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BACKGROUND AND PURPOSE A novel anti-neoplastic gallium complex GaQ(3) (KP46), earlier developed by us, is currently in phase I clinical trial. GaQ(3) induced S-phase arrest and apoptosis via caspase/PARP cleavage in a variety of cancers. However, the underlying mechanism of apoptosis is unknown. Here, we have explored the mechanism(s) of GaQ(3) -induced apoptosis in cancer cells, focusing on p53 and intracellular Ca(2+) signalling. EXPERIMENTAL APPROACH GaQ(3) -induced cytotoxicity and apoptosis were determined in cancer cell lines, with different p53 status (p53(+/+) , p53(-/-) and p53 mutant). Time course analysis of intracellular Ca(2+) calcium release, p53 promoter activation, p53-nuclear/cytoplasmic movements and reactive oxygen species (ROS) were conducted. Ca(2+) -dependent formation of the p53-p300 transcriptional complex was analysed by co-immunoprecipitation and chromatin immunoprecipitation. Ca(2+) signalling, p53, p300 and ROS were serially knocked down to study Ca(2+) -p53-ROS ineractions in GaQ(3) -induced apoptosis. KEY RESULTS GaQ(3) triggered intracellular Ca(2+) release stabilizing p53-p300 complex and recruited p53 to p53 promoter, leading to p53 mRNA and protein synthesis. p53 induced higher intracellular Ca(2+) release and ROS followed by activation of p53 downstream genes including those for the micro RNA mir34a. In p53(-/-) and p53 mutant cells, GaQ(3) -induced Ca(2+) -signalling generated ROS. ROS further increased membrane translocation of FAS and FAS-mediated extrinsic apoptosis. CONCLUSIONS AND IMPLICATIONS This study disclosed a novel mechanism of Ca(2+) -signalling-mediated p53 activation and ROS up-regulation. Understanding the mechanism of GaQ(3) -induced apoptosis will help establish this gallium-based organic compound as a potent anti-cancer drug. [ABSTRACT FROM AUTHOR]

Tumour cells are shown to change shape and lose polarity when they are cultured in 3D, a feature typically associated with tumour progression in vivo, thus making it significant to study cancer cells in an environment that mimics the in vivo milieu. In this study we established hard (MCF7 and MDA-MB-231, breast cancer) and soft (HCT116, colon cancer) 3D cancer tumour models utilizing a blend of P(3HO- co -3HD) and P(3HB). P(3HO- co -3HD) and P(3HB) belong to a group of natural biodegradable polyesters, PHAs, that are synthesised by microorganisms. The 3D PHA scaffolds produced, with a pore size of 30 to 300 µm, allow for nutrients to diffuse within the scaffold and provide the cells with the flexibility to distribute evenly within the scaffold and grow within the pores. Interestingly, by Day 5, MDA-MB-231 showed dispersed growth in clusters, and MCF7 cells formed an evenly dispersed dense layer, while HCT116 formed large colonies within the pockets of the 3D PHA scaffolds. Our results show Epithelial Mesenchymal Transition (EMT) marker gene expression profiles in the hard tumour cancer models. In the 3D-based PHA scaffolds, MDA-MB-231 cells expressed higher levels of Wnt-11 and mesenchymal markers, such as Snail and its downstream gene Vim mRNAs, while MCF7 cells exhibited no change in their expression. On the other hand, MCF7 cells exhibited a significantly increased E-Cadherin expression as compared to MDA-MB-231 cells. The expression levels of EMT markers were comparative to their expression reported in the tumour samples, making them good representative of cancer models. In future these models will be helpful in mimicking hypoxic tumours, in studying gene expression, cellular signalling, angiogenesis and drug response more accurately than 2D and perhaps other 3D models.

