lal-1: a differentially expressed novel gene during proliferation in liver regeneration and in...

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Blackwell Science, LtdOxford, UKGTCGenes to Cells1356-9597© Blackwell Science LtdNovember 2002711Original Articlelal-1: a novel gene in liver regenerationMA Della Fazia et al.

lal-1

: a differentially expressed novel gene during proliferation in liver regeneration and in hepatoma cells

Maria Agnese Della Fazia, Danilo Piobbico, Daniela Bartoli, Marilena Castelli, Stefano Brancorsini, Mariapia Viola Magni and Giuseppe Servillo*

Dipartimento di Scienze Biochimiche e Biotecnologie Molecolari. Sezione Fisiopatologia—Facoltà di Medicina e Chirurgia—Università di Perugia, 06100 Perugia, Italy

Abstract

Background

:

During liver regeneration, 95% of the rest-ing hepatocytes enter in G1/S phase of the cell cycle.A number of hormones, growth factors and cytokineswere identified that activate signal transduction pathwaysplaying a primary role in hepatocyte proliferation.A wide and representative cDNA library containing1.5

××××

10

6

independent clones was constructed fromregenerating liver in order to identify and characterizegene the products which play a crucial role in the firsthours of the proliferative process of liver regeneration.

Results

:

A novel gene expressed in liver regenerationwas cloned by subtractive hybridization. The putative

protein displays in the N

′′′′

-terminal a annexin-likedomain and an aminopeptidase domain. We namedthe novel gene Liver Annexin Like-1 (

lal-1

). The

lal-1

gene is modulated during liver regeneration, inhepatoma cells following physiological stimulationand after cAMP induction.

Conclusion

:

The results indicate that

lal-1

is involvedin liver regeneration and that its expression is finelyregulated during proliferative process. The isolationof

lal-1

paves the way for a further characterizationhelping to assess

lal-1

involvement in cell functionand proliferation.

Introduction

The liver has a remarkable ability to restore its constitu-tive mass a few days after partial hepatectomy (PH)(Michalopoulos & De Frances 1997; Fausto 2001).Indeed, the adult hepatocyte is a normally quiescent,highly differentiated cell. However, in the liver all con-ditions leading to cell loss (physical, chemical or infectiveinjury) induce a proliferative process within hepatocytes.The ability of the liver to change its pattern of growthso abruptly provides a fascinating and meaningful modelwith which to study the molecular regulation of bothnormal and abnormal cell growths (Fausto 2001). Thismodel provides a physiological system to study prolifer-ative processes

in vivo

. DNA synthesis starts at 12–16 hafter PH in rat hepatocytes and gradually increases toreach a maximum at 22–24 h after PH. Within 7–10 daysafter PH, the rat liver has regained its original mass.During liver regeneration 95% of the resting hepatocytes

enter in G1 phase of the cell cycle (Bucher 1963). Anumber of hormones, growth factors and cytokinesactivate signal transduction pathways, playing a primaryrole in hepatocyte proliferation (Michalopoulos 1990;Fausto

et al

. 1995; Diehl & Rai 1996; Taub 1996;Talarmin

et al

. 1999). It has been shown that the DNA-binding activity of Stat3, NF-kB, AP-1 and several othertranscription factors increases during the first hours afterPH. The role of the cytokines IL-6 and TNF that activateStat3, NF-kB and C/EBP was established by using knock-out mice for IL-6 and for TNFR-I genes (Cressman

et al

.1996; Yamada

et al

. 1997).However, the above mentioned factors are not suffi-

cient to trigger liver regeneration. It has been shownthat cAMP levels increased during liver regeneration.Recently, the expression of CREM (cAMP-Responsive-Element Modulator) has been clearly documented inhepatoma cells and in the rat liver at different hours afterPH (Servillo

et al

. 1997; Della Fazia

et al

. 1997). Moreover,the role of CREM has been investigated using mutantmice (Servillo

et al

. 1998). In quiescent cells of the liver,PH-induced proliferation is followed by a plethora ofchanges that occur mainly during the transition from G1

Communicated by

:

Paolo Sassone-Corsi*

Correspondence

: E-mail: [email protected] G. to whom we dedicate this work.

