Jinzhao Hou

Molecular cloning and expression of the gene for a major leucine-rich protein from human hepatoblastoma cells (HEPG2)

SummaryThe human hepatoblastoma cell line, HepG2, exhibits an array of stable properties in culture that have made it a popular cell culture model for studies on regulation of liver-specific gene expression and properties of hepatoma cells. In contrast to other hepatoma cell lines, HepG2 cells overexpress a characteristic detergent-extractable, wheat germ lectin-binding protein with apparent molecular mass of 130 kDa. Using an antibody to screen a phage expression library of HepG2 complementary DNA (cDNA), we identified and cloned a 4734 base pair cDNA which codes for a 130-kDa leucine-rich protein (lrp130) when expressed in transfected cells. The deduced sequence of lrp130 exhibits sequences weakly homologous to the consensus sequence for the ATP binding site in ATP-dependent kinases and the protein kinase C phosphorylation site of the epidermal growth factor receptor. Consistent with the higher levels of expression of lrp130 antigen, Northern hybridization analysis indicated that HepG2 cells express high levels of the major 4.8 kilobase lrp130 mRNA relative to other hepatoma cells. Although currently of unknown function, lrp130 may be of utility as a marker for liver cell lineages represented by the HepG2 cell line.

Heparin-binding fibroblast growth factors and prostate cancer.

Studies of model rat prostate tissue and derived cells indicate the insulin-like (IGF), epidermal growth factor (EGF), transforming growth factor beta (TGF-beta) and heparin-binding fibroblast growth factor (HBGF) families and their receptors may play important roles in regulation of normal prostate cell growth. Tumor cells at different levels in the progression from slow-growing, hormone-dependence to fast-growing, hormone-independence exhibit distinct alterations in expression of specific growth factors and their receptor phenotype. Distinct IGF-I and HBGF mRNAs are constitutively expressed in the mesenchymal cells of slow-tumors, but alteration in HBGF receptor phenotype occurs in the epithelial cells. Fast-tumors exhibit even higher constitutive expression of multiple HBGFs. Splice variants in cDNA for the HBGF receptor in fast-tumors suggest constitutive expression of an intracellular receptor, that together with intracellular HBGFs, may constitute an intracellular autocrine system that is independent of exogenous hormones and growth factors.

Heparin-Binding (Fibroblast) Growth Factor/Receptor Gene Expression in the Prostate

Heparin-binding (fibroblast) growth factors (HBGF) play both autocrine and paracrine roles in growth of normal and tumor prostate epithelial and mesenchymal cells. In the rat, expression of HBGF-1 dominates in normal tissue and slow-growing Dunning prostate tumor tissues. Both HBGF-1 and HBGF-2 are expressed in fast-growing, malignant variants of the Dunning tumor. Expression of HBGF-1 occurs in young normal prostate epithelial cells and disappears with age. Slow-growing Dunning tumor mesenchymal cells, not epithelial cells, constitutively express HBGF-1. HBGF requirement and receptor phenotype is altered in tumor epithelial cells. In contrast to IGF-1 and the Elg/Bek/Cek gene product, a candidate HBGF receptor, neither HBGF-1 nor HBGF-2 expression in normal and tumor tissues increases during androgenstimulated growth. Flg/Bek/Cek is expressed in young prostates, during androgen-stimulated growth of adult prostate, constitutively in tumors, and appears limited to the stromal cell fraction of the slow-growing tumors. Expression is elevated in the fast-growing, highly malignant tumors. Normal and slow-growing tumor epithelial cells have receptor sites for HBGF that may differ from Flg/Bek/Cek. Similar to rat prostate tissue, HBGF-1 expression dominates in young human prostate tissue. However, both HBGF-1 and HBGF-2 mRNA as well as the Flg/Bek/Cek gene are detectable in adult human prostate tissue. These differences in rat and human tissue may reflect differences in the epithelial:stromal cell ratio in prostates of the two species.

Analysis of Serine, Threonine and Tyrosine Phosphorylation Sites with Mass Spectrometry

Publisher Summary This chapter presents an analysis of serine, threonine, and tyrosine phosphorylation sites with mass spectrometry. In a study described in the chapter, an improved purification procedure was used to isolate 100 μg of rhodopsin kinase (RK) from 75 bovine retinas. Soluble-contaminating proteins were washed from bleached rod outer-segment membranes with a low-ionic strength buffer. RK was extracted from the membranes with 0.25% Tween 80 and purified to homogeneity by Heparin–Sepharose chromatography. Recombinant FGF-R1, a tyrosine kinase isoform of the FGF receptor, was purified from baculoviral-infected Sf9 insect cells. For phosphopeptide analysis, lysates from 1–2 × 108 infected insect cells were mixed with 1–2 μg of immunopurified 32P labeled receptor, the mixture absorbed onto heparin-agarose beads, the immobilized protein treated with unlabeled ATP and then fragmented with trypsin while still on the beads. From SDS-PAGE, sequence and audoradiographic analysis of PVDF electroblots, limited proteolysis of RK with endoproteinase Asp-N was shown to remove N- and C-terminal peptides with no 32P remaining associated with 50–55 kDa undigested fragments. One major and two minor radioactive fractions were purified by RP-HPLC.