Glioblastoma is the most common malignant brain tumor in adults. The currently available treatments offer only a palliative survival advantage and the need for effective treatments remains an urgent priority. Activation of the p53 growth suppression/apoptotic pathway is one of the promising strategies in targeting glioma cells. We show that the quinoline derivative chloroquine activates the p53 pathway and suppresses growth of glioma cells in vitro and in vivo in an orthotopic (U87MG) human glioblastoma mouse model. Induction of apoptosis is one of the mechanisms underlying the effects of chloroquine on suppressing glioma cell growth and viability. siRNA-mediated downregulation of p53 in wild-type but not mutant p53 glioblastoma cells substantially impaired chloroquine-induced apoptosis. In addition to its p53-activating effects, chloroquine may also inhibit glioma cell growth via p53-independent mechanisms. Our results clarify the mechanistic basis underlying the antineoplastic effect of chloroquine and reveal its therapeutic potential as an adjunct to glioma chemotherapy.

Transcriptional activation of p53-regulated genes is initiated by sequence-specific DNA binding of p53 to target binding sites. Regulation of sequence-specific DNA binding is complex and occurs at various levels. We demonstrate that DNA topology is an important parameter for regulating the selective and highly specific interaction of p53 with its target binding sites. Specific binding of wild-type p53 is greatly enhanced when cognate binding sites are present in a non-linear stem-loop conformation. The C-terminal domain plays a key role in regulating the specific interactions of p53 with target binding sites in a DNA conformation-dependent manner. The C-terminal domain is required for binding to target sites in a non-linear DNA conformation in contrast to the strong inhibitory effects of the C terminus on p53 interaction with linear DNA. We propose that selective binding of p53 to various promoters may be determined by the DNA conformation within p53 cognate sites.A major function of the tumor suppressor p53 is that of a pleiotropic transcription factor controlling the expression of target genes by either activating or inhibiting the activity of p53-responsive promoters. p53 has several DNA binding activities of which sequence-specific DNA binding (SSDB) 1 (1) is the most important because it is indispensable for the transcriptional activity of the protein. The importance of SSDB is highlighted by the fact that the most frequent p53 mutations found in human cancers are located within the DNA binding domain encoding region (2, 3) and abrogate the SSDB of p53. The specific interaction of p53 with target DNA is complex and tightly regulated. Promoter recognition by p53 is determined by the presence of p53-response elements (PREs), which share homology with the consensus sequence 5Ј-(PuPuPuC(A/T)(T/ A)GPyPyPy) n -3Ј (1). C-terminally unmodified p53 seems to be inactive in SSDB; however, modifications at the C terminus, like binding of the C-terminal-specific antibody PAb421, phosphorylation, or deletion of the 30 C-terminal amino acids, strongly enhance SSDB under certain in vitro conditions (4). The data indicate that the C terminus regulates binding of p53 to target DNA, although the underlying mechanisms are not entirely understood. Several models have been proposed to explain the influence of the C-terminal domain on SSDB by p53. The conformation model postulates that the p53 protein exists in two conformationally distinct forms termed latent (for DNA-binding inactive p53) and activated (for DNA-binding active p53). According to this model, the C-terminal domain in latent p53 interacts directly with the core domain and thereby inhibits SSDB of p53 (5). A conformational switch was proposed as the mechanism that relieves the allosteric inhibition by the C terminus and converts p53 from latent into an activated form. However, such a hypothesis is not supported by the results of structural studies that recently demonstrated that the conformation of DNA binding-inactive and DNA bindingactive p53 forms is largely iden...

