Michael P. Rosenberg

The vascular endothelial growth factor (VEGF) receptor Flt-1 (VEGFR-1) modulates Flk-1 (VEGFR-2) signaling during blood vessel formation.

Mice lacking the vascular endothelial growth factor (VEGF) receptor flt-1 (VEGFR-1) die from vascular overgrowth, caused primarily by aberrant endothelial cell division (Kearney JB, Ambler CA, Monaco KA, Johnson N, Rapoport RG, Bautch VL: Vascular endothelial growth factor receptor Flt-1 negatively regulates developmental blood vessel formation by modulating endothelial cell division. Blood 2002, 99:2397-2407). Because a second high-affinity VEGF receptor, flk-1, produces a positive endothelial proliferation signal, it was logical to ask whether flt-1 affects developmental blood vessel formation by modulating signaling through flk-1. Differentiated embryonic stem cell cultures lacking flt-1 (flt-1-/-) had increased flk-1 tyrosine phosphorylation, indicating that flk-1 signaling is up-regulated in the mutant background. The selective flk-1 inhibitor SU5416 partially rescued the flt-1-/- mutant phenotype, and this rescue was accompanied by a decrease in the relative amount of flk-1 tyrosine phosphorylation. Thus reduced flk-1 signal transduction can partially compensate for the lack of flt-1. The flt-1-/- mutant phenotype was also partially rescued by Flt-1/Fc, a truncated flt-1 that binds and sequesters the VEGF ligand. Taken together, these data show that down-regulation of flk-1 signaling by two different strategies partially rescues the developmental vascular overgrowth seen in the absence of flt-1, and they support a model whereby flt-1 modulates the flk-1 signal at an early point in the pathway.

Why Transgenic and Knockout Animal Models should be used (for Drug Efficacy Studies in Cancer)

The role that preclinical models play in the evaluation of drug efficacy and optimization of lead compounds is an essential one in pharmaceutical companies. Without a robust, dependable animal model of human disease development of structural-activity relationships in the design of better molecules becomes a daunting task. In the cancer arena, while not all chemotherapeutic agents which test positively in mouse models are efficacious in humans, those agents which are efficacious in humans are also effective in mice. The clinical failure of novel chemotherapeutics is often not established until phase II or phase III clinical studies, after costly investments of time and money have been made. Therefore it is highly desirable to have models of human cancers that will more accurately represent human disease and predict clinical outcomes. For this objective, transgenic and knockout mouse and rat models have held great promise, but yet have been underutilized by the pharmaceutical industry, the NCI, and the FDA. The limited use of such models is likely due in part to the failure of many current transgenic and knockout models to exhibit essential qualities of preclinical screening models; validity, reliability, and utility. The in vivo models typically employed by the pharmaceutical industry for preclinical testing of anticancer therapeutics are transplantable mouse (allo-) or human (xeno-) tumor grafts, such as the murine B16BL6 melanoma line or human HT-29 tumor line. Implantation of these tumor lines can be performed subcutaneously, intravenously, or orthotopically, providing a source of versatility. Although widely used by pharmaceutical companies for drug efficacy screening, tumor xenograft models also possess intrinsic disadvantages, making it surprising that genetically engineered models of cancer have seen little use either in conjunction or in lieu of the transplantable tumors. In this review, we will briefly cover the drawbacks of most transgenic models and discuss the features that such models of cancer should ideally possess for preclinical testing of anti-cancer therapies. Despite the shortcomings of transgenic and knockout models of cancer for drug efficacy evaluations, they represent a more natural in vivo history of tumor development. Thus outcomes of compound efficacy in transgenic and knockout mice will likely be more predictive of the clinical outcome. Their use may increase the confidence going into the clinic and reduce the clinical failure rate of novel chemotherapeutic agents. Although pharmaceutical companies have used transgenic and knockout mice, they have primarily been relegated to proof of concept and target identification studies. The basic questions addressed relate to whether a particular gene, wild type or mutant, is involved in the pathogenesis of a particular disease. More commonly, the experiment is performed to determine whether misexpression of a gene product has any consequence on the normal physiological response in a given tissue. For cancer, transgenic and knockout mouse studies often are designed to determine if a gene is involved in tumorigenesis and if so in what tissues. Other types of studies address whether a gene product which plays a role in a specific signaling process contributes to disease in conjunction with other predisposing conditions. Another current use of transgenic and knockout animals is to determine the molecular mode of action of a gene product and the downstream consequences of its misexpression on normal processes. Although these uses of genetically engineered mice may provide valuable information to drug discovery projects, they often do not translate into optimal models for drug testing.

Neurodegenerative Changes Including Altered Tau Phosphorylation and Neurofilament Immunoreactivity in Mice Transgenic for the Serine/Threonine Kinase Mos

Transgenic mice expressing the oncogenic protein-serine/threonine kinase Mos at high levels in the brain display progressive neuronal degeneration and gliosis. Gliosis developed in parallel with the onset of postnatal transgene expression and led to a dramatic increase in the number of astrocytes positive for GFAP, vimentin, and possibly tau. Interestingly, vimentin is normally expressed only in immature or neoplastic astrocytes, but appears to be induced to high levels in Mos-transgenic, mature astrocytes. Mos can activate mitogen activated protein kinase (MAPK) and MAPK has been implicated in Alzheimer-type tau phosphorylation. In the Mos-transgenic brain we found increased levels of phosphorylation at one epitope on tau containing serines 199 and 202 (numbering according to human tau), a pattern similar but not identical to that found in Alzheimers disease. In addition, Mos-transgenic mice express a novel neurofilament-related protein that might be a proteolytic neurofilament heavy chain degradation product. These results suggest that activation of protein phosphorylation in neurons can result in changes in cytoskeletal proteins that might contribute to neuronal degeneration.

Gene knockout and transgenic technologies in risk assessment: The next generation

Transgenic and knockout mice have been proposed as substitutes for one of the standard 2‐yr rodent assays. The advantages of using genetically engineered mouse models is that fewer mice are needed, the time to develop disease is greatly reduced, and the mice are predisposed to developing cancer by virtue of gain or loss of functions. The models currently being used have yielded a large amount of data and have proved to be informative for risk assessment; however, they are still far from ideal. In fact, they inherently do not reflect the complexity of mutation and carcinogenesis in humans. Recent advances in technology and the creation of new knockout mice may produce more useful and more sensitive models. This review covers two recent advances in technology—inducible and regulatable gene expression and targeted genetic modifications in the genome—that will allow us to make better models. I also discuss new gene deletion and transgenic mouse models and their potential impact on risk‐assessment assays. These models are presented in the context of four basic components or events that occur in the multistep process leading to cancer: maintenance of gene expression patterns, genome stability and DNA repair, cell‐cell communication and signaling, and cell‐cycle regulation. Finally, surrogate markers and utility in risk assessment are also discussed. This review is meant to stimulate further discussion in the field and to generate excitement about working toward the next generation of risk‐assessment models. Mol. Carcinog. 20:262–274, 1997.