Convertase PC2 Research Articles

Inhibition of the vacuolar H+-ATPase perturbs the transport, sorting, processing and release of regulated secretory proteins.

Vacuolar H+-ATPases (V-ATPases) are multisubunit enzymes that acidify various intracellular organelles, including secretory pathway compartments. We have examined the effects of the specific V-ATPase inhibitor bafilomycin A1 (Baf) on the intracellular transport, sorting, processing and release of a number of neuroendocrine secretory proteins in primary Xenopus intermediate pituitary cells. Ultrastructural examination of Baf-treated intermediate pituitary cells revealed a reduction in the amount of small dense-core secretory granules and the appearance of vacuolar structures in the trans-Golgi area. Pulse-chase incubations in combination with immunoprecipitation analysis showed that in treated cells, the proteolytic processing of the newly synthesized prohormone proopiomelanocortin, prohormone convertase PC2 and secretogranin III (SgIII) was inhibited, and an intracellular accumulation of intact precursor forms and intermediate cleavage products became apparent. Moreover, we found that treated cells secreted considerable amounts of a PC2 processing intermediate and unprocessed SgIII in a constitutive fashion. Collectively, these data indicate that in the secretory pathway, V-ATPases play an important role in creating the microenvironment that is essential for proper transport, sorting, processing and release of regulated secretory proteins.

Cathepsin-B Fusion Proteins Misroute Secretory Protein Partners Such as the Proprotein Convertase PC2-7B2 Complex toward the Lysosomal Degradation Pathways

A general strategy is presented for the dominant negative reduction in the levels of heterodimeric soluble proteins within the secretory pathway through fusion of one of its partners C-terminal to the lysosomal enzyme cathepsin B (CB). Stable transfectants of CB-7B2 chimeras in AT20 cells result in a drastic reduction of the endogenous levels of its partner, the proprotein convertase PC2. This dominant negative suppressive effect requires active CB. It was partially reversed by NH4Cl, the cell-permeable CB inhibitor CA-074Me, but not by the proteasome inhibitor Lactacystin, suggesting the potential participation of the lysosomal/endosomal degradative pathway in this process.

Processing of synthetic pro-islet amyloid polypeptide (proIAPP) 'amylin' by recombinant prohormone convertase enzymes, PC2 and PC3, in vitro.

Islet amyloid polypeptide (IAPP), amylin, is the constituent peptide of pancreatic islet amyloid deposits which form in islets of Type 2 diabetic subjects. Human IAPP is synthesized as a 67-residue propeptide in islet beta-cells and colocalized with insulin in beta-cell granules. The mature 37-amino acid peptide is produced by proteolysis at pairs of basic residues at the C- and N-termini of the mature peptide. To determine the enzymes responsible for proteolysis and their activity at the potential cleavage sites, synthetic human proIAPP was incubated (0.5-16 h) with recombinant prohormone convertases, PC2 or PC3 at appropriate conditions of calcium and pH. The products were analysed by MS and HPLC. Proinsulin was used as a control and was cleaved by both recombinant enzymes resulting in intermediates. PC3 was active initially at the N-terminal-IAPP junction and later at the C-terminus, whereas initial PC2 activity was at the IAPP-C-terminal junction. Processing at the basic residues within the C-terminal flanking peptide rarely occurred. There was no evidence for substantial competition for the processing enzymes when the combined substrates proinsulin and proIAPP were incubated with both PC2 and PC3. As proinsulin cleavage is sequential in vivo (PC3 active at the B-chain-C-peptide junction, followed by PC2 at A chain-C-peptide junction), these data suggest that proteolysis of proIAPP and proinsulin is coincident in secretory granules and increased proinsulin secretion in diabetes could be accompanied by increased production of proIAPP.

Colocalization of chaperone Cpn60, proinsulin and convertase PC1 within immature secretory granules of insulin-secreting cells suggests a role for Cpn60 in insulin processing.

