Combination of Plant Virus Nanoparticle-Based in Situ Vaccination with Chemotherapy Potentiates Antitumor Response.

Immunotherapeutics are gaining more traction in the armamentarium used to combat cancer. Specifically, in situ vaccination strategies have gained interest because of their ability to alter the tumor microenvironment to an antitumor state. Herein, we investigate whether flexuous plant virus-based nanoparticles formed by the potato virus X (PVX) can be used as an immunotherapeutic for in situ vaccine monotherapy. We further developed dual chemo-immunotherapeutics by incorporating doxorubicin (DOX) into PVX yielding a dual-functional nanoparticle (PVX-DOX) or by coadministration of the two therapeutic regimes, PVX immunotherapy and DOX chemotherapy (PVX+DOX). In the context of B16F10 melanoma, PVX was able to elicit delayed tumor progression when administered as an intratumoral in situ vaccine. Furthermore, the coadministration of DOX via PVX+DOX enhanced the response of the PVX monotherapy through increased survival, which was also represented in the enhanced antitumor cytokine/chemokine profile stimulated by PVX+DOX when compared to PVX or DOX alone. Importantly, coadministered PVX+DOX was better for in situ vaccination than PVX loaded with DOX (PVX-DOX). Whereas the nanomedicine field strives to design multifunctional nanoparticles that integrate several functions and therapeutic regimens into a single nanoparticle, our data suggest a paradigm shift; some therapeutics may need to be administered separately to synergize and achieve the most potent therapeutic outcome. Altogether, our studies show that development of plant viral nanoparticles for in situ vaccines for treatment is a possibility, and dual mechanistic therapeutics can increase efficacy. Nonetheless, combining immunotherapeutics with cytolytic chemotherapy requires detailed investigation to inform optimal integration of cytolytic and immunotherapies and maximize synergy and efficacy.

Immune-mediated regression of established B16F10 melanoma by intratumoral injection of attenuated Toxoplasma gondii protects against rechallenge.

Immune recognition of tumors can limit cancer development, but antitumor immune responses are often blocked by tumor-mediated immunosuppression. Because microbes or microbial constituents are powerful adjuvants to stimulate immune responses, we evaluated whether intratumoral administration of a highly immunogenic but attenuated parasite could induce rejection of an established poorly immunogenic tumor. We treated intradermal B16F10 murine melanoma by intratumoral injection of an attenuated strain of Toxoplasma gondii (cps) that cannot replicate in vivo and therefore is not infective. The cps treatment stimulated a strong CD8(+) T cell-mediated antitumor immune response in vivo that regressed established primary melanoma. The cps monotherapy rapidly modified the tumor microenvironment, halting tumor growth, and subsequently, as tumor-reactive T cells expanded, the tumors disappeared and rarely returned. The treatment required live cps that could invade cells and also required CD8(+) T cells and NK cells, but did not require CD4(+) T cells. Furthermore, we demonstrate that IL-12, IFN-γ, and the CXCR3-stimulating cytokines are required for full treatment efficacy. The treatment developed systemic antitumor immune activity as well as antitumor immune memory and therefore might have an impact against human metastatic disease. The approach is not specific for either B16F10 or melanoma. Direct intratumoral injection of cps has efficacy against an inducible genetic melanoma model and transplantable lung and ovarian tumors, demonstrating potential for broad clinical use. The combination of efficacy, systemic antitumor immune response, and complete attenuation with no observed host toxicity demonstrates the potential value of this novel cancer therapy.

Local hyperthermia treatment of tumors induces CD8(+) T cell-mediated resistance against distal and secondary tumors.

