Eur. J. Biochem. 259, 670±675 (1999) q FEBS 1999

Cloning and expression of the human p8, a nuclear protein with mitogenicactivity

Sophie Vasseur1, Gustavo Vidal Mallo1,2, Fritz Fiedler1,2, Hans BoÈdeker1, Eduardo CaÂnepa3, Silvia Moreno3 and

Juan Lucio Iovanna1

1U.315 INSERM, Marseille, France; 2Institut fuÈr AnaÈsthesie, Klinikum Mannheim, Theodor-Kutzer-Ufer, Mannheim, Germany;3Departamento de QuõÂmica BioloÂgica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria-Pabellon 2,

Argentina

We have previously identified a new rat mRNA, provisionally named p8, which showed a strong, but transient,

induction in the pancreas in response to acute pancreatitis. We report here the cloning and sequencing of the human

p8 cDNA. The human p8 polypeptide is 82 aminoacids long and shows an overall identity of 74% with the rat

counterpart. The complete structure of the p8 gene was also established. The p8 gene comprises three exons

separated by two introns and the gene was mapped to chromosome 16. Analysis of the p8 primary structure

suggested the presence of a bipartite motif of nuclear targeting, indicating that p8 may function within the nucleus.

This presumption has been confirmed by transfection of COS-7 cells with the p8 cDNA followed by

immunostaining with specific antibodies. p8 mRNA expression is induced in some, but not all, cells by serum

starvation and ceramide. To analyze the p8 function, we stably transfected HeLa cells with a p8 expression plasmid,

and measured their growth and their sensitivity to apoptosis. Response to TNFa and staurosporine-induced apoptosis

was not modified by p8 expression. However, cells expressing p8 grew significantly more rapidly.

Keywords: apoptosis; cellular growth; cloning; mRNA; pancreatitis; TNFa.

We have previously reported that gene expression is stronglyaltered in pancreas during the acute phase of pancreatitis [1,2].In fact, during the acute phase of pancreatitis, expression ofgenes encoding pancreatic secretory enzymes, which arepotentially harmful, was generally reduced by the cells, as partof a defence mechanism. Conversely, other genes were stronglyactivated during the acute phase of the disease. Most of thesegenes act to prevent evolution of the acute pancreatitis, whereasothers participate in pancreatic regeneration following pancrea-titis. These results support the view that during acutepancreatitis there is not a mere decrease of pancreatic functionbut a well-defined response of the gland characterized byspecific alterations of protein synthesis. Therefore, identifyingthe genes involved in this benefical response and understandingtheir function could open new therapeutic strategies forpancreatitis treatment. Thus, with a systematic approach toreach that goal we identified a new rat mRNA, provisionallynamed p8 [3], which showed a strong, but transient, induction inthe pancreas in response to acute pancreatitis. We report here thecloning and sequencing of the human p8 mRNA from whichwas deduced the primary structure of the protein. p8 waslocalized in the nucleus by immunohistochemistry analysis andHeLa cells overexpressing p8 grew significantly more rapidlythan control cells. In addition, we have isolated a genomic cloneand have elucidated the exon-intron organization as well as the5 0-flanking sequence of the gene, and finally it was mapped tochromosome 16.

MATERIALS AND METHODS

Cloning of the human p8 mRNA

A human pancreatic cDNA library in the expression vector lgt-11 containing 1.4 £ 106 different recombinant clones wasobtained from Clontech (Palo Alto, CA, USA). The librarywas screened by the plaque screening procedure [4], inconditions adapted to heterologous hybridization. The 2H10insert [3] was used as probe to screen 1 £ 105 recombinantclones. This fragment corresponds to the full length (nucleotides1±602) of the rat p8 [3]. A single clone (hump8) was selectedthrough three rounds of screening. Insert was obtained by EcoRIrestriction, subcloned into the plasmid vector pBluescript andsequenced by Genome Express (Grenoble).

