To characterize genes that become upregulated with malignant transformation of human hepatocytes, a library of monoclonal antibodies was produced against the FOCUS hepatocellular carcinoma cell line. Antibody FB-50 reacted with an antigen that was highly expressed in 4 of 10 primary hepatocellular carcinomas, in all 20 cholangiocarcinomas we studied, and in a variety of transformed cell lines.

The a-ketoglutarate-dependent dioxygenase aspartyl (asparaginyl) J-hydroxylase (EC 1.14. (18,19). A cDNA fragment (nt 1-1033) was deleted from bovine Asp (Asn) /3hydroxylase cDNA by digestion with Pst I and ligated to form pNC3d-1. A 1.4-kb cDNA insert [nt 1034-2498, containing a 233-bp 3' untranslated region (19)] resulting from Xba I/Xho I digestion was isolated and then cloned into an E. coli expression vector, pFLAG-1 (IBI) (20), to form p52-1.Since this 1.4-kb cDNA insert lacked nt 931-1033, a synthetic double-stranded DNA fragment containing these nucleotides, a HindIII site at the 5' end, and a Pst I site at the 3' end was inserted into p52-1 to form the expression plasmid p52. The cDNA reading frame (nt 931-2265) ofp52 was confirmed as follows: p52 was then used to transform E. coli DHSaF'IQ (BRL); it was then purified and the 5' ends ofthe cDNA insert were sequenced. Purified p52 was introduced into both a normal E. coli strain and a protease-deficient strain (BL21; Novagen).Expression of P52. A p52 transformant was grown at 37TC in 3 x LB medium (30 g oftryptone, 15 g of yeast extract, and 5 g of NaCl per liter, pH 7.5) to an OD595 of 0.4-0.6. The culture was diluted 1:40 with 3x LB and incubated at 3rC until an OD595 of 1.5-1.8 was attained. Expression was induced with 1 mM isopropyl P-D-thiogalactopyranoside for 4 hr at 28C.For the purification of P52 (Table 1), 300 g of frozen cellswere resuspended in 1.2 liters of ice-cold lysis buffer (50 mM Tris-HCI, pH 7.5/0.1% Nonidet P-40/3 mM EDTA with protease inhibitors) and lysed. Protease inhibitor concentrations were as follows: aprotinin, 3.0 Ig/ml; leupeptin, 1.5 gg/ml; pepstatin, 2.1 pg/ml; phenylmethylsulfonyl fluoride, 0.6 mM; soybean trypsin inhibitor, 0.01%; benzamidine, 10Abbreviations: EGF, epidermal growth factor; P52, 52-kDa recombinant Asp (Asn) P-hydroxylase.

Ovariectomized (OVX) ewes were assigned to receive vehicle, progesterone (P4, 0.9-g controlled internal drug release vaginal implants), estradiol-17beta (E2, 5 microg/kg bolus + 6 microg kg(-1) day(-1)), or P4 + E2 for 10 days (n = 3/group). Uterine artery endothelial proteins were mechanically isolated on Day 10. The samples were used for protein expression profiling by the Ciphergen Proteinchip system and immunoblotting analysis of endothelial nitric oxide synthase (NOS3, also termed eNOS) and caveolin 1. Uterine artery rings were cut and analyzed by immunohistochemistry to localize NOS3 and caveolin 1 expression. With the use of the IMAC3 protein chip with loading as little as 2 microg protein/sample, many protein peaks could be detected. Compared to vehicle controls, a approximately 133.1-kDa protein was identified to be upregulated by 2- to 4-fold in OVX ewes receiving E2, P4, and their combination, whereas a approximately 22.6-kDa protein was downregulated by 2- to 4-fold in OVX ewes receiving E2 and E2/P4, but not P4 treatments. Western blot analysis revealed that E2, P4, and their combination all increased NOS3 protein, whereas E2 and its combination with P4, but not P4 alone, downregulated caveolin 1 expression. Immunohistochemical analysis revealed that NOS3 was mainly localized in the endothelium and upregulated by E2, whereas caveolin 1 was localized in both endothelium and smooth muscle and downregulated by E2. Thus, our data demonstrate that uterine artery endothelial NOS3 and caveolin 1 are regulated reciprocally by estrogen replacement therapy. In keeping with the facts that E2, but not P4, causes uterine vasodilatation and that E2 and P4 increase NOS3 expression, but only E2 decrease caveolin 1 expression, our current study suggests that both increased NOS3 expression and decreased caveolin 1 expression are needed to facilitate estrogen-induced uterine vasodilatation.

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