Among patients with non-small-cell lung cancer who receive erlotinib, the presence of an EGFR mutation may increase responsiveness to the agent, but it is not indicative of a survival benefit.

Purpose: The epidermal growth factor receptor (EGFR) is a key regulator of growth, differentiation, and survival of epithelial cancers. In a small subset of tumors, the presence of activating mutations within the ATP binding site confers increased susceptibility to gefitinib, a potent tyrosine kinase inhibitor of EGFR. Agents that can inhibit EGFR function through different mechanisms may enhance gefitinib activity in patients lacking these mutations. Mevalonate metabolites play significant roles in the function of the EGFR; therefore, mevalonate pathway inhibitors may potentiate EGFRtargeted therapies.Experimental Design: In this study, we evaluated the effect of lovastatin on EGFR function and on gefitinib activity. Effects on EGFR function were analyzed by Western blot analysis using phosphospecific antibodies to EGFR, AKT, and extracellular signal-regulated kinase. Cytotoxic effects of lovastatin and/or gefitinib were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry.Results: Lovastatin treatment inhibited EGF-induced EGFR autophosphorylation by 24 hours that was reversed by the coadministration of mevalonate. Combining lovastatin and gefitinib treatments showed enhanced inhibition of AKT activation by EGF in SCC9 cells. The combination of 10 Mmol/L lovastatin and 10 Mmol/L gefitinib treatments showed cooperative cytotoxicity in all 8 squamous cell carcinomas, 4 of 4 non -small cell lung carcinoma and 4 of 4 colon carcinoma cell lines tested. Isobologram and flow cytometric analyses of three representative cell lines with wild-type EGFR ATP binding sites confirmed that this combination was synergistic inducing a potent apoptotic response.Conclusions: Taken together, these results show that targeting the mevalonate pathway can inhibit EGFR function. They also suggest the potential utility of combining these clinically relevant therapeutic approaches.

Stable expression of high levels of activated forms of Haras (T24) or v‐Ki‐ras by transfection of Chinese hamster lung fibroblasts (CCL39) yielded cells highly tumorigenic in nude mice. Two classes of transformed cells were distinguished, one with moderate p21 expression (10‐fold increased) had retained growth factor dependency, the second with higher level of p21 (greater than 50‐fold) appeared autonomous for growth. Neither class of transformants expressing Ki‐ras or Ha‐ras displayed a significant basal activity of polyphosphoinositide‐specific phospholipase C, measured either in serum‐starved cells or during exponential growth in the presence of growth factors of the tyrosine kinase family (EGF, FGF, insulin). In the growth‐factor‐dependent class of T24‐Ha‐ras‐transfected cells (clone 39THaB), phospholipase C could be stimulated normally by serum, thrombin and AlF‐4. In the more growth autonomous class (clones 39THaC and 39Ki9), release of inositol phosphates after stimulation with thrombin or serum was drastically reduced. This desensitization, apparently at the receptor level since the response to AlF‐4 persisted, is, however, not specific to ras expression. We observed it to the same degree in polyoma virus‐transformed CCL39 cells. Finally, expression of mutated forms of p21 ras did not abrogate the sensitivity of phospholipase C activation to pertussis toxin. We conclude that the transforming potential of activated forms of p21ras does not result from persistent activation of phospholipase C and that ras GTP‐binding proteins cannot substitute for Gp.

Previous studies demonstrated that growth in DBA/2 mice of MDW4, a wheat germ agglutinin-resistant (WGA9 mutant of the highly metastatic MDAY-D2 DBA/2 mouse tumor, led to the emergence of WGA-sensitive (WGAS) revertants having higher ploidy levels at the site of inoculation as well as at distant visceral metastases. The results implied that MDW4 was nonmetastatic but progressed to become metastatic in vivo only after a cellular change took place which was accompanied by extinction of the WGAr phenotype and acquisition of a higher number of chromosomes. Results presented here provide strong and direct evidence for the underlying mechanism being spontaneous cell fusion in vivo between the MDW4

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A 6-thioguanine-resistant, human lymphoblastoid B-cell line (GM1500 6TG A-ll; IgG secreting) was mutagentreated with low-level V-irradiation and selected for ouabain resistance. One line showing 10,000-fold higher drug resistance, designated KR4, was fused with an Epstein-Barr virus-transformed, cloned, B-lymphocyte cell line (B6) producing antitetanus toxoid (TT) antibody (IgM), and the hybrids were selected in hypoxanthine/aminopterin/thymidine medium containing 10 JIM ouabain. Surviving cells, which arose at an optimal frequency of i0-5, were subcloned by limiting dilution and screened for anti-TT production. Out of395 final subclones, 372 were found positive for anti-TT, and seven that were selected for further study secreted specific antibody (IgM, K chain) at a maximum concentration of 3-6 #g/ml. The differential rate of anti-TT production during the logarithmic phase of cell growth was 15-fold higher in the hybridomas than in the original B6 line. The hybrid nature of the clones was confirmed by karyotype analysis, histocompatibility antigen typing, and expression of secreted and membrane-bound Ig classes. Biosynthetic labeling of the cells revealed that all hybrids secreted both IgM and IgG but that only the IgM class had specificity for TT. Because Epstein-Barr virus is a polyclonal Blymphocyte activator, the technique we applied here may be useful for increasing the recovery of rare antigen-specific B cells in the peripheral blood and for improving the frequency and stability of hybridomas secreting a given antibody.The production of human monoclonal antibodies expressing a desired specificity has proved successful with methods that use either the lymphotropic Epstein-Barr virus (EBV) to immortalize antigen-specific B cells (1)(2)(3)(4)(5) or the classical hybridoma technique, whereby human blood lymphocytes are fused with murine (6) or human myeloma cell lines (7,8). To overcome difficulties inherent in each technique (9), we examined the possibility of combining them. We have described a technique whereby lymphocytes from normal donors immunized with tetanus toxoid (TT) were preselected for antigen-binding cells, subsequently transformed with EBV, and cloned (10). In the present study, an EBV-transformed clone B6 was fused with a human lymphoblastoid cell line ofthe B-cell type (B-LCL), KR-4, to rescue high amounts of anti-TT antibody production. The resulting hybridomas were found to be more stable, to have a higher cloning efficiency, and to secrete =8-fold more specific antibody compared with the parental B6 clone. In addition KR-4 had a 25-fold higher frequency of hybridization with EBVtransformed B cells compared with nontransformed B cells. MATERIALS AND METHODSCell Lines. B6 is an anti-TT antibody-producing, EBV-transformed, cloned cell line that was previously established in our laboratory (10). GM 1500 6TG A-li is a 6-thioguanine-resistant (SGuaR) [hypoxanthine/aminopterin/thymidine (HAT)-sensitive] human B-cell line, which was generously provided by H. Koprowski at The Wistar Insti...

