Activation and proliferation of T cells are tightly regulated during the immune response. We show... more Activation and proliferation of T cells are tightly regulated during the immune response. We show here that kinetics of proliferation of PHA activated T cells follows the expression of cMyc. Expression of p53 is also elevated and remains high several days after activation. To investigate the role of p53 in activated T cells, its expression was further elevated with nultin-3 treatment, a small molecule that dissociates the E3 ubiquitin protein ligase MDM2 from p53. Concomitantly, cMyc expression and proliferation decreased. At the other end of the cMyc-p53 axis, inhibition of cMyc with 10058-F4 led to down regulation of p53, likely through the lower level of cMyc induced p14ARF, which is also known to dissociate the p53-MDM2 complex. Both compounds induced cell cycle arrest and apoptosis. We conclude that the feedback regulation between cMyc and p53 is important for the T cell homeostasis. We also show that the two compounds modulating p53 and cMyc levels inhibited proliferation without abolishing the cytotoxic function, thus demonstrating the dichotomy between proliferation and cytotoxicity in activated T cells.
Activation and proliferation of T cells are tightly regulated during the immune response. We show... more Activation and proliferation of T cells are tightly regulated during the immune response. We show here that kinetics of proliferation of PHA activated T cells follows the expression of cMyc. Expression of p53 is also elevated and remains high several days after activation. To investigate the role of p53 in activated T cells, its expression was further elevated with nultin-3 treatment, a small molecule that dissociates the E3 ubiquitin protein ligase MDM2 from p53. Concomitantly, cMyc expression and proliferation decreased. At the other end of the cMyc-p53 axis, inhibition of cMyc with 10058-F4 led to down regulation of p53, likely through the lower level of cMyc induced p14ARF, which is also known to dissociate the p53-MDM2 complex. Both compounds induced cell cycle arrest and apoptosis. We conclude that the feedback regulation between cMyc and p53 is important for the T cell homeostasis. We also show that the two compounds modulating p53 and cMyc levels inhibited proliferation without abolishing the cytotoxic function, thus demonstrating the dichotomy between proliferation and cytotoxicity in activated T cells.
The role of sirtuins in cancer has recently stimulated both considerable interest and debate. It ... more The role of sirtuins in cancer has recently stimulated both considerable interest and debate. It is becoming clear that some sirtuins deacetylate important tumor suppressors thereby impinging on their activity. Human SirT1, for instance, has been shown to deacetylate p53 in biochemical assays, and growing evidence indicates that it also performs this activity in cells. Since deacetylation of p53 correlates with a decreased p53 transcriptional function, it is conceivable that sirtuin inhibition could lead to improved tumor suppression. There are, however, still many open questions regarding, for example, whether sirtuins deacetylate those lysine residues in p53 that are critical for its activity. Preliminary observations also suggest that sirtuin‐mediated modulation of p53 can also take place indirectly through changes in cellular processes (e.g., nucleolar function and p300 activity) known to affect p53. It also remains unclear whether depletion in the activity of a single sirtuin suffices to stabilize and activate p53 substantially or additional changes in other factors (including other sirtuins) are required. Finally, data from SIRT1‐knockout mice demonstrate that sustained depletion of SirT1 can give rise to genomic instability and that, therefore, SirT1 acts as a tumor suppressor. This observation implies that the safety of therapeutic interventions based on SirT1 inhibition need to be evaluated. Here we review and examine the available data on the regulation of p53 by sirtuins and on the changes in sirtuin function in tumor cells, and discuss whether pharmacological inhibition of sirtuin activity constitutes an adequate approach for cancer treatment.
Malignant melanoma is the most dangerous type of skin cancer. Although recent progress in treatme... more Malignant melanoma is the most dangerous type of skin cancer. Although recent progress in treatment has been achieved, lack of response, drug resistance and relapse remain major problems. The tumor suppressor p53 is rarely mutated in melanoma, yet it is inactive in the majority of cases due to dysregulation of upstream pathways. Thus, we screened for compounds that can activate p53 in melanoma cells. Here we describe effects of the small molecule MJ25 (2-{[2-(1,3-benzothiazol-2-ylsulfonyl)ethyl]thio}-1,3-benzoxazole), which increased the level of p53-dependent transactivation both as a single agent and in combination with nutlin-3. Furthermore, MJ25 showed potent cytotoxicity towards melanoma cell lines, whilst having weaker effects against human normal cells. MJ25 was also identified in an independent screen as an inhibitor of thioredoxin reductase 1 (TrxR1), an important selenoenzyme in the control of oxidative stress and redox regulation. The well-characterized TrxR inhibitor aur...
Lecture Notes of the Institute for Computer Sciences, Social Informatics and Telecommunications Engineering, 2010
The p53 tumour suppressor plays key regulatory roles in various fundamental biological processes,... more The p53 tumour suppressor plays key regulatory roles in various fundamental biological processes, including development, ageing and cell differentiation. It is therefore known as the guardian of the genome and is currently the most extensively studied protein ...
Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigat... more Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acetylation signatures for 19 different KDACIs, covering all 18 human lysine deacetylases. Most KDACIs increased acetylation of a small, specific subset of the acetylome, including sites on histones and other chromatin-associated proteins. Inhibitor treatment combined with genetic deletion showed that the effects of the pan-sirtuin inhibitor nicotinamide are primarily mediated by SIRT1 inhibition. Furthermore, we confirmed that the effects of tubacin and bufexamac on cytoplasmic proteins result from inhibition of HDAC6. Bufexamac also triggered an HDAC6-independent, hypoxia-like response by stabilizing HIF1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. ...
In order to simulate a Superheated-Superconducting detector which is under construction for Parti... more In order to simulate a Superheated-Superconducting detector which is under construction for Particle Physics purposes, we study a bidimensional lattice of small superconducting spheres placed in an external magnetic field. We propose a model to study the diamagnetic interactions among the spheres and solve it using numerical Monte-Carlo techniques. New phenomena are found and the ensuing results are analyzed. Finally it is proposed a qualitative explanation.
Cervical cancer is a major cause of death in women worldwide and is strongly associated with huma... more Cervical cancer is a major cause of death in women worldwide and is strongly associated with human papillomavirus (HPV) infection. Integration of HPV is thought to be a key step in malignant progression, and is associated with loss of regulation of the viral E6 and E7 oncogenes. Leptomycin B (LMB), a nuclear export inhibitor, has previously been shown to induce apoptosis in primary keratinocytes transduced with the HPV 16 E7 or E6/E7 genes, but not in normal cells. We show here that LMB can also induce apoptosis in derivatives of the W12 cell line that contain either episomal or integrated HPV 16. Cells transduced with HPV 16 E7 or E6/E7, and the episomal and integrated W12 derivatives showed distinct temporal expression patterns of the apoptotic markers activated caspase-3 and M30. The expression of both markers occurred later in the episomal derivatives than in either transduced cells or W12 derivatives containing integrated HPV. These findings suggest that, although LMB can induce apoptosis in keratinocytes containing episomal or integrated HPV 16, genome status is likely to influence the response of HPV-associated anogenital lesions to LMB treatment.
In normal cells, p53 is maintained at a low level by ubiquitin-mediated proteolysis, but after ge... more In normal cells, p53 is maintained at a low level by ubiquitin-mediated proteolysis, but after genotoxic insult this process is inhibited and p53 levels rise dramatically. Ubiquitination of p53 requires the ubiquitinactivating enzyme Ubc5 as a ubiquitin conjugation enzyme and Mdm2, which acts as a ubiquitin protein ligase. In addition to the N-terminal region, which is required for interaction with Mdm2, the C-terminal domain of p53 modulates the susceptibility of p53 to Mdm2-mediated degradation. To analyze the role of the C-terminal domain in p53 ubiquitination, we have generated p53 molecules containing single and multiple lysine-toarginine changes between residues 370 and 386. Although wild-type (WT) and mutant molecules show similar subcellular distributions, the mutants display a higher transcriptional activity than WT p53. Simultaneous mutation of lysine residues 370, 372, 373, 381, 382, and 386 to arginine residues (6KR p53 mutant) generates a p53 molecule with potent transcriptional activity that is resistant to Mdm2-induced degradation and is refractory to Mdm2-mediated ubiquitination. In contrast to WT p53, transcriptional activity directed by the 6KR p53 mutant fails to be negatively regulated by Mdm2. Those differences are also manifest in HeLa cells which express the human papillomavirus E6 protein, suggesting that p53 C-terminal lysine residues are also implicated in E6-AP-mediated ubiquitination. These data suggest that p53 C-terminal lysine residues are the main sites of ubiquitin ligation, which target p53 for proteasome-mediated degradation.
Currently, around 11 million people are living with a tumour that contains an inactivating mutati... more Currently, around 11 million people are living with a tumour that contains an inactivating mutation of TP53 (the human gene that encodes p53) and another 11 million have tumours in which the p53 pathway is partially abrogated through the inactivation of other signalling or effector components. The p53 pathway is therefore a prime target for new cancer drug development, and
The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and is associated wi... more The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and is associated with poor prognosis. In such patients, FLT3 tyrosine kinase inhibitors (TKIs) are only partially effective and do not eliminate the leukemia stem cells (LSCs) that are assumed to be the source of treatment failure. Here, we show that the NAD-dependent SIRT1 deacetylase is selectively overexpressed in primary human FLT3-ITD AML LSCs. This SIRT1 overexpression is related to enhanced expression of the USP22 deubiquitinase induced by c-MYC, leading to reduced SIRT1 ubiquitination and enhanced stability. Inhibition of SIRT1 expression or activity reduced the growth of FLT3-ITD AML LSCs and significantly enhanced TKI-mediated killing of the cells. Therefore, these results identify a c-MYC-related network that enhances SIRT1 protein expression in human FLT3-ITD AML LSCs and contributes to their maintenance. Inhibition of this oncogenic network could be an attractive approach for targeting FLT3-IT...
The plum pox potyvirus (PPV) NI. protease expressed from a medium copy number plasmid was able to... more The plum pox potyvirus (PPV) NI. protease expressed from a medium copy number plasmid was able to process an excess of substrate expressed from a high copy number plasmid, in a binary Escherichiu c&expression system. The AB7 NI, protease mutant only partially processed the Nib-CP junction but its efficiency was independent of the amount of substrate. The AB7 mutant essentially did not recognize an artificial cleavage site which was quite efficiently recognized by the wild-type protease. No competitive inhibition of the proteolytic activity by the presence of excess of different protease mutants was observed.
