[Perspectives of genome editing in otorhinolaryngology]. / Perspektiven der Genomeditierung in der Hals-Nasen-Ohren-Heilkunde.

BACKGROUND: Recent advances in DNA sequencing technology have enabled researchers to identify the genetic background underlying human illness. In addition, the latest genome editing technology, CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9), provides great potential to edit genomic DNA sequences precisely with high efficiency. This technology has been evaluated for treatment of genetic diseases in recently published preclinical studies. Since many such genetic disorders can affect functional structures in the head and neck area, the technology bears high therapeutic potential in otorhinolaryngology. OBJECTIVE: In this article, we summarize the concept of CRISPR-Cas9-based therapies, recent achievements in preclinical applications, and future challenges for the implementation of this technology in otolaryngology. MATERIALS AND METHODS: Genetic targeting strategies were analyzed or established using genome sequencing data derived from online databases and literature. RESULTS: Recent research on animal models has shown that genome editing can be used to treat genetic diseases by specifically targeting mutant genomic loci. For example, one preclinical study in the field of otolaryngology has demonstrated that inherited autosomal dominant deafness in mice can be treated using CRISPR-Cas9. Moreover, the same strategies can be used to establish applications for the treatment of head and neck cancer. The greatest challenge appears to be establishment of a system for the safe and efficient delivery of therapeutic nucleotides in clinics. CONCLUSIONS: In theory, genome editing could be used in otolaryngology to target disease-causing genomic loci specifically. However, various challenges have to be overcome until applications can be used clinically.

Efficient animal-serum free 3D cultivation method for adult human neural crest-derived stem cell therapeutics.

Due to their broad differentiation potential and their persistence into adulthood, human neural crest-derived stem cells (NCSCs) harbour great potential for autologous cellular therapies, which include the treatment of neurodegenerative diseases and replacement of complex tissues containing various cell types, as in the case of musculoskeletal injuries. The use of serum-free approaches often results in insufficient proliferation of stem cells and foetal calf serum implicates the use of xenogenic medium components. Thus, there is much need for alternative cultivation strategies. In this study we describe for the first time a novel, human blood plasma based semi-solid medium for cultivation of human NCSCs. We cultivated human neural crest-derived inferior turbinate stem cells (ITSCs) within a blood plasma matrix, where they revealed higher proliferation rates compared to a standard serum-free approach. Three-dimensionality of the matrix was investigated using helium ion microscopy. ITSCs grew within the matrix as revealed by laser scanning microscopy. Genetic stability and maintenance of stemness characteristics were assured in 3D cultivated ITSCs, as demonstrated by unchanged expression profile and the capability for self-renewal. ITSCs pre-cultivated in the 3D matrix differentiated efficiently into ectodermal and mesodermal cell types, particularly including osteogenic cell types. Furthermore, ITSCs cultivated as described here could be easily infected with lentiviruses directly in substrate for potential tracing or gene therapeutic approaches. Taken together, the use of human blood plasma as an additive for a completely defined medium points towards a personalisable and autologous cultivation of human neural crest-derived stem cells under clinical grade conditions.

Morphological characterization of periodontium-derived human stem cells.

The aim of this study has been to characterize adult human somatic periodontium-derived stem cells (PDSCS) isolated from human periodontium and to follow their differentiation after cell culture. PDSCS were isolated from human periodontal tissue and cultured as spheres in serum-free medium. After 10 days the primary spheres were dissociated and the secondary spheres sub-cultured for another 1-2 weeks. Cells from different time points were analyzed, and immunohistochemical and electron microscopic investigations carried out. Histological analysis showed differentiation of spheres deriving from the PDSCS with central production of extracellular matrix beginning 3 days after sub-culturing. Isolated PDSCS developed pseudopodia which contained actin. Tubulin was found in the central portion of the cells. Pseudopodia between different cells anastomosed, indicating intercellular transport. Immunostaining for osteopontin demonstrated a positive reaction in primary spheres and within extracellular matrix vesicles after sub-culturing. In cell culture under serum-free conditions human PDSCS form spheres which are capable of producing extracellular matrix. Further investigations have do be carried out to investigate the capability of these cells to differentiate into osteogenic progenitor cells.

Automation and validation of a rapid method to assess neutrophil and monocyte activation by routine fluorescence flow cytometry in vitro.

