Transcription apparatus of the yeast virus-like elements: Architecture, function, and evolutionary origin.

Extrachromosomal hereditary elements such as organelles, viruses, and plasmids are important for the cell fitness and survival. Their transcription is dependent on host cellular RNA polymerase (RNAP) or intrinsic RNAP encoded by these elements. The yeast Kluyveromyces lactis contains linear cytoplasmic DNA virus-like elements (VLEs, also known as linear plasmids) that bear genes encoding putative non-canonical two-subunit RNAP. Here, we describe the architecture and identify the evolutionary origin of this transcription machinery. We show that the two RNAP subunits interact in vivo, and this complex interacts with another two VLE-encoded proteins, namely the mRNA capping enzyme and a putative helicase. RNAP, mRNA capping enzyme and the helicase also interact with VLE-specific DNA in vivo. Further, we identify a promoter sequence element that causes 5' mRNA polyadenylation of VLE-specific transcripts via RNAP slippage at the transcription initiation site, and structural elements that precede the termination sites. As a result, we present a first model of the yeast virus-like element transcription initiation and intrinsic termination. Finally, we demonstrate that VLE RNAP and its promoters display high similarity to poxviral RNAP and promoters of early poxviral genes, respectively, thereby pointing to their evolutionary origin.

Co-localization of Interleukin-1α and Annexin A2 at the plasma membrane in response to oxidative stress.

Interleukin-1α (IL-1α) and Annexin A2 (AnxA2) are pleiotropic molecules with both intracellular and extracellular roles. They share several characteristics including unconventional secretion aided by S100 proteins, anchoring of the externalized proteins at the outer surface of the plasma membrane and response to oxidative stress. Although IL-1α and AnxA2 have been implicated in a variety of biological processes, including cancer, little is known about the mechanisms of their cellular release. In the present study, employing the non-cancerous breast epithelial MCF10A cells, we demonstrate that IL-1α and AnxA2 establish a close association in response to oxidative stress. Stress conditions lead to translocation of both proteins towards lamellipodia rich in vimentin and association of full-length IL-1α and Tyr23 phosphorylated AnxA2 with the plasma membrane at peripheral sites depleted of F-actin. Notably, membrane-associated IL-1α and AnxA2 preferentially localize to the outer edges of the MCF10A cell islands, suggesting that the two proteins participate in the communication of these epithelial cells with their neighboring cells. Similarly, in U2OS osteosarcoma cell line both endogenous IL-1α and transiently produced IL-1α/EGFP associate with the plasma membrane. While benign MFC10A cells present membrane-associated IL-1α and AnxA2 at the edges of their cell islands, the aggressive cancerous U2OS cells communicate in such manner also with distant cells.

Tristetraprolin affects invasion-associated genes expression and cell motility in triple-negative breast cancer model.

Tristetraprolin (TTP) is an RNA-binding protein that negatively regulates its target mRNAs and has been shown to inhibit tumor progression and invasion. Tumor invasion requires precise regulation of cytoskeletal components, and dysregulation of cytoskeleton-associated genes can significantly alter cell motility and invasive capability. Several genes, including SH3PXD2A, SH3PXD2B, CTTN, WIPF1, and WASL, are crucial components of the cytoskeleton reorganization machinery and are essential for adequate cell motility. These genes are also involved in invasion processes, with SH3PXD2A, SH3PXD2B, WIPF1, and CTTN being key components of invadopodia-specialized structures that facilitate invasion. However, the regulation of these genes is not well understood. This study demonstrates that ectopic expression of TTP in MDA-MB-231 cells leads to decreased mRNA levels of CTTN and SH3PXD2A, as well as defects in cell motility and actin filament organization. Additionally, doxorubicin significantly increases TTP expression and reduces the mRNA levels of cytoskeleton-associated genes, enhancing our understanding of how doxorubicin may affect the transcriptional profile of cells. However, doxorubicin affects target mRNAs differently than TTP ectopic expression, suggesting it may not be the primary mechanism of doxorubicin in breast cancer (BC) treatment. High TTP expression is considered as a positive prognostic marker in multiple cancers, including BC. Given that doxorubicin is a commonly used drug for treating triple-negative BC, using TTP as a prognostic marker in this cohort of patients might be limited since it might be challenging to understand if high TTP expression occurred due to the favorable physiological state of the patient or as a consequence of treatment.

