Incorporation of Ortho- and Meta-Tyrosine Into Cellular Proteins Leads to Erythropoietin-Resistance in an Erythroid Cell Line

Original Paper

Copyright © 2014 S. Karger AG, Basel NonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only.

István Wittmann M.D., Ph.D. 2^nd Department of Medicine and Nephrological Center, Faculty of Medicine University of Pécs, 1. Pacsirta Str., Pécs, 7624 (Hungary)

Tel. +36-72/536-050 Fax +36-72/536-051, E-Mail istvan.wittmann@aok.pte.hu

Incorporation of Ortho- and Meta-

Tyrosine Into Cellular Proteins Leads to Erythropoietin-Resistance in an

Erythroid Cell Line

Esztella Mikolás^a Szilárd Kun^a Boglárka Laczy^a*HUJę$0ROQiU^a Eszter Sélley^a7DPiV.ęV]HJL^b István Wittmann^a

a2^nd Department of Medicine and Nephrological Center; ^bInstitute of Laboratory Medicine, University of Pécs, Pécs, Hungary

Key Words

(U\WKURSRLHWLQUHVLVWDQFH‡&HOOVLJQDOLQJ‡2[LGDWLYHVWUHVV‡7\URVLQHLVRPHUV

Abstract

Background/Aims: Erythropoietin-resistance is an unsolved concern in the treatment of renal anaemia. We aimed to investigate the possible role of ortho- and meta-tyrosine – the hydroxyl free radical products of L-phenylalanine – in the development of erythropoietin-resistance.

Methods: TF-1 erythroblast cell line was used. Cell concentration was determined on day 1; 2 and 3 by two independent observers simultaneously in Bürker cell counting chambers.

Protein concentration was determined with colorimetric method. Para-, ortho- and meta- W\URVLQHOHYHOVZHUHPHDVXUHGXVLQJUHYHUVHSKDVH+3/&ZLWKÁXRUHVFHQFHGHWHFWLRQ8VLQJ Western blot method activating phosphorylation of STAT5 and ERK1/2 were investigated.

Results: We found a time- and concentration-dependent decrease of erythropoietin-induced proliferative activity in case of ortho- and meta-tyrosine treated TF-1 erythroblasts, compared to the para-tyrosine cultured cells. Decreased erythropoietin-response could be regained with a competitive dose of para-tyrosine. Proteins of erythroblasts treated by ortho- or meta- tyrosine had lower para-tyrosine and higher ortho- or meta-tyrosine content. Activating phosphorylation of ERK and STAT5 due to erythropoietin was practically prevented by ortho- or meta-tyrosine treatment. Conclusion: According to this study elevated ortho- and meta-tyrosine content of erythroblasts may lead to the dysfunction of intracellular signaling, resulting in erythropoietin-hyporesponsiveness.

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Introduction

Erythropoietin (EPO) is a hematopoietic growth factor playing major role in the proliferation and differentiation of erythroid cells. Renal anaemia is present in patients suffering in chronic kidney disease (CKD), resulting in the impairment of quality of life. Approximately 15 % of the recombinant-human-EPO (rh-EPO) receiving subjects are hyporesponsive [1]. Several possible pathomechanisms are discussed, such as iron

†‡ϐ‹…‹‡…›ǡ‹ϐŽƒƒ–‹‘ǡ—•‡‘ˆƒ‰‹‘–‡•‹…‘˜‡”–‹‰‡œ›‡‹Š‹„‹–‘”•ǡ—”‡‹…–‘š‹•ǡ

‹•—ˆϐ‹…‹‡–†‹ƒŽ›•‹•ǡŠ›’‡”’ƒ”ƒ–Š›”‘‹†‹•‘”ƒŽ‹‰ƒ…›ȏʹȐǤƒŠ‡‘‰Ž‘„‹–ƒ”‰‡–Ǧ„ƒ•‡†

study, performed by Solomon et al., the decreased response to rh-EPO was associated with higher risk of mortality [3, 4].

”›–Š”‘’‘‹‡–‹Ǧ”‡…‡’–‘”ȋǦȌ‹•ƒ‡„‡”‘ˆ–›’‡…›–‘‹‡”‡…‡’–‘”•—’‡”ˆƒ‹Ž›ǡ

’”‡•‡–‹ƒ˜ƒ”‹‡–›‘ˆ–‹••—‡•ƒ†Šƒ•„‡‡…Šƒ”ƒ…–‡”‹œ‡†‹ƒ™‹†‡”ƒ‰‡‘ˆ…‡ŽŽŽ‹‡•ȏͷǦ ͳʹȐǤǦ„‹†‹‰”‡•—Ž–•‹Ǧ†‹‡”‹œƒ–‹‘ǡʹ–›”‘•‹‡Ǧ‹ƒ•‡’Š‘•’Š‘”›Žƒ–‹‘ǡƒ†

ƒ…–‹˜ƒ–‹‘‘ˆ•‹‰ƒŽ–”ƒ•†—…‡”ƒ†ƒ…–‹˜ƒ–‘”‘ˆ–”ƒ•…”‹’–‹‘ǦͷȋͷȌƒ†—…Ž‡ƒ”ˆƒ…–‘”Ɉ [13]. The stimulus also results in the activation of the Ras/mitogen-activated protein kinase (MAPK) pathway. Erythroid cell proliferation and differentiation is dependent on both the

’ͶʹȀͶͶƒ†–Š‡Ǧƒš‹•ȏͳͶȐǤ

The imbalance between pro- and antioxidant processes leads to the formation of reactive oxygen species (ROS). When excessive amount of hydroxyl radical is present, L-phenylalanine is converted into meta-tyrosine and ortho-tyrosi‡ǡ„‡•‹†‡•–Š‡‡œ›ƒ–‹…

formation of the physiological isomer, para-tyrosine [15]. Gurer-Orhan et al. proved that the concentration-dependent integration of meta-tyrosine into cellular proteins may be a mechanism of cytotoxicity [16]. Ehrlich observed in 1906 the phenomenon, known as concomitant tumor resistance, where a tumor-bearing host inhibits the growth of secondary

–—‘”‹’Žƒ–•‘”‡–ƒ•–ƒ•‹•Ǥ—‰‰‹‡”‘‡–ƒŽǤ‹†‡–‹ϐ‹‡†–Š‡ƒ…–‹˜‡•‡”—ˆ”ƒ…–‹‘”‡•’‘•‹„Ž‡

for this phenomenon containing a mixture of the three isoforms of tyrosine [17]. According to their in vitro and in vivo studies ortho- and meta-tyrosine inhibits tumor growth in a dose dependent manner. Using immunoblot analysis, they found impaired ERK and STAT3

ƒ…–‹˜ƒ–‹‘ ‹ –Š‡ ’”‡•‡…‡ ‘ˆ ‡–ƒǦ–›”‘•‹‡ ȏͳͺȐǤ ‘Žƒ” ‡– ƒŽǤ ˆ‘—† •‹‰‹ϐ‹…ƒ–Ž› Ž‘™‡”

