A transcriptomic signature to predict adjuvant gemcitabine sensitivity in pancreatic adenocarcinoma.

BACKGROUND: Chemotherapy is the only systemic treatment approved for pancreatic ductal adenocarcinoma (PDAC), with a selection of regimens based on patients' performance status and expected efficacy. The establishment of a potent stratification associated with chemotherapeutic efficacy could potentially improve prognosis by tailoring treatments. PATIENTS AND METHODS: Concomitant chemosensitivity and genome-wide RNA profiles were carried out on preclinical models (primary cell cultures and patient-derived xenografts) derived from patients with PDAC included in the PaCaOmics program (NCT01692873). The RNA-based stratification was tested in a monocentric cohort and validated in a multicentric cohort, both retrospectively collected from resected PDAC samples (67 and 368 patients, respectively). Forty-three (65%) and 203 (55%) patients received adjuvant gemcitabine in the monocentric and the multicentric cohorts, respectively. The relationships between predicted gemcitabine sensitivity and patients' overall survival (OS) and disease-free survival were investigated. RESULTS: The GemPred RNA signature was derived from preclinical models, defining gemcitabine sensitive PDAC as GemPred+. Among the patients who received gemcitabine in the test and validation cohorts, the GemPred+ patients had a higher OS than GemPred- (P = 0.046 and P = 0.00216). In both cohorts, the GemPred stratification was not associated with OS among patients who did not receive gemcitabine. Among gemcitabine-treated patients, GemPred+ patients had significantly higher OS than the GemPred-: 91.3 months [95% confidence interval (CI): 61.2-not reached] versus 33 months (95% CI: 24-35.2); hazard ratio 0.403 (95% CI: 0.221-0.735, P = 0.00216). The interaction test for gemcitabine and GemPred+ stratification was significant (P = 0.0245). Multivariate analysis in the gemcitabine-treated population retained an independent predictive value. CONCLUSION: The RNA-based GemPred stratification predicts the benefit of adjuvant gemcitabine in PDAC patients.

Relevance of biopsy-derived pancreatic organoids in the development of efficient transcriptomic signatures to predict adjuvant chemosensitivity in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) patients are frequently treated by chemotherapy. Even if personalized therapy based on molecular analysis can be performed for some tumors, PDAC regimens selection is still mainly based on patients' performance status and expected efficacy. Therefore, the establishment of molecular predictors of chemotherapeutic efficacy could potentially improve prognosis by tailoring treatments. We have recently developed an RNA-based signature that predicts the efficacy of adjuvant gemcitabine using 38 PDAC primary cell cultures. While demonstrated its efficiency, a significant association with the classical/basal-like PDAC spectrum was observed. We hypothesized that this flaw was due to the basal-like biased phenotype of cellular models used in our strategy. To overcome this limitation, we generated a prospective cohort of 27 consecutive biopsied derived pancreatic organoids (BDPO) and include them in the signature identification strategy. As BDPO's do not have the same biased phenotype as primary cell cultures we expect they can compensate one with each other and cover a broader range of molecular phenotypes. We then obtained an improved signature predicting gemcitabine sensibility that was validated in a cohort of 300 resected PDAC patients that have or have not received adjuvant gemcitabine. We demonstrated a significant association between the improved signature and the overall and disease-free survival in patients predicted as sensitive and treated with adjuvant gemcitabine. We propose then that including BDPO along primary cell cultures represent a powerful strategy that helps to overcome primary cell cultures limitations producing unbiased RNA-based signatures predictive of adjuvant treatments in PDAC.

Tumor protein p53-induced nuclear protein (TP53INP1) expression in medullary thyroid carcinoma: a molecular guide to the optimal extent of surgery?