Cloning of the human p8 gene

A genomic library constructed in lDASH, with insertsgenerated by partial digestion with Sau3AI of human lympho-cyte DNA (Stratagene), was screened with the hump8 insertlabeled with [a-32P]dCTP by multiple priming [5]. About5 £ 105 phage plaques of the genomic library were used forplaque hybridization screening [4]. Clones showing positivehybridization were plaque-purified by three rounds of screeningand characterized by restriction enzyme mapping. Restrictionfragments derived from bacteriophages containing the p8 genewere ligated into the plasmid vector pBluescript and sequencedby Genome Express (Grenoble).

Analysis of somatic cell hybrid DNA by polymerase chainreaction (PCR)

To test the specificity for the human genomic p8 gene of the setof oligonucleotides selected, 50 ng of human genomic DNA was

Correspondence to J. L. Iovanna, U.315 INSERM, 46 Bd de la Gaye,

F-13009 Marseille, France. Tel.: + 33 491172176, Fax: + 33 491266219,

E-mail: iovanna@marseille.inserm.fr

Note: The nucleotide sequences reported in this paper were submitted to the

GenBank database with accession numbers AF069073 and AF069074.

(Received 4 June 1998, revised 9 October 1998, accepted 2 November 1998)

q FEBS 1999 Cloning and expression of human p8 (Eur. J. Biochem. 259) 671

amplified by PCR utilizing sense (5 0-CCAGGCATGGTGGCA-GATGTC-3 0) and antisense (5 0-CTGCATCAAGACAA-CAGTTGCC-3 0) primers. The sense primer was located atposition 561±581 in the p8 gene and the antisense primer waslocated at position 1098±1119. Primer sequences are within thefirst and second introns, respectively. Amplification wasperformed in 1£ PCR buffer (50 mm KCl, 10 mm Tris-HCl,pH 8.3, 2 mm MgCl2, and 0.01% gelatin) containing 125 mmdNTP, 1% Me2SO, 25 pmol of each primer, and 2.5 units of Taqpolymerase (Boehringer Mannheim) in a final volume of 50 mL.The reaction protocol was as follows: first cycle, denaturation at94 8C for 2 min, annealing at 55 8C for 2 min and extension for2 min at 72 8C, and the next 30 cycles were denaturation at94 8C for 10 s, annealing at 55 8C for 2 min and extension for2 min at 72 8C. The PCR product was partially sequenced toconfirm the p8 identity. Then, the PCR was used specifically toamplify human p8 sequences in DNA from a panel of somaticcell hybrids. The 20 hybrids used are listed in Table 1. DNAwas prepared from somatic cell hybrids and PCR was carried outusing 50 ng of DNA. The same set of oligonucleotides and PCRconditions were used with the DNA from the somatic cellhybrids. Five microliters were removed and analyzed on 1.5%agarose gel. DNA products were visualized with ethidiumbromide under UV light.

Preparation of anti-human p8 antibodies and immunocyto-chemical localization

A peptide sequence corresponding to aminoacids 62±82(ERKLVTKLQNSERKKRGARR) of human p8 was chemicallysynthesized (Neosystem, France). The purified peptide wasconjugated to ovalbumin and used to immunize New Zealandwhite rabbits at the recommended intervals. Antiserum wascollected by puncture of the ear vein 10 days after the lastinjection.

The full length human p8 cDNA was subcloned into theEcoRI restriction site of the mammalian expression vectorpcDNA3 (Invitrogen), downstream from the CMV promoter.The recombinant plasmid (pcDNA3/hump8) was transfected

into COS-7 cells using the calcium phosphate±DNA copreci-pitation as described previously [6]. The cells were cultivated onglass slides, fixed in 3.7% formaldehyde in NaCl/Pi for 15 minand permeabilized with 0.2% Triton X-100 in NaCl/Pi for5 min. The slides were blocked with 5% non-fat dry milk inNaCl/Pi for 30 min at room temperature, then incubated with theanti-p8 antibody diluted 1 : 1000 for 2 h, followed by incuba-tion (1 h) with goat anti-rabbit IgG (Fc) (Immunotech) anddiaminobenzidine substrate. To test the specificity of theimmunocytochemical reaction, the anti-p8 antibody was sub-stituted by the preimmune serum.

