The dual-function phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is the second most frequently mutated gene in human cancers. PTEN counteracts the functions of many growth factors, the most prevalent of which is insulin-like growth factor II (IGF-II). PTEN expression is stimulated by IGF-II forming a feedback loop. Investigating IGF-binding protein (IGFBP) modulation of IGF-II actions on MCF-7 breast cancer cells, we found that IGFBP-2 also regulates PTEN. The MCF-7 cells were not responsive to high doses of IGF-II due to induction of PTEN, which was not observed with an IGF-II-analog that does not bind to IGFBPs or in the presence of an inhibitor that prevents IGFs associating with IGFBPs. These cells predominantly produce IGFBP-2: blocking IGFBP-2 with a specific antibody, or preventing IGFBP-2 binding to integrins, restored the induction of PTEN and the cells were non-responsive to high doses of the IGF-II-analog. Our findings indicate that breast cancer cells do not respond to high doses of IGF-II due to induction of PTEN, but IGFBP-2, when free from IGF-II can suppress PTEN. Levels of IGFBP-2 are elevated frequently in human tumors: its ability to regulate PTEN could have important implications in relation to therapeutic strategies targeting growth factor pathways.

Plasminogen-activator inhibitor type 1 (PAI-l), 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-Mitre culture. The purified PAI-1 was 80-100% active and was stable upon incubation at 37°C with a half-life of approximately 48 h. At 20°C, PAI-1 activity was stable for a week and at 4°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 highlevel expression and rapid purification under optimum conditions, it is possible to produce active and stable PAI-1 in high yield.Activation of plasminogen to form the serine protease plasmin is central to the processes of fibrinolysis [l], cell migration, angiogenesis and tumour metastasis [2]. Activation of plasminogen is accomplished by either tissue-type or urokinase-type plasminogen activator. Regulation of the system occurs at several levels, including assembly of the components and stimulation of activation on fibrin [l] or at cell surfaces [3, 41. The inhibition of the active enzymes is an important element in the control of the plasminogedplasmin system. Plasminogen-activator inhibitor type 1 (PAI-1) is the main physiological inhibitor of both tissue-type plasminogen activator and urokinase-type plasminogen activator, with association rate constants of the order of 0.1 pM s- ' [5]. PAI-1 is present in a wide variety of tissues and cultured cells, where regulation of its expression by several stimuli has been observed [6]. It is present in the circulation both in cell-free plasma and in platelets, these two pools being clearly distinct [7]. The plasma concentration of PAI-1 is normally low, approximately 20 ng/ml, but higher levels are observed in a range of diseases, suggesting an association with thrombotic disorders [S]. The platelet pool of PAI-1 is released during aggregation, so that it protects the newly formed thrombus against premature dissolution. Platelet PAI-1 contributes to the resistance PAI-1 was identified as a member of the superfamily of serine protease inhibitors known as serpins, on the basis of its sequence [16-181. The serpins are the major class of inhibitors regulating the fibrinolytic, coagulation and complement cascades. They act as pseudosubstrates and form an SDS-stable covalent 1 : 1 complex with their target...

Resistance to antiepidermal growth factor (EGFR) strategies is an emerging clinical problem. Using human colorectal cancer (CRC) cells, we evaluated the involvement of the insulin receptor isoform-A (InsR-A) in de novo resistance to gefitinib, an EGFR tyrosine kinase inhibitor. Challenging the EGFR positive LoVo cells with gefitinib (1 μ M ) resulted in a small (∼18%) inhibition of cell growth and although a modest reduction in phospho (p)EGFR Tyr845 was seen, pEGFR at residues -Tyr1068 and -Tyr1173 were unchanged. LoVo cells produced unprocessed pro-IGF-1R protein, substantial levels of IGF-II mRNA and mature InsR protein, consisting mainly of the InsR-A isoform. Insulin and IGF-II promoted cell growth and pEGFR Tyr845, Tyr1068 and Tyr1173 activity and conversely, the insulin-like growth factor-1 receptor (IGF-1R)/InsR inhibitor ABDP (1 μ M ) inhibited growth and reduced pEGFR activity at all three tyrosine residues. pInsR and pAkt levels were increased after gefitinib treatment. Blocking of pInsR with ABDP enabled gefitinib to markedly reduce pEGFR Tyr845, Tyr1068 and Tyr1173. Short-term gefitinib/ABDP dual treatment was more effective than either agent alone and chronic exposure to this combination resulted in total cell loss after 9 weeks, preventing acquisition of resistance to ABDP. LoVo cells with acquired resistance to ABDP were acutely sensitive to gefitinib. We concluded that InsR-A reduces sensitivity to gefitinib in LoVo CRC cells, thus its co-targeting alongside EGFR can improve the anti-tumour effect of gefitinib.

Variants of human pancreatic carboxypeptidase B (HCPB), with specificity for hydrolysis of C-terminal glutamic acid and aspartic acid, were prepared by site-directed mutagenesis of the human gene and expressed in the periplasm of Escherichia coli. By changing residues in the lining of the S1' pocket of the enzyme, it was possible to reverse the substrate specificity to give variants able to hydrolyse prior to C-terminal acidic amino acid residues instead of the normal C-terminal basic residues. This was achieved by mutating Asp253 at the base of the S1' specificity pocket, which normally interacts with the basic side-chain of the substrate, to either Lys or Arg. The resulting enzymes had the desired reversed polarity and enzyme activity was improved significantly with further mutations at residue 251. The [G251T,D253K]HCPB double mutant was 100 times more active against hippuryl-L-glutamic acid (hipp-Glu) as substrate than was the single mutant, [D253K]HCPB. Triple mutants, containing additional changes at Ala248, had improved activity against hipp-Glu substrate when position 251 was Asn. These reversed-polarity mutants of a human enzyme have the potential to be used in antibody-directed enzyme prodrug therapy of cancer.

, 1988). New types of recombiriant Fv-immunotoxins were constructed in which the V H -V~ heterodimer is stabilized by an interchain disulfide bond engineered into structurally conserved framework residues of VH and VL (Brinkmann et al., 1993; Reiter et al., 19946,~). These types of molecules were termed disulfide stabilized Fvs(dsFvs) and dsFv-immunotoxins. They have good and in some cases improved activity in witro and in vivo cornpared with the corresponding scFv-immunotoxins (Reiter et al., 1994a,d;Benhar and Pastan, 1995). The targeting moiety (in scFv or dsFv form) is usually fused to a truncated form of Pseudornonas exotoxin but can also be fused to diphtheria toxin.