A Proteolytic Fragment from the Central Region of P53 Has Marked Sequence-Specific DNA-Binding Activity When Generated from Wild-Type but Not from Oncogenic Mutant P53-Protein

p53 is a sequence-specific DNA-binding oligomeric protein that can activate transcription from promoters bearing p53-binding sites. Whereas the activation region of p53 has been identified within the amino terminus, the location of the specific DNA-binding domain has not been reported. Thermolysin treatment of p53 protein generates a stable protease-resistant fragment that binds with marked specificity to p53 DNA-binding sites. Amino-terminal sequencing of the fragment located the thermolysin cleavage site to residue 91. Because the fragment does not contain the cdc2 phosphorylation site at Ser-315, we conclude that the the site-specific DNA-binding domain of p53 spans the central region of the protein. The vast majority of the mutations in oncogenically derived p53 proteins are located within this central portion of the molecule. Such mutant p53 proteins exhibit defective sequence-specific DNA-binding. Although thermolysin digestion of mutant p53 proteins generates proteolytic patterns that differ from wild-type protein, one mutant tested, His-273, generates a resistant' fragment that migrates with a similar electrophoretic mobility to the wild-type protease-resistant fragment. Interestingly, although intact mutant His-273 protein binds to DNA at 20-degrees-C, the thermolysin-resistant mutant fragment does not. In addition, the central protease-resistant, site-specific binding region of wild-type p53 does not demonstrate nonspecific DNA-binding. Thus, although sequences outside of the central region of p53 contribute to both nonspecific DNA-binding and oligomerization, they are not required for sequence-specific DNA-binding

A DNA damage signal is required for p53 to activate gadd45

We provide direct evidence that overexpression of p53 is not sufficient for robust p53-dependent activation of the endogenous gadd45 gene. When p53 was induced in TR9-7 cells in the absence of DNA damage, waf1/p21 and mdm2 mRNA levels were increased, but a change in gadd45 mRNA was barely detectable. Activation of the gadd45 gene was observed when camptothecin was added to cells containing p53 in the absence of a further increase in the p53 level. Phosphorylation of p53 at serine 15 and acetylation at lysine 382 were detected after drug treatment. It has been suggested that p53 posttranslational modification is critical during activation. However, inhibition of these modifications by wortmannin was not sufficient to block the transactivation of gadd45. Interestingly, after camptothecin treatment, increased DNase I sensitivity was detected at the gadd45 promoter, suggesting that an undetermined DNA damage signal is involved in inducing chromatin remodeling at the gadd45 promoter while cooperating with p53 to activate gadd45 transcription

Tumor suppressor p53 binds with high affinity to CTG-CAG trinucleotide repeats and induces topological alterations in mismatched duplexes

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 sequence-specific sites or to DNA lesions, the possibility that DNA structure-dependent 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 CTG·CAG 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 CTG·CAG 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 CTG·CAG 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

Differential Toxicity of DNA Adducts of Mitomycin C

The clinically used antitumor agent mitomycin C (MC) alkylates DNA upon reductive activation, forming six covalent DNA adducts in this process. This review focuses on differential biological effects of individual adducts in various mammalian cell cultures, observed in the authors' laboratories. Evidence is reviewed that various adducts are capable of inducing different cell death pathways in cancer cells.This evidence is derived from a parallel study of MC and its derivatives 2,7-diaminomitosene (2,7-DAM) which is the main metabolite of MC and forms two mono-adducts with DNA, and decarbamoyl mitomycin C (DMC), which alkylates and cross-links DNA, predominantly with a chirality opposite to that of the DNA adducts of MC. 2,7-DAM is not cytotoxic and does not activate the p53 pathway while MC and DMC are cytotoxic and able to activate the p53 pathway. DMC is more cytotoxic than MC and can also kill p53-deficient cells by inducing degradation of Checkpoint 1 protein, which is not seen with MC treatment of the p53-deficient cells. This difference in the cell death pathways activated by the MC and DMC is attributed to differential signaling by the DNA adducts of DMC. We hypothesize that the different chirality of the adduct-to-DNA linkage has a modulating influence on the choice of pathway

C. elegans CEP-1/p53 and BEC-1 Are Involved in DNA Repair

p53 is a transcription factor that regulates the response to cellular stress. Mammalian p53 functions as a tumor suppressor. The C. elegans p53, cep-1, regulates DNA-damage induced germline cell death by activating the transcription of egl-1 and ced-13. We used the C. elegans model to investigate how, in the whole animal, different forms of DNA damage can induce p53-dependent versus p53-independent cell death and DNA repair. DNA damage was induced by ultraviolet type C (UVC) radiation, or 10-decarbamoyl mitomycin C (DMC, an agent known to induce mammalian p53-independent cell death). Wild-type or cep-1 loss-of-function mutant animals were assayed for germline cell death and DNA lesions. Wild-type animals displayed greater removal of UVC-lesions over time, whereas cep-1 mutant animals displayed increased UVC-lesion retention. The cep-1 mutation increased UVC-lesion retention directly correlated with a reduction of progeny viability. Consistent with DMC inducing p53-independent cell death in mammalian cells DMC induced a C. elegans p53-independent germline cell death pathway. To examine the influence of wild-type CEP-1 and DNA damage on C. elegans tumors we used glp-1(ar202gf)/Notch germline tumor mutants. UVC treatment of glp-1 mutant animals activated the CEP-1 target gene egl-1 and reduced tumor size. In cep-1(gk138);glp-1(ar202gf) animals, UVC treatment resulted in increased susceptibility to lesions and larger tumorous germlines. Interestingly, the partial knockdown of bec-1 in adults resulted in a CEP-1-dependent increase in germline cell death and an increase in DNA damage. These results strongly support cross-talk between BEC-1 and CEP-1 to protect the C. elegans genome