Cell culture

HeLa, HepG2, SW40, HT29 and TC11 cells were routinelycultivated at 37 8C in a 5% CO2, 95% air atmosphere inDulbecco's modified Eagle's medium containing 10% (v/v)fetal calf serum (Gibco), 4 mm l-glutamine, 50 unit´mL±1 ofpenicillin, and 50 mg´mL±1 streptomycin. When cells reached80±90% confluence, they were dissociated with 0.05% trypsinand 0.02% EDTA in Puck's saline A and replated into 100-mmpetri dishes.

To induce p8 mRNA expression, cells were incubated inpresence of ceramide (20 mm) (Euromedex) or cells were serumstarved for 1, 2, 3, 4 and 5 days. After the different treatments,< 2.5 £ 107 cells were lysed for RNA extraction [7].

Northern blot

Twenty micrograms of RNA was submitted to electrophoresison a 1% agarose gel and vacuum blotted onto Hybond-Nmembranes (Amersham). The filters were hybridized with thecorresponding 32P-labeled probes for 16 h at 65 8C in 5 £ SSPE(SSPE is 180 mm NaCl, 1 mm EDTA, 10 mm NaH2PO4,pH 7.5), 5£ Denhardt's solution, 0.5% SDS and 100 mg´mL±1

single stranded herring sperm DNA. The filters were thenwashed four times for 5 min at room temperature in 2 £ NaCl/Cit, 0.2% SDS, twice for 15 min at 50 8C in 0.2 £ NaCl/Cit,0.2% SDS, and once for 30 min in 0.1 £ NaCl/Cit at 50 8C. The

Table 1. Chromosomal assignment of human p8.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X p8

MOG2C2 + ± + + + ± + + + + + ± ± + + + + + + ± + + + +

MOG2E5 + ± + + + + + + + ± + + + + + + + ± ± + + + +

1aA9498(602+) ± ± ± ± ± + ± ± ± ± ± + ± ± ± ± ± ± ± ± + ± + ±

HORP9�.5 ± ± ± ± ± ± ± ± ± + + + ± + ± ± ± ± ± ± ± + + ±

CTP34B4 + + + ± + + ± + ± ± ± + + + ± + + + ± ± ± ± + +

DUR4�.3 ± ± + ± + ± ± ± ± + + + + + F ± + + ± + + + F ±

CLONE21 ± ± ± ± ± ± + ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

MCP6 ± ± ± ± ± F ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± F ±

CJ9Q(640) ± ± ± ± ± ± ± ± Q ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

TWIN19F6 ± + + + ± + ± + ± ± ± + + ± ± + + ± + + + ± ±

TWIN19F9 + + + + ± + ± + + ± ± + + ± ± + + ± + + + ± ±

TWIN19D12 + ± + + ± + ± + ± ± ± + ± + ± + + + ± + + + ± +

J1CL4 ± ± ± ± ± ± ± ± ± ± + ± ± ± ± ± ± ± ± ± ± ± ± ±

FG10E8EP2�.9 ± + ± ± + ± ± + ± ± ± ± ± ± + ± ± + ± ± + ± + ±

FG10E8EP2�.6 ± ± ± ± ± ± + ± ± ± ± + ± ± + ± ± + ±

FG10E8EP2�.2 ± + ± ± + F ± + F ± ± ± ± + ± ± + ± ± + ± + ±

FG10E8EP2 ± + ± ± + F ± + F ± ± ± ± ± + ± ± + ± ± + ± + ±

FST9/10 ± ± + + ± + ± + + ± + + + + ± ± + ± + ± + + ±

SIF4A31 ± ± ± + + ± ± ± ± ± ± + ± + ± ± ± ± ± ± + +

SIF15P5 ± + ± ± ± + + ± ± + ± ± ± + + ± ± ± ± + ± ± + ±

Concordant +/+ 4 1 4 4 3 4 2 4 1 2 1 3 2 5 2 5 4 4 1 1 3 3 4

Concordant ±/± 14 9 11 12 11 10 13 8 14 11 12 9 13 9 9 15 12 7 15 10 8 10 7

Discordant 2 10 5 4 6 6 5 8 5 7 7 8 5 6 9 0 4 9 4 9 9 7 9

Key: +/±, Chromosome present/absent; F, fragment, Q; long arm only.

672 S. Vasseur et al. (Eur. J. Biochem. 259) q FEBS 1999

human multiple adult tissue Northern blots were purchased fromClontech.