Hypoxia in tumor microenvironment is responsible for resistance to conventional modes of cancer therapeutics. A manganese-clay hybrid compound MHC was shown to generate molecular oxygen in aqueous solution. In this study we have shown that MHC, in hypoxia, causes cancer cell death, through release of molecular oxygen and via p53-dependent apoptosis. MHC treatment of cells results in depletion of mitochondrial membrane potential and inhibition of ROS production, in a cell-specific manner. In hypoxia, the oxygen from MHC releases cells from S-phase arrest thus causing p53-dependent apoptosis. The induction of apoptosis by MHC is higher in p53 Wt/Wt cells when it is compared with p53 Mt/Mt cells. The released oxygen from MHC triggers apoptosis via p53 activation through its enhanced homo-oligomerization, post-translational modifications and nuclear localization. Thus MHC as a cellular oxygen-releasing compound has high potential as a drug for hypoxic tumor regression., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

TAp73, a homologous of tumor suppressor p53, regulates apoptosis in a p53-independent manner and its suppressive as well as stimulatory role in promoting angiogenesis has been reported. It exists in multiple isoforms which varies structurally in their N-terminus and C-terminus region and crucial interplay among them guides the decision of cell survival and death. As molecular chaperones control both stability and degradation of TAp73, selective regulation of p73 isoforms has implication upon developing new therapeutic for hypoxic tumor. We have discovered that under DNA damage carboxy terminus Hsp70 interacting protein (CHIP's) antiapoptotic function is displayed via its E3 ligase activity that inhibits exclusively TAp73α-mediated apoptosis in cancer cell. The decrease in TAp73α level by CHIP as it is supported by increased ubiquitination pattern is reverted back by sh-CHIP. Further, the transactivation of p53-downstream apoptotic genes BAX, PUMA and PIG3 by TAp73α is also shown to be subsequently inhibited by CHIP. The tetratricopeptide TPR-domain of CHIP in its amino-terminus interacts with the carboxy-terminus of TAp73α and ΔNp73α and as a result, U-BOX domain of CHIP in the carboxy-terminus is able to ubiquitinate TAp73α for proteasomal degradation. Due to lack of C-terminus in TAp73β, CHIP fails to interact with and degrade it. In conclusion, we have thus uncovered for the first time a novel mechanism of chaperone-assisted regulation of p73 stability as well as its apoptotic functions by CHIP that might be utilized to develop new anticancer strategies., (© 2019 Wiley Periodicals, Inc.)

Tumor suppressor protein p53 aggregates in the hypoxic core of solid tumors. C terminus of Hsc70-interacting protein (CHIP) displays chaperone as well as E3 ligase activities in both stabilizing and degrading wild-type and mutant p53. In this study, we have discovered that CHIP selectively degrades aggregating mutant p53 under both normal and hypoxic conditions. Silencing of CHIP alleviates degradation of aggregating mutant p53 in both normoxia and hypoxia, but has no significant effect on the level of nonaggregating mutant p53. Although both U-box and TPR domains of CHIP are responsible for p53 degradation, the U-box domain selectively binds to aggregating mutant p53, whereas the TPR domain interacts with nonaggregating mutant p53. The degradation of mutant p53 by CHIP is shown to be via autophagy through K63-linked polyubiquitination. Both in normoxia and under physiological hypoxia, the level of aggregating mutant p53 in the presence of CHIP was reduced threefold, whereas under serum starvation, it was reduced fivefold. Interestingly, both wild-type and mutant p53 interact with and stabilize CHIP at the post-translational level, suggesting a chaperone synergy between p53 and CHIP. This finding may have strong therapeutic significance via selective degradation of oncogenic mutant p53 in regressing hypoxic tumors., (© 2018 Federation of European Biochemical Societies.)

BACE1, a key protein involved in Alzheimer's progression, initiates Aβ42 generation that induce senile plaques in brain. However, the role of chaperone synergy or antagonism on BACE1-mediated amyloid processing is unknown. We have discovered that BACE1 as well as Aβ42 are antagonistically controlled by ER chaperone ORP150 and cellular chaperone CHIP. We have shown ORP150 as a chaperone interacts with and stabilizes BACE1 at post-translational level. Furthermore, ORP150 enhances BACE1-mediated amyloid processing thus masking CHIP-mediated BACE1 degradation. Conversely, siORP150 reversed the chaperone function of ORP150 resulting in BACE1 degradation. ORP150 and CHIP demonstrate antagonism under normal and stress conditions wherein they inversely regulate each other thus affecting BACE1 level. In conclusion, we have uncovered for the first time a phenomenon of chaperone antagonism on BACE1-mediated Aβ42 generation. Future strategy would require both suppression of ORP150 as well as activation of E3-ligase activity of CHIP that might prevent Aβ42 in Alzheimer's disease., (© 2017 Wiley Periodicals, Inc.)

