Role of phosphatidylinositol 4,5-bisphosphate in Ras/Rac-induced disruption of the cortactin-actomyosin II complex and malignant transformation.

Oncogenic Ras mutants such as v-Ha-Ras cause a rapid rearrangement of actin cytoskeleton during malignant transformation of fibroblasts or epithelial cells. Both PI-3 kinase and Rac are required for Ras-induced malignant transformation and membrane ruffling. However, the signal transduction pathway(s) downstream of Rac that leads to membrane ruffling and other cytoskeletal change(s) as well as the exact biochemical nature of the cytoskeletal change remain unknown. Cortactin/EMS1 is the first identified molecule that is dissociated in a Rac-phosphatidylinositol 4,5-biphosphate (PIP2)-dependent manner from the actin-myosin II complex during Ras-induced malignant transformation; either the PIP2 binder HS1 or the Rac blocker SCH51344 restores the ability of EMS1 to bind the complex and suppresses the oncogenicity of Ras. Furthermore, while PIP2 inhibits the actin-EMS1 interaction, HS1 reverses the PIP2 effect. Thus, we propose that PIP2, an end-product of the oncogenic Ras/PI-3 kinase/Rac pathway, serves as a second messenger in the Ras/Rac-induced disruption of the actin cytoskeleton and discuss the anticancer drug potential of PIP2-binding molecules.

Regulation of smooth muscle alpha-actin promoter in ras-transformed cells: usefulness for setting up reporter gene-based assay system for drug screening.

Oncogenic activation of ras results in changes in the transcription of several genes leading to uncontrolled cell growth. In this paper, we demonstrate that transformation of fibroblast cells by the ras oncogene leads to transcriptional repression of the smooth muscle alpha-actin promoter. Transient transfection analysis of plasmids containing the 5' upstream region of the human alpha-actin gene fused to human growth hormone or bacterial chloramphenicol acetyltransferase coding sequences into Rat-2 and ras-transformed Rat-2 (HO6) cells indicates that alpha-actin promoter is repressed in ras-transformed cells. In addition, stable rat fibroblast cell lines expressing human growth hormone or beta-galactosidase under the control of alpha-actin promoter exhibit repressed reporter gene activity following transformation by the ras oncogene. alpha-Actin promoter-driven beta-galactosidase activity is derepressed in revertants of ras-transformed stable cell lines. This revertant cell line expresses elevated levels of ras p21 protein and is resistant to retransformation by Ki and Ha-ras oncogenes. The revertant may have either a defective target protein whose activity is essential for the transforming activity of ras or an activated tumor suppressor gene which can suppress the activity of ras. These results indicate that smooth muscle alpha-actin promoter activity is a sensitive marker to follow phenotypic changes following transformation by ras and subsequent reversion. The advantages of this alpha-actin promoter-reporter gene assay system to screen for drugs that inhibit the transforming activity of ras, either directly or indirectly, are discussed.

SCH 51344 inhibits ras transformation by a novel mechanism.

