Survival Of Mammalian Cells Research Articles

Uptake of substances into living mammalian cells by microwave induced perturbation of the plasma membrane

Delivering foreign molecules and genetic material into cells is a crucial process in life sciences and biotechnology, resulting in great interest in effective cell transfection methods. Importantly, physical transfection methods allow delivery of molecules of different chemical composition and are, thus, very flexible. Here, we investigated the influence of microwave radiation on the transfection and survival of mammalian cells. We made use of an optimized microwave-poration device and analyzed its performance (frequency and electric field strength) in comparison with simulations. We, then, tested the effect of microwave irradiation on cells and found that 18 GHz had the least impact on cell survival, viability, cell division and genotoxicity while 10 GHz drastically impacted cell physiology. Using live-cell fluorescence microscopy and image analysis, we tested the uptake of small chemical substances, which was most efficient at 18 GHz and correlated with electric field strength and frequency. Finally, we were able to obtain cellular uptake of molecules of very different chemical composition and sizes up to whole immunoglobulin antibodies. In conclusion, microwave-induced poration enables the uptake of widely different substances directly into mammalian cells growing as adherent cultures and with low physiological impact.

CRISPR screen identifies the role of RBBP8 in mediating unfolded protein response induced liver damage through regulating protein synthesis

Unfolded protein response (UPR) maintains the endoplasmic reticulum (ER) homeostasis, survival, and physiological function of mammalian cells. However, how cells adapt to ER stress under physiological or disease settings remains largely unclear. Here by a genome-wide CRISPR screen, we identified that RBBP8, an endonuclease involved in DNA damage repair, is required for ATF4 activation under ER stress in vitro. RNA-seq analysis suggested that RBBP8 deletion led to impaired cell cycle progression, retarded proliferation, attenuated ATF4 activation, and reduced global protein synthesis under ER stress. Mouse tissue analysis revealed that RBBP8 was highly expressed in the liver, and its expression is responsive to ER stress by tunicamycin intraperitoneal injection. Hepatocytes with RBBP8 inhibition by adenovirus-mediated shRNA were resistant to tunicamycin (Tm)-induced liver damage, cell death, and ER stress response. To study the pathological role of RBBP8 in regulating ATF4 activity, we illustrated that both RBBP8 and ATF4 were highly expressed in liver cancer tissues compared with healthy controls and highly expressed in Ki67-positive proliferating cells within the tumors. Interestingly, overexpression of RBBP8 in vitro promoted ATF4 activation under ER stress, and RBBP8 expression showed a positive correlation with ATF4 expression in liver cancer tissues by co-immunostaining. Our findings provide new insights into the mechanism of how cells adapt to ER stress through the crosstalk between the nucleus and ER and how tumor cells survive under chemotherapy or other anticancer treatments, which suggests potential therapeutic strategies against liver disease by targeting DNA damage repair, UPR or protein synthesis.

Oxygen-releasing biomaterials for regenerative medicine.

Oxygen is critical to the survival, function and fate of mammalian cells. Oxygen tension controls cellular behavior through metabolic programming, which in turn controls tissue regeneration. A variety of biomaterials with oxygen-releasing capabilities have been developed to provide oxygen supply to ensure cell survival and differentiation for therapeutic efficacy, and to prevent hypoxia-induced tissue damage and cell death. However, controlling the oxygen release with spatial and temporal accuracy is still technically challenging. In this review, we provide a comprehensive overview of organic and inorganic materials available as oxygen sources, including hemoglobin-based oxygen carriers (HBOCs), perfluorocarbons (PFCs), photosynthetic organisms, solid and liquid peroxides, and some of the latest materials such as metal-organic frameworks (MOFs). Additionally, we introduce the corresponding carrier materials and the oxygen production methods and present state-of-the-art applications and breakthroughs of oxygen-releasing materials. Furthermore, we discuss the current challenges and the future perspectives in the field. After reviewing the recent progress and the future perspectives of oxygen-releasing materials, we predict that smart material systems that combine precise detection of oxygenation and adaptive control of oxygen delivery will be the future trend for oxygen-releasing materials in regenerative medicine.

Dync1li1 is required for the survival of mammalian cochlear hair cells by regulating the transportation of autophagosomes.

