Changes in digestive enzyme activities during larval development of leopard grouper (Mycteroperca rosacea).

The leopard grouper is an endemic species of the Mexican Pacific with an important commercial fishery and good aquaculture potential. In order to assess the digestive capacity of this species during the larval period and aid in the formulation of adequate weaning diets, this study aimed to characterize the ontogeny of digestive enzymes during development of the digestive system. Digestive enzymes trypsin, chymotrypsin, acid protease, leucine-alanine peptidase, alkaline phosphatase, aminopeptidase N, lipase, amylase and maltase were quantified in larvae fed live prey and weaned onto a formulated microdiet at 31 days after hatching (DAH) and compared with fasting larvae. Enzyme activity for trypsin, lipase and amylase were detected before the opening of the mouth and the onset of exogenous feeding, indicating a precocious development of the digestive system that has been described in many fish species. The intracellular enzyme activity of leucine-alanine peptidase was high during the first days of development, with a tendency to decrease as larvae developed, reaching undetectable levels at the end of the experimental period. In contrast, activities of enzymes located in the intestinal brush border (i.e., aminopeptidase and alkaline phosphatase) were low at the start of exogenous feeding but progressively increased with larval development, indicating the gradual maturation of the digestive system. Based on our results, we conclude that leopard grouper larvae possess a functional digestive system at hatching and before the onset of exogenous feeding. The significant increase in the activity of trypsin, lipase, amylase and acid protease between 30 and 40 DAH suggests that larvae of this species can be successfully weaned onto microdiets during this period.

Metastasis-associated phosphatase PRL-2 regulates tumor cell migration and invasion.

The phosphatase of regenerating liver (PRL) family, comprising PRL-1, PRL-2 and PRL-3, is a group of prenylated phosphatases that are candidate cancer biomarkers and therapeutic targets. Although several studies have documented that altered expression of PRL-1 or PRL-3 can influence cell proliferation, migration and invasion, there is a dearth of knowledge about the biological functions of PRL-2. Thus, in the current study we have evaluated the role of PRL-2 in cell migration and invasion in human cancer cells. We found that four human lung cancer cells, including A549 cells, overexpress PRL-2 when compared with normal lung cells. PRL-2 knockdown by RNA interference markedly inhibited cell migration and invasion, and this inhibition can be restored by overexpressing the short interference RNA (siRNA)-resistant vector HA-PRL-2m. PRL-2 suppression by siRNA decreased p130Cas and vinculin expression, and decreased extracellular signal-regulated kinase (ERK) phosphorylation, while increasing the phosphorylation of ezrin on tyrosine 146. We found no significant changes in total p53, Akt and c-Src expression levels or their phosphorylation status, suggesting that PRL-2 knockdown could inhibit tumor cell migration and invasion through a Src-independent p130Cas signaling pathway. Ectopic expression of wild-type PRL-2, a catalytic inactive C101S mutant and a C-terminal CAAX deletion revealed a requirement for both the PRL-2 catalytic functionality and prenylation site. Expression of wild-type but not mutant forms of PRL-2 caused ERK phosphorylation and nuclear translocation. These results support a model in which PRL-2 promotes cell migration and invasion through an ERK-dependent signaling pathway.

Astrogliosis and behavioral changes in mice lacking the neutral cysteine protease bleomycin hydrolase.

Bleomycin hydrolase (BLMH) is a multifaceted neutral cysteine protease with a suggested role in antigen presentation, homocysteine-thiolactone metabolism, and Alzheimer's disease pathogenesis. Deletion of the protease in mice results in increased neonatal mortality and dermatopathology. Immunohistochemical and behavioral studies of BLMH knockout mice were undertaken to further evaluate the role of the protease in the brain. No gross abnormalities in the CNS were observed upon preliminary histological examination of B6.129Blmhtm1Geh/J null animals. However, glial fibrillary acid protein immunohistochemistry revealed a global reactive astrogliosis in the aged null animals, indicative of undefined brain pathology. The role of BLMH in the brain was further explored by characterizing the behavioral phenotype of hybrid [129S6-Blmhtm1Geh/JxB6.129 Blmhtm1Geh/J]F1 null and littermate controls using multiple behavioral paradigms. In the water maze, deletion of BLMH resulted in poorer performance during water maze probe trials without detectable effect of the mutation on sensorimotor function. In addition, no age-dependent decline in discriminative performance on probe trials was observed in null animals. These data suggest a physiological non-redundant function for BLMH in the CNS.