BACKGROUND: Medullary thyroid cancer (MTC) is characterized by early regional lymph node metastasis, the presence of which represents a critical obstacle to cure. At present no molecular markers have been successfully integrated into the clinical care of sporadic MTC. The present study was designed to evaluate TP53INP1 expression in MTC and to assess its ability to guide the surgeon to the optimal extent of surgery performed with curative intent. METHODS: Thirty-eight patients with sporadic MTC were evaluated. TP53INP1 immunoexpression was studied on embedded paraffin material and on cytological smears. RESULTS: TP53INP1 was expressed in normal C cells, in C-cell hyperplasia, and in 57.9% of MTC. It was possible to identify two groups of MTC according to the proportion of TP53INP1 expressing tumor cells: group 1 from 0% to <50% and group 2 from 50% to 100% of positive cells. Patients with a decreased expression of TP53INP1 (group 1) had a lower rate of nodal metastasis (18.8% versus 63.4% in group 2; P = 0.009), with only minimal lymph node involvement per N1 patient (2.7% of positive lymph nodes versus 22.9%; P < 0.001) and better outcomes (100% of biochemical cure versus 55.5%; P < 0.001). Patients with distant metastases were only observed in group 2. Cytological samples exhibit similar results to their embedded counterparts. CONCLUSIONS: TP53INP1 immunoexpression appears to be a clinical predictor of lymph node metastasis in MTC. The evaluation of TP53INP1 expression may guide the extent of lymph node dissection in the clinically node-negative neck. These findings require prospective validation.

Rapid PCR cloning and sequence determination of the rat lithostathine gene.

The rat lithostathine gene was isolated from a genomic library using a rapid screening procedure involving PCR amplification. It was characterized over 2.7 kbp of gene sequence and 2.43 kbp of 5'-flanking sequence. The 5'-end of the coding sequence was determined by primer extension of lithostathine mRNA. The lithosathine sequence spanned over six exons. The promoter region of the gene contained the TATAAA and CCAAT consensus sequences 30 and 107 bp upstream of the cap site, respectively. Furthermore, a tract of (TG)22 repeat, with potential Z-DNA conformation, was found at position-1081.

The pancreatitis associated protein III (PAP III), a new member of the PAP gene family.

A third member of the rat pancreatitis associated protein (PAP) gene family is described here. Its messenger RNA was cloned from an intestinal cDNA library and sequenced. The encoded protein, designated PAP III, shows 66% and 63% identity with the rat PAP I and II, respectively. The PAP III gene is constitutively expressed in the small intestine and in the pancreas with acute pancreatitis, but not in the healthy pancreas.

Two transcripts are generated from the pancreatitis associated protein II gene by alternative splicing in the 5' untranslated region.

We have previously reported the coding sequence of the rat PAP II mRNA. We show in this paper the existence in rat pancreas of two forms of PAP II mRNA with identical coding sequence but a different 5'-untranslated region. We demonstrate that this is the result of a differential splicing.

Cdx1 promotes cellular growth of epithelial intestinal cells through induction of the secretory protein PAP I.

Expression of the Cdx1 homeobox gene in epithelial intestinal cells promotes cellular growth and differentiation. Cdx1and the Pancreatitis Associated Protein I (PAP I) are concomitantly expressed in the epithelial cells of the lower part of the intestinal crypts. Because Cdx1 is a transcription factor and PAP I, in other tissues, is a proliferative factor, we looked for a relationship between these two proteins in the intestinal-derived IEC-6 cells. After stable transfection with a Cdx1 expression vector, they produce high levels of the PAP I transcript and protein indicating a functional link between the two genes. Demonstration of Cdx1 binding to the PAP I promoter region and suppression of PAP I induction after deletion of the corresponding sequence indicated that Cdx1 is a transcription factor controlling PAP I gene expression in intestinal cells. By infecting IEC-6 cells with adenoviruses expressing PAP I, we demonstrated that PAP I induces mitosis in these cells. On the other hand, inhibition of the PAP I expression in the IEC-6 Cdxl-expressing cells using an antisense strategy confirmed the requirement of this protein for the effect of Cdx1 on cell growth. Finally, addition of the immunopurified PAP I to the culture medium promotes cell growth of the IEC-6 cells in a dose-dependent manner. Maximal effect was obtained at 1 ng/ml. Taken together these results demonstrate that PAP I is a target of the Cdx1 homeobox gene in intestinal cells which participates in the regulation of intestinal cell growth via an autocrine and/or paracrine mechanism.

Changes in gene expression during pancreatic regeneration: activation of c-myc and H-ras oncogenes in the rat pancreas.