Dync1li1, a subunit of cytoplasmic dynein 1, is reported to play important roles in intracellular retrograde transport in many tissues. However, the roles of Dync1li1 in the mammalian cochlea remain uninvestigated. Here we first studied the expression pattern of Dync1li1 in the mouse cochlea and found that Dync1li1 is highly expressed in hair cells (HCs) in both neonatal and adult mice cochlea. Next, we used Dync1li1 knockout (KO) mice to investigate its effects on hearing and found that deletion of Dync1li1 leads to early onset of progressive HC loss via apoptosis and to subsequent hearing loss. Further studies revealed that loss of Dync1li1 destabilizes dynein and alters the normal function of dynein. In addition, Dync1li1 KO results in a thinner Golgi apparatus and the accumulation of LC3+ autophagic vacuoles, which triggers HC apoptosis. We also knocked down Dync1li1 in the OC1 cells and found that the number of autophagosomes were significantly increased while the number of autolysosomes were decreased, which suggested that Dync1li1 knockdown leads to impaired transportation of autophagosomes to lysosomes and therefore the accumulation of autophagosomes results in HC apoptosis. Our findings demonstrate that Dync1li1 plays important roles in HC survival through the regulation of autophagosome transportation.

Uncovering conditional vulnerabilities in human cancer cells

Forward genetic screens based on either RNAi or CRISPR make it possible to systematically identify genes required for the survival and growth of mammalian cells, and thus offer a powerful approach to characterize protein function and identify potential cancer vulnerabilities. Nonetheless, fitness genes can be context‐dependent, and indeed, CRISPR screens across hundreds of human cell lines have revealed that genetic dependencies can vary with natural cell‐intrinsic diversity. However, while the impact of environmental factors on cell physiology has become better appreciated, there has been little investigation into how nutrient availability affects gene essentiality and, further, most forward genetic screens are performed in vitro using culture media with little relevance to human physiology. We previously developed Human Plasma‐Like Medium (HPLM) – the first systematically designed physiologic medium, which contains over 60 components at concentrations that reflect their average values in human blood. To test the hypothesis that medium composition influences gene essentiality, we performed CRISPR‐based loss‐of‐function screens of blood cancer lines HPLM versus traditional media. Analysis of these data revealed that sets of conditionally essential genes are involved in several biological processes and can vary with both cell‐intrinsic diversity and the combination of basal and serum components that comprise typical media. Moreover, our approach allows for the downstream identification of gene‐nutrient interactions that underlie conditional CRISPR phenotypes, which can in turn uncover conditionally essential protein roles.Additionally, most preclinical therapies fail to achieve translation – a major problem attributed in part to the limited physiologic relevance of existing models. Nonetheless, there has been similarly little systematic investigation into how the composition of culture media might affect drug efficacy. Further, virtually all drug screens, initial hit validation efforts, and studies of drug activity are performed in vitro using traditional media as well. Therefore, we with collaborators at NCATS used a library of approved and (pre)‐clinical drugs to screen blood cancer lines in HPLM versus conventional media. Remarkably, we identified many whose killing activity varied with medium composition, including drugs approved for non‐oncology indications. Indeed, neither the drug‐nutrient interaction nor the mechanism of drug activity in cancer cells is immediately apparent in many such cases.

Interference of Antioxidant Flavonoids with MTT Tetrazolium Assay in a Cell Free System

Introduction: The tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) used extensively to measure the quantitative survival and proliferation of mammalian cells. The analysis is based upon the reduction of MTT by metabolically active cells to insoluble formazan crystals. Flavonoids are a large group of natural compounds found in plants with variable phenolic structures. Flavonoids, as they are potential reducing agents, they act as a free radical scavenger. The aim of the study is to assess the reducing effect of some of the flavonoids on tetrazolium salt and their interference with the colorimetric analysis of MTT. The cell viability obtained from the MTT assay was compared with that of SRB assay in the determination of flavonoids cytotoxicity. 
 Materials and Methods: The present study examined the effect of few bio-flavonoids like Quercetin, EGCG, Rutin and Resveratrol to reduce MTT in the absence of cells under different experimental conditions such as concentration of flavonoids, incubation time and results were compared with SRB assay findings. The study also involves the analysis of flavonoid cytotoxicity on lung cancer cells NCIH-460 and NCIH-522 by MTT and SRB assay to establish the suitable cell viability assay for flavonoids.
 Results: All the flavonoids showed the instant formation of the dark blue formazan salt in the absence of the cells with MTT assay. Whereas SRB assay of flavonoids in the absence of cells, results showed the absorbance similar to that of the blank, indicating that SRB did not interfere with flavonoids in a cell-free system.
 Conclusion: From the results, it is evident that MTT is not a suitable method to determine the effect of flavonoids on cell viability and proliferation as flavonoids itself reduces the MTT to formazan crystals. Study also suggests that SRB assay is more suitable method to determine the effect of flavonoids on cell viability.

