Heat Shock Protein Promoter Research Articles

Changes In Activity of HSPA1A and HSPA1L is Associated with Early Acute Pancreatitis Severity

Introduction: Genome assessment shows promise as a potential method for predicting acute pancreatitis (AP) severity. We hypothesized that Heat Shock Protein 70 (HSP70) single nucleotide polymorphisms (SNPs) and expression changes may play a role in early detection of acute pancreatitis severity. Methods: A total of 57 AP patients and 52 age- and sex-matched healthy controls were studied. Peripheral blood samples from pancreatitis patients were collected upon admission. Two SNPs of the HSP70-gene family were selected. RNA was extracted parallel to genomic DNA from AP patients (N=12) and healthy controls (N=21).Gene expression of two HSP70 family members HSPA1A and HSPA1L was measured using TaqMan gene expression assays via reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results: Major allele frequency in HSPA1A gene was 1 (A>G) for AP patients and 1 (A>G) for controls. For HSPA1A, the gene major allele frequency was 0.672 (A>C) for patients and 0.7 (A>C) for controls, the major allele frequencies were 0.638 (A>G) and 0.672 (G>C) for patients and 0.85 (A>G) and 0.65 (G>C) for controls, respectively. The allele frequencies differed between AP patients with organ failure and those with mild disease. Following gene expression of HSPA1L and SNP relationship analysis, AP patients with heterozygous genotype had higher expression levels as compared to non-AP patients (p=0.014). Conclusions: Our data suggest that polymorphism of the HSP70 promoter region may be a risk factor for developing severe acute pancreatitis. Further study to determine the urine levels of HSP70 protein expression is needed to confirm the protective mechanism of HSP70.

Heat Shock Signaling in Land Plants: From Plasma Membrane Sensing to the Transcription of Small Heat Shock Proteins.

Heat stress events are major factors limiting crop productivity. During summer days, land plants must anticipate in a timely manner upcoming mild and severe temperature. They respond by accumulating protective heat-shock proteins (HSPs), conferring acquired thermotolerance. All organisms synthetize HSPs; many of which are members of the conserved chaperones families. This review describes recent advances in plant temperature sensing, signaling, and response. We highlight the pathway from heat perception by the plasma membrane through calcium channels, such as cyclic nucleotide-gated channels, to the activation of the heat-shock transcription factors (HSFs). An unclear cellular signal activates HSFs, which act as essential regulators. In particular, the HSFA subfamily can bind heat shock elements in HSP promoters and could mediate the dissociation of bound histones, leading to HSPs transcription. Although plants can modulate their transcriptome, proteome, and metabolome to protect the cellular machinery, HSP chaperones prevent, use, and revert the formation of misfolded proteins, thereby avoiding heat-induced cell death. Remarkably, the HSP20 family is mostly tightly repressed at low temperature, suggesting that a costly mechanism can become detrimental under unnecessary conditions. Here, the role of HSP20s in response to HS and their possible deleterious expression at non-HS temperatures is discussed.

Enhanced expression of recombinant miraculin, a tasting modifying protein, in Nicotiana tabacum hairy roots using 18.2 heat shock protein promoter and terminator

Miraculin, a taste modifier, is a protein that was first isolated from miracle fruit (Richadella dulcifica). It can change a sour taste into a sweet taste when sour acids are consumed, although it does not elicit a sweet response. Miraculin may have the potential in industry as a substitute for sugars and as artificial sweeteners. Since the miracle plant has low fruit productivity, mass production of miraculin is limited. Transgenic hairy root culture is a potential alternative system for the mass production of miraculin. In this study, we investigated the expression of recombinant miraculin in tobacco (Nicotiana tabacum) hairy roots. To increase miraculin expression, the heat shock protein 18.2 promoter and terminator were used to drive the expression of miraculin gene in a potential host system. Synthetic miraculin gene was transformed into Nicotiana tobacum leaf explants via Agrobacterium rhizogenes. The transgenic hairy root clones that contained synthetic miraculin gene showed rapid growth and reached maximum growth after 35-day culture. When the expression of miraculin gene was regulated by heat shock protein 18.2 promoter and heat shock protein terminator, the expression of recombinant miraculin increased than the control regulated by CaMV 35S promoter and nopaline synthase terminator. The recombinant miraculin was 19.97 ng per µg of the total soluble protein and equivalently with approximately 2% of the total soluble protein. For the first time, a taste modifying miraculin was successfully expressed in tobacco hairy root. The results in this study have given a promising approach for the application of the transgenic hairy root system to produce recombinant miraculin.

