophages was capable of inducing p53-independent apoptosis when applied to transformed mouse embryonic fibroblasts and was inhibitable by antibodies against TNF-α. Furthermore, in response to direct treatment with TNF-α, both wild-type and p53-deficient tumor cells underwent apoptosis to similar extents and with similar kinetics. Our results suggest that the efficacy of paclitaxel in vivo may be due not only to its microtubule-stabilizing activity, but its ability to activate local release of an apoptosis-inducing cytokine.     

Regulation of p53 stability and p53-dependent apoptosis by NADH quinone oxidoreductase 1

The tumor suppressor gene wild-type p53 encodes a labile protein that accumulates in cells after different stress signals and can cause either growth arrest or apoptosis. One of the p53 target genes, p53-inducible gene 3 (PIG3), encodes a protein with significant homology to oxidoreductases, enzymes involved in cellular responses to oxidative stress and irradiation. This fact raised the possibility that cellular oxidation-reduction events controlled by such enzymes also may regulate the level of p53. Here we show that NADH quinone oxidoreductase 1 (NQO1) regulates p53 stability. The NQO1 inhibitor dicoumarol caused a reduction in the level of both endogenous and gamma-irradiation-induced p53 in HCT116 human colon carcinoma cells. This reduction was prevented by the proteasome inhibitors MG132 and lactacystin, suggesting enhanced p53 degradation in the presence of dicoumarol. Dicoumarol-induced degradation of p53 also was prevented in the presence of simian virus 40 large T antigen, which is known to bind and to stabilize p53. Cells overexpressing NQO1 were resistant to dicoumarol, and this finding indicates the direct involvement of NQO1 in p53 stabilization. NQO1 inhibition induced p53 degradation and blocked wild-type p53-mediated apoptosis in gamma-irradiated normal thymocytes and in M1 myeloid leukemic cells that overexpress wild-type p53. Dicoumarol also reduced the level of p53 in its mutant form in M1 cells. The results indicate that NQO1 plays an important role in regulating p53 functions by inhibiting its degradation.     

Conformation-dependent phosphorylation of p53

Phosphorylation of the p53 tumor suppressor protein is known to modulate its functions. Using bacterially produced glutathione S-transferase (GST)-p53 fusion protein and baculovirus-expressed histidine-tagged p53 (_Hisp53), we have determined human p53 phosphorylation by purified forms of jun-N-kinase (JNK), protein kinase A (PKA), and β subunit of casein kinase II (CKIIβ) as well as by kinases present in whole cell extracts (WCEs). We demonstrate that PKA is potent p53 kinase, albeit, in a conformation- and concentration-dependent manner, as concluded by comparing full-length with truncated forms of p53. We further demonstrate JNK interaction with GST-p53 and the ability of JNK to phosphorylate truncated forms of GST-p53 or full-length _Hisp53. Dependence of phosphorylation on conformation of p53 is further supported by the finding that the wild-type form of p53 (p53^wt) undergoes better phosphorylation by CKIIβ and by WCE kinases than mutant forms of p53 at amino acid 249 (p53²⁴⁹) or 273 (p53²⁷³). Moreover, shifting the kinase reaction’s temperature from 37°C to 18°C reduces the phosphorylation of mutant p53 to a greater extent than of p53^wt. Comparing truncated forms of p53 revealed that the ability of CKIIβ, PKA, or WCE kinases to phosphorylate p53 requires amino acids 97–155 within the DNA-binding domain region. Among three 20-aa peptides spanning this region we have identified residues 97–117 that increase p53 phosphorylation by CKIIβ while inhibiting p53 phosphorylation by PKA or WCE kinases. The importance of this region is further supported by computer modeling studies, which demonstrated that mutant p53²⁴⁹ exhibits significant changes to the conformation of p53 within amino acids 97–117. In summary, phosphorylation-related analysis of different p53 forms in vitro indicates that conformation of p53 is a key determinant in its availability as a substrate for different kinases, as for the phosphorylation pattern generated by the same kinase.     

Wild-type p53 triggers a rapid senescence program in human tumor cells lacking functional p53

The p53 tumor suppressor gene has been shown to play an important role in determining cell fate. Overexpression of wild-type p53 in tumor cells has been shown to lead to growth arrest or apoptosis. Previous studies in fibroblasts have provided indirect evidence for a link between p53 and senescence. Here we show, using an inducible p53 expression system, that wild-type p53 overexpression in EJ bladder carcinoma cells, which have lost functional p53, triggers the rapid onset of G₁ and G₂/M growth arrest associated with p21 up-regulation and repression of mitotic cyclins (cyclin A and B) and cdc2. Growth arrest in response to p53 induction became irreversible within 48-72 h, with cells exhibiting morphological features as well as specific biochemical and ultrastructural markers of the senescent phenotype. These findings provide direct evidence that p53 overexpression can activate the rapid onset of senescence in tumor cells.     

