Juergen Kleinschmidt

Soluble acidic complexes containing histones H3 and H4 in nuclei of Xenopus laevis oocytes

Oocyte nuclei of Xenopus laevis contain nucleosomal-core histones in large amounts and in a soluble, non-chromatin-bound form. Supernatant fractions (100,000 X g) from isolated nuclei are enriched in complexes containing histones H3 and H4, which are of distinct size (5.6S by sucrose gradient centrifugation, approximate molecular weight of 270,000 by gel filtration) and negatively charged (isoelectric at pH 4.4). These complexes bind to DEAE-Sephacel and can be separated from nucleoplasmin. In diverse fractionation experiments, histones H3 and H4 have been found to comigrate with a pair of polypeptides of molecular weight 110,000 that represent the most acidic major protein present in these nuclei. After enrichment by gel filtration, ion exchange chromatography and electrophoresis, this pair of acidic polypeptides has been the only nonhistone protein detected in the histone-complex fraction. We suggest that in the oocyte nucleus, large proportions of the soluble histones H3 and H4 are not contained in complexes of all four nucleosomal-core histones but are differentially associated with specific, very acidic proteins into distinct 5.6S complexes.

MIA (melanoma inhibitory activity) promoter mediated tissue-specific suicide gene therapy of malignant melanoma

Suicide gene therapy of malignant melanoma essentially requires efficient gene transfer and highly selective therapeutic gene expression. To achieve this, recombinant adeno-associated virus (rAAV) particles were constructed containing the tissue-specific promoter of the human melanoma inhibitory activity (hMIA) gene combined with four copies of the enhancer element of the murine tyrosinase gene. Three melanoma and one cervix carcinoma cell line were infected with rAAV particles carrying a reporter gene under control of the enhancer/hMIA promoter in order to determine transcriptional activity and specificity of this system. Viral particles containing the enhancer/hMIA promoter mediated reporter gene activity only in melanoma cells, whereas infection with a cytomegalovirus (CMV)-based promoter construct induced unspecific gene expression. Correspondingly, transient transduction with viral particles bearing the HSVtk gene under the control of the enhancer/MIA promoter elements followed by treatment with ganciclovir (GCV) resulted in growth inhibition only in melanoma cells, whereas the CMV promoter-based construct induced unspecific cytotoxicity. In vivo experiments in nude mice demonstrated that tumors originating from human melanoma cells disappeared after stable, but not transient transduction with vectors bearing the HSVtk gene under the control of the enhancer/hMIA promoter in response to GCV application. In face of higher transduction efficiency, these rAAV particles might therefore be a useful tool for suicide gene therapy of malignant melanoma.

Differential expression of a recombinant adeno-associated virus 2 vector in human CD34 + cells and breast cancer cells

The use of autologous hematopoietic stem cell (HSC) grafts after high-dose chemotherapy protocols may be hampered by contamination of the grafts with tumor cells. Because epithelial cells seem to be the natural hosts of adeno-associated virus 2 (AAV-2), we speculated that epithelial tumor cells in HSC grafts might be selective targets for AAV-2-based vectors. To test this hypothesis, the breast cancer cell lines T47D and MCF-7 were infected with a recombinant AAV-2 vector expressing the green fluorescent protein (GFP) gene; in addition, human CD34+ mobilized peripheral progenitor cells were infected with the same vector. At a multiplicity of infection of 100, only 1.39% ± 0.51% CD34+ cells expressed the GFP gene whereas, 36.06% ± 6.53% of the infected T47D cells and 41.52% ± 3.16% of the infected MCF-7 cells expressed the transduced GFP gene. After further optimizing the transduction procedure by using higher multiplicities of infection (100–500) and preincubation of samples with the tyrosine kinase inhibitor genistein, up to 82.52% and 85.35% GFP+ T47D and MCF-7 cells, respectively, were observed. The GFP fluorescence intensity in transduced mammary tumor cells was up to 3 logs higher than that of transduced CD34+ cells. The differential expression of recombinant AAV-2 vectors in hematopoietic and epithelial tumor cells warrants further research with this vector system, including the use of suicide genes for the purging of autologous HSC grafts.