Background: Previously, we reported that Inauhzin (INZ) induces p53 activity and suppresses tumor... more Background: Previously, we reported that Inauhzin (INZ) induces p53 activity and suppresses tumor growth by inhibiting Sirt1. However, it remains unknown whether INZ may globally affect p53-dependent gene expression or not. Herein, we have conducted microarray and real-time PCR analyses of gene expression to determine the global effect of INZ on human p53-responsive transcriptome. Methodology/Principal Findings: In this study, we conducted microarray analysis followed by PCR validation of general gene expression in HCT116 p53+/+ and HCT116 p532/2 cells treated with or without INZ. Microarray data showed that 324 genes were up-regulated by $2.3-fold and 266 genes were down-regulated by $2-fold in response to INZ treatment in a p53-dependent manner. GO analysis for these genes further revealed that INZ affects several biological processes, including apoptosis (GO:0006915), cell cycle (GO:0007049), immune system process (GO:0002376), and cell adhesion (GO:0007155), which are in line with p53 functions in cells. Also, pathway and STRING analyses of these genes indicated that the p53signaling pathway is the most significant pathway responsive to INZ treatment as predicted, since a number of these p53 target genes have been previously reported and some of them were validated by RT-qPCR. Finally, among the 9 tested and highly expressed genes, ACBD4, APOBEC3C, and FLJ14327 could be novel p53 target genes, for they were up-regulated by INZ in HCT116 p53+/+ cells, but not in HCT116 p532/2 cells. Conclusions/Significance: From our whole genome microarray analysis followed by validation with RT-qPCR, we found that INZ can indeed induce the expression of p53 target genes at a larger scale or globally. Our findings not only verify that INZ indeed activates the p53 signaling pathway, but also provide useful information for identifying novel INZ and/or p53 targets. The global effect of INZ on human p53-responsive transcriptome could also be instrumental to the future design of INZ clinical trials.
The c-Myc oncoprotein promotes cell growth by enhancing ribosomal biogenesis. Overexpression of c... more The c-Myc oncoprotein promotes cell growth by enhancing ribosomal biogenesis. Overexpression of c-Myc and aberrant ribosomal biogenesis lead to deregulated cell growth and tumorigenesis. Hence, c-Myc activity and ribosomal biogenesis must be tightly coordinated during normal homeostasis. We previously found that ribosomal protein L11 inhibits c-Myc activity by blocking the recruitment of its co-activator transformation/transcription domain-associated protein (TRRAP) to the promoter regions of c-Myc target genes that are transcribed by RNA polymerases I and II. In this study, we extended the role of L11 to the regulation of c-Myc-driven transcription of the 5 S rRNA and tRNA genes by RNA polymerase III. L11 co-resided with c-Myc at the 5 S rRNA and tRNA genes and significantly inhibited the binding of TRRAP to these genes. Knocking down endogenous L11 enhanced c-Myc-dependent transcription of these genes. Interestingly, in response to ribosomal stress induced by the treatment of cells with a low dose of actinomycin D or serum starvation, L11 binding to these genes was increased, and inversely TRRAP binding to these genes was decreased. Consistently, knockdown of L11 rescued the reduction of the expression of these genes by the two treatments. These results demonstrate that L11 suppresses c-Myc-dependent and RNA polymerase III-catalyzed transcription of 5 S rRNA and tRNA genes in response to ribosomal stress, ensuring a tight coordination between c-Myc activity and ribosomal biogenesis.
Reactivating the tumor suppressor p53 offers an attractive strategy for developing cancer therapy... more Reactivating the tumor suppressor p53 offers an attractive strategy for developing cancer therapy. We recently identified Inauhzin (INZ) as a novel non-genotoxic p53-activating compound. To develop INZ into a clinically applicable anticancer drug, we have initiated preclinical toxicity studies. Here, we report our study on determining the maximum tolerated dose (MTD) of INZ analog, Inauhzin-C (INZ (C)), following intraperitoneal (i.p.) administration (Phase A) and its toxicity following i.p. administration over a period of 5day dosing plus 2-day recovery (Phase B) in CD-1 mice. The Phase A study showed that the MTD of INZ (C) is 200 mg/kg for female and 250 mg/kg for male, respectively. The Phase B study showed that the administration of INZ (C) via 5-day consecutive i.p. injection is tolerated by female CD-1 mice at all dose levels tested from 50 mg/kg to 120 mg/kg without significant changes in biochemical and pathological parameters in the animals. Together, these results indicate that our previously determined effective dose of INZ at 30-60 mg/kg via i.p. is quite safe to mice, and imply that this compound have the features worthy for further development into a clinically applicable drug.
