RÉSUMÉ
Due to the complex morphology of the prostate, it was hypothesized that precision-cut tissue slices from human prostate would provide a unique in vitro model. Precision-cut slices were generated from zones of human prostate and their viability was assessed under conditions of different media for up to 120 h. Slices were also exposed to several concentrations of CdCI2, which was used as a model toxicant. Maintenance of both stromal and epithelial cells was noted; however, there was a gradual loss of luminal epithelial cells when the medium was not supplemented with dihydrotestosterone (DHT). Minimal leakage of lactate dehydrogenase occurred throughout the incubation. Prostate-specific antigen (PSA) was detected in the medium at all time points, although the rates of secretion fell over time. There was a loss of PSA-positive cells when the medium was not supplemented with DHT, consistent with a loss of luminal cells, whereas PSA-positive cells were maintained in the DHT-supplemented media. A proliferation of basal cells was observed in the presence of media containing 10% fetal bovine serum. Exposure of slices to CdCl2 demonstrated a dose-response effect ranging from proliferation to complete cellular necrosis. Given the retention of stromal-epithelial interactions and the use of acquired human tissue, prostate slices represent a unique in vitro model for investigating human prostate pathobiology.
Sujet(s)
Prostate/cytologie , Chlorure de cadmium/toxicité , Division cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/effets des médicaments et des substances chimiques , Techniques de culture , Humains , Mâle , Modèles biologiques , Prostate/effets des médicaments et des substances chimiques , Prostate/métabolisme , Antigène spécifique de la prostate/métabolismeRÉSUMÉ
Matrilysin is a matrix metalloprotease (MMP) overexpressed in a number of cancers including skin, head and neck squamous cell carcinomas, and prostate and colon adenocarcinomas. Matrilysin has been shown to play a role in the degradation of the basement membrane that separates epithelium from stroma allowing tumor cells to intravasate into the bloodstream and metastasize. Here, we show that an oral squamous cell carcinoma cell line (SCC-25) expresses low levels of promatrilysin when cultured alone. However, when SCC-25 cells are cocultured with human foreskin fibroblasts (HFF), there is a 40-fold induction of promatrilysin expression. We tested whether this induction of promatrilysin expression was due to the release of paracrine factors, cell-cell interactions, or cell-matrix interactions. Our results indicate induced promatrilysin expression is the result of both cell-cell and cell-matrix interactions. We demonstrate that beta1 integrins as well as cadherins, specifically N-cadherin and E-cadherin, are involved in the induction of promatrilysin expression. Our results are of general interest in relation to the regulation of MMP expression through cell surface receptor regulation. Further investigation may lead to the identification of novel targets for suppression of invasion and metastasis in oral tumors.
Sujet(s)
Cadhérines/métabolisme , Carcinome épidermoïde , Derme/cytologie , Antigènes CD29/métabolisme , Matrix metalloproteinase 7/génétique , Tumeurs de la bouche , Technique de Western , Cadhérines/analyse , Cadhérines/génétique , Calcium/métabolisme , Communication cellulaire/physiologie , Chélateurs/pharmacologie , Techniques de coculture , Acide egtazique/pharmacologie , Proenzymes/génétique , Matrice extracellulaire/physiologie , Fibroblastes/cytologie , Fibroblastes/métabolisme , Régulation de l'expression des gènes codant pour des enzymes/effets des médicaments et des substances chimiques , Régulation de l'expression des gènes codant pour des enzymes/physiologie , Régulation de l'expression des gènes tumoraux/effets des médicaments et des substances chimiques , Régulation de l'expression des gènes tumoraux/physiologie , Humains , Metalloendopeptidases/génétique , Peptides/pharmacologie , Cellules cancéreuses en cultureRÉSUMÉ
BACKGROUND: The generation of prostatic cell lines provides in vitro models for experimental studies of the pathogenesis of prostate carcinoma. Therefore, we established and characterized a new human prostate epithelial cell line, PEAZ-1 (prostate epithelial Arizona-1). METHODS: The PEAZ-1 cells were grown from a primary human prostate carcinoma specimen obtained from radical prostatectomy. The isolated cells were characterized by immunobiochemistry, immunohistochemistry, and tumorigenicity studies. RESULTS: PEAZ-1 cells are near diploid, tumorigenic, and androgen independent for cell growth. PEAZ-1 cells express N-cadherin, alpha- and beta-catenins, and p120 at cell-cell contacts, cytoplasmic laminin 5, vinculin, paxillin, and phosphotyrosine at focal adhesions, vimentin, and cytokeratins 8 and 18. They do not express plakoglobin, E-cadherin, and PSA, and do not form desmosomes and hemidesomomes. PEAZ-1 respond to ocadaic acid, a pro-apoptotic agent, by expression of p53. CONCLUSIONS: PEAZ-1 cells is a human prostate cancer cell line that has a number of mesenchymal characteristics.
