Cytotoxicity of pharmaceuticals and their mixtures toward scaffold-free 3D spheroid cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes.

Pharmaceutical residues are widely detected in surface waters all around the world, causing a range of adverse effects on environmental species, such as fish. Besides population level effects (mortality, reproduction), pharmaceutical residues can bioaccumulate in fish tissues resulting in organ-specific toxicities. In this study, we developed in vitro 3D culture models for rainbow trout (Oncorhynchus mykiss) liver cell line (RTH-149) and cryopreserved, primary rainbow trout hepatocytes (RTHEP), and compared their spheroid formation and susceptibility to toxic impacts of pharmaceuticals. The rapidly proliferating, immortalized RTH-149 cells were shown to form compact spheroids with uniform morphology in just three days, thus enabling higher throughput toxicity screening compared with the primary cells that required acclimation times of about one week. In addition, we screened the cytotoxicity of a total of fourteen clinically used human pharmaceuticals toward the 3D cultures of both RTH-149 cells (metabolically inactive) and primary RTHEP cells (metabolically active), to evaluate the impacts of the pharmaceuticals' own metabolism on their hepatotoxicity in rainbow trout in vitro. Among the test substances, the azole antifungals (clotrimazole and ketoconazole) were identified as the most cytotoxic, with hepatic metabolism indicatively amplifying their toxicity, followed by fluoxetine, levomepromazine, and sertraline, which were slightly less toxic toward the metabolically active primary cells than RTH-149 spheroids. Besides individual pharmaceuticals, the 3D cultures were challenged with mixtures of the eight most toxic substances, to evaluate if their combined mixture toxicities can be predicted based on individual substances' half-maximal effect (EC50) concentrations. As a result, the classical concentration addition approach was concluded sufficiently accurate for preliminarily informing on the approximate effect concentrations of pharmaceutical mixtures on a cellular level. However, direct read-across from human data was proven challenging and inexplicit for prediction of hepatotoxicity in fish in vitro.

The material-enabled oxygen control in thiol-ene microfluidic channels and its feasibility for subcellular drug metabolism assays under hypoxia in vitro.

Tissue oxygen levels are known to be critical to regulation of many cellular processes, including the hepatic metabolism of therapeutic drugs, but its impact is often ignored in in vitro assays. In this study, the material-induced oxygen scavenging property of off-stoichiometric thiol-enes (OSTE) was exploited to create physiologically relevant oxygen concentrations in microfluidic immobilized enzyme reactors (IMERs) incorporating human liver microsomes. This could facilitate rapid screening of, for instance, toxic drug metabolites possibly produced in hypoxic conditions typical for many liver injuries. The mechanism of OSTE-induced oxygen scavenging was examined in depth to enable precise adjustment of the on-chip oxygen concentration with the help of microfluidic flow. The oxygen scavenging rate of OSTE was shown to depend on the type and the amount of the thiol monomer used in the bulk composition, and the surface-to-volume ratio of the chip design, but not on the physical or mechanical properties of the bulk. Our data suggest that oxygen scavenging takes place at the polymer-liquid interface, likely via oxidative reactions of the excess thiol monomers released from the bulk with molecular oxygen. Based on the kinetic constants governing the oxygen scavenging rate in OSTE microchannels, a microfluidic device comprising monolithically integrated oxygen depletion and IMER units was designed and its performance validated with the help of oxygen-dependent metabolism of an antiretroviral drug, zidovudine, which yields a cytotoxic metabolite under hypoxic conditions.

Drug glucuronidation assays on human liver microsomes immobilized on microfluidic flow-through reactors.

UDP-glucuronosyltransferases (UGTs), located in the endoplasmic reticulum of liver cells, are an important family of enzymes, responsible for the biotransformation of several endogenous and exogenous chemicals, including therapeutic drugs. However, the phenomenon of 'latency', i.e., full UGT activity revealed by disruption of the microsomal membrane, poses substantial challenges for predicting drug clearance based on in vitro glucuronidation assays. This work introduces a microfluidic reactor design comprising immobilized human liver microsomes to facilitate the study of UGT-mediated drug clearance under flow-through conditions. The performance of the microreactor is characterized using glucuronidation of 8-hydroxyquinoline (via multiple UGTs) and zidovudine (via UGT2B7) as the model reactions. With the help of alamethicin and albumin effects, we show that conducting UGT metabolism assays under flow conditions facilitates in-depth mechanistic studies, which may also shed light on UGT latency.