Discovery and biological evaluation of a new family of potent inhibitors of the dual specificity protein phosphatase Cdc25.

The Cdc25 dual specificity phosphatases have central roles in coordinating cellular signaling processes and cell proliferation, but potent and selective inhibitors are lacking. We experimentally examined the 1990 compound National Cancer Institute Diversity Set and then computationally selected from their 140 000 compound repository 30 quinolinediones of which 8 had in vitro mean inhibitory concentrations <1 microM. The most potent was 6-chloro-7-(2-morpholin-4-ylethylamino)quinoline-5,8-dione (NSC 663284), which was 20- and 450-fold more selective against Cdc25B(2) as compared with VHR or PTP1B phosphatases, respectively. NSC 663284 exhibited mixed competitive kinetics against Cdc25A, Cdc25B(2), and Cdc25C with K(i) values of 29, 95, and 89 nM, respectively. As compared with NSC 663284, the regioisomer 7-chloro-6-(2-morpholin-4-ylethylamino)quinoline-5,8-dione was 3-fold less active against Cdc25B(2) in vitro and less potent as a growth inhibitor of human breast cancer cells. Computational electrostatic potential mapping suggested the need for an electron-deficient 7-position for maximal inhibitor activity. Using a chemical complementation assay, we found that NSC 663284 blocked cellular Erk dephosphorylation caused by ectopic Cdc25A expression.

Involvement of Cdc25A phosphatase in Hep3B hepatoma cell growth inhibition induced by novel K vitamin analogs.

We previously found that K vitamin analogues caused cell growth inhibition in Hep3B hepatoma cells in vitro, which was associated with their inhibitory effects on protein tyrosine-phosphatases. In this study, we show that Cdc25A, a protein phosphatase, was inactivated by novel arylating K vitamin analogues. The inactivation of Cdc25A correlated with their effects on cell growth inhibition. Cyclin-dependent kinase (Cdk) 4, an important regulator for G(1) progression, was found to be tyrosine-phosphorylated by the arylating analogues, and this phosphorylation was correlated with the inhibitory effects of the analogues on Cdc25A activity. Furthermore, Cdk4 dephosphorylation experiments showed that Compound (Cpd) 5, a prototype arylating analogue, inhibited Cdc25A-mediated Cdk4 dephosphorylation, whereas Cpd 26, a nonarylating vitamin K analogue, had no effect on this event. We also examined Cdk4 kinase activity using retinoblastoma protein as a substrate and found that Cpd 5 inhibited retinoblastoma protein phosphorylation in a concentration-dependent manner, indicating that Cdk4 activity was inhibited by Cpd 5 treatment. Moreover, the thiol-antioxidants glutathione and N-acetyl-L-cysteine antagonized the Cpd 5-induced Cdk4 tyrosine phosphorylation, whereas the nonthiol-antioxidants catalase and superoxide dismutase did not. These results suggest that Hep3B cell growth inhibition by these K vitamin analogues may be related in part to inactivation of Cdc25A activity and support the hypothesis that Cdc25A is an attractive target for drugs designed to inhibit cancer cell growth.

New inhibitors of the thioredoxin-thioredoxin reductase system based on a naphthoquinone spiroketal natural product lead.

Natural products of the naphthoquinone spiroketal structural type served as lead structures for the development of novel inhibitors of the thioredoxin-thioredoxin reductase redox system. The most potent compound in this series inhibited thioredoxin with an IC(50) of 350 nM, and many derivatives showed low micromolar activities for growth inhibition against two breast cancer cell lines.

Cysteine 73 in bleomycin hydrolase is critical for amyloid precursor protein processing.

Human bleomycin hydrolase (hBH) is a neutral cysteine protease that may regulate the secretion of soluble amyloid precursor protein (APP) and amyloid beta (A(beta)), which is a major constituent of the Alzheimer's disease-associated amyloid plaques. We have now determined that APP interacts with hBH by using yeast two hybrid methods and in vitro binding studies revealed that APP interacted with a 68 amino acid region that includes the catalytic domain of hBH. Ectopic expression of hBH increased the secretion of A(beta) but not of a second secreted protein, apolipoprotein A-I. Expression of hBH in which the catalytic cysteine 73 was mutated to serine failed to increase A(beta) secretion. These results indicate a critical role for cysteine 73 of hBH in mediating APP processing.