The aim of the present study was to design an automated-gating hematology fluorescence flow cytometry methodology permitting the assessment of neutrophil and monocyte activation in EDTA-anticoagulated whole blood based on cell granularity, lipid membrane components, cell shape and volume, and total cell nucleic acid (NA) compounds. For particularly monitoring the proper functioning of patients' innate immune system as the first line defense against microbial invaders, the suitable test system should be rapid, simple, reliable by yielding reproducible results. It must be validated against established methods, and it must prove to work in selected clinical settings, e.g. in intensive care unit (ICU) environments. The adaptation of a routine hematology cell analyser utilizing fluorescence flow cytometry resulted in a potentially useful system for all requirements. It proved to detect in real-time and in a reliable and reproducible way the main cellular response reactions of neutrophils and monocytes during externally stimulated immune defense. Validation was successful when comparing it to established methods. The quantified activation effects were dose dependent from the applied activating agents. Cellular response kinetics could be measured and described and showed to be in line with the prevailing cell response models. Upon applying the test method to a healthy population of volunteers and a first cohort of ICU patients with and without evident immune depression, the test revealed excellent cellular responses to external activating cytotoxic stimuli (lipopolysaccharide; LPS) for the control group, slightly weaker response from ICU patients without immune depression and no response from patients with evident immune depression.We conclude that routine hematology fluorescence flow cytometry can accurately and reproducibly measure different activation steps of monocytes and polymorphonuclear neutrophilic granulocytes to defined external stimuli. This may potentially be applied as a STAT (Latin statim = immediately) and routine screening and surveillance method for inflammatory diseases.

Neural stem cells, inflammation and NF-kappaB: basic principle of maintenance and repair or origin of brain tumours?

Several recent reports suggest that inflammatory signals play a decisive role in the self-renewal, migration and differentiation of multipotent neural stem cells (NSCs). NSCs are believed to be able to ameliorate the symptoms of several brain pathologies through proliferation, migration into the area of the lesion and either differentiation into the appropriate cell type or secretion of anti-inflammatory cytokines. Although NSCs have beneficial roles, current evidence indicates that brain tumours, such as astrogliomas or ependymomas are also caused by tumour-initiating cells with stem-like properties. However, little is known about the cellular and molecular processes potentially generating tumours from NSCs. Most pro-inflammatory conditions are considered to activate the transcription factor NF-kappaB in various cell types. Strong inductive effects of NF-kappaB on proliferation and migration of NSCs have been described. Moreover, NF-kappaB is constitutively active in most tumour cells described so far. Chronic inflammation is also known to initiate cancer. Thus, NF-kappaB might provide a novel mechanistic link between chronic inflammation, stem cells and cancer. This review discusses the apparently ambivalent role of NF-kappaB: physiological maintenance and repair of the brain via NSCs, and a potential role in tumour initiation. Furthermore, it reveals a possible mechanism of brain tumour formation based on inflammation and NF-kappaB activity in NSCs.

Mathematical model for NF-kappaB-driven proliferation of adult neural stem cells.

Neural stem cells (NSCs) are early precursors of neuronal and glial cells. NSCs are capable of generating identical progeny through virtually unlimited numbers of cell divisions (cell proliferation), producing daughter cells committed to differentiation. Nuclear factor kappa B (NF-kappaB) is an inducible, ubiquitous transcription factor also expressed in neurones, glia and neural stem cells. Recently, several pieces of evidence have been provided for a central role of NF-kappaB in NSC proliferation control. Here, we propose a novel mathematical model for NF-kappaB-driven proliferation of NSCs. We have been able to reconstruct the molecular pathway of activation and inactivation of NF-kappaB and its influence on cell proliferation by a system of nonlinear ordinary differential equations. Then we use a combination of analytical and numerical techniques to study the model dynamics. The results obtained are illustrated by computer simulations and are, in general, in accordance with biological findings reported by several independent laboratories. The model is able to both explain and predict experimental data. Understanding of proliferation mechanisms in NSCs may provide a novel outlook in both potential use in therapeutic approaches, and basic research as well.

Nuclear Factor-kappaB controls the reaggregation of 3D neurosphere cultures in vitro.