Cell adhesion and proliferation on common 3D printing materials used in stereolithography of microfluidic devices.

Three-dimensional (3D) printing has recently emerged as a cost-effective alternative for rapid prototyping of microfluidic devices. The feature resolution of stereolithography-based 3D printing is particularly well suited for manufacturing of continuous flow cell culture platforms. Poor cell adhesion or material-induced cell death may, however, limit the introduction of new materials to microfluidic cell culture. In this work, we characterized four commercially available materials commonly used in stereolithography-based 3D printing with respect to long-term (2 month) cell survival on native 3D printed surfaces. Cell proliferation rates, along with material-induced effects on apoptosis and cell survival, were examined in mouse embryonic fibroblasts. Additionally, the feasibility of Dental SG (material with the most favored properties) for culturing of human hepatocytes and human-induced pluripotent stem cells was evaluated. The strength of cell adhesion to Dental SG was further examined over a shear force gradient of 1-89 dyne per cm2 by using a custom-designed microfluidic shear force assay incorporating a 3D printed, tilted and tapered microchannel sealed with a polydimethylsiloxane lid. According to our results, autoclavation of the devices prior to cell seeding played the most important role in facilitating long-term cell survival on the native 3D printed surfaces with the shear force threshold in the range of 3-8 dyne per cm2.

Healthcare professionals' work engagement in Finnish university hospitals.

BACKGROUND: Concerns about the sufficiency and dedication of the healthcare workforce have arisen as the baby boomer generation is retiring and the generation Y might have different working environment demands. AIMS AND OBJECTIVE: To describe the association between work engagement of healthcare professionals' and its background factors at five Finnish university hospitals. METHODS: Survey data were collected from nurses, physicians and administrative staff (n = 561) at all five university hospitals in Finland. Data were collected using an electronic questionnaire that comprised the Utrecht Work Engagement Scale (9 items) and 13 questions regarding the respondents' backgrounds. Descriptive and correlational analyses were used to examine the data. RESULTS: Most respondents were female (85%) and nursing staff (72%). Baby boomers (49%) were the largest generational cohort. The work engagement composite mean for the total sample was 5.0, indicating high work engagement. Significant differences in work engagement existed only among sex and age groups. The highest work engagement scores were among administrative staff. CONCLUSIONS: Work engagement among healthcare professionals in Finnish university hospitals is high. High work engagement might be explained by suitable job resources and challenges, as well as opportunities provided by a frontline care environment. Attention should especially be paid to meeting the needs of young people entering the workforce to strengthen their dedication and absorption.

Cell-based bioreporter assay coupled to HPLC micro-fractionation in the evaluation of antimicrobial properties of the basidiomycete fungus Pycnoporus cinnabarinus.

CONTEXT: Identification of bioactive components from complex natural product extracts can be a tedious process that aggravates the use of natural products in drug discovery campaigns. OBJECTIVE: This study presents a new approach for screening antimicrobial potential of natural product extracts by employing a bioreporter assay amenable to HPLC-based activity profiling. MATERIALS AND METHODS: A library of 116 crude extracts was prepared from fungal culture filtrates by liquid-liquid extraction with ethyl acetate, lyophilised, and screened against Escherichia coli using TLC bioautography. Active extracts were studied further with a broth microdilution assay, which was, however, too insensitive for identifying the active microfractions after HPLC separation. Therefore, an assay based on bioluminescent E. coli K-12 (pTetLux1) strain was coupled with HPLC micro-fractionation. RESULTS: Preliminary screening yielded six fungal extracts with potential antimicrobial activity. A crude extract from a culture filtrate of the wood-rotting fungus, Pycnoporus cinnabarinus (Jacq.) P. Karst. (Polyporaceae), was selected for evaluating the functionality of the bioreporter assay in HPLC-based activity profiling. In the bioreporter assay, the IC50 value for the crude extract was 0.10 mg/mL. By integrating the bioreporter assay with HPLC micro-fractionation, the antimicrobial activity was linked to LC-UV peak of a compound in the chromatogram of the extract. This compound was isolated and identified as a fungal pigment phlebiarubrone. DISCUSSION AND CONCLUSION: HPLC-based activity profiling using the bioreporter-based approach is a valuable tool for identifying antimicrobial compound(s) from complex crude extracts, and offers improved sensitivity and speed compared with traditional antimicrobial assays, such as the turbidimetric measurement.

Impact of Procyanidins from Different Berries on Caspase 8 Activation in Colon Cancer.

