Accumulation of neoplastic traits prior to spontaneous in vitro transformation of rat cholangiocytes determines susceptibility to activated ErbB-2/Neu

Abstract

Cholangiocarcinoma, a severe form of biliary cancer, has a high mortality rate resulting partially from the advanced stage of disease at earliest diagnosis. A better understanding of the progressive molecular and cellular changes occurring during spontaneous cholangiocarcinogenesis is needed to identify potential biomarkers for diagnosis/prognosis or targets for novel therapeutics. Here, we show that with continued passage (p) in vitro, rat bile duct epithelial cells (BDEC) accumulated neoplastic characteristics that by mid-passage (p31-85) included alterations in morphology, increased growth rate, growth factor independence, decreased cell adhesion, loss of cholangiocyte markers expressed at low passage (p<30), and onset of aneuploidy. At high passage (p>85), BDEC cultures showed increasing numbers of cells expressing activated, tyrosine phosphorylated ErbB-2/Neu, a receptor tyrosine kinase previously reported to be at elevated levels in cholangiocarcinomas. Enrichment for high passage ErbB-2/Neu-positive cells yielded several anchorage-independent sub-lines with elevated levels of activated ErbB-2/Neu and increased expression of cyclooxygenase-2 (COX-2). When injected into immunodeficient beige/nude/xid mice, these sub-lines formed poorly differentiated cystic tumors strongly positive for rat cholangiocyte markers, a finding consistent with a previous report showing the susceptibility of high passage, non-tumorigenic BDEC to transformation by activated ErbB-2/Neu. Mid passage BDEC, in contrast, were resistant to the transforming activity of activated ErbB-2/Neu and remained anchorage dependent in vitro and non-tumorigenic in vivo following stable transfection. Based on these findings, we concluded that during progression to high passage, cultured BDEC undergo preneoplastic changes that enhance their susceptibility to transformation by ErbB-2/Neu. The ability to generate cells at different points in the process of spontaneous neoplastic transformation offers a valuable model system for identifying molecular features that determine whether over-expression of activated ErbB-2/Neu is necessary and sufficient to induce neoplastic conversion.

Characterization of preneoplastic and neoplastic rat mesothelial cell lines: the involvement of TETs, DNMTs, and 5-hydroxymethylcytosine

“…To date, most studies on preneoplastic cell lines have been conducted either on spontaneously transformed normal cells in culture [27] or on immortalized cell clones isolated from biopsies [28]. Additional insights have been obtained from histopathological observations made on both preneoplastic and neoplastic cells [29] and by the use of transgenic animals [30].…”

Differential gene expression profiling of cultured neu-transformed versus spontaneously-transformed rat cholangiocytes and of corresponding cholangiocarcinomas

“…The parent rat cholangiocarcinoma cell lines, BDEneu and BDEsp, used in this study were each generated in our laboratory from an immortalized rat cholangiocyte cell line initially established in the laboratory of Dr. Douglas C. Hixson at Rhode Island Hospital, Providence, RI (Yang et al ., 1993; Rozich et al ., 2010) and have been previously described (Lai et al ., 2005; Sirica et al ., 2008). Cultured BDEneu cells at in vitro passage 14 and BDEsp cells at in vitro passage 11, grown to ≥ 70% confluence in plastic culture dishes, were used in our microarray and QRT-PCR analyses.…”

The cholangiocyte marker, BD. 1, forms a stable complex with CLIP170 and shares an identity with eIF3a, a multifunctional subunit of the eIF3 initiation complex

“…1B). We have shown previously by indirect immunofluorescence that rat BDEC express high levels of BD.1 antigen at low passage ( p <35 passages) but showed a marked decrease in expression levels at mid (p36-84) and high ( p >85) passage (Rozich et al, 2010). As shown by immuno-blot analysis, BD.1 expression in rat PEC also varied with passage number as low ( p 30 passages) and high ( p >70 passages) passage cells showed the strongest and mid-passage (p31-69) the weakest reactivity with MAb BD.1 (Fig.…”

“…Decrease or loss of eIF3a/BD.1 expression by BDEC at mid-passage was also closely paralleled by the onset of aneuploidy, further implicating eIF3a/BD.1 in cell division (Rozich et al, 2010). eIF3a/ BD.1 contains the entire amino acid coding sequence for centrosomin A, a protein required for proper maturation of centrosomes into high capacity microtubule-organizing centers required for driving assemble of the bipolar spindle apparatus during mitosis.…”

“…BDEC were grown in vitro as previously described in medium supplemented with Normocin (0.1 mg/ml, InvivoGen, San Diego, CA) (Rozich et al, 2010). PEC were maintained in a 1:1 mixture of RPMI 1640 (Gibco, Carlsbad, CA) and MCDB 153 (Sigma-Aldrich, St. Louis, MO) supplemented with sodium bicarbonate (1.9 g/L), sodium pyruvate (0.5%), fetal bovine serum (FBS) (5%, Hyclone, Logan, UT), epidermal growth factor (0.02 μg/ml, BD Biosciences, San Jose, CA), bovine pituitary extract (5 μg/ml, BD Biosciences), dexamethasone (2 mM in 95% EtOH), glutamine (1%), gentamycin (0.1 mg/ml, Gibco), ITS (0.25%, BD Biosciences), forskolin (2.5 μg/ml, Calbiochem, San Diego, CA) and Normocin.…”