The β-carboline alkaloid harmine inhibits telomerase activity of MCF-7 cells by down-regulating hTERT mRNA expression accompanied by an accelerated senescent phenotype

The end replication problem, which occurs in normal somatic cells inducing replicative senescence, is solved in most cancer cells by activating telomerase. The activity of telomerase is highly associated with carcinogenesis which makes the enzyme an attractive biomarker in cancer diagnosis and treatment. The indole alkaloid harmine has multiple pharmacological properties including DNA intercalation which can lead to frame shift mutations. In this study, harmine was applied to human breast cancer MCF-7 cells. Its activity towards telomerase was analyzed by utilizing the telomeric repeat amplification protocol (TRAP). Our data indicate that harmine exhibits a pronounced cytotoxicity and induces an anti-proliferation state in MCF-7 cells which is accompanied by a significant inhibition of telomerase activity and an induction of an accelerated senescence phenotype by over-expressing elements of the p53/p21 pathway

Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

The Cancer Genome Atlas (TCGA) data indicate that high MDM2 expression correlates with all subtypes of breast cancer. Overexpression of MDM2 drives breast oncogenesis in the presence of wild-type or mutant p53 (mtp53). Importantly, estrogen-receptor positive (ER+) breast cancers overexpress MDM2 and estrogen mediates this expression. We previously demonstrated that this estrogen-MDM2 axis activates the proliferation of breast cancer cell lines T47D (mtp53 L194F) and MCF7 (wild-type p53) in a manner independent of increased degradation of wildtype p53 (ie, p53-independently). Herein we present data supporting the role of the estrogen-MDM2 axis in regulating cell proliferation and mammary tissue architecture of MCF7 and T47D cells in a p53-independent manner. Inducible shRNA mediated MDM2 knockdown inhibited colony formation in soft agar, decreased mass size and induced lumen formation in matrigel and also significantly reduced mitosis as seen by decreased phospho-histone H3 positive cells. The knockdown of MDM2 in both cell lines decreased Rb phosphorylation and the level of E2F1 protein. This signaling was through the estrogen receptor because fulvestrant (a selective estrogen receptor degrader) decreased MDM2 protein levels and decreased phosphorylation of Rb. Taken together these data indicate that in some ER+ breast cancers the estrogen- MDM2-Rb-E2F1 axis is a central hub for estrogen-mediated p53-independent signal transduction. This is the first indication that estrogen signaling utilizes the estrogen- MDM2 axis to provoke phosphorylation of Rb and increase E2F1 while promoting abnormal mammary architecture

Mutant C. elegans p53 Together with Gain-of-Function GLP-1/Notch Decreases UVC-Damage-Induced Germline Cell Death but Increases PARP Inhibitor-Induced Germline Cell Death

The TP53 gene is mutated in over 50% of human cancers, and the C. elegans p53-1 (cep-1) gene encodes the ortholog CEP-1. CEP-1 is activated by ultraviolet type C (UVC)-induced DNA damage and activates genes that induce germline apoptosis. UVC treatment of gain-of-function glp-1(ar202gf)/Notch tumorous animals reduces germline stem cell numbers (and overall tumor size), while UVC treatment of double-mutant cep-1/p53(gk138);glp-1/Notch(ar202gf) increases DNA damage adducts and stem cell tumor volume. We compared UVC-induced mitotic stem cell death and animal lifespans for the two different C. elegans tumorous strains. C. elegans stem cell compartment death has never been observed, and we used engulfed small stem cells, notable by green fluorescent puncta, to count cell death events. We found UVC treatment of glp-1(ar202gf) animals increased stem cell death and increased lifespan. However, UVC treatment of double-mutant cep-1/p53(gk138);glp- 1/Notch(ar202gf) animals decreased stem cell death, increased tumor volume, and decreased animal lifespan. There are pharmacological agents that induce p53-independent cell death of human cells in culture; and two notable protocols are the PARP-trapping agents of temozolomide plus talazoparib and the nucleoside analogue 8-amino-adenosine. It is important to determine ways to rapidly test for pharmacological agents able to induce p53-independent cell death. We tested feeding cep- 1/p53(gk138);glp-1/Notch(ar202gf) nematodes with either 8-amino-adenosine or temozolomide plus talazoparib and found both were able to decrease tumor volume. This is the first comparison for p53-independent responses in cep-1/p53(gk138);glp-1/Notch(ar202gf) animals and showed UVC DNA damage increased tumor volume and decreased lifespan while PARP inhibition decreased tumor volume

The processing of Holliday junctions by BLM and WRN helicases is regulated by p53.

BLM, WRN, and p53 are involved in the homologous DNA recombination pathway. The DNA structure-specific helicases, BLM and WRN, unwind Holliday junctions (HJ), an activity that could suppress inappropriate homologous recombination during DNA replication. Here, we show that purified, recombinant p53 binds to BLM and WRN helicases and attenuates their ability to unwind synthetic HJ in vitro. The p53 248W mutant reduces abilities of both to bind HJ and inhibit helicase activities, whereas the p53 273H mutant loses these abilities. Moreover, full-length p53 and a C-terminal polypeptide (residues 373-383) inhibit the BLM and WRN helicase activities, but phosphorylation at Ser(376) or Ser(378) completely abolishes this inhibition. Following blockage of DNA replication, Ser(15) phospho-p53, BLM, and RAD51 colocalize in nuclear foci at sites likely to contain DNA replication intermediates in cells. Our results are consistent with a novel mechanism for p53-mediated regulation of DNA recombinational repair that involves p53 post-translational modifications and functional protein-protein interactions with BLM and WRN DNA helicases