Expression of the c-myc and H-ras oncogenes, and of several genes specifically expressed in adult rat pancreas was investigated by monitoring changes in corresponding mRNA concentrations, by dot-blot hybridization, during the early phase of regeneration following subtotal pancreatectomy. The oncogenes c-myc and H-ras were overexpressed after 12-24 and 48 h, respectively, then returned to basal levels. The concentrations of mRNAs encoding amylase, chymotrypsinogen B, and trypsinogen I decreased during the regeneration time. By contrast, proinsulin I mRNA concentration was increased at 12-48 h after surgical resection, and actin mRNA concentration was increased at 12-48 h after surgical resection, and actin mRNA concentration was increased at 12 h after subtotal pancreatectomy and remained elevated thereafter. We concluded that regeneration after subtotal pancreatectomy is accompanied by repression of certain genes that are expressed in differentiated pancreatic tissue, and that derepression of other genes may be necessary for starting and/or maintaining the process of pancreatic regeneration.

Oxidative stress-induced p53 activity is enhanced by a redox-sensitive TP53INP1 SUMOylation.

Tumor Protein p53-Induced Nuclear Protein 1 (TP53INP1) is a tumor suppressor that modulates the p53 response to stress. TP53INP1 is one of the key mediators of p53 antioxidant function by promoting the p53 transcriptional activity on its target genes. TP53INP1 expression is deregulated in many types of cancers including pancreatic ductal adenocarcinoma in which its decrease occurs early during the preneoplastic development. In this work, we report that redox-dependent induction of p53 transcriptional activity is enhanced by the oxidative stress-induced SUMOylation of TP53INP1 at lysine 113. This SUMOylation is mediated by PIAS3 and CBX4, two SUMO ligases especially related to the p53 activation upon DNA damage. Importantly, this modification is reversed by three SUMO1-specific proteases SENP1, 2 and 6. Moreover, TP53INP1 SUMOylation induces its binding to p53 in the nucleus under oxidative stress conditions. TP53INP1 mutation at lysine 113 prevents the pro-apoptotic, antiproliferative and antioxidant effects of TP53INP1 by impairing the p53 response on its target genes p21, Bax and PUMA. We conclude that TP53INP1 SUMOylation is essential for the regulation of p53 activity induced by oxidative stress.

TP53INP1, a tumor suppressor, interacts with LC3 and ATG8-family proteins through the LC3-interacting region (LIR) and promotes autophagy-dependent cell death.

TP53INP1 (tumor protein 53-induced nuclear protein 1) is a tumor suppressor, whose expression is downregulated in cancers from different organs. It was described as a p53 target gene involved in cell death, cell-cycle arrest and cellular migration. In this work, we show that TP53INP1 is also able to interact with ATG8-family proteins and to induce autophagy-dependent cell death. In agreement with this finding, we observe that TP53INP1, which is mainly nuclear, relocalizes in autophagosomes during autophagy where it is eventually degraded. TP53INP1-LC3 interaction occurs via a functional LC3-interacting region (LIR). Inactivating mutations of this sequence abolish TP53INP1-LC3 interaction, relocalize TP53INP1 in autophagosomes and decrease TP53INP1 ability to trigger cell death. Interestingly, TP53INP1 binds to ATG8-family proteins with higher affinity than p62, suggesting that it could partially displace p62 from autophagosomes, modifying thereby their composition. Moreover, silencing the expression of autophagy related genes (ATG5 or Beclin-1) or inhibiting caspase activity significantly decreases cell death induced by TP53INP1. These data indicate that cell death observed after TP53INP1-LC3 interaction depends on both autophagy and caspase activity. We conclude that TP53INP1 could act as a tumor suppressor by inducing cell death by caspase-dependent autophagy.

TP53INP1 decreases pancreatic cancer cell migration by regulating SPARC expression.

Tumor protein 53 induced nuclear protein 1 (TP53INP1) is a p53 target gene that induces cell growth arrest and apoptosis by modulating p53 transcriptional activity. TP53INP1 interacts physically with p53 and is a major player in the p53-driven oxidative stress response. Previously, we demonstrated that TP53INP1 is downregulated in an early stage of pancreatic cancerogenesis and when restored is able to suppress pancreatic tumor development. TP53INP1 downregulation in pancreas is associated with an oncogenic microRNA miR-155. In the present work, we studied the effects of TP53INP1 on cell migration. We found that TP53INP1 inactivation correlates with increased cell migration both in vivo and in vitro. The impact of TP53INP1 expression on cell migration was studied in different cellular contexts: mouse embryonic fibroblast and different pancreatic cancer cell lines. Its expression decreases cell migration by the transcriptional downregulation of secreted protein acidic and rich in cysteine (SPARC). SPARC is a matrix cellular protein, which governs diverse cellular functions and has a pivotal role in regulating cell-matrix interactions, cellular proliferation and migration. SPARC was also showed to be upregulated in normal pancreas and in pancreatic intraepithelial neoplasia lesions in a pancreatic adenocarcinoma mouse model only in the TP53INP1-deficient animals. This novel TP53INP1 activity on the regulation of SPARC expression could explain in part its tumor suppressor function in pancreatic adenocarcinoma by modulating cellular spreading during the metastatic process.

