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Signal transduction in confluent C3H 10T1/2 cells. The role of focal adhesion kinase.

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Miłoszewska J., Trembacz H., Janik P.

2001

vol. 48

issue 1

175-181

The activities of extracellular signal-regulated kinases (ERK1/ERK2) is required for proliferation of several types of cells. The performed analysis showed stimulation of ERK's by fetal calf serum (FCS) or fibronectin in the C3H 10T1/2 cell cultures at logarithmic phase of growth. The ERKs activity was not stimulated in confluent cells. This could not be accounted for a partial down regulation of ERK since its level was stable in both types of cells regardless of their density and kind of stimulation. Searching for ERK upstream elements we studied the integrin receptor gene transcript by RT-PCR and focal adhesion kinase (FAK) by Western blotting and phosphorylation assays. It was found that FCS and fibronectin stimulated phosphorylating activity of FAK in the cells at the logarithmic phase of growth, but were inefficient in the confluent cells. RT-PCR showed the presence of α5 and β1 integrin transcripts, and p125FAK was at the same level regardless of the type of stimulation. These data indicate that the ability of FAK to be activated plays an important role in ERK regulation and, in consequence in proliferation and growth inhibition during confluence.

Construction of a bicistronic proangiogenic expression vector and its application in experimental angiogenesis in vivo.

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Małecki M., Przybyszewska M., Janik P.

2003

vol. 50

issue 3

875-882

Manipulation of angiogenesis in vivo is an example of successful gene therapy strategies. Overexpression of angiogenic genes like VEGF, FGF or PDGF causes new vessel formation and improves the clinical state of patients. Gene therapy is a very promising procedure but requires large amounts of pharmaceutical-grade plasmid DNA. In this regard we have constructed a bicistronic plasmid DNA vector encoding two proangiogenic factors, VEGF165 and FGF-2. The construct (pVIF) contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV) which permits both genes to be translated from a single bicistronic mRNA. The IRES sequence allows for a high efficiency of gene expression in vivo. The pVIF vector was characterized in vitro and in vivo. In vivo angiogenesis studies showed that the bicistronic vector encoding two proangiogenic factors induces the formation of new vessels significantly more than pVEGF165 or pFGF-2 alone. In our opinion the combined proangiogenic approach with VEGF165 and FGF-2 is more powerful and efficient than single gene therapy. We also postulate that IRES sequence can serve as a useful device improving efficiency of gene therapy.

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2 Acta Biochimica Polonica

1 2003

1 2001

2 Janik P.

1 Małecki M.

1 Miłoszewska J.

1 Przybyszewska M.

1 Trembacz H.

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Signal transduction in confluent C3H 10T1/2 cells. The role of focal adhesion kinase.

Construction of a bicistronic proangiogenic expression vector and its application in experimental angiogenesis in vivo.