TUNICAMYCIN INHIBITS CAPILLARY ENDOTHELIAL CELL PROLIFERATION BY INDUCING APOPTOSIS

Targeting dolichol-pathway for generation of new anti-angiogenic therapeutics

Juan A. Martinez, Ivette Torres-Negron, Lilla A. Amigo, Rossely A Roldan, Alba Mendez and Dipak K. Banerjee Department ofBIOchemistry. School ofMediCIne, University of Puerto Rico, San Juan, PR 00936-5067, USA

Abstract:

Bovme adrenal medulla microvascular endothelial cells used in this study undergo cellular proliferation and differentiation upon cultunng in vitro as observed both by light and scanning electron microscopy. Cells also respond to the growth promoting activity of serum and basic fibroblast growth factor (FGF2). Flow cytometnc analysis of a synchronized culture established that cells take 68 hours to complete one cell cycle spendmg 36 hours m the G I phase, 8 hours m the S phase, and 24 hours in the G2 + M phase when cultured In EMEM contaming 2% heat-inactivated fetal bovme serum (FBS). At 10% serum, or m the presence of FGF2 (10 ng/ml - 100 ng/ml) length of the cell cycle IS reduced to 56 hours due to shortening of the G I phase by 12 hours. TUnlcamycm (a glucosamine-containing pyrimidine nucleotide), and an mhlbltor of glucosammyl-l-phosphate (GlcNAc I-P) transferase, the first step of G\c3Man9G\CNAc2-PP-Dol (OSL) biosynthesis is found to inhibit the endothehal cells prohferalton by mducing apoptosis as observed by flow cytometry and DNA laddering. Cell shnnkage, compaction of nucleI, membrane fragmentation, etc., typIcal of apoptotlc response are frequently seen by light mIcroscopy in the presence of tunicamycin. Scannmg electron mIcroscopy also exhibited a considerable amount of cell surface bleb bing. AccumulatIOn of an immunoposltive cell specific asparagine-hnked (N-linked) glycoprotem, Factor VIII:C in the absence of G\C3Man9G\CNAc2-PP-Dol in tUnIcamycm treated cells has been proposed as an apoptotic tnggenng mechanism under the current expenmental conditions.

Angiogenesis: From the Molecular to Integrative Pharmacology Edited by Maragoudakis, Kluwer Academic / Plenum Publishers, New York, 2000

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1.

INTRODUCTION

Our laboratory has made an observation almost a decade ago that Nglycosylation of proteins is increased nearly 3.5-fold, and the Km for Dol-PMan synthase is decreased by ~50% when the capillary endothelial cells (an established cell line from the microvasculature of bovine adrenal medulla) were cultured in the absence of CO 2 (1-4). Dol-P-Man synthase is an essential intermediate in the elongation of MansGlcNAcz-PP-Dol to Man9GlcNAcrPP-Dol (5,6), and an allosteric activator of GlcNAc-lphosphate transferase (7). It has also been demonstrated that Dol-P-Man synthase gene carries a cAMP-dependent protein phosphorylation consensus sequence and its actIvity is regulated by cAMP-dependent protein kinasemediated protein phosphorylation signal (8- 13). We have been interested in establishing a relationship between the dolichol pathway, especially that of Dol-P-Man synthase and angiogenesIs. Earlier studies with amphomycin indicated that inhibItion of Glc3Man9GlcNAcrPP-Dol (OSL) biosynthesis inhibited the endothelial cell prohferatIOn (14). Amphomycin is an undecapeptide from Streptomyces canus, whose N-terminus is blocked due to a fatty acid substitution (15,16). Its binding to Dol-P in the presence of Ca2+ blocks OSL assembly by interfenng with the synthesis of Dol-PP-GlcNAc2, Dol-P-Man, and Dol-PGlc, respectively (17 - 20). This paper while supports the earlier observation of amphomycin, also finds that tunicamycin, an inhibitor of GIcNAc-IP transferase and a blocker of OSL assembly (21) profoundly inhibits the endothelial cell proliferation by inducing apoptosis.

