We have previously reported that gene expression is strongly altered in pancreas during the acute phase of pancreatitis [1, 2]. In fact, during the acute phase of pancreatitis, expression of genes encoding pancreatic secretory enzymes, which are potentially harmful, was generally reduced by the cells, as part of a defence mechanism. Conversely, other genes were strongly activated during the acute phase of the disease. Most of these genes act to prevent evolution of the acute pancreatitis, whereas others participate in pancreatic regeneration following pancreatitis. These results support the view that during acute pancreatitis there is not a mere decrease of pancreatic function but a well-defined response of the gland characterized by specific alterations of protein synthesis. Therefore, identifying the genes involved in this benefical response and understanding their function could open new therapeutic strategies for pancreatitis treatment. Thus, with a systematic approach to reach that goal we 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 mRNA from which was deduced the primary structure of the protein. p8 was localized in the nucleus by immunohistochemistry analysis and HeLa cells overexpressing p8 grew significantly more rapidly than control cells. In addition, we have isolated a genomic clone and have elucidated the exon-intron organization as well as the 5′-flanking sequence of the gene, and finally it was mapped to chromosome 16.
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 p8gene 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 TNFα and staurosporine-induced apoptosis was not modified by p8 expression. However, cells expressing p8 grew significantly more rapidly.
Materials and methods
Cloning of the human p8 mRNA
A human pancreatic cDNA library in the expression vector λgt-11 containing 1.4 × 106 different recombinant clones was obtained from Clontech (Palo Alto, CA, USA). The library was screened by the plaque screening procedure , in conditions adapted to heterologous hybridization. The 2H10 insert  was used as probe to screen 1 × 105 recombinant clones. This fragment corresponds to the full length (nucleotides 1–602) of the rat p8 . A single clone (hump8) was selected through three rounds of screening. Insert was obtained by EcoRI restriction, subcloned into the plasmid vector pBluescript and sequenced by Genome Express (Grenoble).
Cloning of the human p8 gene
A genomic library constructed in λDASH, with inserts generated by partial digestion with Sau3AI of human lymphocyte DNA (Stratagene), was screened with the hump8 insert labeled with [α-32P]dCTP by multiple priming . About 5 × 105 phage plaques of the genomic library were used for plaque hybridization screening . Clones showing positive hybridization were plaque-purified by three rounds of screening and characterized by restriction enzyme mapping. Restriction fragments derived from bacteriophages containing the p8 gene were ligated into the plasmid vector pBluescript and sequenced by Genome Express (Grenoble).
Analysis of somatic cell hybrid DNA by polymerase chain reaction (PCR)
To test the specificity for the human genomic p8 gene of the set of oligonucleotides selected, 50 ng of human genomic DNA was amplified by PCR utilizing sense (5′-CCAGGCATGGTGGCAGATGTC-3′) and antisense (5′-CTGCATCAAGACAACAGTTGCC-3′) primers. The sense primer was located at position 561–581 in the p8 gene and the antisense primer was located at position 1098–1119. Primer sequences are within the first and second introns, respectively. Amplification was performed in 1× PCR buffer (50 m m KCl, 10 m m Tris-HCl, pH 8.3, 2 m m MgCl2, and 0.01% gelatin) containing 125 µm dNTP, 1% Me2SO, 25 pmol of each primer, and 2.5 units of Taq polymerase (Boehringer Mannheim) in a final volume of 50 µL. The reaction protocol was as follows: first cycle, denaturation at 94 °C for 2 min, annealing at 55 °C for 2 min and extension for 2 min at 72 °C, and the next 30 cycles were denaturation at 94 °C for 10 s, annealing at 55 °C for 2 min and extension for 2 min at 72 °C. The PCR product was partially sequenced to confirm the p8 identity. Then, the PCR was used specifically to amplify human p8 sequences in DNA from a panel of somatic cell hybrids. The 20 hybrids used are listed in Table 1. DNA was prepared from somatic cell hybrids and PCR was carried out using 50 ng of DNA. The same set of oligonucleotides and PCR conditions were used with the DNA from the somatic cell hybrids. Five microliters were removed and analyzed on 1.5% agarose gel. DNA products were visualized with ethidium bromide under UV light.
Preparation of anti-human p8 antibodies and immunocytochemical localization
A peptide sequence corresponding to aminoacids 62–82 (ERKLVTKLQNSERKKRGARR) of human p8 was chemically synthesized (Neosystem, France). The purified peptide was conjugated to ovalbumin and used to immunize New Zealand white rabbits at the recommended intervals. Antiserum was collected by puncture of the ear vein 10 days after the last injection.
