Bernd Meyhack

Nuclear Hormone Receptor NR4A2 Is Involved in Cell Transformation and Apoptosis

HeLaHF cells are transformation revertants of cervical cancer HeLa cells and have lost anchorage-independent growth potential and tumorigenicity. Activation of tumor suppressor(s) was implicated previously in this transformation reversion. In this study, expression profiling analysis was carried out to identify potential oncogenes that are down-regulated in HeLaHF cells. We found that all three members of the NR4A1/Nur77/NGFIB orphan nuclear hormone receptor subfamily (NR4A1, NR4A2, and NR4A3) were down-regulated in the HeLaHF revertant. Small interfering RNA-mediated down-regulation of NR4A2 in HeLa cells, either transiently or stably, resulted in reduced anchorage-independent growth that was largely attributable to increased anoikis. Furthermore, down-regulation of NR4A2 as well as NR4A1 promoted intrinsic apoptosis. These phenotypes were also observed in several other experimental cancer cells, suggesting the observed apoptosis suppression is a more general property of NR4A2 and NR4A1. These phenotypes also suggest that the Nur77/NGFIB subfamily of orphan receptors exhibit certain oncogenic functionalities with regards to cell proliferation and apoptosis and could therefore be evaluated as potential cancer therapeutic targets.

One-week, 96-well soft agar growth assay for cancer target validation

Soft agar growth, used to measure cell anchorage-independent proliferation potential, is one of the most important and most commonly used assays to detect cell transformation. However, the traditional soft agar assay is time-consuming, labor-intensive, and plagued with inconsistencies due to individual subjectivity. It does not, therefore, meet the increasing demands of todays oncology drug target screening or validation processes. This report describes an alternative 96-well soft agar growth assay that can function as a replacement for the traditional method and overcomes the aforementioned limitations. It offers the following advantages: a shortened assay duration (1 week instead of 4 weeks) that makes transient transfection or treatment possible; plate reader quantification of soft agar growth (measuring cloning efficiency and colony size); and a significant reduction in required labor. Higher throughput also makes it possible to process large numbers of samples and treatments simultaneously and in a much more efficient manner, while saving precious workspace and overall cost.

The influence of GAP promoter variants on hirudin production, average plasmid copy number and cell growth in Saccharomyces cerevisiae.

SummaryThe yeast Saccharomyces cerevisiae has been engineered to synthesize and secrete desulfato-hirudin (hirudin), a thrombin inhibitor from the leech Hirudo medicinalis. The synthetic gene coding for hirudin was expressed constitutively under the control of four size-variants of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) and cloned into a 2 μ based multicopy yeast vector. The constitutive action of the four promoter variants was confirmed by demonstrating that the expression and secretion of hirudin is growth-related. The different efficiencies of the promoter variants not only affected hirudin expression but also led to changes in several cellular parameters, such as cell growth, average plasmid copy number and plasmid stability. The observed changes show that yeast cells establish a specific equilibrium for each promoter variant. We conclude, that the adjustment of cellular parameters in response to the expression levels of a heterologous protein is regulated by two counteracting selective forces: (1) the need for complementation of the auxotrophic host marker by the plasmid-encoded selection gene which, in the case of dLEU2, requires several plasmid copies; and (2) a selective advantage of cells with a lower copy number enabling them to escape the burden of heterologous protein production.

Synthesis of glia-derived nexin in yeast.

Glia-derived nexin (GDN) is a 43-kDa glycoprotein isolated from rat glioma cell cultures. It promotes neurite extension in cultures of neuroblastoma cells and chick sympathetic neurons. Moreover, GDN is a potent serine protease inhibitor (serpin), belonging to the family of protease nexins. We report here the expression of rat GDN in the Saccharomyces cerevisiae strain GRF18 under the control of the PHO5 promoter. We describe the purification of more than 6 mg total GDN from the cellular extract of 1 liter of yeast culture. The amino acid composition and the sequence of CNBr-fragments of the recombinant protein correlate with the values deduced from the rat GDN cDNA. We provide evidence that the recombinant GDN has exactly the same properties as the glioma-derived protein with respect to its protease-inhibitory activity and its ability to promote the extension of neurites from neuroblastoma cells. The large amounts of recombinant protein obtained from this expression system will allow further biochemical and physiological analysis of GDN and of the serpins in general.

Novel BNIP1 variants and their interaction with BCL2 family members.