SCOPE: The aim of this work is to identify which proapoptotic pathway is induced in human colon cancer cell lines, in contact with proanthocyanidins extracted from various berries. METHODS AND RESULTS: Proanthocyanidins (Pcys) extracted from 11 berry species are monitored for proapoptotic activities on two related human colon cancer cell lines: SW480-TRAIL-sensitive and SW620-TRAIL-resistant. Apoptosis induction is monitored by cell surface phosphatidylserine (PS) detection. Lowbush blueberry extract triggers the strongest activity. When tested on the human monocytic cell line THP-1, blueberry Pcys are less effective for PS externalisation and DNA fragmentation is absent, highlighting a specificity of apoptosis induction in gut cells. In Pcys-treated gut cell lines, caspase 8 (apoptosis extrinsic pathway) but not caspase 9 (apoptosis intrinsic pathway) is activated after 3 hours through P38 phosphorylation (90 min), emphasizing the potency of lowbush blueberry Pcys to eradicate gut TRAIL-resistant cancer cells. CONCLUSION: We highlight here that berries Pcys, especially lowbush blueberry Pcys, are of putative interest for nutritional chemoprevention of colorectal cancer in view of their apoptosis induction in a human colorectal cancer cell lines.

Jagunal homolog 1 is a critical regulator of neutrophil function in fungal host defense.

Neutrophils are key innate immune effector cells that are essential to fighting bacterial and fungal pathogens. Here we report that mice carrying a hematopoietic lineage-specific deletion of Jagn1 (encoding Jagunal homolog 1) cannot mount an efficient neutrophil-dependent immune response to the human fungal pathogen Candida albicans. Global glycobiome analysis identified marked alterations in the glycosylation of proteins involved in cell adhesion and cytotoxicity in Jagn1-deficient neutrophils. Functional analysis confirmed marked defects in neutrophil migration in response to Candida albicans infection and impaired formation of cytotoxic granules, as well as defective myeloperoxidase release and killing of Candida albicans. Treatment with granulocyte/macrophage colony-stimulating factor (GM-CSF) protected mutant mice from increased weight loss and accelerated mortality after Candida albicans challenge. Notably, GM-CSF also restored the defective fungicidal activity of bone marrow cells from humans with JAGN1 mutations. These data directly identify Jagn1 (JAGN1 in humans) as a new regulator of neutrophil function in microbial pathogenesis and uncover a potential treatment option for humans.

JAGN1 deficiency causes aberrant myeloid cell homeostasis and congenital neutropenia.

The analysis of individuals with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling the differentiation, maintenance and decay of neutrophils. We identify 9 distinct homozygous mutations in the JAGN1 gene encoding Jagunal homolog 1 in 14 individuals with SCN. JAGN1-mutant granulocytes are characterized by ultrastructural defects, a paucity of granules, aberrant N-glycosylation of multiple proteins and increased incidence of apoptosis. JAGN1 participates in the secretory pathway and is required for granulocyte colony-stimulating factor receptor-mediated signaling. JAGN1 emerges as a factor that is necessary in the differentiation and survival of neutrophils.

Inherited biallelic CSF3R mutations in severe congenital neutropenia.

Severe congenital neutropenia (SCN) is characterized by low numbers of peripheral neutrophil granulocytes and a predisposition to life-threatening bacterial infections. We describe a novel genetic SCN type in 2 unrelated families associated with recessively inherited loss-of-function mutations in CSF3R, encoding the granulocyte colony-stimulating factor (G-CSF) receptor. Family A, with 3 affected children, carried a homozygous missense mutation (NM_000760.3:c.922C>T, NP_000751.1:p.Arg308Cys), which resulted in perturbed N-glycosylation and aberrant localization to the cell surface. Family B, with 1 affected infant, carried compound heterozygous deletions provoking frameshifts and premature stop codons (NM_000760.3:c.948_963del, NP_000751.1:p.Gly316fsTer322 and NM_000760.3:c.1245del, NP_000751.1:p.Gly415fsTer432). Despite peripheral SCN, all patients had morphologic evidence of full myeloid cell maturation in bone marrow. None of the patients responded to treatment with recombinant human G-CSF. Our study highlights the genetic and morphologic SCN variability and provides evidence both for functional importance and redundancy of G-CSF receptor-mediated signaling in human granulopoiesis.

Very early onset inflammatory bowel disease associated with aberrant trafficking of IL-10R1 and cure by T cell replete haploidentical bone marrow transplantation.

