Mesenchymal Stem Cells Increase Drug Tolerance of A431 Cells Only in 3D Spheroids, Not in 2D Co-Cultures.

Mesenchymal stem cells (MSCs) are an integral part of the tumor microenvironment (TME); however, their role is somewhat controversial: conflicting reports suggest that, depending on the stage of tumor development, MSCs can either support or suppress tumor growth and spread. Additionally, the influence of MSCs on drug resistance is also ambiguous. Previously, we showed that, despite MSCs proliferating significantly more slowly than cancer cells, there are chemotherapeutic drugs which proved to be similarly toxic to both cell types. Here we established 2D co-cultures and 3D co-culture spheroids from different ratios of GFP-expressing, adipose tissue-derived MSCs and A431 epidermoid carcinoma cells tagged with mCherry to investigate the effect of MSCs on cancer cell growth, survival, and drug sensitivity. We examined the cytokine secretion profile of mono- and co-cultures, explored the inner structure of the spheroids, applied MSC-(nutlin-3) and cancer cell-targeting (cisplatin) treatments separately, monitored the response with live-cell imaging and identified a new, double-fluorescent cell type emerging from these cultures. In 2D co-cultures, no effect on proliferation or drug sensitivity was observed, regardless of the changes in cytokine secretion induced by the co-culture. Conversely, 3D spheroids developed a unique internal structure consisting of MSCs, which significantly improved cancer cell survival and resilience to treatment, suggesting that physical proximity and cell-cell connections are required for MSCs to considerably affect nearby cancer cells. Our results shed light on MSC-cancer cell interactions and could help design new, better treatment options for tumors.

Effective synthesis, development and application of a highly fluorescent cyanine dye for antibody conjugation and microscopy imaging.

An asymmetric cyanine-type fluorescent dye was designed and synthesized via a versatile, multi-step process, aiming to conjugate with an Her2+ receptor specific antibody by an azide-alkyne click reaction. The aromaticity and the excitation and relaxation energetics of the fluorophore were characterized by computational methods. The synthesized dye exhibited excellent fluorescence properties for confocal microscopy, offering efficient applicability in in vitro imaging due to its merits such as a high molar absorption coefficient (36 816 M-1 cm-1), excellent brightness, optimal wavelength (627 nm), larger Stokes shift (26 nm) and appropriate photostability compared to cyanines. The conjugated cyanine-trastuzumab was constructed via an effective, metal-free, strain-promoted azide-alkyne click reaction leading to a regulated number of dyes being conjugated. This novel cyanine-labelled antibody was successfully applied for in vitro confocal imaging and flow cytometry of Her2+ tumor cells.

Organic Anion Transporting Polypeptide 3A1 (OATP3A1)-Gated Bio-Orthogonal Labeling of Intracellular Proteins.

Organic anion transporting polypeptides (OATPs) were found to readily deliver membrane impermeable, tetrazine bearing fluorescent probes into cells. This feature was explored in OATP3A1 conditioned bio-orthogonal labeling schemes of various intracellular proteins in live cells. Confocal microscopy and super-resolution microscopy (STED) studies have shown that highly specific and efficient staining of the selected intracellular proteins can be achieved with the otherwise non-permeable probes when OATP3A1 is present in the cell membrane of cells. Such a transport protein linked bio-orthogonal labeling scheme is believed to be useful in OATP3A1 activity-controlled protein expression studies in the future.

Selective Fluorescent Probes for High-Throughput Functional Diagnostics of the Human Multidrug Transporter P-Glycoprotein (ABCB1).

