Sabine Brüsselbach

Cell cycle‐independent induction of apoptosis by the anti‐tumor drug flavopiridol in endothelial cells

The anti‐tumor drug Flavopiridol is a potent inhibitor of cyclin‐dependent kinases (cdks). As a consequence, Flavopiridol‐treated cells arrest in both G1 and G2, but Flavopiridol has also been shown to be cytotoxic for some tumor cell lines. The underlying molecular events are, however, unclear. We now show that Flavopiridol induces apoptosis in human umbilical vein endothelial cells (HUVECs), as judged by the occurrence of classical apoptotic markers, including chromatin condensation, internucleosomal cleavage, DNA fragmentation (TUNEL assay), annexin V binding and poly(ADP‐ribose) polymerase (PARP)‐cleavage. Such induction of apoptosis occurs with equal efficiency in both proliferating and G0/G1‐arrested cells. Because growth‐arrested HUVECs lack cdk2 activity and contain high levels of the cdk inhibitor p27, our observations suggest that cell cycle regulated cdks may not be the only critical target for Flavopiridol‐induced apoptosis. Surprisingly, A549 lung carcinoma cells were clearly dependent on cell proliferation for the induction of cell death, pointing to cell type‐related differences in the mechanism of Flavopiridol action. Int. J. Cancer 77:146–152, 1998.© 1998 Wiley‐Liss, Inc.

Abrogation of c-Raf expression induces apoptosis in tumor cells.

Signal transduction pathways involving the c-Raf protein kinase are frequently activated in tumor cells. We have addressed the relevance of this activation by a loss-of-function approach. An anti-sense phosphorothioate oligonucleotide (ODN) specifically targeted against c-raf mRNA () was used to block c-Raf protein expression in four different cell lines derived from lung, cervical, prostate and colon carcinomas. Concomitant with the abrogation of c-Raf expression we observed the occurrence of classical apoptotic markers, including chromatin condensation, inter-nucleosomal DNA cleavage, annexin V binding and cleavage of PARP, which was followed by cell death, affecting most of the cell population. This induction of apoptosis occurred independent of the p53 status of the cell. These findings demonstrate that c-Raf can protect tumor cells from undergoing programmed cell death, and suggest that the interference with c-Raf expression or function by ODNs or specific drugs could represent a powerful means for improving the efficacy of anti-cancer therapy.

Inhibition of D1, D2, and a cyclin expression in HL-60 cells by the lipid peroxydation product 4-hydroxynonenal

4-Hydroxynonenal (HNE), a product of lipid peroxidation, is an highly reactive aldehyde that, at concentration similar to those found in normal cells, blocks proliferation and induces a granulocytic-like differentiation in HL-60 cells. These effects are accompained by a marked increase in the proportion G0/G1 cells. The mechanisms of HNE action were investigated by analyzing the expression of the cyclins and cyclin-dependent protein kinases (CDKs), controlling the cell cycle progression. Data obtained by exposing cells to dimethyl sulfoxide (DMSO) were used for comparison. 4-Hydroxynonenal downregulated both mRNA and protein contents of cyclins D1, D2, and A until 24 h from the treatments, whereas DMSO inhibited cyclin D1 and D2 expression until the end of experiment (2 days) and induces an increase of cyclin A until 1 day. Cyclins B and E, and protein kinase CDK2 and CDK4 expressions were not affected by HNE, whereas DMSO induced an increase of cyclin E, B, and CDK2 from 8 h to 1 day. These data are in agreement with previous results indicating a different time-course of accumulation in G0/G1 phases of cells treated with HNE and DMSO and suggest that the HNE inhibitory effect on proliferation and cell cycle progression may depend by the downregulation of D1, D2, and A cyclin expression.

Cationized human serum albumin as a non-viral vector system for gene delivery? Characterization of complex formation with plasmid DNA and transfection efficiency.