A pyrazolo-quinoline compound, 6-methoxy-4-[2-[(2-hydroxyethoxyl)-ethyl]amino]-3-methyl-1M-pyrazo lo [3,4-b]quinoline (SCH 51344), was identified based on its ability to derepress human smooth muscle alpha-actin promoter activity in ras-transformed cells. In this study, we show that SCH 51344 reverts several key aspects of ras transformation, such as morphological changes, actin filament organization, and anchorage-independent growth, and also inhibits Val-12 Ras-induced maturation of Xenopus oocytes. SCH 51344 is also a potent inhibitor of the anchorage-independent growth of human tumor lines known to contain multiple genetic alterations in addition to activated ras genes. We have sought to determine whether SCH 51344 disrupts the signaling pathway that activates mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK) in normal and ras-transformed fibroblast cells. NIH 3T3 cells transformed by different oncogenes, which have products that participate at different steps of the Ras signaling pathway, were tested in a soft-agar colony formation assay to determine which step of the pathway is inhibited by SCH 51344. Our results indicate that SCH 51344 inhibits the ability of v-abl, v-mos, H-ras, v-raf, and mutant active MAP kinase kinase-transformed NIH 3T3 cells to grow in soft agar. Only v-fos-transformed cells were found to be resistant to the treatment of SCH 51344. SCH 51344 treatment had very little effect, if any, on the activation of MAP kinase kinase, MAP kinase, and p90RSK activity in response to growth factor stimulation. Treatment of ras-transformed cells with SCH 51344 led to stimulation of serum response factor DNA binding activity and activation of serum response element-dependent gene transcription, accounting for its ability to activate alpha-actin promoter activity in ras-transformed cells. Our results indicate that SCH 51344 inhibits ras transformation by a novel mechanism and acts at a point either downstream or parallel to extracellular signal-regulated kinase-dependent Ras signaling pathway.

Inhibition of angiogenesis and tumor growth by SCH221153, a dual alpha(v)beta3 and alpha(v)beta5 integrin receptor antagonist.

New blood vessel formation is essential for tumor growth and metastatic spread. Integrins alpha(v)beta3 and alpha(v)beta5 are arginine-glycine-aspartic acid-dependent adhesion receptors that play a critical role in angiogenesis. Hence, selective dual alpha(v)beta3 and alpha(v)beta5 antagonists may represent a novel class of angiogenesis and tumor-growth inhibitors. Here, an arginine-glycine-aspartic acid-based peptidomimetic library was screened to identify alpha(v)beta3 antagonists. Selected compounds were then modified to generate potent and selective dual inhibitors of alpha(v)beta3 and alpha(v)beta5 receptors. One of these compounds, SCH 221153, inhibited the binding of echistatin to alpha(v)beta3 (IC50 = 3.2 nM) and alpha(v)beta5 (IC50 = 1.7 nM) with similar potency. Its IC50 values for related alpha(IIb)beta3 and alpha5beta1 receptors were 1294 nM and 421 nM, respectively, indicating that SCH 221153 is highly selective for alpha(v)beta3 and alpha(v)beta5 receptors. In cell-based assays, SCH 221153 inhibited the binding of echistatin to alpha(v)beta3- and alpha(v)beta5-expressing 293 cells and blocked the adhesion of endothelial cells to immobilized vitronectin and fibroblast growth factor 2 (FGF2). SCH 221153, but not the inactive analogue SCH 216687, was effective in inhibiting FGF2 and vascular endothelial growth factor-induced endothelial cell proliferation in vitro with an IC50 equal to 3-10 microM. Angiogenesis induced by FGF2 in the chick chorioallantoic membrane assay was also inhibited by SCH 221153. Finally, SCH 221153 exerted a significant inhibition on tumor growth induced by intradermal or s.c. injection of human melanoma LOX cells in severe combined immunodeficient mice.

Signaling by integrin receptors.

Adhesive interactions are critical for the proliferation, survival and function of all cells. Integrin receptors as the major family of adhesion receptors have been the focus of study for more than a decade. These studies have tremendously enhanced our understanding of the integrin-mediated adhesive interactions and have unraveled novel integrin functions in cell survival mechanisms and in the activation of divergent signaling pathways. The signals from integrin receptors are integrated from those originating from growth factor receptors in order to organize the cytoskeleton, stimulate cell proliferation and rescue cells from matrix detachment-induced programmed cell death. These functions are critical in the regulation of multiple processes such as tissue development, inflammation, angiogenesis, tumor cell growth and metastasis and programmed cell death.

SCH 51344-induced reversal of RAS-transformation is accompanied by the specific inhibition of the RAS and RAC-dependent cell morphology pathway.