GTC_593.fm 183 Thursday, October 10, 2002 2:28 PM

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phase to S phase (Servillo

et al

. 2002). Several genes areactivated in response to the cell proliferation. The preciseactivation and the appropriate concentrations of themare necessary for the progression through the cell cycle(Fausto 2001; Servillo

et al

. 2001).These findings suggest that, although the immediate-

early response gene after PH is necessary for liver regen-eration, an additional later response gene is also required.Therefore, a major undertaking of the present study wasto investigate the mechanisms responsible for the regen-erative response, focusing on molecular changes duringthe liver regeneration following PH.

To better understand the molecular changes in geneexpression from G1 phase to S phase we have screeneda rat regenerating liver cDNA library with cDNA sub-tracted probes derived from rat regenerating livercDNAs (2–18 h after PH) and rat normal liver mRNAs.In the present study we describe the isolation and char-acterization of a novel gene that undergoes up-regulationin regenerating liver and in hepatoma cells. The putativeprotein of the novel gene presents in the N-terminal anannexin-like domain and an aminopeptidase domain.We named the novel isolated gene Liver AnnexinLike-1 (

lal-1

).

Results

Characterization of sequence analysis and up-expressed

lal-1

gene

The analysis of the rat regenerating liver cDNA libraryby means of a subtracted probe enabled us to isolate anovel gene cluster. All isolated genes were tested byNorthern blot analysis to define their expression kineticsduring liver regeneration. A partial sequence of thecloned cDNAs was performed. Our interest was focusedon one of the isolated genes. We named the isolated novelgene Liver Annexin Like-1 (

lal-1

). The

lal-1

nucleotidesequence and the Lal-1 deduced amino acid sequencewas determined. The

lal-1

nucleotide sequence in theNCBI database is under accession no. AF 131077. Theputative Lal-1 protein shows in the N-terminal region asingle annexin domain (18–70 amino acids) and a peptidase_M20 consensus (277–376 amino acids). The

lal-1

spansover a length of 1726 bp with a +1 ATG sequence at63 bp, a coding sequence of 1419 bp and a tandem ofAATAAA Poly A

+

consensus at 1698 bp and 1706 bp.To verify the length of the

lal-1

transcript, we per-formed a primer extension on liver mRNA isolated 18 hafter PH, utilizing a primer close to the 5

′

region of thegene. The results showed the presence of 62 bp of 5

′

untranslated flanking the +1 ATG sequence (Fig. 1).

lal-1

as response gene is increased in liver regeneration

To analyse the expression of

lal-1

in liver regeneration weevaluated liver Poly A

+

mRNAs obtained at differenttime points after rat PH and from sham operated animals.We verified by Northern blot analysis the length of the

lal-1

transcript and its expression following PH. ThemRNAs were hybridized with

lal-1

full length cDNA.The results showed a specific band of almost 1.9 kb, cor-responding in size to the

lal-1

transcript (Fig. 2). Fur-thermore, a rapid induction of gene expression was seenin the first hours of liver regeneration at 2, 5, 18 and 72 hafter PH was detected (Fig. 2). An RNAse protectionassay performed on total regenerating liver RNA con-firmed the variation in

lal-1

expression (data not shown).Changes in

lal-1

gene expression in the liver derivedfrom sham operated animals were undetectable.

lal-1

in human tissues

A human RNA Master Blot containing 50 differenthuman tissues Poly A

+

mRNA was hybridized with anEST fragment of the

lal-1

human cDNA. The resultsshowed

lal-1

expression in all tissues examined (Fig. 3A).However, a widely different gene expression was observedin the various tissues. The most significant

lal-1

expres-sion was detected in the mammary gland and bladder.

Figure 1 Primer extension analysis of the lal-1 transcript. A 32P-end-labelled 35-mer anti-sense oligonucleotide was hybridizedwith 30 µg of regenerating liver total RNA. The arrowheadindicates that the cDNA product is extending to thetranscription start sites of lal-1. The size of the cDNA product(nucleotides) is shown. The size of the cDNA fragment wasdetermined using a set of dideoxy sequencing reactions from aDNA fragment of a plasmid used as marker.