’ƒ”ƒǦ–›”‘•‹‡Ž‡˜‡Žǡƒ†ƒŽ•‘ƒ‘Ǧ•‹‰‹ϐ‹…ƒ–ǡ„—–‘„˜‹‘—•Ž›Š‹‰Š‡”’Žƒ•ƒ‘”–Š‘Ǧ–›”‘•‹‡

level in patients with CKD [19]. According to the recent work of our group, on the one hand

’ƒ”ƒǦ–›”‘•‹‡ Ž‡˜‡Ž ™ƒ• •‹‰‹ϐ‹…ƒ–Ž› Ž‘™‡”ǡ ‘ –Š‡ ‘–Š‡” Šƒ† ‡–ƒǦ ƒ† ‘”–Š‘Ǧ–›”‘•‹‡

Ž‡˜‡Ž•™‡”‡•‹‰‹ϐ‹…ƒ–Ž›Š‹‰Š‡”‹†‹ƒŽ›œ‡†’ƒ–‹‡–•…‘’ƒ”‡†–‘…‘–”‘Ž‰”‘—’•ȋ†ƒ–ƒ—†‡”

publication).

ƒ•‡† ‘ –Š‡•‡ ‘„•‡”˜ƒ–‹‘• ™‡ Š›’‘–Š‡•‹œ‡†ǡ –Šƒ– ‹–‡‰”ƒ–‹‘ ‘ˆ ‡–ƒǦ ƒ† ‘”–Š‘Ǧ tyrosine into cellular proteins may result in the alteration of signal transduction, leading to EPO-hyporesponsiveness.

Materials and Methods

Materials

Unless otherwise noted, chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA). Culture medium products were purchased from Life Technologies (Carlsbad, CA, USA).

Cell culture and treatments

ǦͳȋǦʹͲͲ͵Ȍ‡”›–Š”‘„Žƒ•–•ȋ‡”‹…ƒ›’‡—Ž–—”‡‘ŽŽ‡…–‹‘ǡ‘…˜‹ŽŽ‡ǡǡȌ™‡”‡—•‡†Ǥ

‡ŽŽ•™‡”‡‰”‘™‹Ǧͳ͸ͶͲ‡†‹—ǡ…‘–ƒ‹‹‰ʹ‰ȀŽ‘ˆǦ ǡͳͲ؈‡–ƒŽ…ƒŽˆ•‡”—ƒ†‹š–—”‡

of antibiotics. Prior to experiments, cells were cultured in medium containing indicated amount of para- , ortho-, or meta-tyrosine for 3 days. Tyrosine content of the culture media was stable; no considerable alteration of para-, ortho- or meta-tyrosine concentration could be detected in the absence of cells, during 3 days (data not shown).

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‘”’”‘Ž‹ˆ‡”ƒ–‹‘•–—†‹‡•‹•–‡ƒ†‘ˆǦ ǡ͵ȀŽ”ŠǦ™ƒ•ƒ††‡†—†‡”•‹‹Žƒ”…‘†‹–‹‘•Ǥ ‘”

‡•–‡”„Ž‘––‹‰‡š’‡”‹‡–•ǡƒˆ–‡”ͳʹŠ‘—”•‘ˆ•‡”—ƒ†ˆƒ…–‘”†‡’”‹˜ƒ–‹‘ǡ–”‡ƒ–‡–•™‡”‡’‡”ˆ‘”‡†

„›ƒ††‹–‹‘‘ˆ͵ȀŽ‘ˆ”ŠǦˆ‘”ͳͲ‹—–‡•Ǥ

Analysis of cell proliferation

–ƒ†ƒ”†—„‡”‘ˆ…‡ŽŽ•ȋʹšͳͲ⁵ /ml) was planted onto 60 mm plates in culture medium containing

ƒ††‹–‹‘ƒŽʹͲ‰ȀŽ’ƒ”ƒǦǡ‘”–Š‘Ǧǡ‘”‡–ƒǦ–›”‘•‹‡ǡŽƒ…‹‰Ǧ ǡ™‹–Š‘”™‹–Š‘—–ƒ††‹–‹‘‘ˆ͵ȀŽ

”ŠǦˆ‘”͵†ƒ›•Ǥ‡ŽŽ…‘…‡–”ƒ–‹‘ȋ…‡ŽŽ•ȀρŽȌ™ƒ•†‡–‡”‹‡†‘†ƒ›ͳǢʹƒ†͵„›–™‘‹†‡’‡†‡–

‘„•‡”˜‡”• •‹—Ž–ƒ‡‘—•Ž›ǡ ™‹–Š –Š‡ ƒ’’Ž‹…ƒ–‹‘ ‘ˆ ò”‡” …‡ŽŽ …‘—–‹‰ …Šƒ„‡”•Ǥ ‡ …ƒŽ…—Žƒ–‡† –Š‡

ƒ–Š‡ƒ–‹…ƒŽ‡ƒ‘ˆ–Š‡–™‘‘„•‡”˜‡†…‡ŽŽ…‘—–•Ǥ˜‡–—ƒŽŽ›•ƒ’Ž‡•™‡”‡Ž›œ‡†ǡ–Š‡ƒˆ–‡”•–‘”ƒ‰‡‹ǦͺͲ

oC for one night protein concentration was determined as described below. For concentration-dependence experiments cells were cultured in medium containing para-, ortho-, or meta-tyrosine with the addition of ͲǢʹͲǢͶͲ‘”ͺͲ‰ȀŽ’ƒ”ƒǦ–›”‘•‹‡ˆ‘”͵Ǧ†ƒ›•ǤǦͳ͸ͶͲ‡†‹—…‘–ƒ‹•ʹͲ‰ȀŽ’ƒ”ƒǦ–›”‘•‹‡‘”‹‰‹ƒŽŽ›Ǥ

HPLC (Investigating tyrosine incorporation into cellular proteins)

‘’”‘˜‡‹…‘”’‘”ƒ–‹‘‘ˆ–Š‡†‹ˆˆ‡”‡––›”‘•‹‡‹•‘ˆ‘”•‹–‘…‡ŽŽ—Žƒ”’”‘–‡‹•™‡—•‡†ƒϐŽ—‘”‡•…‡…‡ HPLC-method. After three-day incubation cell culture was terminated. Medium was removed by centrifugation (1000 rpm; 10 min), then cells were washed three times by addition of 1 ml physiological

ƒŽǦ•‘Ž—–‹‘ ƒ† …‡–”‹ˆ—‰ƒ–‹‘ ȋͳͲͲͲ ”’Ǣ ͷ ‹ȌǤ —„•‡“—‡–Ž› ʹͲͲ ρŽ †‹•–‹ŽŽ‡† ™ƒ–‡” ™ƒ• ƒ††‡† –‘

the cells, followed by resuspendation and ultrasound treatment. Thereafter 100 µl 60% trichloro-acetic acid (TCA) was added. Mixture was then vortexed and centrifuged (4000 rpm; 10 min). After removing