PURPOSE: Loss-of-function mutations in IL10 and IL10R cause very early onset inflammatory bowel disease (VEO-IBD). Here, we investigated the molecular pathomechanism of a novel intronic IL10RA mutation and describe a new therapeutic approach of T cell replete haploidentical hematopoietic stem cell transplantation (HSCT). METHODS: Clinical data were collected by chart review. Genotypes of IL10 and IL10R genes were determined by Sanger sequencing. Expression and function of mutated IL-10R1 were assessed by quantitative PCR, Western blot analysis, enzyme-linked immunosorbent assays, confocal microscopy, and flow cytometry. RESULTS: We identified a novel homozygous point mutation in intron 3 of the IL10RA (c.368-10C > G) in three related children with VEO-IBD. Bioinformatical analysis predicted an additional 3' splice site created by the mutation. Quantitative PCR analysis showed normal mRNA expression of mutated IL10RA. Sequencing of the patient's cDNA revealed an insertion of the last nine nucleotides of intron 3 as a result of aberrant splicing. Structure-based modeling suggested misfolding of mutated IL-10R1. Western blot analysis demonstrated a different N-linked glycosylation pattern of mutated protein. Immunofluorescence and FACS analysis revealed impaired expression of mutated IL-10R1 at the plasma membrane. In the absence of HLA-identical donors, T cell replete haploidentical HSCT was successfully performed in two patients. CONCLUSIONS: Our findings expand the spectrum of IL10R mutations in VEO-IBD and emphasize the need for genetic diagnosis of mutations in conserved non-coding sequences of candidate genes. Transplantation of haploidentical stem cells represents a curative therapy in IL-10R-deficient patients, but may be complicated by non-engraftment.

Molecular self-assembly on graphene on SiO2 and h-BN substrates.

One of the suggested ways of controlling the electronic properties of graphene is to establish a periodic potential modulation on it, which could be achieved by self-assembly of ordered molecular lattices. We have studied the self-assembly of cobalt phthalocyanines (CoPc) on chemical vapor deposition (CVD) grown graphene transferred onto silicon dioxide (SiO2) and hexagonal boron nitride (h-BN) substrates. Our scanning tunneling microscopy (STM) experiments show that, on both substrates, CoPc forms a square lattice. However, on SiO2, the domain size is limited by the corrugation of graphene, whereas on h-BN, single domain extends over entire terraces of the underlying h-BN. Additionally, scanning tunneling spectroscopy (STS) measurements suggest that CoPc molecules are doped by the substrate and that the level of doping varies from molecule to molecule. This variation is larger on graphene on SiO2 than on h-BN. These results suggest that graphene on h-BN is an ideal substrate for the study of molecular self-assembly toward controlling the electronic properties of graphene by engineered potential landscapes.

A congenital neutrophil defect syndrome associated with mutations in VPS45.

BACKGROUND: Neutrophils are the predominant phagocytes that provide protection against bacterial and fungal infections. Genetically determined neutrophil disorders confer a predisposition to severe infections and reveal novel mechanisms that control vesicular trafficking, hematopoiesis, and innate immunity. METHODS: We clinically evaluated seven children from five families who had neutropenia, neutrophil dysfunction, bone marrow fibrosis, and nephromegaly. To identify the causative gene, we performed homozygosity mapping using single-nucleotide polymorphism arrays, whole-exome sequencing, immunoblotting, immunofluorescence, electron microscopy, a real-time quantitative polymerase-chain-reaction assay, immunohistochemistry, flow cytometry, fibroblast motility assays, measurements of apoptosis, and zebrafish models. Correction experiments were performed by transfecting mutant fibroblasts with the nonmutated gene. RESULTS: All seven affected children had homozygous mutations (Thr224Asn or Glu238Lys, depending on the child's ethnic origin) in VPS45, which encodes a protein that regulates membrane trafficking through the endosomal system. The level of VPS45 protein was reduced, as were the VPS45 binding partners rabenosyn-5 and syntaxin-16. The level of ß1 integrin was reduced on the surface of VPS45-deficient neutrophils and fibroblasts. VPS45-deficient fibroblasts were characterized by impaired motility and increased apoptosis. A zebrafish model of vps45 deficiency showed a marked paucity of myeloperoxidase-positive cells (i.e., neutrophils). Transfection of patient cells with nonmutated VPS45 corrected the migration defect and decreased apoptosis. CONCLUSIONS: Defective endosomal intracellular protein trafficking due to biallelic mutations in VPS45 underlies a new immunodeficiency syndrome involving impaired neutrophil function. (Funded by the National Human Genome Research Institute and others.).