RAS interacts with multiple targets in the cell and controls at least two signaling pathways, one regulating extracellular signal-regulated kinase (ERK) activation and the other controlling membrane ruffling formation. These two pathways appear to act synergistically to cause transformation. SCH 51344 is a pyrazolo-quinoline derivative identified based on its ability to derepress transformation sensitive alpha-actin promoter in RAS-transformed cells. Previous studies have shown that SCH 51344 is a potent inhibitor of RAS-transformation. However, SCH 51344 had very little effect on the activities of proteins in the ERK pathway, suggesting that it inhibits RAS-transformation by a novel mechanism. In this study, we show that SCH 51344 specifically blocks membrane ruffling induced by activated forms of H-RAS, K-RAS, N-RAS and RAC. Treatment of fibroblast cells with this compound had very little effect on RAS-mediated activation of ERK and JUN kinase activities. SCH 51344 was effective in inhibiting the anchorage-independent growth of Rat-2 fibroblast cells transformed by the three forms of oncogenic RAS and RAC V12. These results indicate that SCH 51344 inhibits a critical component of the membrane ruffling pathway downstream from RAC and suggest that targeting this pathway may be an effective approach to inhibit transformation by RAS and other oncogenes.

Two serum response elements mediate transcriptional repression of human smooth muscle alpha-actin promoter in ras-transformed cells.

The mechanism by which activated ras oncogene expression leads to repression of genes encoding specific actin filament proteins is not understood. However, these changes associated with loss of organized actin filaments, are necessary to maintain the transformed phenotype. The human smooth muscle (sm) alpha-actin promoter is repressed in ras-transformed fibroblast cells and derepressed in revertant cell lines. In this study, we demonstrate that two serum response elements (SREs) present in the alpha-actin promoter are required for transcriptional repression in ras-transformed cells and the two SREs act synergistically to repress heterologous promoters in a ras-transformation dependent manner. Serum response factor (SRF), which can bind to the sm alpha-actin SREs, restores alpha-actin promoter activity in ras-transformed cells. c-Fos, c-Jun and YY1 also repress alpha-actin promoter through SREs, suggesting that these transcription factors may play a role in repressing alpha-actin promoter in ras-transformed cells.

Human smooth muscle alpha-actin gene is a transcriptional target of the p53 tumor suppressor protein.

Smooth muscle (sm) alpha-actin is expressed in vascular smooth muscle cells and fibroblast cells. Its expression is regulated by cell proliferation and repressed during oncogenic transformation. In this study, we demonstrate that p53 activation is associated with a dramatic increase in organized microfilament bundles and an increase in sm alpha-actin mRNA level. Wild-type p53, but not mutant p53, strongly stimulated human sm alpha-actin promoter activity in p53 null cell lines. The sequences homologous to the p53 consensus sequence and to the p53 binding sequence from the muscle creatine kinase, were found within a specific region of the sm alpha-actin promoter. This sequence was sufficient to confer p53-dependent activation to a heterologous promoter and p53 was capable of binding to this sequence as assessed by gel shift analysis. Ionizing irradiation of colorectal tumor cells caused an increase in alpha-actin mRNA level in a p53-dependent manner. Taken together, these results demonstrate that human sm alpha-actin gene is a transcriptional target for p53 tumor suppressor protein and represents the first example of a cytoskeletal gene with a functionally defined p53 response element.

3-Amino-1,2,4-triazole is an inhibitor of protein synthesis on mitoribosomes in Neurospora crassa.