Development of a genetically modified hepatoma cell line with heat-inducible high liver function.

The online version of this article (10.1007/s10616-021-00457-4) contains supplementary material, which is available to authorized users.

Generation of Gene-Engineered Human Hepatoma Cells with Heat-Inducible Liver Functions

Hepatoma cells derived from liver carcinoma are a candidate cell source for bioartificial liver (BAL) systems due to their high proliferative capacity, although liver function of hepatoma cells is considerably low compared with primary hepatocytes. In our previous study, genetically engineered mouse hepatoma cells with inducible high liver function were established by transducing liver-enriched transcription factor (LETF) genes. In this study, we aimed to develop new gene-engineered human hepatoma cells, in which high liver functions are inducible by heat treatment. For this purpose, we constructed a gene expression system for eight LETF genes under control of tetracycline-dependent transactivator (tTA), and the system was introduced into the genome of HepG2-HSP cells, in which a tTA expression system induced by a heat-shock protein promoter with transcriptional amplification was introduced into HepG2 cells. Thus, the heat-inducible tTA promotes LETF genes to induce liver function. Upon the heat treatment of the cells (HepG2-HSP/8F) at 43°C for 30 min, liver functions such as albumin secretion and cytochrome P450 were significantly enhanced. The cells with heat-inducible liver function can be used as a new cell source for various hepatic studies including construction of BAL systems.

Regulation Between HSF1 Isoforms and HSPs Contributes to the Variation in Thermal Tolerance Between Two Oyster Congeners.

Heat shock transcription factor 1 (HSF1) plays an important role in regulating heat shock, which can activate heat shock proteins (HSPs). HSPs can protect organisms from thermal stress. Oysters in the intertidal zone can tolerate thermal stress. The Pacific oyster (Crassostrea gigas gigas) and Fujian oyster (C. gigas angulata)—allopatric subspecies with distinct thermal tolerances—make good study specimens for analyzing and comparing thermal stress regulation. We cloned and compared HSF1 isoforms, which is highly expressed under heat shock conditions in the two subspecies. The results revealed that two isoforms (HSF1a and HSF1d) respond to heat shock in both Pacific and Fujian oysters, and different heat shock conditions led to various combinations of isoforms. Subcellular localization showed that isoforms gathered in the nucleus when exposed to heat shock. The co-immunoprecipitation revealed that HSF1d can be a dimer. In addition, we selected HSPs that are expressed under the heat shock response, according to the RNA-seq and proteomic analyses. For the HSPs, we analyzed the coding part and the promoter sequences. The result showed that the domains of HSPs are conserved in two subspecies, but the promoters are significantly different. The Dual-Luciferase assay showed that the induced expression isoform HSF1d had the highest activity in C. gigas gigas, while the constitutively-expressed HSF1a was most active in C. gigas angulata. In addition, variation in the level of HSP promoters appeared to be correlated with gene expression. We argue that this gene is regulated based on the different expression levels between the two subspecies’ responses to heat shock. In summary, various stress conditions can yield different HSF1 isoforms and respond to heat shock in both oyster subspecies. Differences in how the isoforms and promoter are activated may contribute to their differential expressions. Overall, the results comparing C. gigas gigas and C. gigas angulata suggest that these isoforms have a regulatory relationship under heat shock, providing valuable information on the thermal tolerance mechanism in these commercially important oyster species.

Ectopic expression of nucleolar DEAD-Box RNA helicase OsTOGR1 confers improved heat stress tolerance in transgenic Chinese cabbage.