The ‘ribosomal stress (RS)-p53 pathway’ is triggered by any stressor or genetic alteration that d... more The ‘ribosomal stress (RS)-p53 pathway’ is triggered by any stressor or genetic alteration that disrupts ribosomal biogenesis, and mediated by several ribosomal proteins (RPs), such as RPL11 and RPL5, which inhibit MDM2 and activate p53. Inosine monophosphate (IMP) dehydrogenase 2 (IMPDH2) is a rate-limiting enzyme in de novo guanine nucleotide biosynthesis and crucial for maintaining cellular guanine deoxy- and ribonucleotide pools needed for DNA and RNA synthesis. It is highly expressed in many malignancies. We previously showed that inhibition of IMPDH2 leads to p53 activation by causing RS. Surprisingly, our current study reveals that Inauzhin (INZ), a novel non-genotoxic p53 activator by inhibiting SIRT1, can also inhibit cellular IMPDH2 activity, and reduce the levels of cellular GTP and GTP-binding nucleostemin that is essential for rRNA processing. Consequently, INZ induces RS and the RPL11/RPL5-MDM2 interaction, activating p53. These results support the new notion that INZ ...
Identifying effective small molecules that specifically target the p53 pathway in cancer has been... more Identifying effective small molecules that specifically target the p53 pathway in cancer has been an exciting, though challenging, approach for the development of anti-cancer therapy. We recently identified Inauhzin (INZ) as a novel p53 activator, selectively and efficiently suppressing tumor growth without displaying genotoxicity and with little toxicity to normal cells. In order to reveal the structural features essential for anti-cancer activity of this small molecule, we have synthesized a panel of INZ analogs and evaluated their ability to induce cellular p53 and to inhibit cell growth in cell-based assays. This study as described here leads to the discovery of INZ analog 37 that displays much better potency than INZ in both of p53 activation and cell growth inhibition in several human cancer cell lines including H460 and HCT116 +/+ cells. This INZ analog exhibited much less effect on p53-null H1299 cells and HCT116 2/2 cells, and importantly no toxicity on normal human p53-containing WI-38 cells. Hence, our results not only unveil key chemical features for INZ activity, but also identify the newly synthesized INZ analog 37 as a better small molecule for further development of anti-cancer therapy.
Tight regulation of microtubule (MT) dynamics is essential for proper chromosome movement during ... more Tight regulation of microtubule (MT) dynamics is essential for proper chromosome movement during mitosis. Here we show, using mammalian cells, that structure-specific recognition protein 1 (SSRP1) is a novel regulator of MT dynamics. SSRP1 colocalizes with the spindle and midbody MTs, and associates with MTs both in vitro and in vivo . Purified SSRP1 facilitates tubulin polymerization and MT bundling in vitro . Knockdown of SSRP1 inhibits the growth of MTs and leads to disorganized spindle structures, reduction of K-fibers and midbody fibers, disrupted chromosome movement, and attenuated cytokinesis in vivo . These results demonstrate that SSRP1 is crucial for MT growth and spindle assembly during mitosis.