Apolipoprotein A-I directly interacts with amyloid precursor protein and inhibits A beta aggregation and toxicity.

Amyloid precursor protein (APP) is the source of the neurotoxic amyloid beta (Abeta) peptide associated with Alzheimer's disease. Apolipoprotein A-I (apoA-I), a constituent of high-density lipoprotein complexes, was identified by a yeast two-hybrid system as a strong and specific binding partner of full-length APP (APPfl). This association between apoA-I and APPfl was localized to the extracellular domain of APP (APPextra). Furthermore, the interaction between apoA-I and APPfl was confirmed by coprecipitation using recombinant epitope-tagged APPextra and purified apoA-I. Several functional domains have been identified in APPextra, and we focused on a possible interaction between apoA-1 and the pathologically important Abeta peptide, because APPextra contains the nontransmembrane domain of Abeta. The binding between apoA-I and Abeta was saturable (K(d) = 6 nM), specific, and reversible. APPextra also competed with apoA-I for binding to Abeta. Direct evidence for this interaction was obtained by the formation of an SDS-resistant Abeta-apoA-I complex in polyacrylamide gels. Competitive experiments with apolipoprotein E (isoforms E2 and E4) showed that apoA-I had a higher binding affinity for Abeta. We also found that apoA-I inhibited the beta-sheet formation of Abeta with a mean inhibitory concentration close to that of alpha2-macroglobulin. Finally, we demonstrated that apoA-I attenuated Abeta-induced cytotoxicity. These results suggest apoA-I binds to at least one extracellular domain of APP and has a functional role in controlling Abeta aggregation and toxicity.

Spatial analysis of key signaling proteins by high-content solid-phase cytometry in Hep3B cells treated with an inhibitor of Cdc25 dual-specificity phosphatases.

Protein phosphorylation frequently results in the subcellular redistribution of key signaling molecules, and this spatial change is critical for their activity. Here we have probed the effects of a Cdc25 inhibitor, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, or Compound 5, on the spatial regulation and activation kinetics of tyrosine phosphorylation-dependent signaling events using two methods: (i) high-content, automated, fluorescence-based, solid-phase cytometry and (ii) a novel cellular assay for Cdc25A activity in intact cells. Immunofluorescence studies demonstrated that Compound 5 produced a concentration-dependent nuclear accumulation of phospho-Erk and phospho-p38, but not nuclear factor kappaB. Immunoblot analysis confirmed Erk phosphorylation and nuclear accumulation, and in vitro kinase assays showed that Compound 5-activated Erk was competent to phosphorylate its physiological substrate, the transcription factor Elk-1. Pretreatment of cells with the MEK inhibitor U-0126 prevented the induction by Compound 5 of phospho-Erk (but not phospho-p38) nuclear accumulation and protected cells from the antiproliferative effects of Compound 5. Overexpression of Cdc25A in whole cells caused dephosphorylation of Erk that was reversed by Compound 5. The data show that an inhibitor of Cdc25 increases Erk phosphorylation and nuclear accumulation and support the hypothesis that Cdc25A regulates Erk phosphorylation status.

Antimitotic actions of a novel analog of the fungal metabolite palmarumycin CP1.

The pentacyclic palmarumycins are structurally unique natural products with both antifungal and antibacterial activities but their antineoplastic effects are not well established. We have examined their antiproliferative actions against tumor cells using a temperature-sensitive tsFT210 mouse mammary carcinoma cell line and found that a novel palmarumycin analog, [8-(furan-3-ylmethoxy)-1-oxo-1,4-dihydronaphthalene-4-spiro-2'-naphtho[1",8"-de][1',3'][dioxin] or SR-7, prominently blocked mammalian cell cycle transition in G2/M but not in G1 phase. We found no evidence for inhibition of the critical mitosis-controlling cyclin-dependent kinase Cdk1, or its regulator, the dual specificity phosphatase Cdc25. Moreover, Cdk1 was hypophosphorylated and not directly inhibited by SR-7. SR-7 also failed in vitro to hypernucleate bovine tubulin, did not compete with colchicine for tubulin binding, and only modestly blocked GTP-induced assembly. In addition, SR-7 caused almost equal inhibition of paclitaxel-sensitive and -resistant cell growth. Moreover, unlike benchmark tubulin-disrupting agents, SR-7 did not cause hyperphosphorylation of the antiapoptotic protein Bcl-2. Thus, SR-7 represents a novel chemical structure that can inhibit G2/M transition by a mechanism that appears to be independent of marked tubulin disruption.

