Identification and characterization of human nucleus pulposus cell specific serotypes of adeno-associated virus for gene therapeutic approaches of intervertebral disc disorders.

BACKGROUND: Intervertebral disc (IVD) disorders are often accompanied by painful inflammatory and immunopathological processes. Nucleus pulposus (NP) cells play a pivotal role in maintenance of IVD by organizing the expression of anabolic, catabolic, anti-catabolic and inflammatory cytokines. Human NP cells have been targeted by gene therapeutic approaches using lentiviral or adenoviral systems that could be critical due to genome incorporation or immunological side effects. Adeno-associated viruses (AAVs), which do not express any viral gene and are not linked with any known disease in humans, are attractive gene delivery vectors. However, their lack of specific tissue tropism and preexisting immune response are main problems for therapeutic applications. Heretofore, AAVs have not been studied in human IVD research. Therefore, we attempted to identify NP cell specific AAV serotype by targeting human NP cells with different self-complementary AAV (scAAV) serotypes. Identification and characterization of the proper serotype is crucial to establish less immunogenic and safer gene therapeutic approaches of IVD disorders. METHODS: Preoperative magnetic resonance imaging (MRI) was used for grading of IVD degeneration. NP cells were isolated, cultured with low-glucose and transduced with green fluorescent protein (GFP) packing scAAV serotypes (scAAV1-8) in a dose-dependent manner. scAAV titers were determined by quantitative polymerase chain reaction (qPCR). Transduction efficiencies were determined by fluorescence microscopy and fluorescence-activated cell sorting within 48 days of post-transduction. The 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine NP cell viability. Three-dimensional (3D) cell culture and enzyme-linked immunosorbant assay (ELISA) were performed to examine the expression levels of inflammatory, catabolic and matrix proteins in NP cells. RESULTS: scAAV6, scAAV2 and scAAV3 showed high and prolonged transgene GFP expressions with transdution efficiencies of 98.6 %, 91.5 % and 89.6 % respectively (p ≤ 0.002). Unlike scAAV6, the serotypes scAAV2 and scAAV3 declined the viability of NP cells by about 25 % and 10 % respectively (p ≤ 0.001). Moreover, scAAV6 did not affect the expression of the inflammatory, catabolic and matrix proteins. CONCLUSIONS: As original primary research evaluating AAVs in degenerative human IVDs, this study identified scAAV6 as a proper serotype for high, stable and non-immunogenic target gene expression in human NP cells. The data could be very important to design efficient and safer gene therapeutic approaches of IVD disorders.

Imbalanced protein expression patterns of anabolic, catabolic, anti-catabolic and inflammatory cytokines in degenerative cervical disc cells: new indications for gene therapeutic treatments of cervical disc diseases.

Degenerative disc disease (DDD) of the cervical spine is common after middle age and can cause loss of disc height with painful nerve impingement, bone and joint inflammation. Despite the clinical importance of these problems, in current publications the pathology of cervical disc degeneration has been studied merely from a morphologic view point using magnetic resonance imaging (MRI), without addressing the issue of biological treatment approaches. So far a wide range of endogenously expressed bioactive factors in degenerative cervical disc cells has not yet been investigated, despite its importance for gene therapeutic approaches. Although degenerative lumbar disc cells have been targeted by different biological treatment approaches, the quantities of disc cells and the concentrations of gene therapeutic factors used in animal models differ extremely. These indicate lack of experimentally acquired data regarding disc cell proliferation and levels of target proteins. Therefore, we analysed proliferation and endogenous expression levels of anabolic, catabolic, ant-catabolic, inflammatory cytokines and matrix proteins of degenerative cervical disc cells in three-dimensional cultures. Preoperative MRI grading of cervical discs was used, then grade III and IV nucleus pulposus (NP) tissues were isolated from 15 patients, operated due to cervical disc herniation. NP cells were cultured for four weeks with low-glucose in collagen I scaffold. Their proliferation rates were analysed using 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide. Their protein expression levels of 28 therapeutic targets were analysed using enzyme-linked immunosorbent assay. During progressive grades of degeneration NP cell proliferation rates were similar. Significantly decreased aggrecan and collagen II expressions (P<0.0001) were accompanied by accumulations of selective catabolic and inflammatory cytokines (disintegrin and metalloproteinase with thrombospondin motifs 4 and 5, matrix metalloproteinase 3, interleukin-1ß, interleukin-1 receptor) combined with low expression of anti-catabolic factor (metalloproteinase inhibitor 3) (P<0.0001). This study might contribute to inhibit inflammatory catabolism of cervical discs.