Background: Whether RPL11 directly binds to the zinc finger domain of MDM2 still remains elusive.... more Background: Whether RPL11 directly binds to the zinc finger domain of MDM2 still remains elusive. Results: Mutations of RPL11 or the zinc finger of MDM2 impair the ability of RPL11 to inactivate MDM2 and to activate p53. Conclusion: RPL11 binds directly the zinc finger of MDM2 via hydrophilic residues. Significance: Our study unveils the chemical nature for MDM2-RPL11 interactions. Ribosomal protein L11 (RPL11) has been shown to activate p53 by binding to MDM2 and negating its p53 suppression activity in response to ribosomal stress. Although a mutation at Cys-305 within the zinc finger domain of MDM2 has been shown to drastically impair MDM2 interaction with RPL11 and thus escapes the inhibition by this ribosomal protein, it still remains elusive whether RPL11 inactivates MDM2 via direct action on this zinc finger domain and what is the chemical nature of this specific interaction. To define the roles of the MDM2 zinc finger in association with RPL11, we conducted hydrogen-deuterium exchange mass spectrometry, computational modeling, circular dichroism, and mutational analyses of the zinc finger domain of MDM2 and human RPL11. Our study reveals that RPL11 forms a stable complex with MDM2 in vitro through direct contact with its zinc finger. This binding is disrupted by single mutations of non-cysteine amino acids within the zinc finger domain of MDM2. Basic residues in RPL11 are crucial for the stable binding and RPL11 suppression of MDM2 activity toward p53. These results provide the first line of evidence for the specific interaction between RPL11 and the zinc finger of MDM2 via hydrophilic residues as well as a molecular foundation for better understanding RPL11 inhibition of MDM2 function.
Although $50% of all types of human cancers harbour wild-type TP53, this p53 tumour suppressor is... more Although $50% of all types of human cancers harbour wild-type TP53, this p53 tumour suppressor is often deactivated through a concerted action by its abnormally elevated suppressors, MDM2, MDMX or SIRT1. Here, we report a novel small molecule Inauhzin (INZ) that effectively reactivates p53 by inhibiting SIRT1 activity, promotes p53-dependent apoptosis of human cancer cells without causing apparently genotoxic stress. Moreover, INZ stabilizes p53 by increasing p53 acetylation and preventing MDM2-mediated ubiquitylation of p53 in cells, though not directly in vitro. Remarkably, INZ inhibits cell proliferation, induces senescence and tumour-specific apoptosis, and represses the growth of xenograft tumours derived from p53-harbouring H460 and HCT116 cells without causing apparent toxicity to normal tissues and the tumour-bearing SCID mice. Hence, our study unearths INZ as a novel anti-cancer therapeutic candidate that inhibits SIRT1 activity and activates p53.
mechanisms of nutlin-induced apoptosis in multiple myeloma: Evidence for p53-transcription-depend... more mechanisms of nutlin-induced apoptosis in multiple myeloma: Evidence for p53-transcription-dependent and-independent pathways. Cancer Biol Ther 2010; This issue.
Methods in molecular biology (Clifton, N.J.), 2013
Fluorescence polarization (FP) has become a powerful technique to quantitatively analyze the bind... more Fluorescence polarization (FP) has become a powerful technique to quantitatively analyze the binding of a small soluble fluorescence-labeled probe to a larger soluble protein and its displacement by other molecules. Here, we describe a detailed protocol to identify small molecules capable of targeting p53-MDM2/MDMX interactions using a fluorescence polarization assay with Rhodamine-labeled p53 peptides.
Inherently disparate cell growth and division, which are intimately coupled through a delicate ne... more Inherently disparate cell growth and division, which are intimately coupled through a delicate network of intracellular and extracellular signaling, require ribosomal biogenesis. A number of events imparting instability to ribosomal biogenesis can cause nucleolar stress. In response to this stress, several ribosomal proteins bind to MDM2 and block MDM2-mediated p53 ubiquitination and degradation, resulting in p53-dependent cell cycle arrest. By doing so, the ribosomal proteins play a crucial role in connecting deregulated cell growth with inhibition of cell division. The ribosomal protein-MDM2-p53 signaling pathway provides a molecular switch that may constitute a surveillance network monitoring the integrity of ribosomal biogenesis.