The p53 tumour suppressor protein is regulated by ubiquitin-mediated proteasomal degradation. In ... more The p53 tumour suppressor protein is regulated by ubiquitin-mediated proteasomal degradation. In normal cells p53 is constitutively ubiquitylated by the Mdm2 ubiquitin ligase. When the p53 response is activated by stress signals p53 levels rise due to inhibition of this degradative pathway. Here we show that p53 is modified by the small ubiquitin-like protein SUMO-1 at a single site, K386, in the C-terminus of the protein. Modification in vitro requires only SUMO-1, the SUMO-1 activating enzyme and ubc9. SUMO-1 and ubiquitin modification do not compete for the same lysine acceptor sites in p53. Overexpression of SUMO-1 activates the transcriptional activity of wildtype p53, but not K386R p53 where the SUMO-1 acceptor site has been mutated. The SUMO-1 modification pathway therefore acts as a potential regulator of the p53 response and may represent a novel target for the development of therapeutically useful modulators of the p53 response.
Proceedings of the National Academy of Sciences, 2000
In over 90% of cervical cancers and cancer-derived cell lines, the p53 tumor suppressor pathway i... more In over 90% of cervical cancers and cancer-derived cell lines, the p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV). The HPV E6 protein promotes the degradation of p53 and thus inhibits the stabilization and activation of p53 that would normally occur in response to HPV E7 oncogene expression. Restoration of p53 function in these cells by blocking this pathway should promote a selective therapeutic affect. Here we show that treatment with the small molecule nuclear export inhibitor, leptomycin B, and actinomycin D leads to the accumulation of transcriptionally active p53 in the nucleus of HeLa, CaSki, and SiHa cells. Northern blot analyses showed that both actinomycin D and leptomycin B reduced the amount of HPV E6-E7 mRNA whereas combined treatment with the drugs showed almost complete disappearance of the viral mRNA. The combined treatment activated p53-dependant transcription, and increases in both p21 WAF1͞CIP1 and Hdm2 mRNA were seen. The combined treatment resulted in apoptotic death in the cells, as evidenced by nuclear fragmentation and PARP-cleavage indicative of caspase 3 activity. These effects were greatly reduced by expressing a dominant negative p53 protein. The present study shows that small molecules can reactivate p53 in cervical carcinoma cells, and this reactivation is associated with an extensive biological response, including the induction of the apoptotic death of the cells.
In vitro, high-risk human papillomavirus E6 proteins have been shown, in conjunction with E6-asso... more In vitro, high-risk human papillomavirus E6 proteins have been shown, in conjunction with E6-associated protein (E6AP), to mediate ubiquitination of p53 and its degradation by the 26S proteasome by a pathway that is thought to be analogous to Mdm2-mediated p53 degradation. However, differences in the requirements of E6/E6AP and Mdm2 to promote the degradation of p53, both in vivo and in vitro, suggest that these two E3 ligases may promote p53 degradation by distinct pathways. Using tools that disrupt ubiquitination and degradation, clear differences between E6- and Mdm2-mediated p53 degradation are presented. The consistent failure to fully protect p53 protein from E6-mediated degradation by disrupting the ubiquitin-degradation pathway provides the first evidence of an E6-dependent, ubiquitin-independent, p53 degradation pathway in vivo.
Terminal dierentiation requires cell cycle withdrawal, suggesting the involvement of negative cel... more Terminal dierentiation requires cell cycle withdrawal, suggesting the involvement of negative cell cycle controllers in the process. We have analysed the involvement of the retinoblastoma family of proteins (pRb, p107 and p130) in epidermal proliferation and dierentiation. These proteins play key roles as inhibitors of cell cycle progression and are involved in muscle and neuron dierentiation. We found that during in vitro dierentiation of human HaCaT keratinocytes, pRb, p107 and p130 are sequentially expressed, in contrast to the co-expression observed during cell cycle progression in the same cells. Immuno¯uorescence studies on skin sections revealed the presence of pRb and p107 in basal and suprabasal cell layers, whilst p130 is restricted to cells already committed to dierentiation in the suprabasal compartments. To explore the functional signi®cance of the dierential expression of these proteins, transfection experiments were performed in HaCaT keratinocytes. We observed that the forced over-expression of pRb, p107 or p130 individually did not induce dierentiation of the transfected cells. However, the co-transfection of pRb and p107 induced the expression of early dierentiation markers (keratin k10) and triple transfectants pRb+p107+p130 expressed markers representative of later stages of epidermal dierentiation (involucrin). Finally, we observed that these three proteins repress keratinocyte proliferation, although to a dierent extent (p1074pRb5p130). These results indicate that the members of the pRb family play speci®c, yet coordinated roles during epidermal dierentiation, and that the ordered progression along the dierent stages of this process results from the eects of dierent combinations of these proteins.