Cysteine-rich protein 1 is regulated by transforming growth factor-ß1 and expressed in lung fibrosis.

Transforming growth factor-ß (TGF-ß) is a diverse cytokine regulating growth, apoptosis, differentiation, adhesion, invasion, and extracellular matrix production. Dysregulation of TGF-ß is associated with fibrotic disorders and epithelial-mesenchymal transition, and has been linked with idiopathic pulmonary fibrosis (IPF). Cysteine-rich protein 1 (CRP1) is a small LIM-domain containing protein involved in smooth muscle differentiation. Here, we show that TGF-ß1 increases the expression of CRP1 protein and that CRP1 levels increase in a biphasic fashion. A rapid transient (15-45 min) increase in CRP1 is followed by a subsequent, sustained increase in CRP1 a few hours afterwards that lasts several days. We find that TGF-ß1 regulates the expression of CRP1 through Smad and non-conventional p38 MAPK signaling pathways in a transcription-independent manner and that the induction occurs concomitant with an increase in myofibroblast differentiation. Using CRP1 silencing by shRNA, we identify CRP1 as a novel factor mediating cell contractility. Furthermore, we localize CRP1 to fibroblastic foci in IPF lungs and find that CRP1 is significantly more expressed in IPF as compared to control lung tissue. The results show that CRP1 is a novel TGF-ß1 regulated protein that is expressed in fibrotic lesions and may be relevant in the IPF disease.

LIM-domain proteins in transforming growth factor ß-induced epithelial-to-mesenchymal transition and myofibroblast differentiation.

Epithelial to mesenchymal transition (EMT) is a process during which junctions of the cell-cell contacts are dissolved, actin cytoskeleton is deformed, apical-basolateral cell polarity is lost and cell motility is increased. EMT is needed during normal embryonal development and wound healing, but may also lead to pathogenic transformation and formation of myofibroblasts. Transforming growth factor ß (TGFß) is a multifunctional cytokine promoting EMT and myofibroblast differentiation, and its dysregulation is involved in pathological disorders like cancer and fibrosis. Lin11, Isl-1 and Mec-3 (LIM) domain proteins are associated with actin cytoskeleton and linked to regulation of cell growth, damage signaling, cell fate determination and signal transduction. LIM-domain proteins generally do not bind DNA, but are more likely to function via protein-protein interactions. Despite being a disparate group of proteins, similarities in their functions are observed. In this review we will discuss the role of LIM-domain proteins in TGFß-signaling pathway and in EMT-driven processes. LIM-domain proteins regulate TGFß-induced actin cytoskeleton reorganization, motility and adhesion, but also dissolution of cell-cell junctions during EMT. Finally, the role of LIM-domain proteins in myofibroblasts found in fibrotic foci and tumor stroma will be discussed.

Contrasting DNA damage checkpoint responses in epithelium of the human seminal vesicle and prostate.

BACKGROUND: Prostate and seminal vesicle are two similar hormone responsive human organs that differ dramatically in their cancer incidence. DNA damage response (DDR) is required for maintenance of genomic integrity. METHODS: In this study we investigated the DDR and cell cycle checkpoint activation of these organs using orthotopic cultures of human surgery-derived tissues and primary cultures of isolated prostate and seminal vesicle cells. RESULTS: We find that the activation of ATM signaling pathway by ionizing radiation (IR) was comparable in both tissues. Previously, we have shown that the prostate secretory cells express low levels of histone variant H2AX and phosphorylated H2AX (γH2AX) after IR. Here we demonstrate that H2AX levels are low also in the secretory seminal vesicle cells suggesting that this is a common phenotype of postmitotic cells. We consequently established primary epithelial cell cultures from both organs to compare their DDR. Interestingly, contrary to human prostate epithelial cells (HPEC), primary seminal vesicle epithelial cells (HSVEC) displayed effective cell cycle checkpoints after IR and expressed higher levels of Wee1A checkpoint kinase. Furthermore, HSVEC but not HPEC cells were able to activate p53 and to induce p21 cell cycle inhibitor. DISCUSSION: Our results show that during replication, the checkpoint enforcement is more proficient in the seminal vesicle than in the prostate epithelium cells. This indicates a more stringent enforcement of DDR in replicating seminal vesicle epithelial cells, and suggests that epithelial regeneration combined with sub-optimal checkpoint responses may contribute to high frequency of genetic lesions in the prostate epithelium.

Discovery of dual binding site acetylcholinesterase inhibitors identified by pharmacophore modeling and sequential virtual screening techniques.