Sujet(s)
Tumeurs de la prostate/anatomopathologie , Cellules cancéreuses en culture , Androgènes/pharmacologie , Cadhérines/pharmacologie , Régulation de l'expression des gènes tumoraux , Humains , Immunohistochimie , Intégrines/analyse , Mâle , Prostatectomie , Manipulation d'échantillonsRÉSUMÉ
The alpha(6) integrin is a 140-kDa (nonreduced) laminin receptor. We have identified a novel 70-kDa (nonreduced) form of the alpha(6) integrin called alpha(6)p for the latin word parvus, meaning small. The variant was immunoprecipitated from human cells using four different alpha(6)-specific monoclonal antibodies but not with alpha(3) or alpha(5) antibodies. The alpha(6)p integrin contained identical amino acid sequences within exons 13--25, corresponding to the extracellular "stalk region" and the cytoplasmic tail of the alpha(6) integrin. The light chains of alpha(6) and alpha(6)p were identical as judged by alpha(6)A-specific antibodies and electrophoretic properties. The alpha(6)p variant paired with either beta(1) or beta(4) subunits and was retained on the cell surface three times longer than alpha(6). Reverse transcription/polymerase chain reaction analysis revealed a single polymerase chain reaction product. The alpha(6)p variant was found in human prostate (DU145H, LnCaP, PC3) and colon (SW480) cancer cell lines but not in normal prostate (PrEC), breast cancer (MCF-7), or lung cancer (H69) cell lines or a variant of a prostate carcinoma cell line (PC3-N). Protein levels of alpha(6)p increased 3-fold during calcium-induced terminal differentiation in a normal mouse keratinocyte model system. A novel form of the alpha(6) integrin exists on cell surfaces that contains a dramatically altered extracellular domain.
Sujet(s)
Intégrines/analyse , Séquence d'acides aminés , Humains , Intégrines/composition chimique , Intégrines/génétique , Données de séquences moléculaires , Spécificité d'organeRÉSUMÉ
Human tumor cell progression and metastasis are partially dependent on the ability of a tumor cell to adhere to the proteins of the extracellular matrix (ECM) and survive at the distant location. Six novel D-amino acid-containing peptides were analyzed for their ability to adhere to human prostate tumor cells, support tumor cell adhesion, and inhibit tumor cell adhesion to ECM proteins or human dermal fibroblasts. Of these, two peptides called RZ-3 (kmviywkag) and HYD-1 (kikmviswkg) bound to tumor cell surfaces and compared favorably with the previously reported AG-73 (RKRLQVQLSIRT) L-amino acid peptide, as determined by fluorescence-activated cell sorting analysis. A scrambled peptide derivative of HYD-1, called HYDS-1 (wiksmkivkg), was not active. The RZ-3, HYD-1, and AG-73 peptides supported maximal cancer cell adhesion at 5 microg, 10 microg, and 50 microg/well, respectively. The ECM proteins fibronectin, laminin 1, and collagen IV supported maximal cell adhesion at 1 microg, >10 microg, and 50 microg/well, respectively. Prostate tumor cell adhesion to immobilized RZ-3 and HYD-1 peptides was inhibited by alpha2-6- and beta1-integrin-blocking antibodies. Conversely, tumor cell adhesion to a beta1-integrin-specific antibody was blocked by both RZ-3 and HYD-1. Epithelial cell adhesion to dermal fibroblasts was inhibited by HYD-1 and unaffected by the scrambled peptide, HYDS-1. Cell adhesion to immobilized peptides was unaffected by EDTA. The soluble RZ-3 and HYD-1 peptides inhibited tumor cell adhesion to each of the immobilized four ECM proteins (1.0 microg/well) in a time- and concentration-dependent manner. The IC(50) of the RZ-3 peptide for blocking adhesion to fibronectin, laminin 1, laminin 5, and collagen IV was 2.4 microg, 1.8 microg, 4.6 microg, and 2.8 microg/well, respectively. The IC(50) of the HYD-1 peptide for blocking adhesion to fibronectin, laminin 1, laminin 5, and collagen IV was 6.9 microg, 5.7 microg, >10 microg, and 6.2 microg/well, respectively. Taken together, these results indicate that RZ-3 and HYD-1 are biologically active D-amino acid-containing peptides that can themselves support tumor cell adhesion and can inhibit tumor cell adhesion to immobilized ECM proteins or dermal fibroblasts.