We have previously shown that a 20 amino acid peptide derived from the third ankyrin-like repeat ... more We have previously shown that a 20 amino acid peptide derived from the third ankyrin-like repeat of the p16 CDKN2/INK4a (p16) tumour suppressor protein (residues 84 ± 103 of the human p16 protein) can bind to cdk4 and cdk6 and inhibit cdk4-cyclin D1 kinase activity in vitro as well as block cell cycle progression through G1. Substitution of two valine residues corresponding to amino acids 95 and 96 (V95A and V96A) of the p16 peptide reduces the binding to cdk4 and cdk6 and increases its IC 0.5 for kinase inhibition approximately threefold when linked to the Antennapedia homeodomain carrier sequence. The same mutations increase the IC 0.5 approximately ®vefold in the p16 protein. Substitution of aspartic acid 92 by alanine instead increases the binding of the peptide to cdk4 and cdk6 and the kinase inhibitory activity. The p16 peptide blocks S-phase entry in nonsynchronized human HaCaT cells by approximately 90% at a 24 mM concentration. The V95A and V96A double substitution minimizes the cell cycle inhibitory capacity of the peptide whereas the D92A substitution increases its capacity to block cell cycle progression. A deletion series of the p16 derived peptide shows that a 10 residue peptide still retains cdk4-cyclin D1 kinase and cell cycle inhibitory activity. The p16 peptide inhibited S-phase entry in ®ve cell lines tested, varying between 47 ± 75%, but had only a limited (11%) inhibitory eect in the pRb negative Saos-2 cells at a concentration of 24 mM. Like the full length p16 protein, the p16 peptide does not inhibit cyclin E dependent cdk2 kinase activity in vitro. These data suggest that acute inhibition of CDKcyclin D activity by a peptide derived from the INK4 family will stop cells in late G1 in a pRb dependent fashion.
Thecylindrical inclusion protein ofpotyviruses contains theso-called nucleoside triphosphate bind... more Thecylindrical inclusion protein ofpotyviruses contains theso-called nucleoside triphosphate binding motif, an amino acidsequencemotifpresent inproteins encoded bymostpositive-strand RNA viruses, some double-strand RNA viruses, apparentlyallgroupsofdouble-strand DNAviruses, andalso several single-strand DNA viruses. Further sequenceanalysis hasallowed toinclude thecylindrical inclusion protein ofpotyviruses as a memberofa superfamily ofhelicaselike proteins. Inthis paper we showthatthepurified cylindrical inclusion protein ofplumpox potyvirus interacts withRNA andATP andcopurifies witha nucleic acid-stimulated ATPaseactivity. Toourknowledge, this isthefirst timethat this kindofenzymatic activity has beenexperimentally associated with a positive-strand RNA virus-encoded protein. Several sourcesofinformation haveledtohypotheses abouttheinvolvement ofdifferent protein activities inthe life cycle ofRNA viruses. Recent technical advances have madeitpossible forseveral ofthese proposals t...
Expression of the Epstein-Ban" virus (EBV) latent membrane protein (LMP1) is regulated by virus-a... more Expression of the Epstein-Ban" virus (EBV) latent membrane protein (LMP1) is regulated by virus-and host cell-specific factors. The EBV nuclear antigen 2 (EBNA2) has been shown to transactivate a number of viral and cellular gene promoters including the promoter for the LMP1 gene. EBNA2 is targeted to at least some of these promoters by interacting with a cellular DNA binding protein, RBP-Jlc. In the present report we confirm and extend our previous observation that the LMP1 promoter can be activated by EBNA2 in the absence of the RBP-Jtc-binding sequence in the LMP1 promoter regulatory region (LRS). We show that two distinct LRS regions, -106 to +40 and -176 to -136, contribute to EBNA2 responsiveness. Site-directed mutagenesis analysis of the upstream -176/-136 EBNA2 responsive element revealed that two critical cis-acting elements are required for full promoter function. These same elements analysed by electro-phoretic mobility shift assays define two binding sites recognized by nuclear factors derived from B cells. An octamer-like sequence (-147 to -139) contained overlapping binding sites for an unidentified transcriptional repressor on the one hand and a factor(s) belonging to the POU domain family but distinct from Oct-1 and Oct-2 on the other. An adjacent purine tract (-171 to -155) held a PU.1 binding site, which was also recognized by a related factor. The results suggest that the POU domain protein and either of two PU boxbinding factors bind simultaneously to LRS, creating a ternary complex that might be in part responsible for mediating the transactivation of the LMP1 promoter by EBNA2. There were no qualitative differences between EBV-negative and EBV-positive cells with regard to transcription factor binding to the octamer-like sequence and the PU.1 recognition site, as revealed by electrophoretic mobility shift assays.
The infectivity of plum pox potyvirus (PPV) RNA was decreased by treatment with proteases. Ribonu... more The infectivity of plum pox potyvirus (PPV) RNA was decreased by treatment with proteases. Ribonuclease digestion of iodinated PPV RNA yielded material which had an electrophoretic mobility corresponding to Mr 22000. This protein presumably corresponds to the protease-sensitive structure needed for infectivity. A protein-linked RNase Tl-resistant oligonucleotide, 38 nucleotides long, was sequenced and shown to correspond to the 5' terminus of the RNA by sequence comparison to the RNAs of two other potyviruses, tobacco etch virus and tobacco vein mottling virus. A 12 nucleotide block was found to be completely conserved in the RNAs of the three viruses.