A closed 3D printed microfluidic device for automated growth and differentiation of cerebral organoids from single-cell suspension.

The development of 3D organoids has provided a valuable tool for studying human tissue and organ development in vitro. Cerebral organoids, in particular, offer a unique platform for investigating neural diseases. However, current methods for generating cerebral organoids suffer from limitations such as labor-intensive protocols and high heterogeneity among organoids. To address these challenges, we present a microfluidic device designed to automate and streamline the formation and differentiation of cerebral organoids. The device utilizes microwells with two different shapes to promote the formation of a single aggregate per well and incorporates continuous medium flow for optimal nutrient exchange. In silico simulations supported the effectiveness of the microfluidic chip in replicating cellular microenvironments. Our results demonstrate that the microfluidic chip enables uniform growth of cerebral organoids, significantly reducing the hands-on time required for maintenance. Importantly, the performance of the microfluidic system is comparable to the standard 96-well plate format even when using half the amount of culture medium, and the resulting organoids exhibit substantially developed neuroepithelial buds and cortical structures. This study highlights the potential of custom-designed microfluidic technology in improving the efficiency of cerebral organoid culture.

Photothermal induction of pyroptosis in malignant glioma spheroids using (16-mercaptohexadecyl)trimethylammonium bromide-modified cationic gold nanorods.

Plasmonic photothermal therapy (PPTT) employing plasmonic gold nanorods (GNRs) presents a potent strategy for eradication of tumors including aggressive brain gliomas. Despite its promise, there is a pressing need for a more comprehensive evaluation of PPTT using sophisticated in vitro models that closely resemble tumor tissues, thereby facilitating the elucidation of therapeutic mechanisms. In this study, we exposed 3D glioma spheroids (tumoroids) to (16-mercaptohexadecyl)trimethylammonium bromide-functionalized gold nanorods (MTAB-GNRs) and a near-infrared (NIR) laser. We demonstrate that the photothermal effect can be fine-tuned by adjusting the nanoparticle concentration and laser power. Depending on the selected parameters, the laser can trigger either regulated or non-regulated cell death (necrosis) in both mouse GL261 and human U-87 MG glioma cell lines, accompanied by translocation of phosphatidylserine in the membrane. Our investigation into the mechanism of regulated cell death induced by PPTT revealed an absence of markers associated with classical apoptosis pathways, such as cleaved caspase 3. Instead, we observed the presence of cleaved caspase 1, gasdermin D, and elevated levels of NLRP3 in NIR-irradiated tumoroids, indicating the activation of pyroptosis. This finding correlates with previous observations of lysosomal accumulation of MTAB-GNRs and the known lysosomal pathway of pyroptosis activation. We further confirmed the absence of toxic breakdown products of GNRs using electron microscopy, which showed no melting or fragmentation of gold nanoparticles under the conditions causing regulated cell death. In conclusion, PPTT using coated gold nanorods offers significant potential for glioma cell elimination occurring through the activation of pyroptosis rather than classical apoptosis pathways.

Topological stress triggers persistent DNA lesions in ribosomal DNA with ensuing formation of PML-nucleolar compartment.

PML, a multifunctional protein, is crucial for forming PML-nuclear bodies involved in stress responses. Under specific conditions, PML associates with nucleolar caps formed after RNA polymerase I (RNAPI) inhibition, leading to PML-nucleolar associations (PNAs). This study investigates PNAs-inducing stimuli by exposing cells to various genotoxic stresses. We found that the most potent inducers of PNAs introduced topological stress and inhibited RNAPI. Doxorubicin, the most effective compound, induced double-strand breaks (DSBs) in the rDNA locus. PNAs co-localized with damaged rDNA, segregating it from active nucleoli. Cleaving the rDNA locus with I-PpoI confirmed rDNA damage as a genuine stimulus for PNAs. Inhibition of ATM, ATR kinases, and RAD51 reduced I-PpoI-induced PNAs, highlighting the importance of ATM/ATR-dependent nucleolar cap formation and homologous recombination (HR) in their triggering. I-PpoI-induced PNAs co-localized with rDNA DSBs positive for RPA32-pS33 but deficient in RAD51, indicating resected DNA unable to complete HR repair. Our findings suggest that PNAs form in response to persistent rDNA damage within the nucleolar cap, highlighting the interplay between PML/PNAs and rDNA alterations due to topological stress, RNAPI inhibition, and rDNA DSBs destined for HR. Cells with persistent PNAs undergo senescence, suggesting PNAs help avoid rDNA instability, with implications for tumorigenesis and aging.