STAT3-Specific Single Domain Nanobody Inhibits Expansion of Pathogenic Th17 Responses and Suppresses Uveitis in Mice.

STAT3 activates transcription of genes that regulate cell growth, differentiation, and survival of mammalian cells. Genetic deletion of Stat3 in T cells has been shown to abrogate Th17 differentiation, suggesting that STAT3 is a potential therapeutic target for Th17-mediated diseases. However, a major impediment to therapeutic targeting of intracellular proteins such as STAT3 is the lack of efficient methods for delivering STAT3 inhibitors into cells. In this study, we developed a novel antibody (SBT-100) comprised of the variable (V) region of a STAT3-specific heavy chain molecule and demonstrate that this 15 kDa STAT3-specific nanobody enters human and mouse cells, and induced suppression of STAT3 activation and lymphocyte proliferation in a concentration-dependent manner. To investigate whether SBT-100 would be effective in suppressing inflammation in vivo, we induced experimental autoimmune uveitis (EAU) in C57BL/6J mice by active immunization with peptide from the ocular autoantigen, interphotoreceptor retinoid binding protein (IRBP651-670). Analysis of the retina by fundoscopy, histological examination, or optical coherence tomography showed that treatment of the mice with SBT-100 suppressed uveitis by inhibiting expansion of pathogenic Th17 cells that mediate EAU. Electroretinographic (ERG) recordings of dark and light adapted a- and b-waves showed that SBT-100 treatment rescued mice from developing significant visual impairment observed in untreated EAU mice. Adoptive transfer of activated IRBP-specific T cells from untreated EAU mice induced EAU, while EAU was significantly attenuated in mice that received IRBP-specific T cells from SBT-100 treated mice. Taken together, these results demonstrate efficacy of SBT-100 in mice and suggests its therapeutic potential for human autoimmune diseases.

10 THE ROLE OF DIETARY L-SERINE IN THE REGULATION OF INTESTINAL MUCUS BARRIER DURING INFLAMMATION

Abstract Background Recent accumulating evidence suggests that amino acids have crucial roles in the maintenance of intestinal homeostasis. In inflammatory bowel disease (IBD), amino acid metabolism is changed in both host and the gut microbiota. Among amino acids, L-serine plays a central role in several metabolic processes that are essential for the growth and survival of both mammalian and bacterial cells. However, the role of L-serine in intestinal homeostasis and IBD remains incompletely understood. In this study, we investigated the effect of dietary L-serine on intestinal inflammation in a murine model of colitis. Methods Specific pathogen-free (SPF) mice were fed either a control diet (amino acid-based diet) or an L-serine-deficient diet (SDD). Colitis was induced by the treatment of dextran sodium sulfate (DSS). The gut microbiome was analyzed by 16S rRNA sequencing. We also evaluate the effect of dietary L-serine in germ-free mice and gnotobiotic mice that were colonized by a consortium of non-mucolytic bacterial strains or the consortium plus mucolytic bacterial strains. Results We found that the SDD exacerbated experimental colitis in SPF mice. However, the severity of colitis in SDD-fed mice was comparable to control diet-fed mice in germ-free condition, suggesting that the gut microbiota is required for exacerbation of colitis caused by the restriction of dietary L-serine. The gut microbiome analysis revealed that dietary L-serine restriction fosters the blooms of a mucus-degrading bacterium Akkermansia muciniphila and adherent-invasive Escherichia coli in the inflamed gut. Consistent with the expansion of mucolytic bacteria, SDD-fed mice showed a loss of the intestinal mucus layer. Dysfunction of the mucus barrier resulted in increased intestinal permeability, thereby leading to bacterial translocation to the intestinal mucosa, which subsequently increased the severity of colitis. The increased intestinal permeability and subsequent bacterial translocation were observed in SDD-fed gnotobiotic mice that colonized by mucolytic bacteria. In contrast, dietary L-serine restriction did not alter intestinal barrier integrity in gnotobiotic mice that colonized only by non-mucolytic bacteria. Conclusion Our results suggest that dietary L-serine regulates the integrity of the intestinal mucus barrier during inflammation by limiting the expansion of mucus degrading bacteria.