The effects of the herbicide, 3-amino-1,2,4-triazole, an inhibitor of heme synthesis in rat liver, have been examined in the mold Neurospora crassa. The drug is a potent inhibitor of the growth of the mold and produces biochemical changes identical to those produced by chloramphenicol. 3-Amino-1,2,4-triazole, like chloramphenicol, is a direct and specific inhibitor of protein synthesis on mitoribosomes. A decrease in the levels of mitochondrial proteins which are completely or partly made on mitoribosomes and an accumulation in the levels of mitochondrial proteins of cytosolic origin have been observed. Both drugs depress porphyrin and heme levels, but there is actually an elevation in the levels of delta-aminolevulinate dehydratase, the rate-limiting enzyme of the heme-biosynthetic pathway in Neurospora crassa. In liver the enzyme is present in non-limiting amounts and the levels are depressed under conditions of 3-amino-1,2,4-triazole treatment. In Neurospora crassa the "derepression" of delta-aminolevulinate dehydratase under conditions of 3-amino-1,2,4-triazole or chloramphenicol treatment is only partial because the drugs inhibit protein synthesis on mitoribosomes. It is concluded that an optimal rate of protein synthesis on mitoribosomes is necessary to maintain an adequate rate of heme synthesis.

Expression, purification, characterization and homology modeling of active Akt/PKB, a key enzyme involved in cell survival signaling.

Akt is a serine/threonine kinase that plays a critical role in cell survival signaling and its activation has been linked to tumorigenesis. Up-regulation of Akt as well as its upstream regulator phosphatidylinositol-3 kinase (PI3K) has been found in many tumors and the negative regulator of this pathway PTEN/MMAC is a tumor suppressor. As a target for drug discovery, we have expressed and purified an active Akt1 enzyme from a recombinant baculovirus-infected Sf9 cell culture. Coexpression of Akt1 with the catalytic subunit of PI3K or treatment with okadaic acid during expression was found to generate an active enzyme in the insect cell culture system. We have optimized the kinase activity and developed a simple quantitative kinase assay using biotinylated peptide substrates. Using the purified active enzyme, we have characterized its physical, catalytic and kinetic properties. Since Akt is closely related to protein kinase C (PKC) and protein kinase A, the issue of obtaining selective inhibitors of this enzyme was addressed by comparison of the structures of catalytic domains of Akt and PKC, derived by homology modeling methods. A number of amino acid differences in the ATP binding regions of these kinases were identified, suggesting that selective inhibitors of Akt can be discovered. However, the ATP binding regions are highly conserved in the three isoforms of Akt implying that the discovery of isoform-selective inhibitors would be very challenging.

Chloramine T-induced structural and biochemical changes in echistatin.

Echistatin is a member of the disintegrin family of peptides and a potent inhibitor of platelet aggregation and cell adhesion. Echistatin binds to integrin alpha(v)beta3 and alpha(IIb)beta3 receptors with high affinity. Binding is mediated by an RGD-containing loop maintained in an appropriate conformation by disulfide bridges. In this study, we have compared the binding characteristics of echistatin iodinated by either lactoperoxidase or chloramine T method. We show that echistatin labeled by lactoperoxidase method binds to integrin alpha(v)beta3 receptor with high affinity and in a non-dissociable manner very similar to native echistatin. In contrast, chloramine T-labeled echistatin can rapidly dissociate from the receptor. We demonstrate that chloramine T reaction results in the addition of an extra oxygen to the methionine residue adjacent to the RGD motif in echistatin. Modeling studies and molecular dynamic simulation studies show that the extra oxygen atom on the methionine residue can form hydrogen bonds with the glycine and aspartic acid residues of the RGD motif. These structural changes in echistatin help explain the changes in the binding characteristics of the molecule following chloramine T reaction.

Essential role of Rac GTPase in hydrogen peroxide-induced activation of c-fos serum response element.

In the present study, we investigated whether hydrogen peroxide activates c-fos serum response element (SRE) in Rat-2 fibroblast cells. By transient transfection analysis, exogenous H2O2 stimulated SRE-dependent reporter gene activity in a dose and time-dependent manner. Also, we examined the role of Rac GTPase and phospholipase A2 (PLA2) in the H2O2-induced SRE activation. Either transfection of a dominant negative Rac mutant, RacN17, plasmid or pretreatment of mepacrine, a potent inhibitor of PLA2, blocked H2O2-induced SRE activation dramatically. Together, these findings suggest a critical role of 'Rac and subsequent activation of phospholipase A2' in the signaling pathway of H2O2 to SRE.