Discovery of a 6-Aminobenzo[b]thiophene 1,1-Dioxide Derivative (K2071) with a Signal Transducer and Activator of Transcription 3 Inhibitory, Antimitotic, and Senotherapeutic Activities.

6-Nitrobenzo[b]thiophene 1,1-dioxide (Stattic) is a potent signal transducer and activator of the transcription 3 (STAT3) inhibitor developed originally for anticancer therapy. However, Stattic harbors several STAT3 inhibition-independent biological effects. To improve the properties of Stattic, we prepared a series of analogues derived from 6-aminobenzo[b]thiophene 1,1-dioxide, a compound directly obtained from the reduction of Stattic, that includes a methoxybenzylamino derivative (K2071) with optimized physicochemical characteristics, including the ability to cross the blood-brain barrier. Besides inhibiting the interleukin-6-stimulated activity of STAT3 mediated by tyrosine 705 phosphorylation, K2071 also showed cytotoxicity against a set of human glioblastoma-derived cell lines. In contrast to the core compound, a part of K2071 cytotoxicity reflected a STAT3 inhibition-independent block of mitotic progression in the prophase, affecting mitotic spindle formation, indicating that K2071 also acts as a mitotic poison. Compared to Stattic, K2071 was significantly less thiol-reactive. In addition, K2071 affected cell migration, suppressed cell proliferation in tumor spheroids, exerted cytotoxicity for glioblastoma temozolomide-induced senescent cells, and inhibited the secretion of the proinflammatory cytokine monocyte chemoattractant protein 1 (MCP-1) in senescent cells. Importantly, K2071 was well tolerated in mice, lacking manifestations of acute toxicity. The structure-activity relationship analysis of the K2071 molecule revealed the necessity of the para-substituted methoxyphenyl motif for antimitotic but not overall cytotoxic activity of its derivatives. Altogether, these results indicate that compound K2071 is a novel Stattic-derived STAT3 inhibitor and a mitotic poison with anticancer and senotherapeutic properties that is effective on glioblastoma cells and may be further developed as an agent for glioblastoma therapy.

Graphene Oxide and Polymer Humidity Micro-Sensors Prepared by Carbon Beam Writing.

In this study, novel flexible micro-scale humidity sensors were directly fabricated in graphene oxide (GO) and polyimide (PI) using ion beam writing without any further modifications, and then successfully tested in an atmospheric chamber. Two low fluences (3.75 × 1014 cm-2 and 5.625 × 1014 cm-2) of carbon ions with an energy of 5 MeV were used, and structural changes in the irradiated materials were expected. The shape and structure of prepared micro-sensors were studied using scanning electron microscopy (SEM). The structural and compositional changes in the irradiated area were characterized using micro-Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), Rutherford back-scattering spectroscopy (RBS), energy-dispersive X-ray spectroscopy (EDS), and elastic recoil detection analysis (ERDA) spectroscopy. The sensing performance was tested at a relative humidity (RH) ranging from 5% to 60%, where the electrical conductivity of PI varied by three orders of magnitude, and the electrical capacitance of GO varied in the order of pico-farads. In addition, the PI sensor has proven long-term sensing stability in air. We demonstrated a novel method of ion micro-beam writing to prepare flexible micro-sensors that function over a wide range of humidity and have good sensitivity and great potential for widespread applications.

Frentizole derivatives with mTOR inhibiting and senomorphic properties.