GENE-32. SYNTHETIC LETHAL INTERACTIONS WITH IDH1R132H IN GLIOMA STEM-LIKE CELLS

Abstract Gliomas are the most frequently diagnosed human primary brain tumors. Mutations in Isocitrate Dehydrogenase (IDH) 1 occur in the vast majority of low grade gliomas and secondary high grade glioblastomas. A single amino acid missense mutation in IDH1 at arginine 132 (R132H) is an early event in tumor development. IDH1R132H leads to the production of the oncometabolite 2-R-2-hydroxyglutarate. However the exact roles played by IDH1R132H in the development and malignant transformation of the tumors remain unclear. Further studies are required to determine optimal therapeutic strategies to target the IDH1 mutated subsets of gliomas. New generation high-throughput genetic perturbation technologies make it possible to systematically identify the genes and pathways required for the survival and proliferation of mammalian cells. Herein, we present preliminary results from a CRISPR-dCas9 derived activation to drive the transcriptional activation of more than 23,000 coding genes in both wild type and mutant IDH1 patient-derived pediatric glioma cells. Based on an average of three viability screens per cell type, we analyzed the sgRNA library representation in the IDH1 mutated and non-mutated glioma cultures after the genome wide activation. We identified 1553 candidate genes that upon gain of function trigger the death of glioma cells harboring the IDH1 mutation. The analysis of these results further pinpoints the activation of the Cyclin E1 (CCNE1), the BCL2 Antagonist/Killer 1 (BAK1) and the Homeobox B13 (HOXB13) as the most significant synthetic lethal targets in IDH1R132H glioma cells. Interestingly, results from RNAseq showed a decreased expression of these genes in IDH1 mutated compared to non-mutated glioma cells. Thus, this viability screening aims to elucidate genes that interact with IDH1R123H and play a role in tumor cell fitness. The functional analysis of these candidate genes will allow us to uncover their contribution to the progression of IDH1 mutated gliomas.

Long-Term Culture of Giardia lamblia in Cell Culture Medium Requires Intimate Association with Viable Mammalian Cells.

Giardia lamblia is usually cultured axenically in TYI-S-33, a complex medium which does not permit survival and growth of mammalian cells. Likewise, medium commonly used to maintain and grow mammalian cells does not support healthy trophozoite survival for more than a few hours. The inability to coculture trophozoites and epithelial cells under optimal conditions limits studies of their interactions as well as interpretation of results. Trophozoites of the WB isolate but not the GS isolate were repeatedly adapted to grow stably in long-term cocultures with Caco2, Cos7, and mouse tumor rectal (RIT) cell lines using hybridoma-screened Dulbecco's modified Eagle's medium and 10% fetal calf serum. Giardia did not grow in spent cell culture medium or when separated by a permeable membrane using transwell methodology. Giardia chronically cocultured with specific cell lines became adapted (conditioned). These Giardia cocultures grew better than nonconditioned trophozoites, and the cell lines differed in their ability to support trophozoite growth in the order of RIT > Cos7 > Caco2. Trophozoites conditioned on one cell line and then grown in the presence of a heterologous cell line changed their growth rate to that seen in conditioned Giardia from the heterologous cell line. Trophozoite survival required intimate contact with cells, suggesting that trophozoites obtain an essential nutrient or growth factor from mammalian cells. This may explain why Giardia trophozoites adhere to the small intestinal epithelium during human and animal infections. This coculture system will be useful to understand the complex interactions between the host cells and parasite.