•—’‡”ƒ–ƒ–ǡʹͲͲρŽͳΨ™ƒ•ƒ††‡†ǡˆ‘ŽŽ‘™‡†„›”‡•—•’‡†ƒ–‹‘ǡ˜‘”–‡š‹‰ƒ†ƒ††‹–‹‘‘ˆͳͲͲρŽ͸ͲΨ Ǥƒ’Ž‡•™‡”‡–Š‡…‡–”‹ˆ—‰‡†ȋͶͲͲͲ”’ǢͳͲ‹Ȍǡ•—’‡”ƒ–ƒ–™ƒ•”‡‘˜‡†ƒ†ʹͲͲρŽͳΨ™ƒ•

added. Samples were then resuspendated, treated by ultrasound and added by 100 µl 60% TCA, followed by vortexing. Subsequently precipitate was separated by centrifugation (4000 rpm; 10 min) and 40 µl

„—–Š›Š›†”‘š›Ž–‘Ž—‘ŽȋǢͷͲͲȌǡͶρŽ†‡•ˆ‡”ƒŽȋͶͲͲȌƒ†ͶͲͲρŽͳʹŽ™ƒ•ƒ††‡†–‘‹–ǤŠ‡”‡ƒˆ–‡”

•ƒ’Ž‡•™‡”‡—†‡”‰‘‡ƒ‘˜‡”‹‰Š–Š›†”‘Ž›•‹•‘ͳʹͲιǤƒ’Ž‡•™‡”‡–Š‡…‡–”‹ˆ—‰‡†ȋͷͲͲͲ”’Ǣ ͳͷ ‹ȌǤ Š‡ •—’‡”ƒ–ƒ– ™ƒ• ϐ‹Ž–‡”‡† „› ƒ •›”‹‰‡ ϐ‹Ž–‡” ȋͲǤʹ ρȌ „‡ˆ‘”‡ ƒƒŽ›•‹•Ǥ ‹ƒŽŽ›para-, ortho- and meta-tyrosine levels were determined using reverse phase-HPLC (C₁₈•‹Ž‹…ƒ…‘Ž—ǡʹͷͲšͶȌ™‹–Š

ϐŽ—‘”‡•…‡…‡†‡–‡…–‹‘ȋɉEXαʹ͹ͷǢɉEM=305 nm) as described earlier [19]. Concentrations were calculated using an external standard. We calculated the ratios of para-tyrosine and total-tyrosine, ortho-tyrosine and total-tyrosine and meta-tyrosine and total-tyrosine.

Immunoblot analysis

‘ŽŽ‘™‹‰ ”ŠǦ –”‡ƒ–‡–•ǡ …‡ŽŽ• ™‡”‡ Ž›œ‡† ‹ ”‹•Ǧ”‹–‘ ‡š–”ƒ…–‹‘ „—ˆˆ‡” ȋͳ ”‹•ǦŽǡ ’

͹ǤͶǡͳǤͳͷȋ˜‘ŽȀ˜‘ŽȌΨ”‹–‘ǦͳͲͲǡͷͲͲǡʹͲͲǡͳͲͲ†‹–Š‹‘–Š”‡‹–‘Ž…‘–ƒ‹‹‰–Š‡

’”‘–‡ƒ•‡‹Š‹„‹–‘”•‘ˆͳͲͲƒ†ͲǤͷȋ™–Ȁ˜‘ŽȌΨ’Š‡›Ž‡–Š›Ž•—Žˆ‘›ŽϐŽ—‘”‹†‡ǡŽ‡—’‡’–‹ǡƒ’”‘–‹‹ƒ†

’Š‘•’Šƒ–ƒ•‡‹Š‹„‹–‘”…‘…–ƒ‹ŽʹȀ͵Ȍ‘‹…‡ˆ‘”͵Ͳ‹Ǥ›œ‡†’”‘–‡‹•™‡”‡Šƒ”˜‡•–‡†ǡ…‡–”‹ˆ—‰‡†ȋͳͶͲͲͲ‰ǡ for 15 min, at 4^oȌǡ–Š‡’”‘–‡‹…‘…‡–”ƒ–‹‘‘ˆ–Š‡•—’‡”ƒ–ƒ–™ƒ•ƒ••‡••‡†—•‹‰–Š‡‹‘Ǧƒ†’”‘–‡‹

ƒ••ƒ› ‹– ȋ‡”…—Ž‡•ǡ ǡ Ȍ ™‹–Š „‘˜‹‡ •‡”— ƒŽ„—‹ ȋȌ ƒ• •–ƒ†ƒ”†Ǥ ‘Ž—„‹Ž‹œ‡† ’”‘–‡‹• ™‡”‡

‹š‡†™‹–Šƒ‡Ž‹„—ˆˆ‡”ȋʹȌǡ denaturated (for 5 min, at 90^oC), then were separated on 10% SDS-PAGE

ƒ† –”ƒ•ˆ‡””‡† ‘–‘ ’‘Ž›˜‹›Ž‹†‡‡ †‹ϐŽ—‘”‹†‡ ‡„”ƒ‡ ȋ‹ŽŽ‹’‘”‡ǡ ‹ŽŽ‡”‹…ƒǡ ǡ ȌǤ “—ƒŽ ’”‘–‡‹

Ž‘ƒ†‹‰ȋͷͲǦ͹ͷρ‰Ȍ™ƒ•…‘ϐ‹”‡†„›•–ƒ‹‹‰–Š‡‡„”ƒ‡™‹–Š‘…‡ƒ—ǦǤŠ‡„Ž‘–•™‡”‡„Ž‘…‡†‹

–”‹•„ƒ•‡•ƒŽ‹‡…‘–ƒ‹‹‰ͷȋ™–Ȁ˜‘ŽȌΨƒ†ͲǤͳȋ˜‘ŽȀ˜‘ŽȌΨ™‡‡ǡ–Š‡™‡”‡‹…—„ƒ–‡†™‹–Š’”‹ƒ”›

ƒ–‹„‘†‹‡•ȋͳǣͳͲͲͲǢ‡ŽŽ‹‰ƒŽ‹‰ǡ‡˜‡”Ž›ǡǡȌƒ‰ƒ‹•–’Š‘•’Š‘ǦŠ”ȋʹͲʹȌȀ›”ȋʹͲͶȌƒ†–‘–ƒŽ

ȋ’ͶͶȀͶʹȌǢ’Š‘•’Š‘Ǧ›”ȋ͸ͻͶȌͷƒ†–‘–ƒŽͷ‘”éǦƒ…–‹ˆ‘”‘˜‡”‹‰Š–ƒ–Ͷ^oC. After being washed, the membranes were incubated with appropriate, horseradish peroxidase-conjugated secondary

ƒ–‹„‘†›ȋͳǣʹͲͲͲǢƒ–‹Ǧ”ƒ„„‹–‰ǡ‡ŽŽ‹‰ƒŽ‹‰Ȍˆ‘”͸Ͳ‹ƒ–”‘‘–‡’‡”ƒ–—”‡Ǥˆ–‡”ˆ—”–Š‡”™ƒ•Š‹‰ǡ

‹—‘„Ž‘–•™‡”‡˜‹•—ƒŽ‹œ‡†™‹–Š‡Šƒ…‡†…Š‡‹Ž—‹‡•…‡…‡ȋ—’‡”Ǧ‹‰ƒŽ‡•–‹…‘ǡŠ‡”‘ ‹•Š‡”