Frentizole is immunosuppressive drug with low acute toxicity and lifespan-prolonging effect. Recently, frentizole´s potential to disrupt toxic amyloid ß (Aß) - Aß-binding alcohol dehydrogenase (ABAD) interaction in mitochondria in Alzheimer´s brains has been revealed. Another broadly studied drug with anti-aging and immunosuppressive properties is an mTOR inhibitor - rapamycin. Since we do not yet precisely know what is behind the lifespan-prolonging effect of rapamycin and frentizole, whether it is the ability to inhibit the mTOR signaling pathway, reduction in mitochondrial toxicity, immunosuppressive effect, or a combination of all of them, we have decided within our previous work to dock the entire in-house library of almost 240 Aß-ABAD modulators into the FKBP-rapamycin-binding (FRB) domain of mTOR in order to interlink mTOR-centric and mitochondrial free radical-centric theories of aging and thus to increase the chances of success. Based on the results of the docking study, molecular dynamic simulation and MM-PBSA calculations, we have selected nine frentizole-like compounds (1 - 9). Subsequently, we have determined their real physical-chemical properties (logP, logD, pKa and solubility in water and buffer), cytotoxic/cytostatic, mTOR inhibitory, and in vitro anti-senescence (senolytic and senomorphic) effects. Finally, the three best candidates (4, 8, and 9) have been forwarded for in vivo safety studies to assess their acute toxicity and pharmacokinetic properties. Based on obtained results, only compound 4 demonstrated the best results within in vitro testing, the ability to cross the blood-brain barrier and the lowest acute toxicity (LD50 in male mice 559 mg/kg; LD50 in female mice 575 mg/kg).

Discovery of small molecule mechanistic target of rapamycin inhibitors as anti-aging and anti-cancer therapeutics.

To date, the most studied drug in anti-aging research is the mTOR inhibitor - rapamycin. Despite its almost perfect anti-aging profile, rapamycin exerts one significant limitation - inappropriate physicochemical properties. Therefore, we have decided to utilize virtual high-throughput screening and fragment-based design in search of novel mTOR inhibiting scaffolds with suitable physicochemical parameters. Seven lead compounds were selected from the list of obtained hits that were commercially available (4, 5, and 7) or their synthesis was feasible (1, 2, 3, and 6) and evaluated in vitro and subsequently in vivo. Of all these substances, only compound 3 demonstrated a significant cytotoxic, senolytic, and senomorphic effect on normal and cancerous cells. Further, it has been confirmed that compound 3 is a direct mTORC1 inhibitor. Last but not least, compound 3 was found to exhibit anti-SASP activity concurrently being relatively safe within the test of in vivo tolerability. All these outstanding results highlight compound 3 as a scaffold worthy of further investigation.

Interface Behavior and Interface Tensile Strength of a Hardened Concrete Mixture with a Coarse Aggregate for Additive Manufacturing.

3D concrete printing technology (3DCP) is a relatively new technology that was first established in the 1990s. The main weakness of the technology is the interface strength between the extruded layers, which are deposited at different time intervals. Consequently, the interface strength is assumed to vary in relation to the time of concrete casting. The proposed experimental study investigated the behavior of a hardened concrete mixture containing coarse aggregates that were up to 8 mm in size, which is rather unusual for 3DCP technology. The resulting direct tensile strength at the layer interface was investigated for various time intervals of deposition from the initial mixing of concrete components. To better understand the material behavior at the layer interface area, computed tomography (CT) scanning was conducted, where the volumetric and area analysis enabled validation of the pore size and count distribution in accordance with the layer deposition process. The analyzed CT data related the macroscopic anisotropy and the resulting crack pattern to the temporal and spatial variability that is inherent to the additive manufacturing process at construction scales while providing additional insights into the porosity formation during the extrusion of the cementitious composite. The observed results contribute to previous investigations in this field by demonstrating the causal relationships, namely, how the interface strength development is determined by time, deposition process, and pore size distribution. Moreover, in regard to the printability of the proposed coarse aggregate mixture, the specific time interval is presented and its interplay with interface roughness and porosity is discussed.

Differential effects of serine proteases on the migration of normal and tumor cells: implications for tumor microenvironment.

