cell biochemistry and function

Cell Biochem Funct 2008; 26: 39–42.

Published online 27 December 2006 in Wiley InterScience

(www.interscience.wiley.com) DOI: 10.1002/cbf.1394

The effect of histone deacetylase inhibitor trichostatinA (TSA) on the incorporation of 32P (Pi) and 3H-palmiticacid into the phospholipids of Tetrahymena

Peter Kovacs* and Gyorgy Csaba

Department of Genetics, Cell and Immunobiology, Semmelweis University, Budapest, Hungary

Histone deacetylases (HDACs) are able to control also the acetylation of tubulin. In the present experiments the effect oftrichostatin A (TSA), a HDAC inhibitor was studied on the incorporation of 3H-palmitic acid and 32P to the phospholipids(PI, PIP, PS, PC, PA, PE) of Tetrahymena pyriformis, considering earlier observations on the microtubular system’s influenceon signalling in this unicellular eukaryote. Treatment with 1, 5, or 10mM TSAwas studied. The incorporation of hydrophobictail component, palmitic acid was inhibited in a concentration dependent manner into all the phospholipids, except for PA,where the incorporation was increased. 32P incorporation was also inhibited. The possible relation between the microtubularsystem and signalling is discussed. Copyright # 2006 John Wiley & Sons, Ltd.

key words—Tetrahymena; signal transduction; trichostatin; microtubulus; acetylation

INTRODUCTION

The reversible acetylation/deacetylation of theN-terminal histone tails by specific histone acetylases(HATs) and deacetylases (HDACs) is involved inthe regulation of gene expression,1 and is tightlycoordinated with transcriptional regulation, differen-tiation, and cell cycle progression in mammalian cells.

HDACs are a family of enzymes that catalysethe removal of acetyl groups from core histones,resulting in a change of chromatin structure andgene transcription activity. HDACs have been shownto control also the state of acetylation of manynon-histone proteins, some of which are cytoplasmic(eg. tubulin).2 Reversible acetylation of the e-aminogroup of Lys40 of a-tubulin markedly stabilizes

* Correspondence to: P. Kovacs, Department of Genetics, Cell andImmunobiology, Semmelweis University, Nagyvarad ter 4, P.O. Box370, 1445 Budapest, Hungary. Tel: (þ361)-210-2930. Fax:(þ361)-303-6968. E-mail: kovpet@net.sote.hu

Copyright # 2006 John Wiley & Sons, Ltd.

microtubule structures and may contribute to regulat-ing microtubule dynamics.3

In Tetrahymena—as in other eukaryotes—histoneacetylation has also a very important role. In thevegetative Tetrahymena thermophila cells, its level isdramatically different in the germ line and in thesomatic nuclei. Histone acetylation is developmentallyregulated: both HAT and deacetylase are modulatedduring the course of macronuclear development.4

Tubulin post-translational modifications, for exampleacetylation, play also very important roles: the stabilityof microtubules and the binding of motor proteins areaffected—among other properties—by acetylation.5

Tetrahymena maintains at least 17 distinct microtubulestructures, but expresses only one type of a-tubulin,plus one major and one highly divergent minorb-tubulin proteins encoded by a single a and threeb-tubulin genes, respectively.6 Considering the diver-sity of Tetrahymena microtubules they may derivemainly from association with different microtubule-associated proteins, or from different post-translationalmodifications. Tetrahymena tubulins are secondarilymodified by several mechanisms, including acetylation

Received 11 September 2006Revised 1 November 2006

Accepted 16 November 2006

40 p. kovacs and g. csaba

of a-tubulin at lysine 40 (a modification found in mosteukaryotes).

Trichostatin A (TSA) is a potent and reversibleinhibitor of HDACs;7 it promotes acetylation ofhistones as well as a-tubulin, implying that tubulinacetylation is also controlled by competition betweenacetyltransferases and deacetylases. TSA treatmentinduces developmental arrests in the early stages ofconjugation of Tetrahymena; causes failure of DNAdeletion but not of chromosome breakage.8 In ourearlier work on Tetrahymena, TSA treatments resultedin a spectacular increase in the amount of acetyla-ted-tubulin, but had only a slight effect on the amountof a-tubulin TSA.9

Tetrahymena utilizes many signalling pathwaysanalogous to those of mammalian cells for examplethe inositol phospholipids,10 and provides evidencefor the activity of enzymes such as PLA2 and PLD,which play important roles in the generation ofsecond messengers.11,12 Supposing that—similarly tothe actin cytoskeleton13—also the tubular system ofTetrahymena is in connection with the signallingsystem, we set out to determine the effect of TSA onthe synthesis of phospholipids in Tetrahymena.