The thermostable transketolase from Geobacillus stearothermophilus (TK gst) was successfully engineered for the synthesis of aliphatic acyloins with varying carbon backbone lengths (C 5 À C 10) based on protein structure-guided studies. Efficient TK gst variants were identified with enhanced activities for substrate combinations of aliphatic aldehydes as acceptors together with aliphatic pyruvate homologues as donors. The TK gst single variant L382F was able to catalyze efficiently the transfer of the ketol group from hydroxypyruvate on all targeted aliphatic aldehydes (C 3 À C 8) to give the corresponding 1,3-dihydroxy ketones with good yields and excellent enantioselectivity. The combination of the H102L/H474S mutation previously designed for the improved utilization of aliphatic pyruvate homologues together with a F435I exchange gave the new variant H102L/H474S/F435I, which is able to transfer the acyl goup of 2-oxobutyrate and 2-oxovalerate to aliphatic aldehydes, giving mono hydroxylated ketones.

We have presented kinetic evidence for the occurrence in Escherichia coli K 12 of an active transport system responsible for 2-keto-3-deoxy-~-gluconate uptake. It has been studied in constitutive strains PK4 and CAK 101 since it is completely cryptic in the wild-type strain.1. The accumulation is temperature-dependent, coupled to electron and energy-yielding reactions, and is inhibited by thiol reagents.2. 2-Keto-3-deoxy-~-gluconate is not transported through the phospho-enolpyruvate-transferase system.3. Entry reaction displays saturation kinetics obeying the Michaelis-Menten laws. The K , value has been found to be 0.25 0.05 mM (at 30 "C and pH 7.0), by measuring initial rates of entrance as well as steady-state levels. Both the operator-constitutive PK4 strain and the regulator-constitutive CAKlOl strain exhibit the same affinity but apparent V are 10 and 20nmolx mg-1 x min-l, respectively. 4.The 2-keto-3-deoxy-n-gluconate permeation system is endogenously induced in a strain lacking 2-keto-3-deoxy-~-gluconate kinase, when grown on any intermediary product of the hexuronide catabolic pathway, able to enter the cell and anterior to 2-keto-3-deoxy-~-gluconate in the sequence. We presented further evidence making it very likely that 2-keto-3-deoxy-~-gluconate itself is the true inducer.5 . The constitutive synthesis of the transport system is sensitive to the catabolite repression exerted by glucose and pyruvate.G. By testing a wide number of sugars and derivatives as possible substrates, only D-glUCUr0-nate is a strictly competitive inhibitor (Ki = 1.5 mM). Since a specific hexuronate permease has been described elsewhere, we were led to demonstrate that n-glucuronate could be taken up through both its own specific system with high affinity and through the 2-keto-3-deoxy-~-gluconate specific system with lower affinity.7. The exit reaction rate is first oder with respect to internal concentration, temperaturedependent (exit rate constant kex is 0.0180 min-l a t 30 "C) but is not carrier-mediated. (Fig. I ) that has been discovered by Ashwell et al. [l]. 2-~eto-3-deoxy-~-gluconate is an intermediary product of the D-glucuronate and D-galacturonate catabolic pathwayThe main biochemical as well as physiological aspects of each degradative step of the Escherichia Abbreviations. vi,,, initial rate of the entry reaction; Gin, level of accumulation a t steady-state; vex, initial rate of the exit reaction; kex, exit rate constant.Enzymes. o-Altronate hydrolyase (EC 4.2.1.7); D-mannonate hydrolyase (EC 4.2.1.8); 2-keto-3-deoxy-~-gluconate kinase or ATP: 2-keto-3-deoxy-~-gluconate 6-phosphotransferase (EC 2.7.1.45) ; 2-keto-3-deoxy-6-phospho-~-gluconate aldolase (EC 4.1.2.14).coli K 12 sequence have been studied in this laboratory in order to approach the genetic regulation Two inducible pathways are simultaneously available for the further degradation of 2-keto-3-deoxy-D-glUCOnate. One of them is the phosphorylation into 2-keto-3-deoxy-G-phospho-~-gluconate catalyzed by a specific kinase (step 111) [I, GI, followed by it...