Tissue-dependent factors affect gene delivery to tumors in vivo.

Systemic application of surface-shielded transferrin-polyethylenimine/DNA complexes leads to predominant DNA uptake and gene expression in Neuro2a tumors in syngeneic A/J mice. Similarly, high expression levels were found in Huh-7 and HepG2 human tumor xenografts in SCID mice after systemic application of surface-shielded EGF-PEG-PEI/DNA complexes. Significant DNA uptake but low gene expression were found in the M-3 melanoma while no DNA uptake and no gene expression were found in KB, 518A2, A549, and SW480 xenograft tumor models. To elucidate the reasons for these differences, the tumors were analyzed for vascularization and infiltration of macrophages. Neuro2a, Huh-7, and HepG2 tumors are well vascularized, with a high density of partially immature blood vessels and low numbers of infiltrating macrophages. The M-3 melanoma is well vascularized correlating with significant DNA uptake, however, necrosis and intensive infiltration by macrophages lead to rapid degradation of DNA. In contrast, the KB, 518A2, A549, and SW480 tumors are poorly vascularized, correlating with undetectable DNA uptake and gene expression. Using two different vector systems the data indicate that gene delivery to tumors in vivo is affected by tissue-dependent factors. Uptake of DNA into the tumor depends on vascularization of the tumor, while necrosis and macrophage infiltration may facilitate degradation of the DNA.

Differential expression levels of Par-4 in melanoma.

The pro-apoptotic prostate apoptosis response-4 gene product Par-4 sensitizes prostate cells to the induction of programmed cell death. In this study we examined Par-4 expression in human melanoma cell lines and melanoma metastases. The heterogeneous expression detected prompted us to investigate the biological relevance of Par-4 in a human melanoma xenotransplantation model. Overexpression of Par-4 by transfection decreased tumour development in xenotransplanted A375-C6 melanoma cells in SCID mice and correlated to an increase in tumour cell apoptosis. These data suggest that high expression of the pro-apoptotic protein Par-4 could qualify as a prognostic marker in human melanoma.

Cyclic AMP- and differentiation-dependent regulation of the proximal alphaHCG gene promoter in term villous trophoblasts.

Although the regulatory mechanisms controlling alpha and beta human chorionic gonadotrophin (HCG) expression have been investigated in choriocarcinoma cell model systems, little is known about the regulation of HCG subunit synthesis in non-tumourigenic trophoblasts. We therefore investigated alphaHCG mRNA transcription in villous cytotrophoblasts isolated from term placentae and have shown for the first time that the proximal alphaHCG gene promoter is functional in these cells. By establishing conditions which allow efficient transient transfection of immunopurified cells, we have demonstrated that a 363 bp sequence in the proximal 5' flanking region of the alphaHCG gene is sufficient to direct trophoblast-specific expression of a luciferase reporter. After 12-60 h cultivation, an increase in endogenous alphaHCG mRNA expression could be detected, indicating that aggregated villous trophoblasts undergo biochemical differentiation. Concomitantly, we observed induction of alphaHCG promoter-driven luciferase activity, suggesting that the 363 bp sequence of the proximal 5' flanking region is sufficient to direct differentiation-dependent increase of alphaHCG mRNA. Continuous luciferase expression required functional cAMP-response elements (CREs), since deletion of both recognition sequences eliminated differentiation-dependent transcription of the reporter. Elevation of cAMP values increased transcription of the wild-type construct; however, it did not affect promoter activity of the mutant plasmid. Moreover, we have demonstrated that during in-vitro differentiation, CREs interacted with increasing amounts of phosphorylated activating transcription factor/cyclic AMP response element-binding protein (ATF-1/CREB-1) suggesting that these cAMP-dependent DNA-binding factors are major determinants in regulating alphaHCG gene expression in villous trophoblasts.

In situ detection of protein-DNA interactions in filamentous fungi by in vivo footprinting.

The method described here allows the detection of protein-DNA interactions in vivo in filamentous fungi. We outline culture conditions and conditions of in vivo methylation that permit uniform modification of all cells in an apically growing, non-uniform organism, and subsequent visualization of protected areas by ligation-mediated PCR.

The filamentous fungus Aspergillus niger contains two "differentially regulated" trehalose-6-phosphate synthase-encoding genes, tpsA and tpsB.

