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.
Highlights
Introduction causal links between individual genes and specific cellular changes during tumor progression
Using a rat insulin promoter (RIP)-tva transgene to express the TVA receptor in b cells of the pancreas, we demonstrate that candidate genes can be introduced somatically into developing neoplastic bcell lesions in RIP-Tag; RIP-tva bitransgenic mice by infection with a subgroup A avian retroviral vector, termed RCASBP [13,14], thereby modulating tumorigenesis
By counting and classifying pancreatic lesions (Table 1), we found that (i) the average tumor incidence in bitransgenic mice infected with RCASBP-dominant-negative version of cadherin 1 (dnE-cad) (n 1⁄4 11) was 1.5-fold higher than that in bitransgenic mice injected with the control RCASBP-human placental alkaline phosphatase (ALPP) viruses (n 1⁄4 6); (ii) mice injected with RCASBP-dnE-cad (n 1⁄4 11) exhibited a higher incidence of invasive carcinomas than mice injected with the control viruses (n 1⁄4 6); and (iii) mice injected with RCASBP-dnE-cad had a higher tumor burden than controls (p 1⁄4 0.003, n 1⁄4 6 for each group, Wilcoxon rank sum test)
Summary
Introduction causal links between individual genes and specific cellular changes during tumor progression. Models, mutant genes have been more clearly implicated in the initiation than in the progression of cancer, even when newer methods, such as Cre/loxP-mediated recombination or tetracycline- or estrogen-based gene regulation, are used to produce a particular gene product conditionally in a tissuespecific and time-controlled manner [7,8] It is time-consuming and expensive to generate alterations in the Abbreviations: ALPP, human placental alkaline phosphatase; DMEM, Dulbecco’s Modified Eagle Medium; dnE-cad, a dominant-negative form of cadherin 1; F-actin, filamentous actin; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; GFP, green fluorescent protein; GTPase, guanosine triphosphatase; IC-1, invasive carcinoma type 1; IC-2, invasive carcinoma type 2; IGF, insulin-like growth factor; IP, immunoprecipitation; IT, noninvasive islet tumor; RCASBP, subgroup A replication-competent avian leukosis virus with a splice acceptor and the BryanRSV pol gene; RIP, rat insulin promoter; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis mouse germ line for each gene of interest
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