VAV2 regulates epidermal growth factor receptor endocytosis and degradation.

Vav proteins are guanine nucleotide exchange factors for Rho GTPases that regulate cell adhesion, motility, spreading and proliferation in response to growth factor signalling. In this work, we show that Vav2 expression delayed epidermal growth factor receptor (EGFR) internalization and degradation, and enhanced EGFR, ERK and Akt phosphorylations. This effect of Vav2 on EGFR degradation is dependent on its guanine nucleotide exchange function. Knockdown of Vav2 in HeLa cells enhanced EGFR degradation and reduced cell proliferation. epidermal growth factor stimulation led to co-localization of Vav2 with EGFR and Rab5 in endosomes. We further show that the effect of Vav2 on EGFR stability is modulated by its interaction with two endosome-associated proteins and require RhoA function. Thus, in this work, we report for the first time that Vav2 can regulate growth factors receptor signalling by slowing receptor internalization and degradation through its interaction with endosome-associated proteins.

CIP4 is a new ArgBP2 interacting protein that modulates the ArgBP2 mediated control of WAVE1 phosphorylation and cancer cell migration.

ArgBP2 is a multi-adapter protein involved in signal transduction associated to the cytoskeleton and was shown to regulate the migration and adhesion of pancreatic cancer cells thereby modulating their tumorigenicity. Here we describe the interaction of ArgBP2 with CIP4, a new associated protein identified by yeast two-hybrid. We found that both proteins modulated their reciprocal tyrosine phosphorylation catalyzed by the non-receptor tyrosine kinase c-Abl. We observed that, like ArgBP2, CIP4 directly interacted with WAVE1 and could enhance its phosphorylation by c-Abl. ArgBP2 and CIP4 acted synergistically to increase WAVE1 tyrosine phosphorylation. Finally, we could show that CIP4 was dispensable for the ArgBP2 induced blockade of cell migration whereas its overexpression was deleterious for this important function of ArgBP2.

Characterization of a silencer regulatory element in the rat PAP I gene which confers tissue-specific expression and is promoter-dependent.

Previous analysis of the rat PAP I promoter indicated that the region between nt -180 and -81 possessed silencer activity in cells that did not express PAP I. Based on this finding, we performed a series of experiments to characterize functionally that region and analyze the nuclear proteins interacting with it. Transient transfection assays were conducted in the fibroblast Rat2 cell line, in which PAP I is not expressed, and in the pancreatic cell line AR-42J, expressing PAP I, using the CAT gene as reporter. Experiments in Rat2 cells revealed that the sequence with silencer activity was located within the rep27 region (position -180/-153). Suppressor activity was observed when rep27 was inserted upstream from the core PAP I promoter, in both orientations. By contrast, inserting the rep27 region in front of the promoters of SV40 or thymidine kinase did not affect or weakly enhanced CAT activity. Suppressor activity is therefore position-independent and promoter-dependent. In pancreatic AR-42J cells, rep27 act as a positive element but did not alter CAT expression when inserted in front of the core PAP I promoter or heterologous promoters. Electrophoretic mobility shift assays allowed identification of specific DNA-protein complexes. The shifted complex migrated at the same position with both Rat2 and AR-42J nuclear extracts. Moreover, similar band shifts were obtained with rat nuclear extracts from healthy pancreas, pancreas with acute pancreatitis, liver, kidney, spleen, and small intestine. Results suggest that the rep27 cis-acting element contributes to the tissue specific expression of the PAP I gene. That activity could be mediated by the synergistic action of several transcription factors, one of which being present in all cells.

Identification of a transcriptional regulatory region of the rat pancreatitis-associated protein I (PAP I) gene that confers tissue specificity.