The multidrug transporter ABCB1 (MDR1, Pgp) plays an important role in the absorption, distribution, metabolism, and elimination of a wide range of pharmaceutical compounds. Functional investigation of the ABCB1 expression is also essential in many diseases, including drug-resistant cancer, inflammatory conditions, or Alzheimer disease. In this study, we examined the potential interaction of the ABCB1 multidrug transporter with a group of commercially available viability dyes that are generally considered not to penetrate into intact cells. Here, we demonstrate that the slow cellular accumulation of TO-PRO™-1 (TP1) or TO-PRO™-3 (TP3) was strongly inhibited by ABCB1-dependent dye extrusion. TP1/3 dye accumulation was not affected by the presence of ABCC1 or ABCG2, while this uptake was increased to the level in the ABCB1-negative cells by a specific P-glycoprotein inhibitor, Tariquidar. We suggest that TP compounds can be used as highly sensitive, selective, non-toxic, and stable dyes to examine the functional expression and properties of the ABCB1 multidrug transporter, especially in microplate-based high-throughput flow cytometry assays. In addition, we demonstrate the applicability of the TP dyes to efficiently select and separate even a very low number of Pgp-expressing intact cells.

Cellular expression and function of naturally occurring variants of the human ABCG2 multidrug transporter.

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics; thus the relatively frequent polymorphic and mutant ABCG2 variants in the population may significantly alter disease conditions and pharmacological effects. Low-level or non-functional ABCG2 expression may increase individual drug toxicity, reduce cancer drug resistance, and result in hyperuricemia and gout. In the present work we have studied the cellular expression, trafficking, and function of nine naturally occurring polymorphic and mutant variants of ABCG2. A comprehensive analysis of the membrane localization, transport, and ATPase activity, as well as retention and degradation in intracellular compartments was performed. Among the examined variants, R147W and R383C showed expression and/or protein folding defects, indicating that they could indeed contribute to ABCG2 functional deficiency. These studies and the applied methods should significantly promote the exploration of the medical effects of these personal variants, promote potential therapies, and help to elucidate the specific role of the affected regions in the folding and function of the ABCG2 protein.

A Genetically Encoded Isonitrile Lysine for Orthogonal Bioorthogonal Labeling Schemes.

Bioorthogonal click-reactions represent ideal means for labeling biomolecules selectively and specifically with suitable small synthetic dyes. Genetic code expansion (GCE) technology enables efficient site-selective installation of bioorthogonal handles onto proteins of interest (POIs). Incorporation of bioorthogonalized non-canonical amino acids is a minimally perturbing means of enabling the study of proteins in their native environment. The growing demand for the multiple modification of POIs has triggered the quest for developing orthogonal bioorthogonal reactions that allow simultaneous modification of biomolecules. The recently reported bioorthogonal [4 + 1] cycloaddition reaction of bulky tetrazines and sterically demanding isonitriles has prompted us to develop a non-canonical amino acid (ncAA) bearing a suitable isonitrile function. Herein we disclose the synthesis and genetic incorporation of this ncAA together with studies aiming at assessing the mutual orthogonality between its reaction with bulky tetrazines and the inverse electron demand Diels-Alder (IEDDA) reaction of bicyclononyne (BCN) and tetrazine. Results showed that the new ncAA, bulky-isonitrile-carbamate-lysine (BICK) is efficiently and specifically incorporated into proteins by genetic code expansion, and despite the slow [4 + 1] cycloaddition, enables the labeling of outer membrane receptors such as insulin receptor (IR) with a membrane-impermeable dye. Furthermore, double labeling of protein structures in live and fixed mammalian cells was achieved using the mutually orthogonal bioorthogonal IEDDA and [4 + 1] cycloaddition reaction pair, by introducing BICK through GCE and BCN through a HaloTag technique.

Establishment and Characterization of a Brca1-/-, p53-/- Mouse Mammary Tumor Cell Line.

