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Tri Phosphate-Dependent Doliehol Kinase and Dolichol Phosphatase

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Tri Phosphate-Dependent Doliehol Kinase and Dolichol Phosphatase [CANCER RESEARCH 48. 3418-3424, June 15, 1988] Cytidine 5'-Tri phosphate-dependent Doliehol Kinase and Dolichol Phosphatase Activities and Levels of Dolichyl Phosphate in Microsomal Fractions from Highly Differentiated Human Hepatomas1 Ivan Eggens, Johan Ericsson, and ÖrjanTollbom Department of Pathology at Huddinge Hospital, Karolinska Institute!, S-I4I 86 Huddinge, ¡I.E.], and Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden [J. E., Ö.T.] ABSTRACT synthesis is the free alcohol and that this compound is subse quently phosphorylated to give dolichyl phasphate (15). The Homogenates and microsomal fractions prepared from biopsies of possibility was also raised that the level of dolichyl-P is regu highly differentiated human hepatocellular carcinomas were found to lated by dolichol kinase and dolichol phosphatase activities contain low levels of dolichol in comparison with control tissue. In contrast, the amount of dolichyl phosphate in tumor homogenates was (15). The existence of dolichol mono- and pyrophosphatases have unchanged and actually increased in the microsomal fraction. The pattern of individual polyisoprenoids, both in the free and the phosphorylated been reported by several authors and their activities are found dolichol fractions of hepatomas, did not exhibit any major alterations in several intracellular organelles, although at different levels compared to the control. (16-18). It has also been reported earlier that dolichol can be The rates of incorporation of ['H|mevalonic acid into dolichol and phosphorylated in vitro via a CTP-mediated kinase, which is dolichyl phosphate in hepatomas were low. The dolichol monophospha- situated on the outer surface of microsomal membranes (19, tase activities in microsomal fractions from hepatomas and controls did 20). However, no investigation of the substrate specificities of not show any major differences, whereas the activity of the CTP-depend- the kinase or phosphatase for polyisoprenoid compounds has ent dolichol kinase was increased in tumor microsomes. Glycosylation of endogenous dolichyl phosphate and of total protein using certain nucleo- been performed. tide-activated sugars was found to be slightly elevated in microsomal Recent reports using rat liver tissue found, however, no fractions from the tumor itself when compared to the control. The reasons evidence that dolichol serves as a precursor for dolichyl-P. It for the differences in the levels of polyisoprenoids in hepatomas and was also suggested that the immediate precursors of dolichol control tissue are discussed. and dolichyl-P are the a-unsaturated polyprenol and the cor responding polyprenyl phosphate, respectively (21, 22). An other study using primary cultures of rat hepatocytes indicated INTRODUCTION that dolichyl-P is synthesized primarily through a de novo Several cell lines derived from human hepatocellular carci process and that only about 10% of newly synthesized dolichyl- nomas demonstrate biosynthetic capabilities similar to those of P is produced by phosphorylation of the free alcohol (23). normal liver parenchyma! cells and many of these cell lines are In the present study we have measured and compared the capable of synthesizing and secreting most major plasma pro biosynthesis of dolichyl-P and dolichol as well as certain related teins (1). Nearly all plasma proteins are glycoproteins, the enzyme activities in microsomal fractions from highly differ majority of which are formed by ,/V-linked glycosylation, and entiated hepatomas and from control hepatic tissue. The rea investigations of certain hepatoma lines have revealed defects sons for the differences in dolichyl-P and dolichol levels ob in their glycosylation of different glycoproteins (2, 3). Phospho served are discussed. rylated dolichols are involved in glycoprotein synthesis and it is therefore of interest to study these compounds in hepatomas. MATERIALS AND METHODS These polyisoprenoid compounds are present in all tissues and intracellular membranes, either as a free alcohol, esterified with Isolation and Characterization of the Material. Our material was a fatty acid, and/or in phosphorylated form (4-7). derived from 5 highly differentiated human hepatocellular carcinomas (hepatomas), from the morphologically normal region outside the hep The function of the free alcohol in biological membranes has atoma tumor and the tumor itself, and from 5 normal livers. The not yet been established, but in model membranes dolichol is patients involved were 45-55 years of age. The tissue samples were proposed to influence membrane properties such as fluidity and utilized immediately after surgery and part of the material was then permeability (8-10). Phosphorylated dolichol participates in rapidly frozen to —70°C.Inselecting samples from the tumor, care was the biosynthesis of W-glycosidically linked oligosaccharide taken to exclude fibrotic or hemorrhagic areas. chains