Generation of Arabidopsis protein chips for antibody and serum screening.

Protein array technology has emerged as a new tool to enable ordered screening of proteins for expression and molecular interactions in high throughput. Besides classical solid-phase substrates, such as micro-titre plates and membrane filters, protein arrays have recently been devised with chip-sized supports. Several applications on protein chips have been described, but to our knowledge no studies using plant protein chips were published so far. The aim of this study was to generate Arabidopsis protein chips and to demonstrate the feasibility of the protein chip technology for the investigation of antigen-antibody interactions. Therefore, Arabidopsis cDNAs encoding 95 different proteins were cloned into a GATEWAY-compatible Escherichia coli expression vector. RGS-His6-tagged recombinant proteins were purified in high throughput and robotically arrayed onto glass slides coated either with a nitrocellulose based polymer (FAST slides) or polyacrylamide (PAA slides). Using an anti-RGS-His6 antibody all proteins were detected on the chips. The detection limit was ca. 2-3.6 fmol per spot on FAST slides or 0.1-1.8 fmol per spot on PAA slides. The Arabidopsis protein chips were used for the characterisation of monoclonal antibodies or polyclonal sera. We were able to show that a monoclonal anti-TCP1 antibody and anti-MYB6 and anti-DOF11 sera bound specifically to their respective antigens and did not cross-react with the other 94 proteins including other DOF and MYB transcription factors on the chips. To enable screening of antibodies or other interacting molecules against thousands of Arabidopsis proteins in future, we generated an ordered cDNA expression library and started with high-throughput cloning of full-length cDNAs with GATEWAY technology.

Treatment of colorectal liver metastases by adenoviral transfer of tissue inhibitor of metalloproteinases-2 into the liver tissue.

Metastatic disease is the leading cause of death in cancer patients. Here, we describe a novel gene therapeutic strategy for prevention of metastatic spread by providing a suitable defense mechanism for the target organ. The production of metalloproteinase (MMP) enzymes by cancer cells is critical for local invasion and for infiltration of metastatic cells into distant sites. Using a nude mouse model of colorectal liver metastasis, we have overexpressed the MMP inhibitor, tissue inhibitor of MMP-2 (TIMP-2) in the liver prior to, or following, tumor challenge by metastatic LS174T cells in vivo. Transduction of approximately 50% of hepatocytes resulted in 95% reduction in metastasis after tumor challenge compared with controls. Furthermore, TIMP-2 gene transfer into livers with preexisting metastatic spread resulted in a 77% reduction in tumor cell growth. Our data imply that MMP activity of metastatic cancer cells is required for spread and subsequent tumor growth and that enhancing antiproteolytic defense mechanisms in target organs represents a novel form of cancer gene therapy.

Induction of apoptosis and G2/M arrest by infection with replication-deficient adenovirus at high multiplicity of infection.

Replication-deficient adenoviruses are among the most widely used vectors in gene therapy and are also becoming increasingly popular as analytical tools in basic research. However, significant toxicity of these vectors in vivo has been reported. Here, we show that in an in vitro setting, first generation adenoviruses lead to growth retardation, prolongation of the G2/M phase and induction of apoptosis if applied at a high multiplicity of infection (MOI). These findings were obtained in p53-deficient hepatocytes, derived from knock-out mice (A2 cells) and in several tumor cell lines containing wild-type (wt) or mutant p53. Apoptosis induction was correlated with increased levels of p53 and bax proteins and it was stronger in cells containing wt p53 as compared with cells lacking functional p53. Apoptosis was highly dependent on the MOI used with marked effects starting at an MOI twice as high as needed for 100% gene transfer. Expression of the adenoviral E4 ORF6 gene as well as adenoviral replication were detected in all cell lines infected with first generation adenovirus. Apoptosis could be considerably reduced but not abrogated by UV inactivation of adenovirus, which indicates proapoptotic effects caused by the infection event as well as by residual adenoviral gene expression or adenoviral replication. First generation adenoviruses apparently display proapoptotic activity if used at higher MOIs, which may be of relevance when these vectors are used as analytical or gene therapeutic tools.