Activation of smooth muscle alpha-actin promoter in ras-transformed cells by treatments with antimitotic agents: correlation with stimulation of SRF:SRE mediated gene transcription.

Smooth muscle alpha-actin promoter is repressed in ras-transformed fibroblast cells and derepressed in revertant cells. We have recently shown that serum response factor (SRF) which can bind to the serum response elements (SREs) present in the alpha-actin promoter, can activate alpha-actin promoter activity in ras-transformed cells and suppress transformation by ras. Agents that stimulate SRF expression and alpha-actin promoter activity in ras-transformed cells are expected to be potential candidates as antitumor agents. In this study, we show that treatment of ras-transformed cells with antitumor agents such as taxol, vincristine, vinblastine, colchicine, and nocodazole leads to 5- to 7-fold activation of alpha-actin promoter driven CAT activity, whereas there was very little effect on thymidine kinase promoter driven CAT activity. This activation occurred at subcytotoxic concentrations of these agents and correlated with inhibition of cell cycle progression. Furthermore, these agents stimulated SRF expression in ras-transformed cells, as measured by its SRE binding activity. The increase in alpha-actin expression is accompanied by the restoration of actin filaments into organized bundles. These results suggest a novel mechanism by which antimitotic agents suppress the ras-transformed phenotype.

SCH 51344, an inhibitor of RAS/RAC-mediated cell morphology pathway.

RAS interacts with multiple targets in the cell and controls at least two signaling pathways, one regulating extracellular signal-regulated kinase (ERK) activation and the other controlling membrane ruffling formation. These two pathways appear to act synergistically to cause transformation. Human smooth muscle alpha-actin promoter is repressed in RAS-transformed cells and derepressed in revertant cell lines, suggesting that it is a sensitive marker to follow phenotypic changes in fibroblast cells. SCH 51344 is a pyrazoloquinoline derivative identified on the basis of its ability to derepress alpha-actin promoter in RAS-transformed cells. Previous studies have shown that SCH 51344 is a potent inhibitor of RAS transformation. However, SCH 51344 had very little effect on the activities of proteins in the ERK pathway, suggesting that it inhibits RAS transformation by a novel mechanism. Recently, we have demonstrated that SCH 51344 specifically blocks membrane ruffling induced by activated forms of H-RAS, K-RAS, N-RAS, and RAC. Treatment of fibroblast cells with this compound had very little effect on RAS-mediated activation of ERK and JUN kinase activities. SCH 51344 was effective in inhibiting the anchorage-independent growth of Rat-2 fibroblast cells transformed by the three forms of oncogenic RAS and RAC V12. These results indicate that SCH 51344 inhibits a critical component of the membrane ruffling pathway downstream from RAC and suggest that targeting this pathway may be an effective approach to inhibiting transformation by RAS and other oncogenes.

Drugs targeted against protein kinases.

Current treatments for cancer (surgery, radiation and chemotherapy) are successful for early stage localised disease but have severe side effects. New treatments are needed to increase the cure rate and life expectancy of patients. With the discovery of oncogenes, tumour suppressor genes and an understanding of their role in the development of the malignant disease, a new era of therapy has begun. Cancer is a manifestation of deregulated signalling pathways that mediate cell growth and programmed cell death. Protein kinases are essential elements in these signalling pathways. In the US, Novartis launched Gleevec (imantinib, STI-571) in May 2001 as the first anticancer drug whose mechanism of action is kinase inhibition. In Phase I trials, 23/24 patients with chronic myelogenous leukaemia (CML) had complete remissions and the drug is relatively non-toxic. Herceptin (trastuzumab) is a monoclonal antibody (mAb) against a member of the growth factor receptor family (HER-2/neu) that was launched in 1998 by Genentech for the treatment of breast cancer. Trastuzumab has an excellent antitumour profile, particularly when used in combination with doxorubicin and paclitaxol. These drugs are pioneering the treatment of cancer based on the molecular understanding of the disease. Numerous drugs that target growth factor receptors and their signalling pathways are in advanced clinical trials. Herein, antibodies against receptors and small molecule inhibitors of kinases in signalling pathways will be summarised. Inter-disciplinary preclinical studies have identified chemicals that target specific kinases. We believe that clinical studies of these agents will yield new anticancer agents that target specific diseases and that are less toxic than current agents.