Sujet(s)
Carcinome épidermoïde/anatomopathologie , Protéines de la matrice extracellulaire/métabolisme , Tumeurs de la bouche/anatomopathologie , Oligopeptides/pharmacologie , Tumeurs de la prostate/anatomopathologie , Carcinome épidermoïde/métabolisme , Adhérence cellulaire/effets des médicaments et des substances chimiques , Techniques de coculture , Collagène/métabolisme , Fibroblastes/cytologie , Fibroblastes/métabolisme , Fibronectines/métabolisme , Humains , Intégrines/métabolisme , Laminine/métabolisme , Laminine/pharmacologie , Mâle , Tumeurs de la bouche/métabolisme , Fragments peptidiques/pharmacologie , Tumeurs de la prostate/métabolisme , Peau/cytologie , Cellules cancéreuses en cultureRÉSUMÉ
Interactions between extracellular matrix (ECM) proteins and prostate carcinoma cells provide a dynamic model of prostate tumor progression. Previous work in our laboratory showed that laminin-5, an important member of a family of ECM glycoproteins expressed in the basal lamina, is lost in prostate carcinoma. Moreover, we showed that the receptor for laminin-5, the alpha6beta4 integrin, is altered in prostate tumors. However, the genes that laminin-5 potentially regulates and the significance of its loss of expression in prostate cancer are not known. We selected cDNA microarray as a comprehensive and systematic method for surveying and examining gene expression induced by laminin-5. To establish a definitive role for laminin-5 in prostate tumor progression and understand the significance of its loss of expression, we used a cDNA microarray containing 5289 human genes to detect perturbations of gene expression when DU145 prostate carcinoma cells interacted with purified laminin-5 after 0.5, 6, and 24 h. Triplicate experiments showed modulations of four, 61, and 14 genes at 0.5, 6, and 24 h, respectively. Genes associated with signal transduction, cell adhesion, the cell cycle, and cell structure were identified and validated by northern blot analysis. Protein expression was further assessed by immunohistochemistry. Mol. Carcinog. 30:119-129, 2001.
Sujet(s)
Molécules d'adhérence cellulaire/pharmacologie , Expression des gènes/effets des médicaments et des substances chimiques , Protéines tumorales/biosynthèse , Tumeurs de la prostate/génétique , Technique de Northern , ADN complémentaire/analyse , ADN tumoral/analyse , Analyse de profil d'expression de gènes , Humains , Techniques immunoenzymatiques , Mâle , Séquençage par oligonucléotides en batterie/méthodes , Tumeurs de la prostate/métabolisme , Cellules cancéreuses en culture/effets des médicaments et des substances chimiques ,RÉSUMÉ
BACKGROUND: The alpha6beta4 integrin and its ligand, laminin-5, are essential gene products for the maintenance and remodeling of a stratified epithelium. Apparent loss of polarized alpha6beta4 integrin and laminin-5 protein expression in invasive prostate cancer as compared to normal prostate glands is known to occur. It is unknown whether these alterations occur in prostatic intraepithelial neoplasia (PIN) lesions and whether this combined defect occurs in other epithelial cancers. METHODS: Human prostate tissues containing both normal, PIN, and cancerous regions and normal and cancer tissue from breast and colon were obtained at surgery and examined for beta4 integrin and laminin-5 using standard immunofluorescence staining methods. RESULTS: Both normal prostate glands and PIN lesions contain beta4 integrin and laminin-5. Prostate carcinoma was unique in that both beta4 integrin and laminin-5 expression was uniformly absent. In contrast, the beta4 integrin and its ligand, laminin-5 were detected in all of the colon carcinoma cases and in 60% of the breast carcinomas. CONCLUSIONS: The beta4 integrin and its ligand, laminin-5 are altered during the transition of PIN lesions to invasive prostate carcinoma. These data suggest the loss of these proteins during cancer progression. In both prostate and breast carcinoma, the normal expression pattern of the beta4 integrin and laminin-5 is interrupted, in contrast to the persistent beta4 integrin and laminin-5 expression detected in colon carcinoma.
Sujet(s)
Antigènes de surface/biosynthèse , Carcinomes/métabolisme , Molécules d'adhérence cellulaire/biosynthèse , Intégrines/biosynthèse , Tumeurs de la prostate/métabolisme , Tumeurs du sein/métabolisme , Carcinomes/anatomopathologie , Tumeurs du côlon/métabolisme , Évolution de la maladie , Humains , Intégrine alpha6 bêta4 , Mâle , Microscopie de fluorescence , Prostate/métabolisme , Tumeur intraépithéliale prostate/métabolisme , Tumeur intraépithéliale prostate/anatomopathologie , Tumeurs de la prostate/anatomopathologie , Coloration et marquage ,RÉSUMÉ
A simple method is described for the quantitation of phosphotyrosine signaling in human prostate cell cultures. The phosphotyrosine signals are observed by standard immunohistochemistry techniques, and the resulting digital images are analyzed using the Scion image software program. The signals within the cell adhesion sites are quantitated using the density slice and particle analysis features of the software. The immunohistochemistry results are compared with detection of phosphotyrosine signals using a standard Western blotting procedure with whole cell lysates. The resulting data is converted into graphs using the Sigma Plot Program. This method is illustrated using damage-induced signaling within cell adhesion sites after a low dose of ionizing radiation.
