Biomedical subjects

B Eickhoff

Publications and source records attributed to B Eickhoff.

5 recordsLinked to original sources

PubMed2000

Trichostatin A modulates expression of p21waf1/cip1, Bcl-xL, ID1, ID2, ID3, CRAB2, GATA-2, hsp86 and TFIID/TAFII31 mRNA in human lung adenocarcinoma cells.

Lung adenocarcinoma cells treated for 16 h with trichostatin A (TSA), an inhibitor of histone deacetylases, and untreated cells were analyzed with respect to differential gene expression. Complex hybridization of cDNA arrays revealed repression of Bcl-xL, CRAB2 and TFIID/TAFII31 as well as induction of p21waf1/cip1, GATA-2, hsp86, ID1, ID2 and ID3 mRNA expression, which could be verified by Northern blotting. ID2 induction was further confirmed by Taqman realtime quantitative RT-PCR. The described alterations of gene expression due to TSA renders the lung adenocarcinoma cells susceptible to induction of apoptosis.

Adenocarcinoma↗

PubMed2000

Trichostatin A-mediated regulation of gene expression and protein kinase activities: reprogramming tumor cells for ribotoxic stress-induced apoptosis.

Recently we described a new signal transduction-based tumor therapeutic strategy involving first sensitization of tumor cells by trichostatin A (TSA), an inhibitor of histone deacetylation, and thereafter efficient apoptotic triggering by ribotoxic agents, which activate stress-activated protein kinases. In the present work we investigate the molecular basis of the sensitization step in this therapeutic model system and describe TSA-induced changes in mRNA and protein expression of several candidate genes identified previously by complex hybridization. Furthermore, activities of 15 different protein kinases were followed after TSA application, using a new filter-based technique (PhosphoSpots-Assay). The obtained data suggest that TSA induces pro-apoptotic genes like ID1, ID2, ID3, and down-regulates anti-apoptotic genes like Hsp27 and Bcl-xL, thereby shifting the cellular equilibrium from life to death. Furthermore, activities of calcium/calmodulin-dependent kinase II and protein kinase C, which have been assigned pro-apoptotic function in other systems, are induced.

Apoptosis↗

PubMed1999

Normalization of array hybridization experiments in differential gene expression analysis.

For detecting and confirming differentially expressed genes it is necessary to have a trustworthy reference. So called 'housekeeping genes' are frequently used for this purpose as internal standard. However, if the influence of new experimental conditions is to be analyzed it is not safe to assume a priori that the expression of these genes is not affected. Therefore two synthetic poly(A)-RNAs were generated by PCR and in vitro transcription. They were used as external standards for normalization of northern blots and cDNA arrays where non-regulated genes as internal reference were not available.

Ampicillin Resistance↗

PubMed1999

Human natural immunoglobulin M antibodies induce apoptosis of human neuroblastoma cells by binding to a Mr 260,000 antigen.

Sera of healthy humans contain natural cytotoxic IgM antibodies that specifically recognize a Mr 260,000 antigen (NB-p260) on the surface of human neuroblastoma (NB) cells. Here we demonstrate that anti-NB IgM antibodies prepared from different healthy individuals induce, in all human NB cell lines analyzed thus far, typical morphological and biochemical features of apoptosis including nuclear fragmentation, chromatin condensation, and DNA fragmentation. Both the binding of human anti-NB IgM to NB cells and the induction of apoptosis could be inhibited by preincubation of NB cells with murine IgG raised against purified NB-p260. Furthermore, preincubation of human anti-NB IgM with purified NB-p260 immobilized onto a solid support abolished its ability to induce apoptosis in NB cells. Natural human anti-NB IgM failed to bind to and induce apoptosis in control tumor cell lines that lack expression of NB-p260. The anti-NB IgM-induced apoptotic response was also observed in vivo in xenografted human NB tumors. After a single i.v. injection of anti-NB IgM into nude rats bearing solid NB xenografts, many areas of pyknotic cells with fragmented nuclei were observed that stained positive using the terminal dUTP nick end labeling method. In conclusion, the data demonstrate that natural anti-NB IgM antibodies in the sera of healthy individuals are potent mediators of apoptotic cell death of NB cells both in vitro and in vivo. The NB-p260 antigen was identified as the apoptosis-inducing receptor for anti-NB IgM. Whereas natural anti-NB IgM and NB-p260 may be useful tools for immunotherapy of NB, their biological significance remains to be determined.

Animals↗

PubMed1999

Sensitization of tumor cells to ribotoxic stress-induced apoptotic cell death: a new therapeutic strategy.

We describe a procedure that sensitizes chemotherapy-and tumor necrosis factor-resistant human tumor cell populations in vitro and in nude mouse transplants to the immediate triggering of high rates of cell death by anisomycin, an agent causing activation of stress-activated protein kinases [SAPKs, as defined by P. Cohen (Trends Cell Biol., 7: 353-361, 1997)] including p38/RK and c-jun NH2-terminal kinase homologues, following its binding to ribosomal 28S RNA (M. S. Iordanov et al, Mol. Cell. Biol., 17: 3373-3381, 1997). Sensitization is effected by successive application of an inhibitor of histone deacetylation (trichostatin A, butyrate) and of flavopiridol, known as an inhibitor of cyclin dependent kinases and evaluated presently in clinical trials. Effective concentrations of anisomycin, flavopiridol, and trichostatin A are in the submicromolar range. Tumor cell death can be prevented by epidermal growth factor (EGF), if added before flavopiridol or after anisomycin but not if applied between the additions of these agents, suggesting that flavopiridol interrupts an EGF-activated survival pathway and that anisomycin, besides triggering cell death, guards this pathway against the interference by flavopiridol. In contrast to EGF, dibutyryl-cAMP exerts protection that is flavopiridol-insensitive. For triggering cell death, anisomycin cannot be replaced by DNA- or mitotic spindle-targeted drugs in this system. The present findings, that a combination of transcriptional and signal transduction-targeted modulators sensitizes tumor cells synergetically to stress-mediated triggering of cell death and that ribotoxic stress is more efficient in this respect than genotoxic or spindle-targeted stress, bear important implications for the therapeutic exploitation of cellular stress responses. The stepwise sensitization and triggering of cell death in the present system allow separate analysis and manipulation of processes contributing to cellular death susceptibility and of the mechanism responsible for triggering cell death, thus providing the operational basis for further development of this therapeutic approach.

Animals↗