Cationized human serum albumin (cHSA) could serve as a potential non-viral vector system for gene delivery. Native human serum albumin was cationized by covalent coupling of hexamethylenediamine to the carboxyl groups resulting in a shift of the isoelectric point from pH 4-5 to 7-9. The cationized albumin underwent spontaneous self-assembly with DNA as demonstrated by retardation of CMV-nlacZ plasmid in agarose gel electrophoresis. Photon correlation spectroscopy showed a decrease of complex size with increasing cHSA/plasmid ratios. Under optimized conditions complexes were formed with 230-260 nm mean diameter and a homogenous, narrow size distribution. At room temperature complexes were stable in 0.9% sodium chloride solution pH 7.4 for 1 h without aggregation. Process parameters such as albumin concentration, incubation time, temperature, pH, order of reagent addition, the presence of bivalent ions and the ionic strength of the complexation medium all influenced the complex size. Confocal laser scanning microscopy showed interactions of a Texas Red labeled cationized albumin with cell membranes of ECV 304 cells and an enhanced endocytic uptake compared to native albumin. The potential for introducing exogeneous DNA into cells was shown using NIH 3T3 fibroblasts. Successful, albeit low reporter gene expression could be achieved in the presence of chloroquine. Under in vitro conditions no toxic effect could be observed. In conclusion, cationized albumin may have promise as a non-toxic vector for gene delivery, especially for DNA vaccination.

Highly efficient transduction of endothelial cells by targeted artificial virus-like particles

Targeting the tumor vasculature by gene therapy is a potentially powerful approach, but suitable vectors have not yet been described. We have designed a new type of liposomal vector, based on the composition of anionic retroviral envelopes, that is serum-resistant and nontoxic. These artificial virus-like envelopes (AVEs) were endowed with a cyclic RGD-containing peptide as a targeting device for the avß3-integrin on tumor endothelial cells (ECs). The packaging of plasmid DNA complexed with low-molecular-weight, nonlinear polyethyleneimine into these AVEs yielded artificial virus-like particles (AVPs) that transduced ECs with efficiencies of up to 99%. In contrast, transduction of a variety of other cell types by these RGD–AVPs was comparably inefficient under the same experimental conditions. This EC selectivity was mediated, in part, but not exclusively, by the RGD ligand, as suggested by the reduced, but still relatively high, transduction efficiency seen with AVPs lacking RGD. The interaction of anionic lipids of the AVPs with ECs may therefore contribute to the observed selective and highly efficient transduction of this cell type. These findings suggest that the targeted AVE technology is a useful approach to create highly efficient nonviral vectors. Cancer Gene Therapy (2001) 8, 107–117

Functional domains in cyclin D1 : pRb-kinase activity is not essential for transformation

Although cyclin D1 plays a major role during cell cycle progression and is involved in human tumourigenesis, its domain structure is still poorly understood. In the present study, we have generated a series of cyclin D1 N- and C-terminal deletion constructs. These mutants were used to define the domains required for transformation of rat embryonal fibroblasts (REF) in cooperation with activated Ha-ras and and to establish correlations with defined biochemical properties of cyclin D1. Protein binding and REF assays showed that the region of the cyclin box required for the interaction with CDK4 as well as C-terminal sequences determining protein stability were crucial for transformation. Surprisingly, however, the N-terminal deletion of 20 amino acids which impaired pRb kinase activity did not affect the transforming ability of cyclin D1. Likewise, no effect on transformation was observed with mutants defective in p21CIP interaction. These observations argue against a crucial role of pRb inactivation or p21CIP squelching in cyclin D1-mediated transformation.

Combined transductional and transcriptional targeting of melanoma cells by artificial virus-like particles.

Artificial virus‐like particles (AVPs) represent a novel type of liposomal vector resembling retroviral envelopes. AVPs are serum‐resistant and non‐toxic and can be endowed with a peptide ligand as a targeting device. The vitronectin receptor, αvβ3‐integrin, is commonly upregulated on malignant melanoma cells. In the present study we investigated whether AVPs carrying cyclic peptides with an RGD integrin binding motif (RGD‐AVPs) are suitable for the specific and efficient transduction of human melanoma cells.

A NEW COLLOIDAL LIPIDIC SYSTEM FOR GENE THERAPY

ABSTRACT A novel type of liposomal vector for gene therapy is described (Artificial Virus Particles; AVPs). This vector is based on the composition of retroviral envelopes, serum-resistant and non-toxic and smaller than 200 nm in size. The DNA is condensed using low molecular weight branched PEI. Equipment of these particles with a cyclic RGD peptide ligand as targeting device renders them selective for tumor endothelial and melanoma cells expressing high levels of αvβ3-integrins, and allows for an efficient delivery of the enclosed genetic material. The specificity of the vector system for melanoma cells could be further improved by using a melanocyte-specific tyrosinase promoter to drive transgene expression.