Transfection of the human p8 cDNA, cell proliferation andapoptosis

The pcDNA3/hump8 plasmid was transfected into the HeLacells using the calcium phosphate-DNA coprecipitation asdescribed above. To select for stable transfection, the transgeniccells were cultivated over three weeks in media containing G418(600 mg´mL±1) starting 48 h after transfection. Twenty surviv-ing colonies were picked and expanded in standard culturemedium supplemented with G418 (400 mg´mL±1). RNA wasisolated from the selected clones and their p8 mRNAconcentration analyzed by Northern blot. Elevated levels of p8mRNA were detected in clones p8/3, p8/8, p8/14 and p8/17.

The proliferative response of the p8-transfected HeLa cellswas estimated by [3H]thymidine incorporation. Cells (104 cellsper well) were plated onto 96-well plates and cultivatedovernight under standard conditions. The media was thenremoved and the cells were incubated for 6 h in the presence of1 mCi [Me-3H]thymidine (25 Ci´mmol±1, Amersham). The cellswere then lifted off the plate with trypsin, and cellular DNA wascollected on glass filters (GF/C glass microfilters, Whatman)

with a vacuum filtration cell harvester. Incorporation of tritiumwas measured with a scintillation counter.

For flow cytometric studies, duplicate samples of 5 £ 105

cells were processed for propidium iodide staining as follows:cells were harvested from culture and permeabilized in 500 mLof 50 mg´mL±1 propidium iodide, 0.05% Nonidet P-40, 4kU´mL±1 RNase, in NaCl/Pi. Reagents were obtained as a kitfrom Coultronics France (Margency, France) and used accord-ing to recommendations. After vortexing, samples were allowedto equilibrate at room temperature in the dark for at least 1 hbefore analysis. Propidium iodide fluorescence analysis wasperformed in a flow-cytometer (EPICS, Coulter Corporation).

Cytolysis was quantified by the tetrazolium dye-based MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt) (Promega) assay,which provides a measure of cell viability. Cells were seededat 104 cells per well in quadruplicate in 96-well plates for 16 hprior to the treatment with TNFa (R & D Systems), 0±100 ng´mL±1 alone or in combination with cycloheximide(Sigma), 10 mg´mL±1, for 8 h. Cycloheximide was not cytotoxicat the concentrations used within the time frame of theexperiments. For measurement of cytolysis, MTS was addedto a final concentration of 317 mg´mL±1 per well and incubatedfor 90 min. The absorbance was measured at 490 nm.

Fig. 2. Immunostaining of the p8 in the nucleus of the COS-7 p8-transfected cells. The pcDNA3/hump8 plasmid was transfected into COS-7 cells using the

calcium phosphate-DNA coprecipitation. The cells were cultivated on glass slides, fixed in 3.7% formaldehyde in NaCl/Pi and permeabilized with 0.2% Triton

X-100. After blocking with non-fat dry milk in NaCl/Pi, the slides were incubated with the anti-p8 antibody diluted 1 : 1000 for 2 h, followed by incubation with

goat anti-rabbit IgG and diaminobenzidine substrate. Magnification, 350£.

Fig. 1. Sequence comparison of human (hump8) and rat (ratp8) p8. Boxed areas correspond to aminoacid identities.

q FEBS 1999 Cloning and expression of human p8 (Eur. J. Biochem. 259) 673

RESULTS

Cloning of the human p8 mRNA and structural organizationof the p8 gene

A radiolabeled rat p8 cDNA [3] was used to screen a humanpancreatic cDNA library in lgt-11 at relatively low stringencyas requested by the heterologous nature of the probe. The onlypositive clone after three successive screenings (hump8) wasselected, subcloned into pBluescript and sequenced. A singleopen reading frame was found in the corresponding mRNAsequence. The complete sequence comprised 693 nucleotides,exclusive of the poly A tail. A putative polyadenylation signal(AATAAA) was present 52 nucleotides upstream of the poly A-extension. The p8 polypeptide is 82 amino acids long and showsan overall similarity of 74% with rat p8 (Fig. 1).