The approach of reaggregation involves the regeneration and self-renewal of histotypical 3D spheres from isolated tissue kept in suspension culture. Reaggregated spheres can be used as tumour, genetic, biohybrid and neurosphere models. In addition the functional superiority of 3D aggregates over conventional 2D cultures developed the use of neurospheres for brain engineering of CNS diseases. Thus 3D aggregate cultures created enormous interest in mechanisms that regulate the formation of multicellular aggregates in vitro. Here we analyzed mechanisms guiding the development of 3D neurosphere cultures. Adult neural stem cells can be cultured as self-adherent clusters, called neurospheres. Neurospheres are characterised as heterogeneous clusters containing unequal stem cell sub-types. Tumour necrosis factor-alpha (TNF-alpha is one of the crucial inflammatory cytokines with multiple actions on several cell types. TNF-alpha strongly activates the canonical Nuclear Factor Kappa-B (NF- kappaB) pathway. In order to investigate further functions of TNF in neural stem cells (NSCs) we tested the hypothesis that TNF is able to modulate the motility and/or migratory behaviour of SVZ derived adult neural stem cells. We observed a significantly faster sphere formation in TNF treated cultures than in untreated controls. The very fast aggregation of isolated NSCs (<2h) is a commonly observed phenomenon, though the mechanisms of 3D neurosphere formation remain largely unclear. Here we demonstrate for the first time, increased aggregation and enhanced motility of isolated NSCs in response to the TNF-stimulus. Moreover, this phenomenon is largely dependent on activated transcription factor NF-kappaB. Both, the pharmacological blockade of NF-kappaB pathway by pyrrolidine dithiocarbamate (PDTC) or Bay11-7082 and genetic blockade by expression of a transdominant-negative super-repressor IkappaB-AA1 led to decreased aggregation.

Continuous interleukin-6 application in vivo via macroencapsulation of interleukin-6-expressing COS-7 cells induces massive gliosis.

The inflammatory cytokine interleukin-6 (IL-6) was found in senile plaques of Alzheimer's patients and might be involved in the pathology of Parkinson's disease and multiple sclerosis. Interestingly, an astocytosis is also found in these neurodegenerative disorders. To evaluate the direct effects of IL-6 in vivo on glial cells, we created a new in vivo model. IL-6 and mock-transfected (control group) COS-7 cells were encapsulated in a poly-acryl-nitril membrane for implantation into the rat striatum. Afterward, the host immune reaction to the membrane without encapsulated cells and the biological action of IL-6-producing capsules was evaluated. Animals with an implanted membrane without cells showed a moderate astrocytosis 5 days after the operation. Furthermore, microglia and T-cells could be detected and after 30 days the astrocytosis decreased to a small layer around the membrane. In comparison to the control group, which received a sham operation, our results demonstrate that the response of glial cells is caused by the mechanical damage of the surgical procedure itself rather than due to the introduced membrane material. In contrast, we found a massive proliferation and activation of astrocytes and microglia after 10 days by IL-6-secreting capsules, indicating that IL-6 is involved in the induction of gliosis. Control animals that received encapsulated mock-transfected COS-7 cells showed only a weak response. These data point to an involvement of IL-6 in the proliferation and activation of glial cells as seen in neurodegenerative disorders.

DNA array analysis of the developing rat cerebellum: transforming growth factor-beta2 inhibits constitutively activated NF-kappaB in granule neurons.

The nuclear factor-kappaB (NF-kappaB) pathway is important in neuronal survival and in integration of external signals e.g. cytokines, glutamate, Abeta and nerve growth factor (NGF). During rat cerebellar development NF-kappaB activity is high in granule cells before postnatal day 7 (P7) and declines after P7. Using gene expression profiles, measured by cDNA arrays, up-regulation of transforming growth factor-beta2 (TGF-beta2) was correlated with the developmental down-regulation of NF-kappaB. TGF-beta2 depicted strongest, more than 4-fold, up-regulation in P12 versus P4 cerebella. In situ hybridization and immunohistochemistry confined upregulated TGF-beta2 to granule cells and correlated mRNA and TGF-beta2-protein increase. Finally TGF-beta2 repressed NF-kappaB activity, in an in vitro system resembling migrating cerebellar granule cells. Thus, TGF-beta might fulfill an important role in repressing developmentally activated NF-kappaB in granule neurons.

Retrograde transport of transcription factor NF-kappa B in living neurons.