2. ALTERATION OF ENDOTHELIAL CELL MORPHOLOGY Tumcamycm, a glucosamine-containing pyrimidine nucleoside exists in 16 different homologues, differing mostly in the fatty acid side chain is synthesized by Streptomyces lysosuperificus. (21,22). We exposed a synchronized population of capillary endothelial cells (23) to tunicamycin from Boehringer-Mannheim with no history of protein synthesis inhibition, at various concentrations for different lengths oftime in a complete medium (EMEM) containing 2% fetal bovine serum (heat-inactivated) to evaluate the

199 morphological changes. The cells were monitored by phase-contrast light microscopy as well as by scanning electron microscopy. In controls, cellular proliferation and differentiation were mutually exclusive, but a complete differentiation was observed only after 5 days. Examination of surface morphology by scanning electron microscopy as a function of time established cellular growth, and differentiation into capillary-like structures. Cells cultured in a higher serum concentration (i.e., 10%) duplicated the morphological changes, but took longer time to differentiate. Cells cultured m the presence of tunicamycin (lllg/ml) exhibited cell shrinkage, loss of cell to contact, apparent compaction of nuclei showing condensed pyknotic appearance and membrane fragmentation when analyzed by light lTIlCroSCOpy. Scanning electron microscopy detected considerable surface blebbmg in tunicamycin-treated cells, a morphological change which had never been observed in controls even after culturing for a longer period of hme (Figure 1).

Figure I ScannIng electron microscopy of cells before and after tUnIcamycin treatment. Left panels (top to bottom): cultured for 4 days in 2% serum (x 1,700; -10 ).1m). 4 day:s In 10% serum (x 700; -I 0llm). and 7 days in 2% serum (x 1,100; -10 ).1m), respectIvely. RIght panels (top to bottom): cultured for 4 days In 2% serum (x 1,700; -10m), and 4 days In 2% serum contaInIng 1fig/ml tumcamycIn (x 1,700; -I O).lm).

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3. INHIBITION OF CELL GROWTH AND PROLIFERATION Synchronized cells were cultured in complete EMEM containing fetal bovine serum (l %, 2%, or 10%), or complete EMEM with 2% fetal bovine serum containing 10 ng/ml - 100 ng/ml of FGF2, or containing 5 ng/ml, 10 ng/ml, 1 Jlg/ml, and lO.O Jlg/ml tunicamycin. In each case, the cellular growth and viability was monitored by trypan blue exclusion, by counting the cell number, and by assessing the progression through the cell cycle by flow cytometry. Cells maintained a status quo for the first 32 hours whether cultured in serum alone or in the presence of FGF2. Cells proliferated normally in media containing either 2% or 10% fetal bovine serum, but a maximum cell growth was observed at 10% serum. Similarly, FGF2 at 10 ng/ml - 100 ng/ml stimulated the endothelial cell proliferation by reducing

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Figure 2. Effect of tumcamycin on the induction of apopotosis. Cells were treated with I j.lg/ml of tunicamycm for 32 to 40 hours in EMEM containing 2% serum. DNA laddering: M = 50 bp ladder, T = tumcamycin, C = control; Flow cytometry: A = apoptosis; MI "" GI, M2 '" S, M3 '" G2/M

201 the G 1 phase. When the progression of cells through the cell-cycle was evaluated by flow cytometry, cells cultured in 2% serum needed approximately 68 hours to complete one cycle spending 36 hours in the G 1 phase, 8 hours in the S phase and 24 hours in the G2 + M phase. Presence of either 10% serum, or 10 ng/ml -100 ng/ml FGF2 reduced the time to complete the cell cycle to 56 hours by decreasing the G 1 phase by 12 hours. Cells cultured in the presence of tunicamycin (1 )lg/ml) maintained the same level of growth as controls for 24 hours after which the number started declining, and cells never completed the cell cycle. Flow cytometric evaluation revealed that 70% of the cell population entered into apoptosis (i.e., "programmed cell death") after an exposure to tunicamycin for 32 hours. This was also confirmed by the appearance of laddering when DNA from tunicamycm treated cells was analyzed by agarose gel electrophoresis (Figure 2.) A similar reduction in cell growth has also been observed at all tunicamycin concentrations used in this study (i.e., 5 ng/ml- 10.0 )lg/ml).

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