Two genes encoding trehalose-6-phosphate synthase were cloned from Aspergillus niger. tpsA was cloned using the Saccharomyces cerevisiae GGS1/TPS1 gene as a probe. It encodes a 517-amino acid polypeptide with 64-70% similarity to trehalose-6-phosphate synthase of S. cerevisiae, Kluyveromyces lactis, and Schizosaccharomyces pombe. Its transcription occurs constitutively and is enhanced on carbon-derepressing carbon sources, coinciding with the presence of a CreA-binding nucleotide motif in the 5'-noncoding region of tpsA. Disruption of tpsA only weakly reduces growth on glucose, and neither influences the glucose induction of a low affinity glucose permease nor interferes with the catabolite repression of a pectinase; it causes reduced the heat tolerance of conidia. tpsB was cloned by a polymerase chain reaction-based strategy. Its 480 amino acid sequence showed 76.5% identity to tpsA. Its transcription was hardly detectable at ambient temperatures but was enhanced strongly upon heat shock, which agrees with the presence of several copies of a C4T stress-responsive element in its 5'-upstream sequences. Hence the function of yeast GGS1/TPS1 has been split into two differentially regulated genes in A. niger, of which none appears to be involved in glucose sensing.

Trehalose-6-phosphate synthase A affects citrate accumulation by Aspergillus niger under conditions of high glycolytic flux.

Accumulation of citric acid by Aspergillus niger depends on a high flux through glycolysis. We have investigated the possibility of control of this flux by trehalose 6-phosphate, an inhibitor of hexokinase of Saccharomyces cerevisiae and other eukaryotes (Blasquez et al., FEBS Lett. (1993) 329, 51-54). Hexokinase of A. niger was shown in vitro to be only weakly inhibited by trehalose 6-phosphate (KI 1.5-2 mM). To investigate the in vivo relevance of this inhibition, we used isogenic strains of A. niger, carrying either a disruption or an amplification of the trehalose-6-phosphate synthase A (T6PSA)-encoding gene (ggsA) and exhibiting corresponding differences in T6PSA activity. These strains produced citric acid at comparable rates and with similar yields on 1 or 2.5% (w/v) sucrose. At 5-14% (w/v) sucrose, the ggsA disrupted strain initiated citric acid accumulation earlier, whereas the multicopy strain showed the reverse effect. When sucrose was replaced by lactose, which enabled only low rates of catabolism irrespective of its concentration (1-8%), no differences in the initiation or rate of citric acid accumulation were observed between the three strains. The possible mechanisms by which ggsA controls glycolytic flux in A. niger in the presence of high sugar concentrations are discussed.

Cre1, the carbon catabolite repressor protein from Trichoderma reesei.

In order to investigate the mechanism of carbon catabolite repression in the industrially important fungus Trichoderma reesei, degenerated PCR-primers were designed to amplify a 0.7-bp fragment of the cre1 gene, which was used to clone the entire gene. It encodes a 402-amino acid protein with a calculated M(r) of 43.6 kDa. Its aa-sequence shows 55.6% and 54.7% overall similarity to the corresponding genes of Aspergillus nidulans and A. niger, respectively. Similarity was restricted to the aa-region containing the C2H2 zinc finger and several aa-regions rich in proline and basic amino acids, which may be involved in the interaction with other proteins. Another aa-region rich in the SPXX-motif that has been considered analogous to a region of yeast RGR1p, was instead identified as a domain occurring in several eucaryotic transcription factors. The presence of the cre1 translation product was demonstrated with polyclonal antibodies against Cre1, which identified a protein of 43 (+/- 2) kDa in cell-free extracts from T. reesei. A Cre1 protein fragment from the two zinc fingers to the region similar to the aa-sequence of eucaryotic transcription factors, was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. EMSA and in vitro footprinting revealed binding of the fusion protein to the sequence 5'-GCGGAG-3', which matches well with the A. nidulans consensus sequence for CreA binding (5'-SYGGRG-3'). Cell-free extracts of T. reesei formed different complexes with DNA-fragments carrying this binding sites, and the presence of Cre1 and additional proteins in these complexes was demonstrated. We conclude that T. reesei Cre1 is the functional homologue of Aspergillus CreA and that it binds to its target sequence probably as a protein complex.

Subcellular compartmentation of penicillin biosynthesis in Penicillium chrysogenum. The amino acid precursors are derived from the vacuole.

The cellular localization of the origin of alpha-aminoadipate used in penicillin biosynthesis and the first enzymic step in Penicillium chrysogenum involved, delta-(alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), has been studied. Subcellular fractions were obtained from protoplasts of a high penicillin-producing strain upon lysis by Triton X-100, and vacuoles purified from them. They were identified by the aid of alpha-mannosidase as a marker enzyme, by the presence of polyphosphate, and their ability to sequester [14C]lysin, added to the protoplasts prior to subcellular fractionation. 15.6 and 26.5%, respectively, of 6-[14C]alpha-aminoadipate, and 8.5 and 10.3%, respectively, of [14C]valine added accordingly were also found in the vacuole, and the higher proportion was found in vacuoles isolated from penicillin-producing mycelia. ACVS protein was detected in the membrane as well as the soluble fraction of the purified vacuoles. We propose therefore that ACVS is located either within or bound to the vacuolar membrane, and that the precursor amino acids for penicillin biosynthesis are withdrawn from the vacuolar amino acid pool.