The DEAD-Box RNA helicase OsTOGR1 positively regulates heat stress tolerance in Chinese cabbage. Non-heading Chinese cabbage (Brassica rapa L. ssp. chinensis) is primarily cultivated vegetable crop in Asian countries. Heat stress is one of the major threats for its growth and yield. Numerous regulatory genes in various crops have shown to contribute thermotolerance. Among them, Thermotolerant growth required 1 (TOGR1) is an important DEAD-box RNA helicase. To examine whether its role is conserved in other crops, we constructed pCAMBIA1300-pHSP:OsTOGR1 expression vector driven by the rice small heat shock protein promoter (pHSP17.9) and successfully produced transgenic non-heading Chinese cabbage plants expressing OsTOGR1 gene via Agrobacterium-mediated vacuum infiltration transformation. In total, we generated three independent transgenic cabbage lines expressing TOGR1 gene. Expression and integration of TOGR1 was confirmed by PCR, RT-PCR and qPCR in T1 and T2 generations. The relative leaf electrical conductivity of transgenic seedlings was reduced subjected to high temperature (38°C) compared to heat shock treatment (46°C). In addition, hypocotyl length of transgenic seedlings increased compared to wild-type plants under high temperature and heat shock treatment. Furthermore, the transgenic plants exhibited higher chlorophyll content than wild-type plants under high temperature and heat shock treatment. The transgenic seeds displayed better germination under heat shock treatment. Tested heat stress-responsive genes were also up-regulated in the transgenic plants subjected to high temperature or heat shock treatment. To the best of our knowledge, this is the first report on describing the role of DAED-Box RNA helicases in improving heat stress tolerance of transgenic plants.

Abstract 4062: Chemotherapy-induced therapeutic expression in stem cell vehicles

Abstract Stem cells are being studies as therapeutic vehicles due to their tumor homing properties that make them ideal to track down and attack infiltrating tumor cells that are not easily accessed. In order for stem cells to exert their therapeutic effect, they are engineered with therapeutic payloads that are typically secreted in the vicinity of the tumor. While many have shown that constitutive expression of a therapeutic by a tumor-homing stem cell can potentially treat a variety of cancers, we and others have focused on designing inducible systems that allow us to control the therapeutic expression and thus potentially limit systemic effects. In past work, we created an ultrasound-inducible stem cell that expressed reporter and/or therapeutic genes under the heat shock protein 70 (HSP70) promoter. In the current work, we hypothesize that we can potentially exploit the HSP70 promoter activation that has been reported after cellular stresses induced by standard-of care-chemotherapy, such as temozolomide, that is used to treat Glioblastoma (GBM), a fatal disease with a median survival of only 15 months after diagnosis. To test our hypothesis, we infected bone marrow derived mesenchymal stem cells (MSCs) with a plasmid that expresses GFP and firefly luciferase under the control of the HSP70 promoter and treated them with 100 or 400 ug/ml of temozolomide. We observed significant induction of GFP reporter 24 hours after treatment. We subsequently implanted our stem cells subcutaneously in mice and treated them with temozolomide. 24 hours after treatment, we observed gene activation using bioluminescent and fluorescent imaging. To further test this gene activation in a disease model, we stereotactically implanted our engineered stem cells along with patient derived (PDX) GBM cells intracranially. 5 days post implantation, mice were treated with a single therapeutic dose of temozolomide (200 mg/kg) and sacrificed 24 hours after treatment. After sectioning the brains and examining under fluorescent microscope, we observed significant GFP induction in the temozolomide treated mice compared to untreated controls. In order to demonstrate a biologic effect of induced therapeutics we performed a preliminary experiment where we intracranially implanted engineered stem cells that expressed HSP70 promotor inducible TNFα, a cytokine that we demonstrated can be delivered by stem cells to treat GBM and open the blood brain barrier (BBB) to further systemic therapies. We subsequently induced with 200 mg/kg of systemically administered temozolomide and observed BBB permeability via optical/CT imaging that demonstrated brain deposition of systemically administered cy7-albumin, in contrast to the untreated control. In conclusion, we successfully demonstrated the potential of engineering stem cells to express proteins/therapeutics after induction by temozolomide, a standard-of-care chemotherapy. We are therefore exploring using this strategy to express therapeutics that act in synergy with the inducing chemotherapy agent. Citation Format: Andrei Molotkov, Mikhail Doubrovin, Alessandra Ali, Akiva Mintz. Chemotherapy-induced therapeutic expression in stem cell vehicles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4062.