…‹‡–‹ϐ‹…ǡ ǡ Ȍ ƒ† †‡˜‡Ž‘’‡† ‘ Ǧ”ƒ› ϐ‹Ž• ȋ‘†ƒ ȌǤ ‘–ƒŽ ’”‘–‡‹ Ž‡˜‡Ž• ‘ˆ ͷ ƒ†

™‡”‡‹—‘•–ƒ‹‡†ƒˆ–‡”•–”‹’’‹‰–Š‡„Ž‘–•Ǥ‡•‹–‘‡–”‹…ƒƒŽ›•‡•™‡”‡’‡”ˆ‘”‡†—•‹‰…‹‘ƒ‰‡

•‘ˆ–™ƒ”‡ ȋ ”‡†‡”‹…ǡ ǡ ȌǤ ƒ–ƒ ƒ”‡ ‡š’”‡••‡† ƒ• –Š‡ ”ƒ–‹‘ ‘ˆ ’Š‘•’Š‘”›Žƒ–‡† ƒ† –‘–ƒŽ ͳȀʹ ‘”

ͷǡ…‘””‡…–‡†–‘–‘–ƒŽ…‡ŽŽ—Žƒ”éǦƒ…–‹Ž‡˜‡ŽǤ

y: omanyi 4/29/2014 9:26:54 AM

Statistical analysis

Data are expressed as means±SE or means±SD, as indicated. Analyses were performed as appropriate

—•‹‰ͳ͹ǤͲȋ…ǤǡŠ‹…ƒ‰‘ǡǡȌǤ–ƒ–‹•–‹…ƒŽŽ›•‹‰‹ϐ‹…ƒ–†‹ˆˆ‡”‡…‡•™‡”‡†‡ϐ‹‡†ƒ••‹‰‡†ƒ†

ƒ”‡‹†‹…ƒ–‡†‹–Š‡ϐ‹‰—”‡Ž‡‰‡†•Ǥ‘”ƒŽ†‹•–”‹„—–‹‘™ƒ•˜‡”‹ϐ‹‡†™‹–Š–Š‡ƒ’’Ž‹…ƒ–‹‘‘ˆ‘Ž‘‰‘”‘˜Ȃ

‹”‘˜–‡•–Ǥ…ƒ•‡‘ˆ…‡ŽŽ’”‘Ž‹ˆ‡”ƒ–‹‘•–—†‹‡•ǡƒ†’”‘–‡‹…‘…‡–”ƒ–‹‘‡ƒ•—”‡‡–•

™‹–Š‘ˆ‡””‘‹ǯ•’‘•–ǦŠ‘…–‡•–™ƒ•‡’Ž‘›‡†Ǥ…ƒ•‡‘ˆ‡•–‡”Ǧ„Ž‘–”‡•—Ž–•ǡ’Š‘•’Š‘”›Žƒ–‹‘‘ˆ…‘–”‘Ž•

was taken as 100% and one sample t-test was performed for the comparison versus controls. To compare

–Š‡‡ƒ•„‡–™‡‡–Š‡‰”‘—’•‘ˆ”‡Žƒ–‹˜‡’Š‘•’Š‘”›Žƒ–‹‘•™‹–Š‘ˆ‡””‘‹ǯ•’‘•–ǦŠ‘…–‡•–™ƒ•

used.

Results

Cell proliferation

Panel A of Figure 1 shows the EPO-induced, time-dependent proliferation of TF-1 erythroblasts grown in para-, ortho- or meta-tyrosine containing medium. Culturing TF-1 cells in the presence of ortho- and meta-tyrosine EPO-induced proliferative activity was found to be decreased compared with para-tyrosine cultured cells. Maximal difference between cell counts was observed at day 3 (time curve).

ƒ‡Ž †‡‘•–”ƒ–‡• –Š‡ ‡ˆˆ‡…– ‘ˆ ƒ††‹‰ ‡š–”ƒ Ͳǡ ʹͲǡ ͶͲǡ ‘” ͺͲ ‰ȀŽ ’ƒ”ƒǦ–›”‘•‹‡

into the ortho- or meta-tyrosine supplemented medium on the cell counts at the 3rd day

A C

B

Fig. 1. Effect of para-, ortho-, or meta-tyrosine supplementation on the proliferation of cells cultured in me- dium with or without EPO. Panel A shows the time dependent rise of cell count in para-tyrosine (open bars), ortho-tyrosine (black bars) or meta-tyrosine (grey bars) supplemented medium. (*, P<0.05 vs. para-tyro- sine cultured cells on the same day; n=10). Panel Bshows the alterations of cell counts after incubation for 3-days in media containing ortho- or meta-tyrosine and the indicated additional amount of para-tyrosine (*,P<0.001 vs. para-tyrosine containing medium; n=5). Panel C shows the effect of supplementation by or- tho- or meta-tyrosine on the increment of cellular protein induced by EPO. Results are expressed as the ratio of protein content of EPO and non-EPO (control) cells. (*, P<0.05 vs. para-tyrosine cultured cells; n=10).

yi 014 9:26:54 AM

para-tyrosine in case of ortho-tyrosine cultured control cells (Panel A of Figure 2, left part).

Cells grown in medium containing ortho- or meta-tyrosine and EPO showed less para- tyrosine compared to the para-tyrosine supplemented cells (Panel A of Figure 2, right part).

ƒ‡Ž‘ˆ ‹‰—”‡ʹ†‡‘•–”ƒ–‡•ǡ–Šƒ–‹…‘”’‘”ƒ–‹‘‘ˆ‘”–Š‘Ǧ–›”‘•‹‡™ƒ•Š‹‰Š‡”‹–‘

the proteins of ortho-tyrosine cultured erythroblasts compared to the cells cultured with

‡–ƒǦ‘”’ƒ”ƒǦ–›”‘•‹‡ǡ‹…ƒ•‡‘ˆ‘Ǧȋ…‘–”‘ŽǡŽ‡ˆ–’ƒ”–‘ˆϐ‹‰—”‡Ȍƒ†ȋ”‹‰Š–’ƒ”–

‘ˆϐ‹‰—”‡Ȍ…‡ŽŽ•ǡƒ•™‡ŽŽǤ‘”‡‘˜‡”ǡ‘”–Š‘Ǧ–›”‘•‹‡…‘–‡–™ƒ••‹‰‹ϐ‹…ƒ–Ž›Š‹‰Š‡”‹Ǧ cultured cells than that of non-EPO-treated (control) cells, when both were grown in ortho-

–›”‘•‹‡•—’’Ž‡‡–‡†‡†‹—ȋ…‘’ƒ”‹‰Ž‡ˆ––‘”‹‰Š–’ƒ”–‘ˆϐ‹‰—”‡ȌǤ

‡–ƒǦ–›”‘•‹‡ ‹…‘”’‘”ƒ–‹‘ ™ƒ• •‹‰‹ϐ‹…ƒ–Ž› Š‹‰Š‡” ‹ ‡–ƒǦ–›”‘•‹‡ …—Ž–—”‡†

erythroblasts both in non-EPO (control) and in EPO groups compared to cells cultured in