Many of the mechanisms that control insulin processing and packaging by interaction with different elements along the secretory pathway remain poorly understood. We have investigated the possibility that Cpn60, a member of the heat shock protein family, may be present in rat insulin-secreting cells, participating in the proinsulin-insulin maturation process. Immunofluorescence and high resolution immunocytochemical studies revealed the presence of the Cpn60 protein all along the insulin secretory pathway, being particularly abundant over the proinsulin-containing immature secretory granules. Double-labeling experiments showed associations between Cpn60 and proinsulin, as well as between Cpn60 and PC1 convertase, with a preferential binding to proinsulin. These findings paralleled those of coimmunoprecipitation studies showing the Cpn60 chaperone and the mature form of the PC1 convertase in proinsulin immunoprecipitates, as well as the PC1 in Cpn60 immunoprecipitates from total islet cell extracts. In vitro binding of Cpn60 to proinsulin, insulin and glucagon was also documented. Cpn60, significantly abundant in proinsulin-containing secretory granules where conversion of proinsulin to insulin takes place, and the colocalization of the chaperone with proinsulin and PC1 convertase suggest that the Cpn60 protein may play a role directing precise molecular interactions during insulin processing and/or packaging.

Conversion and storage of somatostatin are established before response to secretagogue stimuli in P19 neurons

In mature neurons, neuropeptides are synthesized via limited proteolysis of propolypeptides by convertases. The bioactive peptides are then stored in secretory granules until they are released extracellularly upon the induction of a fusion between granules and the plasma membrane, in response to secretagogues. We used the mouse P19 embryonic carcinoma cells as a model to determine if the capacities to convert and store neuropeptides and to secrete them in a regulated fashion are established coordinately during neuronal differentiation. We have previously shown that both undifferentiated P19 cells and their neuronal derivatives express the largely distributed furin, PACE4 and PC5 convertases, whereas only neuronal derivatives express the neuroendocrine convertase PC2. In addition, undifferentiated cells displayed furin- rather than PC2-like converting capacities. The present work demonstrates that day 8 P19 neurons mainly convert prosomatostatin (proSS) to somatostatin-14 (SS-14) using HPLC and radioimmunoassay (RIA) analyses, indicating that P19 cells acquire PC2-like converting capacities as a consequence of neuronal differentiation. SS-14 was predominantly intracellular in neuronal cells which were shown to express several granins, markers of granules, by Western blotting. However, cell membrane depolarization with 50 mM K +, a general secretagogue stimulus, evoked the release of SS-14 by day 12, but not by day 8, P19 neurons. The results thus demonstrate that capacities to convert and store neuropeptides can be established before coupling of stimulus-secretion during neuronal differentiation.

Coordinate regulation of neuroendocrine convertase PC2 and peptide 7B2 in P19 neurons

Convertases are proteases responsible for the bioactivation of many proteins and peptides having a potential role in ontogenesis. As a model to study regulation of convertases in embryo, we use the P19 embryonal carcinoma cell line, which can differentiate into various cell types. The expression of convertase PC2 and its specific binding peptide 7B2 are co-induced during neuronal differentiation of P19 cells. We investigated the possibility that expression of both proteins may be coregulated by T3 and dexamethasone, activators of nuclear receptors, isobutylmethylxanthine, and dibutyryl cAMP, activators of protein kinase A, and phorbol 12-myristate 13-acetate, an activator of protein kinase C. Western blotting results show that expression of PC2 and 7B2 can be upregulated by modulators of the protein kinases, and upregulation needs not be strictly stoichiometric.

Interleukin-1beta-induced alteration in a beta-cell phenotype can reduce cellular sensitivity to conditions that cause necrosis but not to cytokine-induced apoptosis.

Previous work has shown that interleukin-1beta (IL-1beta) alters protein expression in beta-cells. This alteration is associated with cell death in isolated rat islets but not in isolated rat beta-cells. We examined whether IL-1beta pretreatment of isolated beta-cells influences their sensitivity to toxic agents. After a 24-h culture with IL-1beta (30 U/ml), beta-cells exhibited a lower expression of the beta-cell-specific protein transcription factor pancreatic and duodenal homeobox gene (PDX)-1, glucose transporter GLUT2, and proinsulin convertase PC2, with a marked reduction (60-70%) in glucose-induced insulin production and selective sensitivity to the toxins alloxan (ALX) and streptozotocin (STZ). On the other hand, the cells presented an increased expression of Mn-superoxide dismutase, heat shock protein 70, inducible heme oxygenase, and inducible nitrite oxide synthase. This IL-1beta-induced alteration in beta-cell phenotype resulted in a reduced cellular sensitivity to the beta-cell-specific toxins ALX and STZ; the production of nontoxic conditions of nitric oxide (NO) also rendered the cells less susceptible to radical-induced damage. Exposure to IL-1beta can thus protect beta-cells against conditions that cause necrosis; however, it did not protect against apoptosis induced by the additional presence of interferon-gamma or tumor necrosis factor-alpha. Release of IL-1beta in the endocrine pancreas is thus not necessarily the cause of massive NO-dependent beta-cell destruction. On the contrary, IL-1beta may protect these cells against necrosis, though with a loss of their characteristic phenotype and homeostatic functions.

