Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes.

Anterior axis duplication in Xenopus induced by the over-expression of the cadherin-binding protein plakoglobin.

Plakoglobin interacts with both classical and desmosomal cadherins. It is closely related to Drosophila aramadillo (arm) gene product; arm acts in the wingless (wg)-signaling pathway to establish segment polarity. In Xenopus, homologs of wg--i.e., wnts, can produce anterior axis duplications by inducing dorsal mesoderm. Studies in Drosophila suggest that wnt acts by increasing the level of cytoplasmic armadillo protein (arm). To test whether simply increasing the level of plakoglobin mimics the effects of exogenous wnts in Xenopus, we injected fertilized eggs with RNA encoding an epitope-tagged form of plakoglobin; this induced both early radial gastrulation and anterior axis duplication. Exogenous plakoglobin accumulates in the nuclei of embryonic cells. Plakoglobin binds to the tail domain of the desmosomal cadherin desmoglein 1. When RNA encoding the tail domain of desmoglein was coinjected with plakoglobin RNA, both the dorsalizing effect and nuclear accumulation of plakoglobin were suppressed. Mutational analysis indicates that the central arm repeat region of plakoglobin is sufficient to induce axis duplication and that this polypeptide accumulates in the nuclei of embryonic cells. These data show that increased plakoglobin levels can, by themselves, generate the intracellular signals involved in the specification of dorsal mesoderm.

Phosphorylated and unphosphorylated forms of human single-stranded DNA-binding protein are equally active in simian virus 40 DNA replication and in nucleotide excision repair.

The trimeric human single-stranded DNA-binding protein (HSSB; also called RP-A) plays an essential role in DNA replication, nucleotide excision repair, and homologous DNA recombination. The p34 subunit of HSSB is phosphorylated at the G1/S boundary of the cell cycle or upon exposure of cells to DNA damage-inducing agents including ionizing and UV radiation. We have previously shown that the phosphorylation of p34 is catalyzed by both cyclin-dependent kinase-cyclin A complex and DNA-dependent protein kinase. In this study, we investigated the effect of phosphorylation of p34 by these kinases on the replication and repair function of HSSB. We observed no significant difference with the unphosphorylated and phosphorylated forms of HSSB in the simian virus 40 DNA replication or nucleotide excision repair systems reconstituted with purified proteins. The phosphorylation status of the p34 subunit of HSSB was unchanged during the reactions. We suggest that the phosphorylated HSSB has no direct effect on the basic mechanism of DNA replication and nucleotide excision repair reactions in vitro, although we cannot exclude a role of p34 phosphorylation in modulating HSSB function in vivo through a yet poorly understood control pathway in the cellular response to DNA damage and replication.

Alterazione della proteostasi a livello del reticolo endoplasmatico: un potenziale target terapeutico nel carcinoma prostatico