’ƒ”ƒǦ‘”‘”–Š‘Ǧ–›”‘•‹‡•—’’Ž‡‡–‡†‡†‹—ȋƒ‡Ž‘ˆ ‹‰—”‡ʹǡŽ‡ˆ–ƒ†”‹‰Š–’ƒ”–ȌǤ Analyses of STAT5 and ERK activation

‘—”‡•–‡”„Ž‘–‡š’‡”‹‡–•–”‡ƒ–‡–™‹–Š‘”–Š‘Ǧƒ†‡–ƒǦ–›”‘•‹‡’”‡˜‡–‡†

the increase of STAT5-phosphorylation induced by EPO in para-tyrosine cultured cells (Panel

ƒ†‘ˆ ‹‰—”‡͵ȌǤŠ‡•ƒ‡‹Š‹„‹–‹‘„›‘”–Š‘Ǧƒ†‡–ƒǦ–›”‘•‹‡™ƒ••‡‡‹…ƒ•‡‘ˆ

’Š‘•’Š‘”›Žƒ–‹‘‘ˆͳƒ†ʹȋƒ‡ŽǦ‘ˆ ‹‰—”‡͵ȌǤ A

C B Fig. 2.Relative para-, ortho- and meta-tyrosi- ne content (i.e. ratios of para-, ortho-, and me- ta-tyrosine/total tyrosine) of cellular proteins

‘ˆ Ǧͳ…‡ŽŽ•ȋƒ‡Žǡƒ†”‡•’‡…–‹˜‡Ž›ȌǤȗǡ P=0.003 vs para-tyrosine cultured erythrob- lasts; §, P<0.001 vs para-tyrosine cultured cells;

†, P<0.001 vs. para- and meta-tyrosine cultured …‡ŽŽ•ǢᓐǡδͲǤͲͲͳ˜•Ǥ‘”–Š‘Ǧ–›”‘•‹‡…—Ž–—”‡†…‘- trol cells; +, P<0.001 vs. para- and ortho-tyrosi- ne cultured erythroblasts; n=10.

of growing with or without rh-EPO (concentration dependence). The ortho- tyrosine induced impairment of EPO- response could be competed by 40 mg/l of para-tyrosine, while in the case of meta-tyrosine at least 60 mg/l of para- tyrosine concentration was necessary.

As shown in Panel C of Figure 1 relative protein content (EPO/non- EPO) of meta- or ortho-tyrosine treated …—Ž–—”‡•™‡”‡•‹‰‹ϐ‹…ƒ–Ž›Ž‘™‡”ǡ–Šƒ

that grown on para-tyrosine.

Tyrosine incorporation into cellular proteins

Treatment of the cells with meta-

–›”‘•‹‡ Ȃ …‘’ƒ”‡† –‘ –Š‡ …—Ž–—”‡

…‘–ƒ‹‹‰’ƒ”ƒǦ–›”‘•‹‡Ȃ†‡…”‡ƒ•‡†–Š‡

para-tyrosine content of cellular proteins in non-EPO (control) experiments. No

•‹‰‹ϐ‹…ƒ– †‹ˆˆ‡”‡…‡ ™ƒ• †‡–‡…–‡† ‹

y: omanyi 4/29/2014 9:26:54 AM

A

C

B

D

E

Fig. 3. Representative immunoblots (Panel A) and densitometric analyses of phosphorylation of STAT5 (Pa-

‡ŽǢα͵ȌǢ‹—‘„Ž‘–•‘ˆͳƒ†ʹȋƒ‡ŽȌƒ††‡•‹–‘‡–”‹…ƒƒŽ›•‹•‘ˆͳȋƒ‡ŽǢαͶȌƒ†

ʹȋƒ‡ŽǢαͶȌǤƒ–ƒƒ”‡‡š’”‡••‡†ƒ•’‡”…‡–‘ˆ—–”‡ƒ–‡†ȋ…‘–”‘ŽȌ…‡ŽŽ•ǤȗǡδͲǤͲͷǢ˜•…‘–”‘Ž

ȋ‘‡Ǧ•ƒ’Ž‡Ǧ–‡•–ȌǢȘǡδͲǤͲͷ˜•’ƒ”ƒȋ‘‡Ǧ™ƒ›ȌǤ

Discussion

–Š‹••–—†›ǡ™‡’”‘˜‡†–Šƒ–‘”–Š‘Ǧƒ†‡–ƒǦ–›”‘•‹‡‹…‘”’‘”ƒ–‡‹–‘…‡ŽŽ—Žƒ”’”‘–‡‹•

‹ƒ†‡–‡…–ƒ„Ž‡ǡ„—–Ȃ…‘’ƒ”‡†–‘–Š‡–‘–ƒŽ…‡ŽŽ—Žƒ”’”‘–‡‹Ǧ„‘—†–›”‘•‹‡Ȃ˜‡”›Ž‘™”ƒ–‹‘

(0.10-0.15%), with a consequent decrease in activation of antiapoptotic and mitogenic

•‹‰ƒŽ‹‰ ’ƒ–Š™ƒ›•Ǥ ƒ†‡“—ƒ–‡ ’Š‘•’Š‘”›Žƒ–‹‘ ‘ˆ ƒ† ͷ ”‡•—Ž–• ‹ †‡ϐ‹…‹‡–

proliferative response to EPO, leading to EPO-hyporesponsiveness of erythroid progenitor cells (Figure 4).

Oxidative stress is proven to play a major role in the pathogenesis of several morbidities,

•—…Š ƒ• †‹ƒ„‡–‹… ‡’Š”‘’ƒ–Š›ǡ ƒ–Š‡”‘•…Ž‡”‘•‹• ‘” ‹•…Š‡‹… Š‡ƒ”– †‹•‡ƒ•‡ ȏʹͲǦʹʹȐǤ ‹‰Š‡”

plasma level or urinary excretion of ortho- and meta-tyrosine, as hydroxylated phenylalanine derivatives, is associated with altered oxidative state, thus they serve as markers of oxidative stress. On the other hand, Ruggiero et al. proved, that ortho- and meta-tyrosine decreases tumor proliferation due to the inhibition of ERK and STAT3 activation. They used meta- and

‘”–Š‘Ǧ–›”‘•‹‡…‘…‡–”ƒ–‹‘•—’–‘ͺʹ͹^Pmol/l. Similarly, we detected decreased ERK and STAT5 phosphorylation in the presence of ortho- and meta-tyrosine.