Combinatorial use of iron oxide nanoparticles (IONPs) and an alternating magnetic field (AMF) can induce local hyperthermia in tumors in a controlled and uniform manner. Heating B16 primary tumors at 43°C for 30 min activated dendritic cells (DCs) and subsequently CD8(+) T cells in the draining lymph node (dLN) and conferred resistance against rechallenge with B16 (but not unrelated Lewis Lung carcinoma) given 7 days post hyperthermia on both the primary tumor side and the contralateral side in a CD8(+) T cell-dependent manner. Mice with heated primary tumors also resisted rechallenge given 30 days post hyperthermia. Mice with larger heated primary tumors had greater resistance to secondary tumors. No rechallenge resistance occurred when tumors were heated at 45°C. Our results demonstrate the promising potential of local hyperthermia treatment applied to identified tumors in inducing anti-tumor immune responses that reduce the risk of recurrence and metastasis. FROM THE CLINICAL EDITOR: Local heating of tumors via iron oxide NPs and an alternating magnetic field led to activation of anti-cancer CD8 T cells, which resulted in resistance against re-challenge and greater resistance to secondary tumors. Similar local heating-based strategies may become an important weapon in enhancing tumor elimination via a naturally existing but attenuated immune response.

Ribosomal protein genes RPS10 and RPS26 are commonly mutated in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA), an inherited bone marrow failure syndrome characterized by anemia that usually presents before the first birthday or in early childhood, is associated with birth defects and an increased risk of cancer. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, in particular craniofacial, upper limb, heart, and urinary system defects that are present in approximately 30%-50% of patients. DBA has been associated with mutations in seven ribosomal protein (RP) genes, RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, and RPS7, in about 43% of patients. To continue our large-scale screen of RP genes in a DBA population, we sequenced 35 ribosomal protein genes, RPL15, RPL24, RPL29, RPL32, RPL34, RPL9, RPL37, RPS14, RPS23, RPL10A, RPS10, RPS12, RPS18, RPL30, RPS20, RPL12, RPL7A, RPS6, RPL27A, RPLP2, RPS25, RPS3, RPL41, RPL6, RPLP0, RPS26, RPL21, RPL36AL, RPS29, RPL4, RPLP1, RPL13, RPS15A, RPS2, and RPL38, in our DBA patient cohort of 117 probands. We identified three distinct mutations of RPS10 in five probands and nine distinct mutations of RPS26 in 12 probands. Pre-rRNA analysis in lymphoblastoid cells from patients bearing mutations in RPS10 and RPS26 showed elevated levels of 18S-E pre-rRNA. This accumulation is consistent with the phenotype observed in HeLa cells after knockdown of RPS10 or RPS26 expression with siRNAs, which indicates that mutations in the RPS10 and RPS26 genes in DBA patients affect the function of the proteins in rRNA processing.

Frameshift mutation in p53 regulator RPL26 is associated with multiple physical abnormalities and a specific pre-ribosomal RNA processing defect in diamond-blackfan anemia.

Diamond-Blackfan anemia (DBA) is an inherited form of pure red cell aplasia that usually presents in infancy or early childhood and is associated with congenital malformations in ∼30-50% of patients. DBA has been associated with mutations in nine ribosomal protein (RP) genes in about 53% of patients. We completed a large-scale screen of 79 RP genes by sequencing 16 RP genes (RPL3, RPL7, RPL8, RPL10, RPL14, RPL17, RPL19, RPL23A, RPL26, RPL27, RPL35, RPL36A, RPL39, RPS4X, RPS4Y1, and RPS21) in 96 DBA probands. We identified a de novo two-nucleotide deletion in RPL26 in one proband associated with multiple severe physical abnormalities. This mutation gives rise to a remarkable ribosome biogenesis defect that affects maturation of both the small and the large subunits. We also found a deletion in RPL19 and missense mutations in RPL3 and RPL23A, which may be variants of unknown significance. Together with RPL5, RPL11, and RPS7, RPL26 is the fourth RP regulating p53 activity that is linked to DBA.

Ribosomal protein L5 and L11 mutations are associated with cleft palate and abnormal thumbs in Diamond-Blackfan anemia patients.