and it was suggested earlier that the level of this com All tumor tissues examined were also macroscopically free of necro pound may be rate limiting for protein glycosylation under sis. All samples were subjected to careful histológica!examination and certain conditions (11-14). those samples which after later examination were found to contain The exact pathways and mechanisms for the biosynthesis and areas of necrosis or fibrosis were not included in this study. Samples were homogenized with an Ultra-Turrax blender prior to regulation of the levels of polyisoprenoids are still not com pletely understood. When dolichol and dolichyl-P2 were studied lipid extraction. Homogenates and microsomal fractions were prepared as described earlier (24, 25). The values presented in the tables are the in sea urchin embryos using radioactively labeled precursors, means of 2 different experiments each involving 5 different liver sam the results indicated that the principal end product of de novo ples handled independently. Each sample consisted of 3 or 4 pieces of tissue picked randomly from the area of interest. Received 9/2/87; revised 2/18/88; accepted 2/26/88. The costs of publication of this article were defrayed in part by the payment Isolation and Quantitation of Dolichyl-P and Dolichol. The tissue was of page charges. This article must therefore be hereby marked advertisement in homogenized, dolichol-23 and dolichyl-23 phosphate were added as accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by Centrala Försöksdjursnämnden,theSwedish internal standards, and lipids were extracted with CMW (1:1:0.3). HC1 was then added to the mixture to give a final concentration of 0.1 M Cancer Society, and the Swedish Medical Research Council. 2The abbreviations used are: dolichyl-P, dolichyl phosphate; CMW, chloro- and acid hydrolysis was performed, tirsi for 60 min at room tempera form:melhanol:water; HPLC, high-performance liquid chromalography. ture, then for 45 min at 55°Cand finally, for 10 min at 100'C. The 3418 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1988 American Association for Cancer Research. DOLICHOL KINASE AND PHOSPHATASE IN HUMAN HEPATOMAS suspension was then neutralized with NaOH and evaporated to dryness. added and the mixture shaken in a vortex and incubated at 37*C for 15 Alkaline hydrolysis was performed with methanol:water:KOH (60%) min. The reaction was stopped by adding 7 ml chloroform:methanol (1:1:0.5) for 45 min at 90°C.The pH was subsequently adjusted to 7.0 (2:1), 1 ml H2O, and 0.5 ml 0.1 M MgCl2. After shaking, this mixture and chloroform was added to obtain a CMW ratio of 3:2:1. After was incubated at 40"C for 20 min. After centrifugation the upper phase washing the lower phase with theoretical upper phase chloro- was discarded and the lower phase washed twice with theoretical upper form:methanol:water (3:48:47) the sample was evaporated to dryness. phase. This washed extract, which contained the polyprenols, was The residue was then dissolved in 200 ^1 CM (2:1) and 10 ml MW evaporated and placed on a silica gel thin-layer chromatography plate. (98:2) containing 20 IHM11.!'( )4 were added. This mixture was placed The plate was developed using hexane:ether:acetic acid (75:30:1.5) and onto a CIS Sep-Pak column which had been equilibrated with 20 DIM the spot containing dolichol (identified using standards) was stained H3PO4 and thereafter washed first with 10 ml MW (98:2) containing with iodine. This spot was scraped off the plate and transferred to 20 mM H3PO4 followed by 10 ml MW (98:2). scintillation vials. One ml methanol was added to the sample, which Dolichol and dolichyl-P were eluted from the CIS Sep-Pak column was then heated to 60°Cfor 30 min and finally, supplemented with 10 with CM (2:1). This fraction was then supplemented with ammonia to ml Aqualuma Plus, whereafter the radioactivity was determined. give a final concentration of 1% and placed onto a silica Sep-Pak Glycosylation of Endogenous Dolichyl-P and Protein. Microsomes (7 column. Dolichol was eluted with CM (2:1) containing 1% ammonia mg protein) were incubated in a total volume of 1 ml. To measure and dolichyl-P with CM (1:1:0.3). The lipid solutions were evaporated glycosylation with GDP-mannose, the medium contained 30 mM Tris- to dryness and the residues dissolved in CM (2:1) (dolichol) or CM HCl, pH 7.8, 1 mM MnCl2, 12.5 mM j3-mercaptoethanol, 0.4% Triton (2:1) containing 40 mM HC1 (dolichyl-P). The samples were then X-100, 2 mM AMP, and 0.4 ^Ci GDP-[MC]mannose (80 mCi/mmol; analyzed by HPLC using a Hewlett-Packard Hypersil ODS 3 pm Radiochemical Centre, Amersham, England). In the case of glycosyla reversed phase column. A convex gradient was used (No. 5, Waters 660 tion with jV-acetylglucosamine, the medium contained 30 mM Tris- solvent program) from the initial 2-propanol-methanol-H2O (40:60:5) HCl, pH 7.8, 2.5 mM EDTA, 12.5 mM jS-mercaptoethanol, 10 mM in pump system A to 40% hexane-2 propanol (70:30) in pump system MnCl2, 1.5 mM ATP, and 0.8 MCiUDP-fCyV-acetylglucosamine (300 B at a flow rate of 1.0 ml/min with a program time of 45 min.
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