A 3D Multiscale Model to Explore the Role of EGFR Overexpression in Tumourigenesis

The epidermal growth factor receptor (EGFR) signalling cascade is one of the main pathways that regulate the survival and division of mammalian cells. It is also one of the most altered transduction pathways in cancer. Acquired mutations in the EGFR/ERK pathway can cause the overexpression of EGFR on the surface of the cell, while others downregulate the inactivation of switched on intracellular proteins such as Ras and Raf. This upregulates the activity of ERK and promotes cell division. We develop a 3D multiscale model to explore the role of EGFR overexpression on tumour initiation. In this model, cells are described as individual objects that move, interact, divide, proliferate, and die by apoptosis. We use Brownian Dynamics to describe the extracellular and intracellular regulations of cells as well as the spatial and stochastic effects influencing them. The fate of each cell depends on the number of active transcription factors in the nucleus. We use numerical simulations to investigate the individual and combined effects of mutations on the intracellular regulation of individual cells. Next, we show that the distance between active receptors increase the level of EGFR/ERK signalling. We demonstrate the usefulness of the model by quantifying the impact of mutational alterations in the EGFR/ERK pathway on the growth rate of in silico tumours.

Robust Thin Film Surface with a Selective Antibacterial Property Enabled via a Cross-Linked Ionic Polymer Coating for Infection-Resistant Medical Applications.

Fabrication of new antibacterial surfaces has become a primary strategy for preventing device-associated infections (DAIs). Although considerable progress has recently been made in reducing DAIs, current antibacterial coating methods are technically complex and do not allow selective bacterial killing. Here, we propose novel anti-infective surfaces made of a cross-linked ionic polymer film that achieve selective bacteria killing while simultaneously favoring the survival of mammalian cells. A one-step polymerization process known as initiated chemical vapor deposition was used to generate a cross-linked ionic polymer film from 4-vinylbenzyl chloride and 2-(dimethylamino) ethyl methacrylate monomers in the vapor phase. In particular, the deposition process produced a polymer network with quaternary ammonium cross-linking sites, which provided the surface with an ionic moiety with an excellent antibacterial contact-killing property. This method confers substrate compatibility, which enables various materials to be coated with ionic polymer films for use in medical implants. Moreover, the ionic polymer-deposited surfaces supported the healthy growth of mammalian cells while selectively inhibiting bacterial growth in coculture models without any detectable cytotoxicity. Thus, the cross-linked ionic polymer-based antibacterial surface developed in this study can serve as an ideal platform for biomedical applications that require a highly sterile environment.

Inhibitory Effects of Novel SphK2 Inhibitors on Migration of Cancer Cells.

Cell migration is an essential process for survival and differentiation of mammalian cells. Numerous diseases are induced or influenced by inappropriate regulation of cell migration, which plays a key role in cancer cell metastasis. In fact, very few anti-metastasis drugs are available on the market. SphKs are enzymes that convert sphingosine to sphingosine-1-phosphate (S1P) and are known to control various cellular functions, including migration of cells. In human, SphK2 is known to promote apoptosis, suppresses cell growth, and controls cell migration; in addition, the specific ablation of SphK2 activity was reported to inhibit cancer cell metastasis. The previously identified SG12 and SG14 are synthetic analogs of sphingoid and can specifically inhibit the functions of SphK2. We investigated the effects of the SphK2 specific inhibitors on the migratory behavior of cells. We investigated how SG12 and SG14 affect cell migration by monitoring both cumulative and individual cell migration behavior using HeLa cells. SG12 and SG14 mutually showed stronger inhibitory effects with less cytotoxicity compared with a general SphK inhibitor, N,N-dimethylsphingosine (DMS). The mechanistic aspects of specific SphK2 inhibition were studied by examining actin filamentation and the expression levels of motility-related genes. The data revealed that SG12 and SG14 resemble DMS in decreasing overall cell motility, but differ in that they differentially affect motility parameters and motility-related signal transduction pathways and therefore actin polymerization, which are not altered by DMS. Our findings show that SphK2 inhibitors are putative candidates for anti-metastatic drugs.