Effects of p53 mutations on apoptosis in mouse intestinal and human colonic adenomas

We have examined the effects of inactivation of the p53 tumor suppressor gene on the incidence of apoptotic cell death in two stages of the adenoma-to-carcinoma progression in the intestine: in early adenomas where p53 mutations are rare and in highly dysplastic adenomas where loss of p53 occurs frequently. Homozygosity for an inactivating germ-line mutation of p53 had no effect on the incidence or the rate of progression of Apc^Min/+-induced adenomas in mice and also did not affect the frequency of apoptosis in the cells of these adenomas. To examine the effect of p53 loss on apoptosis in late-stage adenomas, we compared the incidence of apoptotic cell death before and after the appearance of highly dysplastic cells in human colonic adenomas. The appearance of highly dysplastic cells, which usually coincides during colon tumor progression with loss of heterozygosity at the p53 locus, did not correlate with a reduction in the incidence of apoptosis. These studies suggest that p53 is only one of the genes that determine the incidence of apoptotic in colon carcinomas and that wild-type p53 retards the progression of many benign colonic adenoma to malignant carcinomas by mechanism(s) other than the promotion of apoptosis.     

p53-dependent regulation of MDR1 gene expression causes selective resistance to chemotherapeutic agents

Loss of functional p53 paradoxically results in either increased or decreased resistance to chemotherapeutic drugs. The inconsistent relationship between p53 status and drug sensitivity may reflect p53’s selective regulation of genes important to cytotoxic response of chemotherapeutic agents. We reasoned that the discrepant effects of p53 on chemotherapeutic cytotoxicity is due to p53-dependent regulation of the multidrug resistance gene (MDR1) expression in tumors that normally express MDR1. To test the hypothesis that wild-type p53 regulates the endogenous mdr1 gene we stably introduced a trans-dominant negative (TDN) p53 into rodent H35 hepatoma cells that express P-glycoprotein (Pgp) and have wild-type p53. Levels of Pgp and mdr1a mRNA were markedly elevated in cells expressing TDN p53 and were linked to impaired p53 function (both transactivation and transrepression) in these cells. Enhanced mdr1a gene expression in the TDN p53 cells was not secondary to mdr1 gene amplification and Pgp was functional as demonstrated by the decreased uptake of vinblastine. Cytotoxicity assays revealed that the TDN p53 cell lines were selectively insensitive to Pgp substrates. Sensitivity was restored by the Pgp inhibitor reserpine, demonstrating that only drug retention was the basis for loss of drug sensitivity. Similar findings were evident in human LS180 colon carcinoma cells engineered to overexpress TDN p53. Therefore, the p53 inactivation seen in cancers likely leads to selective resistance to chemotherapeutic agents because of up-regulation of MDR1 expression.     

Cytoplasmically sequestered wild-type p53 protein in neuroblastoma is relocated to the nucleus by a C-terminal peptide

Cytoplasmic sequestration of wild-type p53 protein occurs in a subset of primary human tumors including breast cancer, colon cancer, and neuroblastoma (NB). The sequestered p53 localizes to punctate cytoplasmic structures that represent large protein aggregates. One functional consequence of this blocked nuclear access is impairment of the p53-mediated G₁ checkpoint after DNA damage. Here we show that cytoplasmic p53 from NB cells is incompetent for specific DNA binding, probably due to its sequestration. Importantly, the C-terminal domain of sequestered p53 is masked, as indicated by the failure of a C-terminally directed antibody to detect p53 in these structures. To determine (i) which domain of p53 is involved in the aggregation and (ii) whether this phenotype is potentially reversible, we generated stable NB sublines that coexpress the soluble C-terminal mouse p53 peptide DD1 (amino acids 302–390). A dramatic phenotypic reversion occurred in five of five lines. The presence of DD1 blocked the sequestration of wild-type p53 and relocated it to the nucleus, where it accumulated. The nuclear translocation is due to shuttling of wild-type p53 by heteroligomerization to DD1, as shown by coimmunoprecipitation. As expected, the nuclear heterocomplexes were functionally inactive, since DD1 is a dominant negative inhibitor of wild-type p53. In summary, we show that nuclear access of p53 can be restored in NB cells.     