ƒ‰”‡‡‡–™‹–Š†ƒ–ƒ‘ˆ‹–ƒ—”ƒ‡–ƒŽǤȏͳʹȐǡƒŽ•‘‹‘—”‡š’‡”‹‡–•…—Ž–—”‹‰…‡ŽŽ•

with EPO for 3 days resulted in an approximate 1.7 fold increase in cell counts. Addition of meta- or ortho-tyrosine into the culture medium caused a markedly decreased proliferative activity, leading to lower cell counts. Since, according to the source, duplication time of this …‡ŽŽŽ‹‡‹•ƒ„‘—–ʹʹŠ‘—”ǡ†‹ˆˆ‡”‡…‡•…‘—Ž†„‡ƒŽ”‡ƒ†›‘„•‡”˜‡†‡˜‡ƒˆ–‡”ʹ†ƒ›•‘ˆ…—Ž–—”‹‰

(Panel A of Figure 1). ^{yi 014 9}

:26:54 AM

–Š‡‹””‘Ž‡‹–Š‡’”‘–‡‹†‡‰”ƒ†ƒ–‹‘Ǥ–Š‡‹”‡š’‡”‹‡–•ǡ…‘…‡–”ƒ–‹‘•‘ˆ‡–ƒǦ–›”‘•‹‡

”‡ƒ…Š‡†—’–‘ʹͲͲͲρ‘ŽȀŽȏʹͶȐǤ

As EPO-treatment is widely used among patients suffering in renal anaemia, EPO-

Š›’‘”‡•’‘•‹˜‡‡•• ‹• ƒ …‘‘ …‘…‡”Ǥ Š‡ ƒŽ–‹‘”‡ ‘‰‹–—†‹ƒŽ –—†› ‘ ‰‹‰

‹˜‡•–‹‰ƒ–‡†Š‡ƒŽ–Š›ǡ‘Ǧƒ‡‹…’‡”•‘•ƒ†ˆ‘—†–Šƒ–Ž‡˜‡Ž•”‹•‡™‹–Šƒ‰‡ȏʹͷȐǤVanesse and Berliner•—‰‰‡•–‡†•Š‘”–‡”‡”›–Š”‘…›–‡Ž‹ˆ‡•’ƒƒ•ƒ’‘••‹„Ž‡‡š’Žƒƒ–‹‘ȏʹ͸ȐǤ–Š‡

‘–Š‡”Šƒ†ǡƒ‹‡–ƒŽǤŠ›’‘–Š‡•‹œ‡†ǡ–Šƒ–ƒ‰‹‰”‡•—Ž–•‹‹’ƒ‹”‡–‘ˆ–Š‡‹–‘…Š‘†”‹ƒŽ

electron transport chain and through the leakage of electrons, leads to increased production

‘ˆȏʹ͹ȐǤŠ‡‹”ϐ‹†‹‰•‹†‹…ƒ–‡Ȃ•‹‹Žƒ”Ž›–‘‘—”‘„•‡”˜ƒ–‹‘•Ȃ–Šƒ–ƒ‰‹‰”‡Žƒ–‡†

overproduction may play a role in the decrease of EPO-sensitivity.

—”–Š‡”‘”‡ǡ™‡…‘ϐ‹”‡†–Šƒ–„‡•‹†‡•‹–•™‡ŽŽǦ‘™”‘Ž‡‹—‡”‘—•…‡ŽŽ—Žƒ”•‹‰ƒŽ

–”ƒ•†—…–‹‘’”‘…‡••‡•ȏʹͺȐǡŠ›†”‘š›Žˆ”‡‡”ƒ†‹…ƒŽ‹†—…‡†‘š‹†ƒ–‹˜‡•–”‡••ƒ›„‡‹˜‘Ž˜‡†

in hormone resistances via incorporation of ortho- and meta-tyrosine into the proteins.

ƒ††‹–‹‘ǡMcCullogh et al. observed increased risk for cardiovascular events related

–‘–Š‡—•‡‘ˆŠ‹‰Š‡”Ǧ†‘•‡ǡ‹†‡’‡†‡–‘ˆ–Š‡Š‡‘‰Ž‘„‹Ž‡˜‡Žƒ…Š‹‡˜‡†ȏʹͻȐǤŽ–Š‘—‰Š

Š‹‰Š‡”‡ˆϐ‹…‹‡–Ǧ†‘•‡”‡•—Ž–•‹‘”‡’”‘‹‡–—™ƒ–‡†•‹†‡‡ˆˆ‡…–•ǡ™‡Š›’‘–Š‡•‹œ‡ǡ that this is an epiphenomenon, and higher cardiovascular risk is not triggered by higher plasma EPO-level itself, but rather by the incorporation of the ortho- and meta-tyrosine into

–Š‡…‡ŽŽ—Žƒ”’”‘–‡‹•Ž‡ƒ†‹‰–‘ƒ„‘”ƒŽ…‡ŽŽ—Žƒ”ˆ—…–‹‘•ǤŠ‡•‡ϐ‹†‹‰••Š‘™•‹‹Žƒ”‹–‹‡•

to the role of insulin resistance in mortality instead of role of the necessarily high insulin dosage [30].

Fig. 4. Flowchart of the scheme of the possible subcellu- lar process, how ortho- and meta-tyrosine incorporation leads to the dysfunction of signal pathway mechanisms, resulting in the hyporesponsiveness of erythroid progeni- tor cells.

 –Š‡ •–—†› ‘ˆ Ruggiero et al., inhibition by meta-tyrosine could be reversed with excessive phenylalanine, glutamine, aspartic acid, histidine and glutamate, but not with para-tyrosine itself. On the contrary, we could break through the EPO-resistance by para- tyrosine in a dose dependent manner ȋƒ‡Ž‘ˆ ‹‰—”‡ͳȌǤŠ‡•‡”‡•—Ž–•ƒ›

suggest that para-tyrosine competitively inhibits the integration of ortho- and meta-tyrosine into cellular proteins.

 …ƒ•‡ ‘ˆ ‘”–Š‘Ǧ–›”‘•‹‡ –”‡ƒ–‡–•ǡ 1:1.8 ortho- to para-tyrosine ratio was necessary to regain 50 % of EPO-

•‡•‹–‹˜‹–›Ǥ…ƒ•‡‘ˆ‡–ƒǦ–›”‘•‹‡ǡ–Š‡

”ƒ–‹‘™ƒ•ͳǣʹǤ͸Ǥ

Ǧͳ͸ͶͲ …‡ŽŽ …—Ž–—”‡ ‡†‹—

contains para-tyrosine at twice as high concentration than the normal human serum does. Consequently, the same concentration had to be used in case of ortho- and meta-tyrosine, in order to block EPO-response. We performed a model experiment, demonstrating a total block of the effect of EPO, therefore high doses of tyrosine isomers were

ƒ’’Ž‹‡†Ǥ ‡”–‹ ‡– ƒŽ —•‡† •‹‹Žƒ”

…‘…‡–”ƒ–‹‘• ‘ˆ ‡–ƒǦ–›”‘•‹‡ ȋʹͲǦ ʹ͸ͲɊ‘ŽȀŽȌ–‘•–—†›‹–•‡ˆˆ‡…–‘’Žƒ–

”‘‘–‰”‘™–Šȏʹ͵ȐǤ —”–Š‡”‘”‡ǡ‘†‰‡”•

et al. examined the role of meta-tyrosine incorporation into cellular proteins and

y: omanyi 4/29/2014 9:26:54 AM

Conclusion

—” ϐ‹†‹‰• ’‘‹– ‘—– –Šƒ– ‘”–Š‘Ǧ ƒ† ‡–ƒǦ–›”‘•‹‡ ƒ› „‡ ”‡•’‘•‹„Ž‡ ˆ‘” Ǧ resistance. Furthermore, inhibiting their cellular integration with the physiological