Sulfonylated aminothiazoles as new small molecule inhibitors of protein phosphatases.

Based on a previously identified lead structure, SC-alphaalphadelta9, we have developed a versatile new chemical scaffold that can be readily modified to generate libraries of both Tyr and dual specificity phosphatase inhibitors with reduced molecular weight and lipophilicity. The most potent analogue identified to date, aminothiazole 8z, inhibits the dual specificity phosphatase Cdc25B with a Ki of 4.6+/-0.4 microM and a Hill coefficient of 2.

Reconstitution of apo-superoxide dismutase by nitric oxide-induced copper transfer from metallothioneins.

Little is known about copper transfer from Cu-metallothionein (Cu-MT) to various target proteins, such as apo-SOD, and the potential role of redox mechanisms in this transfer. We studied Cu transfer from Cu-MT to apo/Zn-SOD in a cell-free model system and found that Cu(5)-MT and Cu(10)-MT were able to reconstitute SOD activity only in the presence of a nitric oxide donor, (Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium++ +-1,2-diolate (NOC-15). The percentage of reconstitution by Cu(5)-MT and Cu(10)-MT was 34 and 83%, respectively, compared with that reconstituted by free Cu alone. A Cu chelation assay using bathocuproine disulfonate (BCS) showed that NOC-15 induced release of free Cu from Cu(10)-MT but not from Cu(5)-MT. The transfer of Cu from Cu-MT to apo/Zn-SOD was not accompanied by enhanced Cu-dependent generation of ascorbate radicals or hydroxyl radicals as measured by EPR spectroscopy. We found a 70% decrease in the number of 2,2'-dithiodipyridine titratable SH groups on MT after incubation with NOC-15. Overall, our results suggest that Cu-MT could potentially function in a nitric oxide-dependent pathway for the delivery of Cu to apo-SOD in copper-challenged cells.

Human bleomycin hydrolase regulates the secretion of amyloid precursor protein.

Human bleomycin hydrolase (hBH) is a neutral cysteine protease genetically associated with increased risk for Alzheimer disease. We show here that ectopic expression of hBH in 293APPwt and CHOAPPsw cells altered the processing of amyloid precursor protein (APP) and increased significantly the release of its proteolytic fragment, beta amyloid (Abeta). We also found that hBH interacted and colocalized with APP as determined by subcellular fractionation, in vitro binding assay, and confocal immunolocalization. Metabolic labeling and pulse-chase experiments showed that ectopic hBH expression increased secretion of soluble APPalpha/beta products without changing the half-life of cellular APP. We also observed that this increased Abeta secretion was independent of hBH isoforms. Our findings suggest a regulatory role for hBH in APP processing pathways.

Antitumor imidazolyl disulfide IV-2 causes irreversible G(2)/M cell cycle arrest without hyperphosphorylation of cyclin-dependent kinase Cdk1.

Aberrant function of redox-regulated proteins is a possible cause for cellular transformation and loss of cell cycle control. The small protein thioredoxin has oncogenic properties and controls cell cycle movement through G(1), S, and G(2)/M phases. The redox-active, asymmetrical 1-methylpropyl-2-imidazolyl disulfide (IV-2) has previously been shown to react with and inhibit thioredoxin activity in vitro, the proliferation of human tumor cells in culture, and the growth of tumors in mice. We now examined the effects of IV-2 on cell cycle progression. In synchronized tsFT210 mouse mammary carcinoma cells, IV-2 halted cells in mitosis. In asynchronously growing MCF-7 human breast cancer cells, IV-2 exclusively and irreversibly blocked cells in G(2)/M at concentrations that correlated with its growth inhibitory activity. Neither the closely related, less redox active 2-hydroxy-1-methylpropyl-2-imidazolyl disulfide (AIV-2), which differs from IV-2 only by an additional hydroxyl group, nor the symmetrical diallyl disulfide caused a G(2)/M arrest under these conditions. Furthermore, MCF-7 cells treated with IV-2 showed increased Cdk1 kinase activity and a decrease in Cdk1 tyrosine phosphorylation, indicating that IV-2 did not directly inhibit Cdk1 or Cdc25 activities. IV-2 did, however, increase Bcl-2 phosphorylation. These data suggest that the thioredoxin inhibitor IV-2, despite its simple structure, is able to target redox-sensitive processes that are critical for cell cycle progression through mitosis. The results are also consistent with a role of thioredoxin regulating cell cycle progression through G(2)/M.

