The antiangiogenic protein kinase inhibitors SU5416 and SU6668 inhibit the SCF receptor (c-kit) in a human myeloid leukemia cell line and in acute myeloid leukemia blasts.

SU5416 and SU6668 are potent antiangiogenic small-molecule inhibitors of receptor tyrosine kinases, including those of the vascular endothelial growth factor and platelet-derived growth factor receptor families. The stem cell factor (SCF) receptor, c-kit, is structurally related to these receptors and, although not expressed on mature peripheral blood cells, is expressed in leukemic blasts derived from 60% to 80% of acute myeloid leukemia (AML) patients. The c-kit kinase inhibitory activity of SU5416 and SU6668 was evaluated in MO7E cells, a human myeloid leukemia cell line. Tyrosine autophosphorylation of the receptor, induced by SCF, was inhibited in these cells by SU5416 and SU6668 in a dose-dependent manner (inhibitory concentration of 50% [IC(50)] 0.1-1 microM). Inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, a signaling event downstream of c-kit activation, was also inhibited in a dose-dependent manner. Both compounds also inhibited SCF-induced proliferation of MO7E cells (IC(50) 0.1 microM for SU5416; 0.29 microM for SU6668). Furthermore, both SU5416 and SU6668 induced apoptosis in a dose- and time-dependent manner as measured by the increase in activated caspase-3 and the enhanced cleavage of its substrate poly(ADP-ribose) polymerase. These findings with MO7E cells were extended to leukemic blasts from c-kit(+) patients. In patient blasts, both SU5416 and SU6668 inhibited SCF-induced phosphorylation of c-kit and ERK1/2 and induced apoptosis. These studies indicate that SU5416 and SU6668 inhibit biologic functions of c-kit in addition to exhibiting antiangiogenic properties and suggest that the combination of these activities may provide a novel therapeutic approach for the treatment of AML.

Development of SU5416, a selective small molecule inhibitor of VEGF receptor tyrosine kinase activity, as an anti-angiogenesis agent.

Angiogenesis, or the sprouting of new blood vessels, is a central process in the growth of solid tumors. For many cancers, the extent of vascularization of a tumor is a negative prognostic indicator signifying aggressive disease and increased potential for metastasis. Recent efforts to understand the molecular basis of tumor-associated angiogenesis have identified several potential therapeutic targets, including the receptor tyrosine kinases for the angiogenic factor vascular endothelial growth factor (VEGF). Here we review the approach taken at SUGEN, Inc. to discover and develop small molecule inhibitors of receptor tyrosine kinases as anti-angiogenic agents. We focus on SU5416, a selective inhibitor of VEGF receptors that is currently in clinical development for the treatment of advanced malignancies. Its biochemical, biological and pharmacological properties are reviewed and clinical implications discussed.

Regulated expression of Wnt family members during neuroectodermal differentiation of P19 embryonal carcinoma cells: overexpression of Wnt-1 perturbs normal differentiation-specific properties.

The P19 embryonal carcinoma (EC) cell line represents a useful model system for analysis of neural development and differentiation processes that are difficult to study in mammalian embryos. Since many members of the Wnt family of signaling molecules are expressed in the developing as well as adult nervous system, we have examined expression of these genes in P19 cells. Analysis of the mRNA accumulation profiles for Wnt genes during retinoic acid (RA)-induced neural differentiation of P19 cells showed that nine Wnt family members were expressed in a regulated manner during this process. Most were induced by RA treatment, and some were also expressed in undifferentiated P19 cells. Since Wnt-1 is not expressed in undifferentiated P19 cells but is induced during neuroectodermal differentiation we have generated P19 cell lines that overexpress Wnt-1 in the absence of RA treatment, in order to address the role of Wnt-1 in P19 differentiation. In the presence of ectopic Wnt-1, expression of other endogenous Wnt genes, which serve as early differentiation markers in this system, were induced without RA, which is normally required for appearance of these gene products. Furthermore, ectopic expression of Wnt-1 resulted in a loss of SSEA-1 antigen expression, a marker of undifferentiated P19 cells. Similarly to the parental cell line, addition of RA to P19 cells overexpressing Wnt-1 induced the neuroectodermal pathway, but expression of cell type-specific markers such as MASH-1, HNK-1, and GAP-43 was diminished and the morphology of neuronal processes, stained with an antibody to neurofilament, was abnormal. These data suggest that Wnt-1 itself can induce some aspects of early neuroectodermal differentiation and, furthermore, that the correct timing of Wnt-1 expression is necessary for proper RA-induced expression of the neural phenotype.

Xenopus gamma-crystallin gene expression: evidence that the gamma-crystallin gene family is transcribed in lens and nonlens tissues.