By PCR and EST database searches we have identified three novel BNIP1 splice variants, and found that one of them, BNIP1‐b, contains a highly conserved BH3 domain. The BNIP1 gene has been assigned to chromosome 5q33–34. Using in vitro protein‐protein interaction assays, all BNIP1 variants were shown to interact with BCL2 and also with BCL2L1 (previously Bcl‐xL). These interactions are BH3‐independent. Furthermore, the BNIP1 variants cannot interact with BAX. The results suggest that the BNIP1 variants are novel members of the BCL2 family but function through a mechanism different from other BH3‐only members.

Production of disulfide-linked hirudin dimer by in vitro folding

A simple process of in vitro folding has been developed for the preparation of hirudin dimer. A variant of recombinant hirudin with Asp33 replaced by Cys was expressed in yeast and isolated by HPLC. Crude Cys33‐hirudin contains heterogeneous products that are made of one species of primary sequence. They were together reduced/denatured, and allowed to re‐fold in the sodium bicarbonate buffer (pH 8.3) alone. Active, homogeneous Cys33‐hirudin monomer folded spontaneously with a first order rate constant of 0.05 ± 0.01 min−1, followed by the oxidation of two Cys33 to produce the pure dimer. The folding yield was 90%. On an equal weight basis, both Cys33‐hirudin monomer and the dimer exhibit thrombin inhibitory activity comparable to that of wild‐type hirudin. Due to the presence of an extra cysteine, the folding of active hirudin monomer (formation of three native disulfides) was accelerated by at least 12‐fold.

Expression of rat 5α-reductase in Saccharomyces cerevisiae

Abstract Dihydrotestosterone (DHT) is the principle androgen in certain tissues such as the prostate. DHT is formed from testosterone by the NADPH-dependent enzyme 5α-reductase (5AR). In this paper we report the expression of catalytically active steroid 5AR from the rat in Saccharomyces cerevisiae . A full length cDNA coding for 5AR was isolated from a rat liver cDNA library and fixed in frame to the signal sequence of yeast acid phosphatase. A constitutive short promoter fragment of the acid phosphatase gene ( PHO 5) and the PHO 5 transcriptional terminator were added and the expression cassette ligated into the yeast 2μ vector pDP34. S. cerevisiae transformed with the 5AR expression plasmid pDP34/PHO5AR exhibited about 100-fold more activity per gram wet weight than rat prostate.

Homologous recombination partly restores the secretion defect of underglycosylated acid phosphatase in yeast

Abstract The majority of secreted acid phosphatase in Saccharomyces cerevisiae is encoded by the PHO5 gene. The secretion level of this acid phosphatase is directly determined by its level of glycosylation. Consequently, PHO5-11-encoded acid phosphatase which lacks 11 of 12 glycosylation sites is only poorly secreted. We have isolated and characterized both UV- and EMS-induced variants, which are partly able to restore the secretion of acid phosphatase. Our data indicate that the improved secretion is caused by mitotic intrachromosomal recombination between the PHO5-11 allele and the homologous tandemly repeated PHO3 sequences, resulting in the restoration of glycosylation sites in PHO5-11. Two different recombination mechanisms, unequal sister-chromatid exchange and sister-chromatid gene conversion, are responsible for these alterations of the PHO5-11 locus. Thus, recombination between mutant and wild-type sequences are able to restore the ability of mutant yeast cells to secrete acid phosphatase.

Refolding, isolation and characterization of crystallizable human interferon-α8 expressed in Saccharomyces cerevisiae

Human interferon-α8 was expressed in Saccharomyces cerevisiae and found to accumulate intracellularly in an insoluble form. The protein could be solubilized and converted to a biologically active form with high yield by a denaturation-refolding procedure. The interferon-α8 was further purified to apparent homogeneity by copper-chelate affinity chromatography and anion-exchange chromatography and fully characterized by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, mass spectrometry, circular-dichroism (CD) spectroscopy and specific activity. Secondary-structure predictions from CD spectroscopy indicate that the molecule is correctly folded. Peptide mapping supported the correct sequence and the expected disulfide-bridge connectivity. The purified protein elutes on reversed-phase high-pressure liquid chromatography (RP-HPLC) as two peaks. Electrospray mass spectrometry and N-terminal sequence analysis of the minor component indicated the existence of an N-terminal acetyl group for the later eluting HPLC-component. In anti-viral assays, the two IFN forms were equally active. Hexagonal crystals of this interferon preparation could be obtained. On the basis of the electrophoretic mobility, HPLC profile, and biological activity assay, the crystalline material was judged to be identical to the uncrystallized interferon. Interferon in crystallized form was found to be stable for up to 24 months and, therefore, could be used for long-term storage, particularly for material intended for clinical use.