Breast cancer is the most commonly occurring cancer in women and the second most common cancer overall. By the age of 80, the estimated risk for breast cancer for women with germline BRCA1 or BRCA2 mutations is around 80%. Genetically engineered BRCA1-deficient mouse models offer a unique opportunity to study the pathogenesis and therapy of triple negative breast cancer. Here we present a newly established Brca1-/-, p53-/- mouse mammary tumor cell line, designated as CST. CST shows prominent features of BRCA1-mutated triple-negative breast cancers including increased motility, high proliferation rate, genome instability and sensitivity to platinum chemotherapy and PARP inhibitors (olaparib, veliparib, rucaparib and talazoparib). Genomic instability of CST cells was confirmed by whole genome sequencing, which also revealed the presence of COSMIC (Catalogue of Somatic Mutations in Cancer) mutation signatures 3 and 8 associated with homologous recombination (HR) deficiency. In vitro sensitivity of CST cells was tested against 11 chemotherapy agents. Tumors derived from orthotopically injected CST-mCherry cells in FVB-GFP mice showed sensitivity to cisplatin, providing a new model to study the cooperation of BRCA1-KO, mCherry-positive tumor cells and the GFP-expressing stromal compartment in therapy resistance and metastasis formation. In summary, we have established CST cells as a new model recapitulating major characteristics of BRCA1-negative breast cancers.

Functional characterization of the ABCG2 5' non-coding exon variants: Stem cell specificity, translation efficiency and the influence of drug selection.

ABCG2 is a multidrug transporter with wide substrate specificity, and is believed to protect several cell types from various xenobiotics and endobiotics. This "guardian" function is important in numerous cell types and tissue barriers but becomes disadvantageous by being responsible for the multidrug resistance phenotype in certain tumor cells. ABCG2 regulation at the protein level has already been extensively studied, however, regulation at the mRNA level, especially the functional role of the various 5' untranslated exon variants (5' UTRs) has been elusive. In the present work, we describe a comprehensive characterization of four ABCG2 mRNA variants with different exon 1 sequences, investigate drug inducibility, stem cell specificity, mRNA stability, and translation efficiency. Although certain variants (E1B and E1C) are considered as "constitutive" mRNA isoforms, we show that chemotoxic drugs significantly alter the expression pattern of distinct ABCG2 mRNA isoforms. When examining human embryonic stem cell lines, we provide evidence that variant E1A has an expression pattern coupled to undifferentiated stem cell stage, as its transcript level is regulated parallel to mRNAs of Oct4 and Nanog pluripotency marker genes. When characterizing the four exon 1 variants we found no significant differences in terms of mRNA stabilities and half-lives of the isoforms. In contrast, variant E1U showed markedly lower translation efficiency both at the total protein level or regarding the functional presence in the plasma membrane. Taken together, these results indicate that the different 5' UTR variants play an important role in cell type specific regulation and fine tuning of ABCG2 expression.

Application of fluorescent dye substrates for functional characterization of ABC multidrug transporters at a single cell level.

ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2- and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. © 2016 International Society for Advancement of Cytometry.

KMT2D preferentially binds mRNAs of the genes it regulates, suggesting a role in RNA processing.

Histone lysine methyltransferases (HKMTs) perform vital roles in cellular life by controlling gene expression programs through the posttranslational modification of histone tails. Since many of them are intimately involved in the development of different diseases, including several cancers, understanding the molecular mechanisms that control their target recognition and activity is vital for the treatment and prevention of such conditions. RNA binding has been shown to be an important regulatory factor in the function of several HKMTs, such as the yeast Set1 and the human Ezh2. Moreover, many HKMTs are capable of RNA binding in the absence of a canonical RNA binding domain. Here, we explored the RNA binding capacity of KMT2D, one of the major H3K4 monomethyl transferases in enhancers, using RNA immunoprecipitation followed by sequencing. We identified a broad range of coding and non-coding RNAs associated with KMT2D and confirmed their binding through RNA immunoprecipitation and quantitative PCR. We also showed that a separated RNA binding region within KMT2D is capable of binding a similar RNA pool, but differences in the binding specificity indicate the existence of other regulatory elements in the sequence of KMT2D. Analysis of the bound mRNAs revealed that KMT2D preferentially binds co-transcriptionally to the mRNAs of the genes under its control, while also interacting with super enhancer- and splicing-related non-coding RNAs. These observations, together with the nuclear colocalization of KMT2D with differentially phosphorylated forms of RNA Polymerase II suggest a so far unexplored role of KMT2D in the RNA processing of the nascent transcripts.

Comprehensive investigation of the mutagenic potential of six pesticides classified by IARC as probably carcinogenic to humans.