Extracellular matrix proteins increase the expression of pro-TRH and pro-protein convertase PC1 in fetal hypothalamic neurons in vitro

External clues for neuron development include extracellular matrix (ECM) molecules. To explore ECM influence on the early development of peptide phenotype in the CNS, we have compared pro-TRH levels in primary cultures of rat hypothalamic cells plated either on poly-lysine (PL) (control) or on PL plus one of various ECM molecules at 10 μg/ml. Fetal day 17 cells plated at a density of 1250/mm 2 were grown in a serum free medium made of Neurobasal medium supplemented with B27 (GIBCO). Cultures, consisting mainly of neurons, were analyzed at DIV 2. ECM proteins induced morphological effects in agreement with previously published studies. The amount of pro-TRH per dish, quantified by Western blotting, was increased to 275% for laminin, 191% for fibronectin and 173% for tenascin-C (control=100%); there was no effect of vitronectin. Laminin or fibronectin did not change pro-TRH mRNA or TRH levels but enhanced levels of the pro-protein convertase PC1 suggesting that the ECM molecules did regulate the translational status of pro-TRH. In conclusion, we have shown that some ECM proteins increased pro-TRH level in vitro; this may contribute to the enhancement of pro-TRH levels observed early in vivo in the hypothalamus.

Differential onset of expression of mRNAs encoding proopiomelanocortin, prohormone convertases 1 and 2, and granin family members during Xenopus laevis development

The production of peptide hormones through proteolytic cleavage of prohormones, e.g., proopiomelanocortin (POMC), involves a number of regulated secretory proteins, such as prohormone convertase PC1, PC2 and granin family members, that are co-expressed with the prohormone. Although the expression of these proteins has been well-studied in adult animals, data on their expression during development are limited. We used whole-mount in situ hybridization to visualize POMC mRNA expression in the intermediate and anterior pituitary of Xenopus tadpoles. A more sensitive analysis, namely semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) on total RNA isolated from Xenopus developmental stages, revealed that the expression of POMC, PC1 and PC2 mRNA commenced at stages 13 (neural plate stage), 15 (neural fold stage) and 19 (neural tube stage), respectively, with a gradual increase in their expression levels during further development. Surprisingly, and in contrast to what holds for POMC and the convertases, mRNAs for secretogranin II and III (SgII, SgIII) and 7B2 were not only expressed during neural development, but could already be detected in unfertilized mature oocytes, the first cleavage stages and in blastula-stage embryos. These granins are thus maternally present in Xenopus embryos suggesting that they may have a role during oogenesis and/or early embryonic development.

Long-term elevation of free fatty acids leads to delayed processing of proinsulin and prohormone convertases 2 and 3 in the pancreatic beta-cell line MIN6.

To explore the role of chronically elevated free fatty acids (FFAs) in the pathogenesis of the hyperproinsulinemia of type 2 diabetes, we have investigated the effect of FFAs on proinsulin processing and prohormone convertases PC2 and PC1/PC3 in MIN6 cells cultured in Dulbecco's modified Eagle's medium with or without 0.5 mmol/l FFA mixture (palmitic acid:oleic acid = 1:2). After 7 days of culture, the percent of proinsulin in FFA-exposed cells was increased (25.9 +/-0.3% intracellular and 75.4 +/- 1.2% in medium vs. 13.5 +/-0.2 and 56.2 +/- 4.1%, respectively, in control cells). The biosynthesis and secretion of proinsulin and insulin were analyzed by comparing the incorporation of [3H]Leu and [35S]Met. In pulse-chase studies, proinsulin-to-insulin conversion was inhibited, and proinsulin in the medium was increased by 50% after 3 h of chase, while insulin secretion was decreased by 50% after FFA exposure. Levels of cellular PC2 and PC3 analyzed by Western blotting were decreased by 23 and 15%, respectively. However, PC2, PC3, proinsulin, and 7B2 mRNA levels were not altered by FFA exposure. To test for an effect on the biosynthesis of PC2, PC3, proinsulin, and 7B2, a protein required for PC2 activation, MIN6 cells were labeled with [35S]Met for 10-15 min, followed by a prolonged chase. Most proPC2 was converted after 6 h of chase in control cells, but conversion was incomplete even after 6 h of chase in FFA-exposed MIN6 cells. Media from chase incubations showed that FFA-exposed cells secreted more proPC2 than controls. Similar inhibitory effects were noted on the processing of proPC3, proinsulin, and 7B2. In conclusion, prolonged exposure of beta-cells to FFAs may affect the biosynthesis and posttranslational processing of proinsulin, PC2, PC3, and 7B2, and thereby contribute to the hyperproinsulinemia of type 2 diabetes. The mechanism of inhibition of secretory granule processing by FFAs may be through changes in Ca2+ concentration, the pH in the secretory granules, and/or other factors that may influence the activation and function of the convertases.