[Inhibition of RAS-transformation by SCH51344].

RAS controls at least two signaling pathways, one regulating extracellular signal-regulated kinase (ERK) activation and the other controlling membrane ruffling formation. Activating RAS mutations are commonly found in human tumors, making RAS and its downstream signaling pathways important targets for tumor therapeutics. We have developed a reporter-gene based assay system, utilizing transformation sensitive alpha-actin promoter, to identify compounds that inhibit the transforming activity of RAS either directly or indirectly. SCH51344 is a pyrazolo-quinoline derivative, identified based on its ability to depreprses alpha-actin promoter in RAS-transformed cells and shown to be a potent inhibitor of RAS-transformation. However, this compound had very little effect on the activities of the proteins in the ERK pathway, suggesting that it inhibits RAS-transformation by a novel mechanism and acts on a signaling pathway distinct from ERK pathway. Recently, in collaboration with Dr. Dafna Bar-Sagi's group, we have shown that SCH51344 inhibits membrane ruffling induced by activated forms of H-RAS, K-RAS, N-RAS and RAC. Treatment of fibroblast cells with this compound had very little effect on RAS-mediated activation of ERK and Jun kinase activities. Our results indicate that SCH51344 inhibits a critical component of the membrane ruffling pathway downstream from RAC and suggest that targeting the membrane ruffling pathway may be an effective approach to inhibit transformation by RAS.

Human smooth muscle myosin light chain-2 gene expression is repressed in ras transformed fibroblast cells.

We have previously characterized human smooth muscle myosin light chain (MLC)-2 isoform by complementary DNA cloning and have shown that this isoform is expressed in a number of nonmuscle cells such as fibroblast cells. In this report, we show that when human osteosarcoma derived clonal cells (TE 85 clone F-5) (HOS), which are immortalized and nontumorigenic, undergo transformation following infection by Kirsten murine sarcoma virus (K-HOS) or by a chemical carcinogen [N-methyl-N-nitro-N-nitrosoguanidine (MNNG-HOS)], the smooth muscle MLC-2 mRNA is repressed. Revertants of transformed K-HOS cells (K-HOS312H) show normal levels of smooth muscle MLC-2 mRNA. Transformation of HOS cells by Ha-ras oncogene sequences, either by retroviral infection or by transfection followed by selection for tumorigenic cells in nude mice, results in complete repression of smooth muscle MLC-2 mRNA level. Treatment of HOS cells with tumor promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, results in repression of smooth muscle MLC-2 mRNA. Smooth muscle MLC-2 mRNA level is repressed in many, but not all, transformed cell lines, suggesting that it is not an indirect consequence of transformation but is specific to the agent that brings about transformation. HOS cells synthesize three MLC-2 protein species resolved by the two-dimensional gel electrophoretic system. The identity of the smooth muscle MLC-2 isoform was established by coelectrophoresis of the in vitro synthesized MLC-2 protein corresponding to the cloned complementary DNA in the two-dimensional gel system along with total [35S]methionine labeled HOS cell proteins. Quantitative analysis of MLC-2 isoforms in different HOS cells indicates that the synthesis of smooth muscle MLC-2 isoform is specifically repressed to an undetectable level in ras transformed and MNNG transformed cells and also following treatment with 12-O-tetradecanoylphorbol-13-acetate.