Diamond-Blackfan anemia (DBA), a congenital bone-marrow-failure syndrome, is characterized by red blood cell aplasia, macrocytic anemia, clinical heterogeneity, and increased risk of malignancy. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital anomalies that are present in approximately 30%-50% of patients. The disease has been associated with mutations in four ribosomal protein (RP) genes, RPS19, RPS24, RPS17, and RPL35A, in about 30% of patients. However, the genetic basis of the remaining 70% of cases is still unknown. Here, we report the second known mutation in RPS17 and probable pathogenic mutations in three more RP genes, RPL5, RPL11, and RPS7. In addition, we identified rare variants of unknown significance in three other genes, RPL36, RPS15, and RPS27A. Remarkably, careful review of the clinical data showed that mutations in RPL5 are associated with multiple physical abnormalities, including craniofacial, thumb, and heart anomalies, whereas isolated thumb malformations are predominantly present in patients carrying mutations in RPL11. We also demonstrate that mutations of RPL5, RPL11, or RPS7 in DBA cells is associated with diverse defects in the maturation of ribosomal RNAs in the large or the small ribosomal subunit production pathway, expanding the repertoire of ribosomal RNA processing defects associated with DBA.

Hypertonicity stimulates PGE2 signaling in the renal medulla by promoting EP3 and EP4 receptor expression.

Hypertonicity in the renal medulla stimulates local cyclooxygenase 2 expression, leading to abundant PGE(2) production. Here we found that mRNA expression by the PGE(2)-activated G-protein-coupled receptors, EP3 and EP4 in the renal medulla was decreased by furosemide treatment, a procedure that reduces medullary hypertonicity. When HepG2 cells were cultured in hypertonic conditions by addition of salt or sorbitol, EP3 expression was induced. A specific EP3 agonist inhibited cAMP production, indicating receptor functionality, and this led to a substantial increase in cell survival in hypertonic media. Survival was independent of the SLC5A3 inositol transporter and aldose reductase expression, suggesting that EP3 promoted cell survival under hypertonic conditions independent of cellular organic osmolyte accumulation. Reduced cAMP production did not contribute to increased survival. EP4 expression was stimulated by hypertonicity in MDCK and HepG2 cells, which was associated with increased cAMP production in response to an EP4 agonist. Our study shows that local hypertonicity promotes PGE(2) signaling in the renal medulla by stimulating cognate receptor and cyclooxygenase 2 expression that likely regulates local hemodynamics and tubular transport.Kidney International (2009) 75, 278-284. doi:10.1038/ki.2008.498.

In situ vaccination: Harvesting low hanging fruit on the cancer immunotherapy tree.

After 100 years of debate, it is clear that cancer is recognized by the immune system and this has generated immense interest in cancer immunotherapy. The systemic nature of the immune system gives immunotherapy the ability to treat metastatic disease, which currently requires chemotherapy that frequently fails. Like chemotherapy, most immunotherapy is systemically applied in an effort to generate systemic antitumor immune response. However, local administration of immunostimulatory reagents into a recognized tumor by in situ vaccination (ISV) can also generate systemic antitumor immunity to fight metastatic disease. Conventional vaccines contain antigens and immune adjuvants. With ISV, the tumor itself supplies the antigen and the treatment only applies immune adjuvant directly to the tumor. While current immunotherapy often fails to eliminate cancer because of local immunosuppression mediated by tumors, effective ISV changes the tumor microenvironment from immunosuppressive to immunostimulatory, stimulates presentation of tumor antigens by antigen-presenting cells to T cells, and generates systemic antitumor immunity that promotes antigen-specific effector T-cell attack of both treated and importantly, untreated metastatic tumors. The advantages of ISV are: simple and cost-effective; minimal systemic side effects; feasible and flexible adjuvant delivery; exploiting all tumor antigens in the tumor avoids the need to identify antigens; utilizing all antigens in the tumor minimizes immune escape; and potential synergy when combined with other therapies. This review puts ISV into the broader context of cancer immunotherapy, including the use of nanoparticles, and outlines research needed in order to manifest the potential of ISV for clinical use. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Replication Study: Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis.