Molecular cloning, characterization of cDNA, and distribution of mRNA encoding the frog prohormone convertase PC1

Molecular cloning, characterization of cDNA, and distribution of mRNA encoding the frog prohormone convertase PC1

Molecular cloning, characterization of cDNA, and distribution of mRNA encoding the frog prohormone convertase PC1

Prohormone convertases (PCs) are calcium-dependent serine endoproteases of the subtilisin/kexin family that play a key role in the posttranslational processing of precursors for biologically active peptides. In this study, we have characterized the cDNA encoding PC1 in the European green frog Rana ridibunda. A frog brain cDNA library was screened by using a heterologous probe at low stringency, and a 2.3-kb cDNA clone encoding PC1 was isolated. This cDNA encodes a 736-residue protein with a 26-amino-acid signal peptide. Comparative structural analysis revealed that frog PC1 exhibits a high degree of amino acid identity with its mammalian counterparts, in particular in the subtilisin-like catalytic domain. Northern blot analysis resolved two major transcripts of 3.0 kb and 5.0 kb that were expressed differentially in the brain and pituitary. In situ hybridization studies showed that, in the frog brain, the highest densities of PC1 mRNA are present in the amygdala, the hypothalamus, and the anterior preoptic area. High concentrations of PC1 mRNA also were found in the pars distalis and pars intermedia of the pituitary, whereas the pars nervosa was devoid of hybridization signal. The wide distribution of PC1 mRNA in the brain and pituitary suggests that, in frog, PC1 is involved in the processing of a number of hormone and neuropeptide precursors.

Homotypic Fusion of Immature Secretory Granules during Maturation in a Cell-free Assay

The biogenesis of secretory granules embodies several morphological and biochemical changes. In particular, in neuroendocrine cells maturation of secretory granules is characterized by an increase in size which has been proposed to reflect homotypic fusion of immature secretory granules (ISGs). Here we describe an assay that provides the first biochemical evidence for such a fusion event and allows us to analyze its regulation. The assay reconstitutes homotypic fusion between one population of ISGs containing a [35S]sulfate-labeled substrate, secretogranin II (SgII), and a second population containing the prohormone convertase PC2. Both substrate and enzyme are targeted exclusively to ISGs. Fusion is measured by quantification of a cleavage product of SgII produced by PC2. With this assay we show that fusion only occurs between ISGs and not between ISGs and MSGs, is temperature dependent, and requires ATP and GTP and cytosolic proteins. NSF (N-ethylmaleimide-sensitive fusion protein) is amongst the cytosolic proteins required, whereas we could not detect a requirement for p97. The ability to reconstitute ISG fusion in a cell-free assay is an important advance towards the identification of molecules involved in the maturation of secretory granules and will increase our understanding of this process.