Two genomic clones were obtained in a screening of< 5 £ 105 lDASH clones using hump8 cDNA. The clonesappeared to be similar to each other based on identical mappingby the HindIII, BamHI and PstI digestion of their DNAs and ona Southern assay using hump8 probe (data not shown). Based onthe mapping and sequencing, the p8 gene structure wasestablished. This structure includes three exons interrupted bytwo introns, plus 5 0 and 3 0 untranslated regions. The size ofexons I, II and III is 214, 150 and 329 nucleotides, respectively.The DNA sequence of all the exons agreed perfectly with that ofthe p8 cDNA. All the exon±intron boundary sequencesconformed to the GT/AG rule [8]. The 5 0-flanking region lacksa proximal authentic CAAT or TATA box. Moreover, theinitiator sequence [9], which appears to direct the site ofinitiation and basal level of transcription in many TATA-lesspromoters, was not present.

Mapping of the human p8 gene to chromosome 16

Human DNA sequences were specifically amplified in thehybrid cells, as a product of the expected size of 559 bp and noband was seen using DNA from rat (FAZA), mouse (IR), orhamster (A23) parent cell lines. The results obtained from thehybrid cell lines are summarized in Table 1. The DNA with theappropriate size was obtained in only the hybrid cell lines thatcontained an intact chromosome 16 and was absent from otherhybrid cell lines, indicating assignment of the p8 gene tochromosome 16.

Subcellular localization of p8 in COS-7-p8 cDNA transfectedcells

The p8 has a potential bipartite signal for nuclear targeting atposition 63 (KLVTKLQNSERKKRGA) [10], suggesting thatthe p8 function is in the nucleus. To gain insight into thesubcellular localization of p8, we overexpressed p8 bytransfection in COS-7 cells and studied its localization byindirect immunostaining with antibodies raised against itsCOOH-terminal region (see Materials and Methods). Fig. 2shows that p8 was present almost exclusively in the nucleusconfirming the predictive subcellular targeting. No staining wasobserved in control experiments (data not shown).

RNA blot analysis of p8 mRNA expression

To determine which tissues express p8, we hybridized amultitissue RNA blot with the hump8 probe at high stringency(Fig. 3). A band of < 0.8 kb was detected with abundantexpression in liver, pancreas, prostate, ovary, colon, thyroid,spinal cord, trachea and adrenal gland, with moderate expressionin heart, placenta, lung, skeletal muscle, kidney, testis, smallintestine, stomach and lymph node, and with low expression inbrain, spleen, thymus and bone marrow. No p8 mRNA wasdetected in peripheral blood leukocytes. In addition, the hump8probe detected in several tissues an additional transcript (Fig. 3)whose significance is unclear.

We also studied expression of the p8 mRNA in five differentcell lines. Expression was detected in HepG2 cells but not inHeLa, SW40, HT29 and TC11 (Fig. 4).

Induction of p8 mRNA expression in vitro

HepG2, HeLa, SW40, HT29 and TC11 cells were chosen tostudy p8 mRNA expression after ceramide and serum starvationtreatments as indicated in Material and Methods. Fig. 4 showsthat ceramide treatment induced a strong expression in HeLacells, but not in SW40, HT29 and TC11 cells. In HepG2 cells,which express constitutively p8 mRNA, expression was not

Fig. 4. Expression of p8 mRNA in SW480, TC7, HT29, HeLa and

HepG2 cells. Cells were incubated in presence of ceramide (20 mm) for 24

or cells were serum starved for 1, 2, 3, 4 and 5 days. After the different

treatments cells were lysed for RNA extraction. Identical amounts (20 mg)

of total RNA were blotted after electrophoresis on agarose-formaldehyde

gels. The filter was probed with the 32P-labeled hump8 cDNA and a 28S

ribosomal RNA probe and exposed for 5 days at ± 80 8C. (A) Control cells,

(B) ceramide-treated cells, (C) serum starved for 5 days.

Fig. 3. Tissue distribution of p8 mRNA. Northern hybridization was

carried out on multiple adult tissue mRNA blot (Clontech) with the hump8

cDNA as probe. Molecular mass markers in kb are shown on the left. PBL,

peripheral blood leukocytes.