In patient with Alzheimer's disease (AD), deposition of amyloid-beta Aβ, a proteolytic cleavage of amyloid precursor protein (APP) by β-secretase/BACE1, forms senile plaque in the brain. BACE1 activation is caused due to oxidative stresses and dysfunction of ubiquitin-proteasome system (UPS), which is linked to p53 inactivation. As partial suppression of BACE1 attenuates Aβ generation and AD-related pathology, it might be an ideal target for AD treatment. We have shown that both in neurons and in HEK-APP cells, BACE1 is a new substrate of E3-ligase CHIP and an inverse relation exists between CHIP and BACE1 level. CHIP inhibits ectopic BACE1 level by promoting its ubiquitination and proteasomal degradation, thus reducing APP processing; it stabilizes APP in neurons, thus reducing Aβ. CHIP(U) (box) domain physically interacts with BACE1; however, both U-box and TPR domain are essential for ubiquitination and degradation of BACE1. Further, BACE1 is a downstream target of p53 and overexpression of p53 decreases BACE1 level. In HEK-APP cells, CHIP is shown to negatively regulate BACE1 promoter through stabilization of p53's DNA-binding conformation and its binding upon 5' UTR element (+127 to +150). We have thus discovered that CHIP regulates p53-mediated trans-repression of BACE1 at both transcriptional and post-translational level. We propose that a CHIP-BACE1-p53 feedback loop might control APP stabilization, which could further be utilized for new therapeutic intervention in AD., (© 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Myocardial infarction, an irreversible cardiac tissue damage, involves progressive loss of cardiomyocytes due to p53-mediated apoptosis. Oxygenation is known to promote cardiac survival through activation of NOS3 gene. We hypothesized a dual role for p53, which, depending on oxygenation, can elicit apoptotic death signals or NOS3-mediated survival signals in the infarct heart. p53 exhibited a differential DNA-binding, namely, BAX-p53RE in the infarct heart or NOS3-p53RE in the oxygenated heart, which was regulated by oxygen-induced, post-translational modification of p53. In the infarct heart, p53 was heavily acetylated at Lys(118) residue, which was exclusively reversed in the oxygenated heart, apparently regulated by oxygen-dependent expression of TIP60. The inhibition of Lys(118) acetylation promoted the generation of NOS3-promoting prosurvival form of p53. Thus, oxygenation switches p53-DNA interaction by regulating p53 core-domain acetylation, promoting a prosurvival transcription activity of p53. Understanding this novel oxygen-p53 survival pathway will open new avenues in cardioprotection molecular therapy., (© 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)

A thermo-stable, proteinaceous protease inhibitor (LPI) from Lawsonia inermis is reported. The LPI was purified from Lawsonia inermis seeds by subsequent ammonium sulfate precipitation, ion exchange chromatography (DEAE-Cellulose) and gel permeation chromatography (Sephadex-50). The purified protease inhibitor is effective against a wide range of proteases viz. papain, trypsin, pepsin and metallo-protease. The apparent molecular weight of the protease inhibitor is 19 kDa, determined by SDS-PAGE electrophoresis. The protease inhibitor was found to be stable at 70 degrees C for 30 min. It was also examined for antibacterial activity against Pseudomonas aeruginosa MTCC 7926 and Staphylococcus aureus NCIM 2079; the IC50 values of the purified LPI were 11.4 microg/mL and 16.6 microg/mL respectively.

p53 and calcium signaling are inter-dependent and are known to show both synergistic and antagonistic effects on each other in the cellular environment. However, no molecular mechanism or cellular pathway is known which shows direct regulation between these important cellular signaling molecules. Here we have shown that in cancer cells treated with anti-neoplastic drug GaQ3, p53, there is an increase in intracellular calcium levels by transcriptional regulation of a novel calcium channel gene TRPC6. p53 directly binds to a 22 bp response element in the TRPC6 gene promoter and increase its mRNA and protein expression. Over-expression of TRPC6 results in calcium-dependent apoptotic death and activation of apoptotic genes in a variety of cancer cells. This research work shows that p53 and its transcriptional activity is critical in regulation of calcium signaling and an increase in the intracellular calcium level might be one of the anti-cancer strategies to induce apoptosis in cancer cells.