Nel sesso maschile il carcinoma della prostata (CaP) è la neoplasia più frequente ed è tra le prime cause di morte per tumore. Ad oggi, sono disponibili diverse strategie terapeutiche per il trattamento del CaP, ma, come comprovato dall’ancora alta mortalità, spesso queste sono inefficaci, a causa soprattutto dello sviluppo di fenomeni di resistenza da parte delle cellule tumorali. La ricerca si sta quindi focalizzando sulla caratterizzazione di tali meccanismi di resistenza e, allo stesso tempo, sull’individuazione di combinazioni terapeutiche che siano più efficaci e capaci di superare queste resistenze. Le cellule tumorali sono fortemente dipendenti dai meccanismi connessi con l’omeostasi proteica (proteostasi), in quanto sono sottoposte a numerosi stress ambientali (ipossia, carenza di nutrienti, esposizione a chemioterapici, ecc.) e ad un’aumentata attività trascrizionale, entrambi fattori che causano un accumulo intracellulare di proteine anomale e/o mal ripiegate, le quali possono risultare dannose per la cellula e vanno quindi riparate o eliminate efficientemente. La cellula ha sviluppato diversi sistemi di controllo di qualità delle proteine, tra cui gli chaperon molecolari, il sistema di degradazione associato al reticolo endoplasmatico (ERAD), il sistema di risposta alle proteine non ripiegate (UPR) e i sistemi di degradazione come il proteasoma e l’autofagia. Uno dei possibili bersagli in cellule tumorali secretorie, come quelle del CaP, è rappresentato dal reticolo endoplasmatico (RE), organello intracellulare deputato alla sintesi, al ripiegamento e alle modificazioni post-traduzionali delle proteine di membrana e secrete. Alterazioni della protestasi a livello del RE inducono l’UPR, che svolge una duplice funzione nella cellula: primariamente funge da meccanismo omeostatico e di sopravvivenza, ma, quando l’omeostasi non è più ripristinabile e lo stimolo di attivazione dell’UPR cronicizza, può attivare vie di segnalazione che conducono alla morte cellulare programmata. La bivalenza, tipica dell’UPR, lo rende un bersaglio particolarmente interessante per promuovere la morte delle cellule tumorali: si può, infatti, sfruttare da una parte l’inibizione di componenti dell’UPR per abrogare i meccanismi adattativi e di sopravvivenza e dall’altra si può favorire il sovraccarico dell’UPR con conseguente induzione della via pro-apoptotica. Le catechine del tè verde sono composti polifenolici estratti dalle foglie di Camellia sinesis che possiedono comprovati effetti antitumorali: inibiscono la proliferazione, inducono la morte di cellule neoplastiche e riducono l’angiogenesi, l’invasione e la metastatizzazione di diversi tipi tumorali, tra cui il CaP. Diversi studi hanno osservato come il RE sia uno dei bersagli molecolari delle catechine del tè verde. In particolare, recenti studi del nostro gruppo di ricerca hanno messo in evidenza come il Polyphenon E (estratto standardizzato di catechine del tè verde) sia in grado, in modelli animali di CaP, di causare un’alterazione strutturale del RE e del Golgi, un deficit del processamento delle proteine secretorie e la conseguente induzione di uno stato di stress del RE, il quale causa a sua volta l’attivazione delle vie di segnalazione dell’UPR. Nel presente studio su due diverse linee cellulari di CaP (LNCaP e DU145) e in un nostro precedente studio su altre due linee cellulari (PNT1a e PC3) è stato confermato che il Polyphenon E è capace di indurre lo stress del RE e di determinare l’attivazione delle vie di segnalazione dell’UPR, le quali possono fungere da meccanismo di sopravvivenza, ma anche contribuire a favorire la morte cellulare indotta dalle catechine del tè verde (come nel caso delle PC3). Considerati questi effetti delle catechine del tè verde in qualità di induttori dell’UPR, abbiamo ipotizzato che la combinazione di questi polifenoli bioattivi e degli inibitori del proteasoma, anch’essi noti attivatori dell’UPR, potesse comportare un aggravamento dell’UPR stesso tale da innescare meccanismi molecolari di morte cellulare programmata. Abbiamo quindi studiato l’effetto di tale combinazione in cellule PC3 trattate con epigallocatechina-3-gallato (EGCG, la principale tra le catechine del tè verde) e due diversi inibitori del proteasoma, il bortezomib (BZM) e l’MG132. I risultati hanno dimostrato, diversamente da quanto ipotizzato, che l’EGCG quando associato agli inibitori del proteasoma non produce effetti sinergici, ma che anzi, quando viene addizionato al BZM, causa una risposta simil-antagonistica: si osserva infatti una riduzione della citotossicità e dell’effetto inibitorio sul proteasoma (accumulo di proteine poliubiquitinate) indotti dal BZM, inoltre anche l’induzione dell’UPR (aumento di GRP78, p-eIF2α, CHOP) risulta ridotta nelle cellule trattate con la combinazione di EGCG e BZM rispetto alle cellule trattate col solo BZM. Gli stessi effetti non si osservano invece nelle cellule PC3 trattate con l’EGCG in associazione con l’MG132, dove non si registra alcuna variazione dei parametri di vitalità cellulare e dei marcatori di inibizione del proteasoma e di UPR (rispetto a quelli osservati nel singolo trattamento con MG132). Essendo l’autofagia un meccanismo compensativo che si attiva in seguito all’inibizione del proteasoma o allo stress del RE, abbiamo valutato che ruolo potesse avere tale meccanismo nella risposta simil-antagonistica osservata in seguito al co-trattamento con EGCG e BZM. I nostri risultati hanno evidenziato, in cellule trattate con BZM, l’attivazione di un flusso autofagico che si intensifica quando viene addizionato l’EGCG. Tramite l’inibizione dell’autofagia mediante co-somministrazione di clorochina, è stato possibile stabilire che l’autofagia indotta dall’EGCG favorisce la sopravvivenza delle cellule sottoposte al trattamento combinato tramite la riduzione dell’UPR. Queste evidenze ci portano a concludere che per il trattamento del CaP è sconsigliabile associare le catechine del tè verde con il BZM e che in futuri studi di combinazione di questi polifenoli con composti antitumorali sarà importante valutare il ruolo dell’autofagia come possibile meccanismo di resistenza.

RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. it operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.

Functional identity of receptors for proteolysis-inducing factor on human and murine skeletal muscle

Background: Cachexia in both mice and humans is associated with tumour production of a sulphated glycoprotein called proteolysis-inducing factor (PIF). In mice PIF binds with high affinity to a surface receptor in skeletal muscle, but little is known about the human receptor. This study compares the human PIF receptor with the murine. Methods: Human PIF was isolated from the G361 melanoma and murine PIF from the MAC16 colon adenocarcinoma. The human PIF receptor was isolated from human skeletal muscle myotubes. Protein synthesis and degradation induced by human and murine PIF was studied in human and murine skeletal muscle myotubes. Results: Both the human and murine PIF receptors showed the same immunoreactivity and Mr 40 000. Both murine and human PIF inhibited total protein synthesis and stimulated protein degradation in human and murine myotubes to about the same extent, and this was attenuated by a rabbit polyclonal antibody to the murine PIF receptor, but not by a non-specific rabbit antibody. Both murine and human PIF increased the activity of the ubiquitin-proteasome pathway in both human and murine myotubes, as evidenced by an increased 'chymotrypsin-like' enzyme activity, protein expression of the 20S and 19S proteasome subunits, and increased expression of the ubiquitin ligases MuRF1 and MAFbx, and this was also attenuated by the anti-mouse PIF receptor antibody. Conclusions: These results suggest that the murine and human PIF receptors are identical. © 2014 Cancer Research UK.

Identification and characterization of a membrane receptor for proteolysis-inducing factor on skeletal muscle

Proteolysis-inducing factor (PIF) is a sulfated glycoprotein produced by cachexia-inducing tumors, which induces atrophy of skeletal muscle. PIF has been shown to bind specifically with high affinity (Kd, in nanomolar) to sarcolemma membranes from skeletal muscle of both the mouse and the pig, as well as murine myoblasts and a human muscle cell line. Ligand binding was abolished after enzymatic deglycosylation, suggesting that binding was mediated through the oligosaccharide chains in PIF. Chondroitin sulfate, but not heparan or dermatan sulfate, showed competitive inhibition (Kd, 1.1 × 10-7 mol/L) of binding of PIF to the receptor, suggesting an interaction with the sulfated oligosaccharide chains. Ligand blotting of [ 35S]PIF to triton solublized membranes from C2C 12 cells provided evidence for a binding protein of apparent M r of ∼40,000. Amino acid sequence analysis showed the PIF receptor to be a DING protein. Antisera reactive to a 19mer from the N-terminal amino acid residues of the binding protein attenuated protein degradation and activation of the ubiquitin-proteasome pathway induced by PIF in murine myotubes. In addition, the antisera was highly effective in attenuating the decrease in body weight in mice bearing the MAC16 tumor, with a significant increase in muscle wet weight due to an increase in the rate of protein synthesis, together with a reduction in protein degradation through attenuation of the increased proteasome expression and activity. These results confirm that the PIF binding protein has a functional role in muscle protein atrophy in cachexia and that it represents a potential new therapeutic target. ©2007 American Association for Cancer Research.