674 S. Vasseur et al. (Eur. J. Biochem. 259) q FEBS 1999

modified by the treatment. Serum starvation induced p8 mRNAexpression in HeLa cells when cells were treated for more than24 h. No induction was detected in HT29, SW40 and TC11 cellsuntil 5 days of treatment. When HepG2 cells were serumdeprived, no changes in p8 mRNA expression were observedafter 1 or 2 days; studies could not be carried out for longertimes because of cell death and systematic degradation of theobtained RNA.

p8 Expression promotes cellular growth but does not alterresistance to apoptosis in HeLa cells

To analyze p8 function, we stably transfected HeLa cells with ap8 expression plasmid, and cellular growth and sensitivity toapoptosis were measured in four independent clones. HeLa p8/3,

p8/8, p8/14 and p8/17 clones showed, respectively, a 101, 63, 73and 82% increase in [3H]thymidine incorporation into DNAwhen compared to HeLa nontransfected cells (Fig. 5). Similarresults were obtained when HeLa-pcDNA3 transfected cellswere used as control, suggesting little influence of transfectionand selection, if any, on [3H]thymidine incorporation (data notshown). In addition, to assess the cell cycle effects of p8expression, HeLa and HeLa-p8 transfected cells were grown by96 h and were then permeabilized in the presence of propidiumiodide to stain the nuclear DNA. Flow cytometry profiles ofnuclear DNA content revealed that p8 increase the cell numberin S and G2/M phase in HeLa cells (Fig. 5), confirming andextending results from the [3H]thymidine incorporation experi-ments and previous observations [3].

As most proapototic stimuli induce p8 mRNA expression inHeLa cells (Fig. 4 and data not shown) we looked whether p8expression modified the apoptotic response in these cells. Asobserved with a number of cell types in vitro, HeLa cells aresusceptible to cell death by TNFa when protein synthesis isinhibited [11]. Each of the HeLa cell transfectants was treatedwith TNFa in the presence of cycloheximide for 8 h; after thattime the parental HeLa cell line showed evidence of extensivecell death. The amount of cell death was quantitated by MTSassay, which provides a quantitation of viable cells. Whenassayed by this method, HeLa cells showed near 55% cellsurvival and HeLa p8-transfected clones p8/3, p8/8, p8/14 andp8/17 showed 62, 58, 63 and 53%, respectively (Fig. 6). The celldeath observed was not due to an effect of inhibition of proteinsynthesis, as treatment with cycloheximide alone had minimaleffects on cell survival. Similar results were obtained whenapoptosis was induced with staurosporine (data not shown).

DISCUSSION

We used a strategy based on the assumption that if human p8does exist, human and rat p8 mRNA sequences should be similar

Fig. 5. Effect of p8 expression on cellular proliferation. (A) [3H]Thymi-

dine uptake. The proliferative response of the HeLa tranfectants p8/3, p8/8,

p8/14 and p8/17 and parental HeLa cells was estimated by[3H]thymidine

incorporation. 104 cells/well were plated onto 96-well plates and cultivated

overnight under standard conditions. The media was then removed and the

cells were incubated for 6 h in the presence of 1 mCi [Me-3H]thymidine.

Incorporation of tritium was measured with a scintillation counter. The data

shown are cpm´mg±1 protein for HeLa tranfectants p8/3, p8/8, p8/14 and p8/

17 cells expressed as percentage of HeLa parental, from six experiments. (B)

Flow cytometry studies. Samples of 5 £ 105 cells were processed for

propidium iodide staining using the reagents obtained as a kit from

Coultronics (see Materials and Methods) and fluorescence analysis was

performed in a flow-cytometer. The percentage of cells in G1, S and G2/M is

indicated. Values were obtained as the mean of two independent

experiments.

Fig. 6. Sensitivity of HeLa transfectants to TNFa-mediated apoptosis.

The HeLa tranfectants p8/3, p8/8, p8/14 and p8/17 and parental HeLa cells

were assayed untreated or following treatment with 10 ng´mL±1 TNFa,

10 mg´mL±1 cycloheximide or both for 8 h. Cell survival following

treatments was measured by MTS assay. The data are representative of

eight independent experiments and are expressed as percentage of surviving

relative to untreated cells.

q FEBS 1999 Cloning and expression of human p8 (Eur. J. Biochem. 259) 675

enough to allow detection by heterologous screening. A singlefull-length cDNA (hump8) was selected in the human cDNAlibrary upon screening and sequencing. The protein encoded byhump8 is two amino acids longer than rat p8. The two sequenceshave an overall amino acid identity of 74% (Fig. 1).