Homologous recombination preferentially repairs heat-induced DNA double-strand breaks in mammalian cells

Purpose: Heat shock induces DNA double-strand breaks (DSBs), but the precise mechanism of repairing heat-induced damage is unclear. Here, we investigated the DNA repair pathways involved in cell death induced by heat shock.Materials and methods: B02, a specific inhibitor of human RAD51 (homologous recombination; HR), and NU7026, a specific inhibitor of DNA-PK (non-homologous end-joining; NHEJ), were used for survival assays of human cancer cell lines with different p53-gene status. Mouse embryonic fibroblasts (MEFs) lacking Lig4 (NHEJ) and/or Rad54 (HR) were used for survival assays and a phosphorylated histone H2AX at Ser139 (γH2AX) assay. MEFs lacking Rad51d (HR) were used for survival assays. SPD8 cells were used to measure HR frequency after heat shock.Results: Human cancer cells were more sensitive to heat shock in the presence of B02 despite their p53-gene status, and the effect of B02 on heat sensitivity was specific to the G2 phase. Rad54-deficient MEFs were sensitive to heat shock and showed prolonged γH2AX signals following heat shock. Rad51d-deficient MEFs were also sensitive to heat shock. Moreover, heat shock-stimulated cells had increased HR.Conclusions: The HR pathway plays an important role in the survival of mammalian cells against death induced by heat shock via the repair of heat-induced DNA DSBs.

Reversal of Tetracycline Resistance in Escherichia coli by Noncytotoxic bis(Tryptophan)s.

Nine bis(tryptophan) derivatives (BTs) and two control compounds were synthesized and tested for antimicrobial activity against two Escherichia coli strains and a Staphylococcus aureus strain. The effects of linker type, shape, and conformational rigidity were manifested in dramatic differences in altering tetracycline potency when coadministered with that antibiotic. A reversal of resistance was observed for an E.coli strain having a TetA efflux pump. Survival of mammalian cells was assayed with good result.

Analyses of the modulatory effects of antibacterial silver doped calcium phosphate-based ceramic nano-powder on proliferation, survival, and angiogenic capacity of different mammalian cells in vitro

In this study, the antibacterial, cytotoxic, and angiogenic activities of silver doped calcium phosphate-based inorganic powder (ABT or PAG) were systematically investigated. ABT powders containing varying silver content were fabricated using a wet chemical manufacturing method. Antibacterial efficiencies of the ABT powders were investigated using a standard test with indicator bacteria and yeast. The cytotoxic effects of ABT on three different fibroblast cells and human umbilical vein endothelial cells (HUVECs) were assessed using MTT assay. ABT powder exhibits concentration-related cytotoxicity characteristics. Apoptotic activity, attachment capability, and wound healing effects were examined on fibroblasts. The angiogenic activity of ABT was investigated by tube formation assay in HUVECs; 10 μg ml−1 and 100 μg ml−1 concentrations of the highest metal ion content of ABT did not disrupt the tube formation of HUVECs. All these tests showed that ABT does not compromise the survival of the cells and might impose regeneration ability to various cell types. These results indicate that silver doped calcium phosphate-based inorganic powder with an optimal silver content has good potential for developing new biomaterials for implant applications.

ROS Accumulation by PEITC Selectively Kills Ovarian Cancer Cells via UPR-Mediated Apoptosis.

Unfolded protein response (UPR) is crucial for both survival and death of mammalian cells, which is regulated by reactive oxygen species (ROS) and nutrient depletion. In this study, we demonstrated the effect of ROS-accumulation, induced by β-phenethyl isothiocyanate (PEITC), on UPR-mediated apoptosis in ovarian cancer cells. We used ovarian cancer cell lines, PA-1 and SKOV-3, with different p53 status (wild- and null-type, respectively). PEITC caused increased ROS-accumulation and inhibited proliferation selectively in ovarian cancer cells, and glutathione (GSH) depletion in SKOV-3. However, PEITC did not cause any effect in normal ovarian epithelial cells and peripheral blood mononuclear cells. After 48 h of PEITC treatment (5 μM), apoptotic cell death was shown to increase significantly in the ovarian cancer cells and not in the normal cells. The key regulator of UPR-mediated apoptosis, CHOP/GADD153 and endoplasmic reticulum resident chaperone BiP/GRP78 were parallely up-regulated with activation of two major sensors of the UPR [PERK and ATF-6 in PA-1; PERK, and IRE1α in SKOV-3) in response to ROS accumulation induced by PEITC (5 μM). ROS scavenger, N-acetyl-L-cysteine (NAC), attenuated the effect of PEITC on UPR signatures (P-PERK, IRE1α, CHOP/GADD153, and BiP/GRP78), suggesting the involvement of ROS in UPR-mediated apoptosis. Altogether, PEITC induces UPR-mediated apoptosis in ovarian cancer cells via accumulation of ROS in a cancer-specific manner.