Pesticides are significant environmental pollutants, and many of them possess mutagenic potential, which is closely linked to carcinogenesis. Here we tested the mutagenicity of all six pesticides classified probably carcinogenic (Group 2A) by the International Agency of Research on Cancer: 4,4'-DDT, captafol, dieldrin, diazinon, glyphosate and malathion. Whole genome sequencing of TK6 human lymphoblastoid cell clones following 30-day exposure at subtoxic concentrations revealed a clear mutagenic effect of treatment with captafol or malathion when added at 200 nM or 100 µM initial concentrations, respectively. Each pesticide induced a specific base substitution mutational signature: captafol increased C to A mutations primarily, while malathion induced mostly C to T mutations. 4,4'-DDT, dieldrin, diazinon and glyphosate were not mutagenic. Whereas captafol induced chromosomal instability, H2A.X phosphorylation and cell cycle arrest in G2/M phase, all indicating DNA damage, malathion did not induce DNA damage markers or cell cycle alterations despite its mutagenic effect. Hypersensitivity of REV1 and XPA mutant DT40 chicken cell lines suggests that captafol induces DNA adducts that are bypassed by translesion DNA synthesis and are targets for nucleotide excision repair. The experimentally identified mutational signatures of captafol and malathion could shed light on the mechanism of action of these compounds. The signatures are potentially suitable for detecting past exposure in tumour samples, but the reanalysis of large cancer genome databases did not reveal any evidence of captafol or malathion exposure.

Dynamic ABCG2 expression in human embryonic stem cells provides the basis for stress response.

ABCG2 is a plasma membrane multidrug transporter with an established role in the cancer drug-resistance phenotype. This protein is expressed in a variety of tissues, including several types of stem cell. Although ABCG2 is not essential for life, knock-out mice were found to be hypersensitive to xenobiotics and had reduced levels of the side population of hematopoietic stem cells. Previously we have shown that ABCG2 is present in human embryonic stem cell (hESC) lines, with a heterogeneous expression pattern. In this study we examined this heterogeneity, and investigated whether it is related to stress responses in hESCs. We did not find any difference between expression of pluripotency markers in ABCG2-positive and negative hESCs; however, ABCG2-expressing cells had a higher growth rate after cell separation. We found that some harmful conditions (physical stress, drugs, and UV light exposure) are tolerated much better in the presence of ABCG2 protein. This property can be explained by the transporter function which eliminates potential toxic metabolites accumulated during stress conditions. In contrast, mild oxidative stress in hESCs caused rapid internalization of ABCG2, indicating that some environmental factors may induce removal of this transporter from the plasma membrane. On the basis of these results we suggest that a dynamic balance of ABCG2 expression at the population level has the advantage of enabling prompt response to changes in the cellular environment. Such actively maintained heterogeneity might be of evolutionary benefit in protecting special cell types, including pluripotent stem cells.

Discovery of two new isoforms of the human DUT gene.

In human cells two dUTPase isoforms have been described: one nuclear (DUT-N) and one mitochondrial (DUT-M), with cognate localization signals. In contrast, here we identified two additional isoforms; DUT-3 without any localization signal and DUT-4 with the same nuclear localization signal as DUT-N. Based on an RT-qPCR method for simultaneous isoform-specific quantification we analysed the relative expression patterns in 20 human cell lines of highly different origins. We found that the DUT-N isoform is expressed by far at the highest level, followed by the DUT-M and the DUT-3 isoform. A strong correlation between expression levels of DUT-M and DUT-3 suggests that these two isoforms may share the same promoter. We analysed the effect of serum starvation on the expression of dUTPase isoforms compared to non-treated cells and found that the mRNA levels of DUT-N decreased in A-549 and MDA-MB-231 cells, but not in HeLa cells. Surprisingly, upon serum starvation DUT-M and DUT-3 showed a significant increase in the expression, while the expression level of the DUT-4 isoform did not show any changes. Taken together our results indicate that the cellular dUTPase supply may also be provided in the cytoplasm and starvation stress induced expression changes are cell line dependent.