As part of the Reproducibility Project: Cancer Biology we published a Registered Report (Fiering et al., 2015) that described how we intended to replicate selected experiments from the paper 'Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis' (Goetz et al., 2011). Here we report the results. Primary mouse embryonic fibroblasts (pMEFs) expressing caveolin 1 (Cav1WT) demonstrated increased extracellular matrix remodeling in vitro compared to Cav1 deficient (Cav1KO) pMEFs, similar to the original study (Goetz et al., 2011). In vivo, we found higher levels of intratumoral stroma remodeling, determined by fibronectin fiber orientation, in tumors from cancer cells co-injected with Cav1WT pMEFs compared to cancer cells only or cancer cells plus Cav1KO pMEFs, which were in the same direction as the original study (Supplemental Figure S7C; Goetz et al., 2011), but not statistically significant. Primary tumor growth was similar between conditions, like the original study (Supplemental Figure S7Ca; Goetz et al., 2011). We found metastatic burden was similar between Cav1WT and Cav1KO pMEFs, while the original study found increased metastases with Cav1WT (Figure 7C; Goetz et al., 2011); however, the duration of our in vivo experiments (45 days) were much shorter than in the study by Goetz et al. (2011) (75 days). This makes it difficult to interpret the difference between the studies as it is possible that the cells required more time to manifest the difference between treatments observed by Goetz et al. We also found a statistically significant negative correlation of intratumoral remodeling with metastatic burden, while the original study found a statistically significant positive correlation (Figure 7Cd; Goetz et al., 2011), but again there were differences between the studies in terms of the duration of the metastasis studies and the imaging approaches that could have impacted the outcomes. Finally, we report meta-analyses for each result.

In Situ Vaccination of Tumors Using Plant Viral Nanoparticles.

Viral nanoparticles are self-assembling units that are being developed and applied for a variety of applications. While most clinical uses involve animal viruses, a plant-derived virus, cowpea mosaic virus (CPMV) has been shown to have antitumor properties in mice when applied as in situ vaccine. Here we describe the production and characterization of CPMV and its use as in situ vaccines in the context of cancer. Subsequent analyses to obtain efficacy or mechanistic data are also detailed.

Exploiting Uptake of Nanoparticles by Phagocytes for Cancer Treatment.

Many cancers including ovarian, pancreatic, colon, liver, and stomach cancers are largely confined to the peritoneal cavity. Peritoneal tumors are directly accessible by intraperitoneal injections. Previously we demonstrated that intraperitoneal injection of nanoparticles and subsequent ingestion by tumor-associated phagocytes can be used to either directly impact tumors or stimulate antitumor immune responses. Here we outline methods to specifically utilize iron oxide nanoparticles with the ID8-Defb29/Vegf-A murine ovarian cancer model and discuss the tendency of phagocytes to ingest nanoparticles and the potential of phagocytes to carry nanoparticles to tumors resulting in direct killing of tumor cells or stimulate antitumor immune responses in peritoneal cancers. This basic approach can be modified as needed for different types of tumors and nanoparticles.