HSEAT: A Tool for Plant Heat Shock Element Analysis, Motif Identification and Analysis

Background: Previous methods used to discover cis-regulatory motifs in promoter region of plant genes possess very limited performance, especially for analysis of novel and rare motifs. Different plant genes have differential expression under different environmental or experimental conditions and modular regulation of cis-regulatory sequences in promoter regions of the same or different genes. It has previously been revealed that Heat Shock Proteins (HSPs) creation is correlated with plant tolerance under heat and other stress conditions. Regulation of these HSP genes is controlled by interactions between heat shock factors (HSFs) with cis-acting motifs present in the promoter region of the genes. Differential expression of these HSP genes is because of their unique promoter architecture, cis-acting sequences and their interaction with HSFs. Objective: A versatile promoter analysis tool was proposed for identification and analysis of promoters of HSPs. Methods: Heat Shock Element Analysis Tool (HSEAT) has been implemented in java programming language using pattern recognition approach. This tool has build-in MS access database for storing different motifs. Results: HSEAT has been designed to detect different types of Heat Shock Elements (HSEs) in promoter regions of plant HSPs with integration of complete analysis of plant promoters to the tool. HSEAT is user-friendly, interactive application to discover various types of HSEs e.g. TTC Rich Types, Gap Types and Prefect HSE as well as STRE in HSPs. Here we examined and evaluated some known HSP promoters from different plants using this tool with already available tools. Conclusion: HSEAT has extensive potential to explore conserved or semi-conserved motifs or potential binding sites of different transcription factors for other stress regulating genes. This tool can be found at https://sourceforge.net/projects/heast/.

Functional characterization of the promoter of pearl millet heat shock protein 10 (PgHsp10) in response to abiotic stresses in transgenic tobacco plants

In the present study, the promoter region of the pearl millet heat shock protein 10 (PgHsp10) gene was cloned and characterized. The PgHsp10 promoter (PgHsp10pro) sequence region has all the cis-motifs required for tissue and abiotic stress inducibility. The complete PgHsp10pro (PgHsp10PC) region and a series of 5′ truncations of PgHsp10 (PgHsp10D1 and PgHsp10D2) and an antisense form of PgHsp10pro (PgHsp10AS) were cloned into a plant expression vector (pMDC164) through gateway cloning. All four constructs were separately transformed into tobacco through Agrobacterium-mediated genetic transformation, and PCR-confirmed transgenic plants progressed to T1 and T2 generations. The T2 transgenic tobacco plants comprising all PgHsp10pro fragments were used for GUS histochemical and qRT-PCR assays in different tissues under control and abiotic stresses. The PgHsp10PC pro expression was specific to stem and seedlings under control conditions. Under different abiotic stresses, particularly heat stress, PgHsp10PCpro had relatively higher activity than PgHsp10D1pro, PgHsp10D2pro and PgHsp10ASpro. PgHsp10pro from a stress resilient crop like pearl millet responds positively to a range of abiotic stresses, in particular heat, when expressed in heterologous plant systems such as tobacco. Hence, PgHsp10pro appears to be a potential promoter candidate for developing heat and drought stress-tolerant crop plants.

Genetic Circuits To Detect Nanomaterial Triggered Toxicity through Engineered Heat Shock Response Mechanism.

Biocompatibility assessment of nanomaterials has been of great interest due to their potential toxicity. However, conventional biocompatibility tests fall short of providing a fast toxicity report. We developed a whole cell based biosensor to track biocompatibility of nanomaterials with the aim of providing fast feedback to engineer them with lower toxicity levels. We engineered promoters of four heat shock response (HSR) proteins utilizing synthetic biology approaches. As an initial design, a reporter coding gene was cloned downstream of the selected promoter regions. Initial results indicated that native heat shock protein (HSP) promoter regions were not very promising to generate signals with low background signals. Introducing riboregulators to native promoters eliminated unwanted background signals almost entirely. Yet, this approach also led to a decrease in expected sensor signal upon stress treatment. Thus, a repression based genetic circuit, inspired by the HSR mechanism of Mycobacterium tuberculosis, was constructed. These genetic circuits could report the toxicity of quantum dot nanoparticles in 1 h. Our designed nanoparticle toxicity sensors can provide quick reports, which can lower the demand for additional experiments with more complex organisms.