The full length human p8 cDNA was subcloned into the EcoRI restriction site of the mammalian expression vector pcDNA3 (Invitrogen), downstream from the CMV promoter. The recombinant plasmid (pcDNA3/hump8) was transfected into COS-7 cells using the calcium phosphate–DNA coprecipitation as described previously . The cells were cultivated on glass slides, fixed in 3.7% formaldehyde in NaCl/Pi for 15 min and permeabilized with 0.2% Triton X-100 in NaCl/Pi for 5 min. The slides were blocked with 5% non-fat dry milk in NaCl/Pi for 30 min at room temperature, then incubated with the anti-p8 antibody diluted 1 : 1000 for 2 h, followed by incubation (1 h) with goat anti-rabbit IgG (Fc) (Immunotech) and diaminobenzidine substrate. To test the specificity of the immunocytochemical reaction, the anti-p8 antibody was substituted by the preimmune serum.
HeLa, HepG2, SW40, HT29 and TC11 cells were routinely cultivated at 37 °C in a 5% CO2, 95% air atmosphere in Dulbecco’s modified Eagle’s medium containing 10% (v/v) fetal calf serum (Gibco), 4 m m l-glutamine, 50 unit·mL–1 of penicillin, and 50 µg·mL–1 streptomycin. When cells reached 80–90% confluence, they were dissociated with 0.05% trypsin and 0.02% EDTA in Puck’s saline A and replated into 100-mm petri dishes.
To induce p8 mRNA expression, cells were incubated in presence of ceramide (20 µm) (Euromedex) or cells were serum starved for 1, 2, 3, 4 and 5 days. After the different treatments, ≈ 2.5 × 107 cells were lysed for RNA extraction .
Twenty micrograms of RNA was submitted to electrophoresis on a 1% agarose gel and vacuum blotted onto Hybond-N membranes (Amersham). The filters were hybridized with the corresponding 32P-labeled probes for 16 h at 65 °C in 5 × SSPE (SSPE is 180 m m NaCl, 1 m m EDTA, 10 m m NaH2PO4, pH 7.5), 5× Denhardt’s solution, 0.5% SDS and 100 µg·mL–1 single stranded herring sperm DNA. The filters were then washed four times for 5 min at room temperature in 2 × NaCl/Cit, 0.2% SDS, twice for 15 min at 50 °C in 0.2 × NaCl/Cit, 0.2% SDS, and once for 30 min in 0.1 × NaCl/Cit at 50 °C. The human multiple adult tissue Northern blots were purchased from Clontech.
Transfection of the human p8 cDNA, cell proliferation and apoptosis
The pcDNA3/hump8 plasmid was transfected into the HeLa cells using the calcium phosphate-DNA coprecipitation as described above. To select for stable transfection, the transgenic cells were cultivated over three weeks in media containing G418 (600 µg·mL–1) starting 48 h after transfection. Twenty surviving colonies were picked and expanded in standard culture medium supplemented with G418 (400 µg·mL–1). RNA was isolated from the selected clones and their p8 mRNA concentration analyzed by Northern blot. Elevated levels of p8 mRNA were detected in clones p8/3, p8/8, p8/14 and p8/17.
The proliferative response of the p8-transfected HeLa cells was estimated by [3H]thymidine incorporation. Cells (104 cells per 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 µCi [Me-3H]thymidine (25 Ci·mmol–1, Amersham). The cells were then lifted off the plate with trypsin, and cellular DNA was collected on glass filters (GF/C glass microfilters, Whatman) with a vacuum filtration cell harvester. Incorporation of tritium was 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 µL of 50 µg·mL–1 propidium iodide, 0.05% Nonidet P-40, 4 kU·mL–1 RNase, in NaCl/Pi. Reagents were obtained as a kit from Coultronics France (Margency, France) and used according to recommendations. After vortexing, samples were allowed to equilibrate at room temperature in the dark for at least 1 h before analysis. Propidium iodide fluorescence analysis was performed 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 seeded at 104 cells per well in quadruplicate in 96-well plates for 16 h prior to the treatment with TNFα (R & D Systems), 0–100 ng·mL–1 alone or in combination with cycloheximide (Sigma), 10 µg·mL–1, for 8 h. Cycloheximide was not cytotoxic at the concentrations used within the time frame of the experiments. For measurement of cytolysis, MTS was added to a final concentration of 317 µg·mL–1 per well and incubated for 90 min. The absorbance was measured at 490 nm.
Cloning of the human p8 mRNA and structural organization of the p8 gene
A radiolabeled rat p8 cDNA  was used to screen a human pancreatic cDNA library in λgt-11 at relatively low stringency as requested by the heterologous nature of the probe. The only positive clone after three successive screenings (hump8) was selected, subcloned into pBluescript and sequenced. A single open reading frame was found in the corresponding mRNA sequence. 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 shows an overall similarity of 74% with rat p8 ( Fig. 1).