Activation and proliferation of T cells are tightly regulated during the immune response. We show... more Activation and proliferation of T cells are tightly regulated during the immune response. We show here that kinetics of proliferation of PHA activated T cells follows the expression of cMyc. Expression of p53 is also elevated and remains high several days after activation. To investigate the role of p53 in activated T cells, its expression was further elevated with nultin-3 treatment, a small molecule that dissociates the E3 ubiquitin protein ligase MDM2 from p53. Concomitantly, cMyc expression and proliferation decreased. At the other end of the cMyc-p53 axis, inhibition of cMyc with 10058-F4 led to down regulation of p53, likely through the lower level of cMyc induced p14ARF, which is also known to dissociate the p53-MDM2 complex. Both compounds induced cell cycle arrest and apoptosis. We conclude that the feedback regulation between cMyc and p53 is important for the T cell homeostasis. We also show that the two compounds modulating p53 and cMyc levels inhibited proliferation without abolishing the cytotoxic function, thus demonstrating the dichotomy between proliferation and cytotoxicity in activated T cells.
Activation and proliferation of T cells are tightly regulated during the immune response. We show... more Activation and proliferation of T cells are tightly regulated during the immune response. We show here that kinetics of proliferation of PHA activated T cells follows the expression of cMyc. Expression of p53 is also elevated and remains high several days after activation. To investigate the role of p53 in activated T cells, its expression was further elevated with nultin-3 treatment, a small molecule that dissociates the E3 ubiquitin protein ligase MDM2 from p53. Concomitantly, cMyc expression and proliferation decreased. At the other end of the cMyc-p53 axis, inhibition of cMyc with 10058-F4 led to down regulation of p53, likely through the lower level of cMyc induced p14ARF, which is also known to dissociate the p53-MDM2 complex. Both compounds induced cell cycle arrest and apoptosis. We conclude that the feedback regulation between cMyc and p53 is important for the T cell homeostasis. We also show that the two compounds modulating p53 and cMyc levels inhibited proliferation without abolishing the cytotoxic function, thus demonstrating the dichotomy between proliferation and cytotoxicity in activated T cells.
The role of sirtuins in cancer has recently stimulated both considerable interest and debate. It ... more The role of sirtuins in cancer has recently stimulated both considerable interest and debate. It is becoming clear that some sirtuins deacetylate important tumor suppressors thereby impinging on their activity. Human SirT1, for instance, has been shown to deacetylate p53 in biochemical assays, and growing evidence indicates that it also performs this activity in cells. Since deacetylation of p53 correlates with a decreased p53 transcriptional function, it is conceivable that sirtuin inhibition could lead to improved tumor suppression. There are, however, still many open questions regarding, for example, whether sirtuins deacetylate those lysine residues in p53 that are critical for its activity. Preliminary observations also suggest that sirtuin‐mediated modulation of p53 can also take place indirectly through changes in cellular processes (e.g., nucleolar function and p300 activity) known to affect p53. It also remains unclear whether depletion in the activity of a single sirtuin suffices to stabilize and activate p53 substantially or additional changes in other factors (including other sirtuins) are required. Finally, data from SIRT1‐knockout mice demonstrate that sustained depletion of SirT1 can give rise to genomic instability and that, therefore, SirT1 acts as a tumor suppressor. This observation implies that the safety of therapeutic interventions based on SirT1 inhibition need to be evaluated. Here we review and examine the available data on the regulation of p53 by sirtuins and on the changes in sirtuin function in tumor cells, and discuss whether pharmacological inhibition of sirtuin activity constitutes an adequate approach for cancer treatment.
Malignant melanoma is the most dangerous type of skin cancer. Although recent progress in treatme... more Malignant melanoma is the most dangerous type of skin cancer. Although recent progress in treatment has been achieved, lack of response, drug resistance and relapse remain major problems. The tumor suppressor p53 is rarely mutated in melanoma, yet it is inactive in the majority of cases due to dysregulation of upstream pathways. Thus, we screened for compounds that can activate p53 in melanoma cells. Here we describe effects of the small molecule MJ25 (2-{[2-(1,3-benzothiazol-2-ylsulfonyl)ethyl]thio}-1,3-benzoxazole), which increased the level of p53-dependent transactivation both as a single agent and in combination with nutlin-3. Furthermore, MJ25 showed potent cytotoxicity towards melanoma cell lines, whilst having weaker effects against human normal cells. MJ25 was also identified in an independent screen as an inhibitor of thioredoxin reductase 1 (TrxR1), an important selenoenzyme in the control of oxidative stress and redox regulation. The well-characterized TrxR inhibitor aur...
Lecture Notes of the Institute for Computer Sciences, Social Informatics and Telecommunications Engineering, 2010
The p53 tumour suppressor plays key regulatory roles in various fundamental biological processes,... more The p53 tumour suppressor plays key regulatory roles in various fundamental biological processes, including development, ageing and cell differentiation. It is therefore known as the guardian of the genome and is currently the most extensively studied protein ...
Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigat... more Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acetylation signatures for 19 different KDACIs, covering all 18 human lysine deacetylases. Most KDACIs increased acetylation of a small, specific subset of the acetylome, including sites on histones and other chromatin-associated proteins. Inhibitor treatment combined with genetic deletion showed that the effects of the pan-sirtuin inhibitor nicotinamide are primarily mediated by SIRT1 inhibition. Furthermore, we confirmed that the effects of tubacin and bufexamac on cytoplasmic proteins result from inhibition of HDAC6. Bufexamac also triggered an HDAC6-independent, hypoxia-like response by stabilizing HIF1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. ...
In order to simulate a Superheated-Superconducting detector which is under construction for Parti... more In order to simulate a Superheated-Superconducting detector which is under construction for Particle Physics purposes, we study a bidimensional lattice of small superconducting spheres placed in an external magnetic field. We propose a model to study the diamagnetic interactions among the spheres and solve it using numerical Monte-Carlo techniques. New phenomena are found and the ensuing results are analyzed. Finally it is proposed a qualitative explanation.
Cervical cancer is a major cause of death in women worldwide and is strongly associated with huma... more Cervical cancer is a major cause of death in women worldwide and is strongly associated with human papillomavirus (HPV) infection. Integration of HPV is thought to be a key step in malignant progression, and is associated with loss of regulation of the viral E6 and E7 oncogenes. Leptomycin B (LMB), a nuclear export inhibitor, has previously been shown to induce apoptosis in primary keratinocytes transduced with the HPV 16 E7 or E6/E7 genes, but not in normal cells. We show here that LMB can also induce apoptosis in derivatives of the W12 cell line that contain either episomal or integrated HPV 16. Cells transduced with HPV 16 E7 or E6/E7, and the episomal and integrated W12 derivatives showed distinct temporal expression patterns of the apoptotic markers activated caspase-3 and M30. The expression of both markers occurred later in the episomal derivatives than in either transduced cells or W12 derivatives containing integrated HPV. These findings suggest that, although LMB can induce apoptosis in keratinocytes containing episomal or integrated HPV 16, genome status is likely to influence the response of HPV-associated anogenital lesions to LMB treatment.
In normal cells, p53 is maintained at a low level by ubiquitin-mediated proteolysis, but after ge... more In normal cells, p53 is maintained at a low level by ubiquitin-mediated proteolysis, but after genotoxic insult this process is inhibited and p53 levels rise dramatically. Ubiquitination of p53 requires the ubiquitinactivating enzyme Ubc5 as a ubiquitin conjugation enzyme and Mdm2, which acts as a ubiquitin protein ligase. In addition to the N-terminal region, which is required for interaction with Mdm2, the C-terminal domain of p53 modulates the susceptibility of p53 to Mdm2-mediated degradation. To analyze the role of the C-terminal domain in p53 ubiquitination, we have generated p53 molecules containing single and multiple lysine-toarginine changes between residues 370 and 386. Although wild-type (WT) and mutant molecules show similar subcellular distributions, the mutants display a higher transcriptional activity than WT p53. Simultaneous mutation of lysine residues 370, 372, 373, 381, 382, and 386 to arginine residues (6KR p53 mutant) generates a p53 molecule with potent transcriptional activity that is resistant to Mdm2-induced degradation and is refractory to Mdm2-mediated ubiquitination. In contrast to WT p53, transcriptional activity directed by the 6KR p53 mutant fails to be negatively regulated by Mdm2. Those differences are also manifest in HeLa cells which express the human papillomavirus E6 protein, suggesting that p53 C-terminal lysine residues are also implicated in E6-AP-mediated ubiquitination. These data suggest that p53 C-terminal lysine residues are the main sites of ubiquitin ligation, which target p53 for proteasome-mediated degradation.
Currently, around 11 million people are living with a tumour that contains an inactivating mutati... more Currently, around 11 million people are living with a tumour that contains an inactivating mutation of TP53 (the human gene that encodes p53) and another 11 million have tumours in which the p53 pathway is partially abrogated through the inactivation of other signalling or effector components. The p53 pathway is therefore a prime target for new cancer drug development, and
The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and is associated wi... more The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and is associated with poor prognosis. In such patients, FLT3 tyrosine kinase inhibitors (TKIs) are only partially effective and do not eliminate the leukemia stem cells (LSCs) that are assumed to be the source of treatment failure. Here, we show that the NAD-dependent SIRT1 deacetylase is selectively overexpressed in primary human FLT3-ITD AML LSCs. This SIRT1 overexpression is related to enhanced expression of the USP22 deubiquitinase induced by c-MYC, leading to reduced SIRT1 ubiquitination and enhanced stability. Inhibition of SIRT1 expression or activity reduced the growth of FLT3-ITD AML LSCs and significantly enhanced TKI-mediated killing of the cells. Therefore, these results identify a c-MYC-related network that enhances SIRT1 protein expression in human FLT3-ITD AML LSCs and contributes to their maintenance. Inhibition of this oncogenic network could be an attractive approach for targeting FLT3-IT...
The plum pox potyvirus (PPV) NI. protease expressed from a medium copy number plasmid was able to... more The plum pox potyvirus (PPV) NI. protease expressed from a medium copy number plasmid was able to process an excess of substrate expressed from a high copy number plasmid, in a binary Escherichiu c&expression system. The AB7 NI, protease mutant only partially processed the Nib-CP junction but its efficiency was independent of the amount of substrate. The AB7 mutant essentially did not recognize an artificial cleavage site which was quite efficiently recognized by the wild-type protease. No competitive inhibition of the proteolytic activity by the presence of excess of different protease mutants was observed.