Signaling pathways initiated by beta-hydroxy-beta-methylbutyrate to attenuate the depression of protein synthesis in skeletal muscle in response to cachectic stimuli

To investigate the mechanism by which beta-hydroxy-beta-methylbutyrate (HMB) attenuates the depression of protein synthesis in the skeletal muscle of cachectic mice, a study has been carried out in murine myotubes in the presence of proteolysis-inducing factor (PIF). PIF inhibited protein synthesis by 50% within 4 h, and this was effectively attenuated by HMB (25-50 muM). HMB (50 muM) alone stimulated protein synthesis, and this was attenuated by rapamycin (27 nM), an inhibitor of mammalian target of rapamycin (mTOR). Further evidence for an involvement of this pathway was shown by an increased phosphorylation of mTOR, the 70-kDa ribosomal S6 kinase (p70(S6k)), and initiation factor 4E-binding protein (4E-BP1) and an increased association of eukaryotic initiation factor 2 (eIF4E) with eIF4G. PIF alone induced a transient (1-2 h) stimulation of phosphorylation of mTOR and p70(S6k). However, in the presence of HMB, phosphorylation of mTOR, p70(S6k), and 4E-BP1 was increased, and inactive 4E-BP1-eIF4E complex was reduced, whereas the active eIF4G.eIF4E complex was increased, suggesting continual stimulation of protein synthesis. HMB alone reduced phosphorylation of elongation factor 2, but this effect was not seen in the presence of PIF. PIF induced autophosphorylation of the double-strand RNA-dependent protein kinase (PKR), leading to phosphorylation of eIF2 on the alpha-subunit, which would inhibit protein synthesis. However, in the presence of HMB, phosphorylation of PKR and eIF2alpha was attenuated, and this was also observed in skeletal muscle of cachectic mice administered HMB (0.25 g/kg). These results suggest that HMB attenuates the depression of protein synthesis by PIF in myotubes through multiple mechanisms.

Skeletal muscle atrophy, a link between depression of protein synthesis and increase in degradation

Both proteolysis-inducing factor (PIF) and angiotensin II have been shown to produce a depression in protein synthesis in murine myotubes concomitant with an increased phosphorylation of eukaryotic initiation factor 2 (eIF2α). Both PIF and angiotensin II were shown to induce autophosphorylation of the RNA-dependent protein kinase (PKR), and an inhibitor of this enzyme completely attenuated the depression in protein synthesis and prevented the induction of eIF2α phosphorylation. The PKR inhibitor also completely attenuated the increase in protein degradation induced by PIF and angiotensin II and prevented the increase in proteasome expression and activity. To confirm these results myotubes were transfected with plasmids that express either wild-type PKR, or a catalytically inactive PKR variant, PKRΔ6. Myotubes expressing PKRΔ6 showed no increase in eIF2α phosphorylation in response to PIF or angiotensin II, no depression in protein synthesis, and no increase in protein degradation or increase in proteasome expression. Induction of the ubiquitin-proteasome pathway by PIF and angiotensin II has been linked to activation of the transcription factor nuclear factor-κB (NF-κB). Inhibition of PKR prevented nuclear migration of NF-κB in response to both PIF and angiotensin II, by preventing degradation of the inhibitor protein I-κB. Phosphorylation of PKR and eIF2α was also significantly increased in the gastrocnemius muscle of weight losing mice bearing the MAC16 tumor, suggesting that a similar process may be operative in cancer cachexia. These results provide a link between the depression of protein synthesis in skeletal muscle and the increase in protein degradation. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Changes in nucleic acid and protein levels in atrophying skeletal muscle in cancer cachexia

Background: To investigate factors responsible for muscle loss in cachexia changes in nucleic acid and protein levels have been determined and compared with those induced by a tumour-produced cachectic factor, proteolysis-inducing factor (PIF). Materials and Methods: Mice were transplanted with the MAC16 tumour, while non-tumour bearing mice received PIF (1.5 mg/kg; i.v.) over a 24 h period. Results: There was an exponential decrease in RNA and protein in gastrocnemius muscle with weight loss without an effect on the DNA content. Levels of myosin followed the decrease in total protein, while actin levels remained constant. There was also a significant loss of protein from soleus muscle and spleen, but not from heart, liver and kidney. PIF also produced a significant loss of RNA and protein in spleen and reduced the protein content of soleus muscle. Conclusion: This suggests that PIF may be responsible for changes in protein and RNA content of tissues with the development of cachexia.