Induction of the heat shock response in Arabidopsis by chlorinated 1,4-naphthoquinones

It is known that some plants produce 1,4-naphthoquinones (1,4NQs) to inhibit the growth of other plants as allelochemicals. Here, we report that a chlorinated 1,4NQ enhanced the heat tolerance of Arabidopsis, and this enhancement was associated with induction of the heat shock response (HSR) in the plant. Nine 1,4NQs were subjected to the HSR assay by using the promoter of a small heat shock protein (HSP17.6C-CI) gene of Arabidopsis. The results indicated that chlorinated 1,4NQs, i.e., 2,3-dichloro-1,4-naphthoquinone (DNQ) and 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (DDNQ), apparently showed HSR-inducing activities five to six times greater than a positive control, geldanamycin. However, nonchlorinated 1,4NQs such as lawsone, juglone, plumbagin, and menadione showed much lower activities than DNQ and DDNQ. The administration of DDNQ increased the accumulation of the HSP17.6C-CI and HSP90.1 transcripts as well as of the corresponding proteins in the Arabidopsis seedlings. DDNQ significantly ameliorated the reductions of fresh weight and chlorophyll contents of the plant due to heat. These results suggest that the chlorinated 1,4NQs are potent HSR inducers that can enhance the heat tolerance of plants.

Photothermal-Responsive Conjugated Polymer Nanoparticles for Remote Control of Gene Expression in Living Cells.

Remote control and noninvasive manipulation of cellular bioprocess has received intensive attention as a powerful technology to control cell functions. Here, a strategy is developed to remotely control intracellular gene expression with high spatial and temporal resolutions by using photothermal-responsive conjugated polymer nanoparticles (CPNs) as the transducer under near-infrared light irradiation. After being modified with positive charged peptide, the CPNs with superior photothermal conversion capacity could effectively coat on the surface of living cells and generate localized heat to trigger target gene expression. The heat-inducible heat shock protein-70 promoter starts transcription of downstream EGFP gene in response to heat shock, thus producing green fluorescent protein in the living cells. The combination of heat-inducible gene promoter and photothermal-responsive CPNs provides a method for the development of thermogenetics.

Monoterpenes induce the heat shock response in Arabidopsis.

Monoterpenes are common constituents of essential oils produced by plants. Although it has been reported that monoterpenes enhanced the heat tolerance of plants, the mechanism has not been elucidated. Here, we tested whether 13 monoterpenes promoted the heat shock response (HSR) in Arabidopsis. To assess the HSR-inducing activity of monoterpenes, we produced transgenic Arabidopsis, which has the β-glucuronidase gene driven by the promoter of a small heat shock protein (HSP17.6C-CI) gene. Results indicated that two monocyclic and four bicyclic monoterpenes showed HSR-inducing activities using the reporter gene system. In particular, (-)-perillaldehyde, which is a monocyclic monoterpene, demonstrated the most potent HSR-inducing activity. (-)-Perillaldehyde significantly inhibited the reduction of chlorophyll content by heat shock in Arabidopsis seedlings. Our previous study indicated that chemical HSR inducers such as geldanamycin and sanguinarine inhibited the activity of plant chaperones in vitro. (-)-Perillaldehyde also inhibited chaperone activity, indicating that it might promote the expression of heat shock protein genes by inhibiting chaperones in the plant cell.

Role of Heat Shock Factors in Stress-Induced Transcription.

Heat shock proteins (HSP) are rapidly induced after stresses such as heat shock and accumulate at high concentrations in cells. HSP induction involves primarily a family of heat shock transcription factors (HSF) that bind the heat shock elements of the HSP genes and mediate transcription in trans. We discuss methods for the study of HSP binding to HSP promoters and the consequent increases in HSP gene expression in vitro and in vivo.