The p53 tumour suppressor protein is regulated by ubiquitin-mediated proteasomal degradation. In ... more The p53 tumour suppressor protein is regulated by ubiquitin-mediated proteasomal degradation. In normal cells p53 is constitutively ubiquitylated by the Mdm2 ubiquitin ligase. When the p53 response is activated by stress signals p53 levels rise due to inhibition of this degradative pathway. Here we show that p53 is modified by the small ubiquitin-like protein SUMO-1 at a single site, K386, in the C-terminus of the protein. Modification in vitro requires only SUMO-1, the SUMO-1 activating enzyme and ubc9. SUMO-1 and ubiquitin modification do not compete for the same lysine acceptor sites in p53. Overexpression of SUMO-1 activates the transcriptional activity of wildtype p53, but not K386R p53 where the SUMO-1 acceptor site has been mutated. The SUMO-1 modification pathway therefore acts as a potential regulator of the p53 response and may represent a novel target for the development of therapeutically useful modulators of the p53 response.
Proceedings of the National Academy of Sciences, 2000
In over 90% of cervical cancers and cancer-derived cell lines, the p53 tumor suppressor pathway i... more In over 90% of cervical cancers and cancer-derived cell lines, the p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV). The HPV E6 protein promotes the degradation of p53 and thus inhibits the stabilization and activation of p53 that would normally occur in response to HPV E7 oncogene expression. Restoration of p53 function in these cells by blocking this pathway should promote a selective therapeutic affect. Here we show that treatment with the small molecule nuclear export inhibitor, leptomycin B, and actinomycin D leads to the accumulation of transcriptionally active p53 in the nucleus of HeLa, CaSki, and SiHa cells. Northern blot analyses showed that both actinomycin D and leptomycin B reduced the amount of HPV E6-E7 mRNA whereas combined treatment with the drugs showed almost complete disappearance of the viral mRNA. The combined treatment activated p53-dependant transcription, and increases in both p21 WAF1͞CIP1 and Hdm2 mRNA were seen. The combined treatment resulted in apoptotic death in the cells, as evidenced by nuclear fragmentation and PARP-cleavage indicative of caspase 3 activity. These effects were greatly reduced by expressing a dominant negative p53 protein. The present study shows that small molecules can reactivate p53 in cervical carcinoma cells, and this reactivation is associated with an extensive biological response, including the induction of the apoptotic death of the cells.
In vitro, high-risk human papillomavirus E6 proteins have been shown, in conjunction with E6-asso... more In vitro, high-risk human papillomavirus E6 proteins have been shown, in conjunction with E6-associated protein (E6AP), to mediate ubiquitination of p53 and its degradation by the 26S proteasome by a pathway that is thought to be analogous to Mdm2-mediated p53 degradation. However, differences in the requirements of E6/E6AP and Mdm2 to promote the degradation of p53, both in vivo and in vitro, suggest that these two E3 ligases may promote p53 degradation by distinct pathways. Using tools that disrupt ubiquitination and degradation, clear differences between E6- and Mdm2-mediated p53 degradation are presented. The consistent failure to fully protect p53 protein from E6-mediated degradation by disrupting the ubiquitin-degradation pathway provides the first evidence of an E6-dependent, ubiquitin-independent, p53 degradation pathway in vivo.
Terminal dierentiation requires cell cycle withdrawal, suggesting the involvement of negative cel... more Terminal dierentiation requires cell cycle withdrawal, suggesting the involvement of negative cell cycle controllers in the process. We have analysed the involvement of the retinoblastoma family of proteins (pRb, p107 and p130) in epidermal proliferation and dierentiation. These proteins play key roles as inhibitors of cell cycle progression and are involved in muscle and neuron dierentiation. We found that during in vitro dierentiation of human HaCaT keratinocytes, pRb, p107 and p130 are sequentially expressed, in contrast to the co-expression observed during cell cycle progression in the same cells. Immuno¯uorescence studies on skin sections revealed the presence of pRb and p107 in basal and suprabasal cell layers, whilst p130 is restricted to cells already committed to dierentiation in the suprabasal compartments. To explore the functional signi®cance of the dierential expression of these proteins, transfection experiments were performed in HaCaT keratinocytes. We observed that the forced over-expression of pRb, p107 or p130 individually did not induce dierentiation of the transfected cells. However, the co-transfection of pRb and p107 induced the expression of early dierentiation markers (keratin k10) and triple transfectants pRb+p107+p130 expressed markers representative of later stages of epidermal dierentiation (involucrin). Finally, we observed that these three proteins repress keratinocyte proliferation, although to a dierent extent (p1074pRb5p130). These results indicate that the members of the pRb family play speci®c, yet coordinated roles during epidermal dierentiation, and that the ordered progression along the dierent stages of this process results from the eects of dierent combinations of these proteins.