Mpl ligand prevents lethal myelosuppression by inhibiting p53-dependent apoptosis

This study demonstrates in both stable and inducible BCR-ABL-expressing hematopoietic cells a down-regulation of the major mammalian DNA repair protein DNA-PKcs by BCR-ABL. Similar results were found in BCR-ABL CD34(+) cells from patients with chronic myelogenous leukemia (CML). DNA-PKcs down-regulation is a proteasome-dependent degradation that requires tyrosine kinase activity and is associated with a marked DNA repair deficiency along with increased sensitivity to ionizing radiation. The conjunction of a major DNA repair deficiency and a resistance to apoptosis, both induced by BCR-ABL, provides a new mechanism to explain how secondary genetic alterations can accumulate in CML, eventually leading to blast crisis. The down-regulation of DNA-PKcs was reversible in CD34(+) CML cells suggesting that this approach might offer a novel and powerful therapeutic strategy in this disease, especially to delay the blast crisis. (Blood. 2001;97:2084-2090)     

p19Arf induces p53-dependent apoptosis during Abelson virus-mediated pre-B cell transformation

The Ink4a/Arf locus encodes p16^Ink4a and p19^Arf and is among the most frequently mutated tumor suppressor loci in human cancer. In mice, many of these effects appear to be mediated by interactions between p19^Arf and the p53 tumor-suppressor protein. Because Tp53 mutations are a common feature of the multistep pre-B cell transformation process mediated by Abelson murine leukemia virus (Ab-MLV), we examined the possibility that proteins encoded by the Ink4a/Arf locus also play a role in Abelson virus transformation. Analyses of primary transformants revealed that both p16^Ink4a and p19^Arf are expressed in many of the cells as they emerge from the apoptotic crisis that characterizes the transformation process. Analyses of primary transformants from Ink4a/Arf null mice revealed that these cells bypassed crisis. Because expression of p19^Arf but not p16 ^Ink4a induced apoptosis in Ab-MLV-transformed pre-B cells, p19^Arf appears to be responsible for these events. Consistent with the link between p19^Arf and p53, Ink4a/Arf expression correlates with or precedes the emergence of cells expressing mutant p53. These data demonstrate that p19^Arf is an important part of the cellular defense mounted against transforming signals from the Abl oncoprotein and provide direct evidence that the p19^Arf–p53 regulatory loop plays an important role in lymphoma induction.     

Reactive oxygen species are downstream mediators of p53-dependent apoptosis.

Reactive oxygen species (ROS) have been implicated as potential modulators of apoptosis. Conversely, experiments under hypoxic conditions have suggested that apoptosis could occur in the absence of ROS. We sought to determine whether a central modulator of apoptosis, p53, regulates the levels of intracellular ROS and whether a rise in ROS levels is required for the induction of p53-dependent apoptosis. We transiently overexpressed wild-type p53, using adenoviral gene transfer, and identified cell types that were sensitive or resistant to p53-mediated apoptosis. Cells sensitive to p53-mediated apoptosis produced ROS concomitantly with p53 overexpression, whereas cells resistant to p53 failed to produce ROS. In sensitive cells, both ROS production and apoptosis were inhibited by antioxidant treatment. These results suggest that p53 acts to regulate the intracellular redox state and induces apoptosis by a pathway that is dependent on ROS production.     

Oxidized low-density lipoprotein stimulates p53-dependent activation of proapoptotic Bax leading to apoptosis of differentiated endothelial progenitor cells

Dyslipidemia increases the risks for atherosclerosis in part by impairing endothelial integrity; endothelial progenitor cells (EPCs) play a pivotal role in reendothelialization. In this study, we investigated the mechanism whereby oxidized low-density lipoprotein (oxLDL) affects the function of differentiated EPCs (EDCs). In EDCs expanded in vitro from EPCs isolated from human cord blood, we measured EDC responses to both copper-oxidized LDL and L5, an electronegative LDL minimally oxidized in vivo in patients with hypercholesterolemia. OxLDL induced apoptosis of EDCs and impaired their response to nitric oxide. We found that the key to oxLDL-induced apoptosis in both EDCs and endothelial cells is the induction of a conformational change of Bax, leading to Bax activation without altering its expression. The conformationally changed Bax translocated to the mitochondria and stimulated apoptosis, as Bax knockdown prevented oxLDL-induced apoptosis in EDCs. The activation of Bax is mediated by an increase in p53 and knockdown of p53 abolished oxLDL-induced activation of Bax and apoptosis. OxLDL activated p53 through production of mitochondria-derived reactive oxygen species. In EDCs treated with a recombinant adenovirus expressing superoxide dismutase or N-acetyl-cysteine (but not catalase), the p53-Bax pathway activated by oxLDL was blocked, and apoptosis was prevented. Of importance, treatment of EDC with low-concentration L5 stimulated superoxide dismutase expression, which significantly attenuated apoptosis in EDCs exposed to high-concentration L5. These findings suggest that exposure of EDCs and endothelial cells to either experimentally prepared or naturally occurring modified LDL results in an increased transfer of mitochondria-derived superoxide anion to p53, which stimulates a conformational change in Bax favoring its translocation to the mitochondria with resultant apoptosis of these cells.