Two genomic clones were obtained in a screening of ≈ 5 × 105λDASH clones using hump8 cDNA. The clones appeared to be similar to each other based on identical mapping by the HindIII, BamHI and PstI digestion of their DNAs and on a Southern assay using hump8 probe (data not shown). Based on the mapping and sequencing, the p8 gene structure was established. This structure includes three exons interrupted by two introns, plus 5′ and 3′ untranslated regions. The size of exons I, II and III is 214, 150 and 329 nucleotides, respectively. The DNA sequence of all the exons agreed perfectly with that of the p8 cDNA. All the exon–intron boundary sequences conformed to the GT/AG rule . The 5′-flanking region lacks a proximal authentic CAAT or TATA box. Moreover, the initiator sequence , which appears to direct the site of initiation and basal level of transcription in many TATA-less promoters, was not present.
Mapping of the human p8 gene to chromosome 16
Human DNA sequences were specifically amplified in the hybrid cells, as a product of the expected size of 559 bp and no band was seen using DNA from rat (FAZA), mouse (IR), or hamster (A23) parent cell lines. The results obtained from the hybrid cell lines are summarized in Table 1. The DNA with the appropriate size was obtained in only the hybrid cell lines that contained an intact chromosome 16 and was absent from other hybrid cell lines, indicating assignment of the p8 gene to chromosome 16.
Subcellular localization of p8 in COS-7-p8 cDNA transfected cells
The p8 has a potential bipartite signal for nuclear targeting at position 63 (KLVTKLQNSERKKRGA) , suggesting that the p8 function is in the nucleus. To gain insight into the subcellular localization of p8, we overexpressed p8 by transfection in COS-7 cells and studied its localization by indirect immunostaining with antibodies raised against its COOH-terminal region (see Materials and Methods). Fig. 2 shows that p8 was present almost exclusively in the nucleus confirming the predictive subcellular targeting. No staining was observed in control experiments (data not shown).
RNA blot analysis of p8 mRNA expression
To determine which tissues express p8, we hybridized a multitissue RNA blot with the hump8 probe at high stringency ( Fig. 3). A band of ≈ 0.8 kb was detected with abundant expression in liver, pancreas, prostate, ovary, colon, thyroid, spinal cord, trachea and adrenal gland, with moderate expression in heart, placenta, lung, skeletal muscle, kidney, testis, small intestine, stomach and lymph node, and with low expression in brain, spleen, thymus and bone marrow. No p8 mRNA was detected in peripheral blood leukocytes. In addition, the hump8 probe detected in several tissues an additional transcript ( Fig. 3) whose significance is unclear.
We also studied expression of the p8 mRNA in five different cell lines. Expression was detected in HepG2 cells but not in HeLa, SW40, HT29 and TC11 ( Fig. 4).
Induction of p8 mRNA expression in vitro
HepG2, HeLa, SW40, HT29 and TC11 cells were chosen to study p8 mRNA expression after ceramide and serum starvation treatments as indicated in Material and Methods. Fig. 4 shows that ceramide treatment induced a strong expression in HeLa cells, but not in SW40, HT29 and TC11 cells. In HepG2 cells, which express constitutively p8 mRNA, expression was not modified by the treatment. Serum starvation induced p8 mRNA expression in HeLa cells when cells were treated for more than 24 h. No induction was detected in HT29, SW40 and TC11 cells until 5 days of treatment. When HepG2 cells were serum deprived, no changes in p8 mRNA expression were observed after 1 or 2 days; studies could not be carried out for longer times because of cell death and systematic degradation of the obtained RNA.
p8 Expression promotes cellular growth but does not alter resistance to apoptosis in HeLa cells
To analyze p8 function, we stably transfected HeLa cells with a p8 expression plasmid, and cellular growth and sensitivity to apoptosis were measured in four independent clones. HeLa p8/3, p8/8, p8/14 and p8/17 clones showed, respectively, a 101, 63, 73 and 82% increase in [3H]thymidine incorporation into DNA when compared to HeLa nontransfected cells ( Fig. 5). Similar results were obtained when HeLa-pcDNA3 transfected cells were used as control, suggesting little influence of transfection and selection, if any, on [3H]thymidine incorporation (data not shown). In addition, to assess the cell cycle effects of p8 expression, HeLa and HeLa-p8 transfected cells were grown by 96 h and were then permeabilized in the presence of propidium iodide to stain the nuclear DNA. Flow cytometry profiles of nuclear DNA content revealed that p8 increase the cell number in S and G2/M phase in HeLa cells ( Fig. 5), confirming and extending results from the [3H]thymidine incorporation experiments and previous observations .