Sujet(s)
Phosphotyrosine/analyse , Prostate/composition chimique , Technique de Western , Adhérence cellulaire , Humains , Immunohistochimie , Mâle , Prostate/cytologieRÉSUMÉ
The integrin alpha6beta4, predominantly expressed on tissues of epithelial origin, is known to be variably expressed on carcinomas. The biochemical changes resulting in altered expression during tumor progression are unknown. We have analyzed the expression of alpha6beta4 in a multi-step mouse model of skin carcinogenesis representing normal keratinocyte, benign papilloma and malignant undifferentiated carcinoma. All cell lines expressed the alpha6 integrin exclusively as the alpha6beta4 integrin heterodimer. Analysis of this integrin by flow cytometry and immunoprecipitation of surface labeled proteins revealed that the undifferentiated carcinoma cells have an approximately 75% reduction in surface expression of the integrin as compared with the keratinocyte and papilloma cell lines. The alpha6beta4 integrin which remains expressed on the carcinoma cells is diffusely distributed in the membrane and has an approximately 2.5-fold increased biological turnover as compared with normal keratinocytes. The decreased biological half-life and the loss of polarized expression of alpha6beta4 on the carcinoma cells suggests an altered functional role for the alpha6beta4 integrin on carcinoma cells during tumor progression. These factors may contribute to the known supression of hemidesmosome structures and the increased migration phenotype associated with some epithelial carcinomas.
Sujet(s)
Antigènes de surface/métabolisme , Carcinomes/métabolisme , Régulation de l'expression des gènes tumoraux , Intégrines/métabolisme , Protéines membranaires/métabolisme , Protéines tumorales/métabolisme , Tumeurs cutanées/métabolisme , Animaux , Carcinomes/génétique , Adhérence cellulaire , Mouvement cellulaire , Polarité de la cellule , Évolution de la maladie , Cytométrie en flux , Technique d'immunofluorescence indirecte , Période , Intégrine alpha6 bêta4 , Kératinocytes/métabolisme , Souris , Souris de lignée BALB C , Papillome/génétique , Papillome/métabolisme , Tumeurs cutanées/génétique , Propriétés de surface , Cellules cancéreuses en cultureRÉSUMÉ
In human prostate adenocarcinoma, an association between loss of E-cadherin, increased Gleason score, and extracapsular dissemination has been observed. Further characterization of the E-cadherin/catenin phenotype of human prostate carcinoma cell lines showed loss of E-cadherin and expression of N-cadherin in poorly differentiated prostate carcinoma cell lines (PC-3N derived from PC-3, PC-3, and JCA1). We showed that N-cadherin is concentrated at sites of cell-cell contact in PC-3N cellular extensions. N-cadherin was also expressed in prostate stromal fibroblasts both in vitro and in prostate tissue. Co-cultures of prostate stromal fibroblasts and PC-3N cells showed the immunolocalization of N-cadherin in intercellular contacts. In addition, the isoform expression of the cadherin binding protein p120(ctn) differed in relation to the expression of E- versus N-cadherin by the prostate carcinoma cell lines. The p100 isoform was more highly expressed in E-cadherin-positive carcinoma cell lines, whereas p120 was predominantly expressed only in N-cadherin-positive prostate carcinoma cell lines and prostate stromal fibroblasts. The N-cadherin-positive carcinoma cell line, PC-3N, displayed aggressive invasion into the surface of the diaphragm muscle after intraperitoneal injection of SCID mice. The gain of N-cadherin and loss of E-cadherin by invasive prostate carcinoma cell lines suggests a progression from an epithelial to a mesenchymal phenotype, which may allow for their interaction with surrounding stromal fibroblasts and facilitate metastasis.
Sujet(s)
Adénocarcinome/métabolisme , Cadhérines/biosynthèse , Tumeurs de la prostate/métabolisme , Adénocarcinome/anatomopathologie , Animaux , Cadhérines/métabolisme , Cadhérines/physiologie , Protéines de transport/métabolisme , Caténines , Molécules d'adhérence cellulaire/métabolisme , Agrégation cellulaire/effets des médicaments et des substances chimiques , Cellules cultivées , Protéines du cytosquelette/métabolisme , Humains , Immunotransfert , Immunohistochimie , Mâle , Souris , Souris SCID , Invasion tumorale , Phosphoprotéines/métabolisme , Tumeurs de la prostate/anatomopathologie , ARN messager/biosynthèse , Cellules stromales/métabolisme , Cellules cancéreuses en culture ,RÉSUMÉ
Selection for in vitro drug resistance can result in a complex phenotype with more than one mechanism of resistance emerging concurrently or sequentially. We examined emerging mechanisms of drug resistance during selection with mitoxantrone in the human myeloma cell line 8226. A novel transport mechanism appeared early in the selection process that was associated with a 10-fold resistance to mitoxantrone in the 8226/MR4 cell line. The reduction in intracellular drug concentration was ATP-dependent and ouabain-insensitive. The 8226/MR4 cell line was 34-fold cross-resistant to the fluorescent aza-anthrapyrazole BBR 3390. The resistance to BBR 3390 coincided with a 50% reduction in intracellular drug concentration. Confocal microscopy using BBR 3390 revealed a 64% decrease in the nuclear:cytoplasmic ratio in the drug-resistant cell line. The reduction in intracellular drug concentration of both mitoxantrone and BBR 3390 was reversed by a novel chemosensitizing agent, fumitremorgin C. In contrast, fumitremorgin C had no effect on resistance to mitoxantrone or BBR 3390 in the P-glycoprotein-positive 8226/DOX6 cell line. Increasing the degree of resistance to mitoxantrone in the 8226 cell line from 10 to 37 times (8226/MR20) did not further reduce the intracellular drug concentration. However, the 8226/MR20 cell line exhibited 88 and 70% reductions in topoisomerase II beta and alpha expression, respectively, compared with the parental drug sensitive cell line. This decrease in topoisomerase expression and activity was not observed in the low-level drug-resistant, 8226/MR4 cell line. These data demonstrate that low-level mitoxantrone resistance is due to the presence of a novel, energy-dependent drug efflux pump similar to P-glycoprotein and multidrug resistance-associated protein. Reversal of resistance by blocking drug efflux with fumitremorgin C should allow for functional analysis of this novel transporter in cancer cell lines or clinical tumor samples. Increased resistance to mitoxantrone may result from reduced intracellular drug accumulation, altered nuclear/cytoplasmic drug distribution, and alterations in topoisomerase II activity.