p53 prevents cancer via cell cycle arrest, apoptosis, and the maintenance of genome stability. p53 also regulates energy-generating metabolic pathways such as oxidative phosphorylation (OXPHOS) and glycolysis via transcriptional regulation of SCO2 and TIGAR. SCO2, a cytochrome c oxidase assembly factor, is a metallochaperone which is involved in the biogenesis of cytochrome c oxidase subunit II. Here we have shown that SCO2 functions as an apoptotic protein in tumor xenografts, thus providing an alternative pathway for p53-mediated apoptosis. SCO2 increases the generation of reactive oxygen species (ROS) and induces dissociation of the protein complex between apoptosis signal-regulating kinase 1 (ASK-1) (mitogen-activated protein kinase kinase kinase [MAPKKK]) and its cellular inhibitor, the redox-active protein thioredoxin (Trx). Furthermore, SCO2 induces phosphorylation of ASK-1 at the Thr(845) residue, resulting in the activation of the ASK-1 kinase pathway. The phosphorylation of ASK-1 induces the activation of mitogen-activated protein kinase kinases 4 and 7 (MAP2K4/7) and MAP2K3/6, which switches the c-Jun N-terminal protein kinase (JNK)/p38-dependent apoptotic cascades in cancer cells. Exogenous addition of the SCO2 gene to hypoxic cancer cells and hypoxic tumors induces apoptosis and causes significant regression of tumor xenografts. We have thus discovered a novel apoptotic function of SCO2, which activates the ASK-1 kinase pathway in switching "on" an alternate mode of p53-mediated apoptosis. We propose that SCO2 might possess a novel tumor suppressor function via the ROS-ASK-1 kinase pathway and thus could be an important candidate for anticancer gene therapy.

Cellular stressors are known to inhibit the p53-RPA70 (replication protein A, 70 kDa subunit) complex, and RPA70 increases cellular DNA repair in cancer cells. We hypothesized that regulation of RPA70-mediated DNA repair might be responsible for the inhibition of apoptosis in hypoxic tumours. We have shown that, in cancer cells, hypoxia disrupts the p53-RPA70 complex, thereby enhancing RPA70-mediated NER (nucleotide excision repair)/NHEJ (non-homologous end-joining) repair. In normal cells, RPA70 binds to the p53-NTD (N-terminal domain), whereas this binding is disrupted in hypoxia. Phosphorylation of p53-NTD is a crucial event in dissociating both NTD-RPA70 and p53-RPA70 complexes. Serial mutations at serine and threonine residues in the NTD confirm that p53(Ser15) phosphorylation induces dissociation of the p53-RPA70 complex in hypoxia. DNA-PK (DNA-dependent protein kinase) is shown to induce p53(Ser15) phosphorylation, thus enhancing RPA70-mediated NER/NHEJ repair. Furthermore, RPA70 gene silencing induces significant increases in cellular apoptosis in the resistant hypoxic cancer cells. We have thus elucidated a novel pathway showing how DNA-PK-mediated p53(Ser15) phosphorylation dissociates the p53-RPA70 complex, thus enhancing NER/NHEJ repair, which causes resistance to apoptosis in hypoxic cancer cells. This novel finding may open new strategies in developing cancer therapeutics on the basis of the regulation of RPA70-mediated NER/NHEJ repair.