Identification of new Cdc25 dual specificity phosphatase inhibitors in a targeted small molecule array.

Dual specificity protein phosphatases (DSPases) are key regulators of signal transduction, oncogenesis and the cell cycle. Few potent or specific inhibitors of DSPases, however, are readily available for these pharmacological targets. We have used a combinatorial/parallel synthetic approach to rigidify the variable core region and modify the side chains of 4-(benzyl-(2-[2,5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)- 2-decanoylamino butyric acid (or SC-alphaalphadelta9), which is the most active element in a previously described library of phosphatase inhibitors (Rice, R. L.; Rusnak, J. M.; Yokokawa, F.; Yokokawa, S.; Messner, D. J.; Boynton, A. L.; Wipf, P.; Lazo, J. S. Biochemistry 1997, 36, 15965). Several analogues were identified as effective inhibitors of the protein tyrosine phosphatase (PTPase) PTP1B and the DSPases VHR and Cdc25B2. Two compounds, FY3-alphaalpha09 and FY21-alphaalpha09, were partial competitive inhibitors of Cdc25B2 with Ki values of 7.6+/-0.5 and 1.6+/-0.2 microM, respectively. FY21-alphaalpha09 possessed only moderate activity against PTP1B. Consistent with its in vitro anti-phosphatase activity, FY21-alphaalpha09 inhibited growth in MDA-MB-231 and MCF-7 human breast cancer cell lines. FY21-alphaalpha09 also inhibited the G2/M transition in tsFT210 cells, consistent with Cdc25B inhibition. Several architectural requirements for DSPase inhibition were revealed through modification of the side chain moieties or variable core region of the pharmacophore, which resulted in decreased compound potency. The structure of FY21-alphaalpha09 provides a useful platform from which additional potent and more highly selective phosphatase inhibitors might be generated.

Expert and layperson perceptions of ecosystem risk.

This research examines and compares perceptions held by laypeople and ecologists about risks to ecosystems, particularly risk from global climate change (GCC). A survey elicited perceptions of 31 risk characteristics for 13 GCC and 12 non-GCC risks to ecosystems. Factor analysis was used to examine the structure of layperson and expert risk perceptions. Both experts and laypeople tend to perceive GCC risks to ecosystems as less avoidable and more acceptable than risks from other causes. Compared to laypeople's perceptions, though, experts perceived GCC risks to have slightly lower impacts, be less avoidable, more acceptable, and less understandable than non-GCC risks to ecosystems. These findings may help guide efforts to communicate with laypeople about ecological risks from climate change.

Combinatorial chemistry and contemporary pharmacology.

Both solid- and liquid-phase combinatorial chemistry have emerged as powerful tools for identifying pharmacologically active compounds and optimizing the biological activity of a lead compound. Complementary high-throughput in vitro assays are essential for compound evaluation. Cell-based assays that use optical endpoints permit investigation of a wide variety of functional properties of these compounds including specific intracellular biochemical pathways, protein-protein interactions, and the subcellular localization of targets. Integration of combinatorial chemistry with contemporary pharmacology now represents an important factor in drug discovery and development.

Cdc25 inhibition and cell cycle arrest by a synthetic thioalkyl vitamin K analogue.

A synthetic vitamin K analogue, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or compound 5 (Cpd 5), was found previously to be a potent inhibitor of tumor cell growth. We now demonstrate that Cpd 5 arrested cell cycle progression at both G1 and G2-M. Because of the potential arylating activity of Cpd 5, it might inhibit Cdc25 phosphatases, which contain a cysteine in the catalytic site. To test this hypothesis, we examined the inhibitory activity of Cpd 5 against several cell cycle-relevant protein tyrosine phosphatases and found that Cpd 5 was a potent, selective, and partially competitive inhibitor of Cdc25 phosphatases. Furthermore, Cpd 5 caused time-dependent, irreversible enzyme inhibition, consistent with arylation of the catalytic cysteine in Cdc25. Treatment of cells with Cpd 5 blocked dephosphorylation of the Cdc25C substrate, Cdc2, and its kinase activity. Cpd 5 enhanced tyrosine phosphorylation of both potent regulators of G1 transition, ie., Cdk2 and Cdk4, and decreased the phosphorylation of Rb, an endogenous substrate for Cdk4 kinase. Furthermore, close chemical analogues that lacked in vitro Cdc25 inhibitory activity failed to block cell cycle progression and Cdc2 kinase activity. Cpd 5 did not alter the levels of p53 or the endogenous cyclin-dependent kinase inhibitors, p21 and p16. Our results support the hypothesis that the disruption in cell cycle transition caused by Cpd 5 was attributable to intracellular Cdc25 inhibition. This novel thioalkyl K vitamin analogue could be useful for cell cycle control studies and may provide a valuable pharmacophore for the design of future therapeutics.

