Rapid induction of IGF-IR signaling in normal and tumor tissue following intravenous injection of IGF-I in mice.

The detection of IGF-IR signaling in animal models has important implications for determining the role of this receptor in normal physiology and tumor growth. While many reports have correlated changes in plasma IGF-I levels in vivo with biological responses, few have shown that altered IGF-I levels can directly affect signaling within normal or tumor tissue. Here, we present new data that shows how the intravenous (IV) injection of IGF-I can be used to directly examine IGF signaling at the tissue level. Tail-vein IV injection of IGF-I into mice resulted in a rapid and dose-dependent activation of the IGF-I receptor and downstream phosphorylation of Akt and ERK1/2 in liver, kidney, and mammary gland. Similarly, IV IGF-I rapidly stimulated signaling in HT-29 colorectal and in MCF-7 breast cancer xenografts. This study shows how IV IGF injection can be used to examine the signaling mechanisms used by IGF-IR, in both normal mammary tissue and during tumor growth, and may provide a model for the characterization of IGF inhibitors.

Recombinant single-chain and disulfide-stabilized Fv-immunotoxins that cause complete regression of a human colon cancer xenograft in nude mice.

Monoclonal antibody (MAb) 55.1 specifically recognizes an antigen on the surface of human colon adenocarcinoma cells. We constructed recombinant immunotoxins composed of the heavy- and light-chain variable regions of MAb 55.1 fused to a recombinant form of Pseudomonas exotoxin (PE). The heavy- and light-chain variable regions are stabilized by 2 means. One is by a flexible peptide linker to form a single-chain antigen binding protein (scFv) and the second by an interchain disulfide bond engineered between structurally conserved framework regions. These are termed disulfide stabilized Fvs (dsFv). The 2 Fv forms are fused to truncated forms of PE lacking the cell binding domain. The recombinant scFv- and dsFv-immunotoxins were expressed in E. coli and purified to near homogeneity. The scFv- and dsFv-immunotoxins were shown to be specifically cytotoxic to human colon adenocarcinoma cell lines. The scFv-immunotoxin containing PE38KDEL was more active than the immunotoxin containing PE38 with the native carboxyl terminus (REDLK). However, the PE38KDEL immunotoxin is about 2-fold more toxic in mice, and therefore it does not appreciably increase the therapeutic window in mice. Intravenous administration of the scFv- and dsFv- recombinant immunotoxins caused complete regression of a human colon carcinoma (Colo205) growing subcutaneously in immunodeficient mice. The dsFv-immunotoxin has better antitumor activity compared with its scFv-immunotoxin counterpart.

Purification and characterization of active and stable recombinant plasminogen-activator inhibitor accumulated at high levels in Escherichia coli.

Plasminogen-activator inhibitor type 1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator, is an unusual member of the serine protease inhibitor (serpin) superfamily in that it spontaneously converts to a latent form lacking activity. This latent form can be reactivated by denaturation and refolding, but the activation is usually incomplete and often leads to aggregation of the protein. In this study we have developed a high-level expression system that leads to the accumulation of PAI-1 at 30-50% total microbial protein. We have developed a single-step purification protocol which can be completed in a few hours, yielding approximately 20 mg purified recombinant PAI-1/litre culture. The purified PAI-1 was 80-100% active and was stable upon incubation at 37 degrees C with a half-life of approximately 48 h. At 20 degrees C, PAI-1 activity was stable for a week and at 4 degrees C it retained its activity completely for up to two months. Freezing caused significant loss of activity. The stability of PAI-1 activity was found to be dependent on pH and ionic strength, being most stable at pH 5.6 and at an ionic strength of 1 M salt. We show that by a combination of high-level expression and rapid purification under optimum conditions, it is possible to produce active and stable PAI-1 in high yield.