The mechanism by which signals such as those produced by glutamate are transferred to the nucleus may involve direct transport of an activated transcription factor to trigger long-term transcriptional changes. Ionotropic glutamate receptor activation or depolarization activates transcription factor NF-kappaB and leads to translocation of NF-kappaB from the cytoplasm to the nucleus. We investigated the dynamics of NF-kappaB translocation in living neurons by tracing the NF-kappaB subunit RelA (p65) with jellyfish green fluorescent protein. We found that green fluorescent protein-RelA was located in either the nucleus or cytoplasm and neurites, depending on the coexpression of the cognate inhibitor of NF-kappaB, IkappaB-alpha. Stimulation with glutamate, kainate, or potassium chloride resulted in a redistribution of NF-kappaB from neurites to the nucleus. This transport depended on an intact nuclear localization signal on RelA. Thus, in addition to its role as a transcription factor, NF-kappaB may be a signal transducer, transmitting transient glutamatergic signals from distant sites to the nucleus.

The pro- or anti-apoptotic function of NF-kappaB is determined by the nature of the apoptotic stimulus.

To test whether the behaviour of transcription factor NF-kappaB as a promoter or antagonist of apoptosis depends on the apoptotic stimulus, we determined the influence of NF-kappaB on cell killing elicited by a variety of inducers within a given cell type. Inhibition of NF-kappaB by genetic and pharmacological approaches rendered HeLa cells more susceptible to TNF-alpha-induced cell killing, but protected them almost completely from H2O2- and pervanadate-induced apoptosis. TNF-alpha was unable to protect HeLa from H2O2- and pervanadate-induced apoptosis and further enhanced the cytotoxicity induced by these two adverse agents. Supernatants from HeLa cells stably overexpressing a transdominant negative form of IkappaB-alpha selectively increased the cytotoxicity of TNF-alpha for HeLa cells, suggesting that the enhanced susceptibility of these cells can be attributed to one or more secretable factors. Supershift experiments showed that the various apoptotic stimuli induced the same subset of DNA-binding subunits. Therefore, the nature of the signals elicited by the respective death inducers determines whether NF-kappaB induction leads to apoptosis or survival, suggesting that the manipulation of NF-kappaB activity may provide a new approach to adjuvant therapy in cancer treatment.

Ultrastructural localization of activated NF-kappaB in granule cells of the rat fascia dentata.

The distribution of activated NF-kappaB (p65) was studied in granule cells of the rat fascia dentata using confocal laser scanning microscopy and preembedding immunogold electron microscopy. Activated NF-kappaB, detected with a monoclonal antibody specific for the nuclear localization signal-epitope, was found in proximal dendrites, somata, and nuclei but not in axon terminals of granule cells. Within the nuclei of granule cells, clusters of NF-kappaB immunoreactivity were detected. These data are in line with the proposed function of NF-kappaB as a retrograde signal transducer which transports information from synaptic sites to the nucleus to initiate gene expression.

Constitutive NF-kappa B activity is modulated via neuron-astroglia interaction.

NF-kappa B is found in many neuronal cell types in different states of activity. This study aimed to define which conditions induce constitutive NF-kappa B activity in cultured hippocampal neurons using activity-specific antibody staining. In co-culture with astroglia, hippocampal neurons were devoid of activated NF-kappa B. In these co-cultures, NF-kappa B could not be activated via kainate or glutamate. In contrast, separating neurons from the glial compartment resulted in a time-dependent increase of activated neuronal NF-kappa B. In this line, activation of NF-kappa B by kainate or glutamate is very effective in freshly separated cultures, but inhibited when the cultures are reassembled after stimulation. These findings suggests that a neuronal-glial interaction may regulate gene expression via NF-kappa B.

Optimized protocol for biolistic transfection of brain slices and dissociated cultured neurons with a hand-held gene gun.

DNA-transfer into postmitotic neurons or neuronal tissues has been a major problem in neurobiology. For this aim different methods have been used such as viral infection, microinjection, lipofection or calcium phosphate precipitation. However, using these techniques, very poor transfection efficiency was achieved except for virus-mediated gene transfer. Though viral infections are very efficient, this method is expensive and labor-intensive, especially when recombination is used to prepare viral vectors. Biolistic gene transfer of neurons represents another promising transfection technique. This technique was originally used to transfect plant cells and has been further developed for gene transfer into neurons or neuronal tissues. Up to now, only a few reports are available where successful biolistic gene transfer into neurons or neuronal tissues could be shown. Transfection efficiencies were only about 2%. Most of the previously published experiments were carried out under vacuum conditions using in-chamber gene gun types. Here we describe an improved method for efficient neuronal cell transfection using a hand-held gene gun. Expression vectors could be successfully transferred into dissociated cultured hippocampal neurons, PC12 cells, cultured cerebellar granule cells and cerebellar brain slices. In cerebellar granule cells and hippocampal neurons, transfection efficiencies of about 10% were reached.