Characterization of the human NEK7 interactome suggests catalytic and regulatory properties distinct from those of NEK6.

Human NEK7 is a regulator of cell division and plays an important role in growth and survival of mammalian cells. Human NEK6 and NEK7 are closely related, consisting of a conserved C-terminal catalytic domain and a nonconserved and disordered N-terminal regulatory domain, crucial to mediate the interactions with their respective proteins. Here, in order to better understand NEK7 cellular functions, we characterize the NEK7 interactome by two screening approaches: one using a yeast two-hybrid system and the other based on immunoprecipitation followed by mass spectrometry analysis. These approaches led to the identification of 61 NEK7 interactors that contribute to a variety of biological processes, including cell division. Combining additional interaction and phosphorylation assays from yeast two-hybrid screens, we validated CC2D1A, TUBB2B, MNAT1, and NEK9 proteins as potential NEK7 interactors and substrates. Notably, endogenous RGS2, TUBB, MNAT1, NEK9, and PLEKHA8 localized with NEK7 at key sites throughout the cell cycle, especially during mitosis and cytokinesis. Furthermore, we obtained evidence that the closely related kinases NEK6 and NEK7 do not share common interactors, with the exception of NEK9, and display different modes of protein interaction, depending on their N- and C-terminal regions, in distinct fashions. In summary, our work shows for the first time a comprehensive NEK7 interactome that, combined with functional in vitro and in vivo assays, suggests that NEK7 is a multifunctional kinase acting in different cellular processes in concert with cell division signaling and independently of NEK6.

Molecular insights into microbial β-glucuronidase inhibition to abrogate CPT-11 toxicity.

Bacterial β-glucuronidases expressed by the symbiotic intestinal microbiota appear to play important roles in drug-induced epithelial cell toxicity in the gastrointestinal (GI) tract. For the anticancer drug CPT-11 (irinotecan) and the nonsteroidal anti-inflammatory drug diclofenac, it has been shown that removal of the glucuronide moieties from drug metabolites by bacterial β-glucuronidases in the GI lumen can significantly damage the intestinal epithelium. Furthermore, selective disruption of bacterial β-glucuronidases by small molecule inhibitors alleviates these side effects, which, for CPT-11 {7-ethyl-10-[4-(1-piperidino)-1-piperidino]}, can be dose limiting. Here we characterize novel microbial β-glucuronidase inhibitors that inhibit Escherichia coli β-glucuronidase in vitro with Ki values between 180 nM and 2 μM, and disrupt the enzyme in E. coli cells, with EC50 values as low as 300 nM. All compounds are selective for E. coli β-glucuronidase without inhibiting purified mammalian β-glucuronidase, and they do not impact the survival of either bacterial or mammalian cells. The 2.8 Å resolution crystal structure of one inhibitor bound to E. coli β-glucuronidase demonstrates that it contacts and orders only a portion of the "bacterial loop" present in microbial, but not mammalian, β-glucuronidases. The most potent compound examined in this group was found to protect mice against CPT-11-induced diarrhea. Taken together, these data advance our understanding of the chemical and structural basis of selective microbial β-glucuronidase inhibition, which may improve human drug efficacy and toxicity.

Comparison of Models of Post-Hyperthermia Cell Survival

Several existing mathematical models of the survival of mammalian cells in culture following heating are compared. These models describe the fraction of cells that survive in a normal culture environment following a relatively brief period of heating between approximately 43 °C and 60 °C. The models have been developed either from rate process or mechanistic arguments. Little quantitative comparison between such models has been made using the same sets of data. The models are compared using the Akaike Information Criterion (AICc) after the model parameters have been estimated for two sets of existing data: human prostate cancer cells and Chinese hamster ovary cells. Most of the models capture the cell survival response. Scaled sensitivity coefficients show that some of the models have parameters that are difficult to estimate reliably. Relatively small variations in the AICc suggest that more measurements are needed before ranking the models.