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Elevated gene expression levels were also observed in thethyroid gland and in the spinal cord. Testis, liver, pancreas,brain, heart displayed lower levels of

lal-1

expression(Fig. 3B).

lal-1

was present in all human foetal tissues,with the highest expression in the thymus (Fig. 3C).

lal-1

in proliferating and in cAMP treated H-35 hepatoma cells

To better understand of the role of

lal-1

during liverregeneration, its expression was analysed in an H-35hepatoma cells line induced to proliferate. H-35hepatoma cells were starved in serum-free medium, and

Figure 3 Master blot analysis of lal-1 in different human tissues. DNA: CotDNA was used as a control at two different concentrations.(A) Autoradiography of the human RNA master blot. (B) The AU values in the figures are defined by a ratio where the highestlal-1 expression was taken as 100% (mammary gland Poly A+ mRNA) and the gene expression of Poly A+ mRNA of other tissues.(C) Values of lal-1 expression of foetal tissues defined in AU, as described above. A1: whole brain, A2: amygdala, A3: caudate nucleus,A4: cerebellum, A5: cerebral cortex, A6: frontal lobe, A7: hippocampus, A8: medulla oblungata, B1: occipital lobe, B2: putamen,B3: substantia nigra, B4: temporal lobe, B5: thalamus, B6: nucleus accumbens, B7: spinal cord, C1: heart, C2: aorta, C3: skeletalmuscle, C4: colon, C5: bladder, C6: uterus, C7: prostate, C8: stomach, D1: testis, D2: ovary, D3: pancreas, D4: pituitary gland, D5:adrenal gland, D6: thyroid gland, D7: salivary gland, D8: mammary gland, E1: kidney, E2: liver, E3: small intestine, E4: spleen, E5:thymus, E6: peripheral leucocyte, E7: lymphnode, E8: bone marrow, F1: appendix, F2: lung, F3: trachea, F4: placenta, G1: foetal brain,G2: foetal heart, G3: foetal kidney, G4: foetal liver, G5: foetal spleen, G6: foetal thymus and G7: foetal lung.

Figure 2 Northern blot analysis of lal-1 expression. Poly A+

mRNA (5 µg) was isolated from normal liver (NL) andregenerating liver at 2, 5, 8, 12, 18, 24, 48, 72 h after PH. TherRNA 18S is indicated.


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3 days after starvation (

t

= 0) the cells were induced byserum to proliferate. The cells were harvested at differenttimes after adding the serum to the medium and theRNA was extracted. A Fluorimetric Analysis Cell Sorter(FACS) evaluated the H-35 cell cycle. At

t

= 0–4 hfollowing serum induction, the percentage of hepatomacells in the G

0

/G

1

phase was 85% (Fig. 4A). At

t

= 0,

lal-1

expression showed a detectable amount of mRNA.After 4 h, a Northern blot analysis showed the highestlevel of

lal-1

expression. At 8 h,

lal-1

expressiondecreased and remained unchanged in the followinghours (11–14 h) (Fig. 4B). Analysis of the cell cycle dis-played changes from 0 to 14 h after serum induction.

Fourteen hours after serum induction, there was a sig-nificant (68%) reduction in the number of cells in theG

0

/G

1

phase, whereas the number of cells in the S-phaseincreased by about 25% (Fig. 4A). After 17 h,

lal-1

expression increased again, while the number of G

0

/G

1

cells was even more reduced (50%), showing an S-phasefraction of about 38% of the total cells.

lal-1

expressionafter 24 h from the induction displayed a slight decrease.There were less than 35% of hepatoma cells in the G

0

/G

1

phase, while the fraction of cells in S-phase increasedto 45% and the cells in G

2

/M phase represented 16% ofthe total cells.

Additional experiments were performed where wetreated H-35 hepatoma cells with dbcAMP, and the cellswere then harvested at different times after induction.The RNA was extracted and analysed by Northern blotwith a specific probe for

lal-1

.The results showed a strong cAMP induction of

lal-1

expression at 2 h, and a down-regulation of expression at4 and 8 h, with a second peak at 12 h (Fig. 5).

Cellular localization of Lal-1 protein

To define the cellular localization of Lal-1 protein, weused PCR to determine the coding sequences of

lal-1

and we fused the PCR product in-frame at theN-terminal of Green Fluorescence Protein (GFP) in thepcDNA3.1-NT-GFP vector. The Lal-1-GFP constructwas transfected in COS-1 cells. The transfected COS-1cells displayed a cytoplasmatic localization of Lal-1

Figure 4 H-35 cell cycle and lal-1 expression in H-35 cellsinduced to proliferate. (A) FACS analysis of the H-35 hepatomacell cycle from 0 to 24 h after stimulation by serum. At 0 h the cellsare 85% in resting phase. (B) lal-1 expression in hepatoma cellsfrom 0 to 24 h after serum stimulation. (A) Northern blot analysisof lal-1 expression in H-35 induced to proliferate and recoveredat different times after serum stimulation (18S rRNA is indicated)(top). Nitrocellulose filter showing total RNA loaded (bottom).