Inhibition of NF-kappaB potentiates amyloid beta-mediated neuronal apoptosis.

One mechanism leading to neurodegeneration during Alzheimer's disease (AD) is amyloid beta peptide (Abeta) neurotoxicity. Abeta elicits in cultured central nervous system neurons a biphasic response: a low-dose neurotrophic response and a high-dose neurotoxic response. Previously we reported that NF-kappaB is activated by low doses of Abeta only. Here we show that NF-kappaB activation leads to neuroprotection. In primary neurons we found that a pretreatment with 0.1 microM Abeta-(1-40) protects against neuronal death induced with 10 microM Abeta-(1-40). As a known neuroprotective agent we next analyzed the effect of tumor necrosis factor alpha (TNF-alpha). Maximal activation of NF-kappaB was found with 2 ng/ml TNF-alpha. Pretreatment with TNF-alpha protected cerebellar granule cells from cell death induced by 10 microM Abeta-(1-40). This protection is described by an inverted U-shaped dose response and is maximal with a NF-kappaB-activating dose. The molecular specificity of this protective effect was analyzed by specific blockade of NF-kappaB activation. Overexpression of a transdominant negative IkappaB-alpha blocks NF-kappaB activation and potentiates Abeta-mediated neuronal apoptosis. Our findings show that activation of NF-kappaB is the underlying mechanism of the neuroprotective effect of low-dose Abeta and TNF-alpha. In accordance with these in vitro data we find that nuclear NF-kappaB immunoreactivity around various plaque stages of AD patients is reduced in comparison to age-matched controls. Taken together these data suggest that pharmacological NF-kappaB activation may be a useful approach in the treatment of AD and related neurodegenerative disorders.

Repression of NF-kappaB impairs HeLa cell proliferation by functional interference with cell cycle checkpoint regulators.

NF-kappaB is an inducible transcription factor, which is regulated by interaction with inhibitory IkappaB proteins. Previous studies linked the activity of NF-kappaB to the proliferative state of the cell. Here we have analysed the function of NF-kappaB in the cell cycle. Inhibition of NF-kappaB in HeLa cells by stable overexpression of a transdominant negative IkappaB-alpha protein reduced cell growth. A kinetic analysis of the cell cycle revealed a retarded G1/S transition. The IkappaB-alpha overexpressing cell clones showed a decreased percentage of cells in the S phase and an impaired incorporation of bromodeoxyuridine (BrdU). The amounts of cyclins A, B1, D1, D3, and E were unchanged, but the G1-specific proteins cyclin D2 and cdk2 were strongly elevated in the IkappaB-alpha overexpressing cell clones. These cell clones also displayed an increase in cyclin D1-dependent kinase activity, pointing to a cell cycle arrest at the late G1 phase. IkappaB-alpha overexpression crosstalked to cell cycle checkpoints via a reduction of transcription factor p53 and elevation of p21WAF. Surprisingly, the IkappaB-alpha overexpressing cells showed an enrichment of c-Myc in the nucleoli, although the total amount of c-Myc protein was unchanged. These experiments identify an important contribution of the NF-kappaB/IkappaB system for the growth of HeLa cells.

Improved protocol for a microsphere-adhesion assay on living tissue slices.

Here, we describe a simple microsphere-adhesion assay to characterize adhesive cues on living tissue slices, which we use to study pattern formation in neural tissue. This assay was developed by modifying a cell-adhesion assay on living tissue slices. We replaced dissociated cells by fluorescent microspheres and then coated the microspheres with isolated membranes from these cells. The membrane-coated microspheres were seeded on living tissue slices, and after a short incubation time, nonadherent microspheres were eliminated by washing. Then, the tissue slices with the adherent microspheres were analyzed using epifluorescence microscopy. As an example, it is shown that membrane-coated adherent microspheres were found to be distributed in a characteristic pattern on living slices of hippocampus, mimicking the adhesion pattern of dissociated living cells. The adhesion assay should be suitable to detect and to analyze adhesive cues on living slices of different tissues and, thus, might have numerous applications in tissue research and developmental studies. Here, we describe and discuss a detailed and improved protocol of the microsphere-adhesion assay.