MATERIALS AND METHODS

Materials

TSA, phospholipid chromatographic standards (PI,PIP, PIP2, PE, PS, PC and PA) and tryptone wereobtained from Sigma (St Louis, MO). Yeast extractwas obtained from Oxoid (Unipath, Basingstoke,Hampshire, UK). [9,10-3H] palmitic acid (specificactivity 2 TBq/mM) was obtained from Amersham(Buckingshamshire, UK). 32P Na-orthophosphate(specific activity 76 GBq/mM) was purchased fromIzinta (Budapest, Hungary). Silica gel G TLCaluminium sheets were obtained from Merck (Darm-stadt, Germany). All other chemicals used were ofanalytical grade available from commercial sources.

Tetrahymena cultures

In the experiments, T. pyriformis GL strain was testedin the logarithmic phase of growth. The cells werecultivated at 28oC in 0.1 per cent yeast extractcontaining 1 per cent tryptone medium. Before theexperiments the cells were washed with fresh culturemedium and were resuspended at a concentration of5� 104 cells ml�1.

Copyright # 2006 John Wiley & Sons, Ltd.

Assay of 3H-palmitic acid incorporation into thephospholipids of Tetrahymena

Tetrahymena populations (3 ml) were treated with1mM; 5mM or 10mM TSA, and after 30 min1mCi ml�1 3H-palmitic acid was added to the cellsfor 60 min in the continued presence of TSA.Untreated cells served as controls. After treatmentthe lipid content was extracted by the method of Blighand Dyer.14 The lipids were separated on oxalatepre-treated silica gel G TLC aluminium sheets. Thesolvent was (by volume) chloroform/methanol/H2O¼ 65:35:2.5. Phospholipids were identified byparallel runs of authentic standards of PI, PIP, PIP2,PS, PC, PA and PE. After development the phospho-lipid containing parts of the chromatogram weretransferred to scintillation vials and the radioactivitywas determined by liquid scintillation counting.

Assay of 32P incorporation into the phospholipidsof Tetrahymena

Tetrahymena populations (5 ml) were treatedwith 10mM TSA, and after 30 min, 0.6 MBq ml�1

32P Na-orthophosphate was added for 60 min in thecontinued presence of TSA. The lipid extraction andseparation were done by the method described above.The developed chromatograms were covered byKodak TMG X-ray film. After exposure (�18 h)and development of radiograms, the radioactivityof individual phospholipid spots, and the total amountof incorporated phosphorus, were analysed by a laserdensitometer (Ultroscan GSXL, Pharmacia-LKB,Uppsala, Sweden). The GSXL data file containedthe total amount of incorporated radioactivity (areasum) and the amount of individual phospholipids(individual peaks) as a percentage of total incorpor-ated radioactivity (100 per cent).

RESULTS AND DISCUSSION

Treatments with TSA decreased significantly theincorporation of 3H-palmitic acid into the phospho-lipids of Tetrahymena—in a concentration-dependentmanner—except for PA, where we detected increasedincorporation. (Table 1).

Similarly, TSA-treatments inhibited the incorpora-tion of the hydrophilic head component phosphorusinto all analysed phospholipids of Tetrahymena.(Table 2).

It is difficult to determine whether: (1) treatmentswith drugs affecting the microtubular dynamics also

Cell Biochem Funct 2008; 26: 39–42.