Figure 5 lal-1 expression in cAMP treated H-35 cells. (A) Wild-type H-35 hepatoma cells are treated with cAMP and harvestedat different times after stimulation (18S rRNA is indicated).(B) Nitrocellulose filter showing total loaded RNA.


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protein (Fig. 6). To determine the precise cellularlocalization of Lal-1 we performed Lal-1-GFP proteinco-localization experiments with specific subcellularmarkers (i.e. WGA, MitoTracker) or antibodies thatrecognized specific proteins of subcellular compartments.Using immunofluorescence analysis, we observed theco-localization of Lal-1-GFP and calreticulin (specificendoplasmic reticulum protein) (Fig. 6).

Discussion

We have provided the sequence analysis of a novel genenamed

lal-1

and have shown that it is up-regulated inliver regeneration as well as in the hepatoma H-35 cellline, whose proliferative features closely resemble thoseof the proliferating hepatocyte during liver regeneration.

Liver regeneration is a well-established model ofproliferative process

in vivo

. In the first hours followingpartial hepatectomy the residual hepatocytes are exposedto several stimuli that prompt the cells to proliferate.Modifications in protein synthesis and a rearrangementof the subcellular organelles occur in the cells during thisperiod (Bucher 1963). A wide and representative cDNAlibrary was prepared from rat regenerating liver to identifyand characterize gene products that might play a crucialrole in the first hours of the proliferative process in liverregeneration. Using a screening of the regeneratingliver cDNA library we successfully isolated up-regulated

genes in early response after a 70% hepatectomy. Anucleotide sequence analysis of the isolated clones hasindicated that some of the genes are identical or similarto known genes.

By isolating clones, our interest has been focused onthe

lal-1

up-regulated gene during liver regeneration. Asequence analysis of

lal-1

indicated that the clone isolatedthrough the screening is a novel rat gene. We investigatethe putative role of

lal-1

that was over-expressed in liverregeneration. It should be noted that the

lal-1

transcriptis undetectable in normal liver. Conversely, the levels of

lal-1

mRNA increase after PH, with a rising expressionduring the first few hours post-hepatectomy. At thistime, a large number of hormones, growth factors andcytokines act synergistically to direct liver reconstitution(Michalopoulos & De Frances 1997). Two and five hoursafter PH, the residual hepatocytes showed elevatedcAMP levels that in turn activated transcription factorssuch as CREB and CREM, which play an importantrole in directing the proliferation of residual hepatocytes(Della Fazia

et al. 1998; Servillo et al. 1998). A secondpeak of intracellular cAMP levels occurs 18 h after PH.It is noticeable that the cAMP diphasic kinetic is paral-leled by a similar trend in lal-1 expression that shows twopeaks, coincident with cAMP during liver regeneration.Interestingly, maximal lal-1 expression is also concurrentwith an enhanced expression of early response genes andthe transition from G1/S phase. Another interesting fea-ture of lal-1 expression is that, different from the normalliver, the hepatoma cells such as H-35 cells show high,detectable levels of lal-1 expression. In these cells, lal-1expression is induced rapidly, either in serum-starvedcells induced to proliferate or in cells treated with cAMP.Moreover, we observed that in hepatoma cells a secondpeak of lal-1 expression occurs 17 h after PH which iscoincident with the G1/S phase transition of H-35.

During the characterization of lal-1, Gingras et al. (1999)isolated from human blood plasma a similar protein thathas been identified as Glutamate Carboxypeptidase (PGCP)homologous to Prostate Specific Membrane Antigen(PSMA) (Heston 1996). The deduced amino acid sequenceof lal-1 shows a significant homology with a Prostate-Specific Membrane Antigen (PSMA) gene. PSMA is atype II integral membrane glycoprotein which is highlyexpressed in prostate cancer cells. It has recently beenshown that PSMA is expressed in the neovasculature ofnon-prostatic primary malignancy neovasculature renalcarcinoma (Chang et al. 2001). At the present time,similar pathophysiological correlations for lal-1 expressionhave not been disclosed. The clear data that lal-1 isinvolved during liver regeneration and that its expressionis modulated during proliferative process prompt us to

Figure 6 Lal-1 protein cellular localization. Immunofluorescencephotomicrographs of transfected COS-7 cells expressing GFP-tag-Lal-1. (A) Fluorescence analysis in transfected cells to detectGFP. (B) Cells labelled with rabbit anti-Calreticulin Ig andsecondary Cy3-conjugated sheep anti-rabbit IgG. (C) Cellsstained with DAPI. (D): Merge of A, B and C.