One of the most important characteristics of p8 gene is a verylow level of expression in normal pancreas and a dramaticincrease during the acute phase of pancreatitis [3]. Contrary tothe pancreas, p8 mRNA is expressed at high levels in severalnormal tissues as previously reported [3]. In this work, we haveused a multitissue RNA blot (commercially available) to studyhuman p8 mRNA distribution. Expression in human and rat p8correlate well for all tissues except for pancreas. Whereasnormal rat pancreas expresses low levels of p8 mRNA, humanpancreas seems to express very high levels. In our previous work[3] we have shown that p8 is rapidly and strongly activated inpancreas in response to minor pancreatic injuries. We speculatedthat pancreatic RNA from multitissue RNA blot could bederived from a suffering pancreas. We therefore studied p8mRNA expression in seven pancreatic RNA samples obtainedfrom human with non-symptomatic pancreatic diseases andfound that three of these expressed very low levels, as expected,but two samples expressed moderate and two samples showedhigh levels of this transcript (data not shown), suggesting avariability among samples and supporting the idea that p8mRNA is strongly activated in response to a minor injury.

Analysis of the p8 primary structure suggested the presence ofa bipartite motif of nuclear targeting in its COOH-terminalregion indicating that p8 may function within the nucleus. Thispresumption has been confirmed by transfection of the p8 cDNAinto COS-7 cells followed by immunostaining with specificantibodies (Fig. 2). Most nucleoprotein functions are regulatedby phosphorylation/dephosphorylation. Preliminary results ob-tained by incubating COS-7 cells with 32P indicate that p8 is infact a phosphoprotein (data not shown), but the sites ofphosphorylation and the kinases involved remain to bedetermined.

In our previous work, we have observed that p8 mRNAexpression was induced in response to various, although not all,proapoptotic stimuli [3]. Therefore, we speculated for aproapoptotic or an antiapoptotic function for p8. In this workwe found that p8 expression does not alter the apoptotic effect ofTNFa or staurosporine on HeLa cells (Fig. 6). However, as forCOS-7 and pancreatic AR4-2 J cells, p8 expression in HeLacells promotes a significant cellular growth (Fig. 5), stronglysuggesting a cellular growth promoting function for p8 ratherthan an apoptosis-related function. Interestingly, p8 mRNA isconstitutively expressed in some tissues and cells (Fig. 3), butits expression was only detected after induction in others.Moreover, this transcript was not detected in a few number oftissues or cells. These findings suggest a nonvital function forthis molecule. Because p8 is expressed in response toproapoptotic stimuli and promotes cellular growth, we canassume that p8 could participate in the response to someproapoptotic stimuli by promoting cellular growth in a way thathelps the tissue counteract diverse injuries.

While writing this paper, we learned that The Institute ofGenomic Research (TIGR) deposited the complete nucleotide

sequence of a BAC clone (GenBank accession numberAC002425) which contains the p8 gene sequence. Thatsequence agrees perfectly with our sequence and confirms ourchromosomal assignment. Moreover, that work defines moreprecisely the localization of the p8 gene within chromosome 16,at position p11.2. Interestingly, this region is frequentlyamplified in breast tumors [12,13]. The implication of the p8,as a growth promoting molecule, in this tumor will beinvestigated.

ACKNOWLEDGEMENTS

We acknowledge Dr J. C. Dagorn for insightful discussions. G.V.M. is a

fellow of the Humboldt Foundation and H.B. was supported by a fellowship

from the Fondation pour la Recherche MeÂdicale. F.F. was supported by a

grant from Forschungsfond der FakultaÈt fuÈr Klinische Medizin Mannheim

and E.C and S.M. are supported by grants from CONICET and Agencia

Nacional de Promocion Cientifica y Tecnologica. We thank Dr D. M.

Swallow for providing the DNA from the somatic cell hybrids.

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