The core promoter of protein-encoding genes plays a central role in regulating transcription. M1B... more The core promoter of protein-encoding genes plays a central role in regulating transcription. M1BP is a transcriptional activator that associates with a core promoter element known as Motif 1 that resides at thousand of genes in Drosophila. To gain insight into how M1BP functions, we identified an interacting protein called GFZF. GFZF had been previously identified in genetic screens for factors involved in maintenance of hybrid inviability, the G2-M DNA damage checkpoint, and RAS/MAPK signaling but its contribution to these processes was unknown. Here, we show that GFZF resides in the nucleus and functions as a transcriptional co-activator. In addition, we show that GFZF is a glutathione S-transferase(GST). Thus, GFZF is the first transcriptional co-activator with intrinsic GST activity, and its identification as a transcriptional co-activator provides an explanation for its role in numerous biological processes.
It has been shown that p300 binds to MDM2 and leads to down-regulation of the p53 function. Howev... more It has been shown that p300 binds to MDM2 and leads to down-regulation of the p53 function. However, it remains unclear whether the acetylase activity of p300 is necessary for regulating MDM2 stability. In this study, we address this issue. First, p300 did not acetylate MDM2 in solution and in cells. Second, overexpression of p300 in cells increased the level of the MDM2 protein but not its mRNA. Similarly, the acetylase-defective p300 AT2 mutant stabilized the MDM2 protein as well. Consistently, the deacetylase inhibitor, trichostatin A, did not significantly affect the half-life of the endogenous MDM2 protein, whereas p300 enhanced the halflife of MDM2. Finally, both wild type and acetylase-defective mutant p300 proteins associated with MDM2 in nuclear body-like structures where MDM2 might be protected from proteasomal degradation. Thus, these results suggest that p300 appears to stabilize MDM2 by retaining this protein in a specific nuclear structure rather than by acetylating it.
Several ribosomal proteins (RPs) in response to various ribosomal stressors have been shown to pl... more Several ribosomal proteins (RPs) in response to various ribosomal stressors have been shown to play a critical role in p53-dependent regulation of cell cycle arrest, apoptosis and tumor suppression. Here, we report ribosomal protein L22 (RPL22/eL22) as a novel p53 activator highly mutated (mostly deletion mutation) in various types of human cancers, but not essential for ribosomal biogenesis in normal cells. Ectopic expression of RPL22/eL22 suppressed the colony formation of cancer cells in a p53-dependent manner, whereas knockdown of RPL22/eL22 significantly compromised p53 activation by Actinomycin D, rescuing p53-induced G1/G0 cell cycle arrest. Interestingly, human tumors with RPL22/eL22 deletion appeared to sustain wild type p53. Mechanistically, RPL22/eL22 bound to MDM2 acidic domain and inhibited MDM2-mediated p53 ubiquitination and degradation, hence extending the half-life of p53. Ribosome-profiling analysis revealed that induction of ribosomal stress by Actinomycin D leads...
Protein ubiquitylation has been demonstrated to play a vital role not only in mediating protein t... more Protein ubiquitylation has been demonstrated to play a vital role not only in mediating protein turnover but also in modulating protein activity. The stability and activity of the tumor suppressor p53 and of the oncoprotein c-Myc are no exception. Both are regulated through independent ubiquitylation by several E3 ubiquitin ligases. Interestingly, p53 and c-Myc are functionally connected by some of these E3 enzymes and their regulator ARF, although these proteins play opposite roles in controlling cell growth and proliferation. The balance of this complex ubiquitylation network and its disruption during oncogenesis will be the topics of this review.
American journal of clinical and experimental urology, 2014
Methoxyacetic acid (MAA) is a primary metabolite of ester phthalates that are used in production ... more Methoxyacetic acid (MAA) is a primary metabolite of ester phthalates that are used in production of consumer products and pharmaceutical products. MAA causes embryo malformation and spermatocyte death through inhibition of histone deacetylases (HDACs). Little is known about MAA's effects on cancer cells. In this study, two immortalized human normal prostatic epithelial cell lines (RWPE-1 and pRNS-1-1) and four human prostate cancer cell lines (LNCaP, C4-2B, PC-3, and DU-145) were treated with MAA at different doses and for different time periods. Cell viability, apoptosis, and cell cycle analysis were performed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR, Western blot, and chromatin immunoprecipitation analyses. We found that MAA dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. MAA-induced apoptosis was due to down-regulation of the anti-apop...