A combinatorial relative mass value evaluation of endogenous bioactive proteins in three-dimensional cultured nucleus pulposus cells of herniated intervertebral discs: identification of potential target proteins for gene therapeutic approaches.

Painful degenerative disc diseases have been targeted by different biological treatment approaches. Nucleus pulposus (NP) cells play a central role in intervertebral disc (IVD) maintenance by orchestrating catabolic, anabolic and inflammatory factors that affect the extracellular matrix. IVD degeneration is associated with imbalances of these factors, resulting in a catabolic inflammatory metabolism. Therefore, accurate knowledge about their quantity and quality with regard to matrix synthesis is vital for a rational gene therapeutic approach. NP cells were isolated from 63 patients operated due to lumbar disc herniation (mean age 56 / range 29 - 84 years). Then, three-dimensional culture with low-glucose was completed in a collagen type I scaffold for four weeks. Subsequently cell proliferation evaluation was performed using 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and intracellular concentration of 28 endogenously expressed anabolic, catabolic, inflammatory factors and relevant matrix proteins was determined by enzyme-linked immunosorbent assay. Specimen-related grades of degeneration were confirmed by preoperative magnetic resonance imaging. Independent from gender, age and grade of degeneration proliferation rates remained similar in all groups of NP cells. Progressive grades of degeneration, however, showed a significant influence on accumulation of selective groups of factors such as disintegrin and metalloproteinase with thrombospondin motifs 4 and 5, matrix metalloproteinase 3, metalloproteinase inhibitor 1 and 2, interleukin-1ß and interleukin-1 receptor. Along with these changes, the key NP matrix proteins aggrecan and collagen II decreased significantly. The concentration of anabolic factors bone morphogenetic proteins 2, 4, 6 and 7, insulin-like growth factor 1, transforming growth factor beta 1 and 3, however, remained below the minimal detectable quantities. These findings indicate that progressive degenerative changes in NP may be problematic with regard to biologic treatment strategies. Hence, gene therapeutic interventions regulating relevant bioactive factors identified in this work might contribute to the development of regenerative treatment approaches for degenerative disc diseases.

Gene ercA, encoding a putative iron-containing alcohol dehydrogenase, is involved in regulation of ethanol utilization in Pseudomonas aeruginosa.

Several two-component regulatory systems are known to be involved in the signal transduction pathway of the ethanol oxidation system in Pseudomonas aeruginosa ATCC 17933. These sensor kinases and response regulators are organized in a hierarchical manner. In addition, a cytoplasmic putative iron-containing alcohol dehydrogenase (Fe-ADH) encoded by ercA (PA1991) has been identified to play an essential role in this regulatory network. The gene ercA (PA1991) is located next to ercS, which encodes a sensor kinase. Inactivation of ercA (PA1991) by insertion of a kanamycin resistance cassette created mutant NH1. NH1 showed poor growth on various alcohols. On ethanol, NH1 grew only with an extremely extended lag phase. During the induction period on ethanol, transcription of structural genes exa and pqqABCDEH, encoding components of initial ethanol oxidation in P. aeruginosa, was drastically reduced in NH1, which indicates the regulatory function of ercA (PA1991). However, transcription in the extremely delayed logarithmic growth phase was comparable to that in the wild type. To date, the involvement of an Fe-ADH in signal transduction processes has not been reported.

Targeting Id1 and Id3 by a specific peptide aptamer induces E-box promoter activity, cell cycle arrest, and apoptosis in breast cancer cells.