Tumor-host interactions

A number of malignant tumors interact with the host to cause a syndrome of cachexia, characterized by extensive loss of adipose tissue and skeletal muscle mass, but with preservation of proteins in visceral tissues. Although anorexia is frequently present, the body composition changes in cancer cachexia cannot be explained by nutritional deprivation alone. Loss of skeletal muscle mass is a result of depression in protein synthesis and an increase in protein degradation. The main degradative pathway that has been found to have increased expression and activity in the skeletal muscle of cachectic patients is the ubiquitin-proteasome proteolytic pathway. Cachexia-inducing tumors produce catabolic factors such as proteolysis-inducing factor (PIF), a 24 kDa sulfated glycoprotein, which inhibit protein synthesis and stimulate degradation of intracellular proteins in skeletal muscle by inducing an increased expression of regulatory components of the ubiquitin-proteasome proteolytic pathway. While the oligosaccharide chains in PIF are required to initiate protein degradation the central polypeptide core may act as a growth and survival factor. Only cachexia-inducing tumors are capable of elaborating fully glycosylated PIF, and the selectivity of production possibly rests with the acquisition of the necessary glycosylating enzymes, rather than expressing the gene for the polypeptide core. Loss of adipose tissue is probably the result of an increase in catabolism rather than a defect in anabolism. A lipid mobilizing factor (LMF), identical with the plasma protein Zn-α2-glycoprotein (ZAG) is found in the urine of cachectic cancer patients and is produced by tumors causing a decrease in carcass lipid. LMF causes triglyceride hydrolysis in adipose tissue through a cyclic AMP-mediated process by interaction with a β3-adrenoreceptor. Thus, by producing circulating factors certain malignant tumors are able to interfere with host metabolism even without metastasis to that particular site. © 2004 Wiley-Liss, Inc.

Effect of eicosapentaenoic acid, protein and amino acids on protein synthesis and degradation in skeletal muscle of cachectic mice

Atrophy of skeletal muscle reduces both the quality and quantity of life of patients with cancer cachexia. Loss of muscle mass is thought to arise from a reduction in protein synthesis combined with an enhanced rate of protein degradation, and few treatments are available to counteract this process. Eicosapentaenoic acid (EPA) has been shown to attenuate the enhanced protein degradation, but to have no effect on protein synthesis. This study examines the effect of EPA combined with a protein and amino-acid supplementation on protein synthesis and degradation in gastrocnemius muscle of mice bearing the cachexia-inducing MAC16 tumour. Muscles from cachectic mice showed an 80% reduction in protein synthesis and about a 50-fold increase in protein degradation compared with muscles from nontumour-bearing mice of the same age and weight. Treatment with EPA (1 g kg-1) daily reduced protein degradation by 88%, but had no effect on protein synthesis. Combination of EPA with casein (5.35 g kg-1) also had no effect on protein synthesis, but when combined with the amino acids leucine, arginine and methionine there was almost a doubling of protein synthesis. The addition of carbohydrate (10.7 g kg-1) to stimulate insulin release had no additional effect. The combination involving the amino acids produced almost a doubling of the ratio of protein synthesis to protein degradation in gastrocnemius muscle over that of EPA alone. No treatment had a significant effect on tumour growth rate, but the inclusion of amino acids had a more significant effect on weight loss induced by the MAC16 tumour than that of EPA alone. The results suggest that combination therapy of cancer cachexia involving both inhibition of the enhanced protein degradation and stimulation of the reduced protein synthesis may be more effective than either treatment alone. © 2004 Cancer Research UK.