We have previously shown that a 20 amino acid peptide derived from the third ankyrin-like repeat ... more We have previously shown that a 20 amino acid peptide derived from the third ankyrin-like repeat of the p16 CDKN2/INK4a (p16) tumour suppressor protein (residues 84 ± 103 of the human p16 protein) can bind to cdk4 and cdk6 and inhibit cdk4-cyclin D1 kinase activity in vitro as well as block cell cycle progression through G1. Substitution of two valine residues corresponding to amino acids 95 and 96 (V95A and V96A) of the p16 peptide reduces the binding to cdk4 and cdk6 and increases its IC 0.5 for kinase inhibition approximately threefold when linked to the Antennapedia homeodomain carrier sequence. The same mutations increase the IC 0.5 approximately ®vefold in the p16 protein. Substitution of aspartic acid 92 by alanine instead increases the binding of the peptide to cdk4 and cdk6 and the kinase inhibitory activity. The p16 peptide blocks S-phase entry in nonsynchronized human HaCaT cells by approximately 90% at a 24 mM concentration. The V95A and V96A double substitution minimizes the cell cycle inhibitory capacity of the peptide whereas the D92A substitution increases its capacity to block cell cycle progression. A deletion series of the p16 derived peptide shows that a 10 residue peptide still retains cdk4-cyclin D1 kinase and cell cycle inhibitory activity. The p16 peptide inhibited S-phase entry in ®ve cell lines tested, varying between 47 ± 75%, but had only a limited (11%) inhibitory eect in the pRb negative Saos-2 cells at a concentration of 24 mM. Like the full length p16 protein, the p16 peptide does not inhibit cyclin E dependent cdk2 kinase activity in vitro. These data suggest that acute inhibition of CDKcyclin D activity by a peptide derived from the INK4 family will stop cells in late G1 in a pRb dependent fashion.
Thecylindrical inclusion protein ofpotyviruses contains theso-called nucleoside triphosphate bind... more Thecylindrical inclusion protein ofpotyviruses contains theso-called nucleoside triphosphate binding motif, an amino acidsequencemotifpresent inproteins encoded bymostpositive-strand RNA viruses, some double-strand RNA viruses, apparentlyallgroupsofdouble-strand DNAviruses, andalso several single-strand DNA viruses. Further sequenceanalysis hasallowed toinclude thecylindrical inclusion protein ofpotyviruses as a memberofa superfamily ofhelicaselike proteins. Inthis paper we showthatthepurified cylindrical inclusion protein ofplumpox potyvirus interacts withRNA andATP andcopurifies witha nucleic acid-stimulated ATPaseactivity. Toourknowledge, this isthefirst timethat this kindofenzymatic activity has beenexperimentally associated with a positive-strand RNA virus-encoded protein. Several sourcesofinformation haveledtohypotheses abouttheinvolvement ofdifferent protein activities inthe life cycle ofRNA viruses. Recent technical advances have madeitpossible forseveral ofthese proposals t...
Expression of the Epstein-Ban" virus (EBV) latent membrane protein (LMP1) is regulated by virus-a... more Expression of the Epstein-Ban" virus (EBV) latent membrane protein (LMP1) is regulated by virus-and host cell-specific factors. The EBV nuclear antigen 2 (EBNA2) has been shown to transactivate a number of viral and cellular gene promoters including the promoter for the LMP1 gene. EBNA2 is targeted to at least some of these promoters by interacting with a cellular DNA binding protein, RBP-Jlc. In the present report we confirm and extend our previous observation that the LMP1 promoter can be activated by EBNA2 in the absence of the RBP-Jtc-binding sequence in the LMP1 promoter regulatory region (LRS). We show that two distinct LRS regions, -106 to +40 and -176 to -136, contribute to EBNA2 responsiveness. Site-directed mutagenesis analysis of the upstream -176/-136 EBNA2 responsive element revealed that two critical cis-acting elements are required for full promoter function. These same elements analysed by electro-phoretic mobility shift assays define two binding sites recognized by nuclear factors derived from B cells. An octamer-like sequence (-147 to -139) contained overlapping binding sites for an unidentified transcriptional repressor on the one hand and a factor(s) belonging to the POU domain family but distinct from Oct-1 and Oct-2 on the other. An adjacent purine tract (-171 to -155) held a PU.1 binding site, which was also recognized by a related factor. The results suggest that the POU domain protein and either of two PU boxbinding factors bind simultaneously to LRS, creating a ternary complex that might be in part responsible for mediating the transactivation of the LMP1 promoter by EBNA2. There were no qualitative differences between EBV-negative and EBV-positive cells with regard to transcription factor binding to the octamer-like sequence and the PU.1 recognition site, as revealed by electrophoretic mobility shift assays.
The infectivity of plum pox potyvirus (PPV) RNA was decreased by treatment with proteases. Ribonu... more The infectivity of plum pox potyvirus (PPV) RNA was decreased by treatment with proteases. Ribonuclease digestion of iodinated PPV RNA yielded material which had an electrophoretic mobility corresponding to Mr 22000. This protein presumably corresponds to the protease-sensitive structure needed for infectivity. A protein-linked RNase Tl-resistant oligonucleotide, 38 nucleotides long, was sequenced and shown to correspond to the 5' terminus of the RNA by sequence comparison to the RNAs of two other potyviruses, tobacco etch virus and tobacco vein mottling virus. A 12 nucleotide block was found to be completely conserved in the RNAs of the three viruses.
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Papers by Sonia Lain