Aims: p53 is known to induce apoptotic and necrotic cell death in response to stress, although the mechanism of these pathways is unknown. The aim of this study was to determine the molecular mechanism that determines p53's decision to select the apoptotic or necrotic mode of cell death., Results: Gold nanoparticles (Au-NPs) induced both apoptosis and necrosis in cancer cells in a p53-dependent manner. In cells undergoing apoptosis and necrosis, differential patterns of reactive oxygen species (ROS) generation were observed that leads to the activation of two different sets of p53-interacting kinases and acetylases. The differential activation of cellular kinases and acetylases caused dissimilar patterns of p53 phosphorylation and acetylation. In apoptotic cells, p53 was post-translationally modified in the core-domain, whereas in necrotic cells, it was modified at both N- and C-terminii, thus displaying differential DNA-binding activity. Au-NP10 and Au-NP80 activated fifty apoptotic and fifty nine necrotic p53-downstream genes, respectively. Both Au-NP10 and Au-NP80 showed HCT (p53+/+) tumor regression in mice xenografts., Innovation: This study established for the first time that, in cancer cells, Au-NP-mediated apoptosis and necrosis are controlled by differential activation of p53 and its downstream genes. Further, both Au-NP10 and Au-NP80 were shown to regress HCT (p53+/+) tumors via apoptotic and necrotic pathways, respectively., Conclusion: Au-NP-mediated p53 activation at both transcription and proteome level, through ROS-mediated p53 post-translational modification pattern, is responsible for tumor regression, which may further find wider application of nanoparticles in cancer therapy.

Mutant (Mt) p53 abrogates tumor suppression functions of wild-type (WT) p53 through mutant-specific, gain-of-function effects, and patients bearing Mt p53 are chemoresistant. The dominant negative effect of p53 mutants results from their aggregation propensity which causes co-aggregation of WT p53. We explored the mechanism of p53 inactivation in hypoxia and hypothesized whether WT p53 could rescue Mt p53 in hypoxic tumors. WT p53 exists in mutant conformation in hypoxic core of MCF-7 solid tumors, and its conformation is oxygen-dependent. Under simulated hypoxia in cells, WT p53 undergoes conformational change in acquiring mutant conformation. An in vivo chaperone assay shows that WT p53 functions as a molecular chaperone in rescuing conformational and structural p53 mutants in cancer cells both at the transcription and proteome levels. WT p53 chaperone therapy is further shown to cause significant regression of tumor xenografts through reconversion of the mutant phenotype to wild-type p53. The chaperone function of WT p53 is directly linked to the induction of apoptosis in both cancer cells and tumor xenografts. As oncogenic p53 mutants are linked to chemoresistance in hypoxic tumors, p53 chaperone therapy will introduce new dimensions to existing cancer therapeutics. We propose that in cancer cells, WT p53 chaperoning may either exist as a cellular event to potentially reverse the dominant negative effect of its oncogenic mutants or to stabilize yet unidentified factors.

p53 is well known as the "guardian of the genome" for differentiated and neoplastic cells. p53 induces cell-cycle arrest and cell death after DNA damage and thus contributes to the maintenance of genomic stability. In addition to this tumor suppressor function for pro-oncogenic cells, p53 also plays an important role as the central regulator of stress response by maintaining cellular homeostasis at the molecular and biochemical level. p53 regulates aerobic respiration at the glycolytic and oxidative phosphorylation (OXPHOS) steps via transcriptional regulation of its downstream genes TP53-induced glycolysis regulator (TIGAR) and synthesis of cytochrome c oxidase (SCO2). p53 negatively regulates glycolysis through activation of TIGAR (an inhibitor of the fructose-2,6-bisphosphate). On the contrary p53 positively regulates OXPHOS through upregulation of SCO2, a member of the COX-2 assembly involved in the electron-transport chain. It is interesting to notice that p53 antagonistically regulates the inter-dependent glycolytic and OXPHOS cycles. It is important to understand whether the p53-mediated transcriptional regulation of TIGAR and SCO2 is temporally segregated in cancer cells and what is the relation between these paradoxical regulations of glycolytic pathway with the tumor suppressor activity of p53. In this review we will elucidate the importance of p53-mediated regulation of glycolysis and OXPHOS and its relation with the tumor suppressor function of p53. Further since cellular metabolism shares great relation with the process of aging we will also try and establish the role of p53 in regulation of aging via its transcriptional control of cellular metabolism.