Nitric oxide dissociates lipid oxidation from apoptosis and phosphatidylserine externalization during oxidative stress.

Oxidative stress in biological membranes can regulate various aspects of apoptosis, including phosphatidylserine (PS) externalization. It is not known, however, if the targets for these effects are lipids or proteins. Nitric oxide (NO), a bifunctional modulator of apoptosis, has both antioxidant and prooxidant potential. We report here that the NO donor PAPANONOate completely protected all phospholipids, including PS, from oxidation in HL-60 cells treated with 2,2'-azobis(2,4-dimethylisovaleronitrile) (AMVN), presumably via the ability of NO to react with lipid-derived peroxyl radicals and terminate the propagation of lipid peroxidation. PAPANONOate, however, had no effect on PS externalization or other markers of apoptosis following AMVN. Therefore, PS oxidation is not required for PS externalization during AMVN-induced apoptosis. PS externalization was accompanied by inhibition of aminophospholipid translocase (APT). NO potentiated AMVN inhibition of APT. Treatment with PAPANONOate alone produced modest (20%) inhibition of APT without PS externalization. NO did not reverse AMVN-induced oxidation of glutathione and protein thiols. We speculate that APT was sensitive to AMVN and/or NO via modification of protein thiols critical for functional activity. Therefore, the lipoprotective effects of NO were insufficient to prevent PS externalization and apoptosis following oxidative stress. Other targets such as protein thiols may be important redox-sensitive regulators of apoptosis initiation and execution. Thus, in the absence of significant peroxynitrite formation, NO's antioxidant effects are restricted to protection of lipids, while modification of protein substrates continues to occur.

In vivo antitumor activity and induction of insulin-like growth factor-1-resistant apoptosis by SC-alphaalphadelta9.

We previously showed that SC-alphaalphadelta9 (4-(benzyl-(2-[(2, 5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylami no butyric acid) is a novel antiphosphatase agent that selectively inhibits the growth of transformed cells in culture and affects elements of insulin-like growth factor-1 (IGF-1) signaling. We now show that SC-alphaalphadelta9 induces IGF-1-resistant apoptosis and kills tumor cells in vivo. In cultured murine 32D cells, SC-alphaalphadelta9 induced concentration-dependent apoptosis that was blocked by ectopic Bcl-2 expression. No apoptosis was detected in 32D cells treated with the congener SC-alpha109, which lacks the ability to disrupt IGF-1 signaling. After interleukin-3 withdrawal or etoposide treatment, exogenous IGF-1 prevented apoptosis and elevated levels of Cdc2, a biochemical indicator of a functional IGF-1 receptor pathway. In contrast, exogenous IGF-1 did not prevent apoptosis or loss of Cdc2 expression caused by SC-alphaalphadelta9. Furthermore, IGF-1 receptor overexpression failed to protect cells against SC-alphaalphadelta9-induced apoptosis. Kinetic analyses demonstrated that Cdc2 down-regulation after SC-alphaalphadelta9 treatment preceded both apoptosis and loss of the IGF-1 receptor, indicating that loss of Cdc2 was a direct effect of SC-alphaalphadelta9 treatment and not secondary to cell death. IGF-1 receptor autophosphorylation studies indicated that SC-alphaalphadelta9 did not interact directly with the IGF-1 receptor nor bind to the growth factor itself, suggesting a site of action distal to the IGF-1 receptor. In the SCCVII murine tumor model, a single i.p. injection of SC-alphaalphadelta9 caused a dose-dependent decrease in clonogenic cell survival. The IC(50) of SC-alphaalphadelta9 was 35 mg/kg, comparable to 25 mg/kg carboplatin. The ability to induce IGF-1-resistant apoptosis distinguishes SC-alphaalphadelta9 from other apoptosis-inducing agents and suggests compounds of this class deserve further study as potential anticancer agents.