As most proapototic stimuli induce p8 mRNA expression in HeLa cells ( Fig. 4 and data not shown) we looked whether p8 expression modified the apoptotic response in these cells. As observed with a number of cell types in vitro, HeLa cells are susceptible to cell death by TNFα when protein synthesis is inhibited . Each of the HeLa cell transfectants was treated with TNFα in the presence of cycloheximide for 8 h; after that time the parental HeLa cell line showed evidence of extensive cell death. The amount of cell death was quantitated by MTS assay, which provides a quantitation of viable cells. When assayed by this method, HeLa cells showed near 55% cell survival and HeLa p8-transfected clones p8/3, p8/8, p8/14 and p8/17 showed 62, 58, 63 and 53%, respectively ( Fig. 6). The cell death observed was not due to an effect of inhibition of protein synthesis, as treatment with cycloheximide alone had minimal effects on cell survival. Similar results were obtained when apoptosis was induced with staurosporine (data not shown).
We used a strategy based on the assumption that if human p8 does exist, human and rat p8 mRNA sequences should be similar enough to allow detection by heterologous screening. A single full-length cDNA (hump8) was selected in the human cDNA library upon screening and sequencing. The protein encoded by hump8 is two amino acids longer than rat p8. The two sequences have an overall amino acid identity of 74% ( Fig. 1).
One of the most important characteristics of p8 gene is a very low level of expression in normal pancreas and a dramatic increase during the acute phase of pancreatitis . Contrary to the pancreas, p8 mRNA is expressed at high levels in several normal tissues as previously reported . In this work, we have used a multitissue RNA blot (commercially available) to study human p8 mRNA distribution. Expression in human and rat p8 correlate well for all tissues except for pancreas. Whereas normal rat pancreas expresses low levels of p8 mRNA, human pancreas seems to express very high levels. In our previous work  we have shown that p8 is rapidly and strongly activated in pancreas in response to minor pancreatic injuries. We speculated that pancreatic RNA from multitissue RNA blot could be derived from a suffering pancreas. We therefore studied p8 mRNA expression in seven pancreatic RNA samples obtained from human with non-symptomatic pancreatic diseases and found that three of these expressed very low levels, as expected, but two samples expressed moderate and two samples showed high levels of this transcript (data not shown), suggesting a variability among samples and supporting the idea that p8 mRNA is strongly activated in response to a minor injury.
Analysis of the p8 primary structure suggested the presence of a bipartite motif of nuclear targeting in its COOH-terminal region indicating that p8 may function within the nucleus. This presumption has been confirmed by transfection of the p8 cDNA into COS-7 cells followed by immunostaining with specific antibodies ( Fig. 2). Most nucleoprotein functions are regulated by phosphorylation/dephosphorylation. Preliminary results obtained by incubating COS-7 cells with 32P indicate that p8 is in fact a phosphoprotein (data not shown), but the sites of phosphorylation and the kinases involved remain to be determined.
In our previous work, we have observed that p8 mRNA expression was induced in response to various, although not all, proapoptotic stimuli . Therefore, we speculated for a proapoptotic or an antiapoptotic function for p8. In this work we found that p8 expression does not alter the apoptotic effect of TNFα or staurosporine on HeLa cells ( Fig. 6). However, as for COS-7 and pancreatic AR4-2 J cells, p8 expression in HeLa cells promotes a significant cellular growth ( Fig. 5), strongly suggesting a cellular growth promoting function for p8 rather than an apoptosis-related function. Interestingly, p8 mRNA is constitutively expressed in some tissues and cells ( Fig. 3), but its expression was only detected after induction in others. Moreover, this transcript was not detected in a few number of tissues or cells. These findings suggest a nonvital function for this molecule. Because p8 is expressed in response to proapoptotic stimuli and promotes cellular growth, we can assume that p8 could participate in the response to some proapoptotic stimuli by promoting cellular growth in a way that helps the tissue counteract diverse injuries.
While writing this paper, we learned that The Institute of Genomic Research (TIGR) deposited the complete nucleotide sequence of a BAC clone (GenBank accession number AC002425) which contains the p8 gene sequence. That sequence agrees perfectly with our sequence and confirms our chromosomal assignment. Moreover, that work defines more precisely the localization of the p8 gene within chromosome 16, at position p11.2. Interestingly, this region is frequently amplified in breast tumors [12, 13]. The implication of the p8, as a growth promoting molecule, in this tumor will be investigated.
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 Médicale. F.F. was supported by a grant from Forschungsfond der Fakultät für 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.