The supporting role of proteases in tumor progression and invasion is well known; however, the use of proteases as therapeutic agents has also been demonstrated. In this article, the authors report on the differential effects of exogenous serine proteases on the motility of tumor and normal cells. The treatment of normal and tumor cells with a single dose of pancreatic serine proteases, trypsin (TR) and chymotrypsin (CH), leads to a concentration-dependent response by cells, first accelerating and then slowing mobility. Tumor cells are 10 to 20 times more sensitive to exogenous TR/CH, suggesting that a single dose of proteases may cause discordant movements of normal and tumor cells within the tumor environment. The inhibitory effects of TR on cell motility are contradicted by thrombin (TH), particularly in the regulation of normal cells' migration. The purpose of this investigation was to ascertain the role of protease-activated receptors (PARs) in terms of normal and tumor cell motility. Duplicate treatments with proteases resulted in diminished mobility of both normal and tumor cells. Repeated application of TR and TH in 1-hour treatment intervals initially desensitizes cell surface PARs. However, cell surface PARs reappear regardless of subsequent protease treatments in both normal and tumor cells. The resensitization process is retarded in tumor cells when compared with normal cells. This is evidenced by lower expression of PARs as well as by their relocalization at the tumor cell surfaces. Under these conditions, normal cells remain responsive to exogenous proteases in terms of cell motility. Exogenous proteases do not modulate motility of repeatedly stimulated tumor cells, and consequently, the migration of tumor cells appears disconnected from the PAR signaling pathways. The use of activating peptides in lieu of the cognate proteases for a given PAR system indicated that proteases may act through additional targets not regulated by PAR signaling. We hypothesize that the divergent migration patterns of normal and tumor cells due to exposure to proteases is in part mediated by PARs. Thus, treatment with exogenous proteases may cause rearrangement of the tumor and stromal cells within the tumor microenvironment. Such topographical effects may lead to the inhibition of tumor progression and metastasis development.

Proenzyme therapy of cancer.

Proteases and their inhibitors have long been investigated in numerous tumor systems, and at the tumor growing front, their balance has been universally found to be shifted towards higher proteolytic activities. However, out of many promising serine and metalloproteinase inhibitors, none are included in cancer treatment regimens at present. The current search for active antiproteolytic compounds is in contrast to the classical approach developed by John Beard, who suggested treating advanced cancer by fresh pancreatic extracts whose antitumor activity was based on their proteolytic potential. We followed John Beard's recommendations by using purified pancreatic proenzymes/enzymes, trypsinogen/trypsin (TG/TR), chymotrypsinogen/chymotrypsin (CG/CH) and amylase (AM). The mixture of these enzymatic activities produces potent antimetastatic and antitumor effects in cellular, animal and human systems. The treatment of cultured tumor cells with TR and CH at nanomolar [corrected] concentrations, comparable to those achieved in the blood of the patients, causes complete arrest of the directional movement of metastatic cells. Conversely, the same treatment of normal cells results in enhanced motility and an accelerated closure of the gap created in cell monolayers. Further, treatment of cells with serine proteases results in the formation of cellular 3-dimensional structures such as lamellae, cell streams and aggregates. In some cell types, the aggregates are compacted via cadherin-based cell-cell communication systems and form compact spheroids. In the highly metastatic cells with lower cadherin expression, the ability to form spheroids also diminishes. Tumor cells unable to form spheroids when treated with proteases are subject to elimination by apoptosis. In contrast, a large proportion of cells that form spheroids remain viable, although they are metabolically suppressed. Protease-treated tumor cells contain a disrupted actin cytoskeleton and exhibit a loss of front-to-back polarity. We hypothesize that the provision of zymogens, rather than the enzymes, was of crucial importance to the clinical effectiveness in the human trials conducted by Beard and his co-workers. The precursor nature of the active enzymes may offer protection against numerous serpins present in the tissues and blood. Experimental evidence supports the assertion that the conversion from proenzyme to enzyme occurs selectively on the surface of the tumor cells, but not on normal cells. We believe that this selectivity of activation is responsible for the antitumor/antimetastatic effect of proenzyme therapy and low toxicity to normal cells or tumor host. Elevated levels of endostatin and angiostatin appear in the blood of TG/CG/AM-treated tumor-bearing mice, but not in tumor mice treated with the vehicle alone or in proenzyme-treated tumor-free mice. These findings support the conclusion that proteolysis is the active mechanism of the proenzyme treatment. Future studies will focus on the molecular mechanisms of the proenzyme therapy including the identification of molecular target(s) on the tumor cells. In conclusion, we have discovered that proenzyme therapy, mandated first by John Beard nearly one hundred years ago, shows remarkable selective effects that result in growth inhibition of tumor cells with metastatic potential.

Transformation of MC3T3-E1 cells following stress and transfection with pSV2neo plasmid.

