Assessing Tumor Progression Factors by Somatic Gene Transfer into a Mouse Model: Bcl-xL Promotes Islet Tumor Cell Invasion

Abstract

Tumors develop through multiple stages, implicating multiple effectors, but the tools to assess how candidate genes contribute to stepwise tumor progression have been limited. We have developed a novel system in which progression of phenotypes in a mouse model of pancreatic islet cell tumorigenesis can be used to measure the effects of genes introduced by cell-type-specific infection with retroviral vectors. In this system, bitransgenic mice, in which the rat insulin promoter (RIP) drives expression of both the SV40 T antigen (RIP-Tag) and the receptor for subgroup A avian leukosis virus (RIP-tva), are infected with avian viral vectors carrying cDNAs encoding candidate progression factors. Like RIP-Tag mice, RIP-Tag; RIP-tva bitransgenic mice develop isolated carcinomas by ∼14 wk of age, after progression through well-defined stages that are similar to aspects of human tumor progression, including hyperplasia, angiogenesis, adenoma, and invasive carcinoma. When avian retroviral vectors carrying a green fluorescent protein marker were introduced into RIP-Tag; RIP-tva mice by intra-cardiac injection at the hyperplastic or early dysplastic stage of tumorigenesis, approximately 20% of the TVA-positive cells were infected and expressed green fluorescent proteins as measured by flow cytometry. Similar infection with vectors carrying cDNA encoding either of two progression factors, a dominant-negative version of cadherin 1 (dnE-cad) or Bcl-xL, accelerated the formation of islet tumors with invasive properties and pancreatic lymph node metastasis. To begin studying the mechanism by which Bcl-xL, an anti-apoptotic protein, promotes invasion and metastasis, RIP-Tag; RIP-tva pancreatic islet tumor cells were infected in vitro with RCASBP-Bcl-xL. Although no changes were observed in rates of proliferation or apoptosis, Bcl-xL altered cell morphology, remodeled the actin cytoskeleton, and down-regulated cadherin 1; it also induced cell migration and invasion, as evaluated using two-chamber transwell assays. In addition, myosin Va was identified as a novel Bcl-xL-interacting protein that might mediate the effects of Bcl-xL on tumor cell migration and invasion.

Distinct functions of macrophage-derived and cancer cell-derived cathepsin Z combine to promote tumor malignancy via interactions with the extracellular matrix

“…CtsZ heterozygous mice were backcrossed into the C57BL/6 background for nine generations before crossing to RT2 mice. The RIP-Tva mice (Du et al 2007) were a kind gift of Dr. Nancy Du and Dr. Harold Varmus and were crossed with RT2 mice to generate RIP-Tag; RIP-Tva mice in a wild-type or CtsZ À/À background. b-Actin GFP transgenic mice (Okabe et al 1997) in the C57BL/6 background were purchased from Jackson Laboratories.…”

“…We crossed RT2 animals with RIP-Tva transgenic mice, which allowed for retroviral-based gene delivery specifically to Tva + tumor-initiating b cells of the pancreas (Du et al 2007). Intracardiac injection of RIP-Tva; RT2 mice with RCAS viruses prior to tumor development, at 7 wk of age, has been shown to result in successful infection of hyperplastic cells (Du et al 2007).We delivered either RCAS-CtsZ or the control RCAS-GFP virus into recipient RIP-Tva; CtsZ À/À RT2 animals as schematized in Figure 4A. We first confirmed RT2 mice injected with the RCAS-CtsZ virus that were categorized according to expression of CtsZ as in C. Quantification was performed and showed a significant increase in proliferation in tumors re-expressing CtsZ (n = 36 tumors negative for CtsZ expression; n = 18 tumors positive for CtsZ expression).…”

“…We crossed RT2 animals with RIP-Tva transgenic mice, which allowed for retroviral-based gene delivery specifically to Tva + tumor-initiating b cells of the pancreas (Du et al 2007). Intracardiac injection of RIP-Tva; RT2 mice with RCAS viruses prior to tumor development, at 7 wk of age, has been shown to result in successful infection of hyperplastic cells (Du et al 2007).…”

A Cre-loxP based mouse model for conditional somatic gene expression and knock-down in vivo using avian retroviral vectors

“…By taking advantage of the increasing number of Cre-expressing mouse lines and Cre expression strategies (35), our model allows Cre-inducible TVA expression and RCASBP(A)-mediated gene transfer in a wide range of different tissue types and cell populations and therefore overcomes major limitations of existing mouse lines that use tissue-specific promoters for transgenic TVA expression (9,10,12,13,15,(17)(18)(19). Thus, our mouse line opens up many possibilities for analysis of gene function in a time-controlled and tissue-specific fashion in vivo.…”

“…The RCAS-TVA somatic gene transfer system has been used in a variety of murine models in vivo (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), and the advantages and disadvantages are well documented (2)(3)(4). In particular, the system has been widely used to model sporadic human cancer in mice.…”