Myristoylated p110α Causes Embryonic Death Due to Developmental and Vascular Defects.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates many important cellular functions. The functional impact of deregulating the PIK3CA gene, encoding the p110α catalytic subunit of PI3K, is validated by frequent gain of function mutations in a range of human cancers. We generated a mouse model with an inducible constitutively active form of PI3K. In this model Cre recombinase activates expression of a myristoylated form of p110α (myr-p110α). The myristoylated version of p110α brings the protein to the cytoplasmic side of the cell membrane, which mimics the normal activation mechanism for the p110α catalytic subunit and activates the PI3K enzyme. Constitutively activated PI3K signaling induced by myr-p110α in all cells of the developing mouse caused lethality during embryonic development. Transgenic Cre;myr-p110α heterozygous embryos displayed morphological malformation and poor vascular development with extremely dilated blood vessels and hemorrhage in the embryo and the extraembryonic yolk sac. Previous studies demonstrated that loss of p110α during embryonic development causes angiogenic disruption and here we show that constitutive activation of p110α by gain of function mutation during development also disrupts vasculogenesis/angiogenesis in what appears to be a similar manner. These finding demonstrate the importance of tight regulation of PI3K signaling during embryonic vasculogenesis/angiogenesis..

Structure of human PRL-3, the phosphatase associated with cancer metastasis.

PRL-3, a novel class protein of prenylated tyrosine phosphatase, is important in cancer metastasis. Due to its high levels of expression in metastatic tumors, PRL-3 may constitute a useful marker for metastasis and might be a new therapeutic target. Here, we present the solution structure of the phosphatase domain of a human PRL-3 (residues 1-162) in phosphate-free state. The nuclear magnetic resonance (NMR) structure of PRL-3 is similar to that of other known phosphatases with minor differences in the secondary structure. But the conformation and flexibility of the loops comprising the active site differ significantly. When phosphate ions or sodium orthovanadate, which is a known inhibitor, are added to the apo PRL-3, the NMR signals from the residues in the active site appeared and could be assigned, indicating that the conformation of the residues has been stabilized.

Stimulating antitumor immunity with nanoparticles.

A variety of strategies, have been applied to cancer treatment and the most recent one to become prominent is immunotherapy. This interest has been fostered by the demonstration that the immune system does recognize and often eliminate small tumors but tumors that become clinical problems block antitumor immune responses with immunosuppression orchestrated by the tumor cells. Methods to reverse this tumor-mediated immunosuppression will improve cancer immunotherapy outcomes. The immunostimulatory potential of nanoparticles (NPs), holds promise for cancer treatment. Phagocytes of various types are an important component of both immunosuppression and immunostimulation and phagocytes actively take up NPs of various sorts, so NPs are a natural system to manipulate these key immune regulatory cells. NPs can be engineered with multiple useful therapeutic features, such as various payloads such as antigens and/or immunomodulatory agents including cytokines, ligands for immunostimulatory receptors or antagonists for immunosuppressive receptors. As more is learned about how tumors suppress antitumor immune responses the payload options expand further. Here we review multiple approaches of NP-based cancer therapies to modify the tumor microenvironment and stimulate innate and adaptive immune systems to obtain effective antitumor immune responses.

Phagocytes mediate targeting of iron oxide nanoparticles to tumors for cancer therapy.

Nanotechnology has great potential to produce novel therapeutic strategies that target malignant cells through the ability of nanoparticles to get access to and be ingested by living cells. However its specificity for accumulation in tumors, which is the key factor that determines its efficacy, has always been a challenge. Here we tested a novel strategy to target and treat ovarian cancer, a representative peritoneal cancer, using iron oxide nanoparticles (IONPs) and an alternating magnetic field (AMF). Peritoneal tumors in general are directly accessible to nanoparticles administered intraperitoneally (IP), as opposed to the more commonly attempted intravenous (IV) administration. In addition, tumor-associated immunosuppressive phagocytes, a predominant cell population in the tumor microenvironment of almost all solid tumors, and cells that are critical for tumor progression, are constantly recruited to the tumor, and therefore could possibly function to bring nanoparticles to tumors. Here we demonstrate that tumor-associated peritoneal phagocytes ingest and carry IONPs specifically to tumors and that these specifically delivered nanoparticles can damage tumor cells after IONP-mediated hyperthermia generated by AMF. This illustrates therapeutic possibilities of intraperitoneal (IP) injection of nanoparticles and subsequent ingestion by tumor-associated phagocytes, to directly impact tumors or stimulate antitumor immune responses. This approach could use IONPs combined with AMF as done here, or other nanoparticles with cytotoxic potential. Overall, the data presented here support IP injection of nanoparticles to utilize peritoneal phagocytes as a delivery vehicle in association with IONP-mediated hyperthermia as therapeutic strategies for ovarian and other peritoneal cancers.