Sujet(s)
Antinéoplasiques/toxicité , Multirésistance aux médicaments , Résistance aux médicaments antinéoplasiques , Mitoxantrone/toxicité , Adénosine triphosphate/métabolisme , Transport biologique , Noyau de la cellule/anatomopathologie , Survie cellulaire/effets des médicaments et des substances chimiques , Cytoplasme/anatomopathologie , Humains , Indoles/toxicité , Cinétique , Microscopie confocale , Mitoxantrone/pharmacocinétique , Myélome multiple , Mycotoxines/toxicité , Ouabaïne/pharmacologie , Cellules cancéreuses en cultureRÉSUMÉ
Integrin-mediated adhesion influences cell survival and may prevent programmed cell death. Little is known about how drug-sensitive tumor cell lines survive initial exposures to cytotoxic drugs and eventually select for drug-resistant populations. Factors that allow for cell survival following acute cytotoxic drug exposure may differ from drug resistance mechanisms selected for by chronic drug exposure. We show here that drug-sensitive 8226 human myeloma cells, demonstrated to express both VLA-4 (alpha4beta1) and VLA-5 (alpha5beta1) integrin fibronectin (FN) receptors, are relatively resistant to the apoptotic effects of doxorubicin and melphalan when pre-adhered to FN and compared with cells grown in suspension. This cell adhesion mediated drug resistance, or CAM-DR, was not due to reduced drug accumulation or upregulation of anti-apoptotic Bcl-2 family members. As determined by flow cytometry, myeloma cell lines selected for drug resistance, with either doxorubicin or melphalan, overexpress VLA-4. Functional assays revealed a significant increase in alpha4-mediated cell adhesion in both drug-resistant variants compared with the drug-sensitive parent line. When removed from selection pressure, drug-resistant cell lines reverted to a drug sensitive and alpha4-low phenotype. Whether VLA-4-mediated FN adhesion offers a survival advantage over VLA-5-mediated adhesion remains to be determined. In conclusion, we have demonstrated that FN-mediated adhesion confers a survival advantage for myeloma cells acutely exposed to cytotoxic drugs by inhibiting drug-induced apoptosis. This finding may explain how some cells survive initial drug exposure and eventually express classical mechanisms of drug resistance such as MDR1 overexpression.
Sujet(s)
Apoptose/génétique , Résistance aux médicaments antinéoplasiques/génétique , Régulation de l'expression des gènes tumoraux , Intégrines/génétique , Myélome multiple/génétique , Myélome multiple/anatomopathologie , Antinéoplasiques , Apoptose/effets des médicaments et des substances chimiques , Adhérence cellulaire/génétique , Doxorubicine/pharmacologie , Fibronectines/métabolisme , Humains , Melphalan/pharmacologie , Myélome multiple/métabolisme , Cellules cancéreuses en cultureRÉSUMÉ
Matrilysin, gelatinase A and gelatinase B are matrix metalloproteinases (MMPs) implicated in normal and pathological processes that require remodelling of the extracellular matrix. In human prostate tissue, matrilysin is synthesized in ducts surrounded by inflammatory cells, and focally in prostate carcinoma, but not in normal glands. Gelatinase B expression is restricted to inflammatory cells. Gelatinase A can be found in both benign and malignant prostate tissue. MMP activities are regulated by their transition from latent to activated forms, as well as by the presence of tissue inhibitors of metalloproteinases (TIMPs). We investigated whether matrilysin can activate progelatinases A and B in the presence of their bound inhibitors TIMP2 and TIMP1 respectively. Incubation of progelatinase B-TIMP1 complex with active matrilysin resulted in 78 and 68 kDa active forms, as measured by SDS-PAGE and enzyme activity assays. TIMP-free gelatinase B was also activated by matrilysin. In addition, activation of progelatinase B by matrilysin was demonstrated in the conditioned medium of phorbol ester-treated HT1080 cells, confirming the results obtained in the in vitro experiments. In contrast, matrilysin did not proteolytically cleave gelatinase A-TIMP2 complex, but led to a transient increase in gelatinolytic activity of the proenzyme. Matrilysin did not enhance the autocatalytic conversion of its own proform. The data presented here suggest that matrilysin participates in a proteolytic cascade and can activate gelatinases in the presence of TIMPs.
