The p53 protein is kept labile under normal conditions. This regulation is governed largely by its major negative regulator, Mdm2. In response to stress however, p53 accumulates and becomes activated. For this to occur, the inhibitory effects of Mdm2 have to be neutralized. Here we investigated the role of the promyelocytic leukemia protein (PML) in the activation of p53 in response to stress. We found that PML is critical for the accumulation of p53 in response to DNA damage under physiological conditions. PML protects p53 from Mdm2-mediated ubiquitination and degradation, and from inhibition of apoptosis. PML neutralizes the inhibitory effects of Mdm2 by prolonging the stress-induced phosphorylation of p53 on serine 20, a site of the checkpoint kinase 2 (Chk2). PML recruits Chk2 and p53 into the PML nuclear bodies and enhances p53/Chk2 interaction. Our results provide a novel mechanistic explanation for the cooperation between PML and p53 in response to DNA damage.

The Cannabis plant contains over 100 phytocannabinoids and hundreds of other components. The biological effects and interplay of these Cannabis compounds are not fully understood and yet influence the plant’s therapeutic effects. Here we assessed the antitumor effects of whole Cannabis extracts, which contained significant amounts of differing phytocannabinoids, on different cancer lines from various tumor origins. We first utilized our novel electrospray ionization liquid chromatography mass spectrometry method to analyze the phytocannabinoid contents of 124 Cannabis extracts. We then monitored the effects of 12 chosen different Cannabis extracts on 12 cancer cell lines. Our results show that specific Cannabis extracts impaired the survival and proliferation of cancer cell lines as well as induced apoptosis. Our findings showed that pure (-)-Δ 9 - trans -tetrahydrocannabinol (Δ 9 -THC) did not produce the same effects on these cell lines as the whole Cannabis extracts. Furthermore, Cannabis extracts with similar amounts of Δ 9 -THC produced significantly different effects on the survival of specific cancer cells. In addition, we demonstrated that specific Cannabis extracts may selectively and differentially affect cancer cells and differing cancer cell lines from the same organ origin. We also found that cannabimimetic receptors were differentially expressed among various cancer cell lines and suggest that this receptor diversity may contribute to the heterogeneous effects produced by the differing Cannabis extracts on each cell line. Our overall findings indicate that the effect of a Cannabis extract on a specific cancer cell line relies on the extract’s composition as well as on certain characteristics of the targeted cells.

The promyelocytic leukemia (PML) tumor suppressor is essential for the formation of PML nuclear bodies (NBs). PML and PMLNBs have been implicated in the regulation of growth inhibition, senescence and apoptosis. PML is activated in response to stress signals and is downregulated in certain human cancers. However, the factors mediating PML stability are incompletely understood. Here we demonstrate that a catalytically active form of the mammalian E3 ligase E6AP (HPV E6-associated protein) acts to reduce the half-life of the PML protein by promoting its degradation in the proteasome. E6AP mediates the ubiquitination of PML in an in vitro ubiquitination assay. E6AP and PML interact at physiological levels and colocalize in PML-NBs. Importantly, PML protein expression is elevated in multiple organs and cell types from E6AP null mice and in lymphoid cells is associated with increased number and intensity of PML-NBs. This PML elevation is enhanced in response to DNA damage. Our results identify E6AP as an important regulator of PML and PML-NBs. The promyelocytic leukemia (PML) tumor suppressor is implicated in the regulation of cell-cycle progression, premature senescence (triggered by oncogenic Ras) and apoptosis (reviewed by Bernardi and Pandolfi 1 ). Deregulation of PML can be oncogenic. PML-RARa contributes to acute promyelocytic leukemia (APL) 2,3 and downregulation of PML was observed in multiple human cancers. 4 PML knockout (KO) mice are resistant to lethal doses of ionizing irradiation (IR), and exhibit genomic instability and enhanced susceptibility to tumorigenesis upon exposure to carcinogens 1,5 or in the context of additional oncogenic events (e.g., a loss of Pten 6 ). The myriad of PML functions have been linked to its function in PML nuclear body (PML-NB) formation. 7 These are dynamic structures whose configuration and composition are modified in response to specific stress signals. 8 However, their mechanisms of action are only partially understood and are believed to be mediated by key proteins that are recruited to these structures, including pRb, SUMO, Daxx and p53. 9 The regulation of certain proteins, such as p53, is associated with the PML-NBs. 9 Stress stimuli that have been identified as activators of PML and PML-NBs include interferon (a and g), DNA damage, oncogenic stress and viral infection (reviewed in references 1,8,10 ). In contrast, exposure of cells to arsenic trioxide (As 2 O 3 ) or retinoic acid promotes PML degradation. 11 In contrast, the overexpression of specific cellular ubiquitin ligases, such as the Siah protein, 12 or certain viral ubiquitin ligases, such as the ICP0 regulatory protein of herpesvirus-1, 10 promotes the proteasomal degradation of PML and PML-RARa. However, the nature of the underlying mechanism of this degradation and whether it occurs under physiological conditions is not known. Recently, a function for CK2-mediated phosphorylation of PML (on serine 517) in the control of its protein stability was demonstrated. 13 In addition to phosphorylation, the covalen...