‹•‘‡” Ȃ ’ƒ”ƒǦ–›”‘•‹‡ Ȃ –Š‡ ”‡•‹•–ƒ…‡ …ƒ „‡ „”‘‡ –Š”‘—‰Šǡ ’”‡•‡”˜‹‰ –Š‡ ƒ†‡“—ƒ–‡

‹–‘‰‡‹…ƒ…–‹˜‹–›Ǥ‡˜‡”–Š‡Ž‡••ǡˆ—”–Š‡”‹˜‹–”‘ƒ†ƒ‹ƒŽ‡š’‡”‹‡–•Ȃƒ†‹…ƒ•‡‘ˆ

–Š‡‹”’‘•‹–‹˜‹–›ȂŠ—ƒ‡šƒ‹ƒ–‹‘•ƒ”‡‡…‡••ƒ”›–‘†‡–‡”‹‡–Š‡”‘Ž‡‘ˆ’ƒ”ƒǦ–›”‘•‹‡

treatment in the management of EPO-resistance.

Disclosure Statement

‘…‘’‡–‹‰ϐ‹ƒ…‹ƒŽ‹–‡”‡•–•‡š‹•–Ǥ

Acknowledgements

This research was supported by the European Union and the State of Hungary, co- ϐ‹ƒ…‡†„›–Š‡—”‘’‡ƒ‘…‹ƒŽ —†‹–Š‡ˆ”ƒ‡™‘”‘ˆͶǤʹǤͶǤȀʹǦͳͳǦͳǦʹͲͳʹǦ ͲͲͲͳǮƒ–‹‘ƒŽš…‡ŽŽ‡…‡”‘‰”ƒǯǤ

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yi 014 9:26:54 AM

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‡—œƒ”†ǡ‹•Š‹‡–ƒŽǣͺͳʹǣƒ’Ž—”‹’‘–‡–Š—ƒ…‡ŽŽŽ‹‡™‹–Š•’‘–ƒ‡‘—•‡”›–Š”‘‹†–‡”‹ƒŽ

ƒ–—”ƒ–‹‘ǤŽ‘‘†ͳͻͺͻǢ͹͵ǣʹͲͲ͵ǦʹͲͳ͵Ǥ

ͳʹ ‹–ƒ—”ƒǡ‘Œ‘ǡ—™ƒ‹ǡŠ‹„ƒǡ‹›ƒœ‘‘ǡ”ƒ„‡ǡƒƒ— ǣ†‡–‹ϐ‹…ƒ–‹‘ƒ†ƒƒŽ›•‹•‘ˆŠ—ƒ

‡”›–Š”‘’‘‹‡–‹”‡…‡’–‘”•‘ƒˆƒ…–‘”Ǧ†‡’‡†‡–…‡ŽŽŽ‹‡ǡ ǦͳǤŽ‘‘†ͳͻͺͻǢ͹͵ǣ͵͹ͷǦ͵ͺͲǤ ͳ͵ ‹––Š—Šǡ—‡ŽŽ‡ ǡ‹Ž˜‡‘‹‡ǡ‹ǡƒ‰ǡ‹—”ƒǡŠŽ‡ǣʹƒ••‘…‹ƒ–‡•™‹–Š–Š‡

erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with

‡”›–Š”‘’‘‹‡–‹Ǥ‡ŽŽͳͻͻ͵Ǣ͹Ͷǣʹʹ͹Ǧʹ͵͸Ǥ

ͳͶ ƒ–‘™‹…ŠǣŠ‡”›–Š”‘’‘‹‡–‹‡…‡’–‘”ǣ‘Ž‡…—Žƒ”–”—…–—”‡ƒ†‡ƒ–‘’‘‹‡–‹…‹‰ƒŽ‹‰ƒ–Š™ƒ›•Ǥ

˜‡•–‹‰‡†ʹͲͳͳǢͷͻǣͳͲ͸͹ǦͳͲ͹ʹǤ

ͳͷ ‘‘Žˆǡƒ—„‘˜‹…ǡŠƒǦ‡”›ǣŠ‡‘Ǧ‡œ›‹…Š›†”‘š›Žƒ–‹‘‘ˆ’Š‡›ŽƒŽƒ‹‡–‘–›”‘•‹‡„›

ʹǦƒ‹‘ǦͶǦŠ›†”‘š›Ǧ͸ǡ͹Ǧ†‹‡–Š›ŽǦͷǡ͸ǡ͹ǡͺǦ–‡–”ƒŠ›†”‘’–‡”‹†‹‡Ǥ‹‘…Š‡ͳͻ͹ͳǢͳʹͷǣͷ͸ͻǦͷ͹ͶǤ

ͳ͸ —”‡”Ǧ”Šƒǡ”…ƒŽǡƒ”‡ǡ‡ƒ–Š—”ǡ”Šƒǡ‡‹‡…‡ǣ‹•‹…‘”’‘”ƒ–‹‘‘ˆˆ”‡‡Ǧ–›”‘•‹‡

‹–‘…‡ŽŽ—Žƒ”’”‘–‡‹•ǣƒ’‘–‡–‹ƒŽ…›–‘–‘š‹…‡…Šƒ‹•ˆ‘”‘š‹†‹œ‡†ƒ‹‘ƒ…‹†•Ǥ‹‘…Š‡ʹͲͲ͸Ǣ͵ͻͷǣʹ͹͹Ǧ ʹͺͶǤ

ͳ͹ —‰‰‹‡”‘ǡ”—œœ‘ǡŠ‹ƒ”‡ŽŽƒǡ—•–—‘ƒ„ƒ†ǡ‡‹••ǡƒ•“—ƒŽ‹‹ǣ‘…‘‹–ƒ––—‘””‡•‹•–ƒ…‡ǣ

–Š‡”‘Ž‡‘ˆ–›”‘•‹‡‹•‘‡”•‹–Š‡‡…Šƒ‹••‘ˆ‡–ƒ•–ƒ•‡•…‘–”‘ŽǤƒ…‡”‡•ʹͲͳʹǢ͹ʹǣͳͲͶ͵ǦͳͲͷͲǤ ͳͺ —‰‰‹‡”‘ǡ”—œœ‘ǡŠ‹ƒ”‡ŽŽƒǡ†‹‹ƒ‹ǡ•–—”‹œǡ‹•‡•ǡ’‡œ‹ƒŽ‡ǡ‡‹••ǡ—•–—‘ƒ„ƒ†

OD, Pasqualini CD: Tyrosine isomers mediate the classical phenomenon of concomitant tumor resistance.