Sujet(s)
Gelatinases/métabolisme , Metalloendopeptidases/pharmacologie , Inhibiteur tissulaire des métalloprotéinases/métabolisme , Collagenases/métabolisme , Activation enzymatique/physiologie , Proenzymes/métabolisme , Gélatine/métabolisme , Humains , Matrix metalloproteinase 2 , Matrix metalloproteinase 7 , Matrix metalloproteinase 9 , Metalloendopeptidases/métabolisme , Liaison aux protéines , 12-Myristate-13-acétate de phorbol/pharmacologie , Inhibiteur tissulaire de métalloprotéinase-1/métabolisme , Inhibiteur tissulaire de métalloprotéinase-2/métabolisme , Cellules cancéreuses en cultureRÉSUMÉ
Overexpression of the matrix metalloproteinase matrilysin and the absence of beta 4 integrin are two features characteristic of human prostate carcinoma. In the following study we demonstrate that the beta 4 integrin, but not the alpha 6 or beta 1 integrin subunits, is cleaved by matrilysin in vitro. A specific fragment of 90 kDa is generated using matrilysin, which is not observed with other proteases. Two putative cleavage sites for matrilysin within the extracellular domain of the beta 4 integrin at residues 107 (isoleucine, prior to the ligand-binding region) and 417 (leucine, prior to cysteine-rich region) are identified by sequence comparisons with known matrilysin substrates. The selective cleavage of the beta 4 integrin by matrilysin may partly explain the loss of beta 4 integrin expression in invasive prostate carcinoma.
Sujet(s)
Antigènes CD/métabolisme , Metalloendopeptidases/métabolisme , Metalloendopeptidases/pharmacologie , Tumeurs de la prostate , Antigènes CD/composition chimique , Antigènes de surface/métabolisme , Calpain/métabolisme , Calpain/pharmacologie , Protéines de la matrice extracellulaire/métabolisme , Protéines de la matrice extracellulaire/pharmacologie , Humains , Intégrine bêta4 , Intégrines/métabolisme , Mâle , Matrix metalloproteinase 7 , Structure tertiaire des protéines , Spécificité du substrat , Trypsine/métabolisme , Trypsine/pharmacologie , Cellules cancéreuses en culture/composition chimique , Cellules cancéreuses en culture/enzymologieRÉSUMÉ
The p53 tumor suppressor is a transcription factor that regulates several pathways, which function collectively to maintain the integrity of the genome. Nuclear localization is critical for wild-type function. However, the signals that regulate subcellular localization of p53 have not been identified. Here, we examine the effect of ionizing radiation on the subcellular localization of p53 in two cell lines in which p53 is normally sequestered in the cytoplasm and found that ionizing radiation caused a biphasic translocation response. p53 entered the nucleus 1-2 h postirradiation (early response), subsequently emerged from the nucleus, and then again entered the nucleus 12-24 h after the cells had been irradiated (delayed response). These changes in subcellular localization could be completely blocked by the free radical scavenger, WR1065. By comparison, two DNA-damaging agents that do not generate free radicals, mitomycin C and doxorubicin, caused translocation only after 12-24 h of exposure to the drugs, and this effect could not be inhibited by WR1065. Hence, although all three DNA-damaging agents induced relocalization of p53 to the nucleus, only the translocation caused by radiation was sensitive to free radical scavenging. We suggest that the free radicals generated by ionizing radiation can signal p53 translocation to the nucleus.
Sujet(s)
Noyau de la cellule/effets des radiations , Rayons gamma , Transduction du signal/effets des radiations , Protéine p53 suppresseur de tumeur/métabolisme , Protéine p53 suppresseur de tumeur/effets des radiations , Animaux , Transport biologique/effets des radiations , Lignée cellulaire , Radicaux libres/effets des radiations , Humains , Liquide intracellulaire/métabolisme , Liquide intracellulaire/effets des radiations , Souris , Cellules cancéreuses en cultureRÉSUMÉ
One major obstacle to the successful treatment of epithelial derived tumors, such as breast and prostate carcinoma, is the presence of a multiple drug resistance phenotype. The drug resistance which is observed in growing epithelial derived cancer cells could either be an intrinsic, selected and/or an acquired characteristic. A survey of the survival data from several laboratories suggests that epithelial derived tumor cells, which have never been challenged with damaging agents, are in some cases 10 to 2,000 times more resistant to various chemotherapeutic agents as compared to hematopoietic cell lines. An intrinsic characteristic of epithelial cells is their resistance to the lethal effects of multiple types of damaging agents. A major feature of epithelial derived tumors is the expression of the intermediate filament type proteins known as cytokeratin. The simplest cytokeratin combination, cytokeratin 8 and 18, is a major cytoplasmic element within the cells of epithelial derived tumors. Earlier work showed that cytokeratin could be modified by mitoxantrone, a chemotherapeutic agent used in the treatment of breast cancer. Increasing data indicates that the intrinsic drug resistance phenotype is due in part to the presence of continued expression of the cytokeratin 8 and 18. The cytokeratin dependent drug resistance (C-MDR) has been observed in two different cell types that were engineered to contain cytokeratin 8 and 18 expression. The cytokeratin monomers are known to self assemble into intermediate filament networks as shown by numerous basic studies. Experiments using transfected cell lines which are unable to assemble networks indicated that C-MDR does not depend upon the formation of an intermediate filament network. Selection of cytokeratin network defective tumor cells did not increase their sensitivity to chemotherapeutic agents. These data are interesting since it suggests that the C-MDR phenotype is not dependent upon the structural nature (i.e. network forming ability) of the cytokeratin. Our current working hypothesis is that the interaction of the damaging agent with cytokeratin may initiate signaling response(s) for cell survival.