Platelets are characterized as a systemic tool to elucidate mitochondria-allied perturbance in neurological diseases. The authors studied ultrastructural changes in platelets and platelet mitochondria using a case-control approach in amyotrophic lateral sclerosis (ALS). Subjects were sporadic ALS cases (n = 22) and age- and sex-matched controls (n = 16). Phlebotomy was performed, platelet concentrates (PCs) were prepared, and mitochondria were extracted. PCs and mitochondria were processed for ultrastructure study using transmission electron microscopy. Image analysis was done using Image-J. Transmission electron microscopy demonstrated both qualitative and quantitative variations in ALS platelets and platelet mitochondria. Heterogeneous distribution of granules, formation of vacuoles, blebs, pseudopodia, loose demarcation of cell membrane with a significant increase in area (20.3%), perimeter (17.82%), integrated density (21.44%), electron-lucent granules (41.79%), and vacuoles (36.58%) were observed in ALS platelets. Conversely, control platelets exhibited an increase of circularity (11.7%) and electron-dense granules (36.89%). In parallel, nonuniformity of matrix, faint cristae, greater lysosomal bodies, and lesser intramitochondrial granules were seen in ALS platelet mitochondria. Significantly greater area (26.88%), perimeter (15%), circularity (3.76%), and integrated density (25.18%) were observed in control platelet mitochondria. Ultastructural divergence in platelets of ALS patients underlines a potential dependence of platelets on modest mitochondrial functioning. These observations also support the view that systemic involvement might be a novel feature in ALS pathophysiology.

Role of platelets have been evinced as a systemic tool in a variety of neurological disorders. Oxidative phosphorylation contributes approximately 80% of total adenosine-tri-phosphate (ATP) production in resting platelets suggesting potential dependence of platelets on modest mitochondrial functioning. Since mitochondria play a pivotal role in regulating metabolic and apoptotic pathways in various neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), we assessed mitochondrial membrane potential (MMP) associated alterations and apoptotic status of platelet mitochondria in ALS patients using case-control approach. Confocal microscopy reflected heterogeneous distribution of JC-1 aggregates and monomers indicating altered MMP in ALS platelets. Our flow cytometry results confirmed greater percentage of mitochondrial depolarization in ALS platelets. Greater exposure of phosphatidyl serine (PS) residue vindicated by annexin V binding and lesser accumulation of mitotracker red in mitochondrial matrix demonstrated initiation of apoptosis in ALS platelets. Our findings corroborate mitochondrial abnormalities such as perturbance of MMP, mitochondrial depolarization, and apoptosis in ALS platelet mitochondria. In conclusion, our study further evinces the involvement of mitochondrial dysfunction in the pathogenesis of ALS and suggests implication of cell death in peripheral tissues apart from motor neurons in ALS.

Background: The intrinsically disordered N-ter domain (NTD) of p53 encompasses approximately hundred amino acids that contain a transactivation domain (1-73) and a proline-rich domain (64-92) and is responsible for transactivation function and apoptosis. It also possesses an auto-inhibitory function as its removal results in remarkable reduction in dissociation of p53 from DNA., Principal Findings/methodology: In this report, we have discovered that p53-NTD spanning amino acid residues 1-125 (NTD125) interacted with WT p53 and stabilized its wild type conformation under physiological and elevated temperatures, both in vitro and in cellular systems. NTD125 prevented irreversible thermal aggregation of heat denatured p53, enhanced p21-5'-DBS binding and further restored DBS binding activity of heat-denatured p53, in vitro, in a dose-dependent manner. In vivo ELISA and immunoprecipitation analysis of NTD125-transfected cells revealed that NTD125 shifted equilibrium from p53 mutant to wild type under heat stress conditions. Further, NTD125 initiated nuclear translocation of cytoplasmic p53 in transcriptionally active state in order to activate p53 downstream genes such as p21, Bax, PUMA, Noxa and SUMO., Conclusion/significance: Here, we showed that a novel chaperone-like activity resides in p53-N-ter region. This study might have significance in understanding the role of p53-NTD in p53 stabilization, conformational activation and apoptosis under heat-stress conditions.