Genetic polymorphisms and decreased protein expression of ABCG2 urate transporters are associated with susceptibility to gout, disease severity and renal-overload hyperuricemia.

Gout is a common crystal induced disease of high personal and social burden, characterised by severe arthritis and comorbidity if untreated. Impaired function of ABCG2 transporter is causative in gout and may be responsible for renal-overload type hyperuricemia. Despite its importance, there is limited information on how clinical parameters correlate with protein expression and that with genetic changes. Urate and clinical parameters of 78 gouty patients and healthy controls were measured among standardised circumstances from a Hungarian population. ABCG2 membrane expression of red blood cells was determined by flow cytometry-based method and SNPs of this protein were analysed by TaqMan-based qPCR. The prevalence of ABCG2 functional polymorphisms in gouty and control patients were 32.1 and 13.7%, respectively. Most common SNP was Q141K while one sample with R236X, R383C and the lately described M71V were found in the gouty population. These polymorphisms showed strong linkage with decreased protein expression while the latter was also associated with higher fractional urate excretion (FUE) and urinary urate excretion (UUE). This study firstly evaluated ABCG2 protein expression in a clinically defined gouty population while also proving its associations between ABCG2 genetic changes and renal-overload hyperuricemia. The paper also highlighted relations between ABCG2 SNPs, gout susceptibility and disease severity characterised by an early onset disease with frequent flares and tophi formation.

Expression, Function and Trafficking of the Human ABCG2 Multidrug Transporter Containing Mutations in an Unstructured Cytoplasmic Loop.

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics, contributes to cancer drug resistance and the development of gout. In this work, we have analyzed the effects of selected variants, residing in a structurally unresolved cytoplasmic region (a.a. 354-367) of ABCG2 on the function and trafficking of this protein. A cluster of four lysines (K357-360) and the phosphorylation of a threonine (T362) residue in this region have been previously suggested to significantly affect the cellular fate of ABCG2. Here, we report that the naturally occurring K360del variant in human cells increased ABCG2 plasma membrane expression and accelerated cellular trafficking. The variable alanine replacements of the neighboring lysines had no significant effect on transport function, and the apical localization of ABCG2 in polarized cells has not been altered by any of these mutations. Moreover, in contrast to previous reports, we found that the phosphorylation-incompetent T362A, or the phosphorylation-mimicking T362E variants in this loop had no measurable effects on the function or expression of ABCG2. Molecular dynamics simulations indicated an increased mobility of the mutant variants with no major effects on the core structure of the protein. These results may help to decipher the potential role of this unstructured region within this transporter.

Synthesis and Application of Two-Photon Active Fluorescent Rhodol Dyes for Antibody Conjugation and In Vitro Cell Imaging.

A novel family of julolidine-containing fluorescent rhodols equipped with a wide variety of substituents was synthesized in a versatile two-step process. The prepared compounds were fully characterized and exhibited excellent fluorescence properties for microscopy imaging. The best candidate was conjugated to the therapeutic antibody trastuzumab through a copper-free strain-promoted azide-alkyne click reaction. The rhodol-labeled antibody was successfully applied for in vitro confocal and two-photon microscopy imaging of Her2+ cells.

Hydrogen peroxide production by epidermal dual oxidase 1 regulates nociceptive sensory signals.

Keratinocytes of the mammalian skin provide not only mechanical protection for the tissues, but also transmit mechanical, chemical, and thermal stimuli from the external environment to the sensory nerve terminals. Sensory nerve fibers penetrate the epidermal basement membrane and function in the tight intercellular space among keratinocytes. Here we show that epidermal keratinocytes produce hydrogen peroxide upon the activation of the NADPH oxidase dual oxidase 1 (DUOX1). This enzyme can be activated by increasing cytosolic calcium levels. Using DUOX1 knockout animals as a model system we found an increased sensitivity towards certain noxious stimuli in DUOX1-deficient animals, which is not due to structural changes in the skin as evidenced by detailed immunohistochemical and electron-microscopic analysis of epidermal tissue. We show that DUOX1 is expressed in keratinocytes but not in the neural sensory pathway. The release of hydrogen peroxide by activated DUOX1 alters both the activity of neuronal TRPA1 and redox-sensitive potassium channels expressed in dorsal root ganglia primary sensory neurons. We describe hydrogen peroxide, produced by DUOX1 as a paracrine mediator of nociceptive signal transmission. Our results indicate that a novel, hitherto unknown redox mechanism modulates noxious sensory signals.