Sujet(s)
Multirésistance aux médicaments , Filaments intermédiaires/physiologie , Animaux , Humains , Tumeurs/traitement médicamenteux , Tumeurs/métabolismeRÉSUMÉ
A new human myeloma cell line, 8226/MDR10V, was selected from a P-glycoprotein-positive cell line, 8226/Dox40, in the continuous presence of doxorubicin and verapamil. MDR10V cells are 13-fold more resistant to doxorubicin and 4-fold more resistant to vincristine than the parent cell line, Dox40. Chemosensitizers are also less effective in reversing resistance in the MDR10V compared to the Dox40 cells. Despite higher resistance to cytotoxic agents, MDR10V expresses 40% less P-glycoprotein in the plasma membrane compared to Dox40; however, total cellular P-glycoprotein is the same in both cell lines. Confocal immunofluorescence microscopy shows 2.5-fold more P-glycoprotein in the cytoplasm of MDR10V cells as compared to Dox40 cells. The cytoplasmic location of P-glycoprotein in the MDR10V cells is associated with a redistribution of doxorubicin. In Dox40 cells, doxorubicin is concentrated in the nucleus, whereas in MDR10V cells, 90% of doxorubicin is found in the cytoplasm. In the presence of equivalent intracellular doxorubicin, there was a decrease in DNA-protein crosslinks in the MDR10V cell line compared to the Dox40 cell line. This finding is in agreement with the intracellular doxorubicin fluorescence studies showing less doxorubicin in the nuclei of MDR10V cells compared to Dox40 cells. Verapamil is less effective in increasing doxorubicin accumulation in the nuclei of MDR10V cells compared to Dox40 cells. Processing of P-glycoprotein from the endoplasmic reticulum to the medial Golgi was identical between the two cell lines as determined by endoglycosidase H sensitivity of newly sensitized P-glycoprotein. No mutations were found in MDR1 cDNA from MDR10V cells compared to Dox40 cells. These results suggest that resistance to chemosensitizing agents plus cytotoxic drugs is associated with a redistribution of P-glycoprotein from the plasma membrane to the cytoplasm, which in turn reduces the amount of cytotoxic drug reaching the nucleus.
Sujet(s)
Glycoprotéine P/analyse , Antinéoplasiques/pharmacologie , Cytoplasme/composition chimique , Doxorubicine/pharmacologie , Multirésistance aux médicaments , Résistance aux médicaments antinéoplasiques , Protéines tumorales/analyse , Vérapamil/pharmacologie , Transport biologique actif/effets des médicaments et des substances chimiques , Membrane cellulaire/composition chimique , Noyau de la cellule/métabolisme , Humains , Microscopie confocale , Microscopie de fluorescence , Myélome multiple/métabolisme , Myélome multiple/anatomopathologie , Cellules cancéreuses en cultureRÉSUMÉ
Laminin 5 is an extracellular matrix protein integral to the formation of the hemidesmosomes that attach normal basal cells to the underlying basal lamina. We have shown that these hemidesmosomal complexes are lost in prostate carcinoma, possibly allowing malignant cells to detach from the anchoring structures and then to invade and migrate through the adjacent tissue. Our previous immunohistochemical studies of normal and malignant human prostate tissue demonstrated that the laminin subchains alpha 1, alpha 2, beta 1, beta 2, gamma 1, and gamma 2 were all expressed as normal components of the basal lamina surrounding prostate glands. Although most of these subchains were also expressed by the de novo basal lamina synthesized by prostate carcinoma, the gamma 2 subchain of laminin 5 was not detected. In an effort to investigate the role laminin 5 plays in the tumorigenesis of prostate carcinoma, the protein expression of the three subchains of laminin 5 (alpha 3, beta 3, and gamma 2) was compared in normal prostate, prostatic intraepithelial neoplasia, and invasive carcinoma using immunohistochemistry. The results showed that the protein for the alpha 3 subchain of laminin 5 is retained by both normal prostate epithelium and prostate carcinoma, but the beta 3 and the gamma 2 subchains were not detected in invasive carcinoma. Despite the absence of the gamma 2 protein, however, the carcinoma cells continued to express substantial amounts of the gamma 2 mRNA. Although it is unclear how the gene for the gamma 2 subchain of laminin 5 is regulated, results of this study suggest that there is a post-transcriptional defect in the expression of the gamma 2 subchain that occurs during the progression from a premalignant lesion to invasive carcinoma. As laminin 5 is a component of the anchoring filaments, the failure to express the gamma 2 subchain may contribute to the failure to form anchoring filaments and hemidesmosomes. This failure of hemidesmosome formation results in a less stable epithelial-stromal junction, which may allow malignant cells more potential to invade and spread through adjacent structures.
