Biomedical subjects

A D Laird

Publications and source records attributed to A D Laird.

7 recordsLinked to original sources

PubMed2000

SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors.

Vascular endothelial growth factor, fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) and their cognate receptor tyrosine kinases are strongly implicated in angiogenesis associated with solid tumors. Using rational drug design coupled with traditional screening technologies, we have discovered SU6668, a novel inhibitor of these receptors. Biochemical kinetic studies using isolated Flk-1, FGF receptor 1, and PDGF receptor beta kinases revealed that SU6668 has competitive inhibitory properties with respect to ATP. Cocrystallographic studies of SU6668 in the catalytic domain of FGF receptor 1 substantiated the adenine mimetic properties of its oxindole core. Molecular modeling of SU6668 in the ATP binding pockets of the FIk-1/KDR and PDGF receptor kinases provided insight to explain the relative potency and selectivity of SU6668 for these receptors. In cellular systems, SU6668 inhibited receptor tyrosine phosphorylation and mitogenesis after stimulation of cells by appropriate ligands. Oral or i.p. administration of SU6668 in athymic mice resulted in significant growth inhibition of a diverse panel of human tumor xenografts of glioma, melanoma, lung, colon, ovarian, and epidermoid origin. Furthermore, intravital multifluorescence videomicroscopy of C6 glioma xenografts in the dorsal skinfold chamber model revealed that SU6668 treatment suppressed tumor angiogenesis. Finally, SU6668 treatment induced striking regression of large established human tumor xenografts. Investigations of SU6668 activity in cancer patients are ongoing in Phase I clinical trials.

3T3 Cells↗

PubMed2000

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.

Angiogenesis Inhibitors↗

PubMed1999

Characterization of Raf-1 activation in mitosis.

We have used site-directed mutagenesis to explore the mechanisms underlying Raf-1 activation in mitosis, and we have excluded most previously characterized activating interactions. Our results indicate that the primary locus of activation lies in the carboxyl-half of the molecule, although the extent of activation can be influenced by the amino-proximal region, particularly by the Raf-1 zinc finger. We also found that Raf-1 is hyperphosphorylated in mitosis at multiple sites within residues 283-302 and that these hyperphosphorylations are not required for activation. In addition, neither Mek1 nor Mek2 are stably activated in coordination with Raf-1 in nocodazole-arrested cells. Overall, the data suggest that the mechanism(s) responsible for activating Raf-1 during mitosis, and the subsequent downstream effects, are distinct from those involved in growth factor stimulation.

14-3-3 Proteins↗

PubMed1995

Raf-1 is activated during mitosis.

The Raf-1 serine/threonine kinase transmits proliferative and developmental signals to downstream effectors in the mitogen-activated protein kinase cascade. Studies to date have concentrated on its role in growth factor-mediated activation of the cell cycle. We show here that Raf-1 kinase activity is also highly stimulated during mitosis, with an attendant distinctive electrophoretic mobility shift due to hyperphosphorylation. These results suggest that Raf-1 may play a role in traversal of at least two distinct phases of the cell cycle.

3T3 Cells↗

PubMed1995

Liver regeneration. 2. Role of growth factors and cytokines in hepatic regeneration.

During liver regeneration quiescent hepatocytes undergo one or two rounds of replication and then return to a nonproliferative state. Growth factors regulate this process by providing both stimulatory and inhibitory signals for cell proliferation. EGF, TGF alpha, and HGF stimulate DNA synthesis in hepatocytes in vivo and in culture but the sensitivity of cultured hepatocytes to the mitogenic effects of these factors is much higher than that of quiescent hepatocytes in intact livers. We have proposed that after partial hepatectomy, hepatocytes enter a state of replicative competence ("priming") before they can fully respond to growth factors. The priming step is an initiating event in liver regeneration that involves the activation and DNA binding of NF-kappa B and other transcription factors, which could be induced by TNF or other cytokines. EGF, TGF alpha, and HGF have major effects on liver growth. TGF alpha expression correlates with hepatocyte DNA synthesis during liver development and growth and the constitutive expression of the factor confers proliferative activity to adult hepatocytes in vivo and in culture. The data indicate that the activity of stimulatory and inhibitory growth factors such as TGF beta 1 and activin is low in normal livers but that the expression of both types of factors increase during liver regeneration.

Adult↗

PubMed1994

Inhibition of tumor growth in liver epithelial cells transfected with a transforming growth factor alpha antisense gene.

Transforming growth factor alpha (TGF alpha) overexpression is associated with human hepatocellular carcinoma and with transformation of rat liver epithelial cell lines. In transgenic mice TGF alpha overexpression in the liver induces hepatocyte proliferation and leads to the development of tumors. Using a transformed rat liver epithelial cell line which can give rise to hepatocellular carcinomas, we used antisense genes to examine the importance of TGF alpha in tumor growth. Two different rat TGF alpha complementary DNA fragments were cloned in the antisense orientation into a thymidine kinase minigene downstream of a retroviral long terminal repeat. Cell lines that stably expressed the more effective construct, which contained a fragment that spanned the TGF alpha start codon, exhibited a 4-fold reduction in TGF alpha secretion relative to cell lines that expressed the thymidine kinase minigene alone. Following introduction into nude mice of 2 x 10(5) cells the control cell lines grew rapidly to produce large, highly cellular tumors by 5-6 weeks following injection, whereas with the antisense cell lines tumor growth was delayed so that tumors needed an additional 5 weeks to reach the same size. A high level of growth inhibition was also evident following injection of 2 x 10(6) cells, although the delay in tumor growth from antisense lines was shortened to about 3 weeks. Furthermore, tumors produced by 3 of the 4 antisense cell lines tested were fibrotic and hypocellular relative to those produced by the control cell lines. Growth of tumors from the antisense cell lines was associated with a decline in antisense RNA expression. In contrast, tumors generated from the control cell lines maintained high levels of expression of the control thymidine kinase minigene. These data demonstrate that tumor growth from highly tumorigenic liver cells can be inhibited by disrupting their ability to produce TGF alpha.

Animals↗

PubMedDate not supplied

Oncoprotein signalling and mitosis.

Studies of the roles of oncoproteins in cell cycle progression have concentrated on G1 because transformation is frequently associated with loss of G1 checkpoint control. However, it has become evident that G2 and mitotic checkpoints are often compromised in transformed cells and that many tumour suppressor proteins and oncoprotein kinases regulate and/or are activated in G2 and M. Disruption of p53 and ATM tumour suppressor protein functions can eliminate G2 and M checkpoints. The Src family kinases are activated in mitosis and collectively play an indispensable role in progression through G2/M. In addition, evidence suggests that Mos and elements of the Ras/Raf/MAPK cascade are also active in mitosis and appear likely to regulate G2 and/or M. Potential targets of these kinases include likely regulators of gene expression and microtubule dynamics such as Sam68 and Oncoprotein 18/stathmin. The ability of some oncoproteins to perturb orderly progression through both G1 and/or S and G2 and/or M is probably important for transformation.

Animals↗