Dual binding site acetylcholinesterase (AChE) inhibitors are promising for the treatment of Alzheimer's disease (AD). They alleviate the cognitive deficits and AD-modifying agents, by inhibiting the ß-amyloid (Aß) peptide aggregation, through binding to both the catalytic and peripheral anionic sites, the so called dual binding site of the AChE enzyme. In this Letter, chemical features based 3D-pharmacophore models were developed based on the eight potent and structurally diverse AChE inhibitors (I-VIII) obtained from high-throughput in vitro screening technique. The best 3D-pharmacophore model, Hypo1, consists of two hydrogen-bond acceptor lipid, one hydrophobe, and two hydrophobic aliphatic features obtained by Catalyst/HIPHOP algorithm adopted in Discovery studio program. Hypo1 was used as a 3D query in sequential virtual screening study to filter three small compound databases. Further, a total of nine compounds were selected and followed on in vitro analysis. Finally, we identified two leads--Specs1 (IC(50)=3.279 µM) and Spec2 (IC(50)=5.986 µM) dual binding site compounds from Specs database, having good AChE enzyme inhibitory activity.

Potency determinations of acetylcholinesterase inhibitors using Ellman's reaction-based assay in screening: Effect of assay variants.

In primary drug discovery screenings and potency determinations of active acetylcholinesterase (AChE) inhibitors, different variations of the Ellman's reaction-based assay are extensively applied. However, these are prone to produce variable results. Here we studied how assay variants differing in the order of reagent addition and substrate concentrations influence potency measurements of AChE inhibitors. Three model compounds were used: tacrine, physostigmine, and a newly reported inhibitor, 1-[5-[4-[(hexahydro-1H-azepin-1-yl)carbonyl]-1-piperidinyl]-1,3,4-thiadiazol-2-yl]-2-pyrrolidinone. Different patterns of potency changes related to the different inhibition mechanisms of the compounds were detected. Recognizing this, better judgment can be applied when publishing results and selecting optimal screening assays.

Molecular Docking Guided Comparative GFA, G/PLS, SVM and ANN Models of Structurally Diverse Dual Binding Site Acetylcholinesterase Inhibitors.

Recently discovered 42 AChE inhibitors binding at the catalytic and peripheral anionic site were identified on the basis of molecular docking approach, and its comparative quantitative structure-activity relationship (QSAR) models were developed. These structurally diverse inhibitors were obtained by our previously reported high-throughput in vitro screening technique using 384-well plate's assay based on colorimetric method of Ellman. QSAR models were developed using (i) genetic function algorithm, (ii) genetic partial least squares, (iii) support vector machine and (iv) artificial neural network techniques. The QSAR model robustness and significance was critically assessed using different cross-validation techniques on test data set. The generated QSAR models using thermodynamic, electrotopological and electronic descriptors showed that nonlinear methods are more robust than linear methods, and provide insight into the structural features of compounds that are important for AChE inhibition.

Ultraviolet B radiation regulates cysteine-rich protein 1 in human keratinocytes.

BACKGROUND: Cysteine-rich protein 1 (CRP1) is a growth-inhibitory cytoskeletal protein that is induced by ultraviolet (UV) C radiation radiation in fibroblasts. Our aim was to investigate the effects of UV radiation on CRP1 in keratinocytes, the main cell type subjected to UV radiation in the human body. METHODS: The effects of physiologically relevant doses of UVB radiation on CRP1 protein levels were studied in cultured primary keratinocytes and transformed cell lines (HaCaT, A-431) by immunoblotting. UVB-induced keratinocyte apoptosis was assessed by flow cytometry and monitoring caspase activity. Expression of CRP1 in human skin in vivo was studied by immunohistochemistry in samples of normal skin, actinic keratosis (AK) representing UV-damaged skin and squamous cell carcinoma (SCC), a UV-induced skin cancer. RESULTS: CRP1 expression increased by UVB radiation in primary but not in immortalized keratinocytes. Upon high, apoptosis-inducing doses of UV radiation, CRP1 was cleaved in a caspase-dependent manner. In normal skin, CRP1 was expressed in smooth muscle cells, vasculature, sweat glands, sebaceous glands and hair root sheath, but very little CRP1 was present in keratinocytes. CRP1 expression was elevated in basal cells in AK but not in SCC. CONCLUSION: CRP1 expression is regulated by UVB in human keratinocytes, suggesting a role for CRP1 in the phototoxic responses of human skin.