We have previously characterized the rat pancreatitis-associated protein I (PAP I) gene by nucleotide sequencing. We describe in this paper its promoter region by analysing the regulatory functions associated with the DNA sequence comprising nt -1253 to + 10 of the gene. That sequence strongly promoted the transcription of the promotorless chloramphenicol acetyltranferase (CAT) gene in cells of pancreatic origin (AR-42J) but not in cells of non-pancreatic origin (Rat 2 and IEC 6). The influence on CAT expression of stepwise 5' deletions in the promoter sequence was monitored in the three cell lines. In pancreatic AR-42J cells, deletion down to position -926 did not affect significantly the expression of the reporter gene. Deletion to nt -685 caused about a 30% decrease in expression. Extending the deletion to nt -444 did not have any additional effect, but a further deletion to nt -180, resulted in a reduction to about 25%. Moreover, deletion from nt -180 to -118 resulted in a further reduction to about one-third of that. Finally, deletion down to nt -61 further reduced activity by a factor of 3, although it remained above background. These results suggest the presence of several positive cis-acting elements in the PAP I promoter. In non-pancreatic cells, CAT expression remained very low when the promoter was deleted down to nt -180. Yet, deletion from -180 to -118 significantly increased CAT expression, suggesting suppression of a negative cis-acting element. Further deletion down to nt -61 decreased CAT activity by a factor of 5. The region between nt -180 and -61 was subjected to footprint analysis. A similar pattern of DNase protection was obtained with AR-42J and Rat 2 nuclear extracts, the only protected region extending from nt -125 to -95. That region was further analysed by inserting the nt -180 to -81 fragment, in both orientations, upstream of thymidine kinase (TK) or simian virus 40 (SV40) promoter-CAT constructs. In all cases CAT expression was increased in pancreatic cells but reduced in Rat 2 cells. These results indicated the presence of cell-specific positive and negative elements within that region.

PAP gene transcription induced by cycloheximide in AR4-2J cells involves ADP-ribosylation.

We report in this paper that cycloheximide induces PAP mRNA expression in the pancreatic acinar cell line AR4-2J in a dose- and time-dependent manner. We analyzed whether stabilization of the PAP mRNA or the direct induction of its transcription contributed to the induction of PAP mRNA expression by the drug. We first infected the cells, which do not express PAP mRNA constitutively, with a recombinant adenovirus in which the PAP cDNA was subcloned downstream of the CMV promotor, to obtain high levels of transcript. Then, transcription was pharmacologically blocked, the cells were treated with cycloheximide, and the PAP mRNA concentration was monitored over 8 h by Northern blot. PAP mRNA concentration remained unchanged for 4 h and then decreased in both cycloheximide-treated and control cells, ruling out a significant contribution of posttranscriptional regulation in cycloheximide induction. Direct regulation of gene transcription is therefore likely and we investigated whether it could involve ADP-ribosylation. Cycloheximide-induced cells were treated with two chemical inhibitors of poly(ADP-ribose) polymerase. 3-Aminobenzamide inhibited 75% of PAP gene induction and 4-hydroxyquinazolone, the highly specific inhibitor of the enzyme, blocked almost completely PAP expression, suggesting that ADP-ribosylation was indeed required for the upregulation of PAP gene expression by cycloheximide.

Serum from rats with acute pancreatitis induces expression of the PAP mRNA in the pancreatic acinar cell line AR-42J.

PAP is a pancreatic secretory protein expressed in the pancreas during the acute phase of pancreatitis. We have investigated the effect of the serum from rats with acute pancreatitis (SAP) on the expression of the PAP mRNA in AR-42J cells. PAP mRNA is strongly induced by SAP in a dose-dependent manner. This induction is abolished by preheating the SAP or diminished by treating the cells with cycloheximide. In addition, amylase but not actin mRNA expression was induced by a different SAP factor. We transfected the AR-42J cells with a chimeric gene containing 1.2 kbp 5'-flanking region of the PAP promoter linked to the CAT reporter gene. The CAT activity was significatively increased in the cells, on treating them with SAP. Our results show: first, SAP contains factors responsible for the PAP mRNA expression and secondly, the cis-acting elements are localized within the 1.2 kbp upstream region of the transcription initiation site.

Identification of a second rat pancreatitis-associated protein. Messenger RNA cloning, gene structure, and expression during acute pancreatitis.