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hypothesize a specific role during the vasculature processof regenerating liver. Although our results certainly donot include all changes of gene expression occurring ingene response during liver regeneration, the gene that wehave isolated provides information for further character-ization and understanding of the gene in cell functionand proliferation.

Experimental proceduresCells and culture

Reuber H-35 rat hepatoma cells (Reuber subline) and COS-1cells were cultured in Dulbecco’s Modified Eagle’s medium(DMEM) supplemented with 10% foetal calf serum (ClontechPalo Alto, CA, USA) (Taub et al. 1987) and 1% penicillin/strep-tomycin. The Reuber H-35 cells subline was a generous gift of DrJ.W. Koontz (University of Tennessee, Knoxville).

H-35 cells were starved in serum-free medium, and after 3 daysof starvation the cells were induced to proliferate by serum addi-tion. The time point at which H-35 cells received serum afterstarvation was taken as t = 0. The cells were harvested at differenttimes (2, 4, 8, 11, 14, 17, 24 h) after adding the serum to themedium and the RNA was extracted for lal-1 gene expression evalu-ation. The H-35 cell cycle was evaluated by Fluorimetric AnalysisCell Sorter (FACS), labelling the cells with propidium iodide.

The H-35 hepatoma cells were treated with 8-dCTP-cAMP(Sigma) (0.005 mg/mL medium) according to Sasaki et al. (1984).The cells were collected at 2, 4, 8, 12 and 24 h after cAMP induc-tion and the RNA was extracted for evaluation of lal-1 geneexpression.

Animals

The experiments were performed on 3-month-old maleSprague–Dawley rats. Adult male rats (body weight 220–250 g)were purchased from Harlan-Nossan. The animals were raisedaccording NIH guidelines. Rats were maintained in a 12 : 12 hlight/dark cycle, and food and water were provided ad libitum.

Liver resection of the left and median lobes was performedbetween 8 a.m. and 12 a.m. under deep ether anaesthesia, remov-ing about 70% of the liver mass (Higgins & Anderson 1931). As acontrol, a Sham Operation (SO) was performed (transverseabdominal incision followed by digital manipulation of the livers).Animals were sacrificed at 2, 5, 8, 12, 18, 24, 48 and 72 h after PH.The liver was then quickly removed and frozen in liquid nitrogenfor further processing (Della Fazia et al. 1992).

Four animals were used for each time point and the livers werepooled prior to analysis.

RNA isolation

RNA was isolated from H-35 hepatoma cells as described byChomczynski & Sacchi (1987). Liver RNA was obtained byhomogenizing the tissue in guanidium thiocyanate solution

followed by CsCl step gradient centrifugation (Chirgwin et al.1979). Poly A+ RNA was purified as previously described (DellaFazia et al. 1992).

Construction and screening of cDNA library

A pool of 10 µg of liver Poly A+ RNA (experimental points: 2, 5,8, 12, 18 h after PH) was used to construct a lambda gt11 phageregenerating of liver cDNA library. The double strand cDNA wasprepared using a cDNA kit (Gibco-BRL, Life Technologies, Pais-ley, UK) and then cloned in the EcoRI arms of lambda gt11 phage.A large and representative cDNA library containing 1.5 × 106 inde-pendent clones was obtained from regenerating liver to identifyand characterize gene products. The library was spread at low celldensity and was then screened with subtracted probes obtained bycDNA of regenerating liver and 10 times normal liver mRNA(Subtractor kit, Invitrogen, CA, USA).

The positive colonies were amplified and the phages wereisolated. An aliquot of the cDNA library was subjected to restric-tion digestion with EcoRI. The fragment corresponding to thecDNA insert was purified by electrophoresis analysis and radiola-belled by random-primed DNA (Ambion, Austin, TX, USA) syn-thesis and used as a probe in a standard Northern blotting analysis.

Sequence analysis of lal-1

The phages derived from the confirmed positive colonies werecloned in pBluescript vector and then subjected to bidirectionalpartial DNA sequencing (using T3 and T7 primers) by the Sangermethod (Sequenase kit; Amersham Life Science, Uppsala, Sweden).