Inhibitors of differentiation or DNA binding (Id) proteins have been shown to be involved in tumor growth, invasiveness, metastasis, and angiogenesis. Overexpression of Id proteins, especially Id1, correlates with unfavorable clinical prognosis. Thus, they are attractive molecular targets for anticancer therapy. Overexpression of Id proteins mediates breast cancer metastasis to lung. Targeting Id1 and Id3 expression in breast cancer cells reduces breast cancer metastasis in animal models. Different breast tumors failed to grow and/or metastasize in Id1 (+/-) Id3 (-/-) mice. Id1 and Id3 preferentially dimerize with the key regulatory E-proteins which inhibit the expression of different tumor suppressor genes. Nevertheless, the inhibition of tumorigenic activities of Id1 and Id3 at protein level has never been studied. Here, we isolated a novel peptide aptamer, Id1/3-PA7, specifically interacting with Id1 and Id3 from randomized combinatorial expression library using yeast and mammalian two-hybrid systems. Intracellular delivered Id1/3-PA7 co-localized to Id1 and Id3 and interfered with their functions. It repressed E47 protein sequestration by Id1 and Id3, activated the E-box promoter and increased the expression level of cyclin-dependent kinase inhibitors (CDKN1A and CDKN1B) in a dose-dependent fashion, paralleled by the cleavage of poly ADP ribose polymerase (PARP). These effects were counteracted by ectopically overexpressed Id1 and Id3. Peptide aptamer Id1/3-PA7 induced cell cycle arrest and apoptosis in breast cancer cells MCF7 and MDA-MB-231. In conclusion, Id1/3-PA7 could represent a nontoxic exogenous agent that can significantly provoke antiproliferative and apoptotic effects in breast cancer cells, which are associated with deregulated expression of Id1 and Id3.

A complex regulatory network controls aerobic ethanol oxidation in Pseudomonas aeruginosa: indication of four levels of sensor kinases and response regulators.

In addition to the known response regulator ErbR (former AgmR) and the two-component regulatory system EraSR (former ExaDE), three additional regulatory proteins have been identified as being involved in controlling transcription of the aerobic ethanol oxidation system in Pseudomonas aeruginosa. Two putative sensor kinases, ErcS and ErcS', and a response regulator, ErdR, were found, all of which show significant similarity to the two-component flhSR system that controls methanol and formaldehyde metabolism in Paracoccus denitrificans. All three identified response regulators, EraR (formerly ExaE), ErbR (formerly AgmR) and ErdR, are members of the luxR family. The three sensor kinases EraS (formerly ExaD), ErcS and ErcS' do not contain a membrane domain. Apparently, they are localized in the cytoplasm and recognize cytoplasmic signals. Inactivation of gene ercS caused an extended lag phase on ethanol. Inactivation of both genes, ercS and ercS', resulted in no growth at all on ethanol, as did inactivation of erdR. Of the three sensor kinases and three response regulators identified thus far, only the EraSR (formerly ExaDE) system forms a corresponding kinase/regulator pair. Using reporter gene constructs of all identified regulatory genes in different mutants allowed the hierarchy of a hypothetical complex regulatory network to be established. Probably, two additional sensor kinases and two additional response regulators, which are hidden among the numerous regulatory genes annotated in the genome of P. aeruginosa, remain to be identified.

Elevated endogenous expression of the dominant negative basic helix-loop-helix protein ID1 correlates with significant centrosome abnormalities in human tumor cells.

BACKGROUND: ID proteins are dominant negative inhibitors of basic helix-loop-helix transcription factors that have multiple functions during development and cellular differentiation. Ectopic (over-)expression of ID1 extends the lifespan of primary human epithelial cells. High expression levels of ID1 have been detected in multiple human malignancies, and in some have been correlated with unfavorable clinical prognosis. ID1 protein is localized at the centrosomes and forced (over-)expression of ID1 results in errors during centrosome duplication. RESULTS: Here we analyzed the steady state expression levels of the four ID-proteins in 18 tumor cell lines and assessed the number of centrosome abnormalities. While expression of ID1, ID2, and ID3 was detected, we failed to detect protein expression of ID4. Expression of ID1 correlated with increased supernumerary centrosomes in most cell lines analyzed. CONCLUSIONS: This is the first report that shows that not only ectopic expression in tissue culture but endogenous levels of ID1 modulate centrosome numbers. Thus, our findings support the hypothesis that ID1 interferes with centrosome homeostasis, most likely contributing to genomic instability and associated tumor aggressiveness.