Background: Previously, we reported that Inauhzin (INZ) induces p53 activity and suppresses tumor... more Background: Previously, we reported that Inauhzin (INZ) induces p53 activity and suppresses tumor growth by inhibiting Sirt1. However, it remains unknown whether INZ may globally affect p53-dependent gene expression or not. Herein, we have conducted microarray and real-time PCR analyses of gene expression to determine the global effect of INZ on human p53-responsive transcriptome. Methodology/Principal Findings: In this study, we conducted microarray analysis followed by PCR validation of general gene expression in HCT116 p53+/+ and HCT116 p532/2 cells treated with or without INZ. Microarray data showed that 324 genes were up-regulated by $2.3-fold and 266 genes were down-regulated by $2-fold in response to INZ treatment in a p53-dependent manner. GO analysis for these genes further revealed that INZ affects several biological processes, including apoptosis (GO:0006915), cell cycle (GO:0007049), immune system process (GO:0002376), and cell adhesion (GO:0007155), which are in line with p53 functions in cells. Also, pathway and STRING analyses of these genes indicated that the p53signaling pathway is the most significant pathway responsive to INZ treatment as predicted, since a number of these p53 target genes have been previously reported and some of them were validated by RT-qPCR. Finally, among the 9 tested and highly expressed genes, ACBD4, APOBEC3C, and FLJ14327 could be novel p53 target genes, for they were up-regulated by INZ in HCT116 p53+/+ cells, but not in HCT116 p532/2 cells. Conclusions/Significance: From our whole genome microarray analysis followed by validation with RT-qPCR, we found that INZ can indeed induce the expression of p53 target genes at a larger scale or globally. Our findings not only verify that INZ indeed activates the p53 signaling pathway, but also provide useful information for identifying novel INZ and/or p53 targets. The global effect of INZ on human p53-responsive transcriptome could also be instrumental to the future design of INZ clinical trials.
The c-Myc oncoprotein promotes cell growth by enhancing ribosomal biogenesis. Overexpression of c... more The c-Myc oncoprotein promotes cell growth by enhancing ribosomal biogenesis. Overexpression of c-Myc and aberrant ribosomal biogenesis lead to deregulated cell growth and tumorigenesis. Hence, c-Myc activity and ribosomal biogenesis must be tightly coordinated during normal homeostasis. We previously found that ribosomal protein L11 inhibits c-Myc activity by blocking the recruitment of its co-activator transformation/transcription domain-associated protein (TRRAP) to the promoter regions of c-Myc target genes that are transcribed by RNA polymerases I and II. In this study, we extended the role of L11 to the regulation of c-Myc-driven transcription of the 5 S rRNA and tRNA genes by RNA polymerase III. L11 co-resided with c-Myc at the 5 S rRNA and tRNA genes and significantly inhibited the binding of TRRAP to these genes. Knocking down endogenous L11 enhanced c-Myc-dependent transcription of these genes. Interestingly, in response to ribosomal stress induced by the treatment of cells with a low dose of actinomycin D or serum starvation, L11 binding to these genes was increased, and inversely TRRAP binding to these genes was decreased. Consistently, knockdown of L11 rescued the reduction of the expression of these genes by the two treatments. These results demonstrate that L11 suppresses c-Myc-dependent and RNA polymerase III-catalyzed transcription of 5 S rRNA and tRNA genes in response to ribosomal stress, ensuring a tight coordination between c-Myc activity and ribosomal biogenesis.
Reactivating the tumor suppressor p53 offers an attractive strategy for developing cancer therapy... more Reactivating the tumor suppressor p53 offers an attractive strategy for developing cancer therapy. We recently identified Inauhzin (INZ) as a novel non-genotoxic p53-activating compound. To develop INZ into a clinically applicable anticancer drug, we have initiated preclinical toxicity studies. Here, we report our study on determining the maximum tolerated dose (MTD) of INZ analog, Inauhzin-C (INZ (C)), following intraperitoneal (i.p.) administration (Phase A) and its toxicity following i.p. administration over a period of 5day dosing plus 2-day recovery (Phase B) in CD-1 mice. The Phase A study showed that the MTD of INZ (C) is 200 mg/kg for female and 250 mg/kg for male, respectively. The Phase B study showed that the administration of INZ (C) via 5-day consecutive i.p. injection is tolerated by female CD-1 mice at all dose levels tested from 50 mg/kg to 120 mg/kg without significant changes in biochemical and pathological parameters in the animals. Together, these results indicate that our previously determined effective dose of INZ at 30-60 mg/kg via i.p. is quite safe to mice, and imply that this compound have the features worthy for further development into a clinically applicable drug.