Effects of heat and cadmium exposure on stress-related responses in the liver of female zebrafish: Heat increases cadmium toxicity

In this study, female zebrafish (Danio rerio) were exposed to 26°C or 34°C, 0 or 197μg/L cadmium (Cd), singly or in combination for 7days. Multiple stress-related indicators were evaluated in the liver. Mortality, lipid peroxidation (LPO) and ultrastructural damage increased significantly by Cd exposure alone, and were not affected by heat alone. Interestingly, the combined exposure increased LPO, ultrastructural damage, and mortality compared with Cd exposure alone. The results indicated that elevated temperature increased Cd toxicity, which could be explained by several reasons. Firstly, Cd-exposed fish failed to activate the antioxidant defense system under heat stress. Secondly, expression levels of heat shock protein 70 (HSP70) were not significantly up-regulated by heat in Cd-exposed fish but increased by 117 times in Cd-free fish. Besides, hypermethylation of heat shock factor (HSF) binding motif in HSP70 promoter was observed during the combined exposure, indicating that simultaneous exposure may have partially suppressed the cytoprotective up-regulation of HSP70. Thirdly, heat induced an immunosuppressive effect in Cd-exposed fish, as reflected by the reduced mRNA and activity levels of nitric oxide synthase (iNOS) and interleukin-1β (IL-1β) expression levels. Finally, heat down-regulated Zir-, Irt-like protein 8 (ZIP8) and copper transporter 1 (CTR1) and up-regulated metallothioneins (MTs) in Cd-exposed fish, possibly suggesting Cu and Zn depletion and Cd accumulation. Hence, our data provide evidences that warmer temperatures can potentiate Cd toxicity, involved in the regulation of gene transcription, enzymatic activity, and DNA methylation. We found that heat indicators showed varied sensitivity between normal and Cd-exposed fish, emphasizing that the field metal pollution should be carefully considered when evaluating effects of climate change.

Surface chemistry for cytosolic gene delivery and photothermal transgene expression by gold nanorods

Light-inducible gene regulation has great potential for remote and noninvasive control of the fate and function of target cells. One method to achieve such control is delivery of heat shock protein (HSP) promoter-driven protein expression vectors and photothermal heaters into the cells, followed by activation by illumination. In this study, we show that gold nanorods (AuNRs) functionalized with two conventional lipids, oleate and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), are capable of efficient transfection and quick photoactivation of the HSP promoter. Use of our AuNRs (DOTAP-AuNRs) was comparable to Lipofectamine 2000 in terms of transfection efficiency, while lower in cytotoxicity. Subsequent near-infrared laser (NIR) illumination of the cells transfected by DOTAP-AuNRs for 10 s induced time- and site-specific transgene expression without significant phototoxicity, to a degree similar to that of heating the entire culture dish for 30 min. Our mechanistic studies suggest that efficient transfection and quick photoactivation of the HSP promoter (HSP70b’) are due to the promoted endosomal escape of DOTAP-AuNRs. We propose a novel protocol for NIR-inducible, site-directed gene expression using an unprecedented complex of the three conventional components capable of both transfection and photothermal heating.

Preheating mitigates cadmium toxicity in zebrafish livers: Evidence from promoter demethylation, gene transcription to biochemical levels

The working hypothesis for this study was that moderate heat stress would alleviate the deleterious effects of subsequent cadmium (Cd) exposure on fish. Thus, zebrafish (Danio rerio) were subjected to water maintained at 26°C and 34°C for 4days, and then exposed to 0 or 200μg/L Cd for 1 week at 26°C. Multiple indicators were measured from livers of zebrafish at different levels, including DNA, RNA, protein and enzymatic activity associated with oxidative stress, inflammation and metal transport. The ameliorative effect of preheatinging on Cd toxicity was demonstrated. In the Cd-exposed groups, preheating decreased mortality and lipid peroxidation, increased activity levels of catalase (CAT) and copper/zinc-superoxide dismutase (Cu/Zn-SOD), and up-regulated mRNA levels of heat shock protein 70 (HSP70) and heat shock factor 2 (HSF2). Preheating also mitigated Cd-induced increases in protein and mRNA levels of metallothioneins (MTs), and mRNA levels of several inflammation-related genes. Furthermore, preheating alone dramatically up-regulated mRNA levels of genes related to antioxidant and immune defenses, zinc and copper transporters, protein folding, and reduced methylation levels in the HSF binding motif of the HSP70 promoter. Overall, preheating-induced accumulation of transcripts via demethylation might support the rapid defense responses at post-transcriptional levels caused by subsequent Cd exposure, indicating an adaptive mechanism for organisms exposed to one mild stressor followed by another.