IntroductionA link between proteasomal degradation and cancer development has been established and a general proteasome inhibitor, Velcade (bortezomib), is in clinical use for the treatment of multiple myeloma and mantle cell lymphoma. 1,2 Deregulation of E3 ubiquitin ligases can be sufficient to suppress the expression and function of key tumor suppressors. For example, the inhibition of p53 as a consequence of Mdm2 amplification is frequently observed in human sarcomas and retinoblastoma. [3][4][5] Interestingly, in human papilloma virus (HPV)-infected cells the suppression of p53 is not achieved by Mdm2, but rather by the cellular E6AP (E6-associated protein) ubiquitin ligase, which is recruited to p53 by the HPV-E6 protein. [6][7][8] E6AP is encoded by the UBE3A locus, which is mutated in Angelman syndrome (AS), a human neuro-developmental disorder. 9 E6AP was the first mammalian ubiquitin E3 ligase to be identified. It is the prototype of the subfamily of E3 ligases that covalently bind ubiquitin and are characterized by a C-terminal HECT (homologous to the E6AP C terminus) domain. 10 We recently demonstrated that E6AP regulates the stability of the promyelocytic leukemia (PML) protein and the formation of PML nuclear bodies (PML-NBs). 11 PML is a tumor suppressor that was identified as a consequence of the chromosomal translocation of its gene in acute promyelocytic leukemia (APL). 12 Consistent with the role of PML as a tumor suppressor, PML deficient mice showed abnormally increased susceptibility to carcinogen 13,14 and oncogene-induced tumorigenesis. 15 Importantly, PML expression was found to be down-regulated or lost in a variety of human cancer types, including prostate, breast, and colon adenocarcinomas. 16,17 PML protein and the PML-NBs were found to play critical roles in cellular stress responses, including those that elicit apoptosis or cellular senescence. 18-21 Cellular senescence is emerging as an important mechanism for tumor suppression. 22,23 It represents a profound arrest of cellular proliferation, accompanied by a distinct set of alterations in the cellular phenotype, such as the formation of senescence-associated heterochromatin foci (SAHF, eg, H3K9me3) and up-regulation of certain inhibitors of cell growth, such as p21, PAI-1, and p16. 24 In this study, we explored the role of the E6AP-PML axis in HPV-independent cancer development. We chose pre-B/B-cell lymphomagenesis as a model For personal use only. on May 7, 2018. by guest www.bloodjournal.org From because of the high frequency of PML down-regulation in nonHodgkin lymphomas (NHLs). 16 For this purpose we used the well established E-myc transgenic mice, a mouse model for Burkitt lymphoma and other NHLs. 25 We found that the loss of one allele of E6AP significantly delayed Myc-driven B-cell lymphomagenesis and this was accompanied by elevated PML expression and the induction of cellular senescence. Importantly, E6AP expression was observed to be elevated in human Burkitt lymphoma and cell lines derived from these tumors. Our findi...

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