The ‘ribosomal stress (RS)-p53 pathway’ is triggered by any stressor or genetic alteration that d... more The ‘ribosomal stress (RS)-p53 pathway’ is triggered by any stressor or genetic alteration that disrupts ribosomal biogenesis, and mediated by several ribosomal proteins (RPs), such as RPL11 and RPL5, which inhibit MDM2 and activate p53. Inosine monophosphate (IMP) dehydrogenase 2 (IMPDH2) is a rate-limiting enzyme in de novo guanine nucleotide biosynthesis and crucial for maintaining cellular guanine deoxy- and ribonucleotide pools needed for DNA and RNA synthesis. It is highly expressed in many malignancies. We previously showed that inhibition of IMPDH2 leads to p53 activation by causing RS. Surprisingly, our current study reveals that Inauzhin (INZ), a novel non-genotoxic p53 activator by inhibiting SIRT1, can also inhibit cellular IMPDH2 activity, and reduce the levels of cellular GTP and GTP-binding nucleostemin that is essential for rRNA processing. Consequently, INZ induces RS and the RPL11/RPL5-MDM2 interaction, activating p53. These results support the new notion that INZ ...
Identifying effective small molecules that specifically target the p53 pathway in cancer has been... more Identifying effective small molecules that specifically target the p53 pathway in cancer has been an exciting, though challenging, approach for the development of anti-cancer therapy. We recently identified Inauhzin (INZ) as a novel p53 activator, selectively and efficiently suppressing tumor growth without displaying genotoxicity and with little toxicity to normal cells. In order to reveal the structural features essential for anti-cancer activity of this small molecule, we have synthesized a panel of INZ analogs and evaluated their ability to induce cellular p53 and to inhibit cell growth in cell-based assays. This study as described here leads to the discovery of INZ analog 37 that displays much better potency than INZ in both of p53 activation and cell growth inhibition in several human cancer cell lines including H460 and HCT116 +/+ cells. This INZ analog exhibited much less effect on p53-null H1299 cells and HCT116 2/2 cells, and importantly no toxicity on normal human p53-containing WI-38 cells. Hence, our results not only unveil key chemical features for INZ activity, but also identify the newly synthesized INZ analog 37 as a better small molecule for further development of anti-cancer therapy.
Tight regulation of microtubule (MT) dynamics is essential for proper chromosome movement during ... more Tight regulation of microtubule (MT) dynamics is essential for proper chromosome movement during mitosis. Here we show, using mammalian cells, that structure-specific recognition protein 1 (SSRP1) is a novel regulator of MT dynamics. SSRP1 colocalizes with the spindle and midbody MTs, and associates with MTs both in vitro and in vivo . Purified SSRP1 facilitates tubulin polymerization and MT bundling in vitro . Knockdown of SSRP1 inhibits the growth of MTs and leads to disorganized spindle structures, reduction of K-fibers and midbody fibers, disrupted chromosome movement, and attenuated cytokinesis in vivo . These results demonstrate that SSRP1 is crucial for MT growth and spindle assembly during mitosis.