Crystallins, the major gene products of the lens, accumulate to high levels during the differentiation of the vertebrate lens. Although crystallins were traditionally thought to be lens specific, it has recently been shown that some are also expressed at very low levels in nonlens tissues. We have examined the embryonic expression pattern of gamma-crystallins, the most abundant crystallins of the embryonic lens in Xenopus laevis. The expression profile of five Xenopus gamma-crystallin genes mirrors the pattern of lens differentiation in X. laevis, exhibiting on average a 100-fold increase between tailbud and tadpole stages. Four of these genes are also ubiquitously expressed outside the lens at a very low level, the first demonstration of nonlens expression of any gamma-crystallin gene; expression of the remaining gene was not detected outside the head region, thus suggesting that there may be two classes of gamma-crystallin genes in X. laevis. Predictions regarding control mechanisms responsible for this dual mode of expression are discussed. This study raises the question of whether any crystallin, on stringent examination, will be found exclusively in the lens.

Wnt family proteins are secreted and associated with the cell surface.

Members of the Wnt gene family are proposed to function in both normal development and differentiation as well as in mammary tumorigenesis. To understand the function of Wnt proteins in these two processes, we present here a biochemical characterization of seven Wnt family members. For these studies, AtT-20 cells, a neuroendocrine cell line previously shown to efficiently process and secrete Wnt-1, was transfected with expression vectors encoding Wnt family members. All of the newly characterized Wnt proteins are glycosylated, secreted proteins that are tightly associated with the cell surface or extracellular matrix. We have also identified native Wnt proteins in retinoic acid-treated P19 embryonal carcinoma cells, and they exhibit the same biochemical characteristics as the recombinant proteins. These data suggest that Wnt family members function in cell to cell signaling in a fashion similar to Wnt-1.

Characterization of Xenopus laevis gamma-crystallin-encoding genes.

In order to gain insight into crystallin (Cry)-encoding gene (cry) evolution and developmental function, we have determined the gene structure and sequence of several Xenopus laevis gamma-cry. These encode the most abundant Cry in the embryonic lens. Four of the X. laevis gamma-cry, which are part of a multigene family, were isolated from a X. laevis genomic library and demonstrated to have the same gene structure as gamma-cry from other vertebrates, thereby providing further evidence that the split between beta and gamma members of the beta gamma cry family occurred relatively early in evolution. Sequence comparisons indicate that these X. laevis genes share 88-90% nucleotide sequence identity in the protein coding regions, which is slightly higher than the identity observed between gamma-cry of other species. The 5' upstream regions of X. laevis gamma-cry contain a few short stretches of homology and one putative promoter element conserved among all cry genes but lack other regions common to gamma-cry promoters from other organisms. The deduced amino acid sequences of all four genes and one cDNA suggest that the structure of X. laevis gamma-Cry is highly conserved with that of other vertebrate gamma-Cry, as deduced from the known three-dimensional structure of bovine gamma B Cry.

Molecular cloning and physical mapping of the Neurospora crassa 74-OR23-1A mitochondrial genome.

To provide for thorough sampling of the Neurospora crassa mitochondrial genome for evolutionary studies, recombinant plasmids containing each of the EcoRI digestion fragments of the genome were assembled and used to map the locations of 89 additional restriction endonuclease cleavage sites, representing 10 newly mapped enzymes and 2 previously unmapped HincII sites. Data used to locate new restriction sites were obtained from digestions of whole mitochondrial DNA, digestions of the cloned EcoRI mitochondrial DNA fragments and hybridizations between new restriction fragments and the cloned fragments. Length measurements of the total genome and of EcoRI fragment 1 are larger than commonly reported.

An evolutionary comparison of homologous mitochondrial plasmid DNAs from three Neurospora species.

We have discovered a mitochondrial DNA plasmid in N. crassa 516 (Roanoke, LA) which is homologous to those previously described from N. intermedia 435 (Fiji) and N. tetrasperma 2510 (Hanalei, HA). Subsequent analysis by DNA-DNA hybridization showed that 6 of 14 other Louisiana N. crassa isolates possessed plasmids homologous to these three plasmids, but at lower copy number. Plasmids from the three named strains were studied to examine possible plasmid diversity within each isolate, the extent of the homology between the plasmids, and the possibility that these plasmids could be inherited separately from their host mitochondria. Comparison of cloned plasmids and covalently closed circular mitochondrial DNA showed that only one plasmid line was present in each of the three intensively studied isolates. DNA-DNA hybridization and restriction endonuclease site mapping showed that the mitochondrial plasmids from the three species were very similar; most of the variation was due to presumed nucleotide substitutions. Plasmids judged identical by our analysis were found in different species. The distribution of the homologous plasmids in nature and the presence of these identical plasmids in different species, suggested that these plasmids could be transmitted between isolates independently of their host mitochondria.