Sujet(s)
Carcinomes/métabolisme , Molécules d'adhérence cellulaire/métabolisme , Prostate/métabolisme , Tumeurs de la prostate/métabolisme , Technique de Northern , Technique de Western , Épithélioma in situ/métabolisme , Humains , Immunohistochimie , Mâle , ARN messager/métabolisme ,RÉSUMÉ
Tumor cell progression is dependent in part on the successful adhesive interactions of the cells with the extracellular matrix. In this study, a new approach is described to isolate linear peptide ligand candidates involved in cellular adhesion. A synthetic combinatorial peptide library based on the 'one-bead-one-peptide' concept was incubated with live human prostate cancer cells for 90 min at 37 degrees C. The peptide bead coated with a monolayer of cells was then isolated for microsequencing. The DU145 (DU-H) cells were chosen since they have been previously characterized as containing elevated levels of a laminin receptor for cell adhesion, the alpha 6 beta 1 integrin on the cell surface. The use of a function-blocking antibody (GoH3) allows for the detection of peptides which are alpha 6-specific ligand candidates. From two different libraries (linear 9-mer and 11-mer) of a total of 1,500,000 beads, 68 peptide beads containing attached cells were isolated. These positive beads were then retested to determine the ability of the GoH3 antibody to block binding of the cells to the peptide beads. The alpha 6 integrin candidate peptide beads (five in total) were recovered and two of the beads were microsequenced. These two peptides, RU-1 (LNIVS-VNGRHX) and RX-1 (DNRIRLQAKXX), resemble the previously reported active peptide sequences (GD-2 and AG-73) from native laminin. The RU-1, RX-1 and AG-73 peptides were tested for their ability to support cell attachment and to bind the cell surface of DU-H prostate carcinoma cells in suspension using fluorescence-activated cell-sorting (FACS) analysis. Both RU-1 and AG-73 peptides supported cellular attachment within 1 h. In contrast, after 1 h, EHS laminin supported both cellular attachment and spreading. The RX-1 peptide exhibited only weak binding to the DU-H prostate carcinoma cells. FACS analysis indicated that AG-73 peptide attached to tumor cell surfaces over a range of concentrations, whereas the RU-1 peptide showed a homogeneous concentration required for attachment. The described strategy for screening a random peptide library offers three advantages: (i) ligands for conformationally sensitive receptors of adhesion can be isolated using live cells; (ii) specific binding can be selected for using function-blocking antibodies; and (iii) peptides supporting adhesion independent of spreading properties can be distinguished. In principle, specific adhesive peptides without prior knowledge of the sequence could be isolated for any epithelial cell surface receptor for which a function-blocking reagent is available.
Sujet(s)
Molécules d'adhérence cellulaire/isolement et purification , Banque de peptides , Peptides/composition chimique , Antigènes CD/métabolisme , Carcinomes , Adhérence cellulaire , Molécules d'adhérence cellulaire/physiologie , Cytométrie en flux , Humains , Intégrine alpha6 , Intégrines/métabolisme , Mâle , Microsphères , Peptides/synthèse chimique , Peptides/physiologie , Tumeurs de la prostate , Liaison aux protéines , Propriétés de surface , Cellules cancéreuses en cultureRÉSUMÉ
The cytokeratin network is an abundant cytoplasmic system whose function is largely unknown. Recently, we have found that the introduction of a cytokeratin network into eukaryotic cells results in a drug resistance phenotype. The current study was undertaken to determine the universal nature of this phenomenon by investigating the survival response of two different cell lines to six different DNA-damaging agents using two different assays of cell survival. To correlate our in vitro assays of survival with known in vivo responses to DNA damage, we compared the apoptotic response of cytokeratin-positive and cytokeratin-negative cell lines. The results show that the introduction of a cytoskeletal network confers a resistant phenotype to mitoxantrone, doxorubicin, melphalan, bleomycin, and mitomycin C in the different cytokeratin-positive cell lines. No survival advantage was noted when damage was conferred by cisplatin or UV irradiation. We found the cytokeratin-positive cell lines were protected from apoptosis, while the cell lines without cytokeratins showed apoptosis in response to mitoxantrone exposure. Cytokeratin-dependent drug resistance is observed in different cell lines but is not observed with all DNA-damaging agents. The data suggest that the mechanism of this drug resistance may be attributed, in part, to a cytokeratin-conferred protection against apoptosis.