Restoration of Smad4 in BxPC3 Pancreatic Cancer Cells Attenuates Proliferation without Altering Angiogenesis

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive human malignancy in which the transforming growth factor beta (TGF-beta) signal transducer, Smad4, is commonly mutated or deleted. BxPC3 human pancreatic cancer cells exhibit a homozygous deletion of the Smad4 gene, yet are growth inhibited by TGF-beta1. In the present study, we sought to determine whether reintroduction of Smad4 into BxPC3 cells alters their behavior in vitro and in vivo. Sham transfected and Smad4 expressing BxPC3 cells exhibited similar responses to TGF-beta1 with respect to p21 upregulation, hypophosphorylation of the RB protein, Smad2 phosphorylation, and Smad2/3 nuclear translocation. TGF-beta1 did not alter p27 expression, and silencing of p21 with an appropriate siRNA markedly attenuated TGF-beta1-mediated growth inhibition. Nonetheless, the presence of Smad4 was associated in vitro with a more prolonged doubling time, enhanced sensitivity to the growth inhibitory actions of exogenous TGF-beta1, and a more flattened cellular morphology. In vivo, Smad4 expression resulted in delayed tumor growth and decreased cellular proliferation, without effects on either apoptosis or angiogenesis. These findings indicate that, in spite of the absence of Smad4, growth inhibition in BxPC3 cells by TGF-beta1 is dependent on p21 upregulation and maintenance of RB in a hypophosphorylated, active state. Moreover, the presence of a functional Smad4 attenuates the capacity of BxPC3 cells to proliferate in vivo. However, this effect is transient, indicating that Smad4 growth inhibitory actions are circumvented in the later stages of pancreatic tumorigenicity.

Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs

“…Nuclear and cytoplasmic fractions were purified from mouse Hepa-1c1c7 (Hankinson et al 1991) and human BxPC3 cells (Yasutome et al 2005), from which total RNA (Schwanekamp et al 2006) and proteins (Yasutome et al 2005) were isolated from the same fractions using TRI reagent (Molecular Research Center). Two approaches were taken to demonstrate that the first criterion was satisfied, in which clean nuclear and cytoplasmic fractions were isolated (Fig.…”

“…Two diverse cell lines and experimental conditions were selected in order to more broadly test our hypothesis. The first set of experiments was a comparison of vehicle-treated and TCDDtreated mouse Hepa-1c1c7 hepatocyte cells (Hankinson et al 1991), and the second experimental set compared the relative RNA levels of sham vector (SMAD4 À/À ) and SMAD4-positive (Yasutome et al 2005) human BxPC3 pancreatic cancer cell lines (Tan et al 1986). The microarray results for selected genes were confirmed by quantitative real-time polymerase chain reaction assays using SYBR green and designed primers (Supplemental Tables S1, S2; Guo et al 2004).…”

“…To highlight the potential power of using cytoplasmic RNA, only the cytoplasmic RNA fraction of BxPC3 cells from the GenMAPP analysis (Table 3) revealed the involvement of the TGF-b signaling pathway, which would be expected in two cell types that differ only in the expression of the SMAD4 gene (Yasutome et al 2005). The findings showed that two genes in the TGF-b signaling pathway, BMP4 (NM_001202) and LEF1 (NM_016269) (Supplemental Tables S5, S6), were up-regulated by SMAD4 in BxPC3 cells.…”

The Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid Induces Growth Inhibition and Enhances Gemcitabine-Induced Cell Death in Pancreatic Cancer

“…Flow cytometric data were acquired using a FACSCan analysis system equipped with a FACStation, MAC PowerPC computer and CellQuest Acquisition software from Becton Dickinson (San Jose, CA). Data were analyzed using ModFit LT 2.0 software from Verity Software House (Topsham, ME) as reported previously (26).…”

“…To block phosphatase activity, slides were incubated for 15 min with 20 mmol/L NaF/2 mmol/L sodium orthovanadate/PBS before fixation and fixed with paraformaldehyde containing the same phosphatase inhibitors. All images were captured using Zeiss LSM 510 META confocal system and LSM release 3.2 software (Carl Zeiss, Inc., Thornwood, NY) as reported previously (26).…”