Background: Whether RPL11 directly binds to the zinc finger domain of MDM2 still remains elusive.... more Background: Whether RPL11 directly binds to the zinc finger domain of MDM2 still remains elusive. Results: Mutations of RPL11 or the zinc finger of MDM2 impair the ability of RPL11 to inactivate MDM2 and to activate p53. Conclusion: RPL11 binds directly the zinc finger of MDM2 via hydrophilic residues. Significance: Our study unveils the chemical nature for MDM2-RPL11 interactions. Ribosomal protein L11 (RPL11) has been shown to activate p53 by binding to MDM2 and negating its p53 suppression activity in response to ribosomal stress. Although a mutation at Cys-305 within the zinc finger domain of MDM2 has been shown to drastically impair MDM2 interaction with RPL11 and thus escapes the inhibition by this ribosomal protein, it still remains elusive whether RPL11 inactivates MDM2 via direct action on this zinc finger domain and what is the chemical nature of this specific interaction. To define the roles of the MDM2 zinc finger in association with RPL11, we conducted hydrogen-deuterium exchange mass spectrometry, computational modeling, circular dichroism, and mutational analyses of the zinc finger domain of MDM2 and human RPL11. Our study reveals that RPL11 forms a stable complex with MDM2 in vitro through direct contact with its zinc finger. This binding is disrupted by single mutations of non-cysteine amino acids within the zinc finger domain of MDM2. Basic residues in RPL11 are crucial for the stable binding and RPL11 suppression of MDM2 activity toward p53. These results provide the first line of evidence for the specific interaction between RPL11 and the zinc finger of MDM2 via hydrophilic residues as well as a molecular foundation for better understanding RPL11 inhibition of MDM2 function.
Although $50% of all types of human cancers harbour wild-type TP53, this p53 tumour suppressor is... more Although $50% of all types of human cancers harbour wild-type TP53, this p53 tumour suppressor is often deactivated through a concerted action by its abnormally elevated suppressors, MDM2, MDMX or SIRT1. Here, we report a novel small molecule Inauhzin (INZ) that effectively reactivates p53 by inhibiting SIRT1 activity, promotes p53-dependent apoptosis of human cancer cells without causing apparently genotoxic stress. Moreover, INZ stabilizes p53 by increasing p53 acetylation and preventing MDM2-mediated ubiquitylation of p53 in cells, though not directly in vitro. Remarkably, INZ inhibits cell proliferation, induces senescence and tumour-specific apoptosis, and represses the growth of xenograft tumours derived from p53-harbouring H460 and HCT116 cells without causing apparent toxicity to normal tissues and the tumour-bearing SCID mice. Hence, our study unearths INZ as a novel anti-cancer therapeutic candidate that inhibits SIRT1 activity and activates p53.
mechanisms of nutlin-induced apoptosis in multiple myeloma: Evidence for p53-transcription-depend... more mechanisms of nutlin-induced apoptosis in multiple myeloma: Evidence for p53-transcription-dependent and-independent pathways. Cancer Biol Ther 2010; This issue.
Methods in molecular biology (Clifton, N.J.), 2013
Fluorescence polarization (FP) has become a powerful technique to quantitatively analyze the bind... more Fluorescence polarization (FP) has become a powerful technique to quantitatively analyze the binding of a small soluble fluorescence-labeled probe to a larger soluble protein and its displacement by other molecules. Here, we describe a detailed protocol to identify small molecules capable of targeting p53-MDM2/MDMX interactions using a fluorescence polarization assay with Rhodamine-labeled p53 peptides.
Inherently disparate cell growth and division, which are intimately coupled through a delicate ne... more Inherently disparate cell growth and division, which are intimately coupled through a delicate network of intracellular and extracellular signaling, require ribosomal biogenesis. A number of events imparting instability to ribosomal biogenesis can cause nucleolar stress. In response to this stress, several ribosomal proteins bind to MDM2 and block MDM2-mediated p53 ubiquitination and degradation, resulting in p53-dependent cell cycle arrest. By doing so, the ribosomal proteins play a crucial role in connecting deregulated cell growth with inhibition of cell division. The ribosomal protein-MDM2-p53 signaling pathway provides a molecular switch that may constitute a surveillance network monitoring the integrity of ribosomal biogenesis.