A cDNA fragment of about 1800 bp corresponding to lal-1 wassequenced using T3 and T7 primers. Additional primers were usedto sequence in double strand lal-1. All positive clones were sub-jected to a computerized search for homologies to all nucleotidesequences in the updated world-wide Gene Data Bank (NCBI)databases. The lal-1 nucleotide sequence database is registeredunder accession no. AF 131077.

Primer extension analysis

Primer extension was performed with 30 µg of liver total RNA(12 h after PH). The anti-sense primer used spanned from 140 to108 bp over gene sequence. The primer was radiolabelled byend-labelling with [γ-32P]ATP and the synthesis of lal-1 wasperformed as previously described (Della Fazia et al. 1998). As a pre-cise marker, a set of dideoxy sequencing reaction of known DNAfragment was used.

Northern blot and Master blot analyses

Northern blot analysis of regenerating livers was performed usingPoly A+ mRNA at 2, 5, 8, 12, 18, 24, 48 and 72 h after PH (Maniatiset al. 1989). Regenerating liver Poly A+ mRNA (5 µg) waselectrophoresed in a denaturing 2.2 m formaldeyde 1% agarosegel, transferred and fixed to nitrocellulose membrane (Schleicher& Schuell, Dassel, Germany). The full length of lal-1 was labelled


lal-1: a novel gene in liver regeneration

© Blackwell Science Limited Genes to Cells (2002) 7, 1183–1190 1189

[α-32P]dCTP using a random primed labelling kit (Ambion, Aus-tin, TX, USA) and used as a hybridization probe in Northernblot analysis. Hybridization was carried out according to standardtechniques (Maniatis et al. 1989).

Human RNA Master Blot (Clontech, Palo Alto, CA, USA)containing Poly A+ mRNA of 50 different human tissues washybridized according to the manufacturer’s instruction with ESTno. AA452391, corresponding to the human lal-1 gene. Thehousekeeping gene expression quantified the Poly A+ mRNA ofthe different tissues spotted on to a nylon filter. Hybridizationsignals were quantified in Arbitrary Units (AU) using a densito-metric analyser. The tissue expressing the highest level of lal-1 wasindicated as 100% with respect to the expression of the lal-1 of theother tissues. Autoradiography was acquired with an imagescanner using a Photo-touch program and analysed by NIH Imageprogram 1.58. All immunostaining experiments were repeated atleast five times with similar results.

Cellular localization of Lal-1 protein

A fragment of 1419 bp—corresponding to the coding sequenceof lal-1—was amplified by PCR and then fused in-frame at theC-terminal sequence of Green Fluorescence Protein (GFP) inpcDNA3.1-NT-GFP vector (Invitrogen). A double strand sequenceanalysis of the Lal-1-GFP construct was performed. Lal-1-GFPDNA was transfected in COS-1 cells. The efficiency transfectionwas confirmed by Western blot with a specific primary anti-GFPantibody.

COS-1 cells were washed in PBS, fixed for 15 min in 4%formaldehyde, rinsed in PBS and permeabilized in 0.2% Triton/PBS for 5 min. Following three washes in PBS, the cells werepre-incubated in 3% BSA and then incubated with the specificprimary antibody (dilution 1 : 200). A rabbit polyclonal antibodyanti-calreticulin (StressGen Biotechnology Corp., Victoria,Canada) and an antibody anti-Cyt-c3 (dilution 1 : 200) were used asprimary antibodies for 2 h at room temperature. After threewashes in PBS, the cells were incubated with a 1 : 1000 of anti-rabbit Cy-3 conjugated secondary antibody in 3% BSA for 1 h atroom temperature. After fixation, the COS-1 cells were treatedwith TRITC-labelled WGA lectin (3 µg/mL). Cells were washedthree times in PBS, mounted on slides and then examined with aZeiss Axioplan fluorescence microscope. The images were acquiredby using a Spot-2 cooled camera (Diagnostic Instruments). In theCOS-1 cells, negative controls were performed using blockingserum in place of the primary antibody.

AcknowledgementsWe would like to thank the other members of our laboratory:V. Pettirossi, S. Pieroni and S. Luzi, for help and discussion, andN. Paolocci for a critical reading of the manuscript. Thanks areextended to E. Ayroldi for the FACS analysis and to J.W. Koontzfor the gift of the H-35 hepatoma cells. This work was supportedby grants from the Ministero dell’Istruzione dell’Università e dellaRicerca Scientifica (MIUR) and the Associazione Italiana per laRicerca sul Cancro (AIRC).

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Received: 31 May 2002 Accepted: 27 August 2002


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