The pancreatitis-associated protein (PAP) is a lectin-related secretory protein present in small amounts in the rat pancreas and overexpressed during the acute phase of pancreatitis. On the other hand, PAP is constitutively expressed in the intestinal tract but not in other tissues. We cloned from a pancreatic cDNA library two overlapping cDNAs encoding a protein structurally related to PAP. This second PAP, which was called PAP II, was the same size as the original PAP (PAP I) and showed 74.3% amino acid homology. Studies on gene expression demonstrated that PAP II mRNA concentration increased within 6 h following induction of pancreatitis, reached maximal levels (> 200 times control values) at 24-48 h, and decreased thereafter, similar to PAP I. However, PAP II mRNA could not be detected in the intestinal tract or in other tissues. We also isolated a PAP II genomic DNA fragment which was characterized over 2.7 kb of gene sequence and 1.9 kb of 5' flanking sequence. The 5' end of the coding sequence was determined by primer extension of the PAP II mRNA. The PAP II coding sequence spanned six exons separated by five introns. Several potential regulatory elements were identified in the promoter region, including two glucocorticoid-response elements and one IL-6-response element. Antibodies raised to a synthetic peptide of PAP II detected a single band in Western blot analysis of the pancreatic secretory proteins from rats with pancreatitis, with a M(r) compatible with the theoretical M(r) of PAP II.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural organization of the gene encoding the rat pancreatitis-associated protein. Analysis of its evolutionary history reveals an ancient divergence from the other carbohydrate-recognition domain-containing genes.

Rat pancreatitis-associated protein (PAP) mRNA is barely detectable in normal pancreas and overexpressed during acute pancreatitis (Iovanna, J., Orelle, B., Keim, V., and Dagorn J.-C. (1991) J. Biol. Chem. 266, 24664-24669). RNA amplification by reverse-transcriptase-coupled polymerase chain reaction showed that PAP mRNA was constitutively expressed in duodenum, jejunum, and ileum, at similar levels as in pancreas during the acute phase of pancreatitis. A weak expression was also detected in several other tissues. The rat PAP gene was isolated from a genomic library and characterized over 3.2 kilobases of gene sequence and 1.2 kilobases of 5'-flanking sequence. The 5' end of the coding sequence was determined by primer extension of the PAP transcript. Several potential regulatory elements were identified in the promoter region, including a pancreas-specific consensus sequence, two Pan1 (pancreas-specific) transcription activators, two IL-6 response elements, and one glucocorticoid response element. The PAP coding sequence spanned over six exons. The first three exons encoded the 5'-untranslated region of the mRNA, the signal peptide, and 39 amino acids of the NH2-terminal end of the mature protein, respectively. The other three exons encoded a domain of the protein with significant homology to the carbohydrate-recognition domain of animal lectins. Sequence comparison of the PAP gene with 13 carbohydrate-recognition domain-containing genes revealed that they derived from the same ancestor gene. Position of introns within the carbohydrate-recognition domain were different, however, suggesting that PAP belongs to a new group of lectins. These results support the hypothesis that genes encoding PAP and other lectins evolved from a common ancestor gene by intron gain.

Cloning, expression and chromosomal localization of the rat pancreatitis-associated protein III gene.

PAP III belongs to the family of pancreatitis-associated proteins, recently characterized as pancreatic secretory proteins structurally related to C-type lectins, and whose expression is induced during the acute phase of pancreatitis. In this paper, we describe the cloning, characterization and chromosomal localization of the rat PAP III gene. The gene was isolated from a genomic library using a PCR-based method and characterized over 2.5 kb of gene sequence and 1.7 kb of 5'-flanking sequence. The 5' end of the coding sequence was determined by primer extension of the PAP III transcript. The PAP III coding sequence spanned over six exons. We found striking similarities between PAP III and PAP I and II genes, in genomic organization as well as in promoter sequences. Moreover, the rat PAP III gene was mapped to chromosome 4 using mouse-rat hybrid cells, a localization which coincides with that of the PAP I and II genes. The three genes could therefore derive from the same ancestral gene by duplication. Expression of the PAP III gene was compared with that of PAPs I and II. Expression levels were similar in pancreas, where PAP III mRNA concentration increased within 6 h following induction of pancreatitis, reached maximal levels (> 200 times control values) at 24-48 h, and decreased thereafter. In the intestinal tract, where PAP II is not expressed, the pattern of PAP III expression was comparable with that of PAP I; fasting induced a decrease in its mRNA concentration by more than 80%, which could be reversed within 6 h upon feeding. PAP III is therefore a new member of the PAP gene family, more closely related to the PAP I gene.