Activation of NF-kappa B by reactive oxygen intermediates in the nervous system.

Nuclear factor kappa B (NF-kappa B) is a transcription factor crucially involved in glial and neuronal function. NF-kappa B is ubiquitously distributed within the nervous system, and its inducible activity can be discerned from constitutive activity. Prototypic inducible NF-kappa B in the nervous system is composed of the DNA-binding subunits p50 and p65 complexed with an inhibitory I kappa B-alpha molecule. A number of signals from the cell surface can lead to rapid activation of NK-kappa B, thus releasing the inhibition by I kappa B. This activates translocation of NF-kappa B to the nucleus, where it binds to kappa B motifs of target genes and activates transcription. Previous findings have identified reactive oxygen intermediates (ROI) as a common denominator of NF-kappa B activating signals. More specifically, hydrogen peroxide (H2O2) might be used as second messenger in the NF-kappa B system, despite its cytotoxicity. Analysis of pathways leading to NF-kappa B activation in the nervous system has identified a number of ROI-dependent pathways such as cytokine- and neurotrophin-mediated activation, glutamatergic signal transduction, and various diseases with crucial ROI involvement (e.g., Alzheimer's disease, Parkinson's disease, experimental autoimmune encephalomyelitis, multiple sclerosis, amyotrophic lateral sclerosis, and injury). A number of NF-kappa B-specific target genes contribute to the production of ROI or are involved in detoxification of ROIs. In this review, possible mechanisms and regulatory pathways of ROI-mediated NF-kappa B activation are discussed.

Lamina-specific cell adhesion on living slices of hippocampus.

Laminar distribution of fiber systems is a characteristic feature of hippocampal organization. Ingrowing afferents, e.g. the fibers from the entorhinal cortex, terminate in specific layers, which implies the existence of laminar recognition cues. To identify cues that are involved in the laminar segregation of fiber systems in the hippocampus, we used an in vitro assay to study the adhesion of dissociated entorhinal cells on living hippocampal slices. Here we demonstrate that dissociated entorhinal cells adhere to living hippocampal slices with a lamina-specific distribution that reflects the innervation pattern of the entorhino-hippocampal projection. In contrast, laminae which are not invaded by entorhinal fibers are a poor substrate for cell adhesion. Lamina-specific cell adhesion does not require the neural cell adhesion molecule or the extracellular matrix glycoprotein reelin, as revealed in studies with mutants. However, the pattern of adhesive cues in the reeler mouse hippocampus mimics characteristic alterations of the entorhinal projection in this mutant, suggesting a role of layer-specific adhesive cues in the pathfinding of entorhinal fibers. Lamina-specific cell adhesion is independent of divalent cations, is abolished after cryofixation or paraformaldehyde fixation and is recognized across species. By using a novel membrane adhesion assay, we show that lamina-specific cell adhesion can be mimicked by membrane-coated fluorescent microspheres. Recognition of the adhesive properties of different hippocampal laminae by growing axons, as either a growth permissive or a non-permissive substrate, may provide a developmental mechanism underlying the segregation of lamina-specific fiber projections.

Isolation of a hypoxia-induced cDNA with homology to the mammalian growth-related protein p23.

Hypoxia leads to a cellular stress reaction and can induce transcription of immediate early genes, such as c-fos. We have generated a differential cDNA cloning strategy to isolate further hypoxia-induced genes. We report on the identification and characterization of a novel transcript (cDNA clone pSH16), which is inducible 12-fold in HeLa cells after 50 min of exposure to hypoxia. Sequence analysis revealed a hybrid transcript with high homology to the mammalian growth-related protein p23 mRNA fused to mitochondrial 16S rRNA. Complete homology was found for the coding region, whereas the 3'-untranslated part of the hypoxia-induced p23 sequence was elongated and carried an ATTTA box and a second consensus poly(A) signal in addition. The functional integrity of the pSH16 mRNA was verified by cell-free translation. Hypoxia induced the expression of both fusion partners, p23 and 16S rRNA, separately. In contrast to the hypoxia-induced expression in HeLa cells, we found constitutive high-level expression in breast and cervix tissue. No further upregulation of p23 transcripts was detectable in primary tumors of the breast and cervix. These data provide first evidence for a hypoxia-induced upregulation of the mammalian growth-related protein p23, which might be relevant for understanding of the physiology of hypoxic conditions in tumors.