ƒ…‡”‡•ʹͲͳͳǢ͹ͳǣ͹ͳͳ͵Ǧ͹ͳʹͶǤ

ͳͻ ‘Žž”ǡƒ‰‡”ǡƒ”×ǡל‡‰‹ǡ‘Šž•ǡ‘…•‹•ǡƒ–—•ǡƒ‰‡”ǡƒƒ•×ǡƒœž

ǡƒ…œ›ǡƒ‰›ǡ‹––ƒǣ”‹ƒ”›‘”–Š‘Ǧ–›”‘•‹‡‡š…”‡–‹‘‹†‹ƒ„‡–‡•‡ŽŽ‹–—•ƒ†”‡ƒŽˆƒ‹Ž—”‡ǣ

‡˜‹†‡…‡ˆ‘”Š›†”‘š›Ž”ƒ†‹…ƒŽ’”‘†—…–‹‘Ǥ‹†‡›–ʹͲͲͷǢ͸ͺǣʹʹͺͳǦʹʹͺ͹Ǥ

ʹͲ ‹ƒǣϐŽƒƒ–‹‘ƒ†‘š‹†ƒ–‹˜‡•–”‡••‹†‹ƒ„‡–‹…‡’Š”‘’ƒ–Š›ǣ‡™‹•‹‰Š–•‘‹–•‹Š‹„‹–‹‘ƒ•‡™

–Š‡”ƒ’‡—–‹…–ƒ”‰‡–•Ǥ‹ƒ„‡–‡•‡•ʹͲͳ͵ǢʹͲͳ͵ǣʹͶͺͷ͸͵Ǥ

ʹͳ ƒ‰‡–ƒǡ”‡…‘ǡƒ‡–ƒ‘ǡƒ”–‡ŽŽ‹ ǣš‹†ƒ–‹˜‡•–”‡••ƒ†‹…”‘•‹˜ƒ•…—Žƒ”†‹•‡ƒ•‡•Ǥ–‘Ž

…‹ʹͲͳ͵ǢͳͶǣͳ͹͵ͳͻǦͳ͹͵Ͷ͸Ǥ

ʹʹ ‹•”ƒǡƒ”™ƒ–ǡŠƒ—‹ǡ—–‡Œƒǡ—–‡Œƒǣš‹†ƒ–‹˜‡•–”‡••ƒ†‹•…Š‡‹…›‘…ƒ”†‹ƒŽ•›†”‘‡•Ǥ

‡†…‹‘‹–ʹͲͲͻǢͳͷǣʹͲͻǦʹͳͻǤ

ʹ͵ ‡”–‹ǡ‡•–‘ǡ—ƒ‰ǡƒ†‡”ǡ™‡•ǡ‡‹™ƒŽ†ǡ…Š”‘‡†‡” ǣ”ƒ••”‘‘–•…Š‡‹•–”›ǣ‡–ƒǦ

–›”‘•‹‡ǡƒŠ‡”„‹…‹†ƒŽ‘’”‘–‡‹ƒ‹‘ƒ…‹†Ǥ”‘…ƒ–Ž…ƒ†…‹ʹͲͲ͹ǢͳͲͶǣͳ͸ͻ͸ͶǦͳ͸ͻ͸ͻǤ

ʹͶ ‘†‰‡”•ǡƒ‰ǡ —ǡ‡ƒǣ‹‘•›–Š‡–‹…‹…‘”’‘”ƒ–‹‘‘ˆ‘š‹†‹œ‡†ƒ‹‘ƒ…‹†•‹–‘’”‘–‡‹•ƒ†

–Š‡‹”…‡ŽŽ—Žƒ”’”‘–‡‘Ž›•‹•Ǥ ”‡‡ƒ†‹…‹‘Ž‡†ʹͲͲʹǢ͵ʹǣ͹͸͸Ǧ͹͹ͷǤ

ʹͷ ”•ŠŽ‡”ǡŠ‡‰ǡ…‡Ž˜‡›ǡ”–œǡ‡†—Ž—”‹ǡ‡…•‘ǡƒ—„ǡ”ƒ–ǡ ‡””—……‹ǡ‘‰‘ǣ

‡”—‡”›–Š”‘’‘‹‡–‹ƒ†ƒ‰‹‰ǣƒŽ‘‰‹–—†‹ƒŽƒƒŽ›•‹•Ǥ‡”‹ƒ–”‘…ʹͲͲͷǢͷ͵ǣͳ͵͸ͲǦͳ͵͸ͷǤ

ʹ͸ ƒƒ••‡ǡ‡”Ž‹‡”ǣ‡‹ƒ‹‡Ž†‡”Ž›’ƒ–‹‡–•ǣƒ‡‡”‰‹‰’”‘„Ž‡ˆ‘”–Š‡ʹͳ•–…‡–—”›Ǥ‡ƒ–‘Ž‘‰›

‘…‡ƒ–‘Ž†—…”‘‰”ƒʹͲͳͲǢʹͲͳͲǣʹ͹ͳǦʹ͹ͷǤ

ʹ͹ ƒ‹ ǡƒ„‹‘˜‹–…Šǡ‰˜ƒ”‹ǣ‹–‘…Š‘†”‹ƒƒ†…ƒ”†‹‘˜ƒ•…—Žƒ”ƒ‰‹‰Ǥ‹”…‡•ʹͲͳʹǢͳͳͲǣͳͳͲͻǦͳͳʹͶǤ ʹͺ ƒŽ‘ǡ‡‹„ˆ”‹–œǡ‘…‘Žǡ”‘‹ǡƒœ—”ǡ‡Ž•‡”ǣ ”‡‡”ƒ†‹…ƒŽ•ƒ†ƒ–‹‘š‹†ƒ–•‹‘”ƒŽ

’Š›•‹‘Ž‘‰‹…ƒŽˆ—…–‹‘•ƒ†Š—ƒ†‹•‡ƒ•‡Ǥ–‹‘…Š‡‡ŽŽ‹‘ŽʹͲͲ͹Ǣ͵ͻǣͶͶǦͺͶǤ

ʹͻ …—ŽŽ‘—‰Šǡƒ”Šƒ”–ǡ”‹‰ǡ‡††ƒǡƒ’’ǡƒ–‡Žǡ‹‰Šǡœ…œ‡…ŠǡƒŽ‹ˆˆǣ

ƒ”†‹‘˜ƒ•…—Žƒ”–‘š‹…‹–›‘ˆ‡’‘‡–‹ǦƒŽˆƒ‹’ƒ–‹‡–•™‹–Š…Š”‘‹…‹†‡›†‹•‡ƒ•‡Ǥ‡’Š”‘ŽʹͲͳ͵Ǣ͵͹ǣͷͶͻǦ 558.

͵Ͳ —•ǡ‘›‘ǡ‘ƒ‘—ǣ•—Ž‹”‡•‹•–ƒ…‡’”‡†‹…–•‘”–ƒŽ‹–›‹‘†‹ƒ„‡–‹…‹†‹˜‹†—ƒŽ•‹–Š‡ǤǤ

‹ƒ„‡–‡•ƒ”‡ʹͲͳͲǢ͵͵ǣͳͳ͹ͻǦͳͳͺͷǤ

y: omanyi 4/29/2014 9:26:54 AM