Characterization of the cDNA encoding the prohormone convertase PC2 and localization of the mRNA in the brain of the frog Rana ridibunda

A number of precursors for neuropeptides have recently been cloned in amphibians, but little is known concerning the endoproteases responsible for the processing of these precursors. Here we report on the molecular cloning of the cDNA encoding the proprotein convertase PC2 and the distribution of the corresponding mRNA in the European green frog Rana ridibunda. The full cDNA structure (2125 bp) was obtained from the analysis of the PCR products combined with the sequence from a clone isolated from a frog pituitary cDNA library. The deduced amino acid sequence revealed that frog PC2 comprises 636 amino acid residues including a 22-residue signal peptide. RT-PCR analysis showed that PC2 is expressed not only in the brain and pituitary but also in various peripheral organs including the pancreas, stomach, intestine, liver, kidney and testis. In situ hybridization histochemistry revealed that, in the central nervous system, PC2 mRNA is widely distributed, the highest concentrations being found in the pallium, the anterior preoptic area, the hypothalamus and the medial amygdala. High levels of PC2 mRNA were also detected in the intermediate lobe of the pituitary. The overall distribution of PC2 mRNA in the frog brain is consistent with its involvement in the processing of a number of neuropeptide and hormone precursors.

Synthesis and differentially regulated processing of proinsulin in developing chick pancreas, liver and neuroretina

Regulated preproinsulin gene expression in non-pancreatic tissues during development has been demonstrated in rodents, Xenopus and chicken. Little is known, however, about the synthesis and processing of the primary protein product, proinsulin, in comparison with these events in pancreas. Using specific antisera and immunocytochemistry, immunoblot and HPLC criteria, we characterize the differential processing of proinsulin in developing neuroretina, liver and pancreas. The chick embryo pancreas expresses the convertase PC2, and largely processes proinsulin to insulin. In contrast, little or no mature PC2 is present in embryonic liver and neuroretina and the (pro)insulin immunoactivity identified is predominantly proinsulin.

PC5-A-mediated Processing of Pro-neurotensin in Early Compartments of the Regulated Secretory Pathway of PC5-transfected PC12 Cells

Among the members of the proprotein convertase (PC) family, PC1 and PC2 have well established roles as prohormone convertases. Another good candidate for this role is PC5-A that has been shown to be present in the regulated secretory pathway of certain neuroendocrine tissues, but evidence that it can process prohormones is lacking. To determine whether PC5-A could function as a prohormone convertase and to compare its cleavage specificity with that of PC1 and PC2, we stably transfected the rat pheochromocytoma PC12 cell line with PC5-A and analyzed the biosynthesis and subcellular localization of the enzyme, as well as its ability to process pro-neurotensin/neuromedin N (pro-NT/NN) into active peptides. Our data showed that in transfected PC12 cells, PC5-A was converted from its 126-kDa precursor form into a 117-kDa mature form and, to a lesser extent, into a C-terminally truncated 65-kDa form of the 117-kDa product. Metabolic and immunochemical studies showed that PC5-A was sorted to early compartments of the regulated secretory pathway where it colocalized with immunoreactive NT. Furthermore, pro-NT/NN was processed in these compartments according to a pattern that differed from that previously described in PC1- and PC2-transfected PC12 cells. This pattern resembled that previously reported for pro-NT/NN processing in the adrenal medulla, a tissue known to express high levels of PC5-A. Altogether, these data demonstrate for the first time the ability of PC5-A to function as a prohormone convertase in the regulated secretory pathway and suggest a role for this enzyme in the physiological processing of pro-NT/NN.

The pro-protein convertase PC1 is induced in the transected sciatic nerve and is present in cultured Schwann cells: comparison with PC5, furin and PC7, implication in pro-BDNF processing

Injury of peripheral nerves induces expression of several pro-protein convertases (PCs) involved in processing of precursor proteins into their diverse active end-products. In this study, the focus was on convertase PC1 which, although undetectable in control nerves, is strongly induced in injured nerves. High concentrations of PC1 mRNA of 9.0, 5.5, 3.0, 2.5 and 1.6 kb were observed on day 4 post-lesion in proximal and distal segments. By in situ hybridization PC1 mRNA was detected in most of endoneurial cells, which were further identified by immunocytochemistry as myelin 2′,3′-cyclic nucleotide 3′-phosphodiesterase containing Schwann cells. PC1 mRNA and protein were also present in cultured Schwann cells also containing convertases PC5, furin and PC7 as well as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Mostly unprocessed pro-NGF of 35 kDa and pro-BDNF of 35 kDa were found on Western blotting of Schwann cells. Expression of exogenous neurotrophins by infection with vaccinia virus vector showed that mouse pro-NGF and rat pro-BDNF are cleaved intracellularly on smaller forms of 13.5 kDa NGF and 14 kDa BDNF. Infection experiments demonstrated that Schwann cells contain active processing enzymes. In conclusion, this work provides in vivo evidence of the presence of several PCs in the injured rat sciatic nerve and ex vivo in cultured Schwann cells.