Despite the loss of sequence-specific DNA binding, mutant p53 (mutp53) proteins can induce or repress transcription of mutp53-specific target genes. To date, the molecular basis for transcriptional modulation by mutp53 is not understood, but increasing evidence points to the possibility that specific interactions of mutp53 with DNA play an important role. So far, the lack of a common denominator for mutp53 DNA binding, i.e. the existence of common sequence elements, has hampered further characterization of mutp53 DNA binding. Emanating from our previous discovery that DNA structure is an important determinant of wild-type p53 (wtp53) DNA binding, we analyzed the binding of various mutp53 proteins to oligonucleotides mimicking non-B DNA structures. Using various DNA-binding assays we show that mutp53 proteins bind selectively and with high affinity to non-B DNA. In contrast to sequence-specific and DNA structure-dependent binding of wtp53, mutp53 DNA binding to non-B DNA is solely dependent on the stereo-specific configuration of the DNA, and not on DNA sequence. We propose that DNA structure-selective binding of mutp53 proteins is the basis for the well-documented interaction of mutp53 with MAR elements and for transcriptional activities mediates by mutp53.

BackgroundIn analogy to normal stem cell differentiation, the current cancer stem cell (CSC) model presumes a hierarchical organization and an irreversible differentiation in tumor tissue. Accordingly, CSCs should comprise only a small subset of the tumor cells, which feeds tumor growth. However, some recent findings raised doubts on the general applicability of the CSC model and asked for its refinement.Methodology/Principal FindingsIn this study we analyzed the CSC properties of mammary carcinoma cells derived from transgenic (WAP-T) mice. We established a highly tumorigenic WAP-T cell line (G-2 cells) that displays stem-like traits. G-2 cells, as well as their clonal derivates, are closely related to primary tumors regarding histology and gene expression profiles, and reflect heterogeneity regarding their differentiation states. G-2 cultures comprise cell populations in distinct differentiation states identified by co-expression of cytoskeletal proteins (cytokeratins and vimentin), a combination of cell surface markers and a set of transcription factors. Cellular subsets sorted according to expression of CD24a, CD49f, CD61, Epcam, Sca1, and Thy1 cell surface proteins, or metabolic markers (e.g. ALDH activity) are competent to reconstitute the initial cellular composition. Repopulation efficiency greatly varies between individual subsets and is influenced by interactions with the respective complementary G-2 cellular subset. The balance between differentiation states is regulated in part by the transcription factor Sox10, as depletion of Sox10 led to up-regulation of Twist2 and increased the proportion of Thy1-expressing cells representing cells in a self-renewable, reversible, quasi-mesenchymal differentiation state.Conclusions/SignificanceG-2 cells constitute a self-reproducing cancer cell system, maintained by bi- and unidirectional conversion of complementary cellular subsets. Our work contributes to the current controversial discussion on the existence and nature of CSC and provides a basis for the incorporation of alternative hypotheses into the CSC model.

Mutant, but not wild-type p53 binds with high affinity to a variety of MAR-DNA elements (MARs), suggesting that MAR-binding of mutant p53 relates to the dominantoncogenic activities proposed for mutant p53. MARs recognized by mutant p53 share AT richness and contain variations of an AATATATTT ''DNA-unwinding motif,'' which enhances the structural dynamics of chromatin and promotes regional DNA base-unpairing. Mutant p53 specifically interacted with MARderived oligonucleotides carrying such unwinding motifs, catalyzing DNA strand separation when this motif was located within a structurally labile sequence environment. Addition of GCclamps to the respective MAR-oligonucleotides or introducing mutations into the unwinding motif strongly reduced DNA strand separation, but supported the formation of tight complexes between mutant p53 and such oligonucleotides. We conclude that the specific interaction of mutant p53 with regions of MAR-DNA with a high potential for base-unpairing provides the basis for the high-affinity binding of mutant p53 to MAR-DNA.