Methyl CpG level at distal part of heat-shock protein promoter HSP70 exhibits epigenetic memory for heat stress by modulating recruitment of POU2F1-associated nucleosome-remodeling deacetylase (NuRD) complex.

Depending on its stringency, exposure to heat in early life leads to either resilience or vulnerability to heat stress later in life. We hypothesized that epigenetic alterations in genes belonging to the cell proteostasis pathways are attributed to long-term responses to heat stress. Epigenetic regulation of the mRNA expression of the molecular chaperone heat-shock protein (HSP) 70 (HSPA2) was evaluated in the chick hypothalamus during the critical period of thermal-control establishment on day 3 post-hatch and during heat challenge on day 10. Both the level and duration of HSP70 expression during heat challenge a week after heat conditioning were more pronounced in chicks conditioned under harsh versus mild temperature. Analyzing different segments of the promoter invitro indicated that methylation of a distal part altered its transcriptional activity. In parallel, DNA-methylation level of this segment invivo was higher in harsh- compared to mild-heat-conditioned chicks. Hypermethylation of the HSP70 promoter in high-temperature-conditioned chicks was accompanied by a reduction in both POU Class 2 Homeobox 1 (POU2F1) binding and recruitment of the nucleosome remodeling deacetylase (NuRD) chromatin-remodeling complex. As a result, histone H3 acetylation levels at the HSP70 promoter were higher in harsh-temperature-conditioned chicks than in their mild-heat-conditioned counterparts. These results suggest that methylation level of a distal part of the HSP70 promoter and POU2F1 recruitment may reflect heat-stress-related epigenetic memory and may be useful in differentiating between individuals that are resilient or vulnerable to stress.

Radiofrequency hyperthermia promotes the therapeutic effects on chemotherapeutic-resistant breast cancer when combined with heat shock protein promoter-controlled HSV-TK gene therapy: Toward imaging-guided interventional gene therapy.

ObjectiveGene therapy is a frontier in modern medicine. In the present study, we explored a new technique for the effective treatment of multidrug-resistant (MDR) breast cancer by combining fully the advantages of multidisciplinary fields, including image-guided minimally invasive interventional oncology, radiofrequency technology, and direct intratumoral gene therapy.ResultsCombination treatment with PHSP-TK plus RFH resulted in significantly higher TK gene transfection/expression, as well as a lower cell proliferation rate and a higher cell apoptosis index, than those of control groups. In vivo validation experiments with MRI confirmed that combination therapy resulted in a significant reduction of relative tumor volume compared with those of control animals, which was supported by the results of histologic and apoptosis analyses.Materials and methodsThe heat shock protein promoter (PHSP) was used to precisely control the overexpression of thymidine kinase (TK) (PHSP-TK). Serial in vitro experiments were performed to confirm whether radiofrequency hyperthermia (RFH) could enhance PHSP-TK transfection and expression in a MDR breast cancer cell line (MCF7/Adr). Serial in vivo experiments were then carried out to validate the feasibility of the new technique, termed interventional RFH-enhanced direct intratumoral PHSP-TK gene therapy. The therapeutic effect of combination therapy was evaluated by MRI and confirmed by subsequent laboratory correlation.ConclusionsThis study has established “proof-of-principle” of a new technique, interventional RFH-enhanced local gene therapy for MDR breast cancer, which may open new avenues for the effective management of MDR breast cancers via the simultaneous integration of interventional oncology, RF technology, and direct intratumoral gene therapy.