Manipulation of disulfide bonds differentially affects the intracellular transport, sorting, and processing of neuroendocrine secretory proteins.

To investigate if the prevention of disulfide bond formation affects the intracellular transport, sorting, and processing of a distinct set of neuroendocrine proteins in the regulated secretory pathway, we have treated Xenopus intermediate pituitaries with the thiol-reducing agent dithiothreitol. Pulse-chase incubations in combination with immunoprecipitation analysis were used to monitor the fates of the prohormone proopiomelanocortin (POMC), prohormone convertase PC2 and its helper protein 7B2, as well as secretogranin III. Manipulation of the disulfide bonds in POMC and proPC2 blocked their transport to the trans-Golgi network and strongly inhibited their processing. Reduction of the single disulfide bond in 7B2 did not disturb its transport and cleavage, but caused its missorting to the constitutive secretory pathway. Moreover, the liaison between proPC2 and 7B2 was prevented. Dithiothreitol did not affect transport, sorting, and cleavage of secretogranin III, which lacks disulfide bonds. When the reducing agent was washed away, POMC processing, proPC2 maturation, and the association between proPC2 and 7B2 were reestablished. Collectively, our findings indicate that manipulation of disulfide bonds differentially affects the fates of neuroendocrine proteins during their transit through the secretory pathway.

Residues unique to the pro-hormone convertase PC2 modulate its autoactivation, binding to 7B2 and enzymatic activity

The prohormone convertase PC2 is one of the major subtilisin/kexin-like enzymes responsible for the formation of small bioactive peptides in neural and endocrine cells. This convertase is unique among the members of the subtilisin/kexin-like mammalian serine proteinase family in that it undergoes zymogen processing of its inactive precursor proPC2 late along the secretory pathway and requires the help of a PC2-specific binding protein known as 7B2. We hypothesized that some of these unique properties of PC2 are dictated by the presence of PC2-specific amino acids, which in the six other known mammalian convertases are otherwise conserved but distinct. Accordingly, six sites were identified within the catalytic segment of PC2. Herein we report on the site-directed mutagenesis of Tyr 194 and of the oxyanion hole Asp 309 and the consequences of such mutations on the cellular expression and enzyme activity of PC2. The data show that the Y194D mutation markedly increases the ex vivo ability of PC2 to process proopiomelanocortin (POMC) into β-endorphin in cells devoid of 7B2, e.g. BSC40 cells. In these cells, expression of native PC2 does not result in the secretion of measurable in vitro activity against a pentapeptide fluorogenic substrate. In contrast, secreted Y194D-PC2 exhibited significant enzymatic activity, even in the absence of 7B2. Based on co-immunoprecipitations and Western blots, binding assays indicate that Tyr 194 participates in the interaction of PC2 with 7B2, and that the oxyanion hole Asp 309 is critical for the binding of proPC2 with pro7B2.

Pro-neurotensin/Neuromedin N Expression and Processing in Human Colon Cancer Cell Lines

The regulatory peptide neurotensin NT has been proposed to exert an autocrine trophic effect on human colon cancers. In the present study, pro-neurotensin/neuromedin N (proNT/NN) expression and processing were investigated in 13 human colon cancer cell lines using a combination of radioimmunoassay and HPLC techniques. All 13 cell lines displayed low to moderate levels of proNT/NN ranging from 10 to 250 fmol/mg protein. However, only 6 (HCT8, LoVo, HT29, C119A, LS174T, and coloDM320) processed the precursor. Three of the latter (HCT8, LS174T, and coloDM320) were analysed in detail with regard to proNT/NN processing pattern and were found to produce NT and large precursor fragments ending with the NT or NN sequence. They had no detectable level of NN. Such a processing pattern resembles that generated by the prohormone convertase PC5. Northern and Western blot analysis of prohormone convertase expression in the 3 cell lines revealed that they were devoid of PC1 and PC2, whereas they all expressed PC5. These data indicate that proNT/NN is a good marker of human colon cancer cell lines while NT is found in only about half of the cell lines. They also suggest that, in addition to NT, several proNT/NN-derived products, possibly generated by PC5, might exert an autocrine positive effect on human colon cancer growth.