Signal transduction pathways involved in proteolysis-inducing factor induced proteasome expression in murine myotubes

The proteolysis-inducing factor (PIF) is produced by cachexia-inducing tumours and initiates protein catabolism in skeletal muscle. The potential signalling pathways linking the release of arachidonic acid (AA) from membrane phospholipids with increased expression of the ubiquitin-proteasome proteolytic pathway by PIF has been studied using C2C12 murine myotubes as a surrogate model of skeletal muscle. The induction of proteasome activity and protein degradation by PIF was blocked by quinacrine, a nonspecific phospholipase A2 (PLA2) inhibitor and trifluroacetyl AA, an inhibitor of cytosolic PLA2. PIF was shown to increase the expression of calcium-independent cytosolic PLA2, determined by Western blotting, at the same concentrations as those inducing maximal expression of 20S proteasome α-subunits and protein degradation. In addition, both U-73122, which inhibits agonist-induced phospholipase C (PLC) activation and D609, a specific inhibitor of phosphatidylcholine-specific PLC also inhibited PIF-induced proteasome activity. This suggests that both PLA 2 and PLC are involved in the release of AA in response to PIF, and that this is important in the induction of proteasome expression. The two tyrosine kinase inhibitors genistein and tryphostin A23 also attenuated PIF-induced proteasome expression, implicating tyrosine kinase in this process. PIF induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) at the same concentrations as that inducing proteasome expression, and the effect was blocked by PD98059, an inhibitor of MAPK kinase, as was also the induction of proteasome expression, suggesting a role for MAPK activation in PIF-induced proteasome expression. © 2003 Cancer Research UK.

Increased expression of the ubiquitin-proteasome pathway in murine myotubes by proteolysis-inducing factor (PIF) is associated with activation of the transcription factor NF-kappa B

Proteolysis-inducing factor (PIF), isolated from a cachexia-inducing murine tumour, has been shown to stimulate protein breakdown in C 2C12 myotubes. The effect was attenuated by the specific proteasome inhibitor lactacystin and there was an elevation of proteasome 'chymotrypsin-like' enzyme activity and expression of 205 proteasome α-subunits at concentrations of PIF between 2 and 16 nM. Higher concentrations of PIF had no effect. The action of PIF was attenuated by eicosapentaenoic acid (EPA) (50 μM). At a concentration of 4 nM, PIF induced a transient decrease in IκBα levels after 30 min incubation, while no effect was seen at 20 nM PIF. The level of IκBα, an NF-κB inhibitory protein, returned to normal after 60 min. Depletion of IκBα from the cytosol was not seen in myotubes pretreated with EPA, suggesting that the NF-κB/IκB complex was stabilised. At concentrations between 2 and 8 nM, PIF stimulated an increased nuclear migration of NF-κB, which was not seen in myotubes pretreated with EPA. The PIF-induced increase in chymotrypsin-like enzyme activity was also attenuated by the NF-κB inhibitor peptide SN50, suggesting that NF-κB may be involved in the PIF-induced increase in proteasome expression. The results further suggest that EPA may attenuate protein degradation induced by PIF, at least partly, by preventing NF-κB accumulation in the nucleus. © 2003 Cancer Research UK.

Expression of the human cachexia-associated protein (HCAP) in prostate cancer and in a prostate cancer animal model of cachexia

Prostate cancer (CaP) patients with disseminated disease often suffer from severe cachexia, which contributes to mortality in advanced cancer. Human cachexia-associated protein (HCAP) was recently identified from a breast cancer library based on the available 20-amino acid sequence of proteolysis-inducing factor (PIF), which is a highly active cachectic factor isolated from mouse colon adenocarcinoma MAC16. Herein, we investigated the expression of HCAP in CaP and its potential involvement in CaP-associated cachexia. HCAP mRNA was detected in CaP cell lines, in primary CaP tissues and in its osseous metastases. In situ hybridization showed HCAP mRNA to be localized only in the epithelial cells in CaP tissues, in the metastatic foci in bone, liver and lymph node, but not in the stromal cells or in normal prostate tissues. HCAP protein was detected in 9 of 14 CaP metastases but not in normal prostate tissues from cadaveric donors or patients with organ-confined tumors. Our Western blot analysis revealed that HCAP was present in 9 of 19 urine specimens from cachectic CaP patients but not in 19 urine samples of noncachectic patients. HCAP mRNA and protein were also detected in LuCaP 35 and PC-3M xenografts from our cachectic animal models. Our results demonstrated that human CaP cells express HCAP and the expression of HCAP is associated with the progression of CaP and the development of CaP cachexia. © 2003 Wiley-Liss, Inc.