The core promoter of protein-encoding genes plays a central role in regulating transcription. M1B... more The core promoter of protein-encoding genes plays a central role in regulating transcription. M1BP is a transcriptional activator that associates with a core promoter element known as Motif 1 that resides at thousand of genes in Drosophila. To gain insight into how M1BP functions, we identified an interacting protein called GFZF. GFZF had been previously identified in genetic screens for factors involved in maintenance of hybrid inviability, the G2-M DNA damage checkpoint, and RAS/MAPK signaling but its contribution to these processes was unknown. Here, we show that GFZF resides in the nucleus and functions as a transcriptional co-activator. In addition, we show that GFZF is a glutathione S-transferase(GST). Thus, GFZF is the first transcriptional co-activator with intrinsic GST activity, and its identification as a transcriptional co-activator provides an explanation for its role in numerous biological processes.
It has been shown that p300 binds to MDM2 and leads to down-regulation of the p53 function. Howev... more It has been shown that p300 binds to MDM2 and leads to down-regulation of the p53 function. However, it remains unclear whether the acetylase activity of p300 is necessary for regulating MDM2 stability. In this study, we address this issue. First, p300 did not acetylate MDM2 in solution and in cells. Second, overexpression of p300 in cells increased the level of the MDM2 protein but not its mRNA. Similarly, the acetylase-defective p300 AT2 mutant stabilized the MDM2 protein as well. Consistently, the deacetylase inhibitor, trichostatin A, did not significantly affect the half-life of the endogenous MDM2 protein, whereas p300 enhanced the halflife of MDM2. Finally, both wild type and acetylase-defective mutant p300 proteins associated with MDM2 in nuclear body-like structures where MDM2 might be protected from proteasomal degradation. Thus, these results suggest that p300 appears to stabilize MDM2 by retaining this protein in a specific nuclear structure rather than by acetylating it.
Several ribosomal proteins (RPs) in response to various ribosomal stressors have been shown to pl... more Several ribosomal proteins (RPs) in response to various ribosomal stressors have been shown to play a critical role in p53-dependent regulation of cell cycle arrest, apoptosis and tumor suppression. Here, we report ribosomal protein L22 (RPL22/eL22) as a novel p53 activator highly mutated (mostly deletion mutation) in various types of human cancers, but not essential for ribosomal biogenesis in normal cells. Ectopic expression of RPL22/eL22 suppressed the colony formation of cancer cells in a p53-dependent manner, whereas knockdown of RPL22/eL22 significantly compromised p53 activation by Actinomycin D, rescuing p53-induced G1/G0 cell cycle arrest. Interestingly, human tumors with RPL22/eL22 deletion appeared to sustain wild type p53. Mechanistically, RPL22/eL22 bound to MDM2 acidic domain and inhibited MDM2-mediated p53 ubiquitination and degradation, hence extending the half-life of p53. Ribosome-profiling analysis revealed that induction of ribosomal stress by Actinomycin D leads...
Protein ubiquitylation has been demonstrated to play a vital role not only in mediating protein t... more Protein ubiquitylation has been demonstrated to play a vital role not only in mediating protein turnover but also in modulating protein activity. The stability and activity of the tumor suppressor p53 and of the oncoprotein c-Myc are no exception. Both are regulated through independent ubiquitylation by several E3 ubiquitin ligases. Interestingly, p53 and c-Myc are functionally connected by some of these E3 enzymes and their regulator ARF, although these proteins play opposite roles in controlling cell growth and proliferation. The balance of this complex ubiquitylation network and its disruption during oncogenesis will be the topics of this review.
American journal of clinical and experimental urology, 2014
Methoxyacetic acid (MAA) is a primary metabolite of ester phthalates that are used in production ... more Methoxyacetic acid (MAA) is a primary metabolite of ester phthalates that are used in production of consumer products and pharmaceutical products. MAA causes embryo malformation and spermatocyte death through inhibition of histone deacetylases (HDACs). Little is known about MAA's effects on cancer cells. In this study, two immortalized human normal prostatic epithelial cell lines (RWPE-1 and pRNS-1-1) and four human prostate cancer cell lines (LNCaP, C4-2B, PC-3, and DU-145) were treated with MAA at different doses and for different time periods. Cell viability, apoptosis, and cell cycle analysis were performed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR, Western blot, and chromatin immunoprecipitation analyses. We found that MAA dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. MAA-induced apoptosis was due to down-regulation of the anti-apop...
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