Purpose: To investigate homology and stress response of p53 (a 53 kDa tumor suppressor protein) orthologue in Sf9 Lepidopteran insect cell line that exhibits very high radioresistance., Materials and Methods: Western immunoblotting, immunoprecipitation, degenerate RT-PCR (reverse transcription-polymerase chain reaction), electrophoretic gel mobility shift assay, flow cytometry and immuno-fluorescence microscopy were used for characterizing structural and functional features of Sfp53 (Spodoptera frugiperda p53) in gamma-irradiated or etoposide-treated Sf9 insect and BMG-1 (brain malignant glioma) human cells. Cells were pre-treated with caffeine for inhibiting ATM/ATR (ataxia-telangiectasia mutated protein/ATM and Rad-3-related protein) activation, wherever required., Results: A 47-49 kDa protein band was observed with antibodies against three different epitopes, demonstrating conservation of respective domains in Sfp53. Immunoprecipitation also yielded similar-sized protein. Degenerate RT-PCR resulted in product of same size in both cell lines. Similar gel mobility shift of p53-binding oligonucleotide with BMG-1 and Sf9 cell lysates indicated analogous transcriptional activity of Sfp53. Constitutive Sfp53 level was higher than hp53 (human p53) and showed primarily cytoplasmic localization. Radiation-induced accumulation was considerably less in Sf9 even as an analogous ATM/ATR-dependent nuclear translocation was observed following gamma-irradiation and etoposide., Conclusions: A smaller-sized Sfp53 orthologue shows highly conserved native structure with DNA-binding, N-terminus and C-terminus domains, and has analogous p53 transcriptional activity. While its nuclear translocation and ATM/ATR dependence were similar to hp53, the cytoplasmic localization and subdued accumulation following gamma-irradiation indicate functional differences from human cells.

The monoesters of curcumin, a symmetric diphenol with valine and glycine have been prepared by a novel solid phase synthesis and its diesters with valine, glutamic acid and demethylenated piperic acid have been prepared by solution phase method. The assessment of their antimicrobial and anticancer (antiproliferative) activities suggested that diesters of curcumin are relatively more active than curcumin itself due to their increased solubility, slow metabolism and better cellular uptake. Furthermore, significant observation was that monoesters of curcumin have even better antimicrobial activity than their corresponding diesters, emphasizing the role of free phenolic group. The conjugate of curcumin with demethylenated piperic acid in which methylenedioxy ring was open also shows enhanced activity than the corresponding piperic acid conjugate, emphasizing the role of free phenolics in the transport or in the binding processes.

Wild type p53 exists in a constant state of equilibrium between wild type and mutant conformation and undergoes conformational changes at elevated temperature. We have demonstrated that the co-chaperone CHIP (carboxyl terminus of Hsp70-interacting protein), which suppressed aggregation of several misfolded substrates and induced the proteasomal degradation of both wild type and mutant p53, physically interacts with the amino terminus of WT53 and prevented it from irreversible thermal inactivation. CHIP preferentially binds to the p53 mutant phenotype and restored the DNA binding activity of heat-denatured p53 in an ATP-independent manner. In cells under elevated temperatures that contained a higher level of p53 mutant phenotype, CHIP restored the native-like conformation of p53 in the presence of geldanamycin, whereas CHIP-small interfering RNA considerably increased the mutant form. Further, under elevated temperatures, the levels of CHIP and p53 were higher in nucleus, and chromatin immunoprecipitation shows the presence of p53 and CHIP together upon the DNA binding site in the p21 and p53 promoters. We propose that CHIP might be a direct chaperone of wild type p53 that helps p53 in maintaining wild type conformation under physiological condition as well as help resurrect p53 mutant phenotype into a folded native state under stress condition.

A deoxy 11-mer oligonucleotide 5'-GTTAGGGTTAG-3', complementary to a repeat sequence of human telomerase RNA template has been linked through phosphate and a C-2 linker to a bioactive tetraglycine conjugate of curcumin, a well-known antitumor herbal spice component of turmeric. This molecule has been transfected into KB and HeLa cell lines and found to affect cell growth in the former. This DNA-curcumin-tetraglycine acts as a prodrug being targeted by antisense mechanism to telomerase.