Downregulation of renal sodium transporters and tonicity-responsive enhancer binding protein by long-term treatment with cyclosporin A.

Tonicity-responsive enhancer binding protein (TonEBP) is a transcriptional activator that is regulated by ambient tonicity. TonEBP protects the renal medulla from the deleterious effects of hyperosmolality and regulates the urinary concentration by stimulating aquaporin-2 and urea transporters. The therapeutic use of cyclosporin A (CsA) is limited by nephrotoxicity that is manifested by reduced GFR, fibrosis, and tubular defects, including reduced urinary concentration. It was reported recently that long-term CsA treatment was associated with decreased renal expression of TonEBP target genes, including aquaporin-2, urea transporter, and aldose reductase. This study tested the hypothesis that long-term CsA treatment reduces the salinity/tonicity of the renal medullary interstitium as a result of inhibition of active sodium transporters, leading to downregulation of TonEBP. CsA treatment for 7 d did not affect TonEBP or renal function. Whereas expression of sodium transporters was altered, the medullary tonicity seemed unchanged. Conversely, 28 d of CsA treatment led to downregulation of TonEBP and overt nephrotoxicity. The downregulation of TonEBP involved reduced expression, cytoplasmic shift, and reduced transcription of its target genes. This was associated with reduced expression of active sodium transporters-sodium/potassium/chloride transporter type 2 (NKCC2), sodium/chloride transporter, and Na(+),K(+)-ATPase-along with increased sodium excretion and reduced urinary concentration. Infusion of vasopressin restored the expression of NKCC2 in the outer medulla as well as the expression and the activity of TonEBP. It is concluded that the downregulation of TonEBP in the setting of long-term CsA administration is secondary to the reduced tonicity of the renal medullary interstitium.

Downregulation of renal TonEBP in hypokalemic rats.

Hypokalemia causes a significant decrease in the tonicity of the renal medullary interstitium in association with reduced expression of sodium transporters in the distal tubule. We asked whether hypokalemia caused downregulation of the tonicity-responsive enhancer binding protein (TonEBP) transcriptional activator in the renal medulla due to the reduced tonicity. We found that the abundance of TonEBP decreased significantly in the outer and inner medullas of hypokalemic rats. Underlying mechanisms appeared different in the two regions because the abundance of TonEBP mRNA was lower in the outer medulla but unchanged in the inner medulla. Immunohistochemical examination of TonEBP revealed cell type-specific differences. TonEBP expression decreased dramatically in the outer and inner medullary collecting ducts, thick ascending limbs, and interstitial cells. In the descending and ascending thin limbs, TonEBP abundance decreased modestly. In the outer medulla, TonEBP shifted to the cytoplasm in the descending thin limbs. As expected, transcription of aldose reductase, a target of TonEBP, was decreased since the abundance of mRNA and protein was reduced. Downregulation of TonEBP appeared to have also contributed to reduced expression of aquaporin-2 and UT-A urea transporters in the renal medulla. In cultured cells, expression and activity of TonEBP were not affected by reduced potassium concentrations in the medium. These data support the view that medullary tonicity regulates expression and nuclear distribution of TonEBP in the renal medulla in cell type-specific manners.

Comparison of experimental lung injury from acute renal failure with injury due to sepsis.