Stimulus-induced expression of the ABCG2 multidrug transporter in HepG2 hepatocarcinoma model cells involves the ERK1/2 cascade and alternative promoters.

The ATP-binding cassette G subfamily member ABCG2 protein is involved in drug resistance of various types of cancer including hepatocellular carcinoma (HCC). The transcriptional regulation of the ABCG2 gene was shown to depend on various transcription factors, and three alternative promoters were described. Here we aimed to decipher the role of hepatocyte growth factor (HGF) and the related kinase cascades on the expression of ABCG2 and the role of the different promoters in this process in the HepG2 human HCC cell line. We observed that HGF treatment increased the amount of ABCG2 on the cell surface in parallel with an increased ABCG2 transcription. ABCG2 mRNA expression was also increased by EGF, oxidative stress or activation of the aryl hydrocarbon receptor, while decreased by TGFb. Treatment with U0126, a specific inhibitor of the ERK1/2 cascade, prevented the HGF and the oxidative stress induced ABCG2 upregulation. We also show that the regulation of ABCG2 by various modulators involve specific alternative promoters. In conclusion, we demonstrate a unique role of the ERK1/2 cascade on ABCG2 modulation in HepG2, and the differential use of the alternative ABCG2 promoters in this cell line. This study reveals the molecular participants of ABCG2 overexpression as new potential treatment targets in HCC.

Establishment and characterization of a novel human induced pluripotent stem cell line stably expressing the iRFP720 reporter.

Stem cell therapy has great potential for replacing beta-cell loss in diabetic patients. However, a key obstacle to cell therapy's success is to preserve viability and function of the engrafted cells. While several strategies have been developed to improve engrafted beta-cell survival, tools to evaluate the efficacy within the body by imaging are limited. Traditional labeling tools, such as GFP-like fluorescent proteins, have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent this limitation, a near-infrared fluorescent mutant version of the DrBphP bacteriophytochrome, iRFP720, has been developed for in vivo imaging and stem/progenitor cell tracking. Here, we present the generation and characterization of an iRFP720 expressing human induced pluripotent stem cell (iPSC) line, which can be used for real-time imaging in various biological applications. To generate the transgenic cells, the CRISPR/Cas9 technology was applied. A puromycin resistance gene was inserted into the AAVS1 locus, driven by the endogenous PPP1R12C promoter, along with the CAG-iRFP720 reporter cassette, which was flanked by insulator elements. Proper integration of the transgene into the targeted genomic region was assessed by comprehensive genetic analysis, verifying precise genome editing. Stable expression of iRFP720 in the cells was confirmed and imaged by their near-infrared fluorescence. We demonstrated that the reporter iPSCs exhibit normal stem cell characteristics and can be efficiently differentiated towards the pancreatic lineage. As the genetically modified reporter cells show retained pluripotency and multilineage differentiation potential, they hold great potential as a cellular model in a variety of biological and pharmacological applications.

PEAR, a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells.

Prime editing is a recently developed CRISPR/Cas9 based gene engineering tool that allows the introduction of short insertions, deletions, and substitutions into the genome. However, the efficiency of prime editing, which typically achieves editing rates of around 10%-30%, has not matched its versatility. Here, we introduce the prime editor activity reporter (PEAR), a sensitive fluorescent tool for identifying single cells with prime editing activity. PEAR has no background fluorescence and specifically indicates prime editing events. Its design provides apparently unlimited flexibility for sequence variation along the entire length of the spacer sequence, making it uniquely suited for systematic investigation of sequence features that influence prime editing activity. The use of PEAR as an enrichment marker for prime editing can increase the edited population by up to 84%, thus significantly improving the applicability of prime editing for basic research and biotechnological applications.