DOI: 10.1002/cbf

Table 1. Incorporation of 3H-palmitic acid into the phospholipids of Tetrahymena

PIP2 PIP PI PCþPS PE PA

Control 192� 2.33 409� 3.33 553� 2.88 1372� 8.69 1333� 3.84 76� 0.11TSA 1mM 117� 1.56� 184� 1.92� 126� 1.13� 137� 1.07� 401� 2.73� 116� 0.67y

TSA 5mM 41� 0.6� 46� 0.73� 47� 0.32� 54� 0.99� 61� 0.58� 155� 0.95�

TSA 10mM 24� 0.72� 22� 0.19� 29� 0.22� 40� 0.71� 54� 0.34� 237� 1.38�

The data (cpm) represent mean (�SD) from independent experiments.PI, phosphatydylinositol; PIP, phosphatydylinositol-4-phosphate; PIP2, phosphatydylinositol-4,5-bisphosphate; PC, phosphatydylcholine;PS, phosphatydylserine; PE, phosphatydylethanolamine; PA, phosphatidic acid.�p< 0.01 to the control.yp< 0.05 to the control.

effect of tsa on the phospholipids of TETRAHYMENA 41

affect the different functions in a more direct waywhich lead to the different effects observed by us, or(2) microtubular defects are secondary effects ofan impaired phospholipid signalling system or (3) aconsequence of another disturbed phenomena, forexample reduced food uptake. These treatmentsresult in alterations of numerous diverse systems;for example disruption of the cytoskeleton (e.g.with nocodazole) blocks agonist-elicited Ca2þ

mobilization, but this effect does not originate fromdiminished PLC activity or impaired function ofthe IP3 receptor. The cytoskeletal disruption pre-sumably alters the spatial relationship betweenPLC and IP3 receptor.15 Tubulin forms the micro-tubule and regulates certain G-protein-mediatedsignalling pathways. Both functions rely on theGTP-binding properties of tubulin. Signal transduc-tion through Gaq-regulated phospholipase Cb1

(PLCb1) is activated by tubulin through a directtransfer of GTP from tubulin to Gaq. Tubulin,depending on local membrane concentration, mayserve as a positive or negative regulator of phosphoi-nositide hydrolysis. Rapid changes in membrane lipidcomposition or in the cytoskeleton may modifyneuronal signalling through such a mechanism.16

Structural proteins, such as tubulin, can participate incellular signalling also by communicating through

Table 2. Incorporation of Pi into the phospholipids of Tetrahymena

Area sum (AU) PIP2 PIP

Control 44.5� 3.7 0.55� 0.1 1.0� 0.08TSA 10mM 18.35� 2.2� — —

The data represent mean (�SD) from independent experiments. Area suof individual phospholipids are given as percentages of total incorpor�p< 0.01 to the control.

Copyright # 2006 John Wiley & Sons, Ltd.

physical forces. By virtue of its interaction withthe sub-membranous network of cytoskeletal proteins,perturbation in one locus, can transmit large con-formation changes to other points. Thus, GTP bindingto membrane-associated tubulin may lead to aconformational change in receptors or G-proteins.This may, in turn, influence the binding of an agonistto its receptor. On the other hand, subsequent toagonist-induced translocation of G-proteins from themembrane compartment to the cytosol, these mayaffect microtubule formation.17 G-protein activatingfluorides influence the synthesis/metabolism of phos-pholipids of Tetrahymena.18 The disturbance ofthe signalling system (eg. inositol phospholipids) ismanifested on the cytoskeleton and skeleton-dependent function of Tetrahymena.19 The alteredtubulin acetylation caused by TSA presumablyinfluences the association of MAPs, and this alterationconceivably influences also the communicationbetween the tubules and the components of signallingsystems. These alterations have effects also on thesynthesis of phospholipids.

The data arising from the present experimentsindicate that disturbance/modification of skeletalelements, such as tubulin, can cause alterations inthe metabolism of phospholipids of Tetrahymena;molecules which play important roles in signalling.

PI PSþ PC PE PA

2.05� 0.27 21.22� 1.65 32.5� 3.8 10.7� 1.10.88� 0.09� 9.32� 0.87� 14.2� 1.3� 1.3� 0.1�

m: total incorporated radioactivity in arbitrary units (AU). The valuesated activity as 100.

Cell Biochem Funct 2008; 26: 39–42.

DOI: 10.1002/cbf

42 p. kovacs and g. csaba

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Cell Biochem Funct 2008; 26: 39–42.

DOI: 10.1002/cbf