The apo(a) [apolipoprotein(a)] gene is responsible for variations in plasma lipoprotein(a), high levels of which are a risk factor for atherosclerosis and myocardial infarction. The apo(a) promoter stimulates the expression of reporter genes in HepG2 cells, but not in HeLa cells. In the present study, we demonstrate that the 1.4 kb apo(a) promoter comprises two composite regulatory regions: a distal negative regulatory module (positions -1432 to -716) and a proximal tissue-specific module (-716 to -616). The distal negative regulatory module contains two strong negative regulatory regions [polymorphic PNR (pentanucleotide repeat region) and NREbeta (negative regulatory element beta)], which sandwich the postive regulatory region PREbeta (positive regulatory element beta). The PNR was shown to bind to transcription factors in a tissue-specific manner, whereas the ubiquitous transcription factors hepatocyte nuclear factor 3alpha and GATA binding protein 4 bound to NREbeta to repress gene transcription. The proximal tissue-specific module contains two regulatory elements: an activating region (PREalpha) that activates transcription in HepG2 cells, and NREalpha, which is responsible for repressing the apo(a) gene in HeLa cells. NREalpha binds to a HeLa-specific repressor. These multiple regulatory elements might work co-operatively to finely regulate apo(a) gene expression. Although the tissue-specific module is required for apo(a) gene activation and repression in a tissue-specific manner, the combinatorial interplay of the distal and proximal regulators might define the complex pathway(s) of apo(a) gene regulation.

The high plasma levels of lipoprotein(a) [Lp(a)] are associated with atherosclerosis. The apo(a) gene is responsible for the variance of Lp(a) concentration and its expression is liver-specific. By 5'-deletion analysis, we, in a luciferase gene reporter assay, have identified a 64-bp AT-rich region of upstream apo(a) gene (-703 to -640) that binds to multiple liver-specific factors. The 64 bp cis-element contained three dyad symmetry elements (DSEs) that are crucial for synergistic binding to the factors. We have demonstrated that both DSE-2 and -3 together are responsible for factor binding in vitro, and for gene activation in liver cells. Further, we have purified one of the UV cross-linked DNA-protein complexes to homogeneity by streptavidin magnetic bead chromatography. The identification of a further upstream negative regulatory region (-1432 to -704) led us to predict that as yet unidentified transcriptional repressor(s) might also repress apo(a) gene transcription., ((C)2002 Elsevier Science (USA).)

High plasma lipoprotein(a) [Lp(a)] levels have been implicated as an independent risk factor for coronary artery disease in Caucasians, Chinese, Africans, and Indians. Apo(a) that evolved from a duplicated plasminogen gene during recent primate evolution is responsible for the concentration of Lp(a) in the artery wall leading to atherosclerosis, by virtue of its ability to bind to the extracellular matrix and its role in stimulating the proliferation and migration of human smooth muscle cells. Several types of polymorphisms, size as well as sequence changes both in the coding and regulatory sequences, have been reported to influence the variability of Lp(a) concentration. Apo(a) exhibits genetic size polymorphism varying between 300 and 800 kDa that could be attributed to the number of k-4 VNTR (variable number of transcribed kringle-4 repeats). An inverse relationship between Lp(a) level and apo(a) allele sizes is a general trend in all ethnic populations although apo(a) allele size distribution could be significantly variable in ethnic types. A negative correlation between the number of pentanucleotide TTTTA(n) repeat (PNR) sequences in the regulatory region of the apo(a) gene and Lp(a) level has also been observed in Caucasians and Indians, but not in African Americans. However, a significant linkage disequilibrium was noted between the PNR number and k-4 VNTR. In order to correlate the role of apo(a) gene polymorphisms to apo(a) gene regulation, we have proposed that liver-specific transcriptional activators and repressors might contribute to the differential expression of apo(a) gene, in an individual-specific manner., (Copyright 2000 Academic Press.)

High plasma levels of Lp(a), a low-density lipoprotein particle with an attached apo(a), are associated with the development of atherosclerosis. We present two simultaneous mutations in the apo(a) promoter (A/G(-418) and C/T(-384)) in healthy Indians with a low Lp(a) level (<5 mg/dl). No such mutations were detected in coronary artery disease positive individuals with very high Lp(a) levels (>200 mg/dl). The mutations described here might be useful in understanding the transcriptional regulation of the apo(a) gene., (Copyright 2000 Academic Press.)