A continuous cell line, MC3T3-E1 cells, originally derived from murine calvaria bones, loses its osteogenic properties as a result of extended passage number under stress conditions. These aged/stressed MC3T3-S cells, although nontumorigenic, do not display some of the osteogenic properties characteristic of the MC3T3-E1 cells. Altered properties include low expression of alkaline phosphatase, diminished collagen synthesis and inability to form mineralized nodules in vitro. We attempted to reactivate these osteogenic properties by transfections with a pSV2neo plasmid containing the TGFbeta1 gene. During these experiments we found that transfected MC3T3-S cells not only acquired high alkaline phosphatase activity and a potent mineralization potential, but also properties akin to the transformed state, such as ability to grow in soft agar and ability to produce tumors in immunodeficient animals. Further analysis showed that the TGFbeta1 gene is not required and that the changes can be introduced by transfections with pSV2neo alone. In contrast, MC3T3-S cells transfected with pcDNA3 (a plasmid containing only the SV40 origin of replication, early promoter, enhancer and polyadenylation signals) or mock-transfected MC3T3-S cells did not show any transformation traits. The results identify two additional SV40 fragments present in pSV2neo (SV40 virus sequence; Genbank accession number: NC_001669: 4100-4191 and 2668-2774) as functional elements contributing to the transformation of aged/stressed and immortalized osteoblastic cells. These findings are analogous to earlier reports describing the cell modifying potential of pSV2neo. We conclude that stressed and aged MC3T3-S can be transformed by transfection with pSV2neo and that such cells acquire not only the tumorigenic potential but exhibit also some of the osteogenic properties characteristic of the parent MC3T3-E1 cells.

N-terminal domain of nuclear IL-1α shows structural similarity to the C-terminal domain of Snf1 and binds to the HAT/core module of the SAGA complex.

Interleukin-1α (IL-1α) is a proinflammatory cytokine and a key player in host immune responses in higher eukaryotes. IL-1α has pleiotropic effects on a wide range of cell types, and it has been extensively studied for its ability to contribute to various autoimmune and inflammation-linked disorders, including rheumatoid arthritis, Alzheimer's disease, systemic sclerosis and cardiovascular disorders. Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes. Despite the importance of IL-1α, little is known regarding its binding targets and functions in the nucleus. We took advantage of the histone acetyltransferase (HAT) complexes being evolutionarily conserved from yeast to humans and the yeast SAGA complex serving as an epitome of the eukaryotic HAT complexes. Using gene knock-out technique and co-immunoprecipitation of the IL-1α precursor with TAP-tagged subunits of the yeast HAT complexes, we mapped the IL-1α-binding site to the HAT/Core module of the SAGA complex. We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively. This finding is further supported with the ability of the IL-1α precursor to partially rescue growth defects of snf1Δ yeast strains on media containing 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of His3. Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly.

Constitutive and ligand-induced nuclear localization of oxytocin receptor.

Oxytocin receptor (OTR) is a membrane protein known to mediate oxytocin (OT) effects, in both normal and neoplastic cells. We report here that human osteosarcoma (U2OS, MG63, OS15 and SaOS2), breast cancer (MCF7), and primary human fibroblastic cells (HFF) all exhibit OTR not only on the cell membrane, but also in the various nuclear compartments including the nucleolus. Both an OTR-GFP fusion protein and the native OTR appear to be localized to the nucleus as detected by transfection and/or confocal immunofluorescence, respectively. Treatment with oxytocin causes internalization of OTR and the resulting vesicles accumulate in the vicinity of the nucleus and some of the perinuclear OTR enters the nucleus. Western blots indicate that OTR in the nucleus and on the plasma membrane are likely to be the same biochemical and immunological entities. It appears that OTR is first visible in the nucleoli and subsequently disperses within the nucleus into 4-20 spots while some of the OTR diffuses throughout the nucleoplasm. The behaviour and kinetics of OTR-GFP and OTR are different, indicating interference by GFP in both OTR entrance into the nucleus and subsequent relocalization of OTR within the nucleus. There are important differences among the tested cells, such as the requirement of a ligand for transfer of OTR in nuclei. A constitutive internalization of OTR was found only in osteosarcoma cells, while the nuclear localization in all other tested cells was dependent on ligand binding. The amount of OTR-positive material within and in the vicinity of the nucleus increased following a treatment with oxytocin in both constitutive and ligand-dependent type of cells. The evidence of OTR compartmentalization at the cell nucleus (either ligand-dependent or constitutive) in different cell types suggests still unknown biological functions of this protein or its ligand and adds this G-protein-coupled receptor to other heptahelical receptors displaying this atypical and unexpected nuclear localization.