DNA binding is central to the ability of p53 to function as a tumor suppressor. In line with the remarkable functional versatility of p53, which can act on DNA as a transcription, repair, recombination, replication, and chromatin accessibility factor, the modes of p53 interaction with DNA are also versatile. One feature common to all modes of p53-DNA interaction is the extraordinary sensitivity of p53 to the topology of its target DNA. Whereas the strong impact of DNA topology has been demonstrated for p53 binding to sequencespecific sites or to DNA lesions, the possibility that DNA structuredependent recognition may underlie p53 interaction with other types of DNA has not been addressed until now. We demonstrate for the first time that conformationally flexible CTG⅐CAG trinucleotide repeats comprise a novel class of p53-binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in vivo. Our major finding is that p53 binds to CTG⅐CAG tracts by different modes depending on the conformation of DNA. Although p53 binds preferentially to hairpins formed by either CTG or CAG strands, it can also bind to linear forms of CTG⅐CAG tracts such as canonic B DNA or mismatched duplex. Intriguingly, by binding to a mismatched duplex p53 can induce further topological alterations in DNA, indicating that p53 may act as a DNA topology-modulating factor.Cells are equipped with extraordinarily sensitive surveillance systems, which are able to monitor genomic DNA for the appearance of any unprogrammed alterations that may pose a danger to the structural integrity of the genome (1-4). The appearance of just one or two double strand breaks in a cell is sufficient for rapidly putting the system into a "high alert" mode, marked by the activation of signaling pathways that are responsive to DNA damage (5-7). The ability to function either in a "stand by" or in an activated high alert mode is also a hallmark of the tumor suppressor p53, one of the key factors involved in the maintenance of genomic integrity (8 -11). Activation of p53 is an essential component of the global response of the cell to acute genotoxic insults that leads to rapid stabilization of the p53 protein and to p53 functioning in a high alert mode (12, 13). As a DNA damage-inducible factor, p53 can be potently activated by various types of genotoxic stimuli (9,14,15). A causative relationship between the occurrence of damaged DNA and the activation of the p53 response has been established by studies in a cell-free system demonstrating that sequence-specific DNA binding of p53 (p53-SSDB) can be potently stimulated by free DNA ends (16). In vitro DNA binding analyses further revealed that the C-terminal DNAbinding domain of p53 can bind selectively to some types of aberrant DNA structures in a sequence-independent manner (17-19). These findings led to the proposal that p53 may recruit cellular repair factors to the sites of damage by directly binding to DNA lesions in a DNA structure-dependent fashion (20, 21). Notably, it appears that such sequence-indep...

We recently reported that murine MethA mutant but not wild-type p53 specifically binds to MAR-DNA elements (MARs) with high affinity. Here we show that this DNA binding activity is exerted not only by MethA mutant p53 but also by other murine mutant p53 proteins isolated from the transformed murine BALB/c cell lines 3T3tx and T3T3 and differing in their conformational status. High affinity MAR-DNA binding was not restricted to the Xbal-IgE-MAR-DNA fragment from the murine immunoglobulin heavy chain gene enhancer locus [Cockerill et al. (1987): J Biol Chem 262:5394-5397] used in previous studies, as MethA p53 also specifically interacted with other A/T-rich bona fide MARs. Not only murine but also human mutant p53 proteins carrying the mutational hot spot amino acid exchanges 175Arg-->His, 273Arg-->Pro, or 273Arg-->His bound to the Xbal-IgE-MAR-DNA fragment. We therefore conclude that high affinity MAR-DNA binding is a property common to a variety of mutant p53 proteins.

In this report we analyzed the impact of interleukin-4 (IL-4) on tumor-associated simian virus 40 (SV40) large T-antigen (TAg)-specific CD8+ cytotoxic T cells during rejection of syngeneic SV40 transformed mKSA tumor cells in BALB/c mice. Strikingly, challenge of naïve mice with low doses of mKSA tumor cells revealed a CD8+ T cell-dependent prolonged survival time of naïve IL-4-/- mice. In mice immunized with SV40 TAg we observed in IL-4-/- mice, or in wild type mice treated with neutralizing anti-IL-4 monoclonal antibody, a strongly enhanced TAg-specific cytotoxicity of tumor associated CD8+ T cells. The enhanced cytotoxicity in IL-4-/- mice was accompanied by a significant increase in the fraction of CD8+ tumor associated T-cells expressing the cytotoxic effector molecules granzyme A and B and in granzyme B-specific enzymatic activity. The data suggest that endogenous IL-4 can suppress the generation of CD8+ CTL expressing cytotoxic effector molecules especially when the antigen induces only a very weak CTL response.