Suppression of Prostate Carcinoma Cell Invasion by Expression of Antisense L-Plastin Gene

Abstract

Based on the finding that gene expression for the actin-bundling protein L-plastin is inducible by androgen and that L-plastin is overexpressed in malignant epithelium of the prostate, we examined the functional consequences of L-plastin down-regulation in prostate carcinoma cell lines by both transfection and retroviral infection. We constructed retroviral vectors to express two different regions of the L-plastin gene, a 1713-bp 3'-coding portion and a 163-bp 5'-untranslated region, both in antisense orientation. Introduction of either constructs into prostate carcinoma cell lines, PC-3 and its isogenic but metastatic variant PC-3M cells, reduced the growth rates of both cell lines. In vitro invasion and motility of PC-3 and PC-3M cells were drastically suppressed (approximately 10-fold) by the expression of the antisense constructs. Evidence was obtained to indicate that L-plastin protein levels were indeed decreased by the antisense expression. The antisense construct for the 5'-untranslated region with the most unique sequence for the L-plastin gene was more effective in down-regulation efficiency compared with the larger antisense construct in the coding region, which maintains homology to other members of the plastin gene family. Cells infected with the 163-bp antisense virus, which were also tested in a nude mouse diaphragm invasion model, showed suppression of in vivo invasion of both PC-3 and PC-3M cells. These results suggested that overexpression of L-plastin might be functionally involved in prostate cancer invasion and metastasis, and raised the possibility that L-plastin gene-specific antisense delivery could potentially be a useful approach to interfere with prostate cancer progression in vivo.

Phosphorylation on Ser5 increases the F-actin-binding activity of L-plastin and promotes its targeting to sites of actin assembly in cells

“…This is consistent with previous data showing that L-plastin repression by an RNA antisense approach decreased the invasive capacity of prostate carcinoma cells (Zheng et al, 1999). Cell invasion depended on Ser5 phosphorylation, suggesting that targeting of L-plastin to peripheral actin-rich membrane extensions and adhesions was required for this process.…”

“…L-plastin is also detected in solid tumours of epithelial and mesenchymal origin and was proposed to play a role in tumour cell invasion (Zheng et al, 1999). Among the three plastin isoforms, L-plastin has the unique property of being phosphorylated in response to signals triggering the activation of the immune response, cell migration and proliferation.…”

“…Since L-plastin has been associated with cell invasion (Zheng et al, 1999), we expressed wild-type L-plastin or phosphorylation variants in human embryonic kidney HEK293T cells that do not produce endogenous L-plastin (data not shown) and investigated their invasive capacity in an in vitro collagen assay De Corte et al, 2002). This semi-quantitative assay measures to what extent cells can migrate through a 3D-collagen gel and mimic invasion of tumour cells in the stroma.…”

Phosphorylation of ectopically expressed L‐plastin enhances invasiveness of human melanoma cells

“…Thus, expression of antisense L-plastin constructs reduced the invasiveness of the cells in vitro. 46 However, data regarding the functional importance of L-plastin phosphorylation were not analyzed in this study. Our data clearly demonstrate that not only expression but also phosphorylation of L-plastin is required to enhance melanoma cell invasion.…”

Increased Expression of Osteopontin Contributes to the Progression of Prostate Cancer

“…Following a 24-hour pretreatment in medium containing 0.5% serum in the presence or absence of 5 Ag/mL osteopontin antibody (R&D Systems, Minneapolis, MN), the cells (10 5 ) were added to the upper chamber, correspondingly with or without 5 Ag/mL osteopontin antibody, and incubated at 37jC for 24 hours. Invasion of the cells through the membrane was detected by staining with hematoxylin and counted as previously described (18,19).…”