BACKGROUND: Acute renal failure (ARF) and acute respiratory distress syndrome (ARDS) coexist frequently, and the mortality rate of this combination is very high. It is well established that cytokines and chemokines play a major role in the pathogenesis of ARDS. In addition, heat shock proteins (HSPs) have been shown to be protective against ARDS. OBJECTIVES: The purpose of this study was to investigate the pathophysiology of ARDS in two different conditions, sepsis and ARF. METHODS: We examined five different rat animal models including sham-operated control, sepsis and three ARF models induced by renal ischemia/reperfusion injury, bilateral nephrectomy or bilateral ligation of renal pedicles. We analyzed pulmonary histology, pulmonary vascular permeability, cellular infiltration, and expression of cytokines, chemokines and HSPs. RESULTS: Like sepsis, the three forms of ARF led to ARDS, as manifested by increased pulmonary vascular permeability and histological changes consistent with ARDS. On the other hand, ARF and sepsis differed in that ARF was associated with markedly lower levels of pulmonary cellular infiltration. Furthermore, while pulmonary expression of tumor necrosis factor-alpha increased in sepsis, cytokine-induced neutrophil chemoattractant 2 increased in nephrectomized rats indicating that different inflammatory mediators were involved in the injury mechanism. Finally, pulmonary expression of multiple HSPs including HSP27-1, HSP70, HSP70-4, HSP70-8 and HSP90 was significantly different between the two conditions. CONCLUSIONS: We conclude that the pathophysiology of ARDS following ARF is distinct from that in sepsis. ARF-induced ARDS is characterized by a low level of cellular infiltration, induction of cytokine-induced neutrophil chemoattractant 2, and a discrete expression profile of HSPs.

Mre11-Rad50-Nbs1 complex is activated by hypertonicity.

When exposed to hypertonic conditions, cells accumulate double-strand DNA breaks (DSBs) like they are exposed to ionizing radiation. It has been proposed that inactivation of the Mre11-Rad50-Nbs1 (MRN) complex due to nuclear exit is responsible for the accumulation of DSBs as cells fail to repair DSBs produced during normal cellular activity. In this study, we examined the MRN complex in cells switched to hypertonicity. Surprisingly, we found that the MRN complex stayed in the nucleus and remained intact in response to hypertonicity. In fact, the MRN complex was dramatically activated after 4 h of switch to hypertonicity in a dose-dependent manner as shown by formation of foci. Activation of ATM and the MRN complex by hypertonicity and bleomycin was additive as was activation of their downstream targets including gammaH2AX and Chk2 indicating that the cellular response to DSB was intact in hypertonic conditions. Activation of Chk2 in response to hypertonicity was not observed in mutant cells with functionally impaired MRN complex confirming that they are in the same pathway. After 20 h of a switch to hypertonicity, MRN foci and gammaH2AX returned to a control level, suggesting that cells adapted to hypertonicity by repairing DNA. We conclude that cells respond normally to DSB and repair the DNA damages induced by hypertonicity.

Multiple domains of TonEBP cooperate to stimulate transcription in response to hypertonicity.

Tonicity-responsive enhancer binding protein (TonEBP), also known as NFAT5, belongs to the Rel family of transcriptional activators. In the kidney medulla and thymus, TonEBP plays a major role in protecting renal cells and T cells from the deleterious effects of ambient hypertonicity. TonEBP is stimulated by hypertonicity via several pathways: increased expression of protein, nuclear translocation, and increased transactivation. In this study, we identified five domains of TonEBP involved in transactivation. The two conserved glutamine repeats were not involved in transactivation. There were three activation domains that could stimulate transcription independently. In addition, there were two modulation domains that potentiated the activity of the activation domains. One of the activation domains is unique to a splice isoform that is more active than others, indicating that alternative splicing can affect the activity of TonEBP. Another activation domain and one of the modulation domains were stimulated by hypertonicity. All the five domains acted in synergy in every combination. Although overall phosphorylation of TonEBP increased in response to hypertonicity, phosphorylation of the activation and modulation domains did not increase in isolation. In sum, TonEBP possesses far more elaborate domains involved in transactivation compared with other Rel proteins.