RESUMEN
BACKGROUND: We previously reported that an ambient aspartic proteinase is crucial to desquamation of the stratum corneum at pH 5. Identification of this aspartic proteinase by using enzyme inhibitors suggested it to be cathepsin D, although we could not exclude cathepsin E. OBJECTIVES: To determine the identity of this aspartic proteinase and its distribution within the stratum corneum. METHODS: We measured enzyme activities of cathepsin D and cathepsin E in the salt and detergent extracts from callus stratum corneum, using a fluorogenic peptide as a substrate and comparing the effect of addition of Ascaris pepsin inhibitor (specific for cathepsin E) with that of pepstatin A (which inhibits both cathepsin D and cathepsin E). Both enzymes were then extracted and purified from plantar stratum corneum samples and identified by Western blotting. Immunofluorescence microscopy was used to investigate the localization of proteinases within human plantar stratum corneum sample sections. RESULTS: We found that 20% of total aspartic proteinase activity could be attributed to cathepsin E, the remainder to cathepsin D. Two subunits of cathepsin D were identified, a mature active form at 33 kDa and an intermediate active form at 48 kDa; cathepsin E was also identified at 48 kDa, although in a stained band 10-fold weaker in the immunoblot. Immunofluorescence microscopy showed the antibody to cathepsin D to be localized in the lipid envelopes of the stratum corneum, whereas that to cathepsin E stained the tissue diffusely. The labelling for cathepsin D was similar to that observed for desmosomes, and immunoelectron microscopy confirmed that cathepsin D was present on desmosomes. On the other hand, cathepsin E occurred intracellularly within the squames. CONCLUSIONS: We conclude that cathepsin D, and not cathepsin E, causes desquamation by degrading desmosomes.
Asunto(s)
Catepsina D/metabolismo , Catepsina E/metabolismo , Desmosomas/metabolismo , Epidermis/metabolismo , Catepsina D/aislamiento & purificación , Catepsina E/aislamiento & purificación , Desmosomas/inmunología , Desmosomas/ultraestructura , Epidermis/inmunología , Epidermis/ultraestructura , Talón , HumanosRESUMEN
Zinc-alpha(2)-glycoprotein (Znalpha(2)gp) is widely distributed in body fluids and epithelia. Its expression in stratified epithelia increases with differentiation. We previously showed that Zn alpha(2)gp has ribonuclease activity, and that squamous tumor cells grown on a matrix of Znalpha(2)gp were growth-inhibited. Here we demonstrate, both by adding Znalpha(2)gp to the culture medium and, more unequivocally, by stably transfecting SiHa cells with Znalpha(2)gp cDNA, that the introduction of Znalpha(2)gp into SiHa tumor cells reduces proliferation. In response to Znalpha(2)gp, we find an accumulation of the cell population in G(2)/M by flow cytometry, paralleling the reduction of proliferation. In order to distinguish growth inhibition by cell cycle arrest from that produced by apoptosis or differentiation, we examine by RT-PCR how Znalpha(2)gp affects the expression of genes commonly used as markers of these properties. No changes are observed for PCNA, p53, c-myc, or bcl-2. Only cdc2 expression responds to Znalpha(2)gp, with a reduction of up to over a factor of two. Cdc2 is the only cyclin-dependent kinase regulating the G(2)/M transition without redundancy and is required as a rate-limiting step in the cell cycle. Its increased expression has been directly linked to increased proliferation and decreased differentiation of advanced tumors; conversely, its downregulation by Znalpha(2)gp might hinder tumor progression. J. Cell. Biochem. Suppl. 36: 162-169, 2001.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Glicoproteínas/metabolismo , Proteínas de Plasma Seminal , Western Blotting , Proteína Quinasa CDC2/genética , División Celular , Regulación hacia Abajo , Citometría de Flujo , Humanos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Células Tumorales Cultivadas , Zn-alfa-2-GlicoproteínaRESUMEN
Psoriasis is a T cell-mediated inflammatory disease characterized by hyperproliferation and by aberrant differentiation. We found cathepsin D and zinc-alpha(2)-glycoprotein, two catalytic enzymes associated with apoptosis and desquamation, to be present in the stratum corneum of the normal epidermis but absent from the psoriatic plaque. Psoriasis is characterized by an altered response to interferon-gamma (IFN-gamma), including the induction of apoptosis in normal but not in psoriatic keratinocytes, often with opposite effects on gene expression of suprabasal proteins. We found that IFN-gamma binding and signaling were attenuated in psoriasis: The IFN-gamma receptor, the signal transducer and activator of transcription STAT-1, and the interferon regulatory factor IRF-1 were strongly up-regulated by IFN-gamma in normal keratinocytes, but not in psoriatic ones. IFN-gamma strongly up-regulated the expression of the catalytic enzymes cathepsin D and zinc-alpha(2)-glycoprotein in normal keratinocytes but down-regulated them in psoriatic ones; the reverse was true of the apoptotic suppressor bcl-2. We believe that the aberrant response to IFN-gamma plays a central role in the pathophysiology of psoriasis, particularly the disruption of apoptosis and desquamation.
Asunto(s)
Catepsina D/genética , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas/genética , Interferón gamma/farmacología , Queratinocitos/metabolismo , Psoriasis/metabolismo , Proteínas de Plasma Seminal , Células Cultivadas , Proteínas de Unión al ADN/genética , Epidermis/efectos de los fármacos , Epidermis/inmunología , Epidermis/metabolismo , Humanos , Factor 1 Regulador del Interferón , Queratinocitos/efectos de los fármacos , Queratinocitos/inmunología , Fosfoproteínas/genética , Psoriasis/inmunología , Receptores de Interferón/genética , Factor de Transcripción STAT1 , Transducción de Señal , Transactivadores/genética , Zn-alfa-2-Glicoproteína , Receptor de Interferón gammaRESUMEN
Even though the skin surface is acidic (about pH 5), most in vitro studies on desquamation have been performed at alkaline pH. We demonstrate that the standard in vitro model system, which achieves squame shedding upon incubation of plantar stratum corneum for 1 day in an alkaline buffer that must include a chelating agent, can be extended to a more realistic model in which the incubation is for 4 days, at varying pHs from 5 to 8, without exogenous chelators. Desmoglein I from stratum corneum was degraded by the squames shed at pH 5 as well as at pH 8. Squame shedding was inhibited to varying extents by the addition of proteinase inhibitors, whose specificity suggested that the crucial enzymatic activity at pH 8 was a chymotrypsin-like serine proteinase, while a similar activity at pH 5 was accompanied by an aspartic proteinase activity of comparable strength. Four degradation peaks were observed when the insulin B chain was reacted with shed squames at pH 5. Two of these peptides were suppressed by the addition of phenylmethylsulphonyl fluoride, the other two by pepstatin A; chymostatin inhibited all four, but E-64 and leupeptin showed no effect. The implied specificity was confirmed by reacting the insulin (without squames) with the standard enzymes human liver cathepsin D and pancreatic chymotrypsin, reproducing the expected degradation products. These results suggest that epidermal desquamation at acidic pH requires two proteolytic activities, one of which is an analogue of chymotrypsin and the other of cathepsin D. Endogenous proteinases corresponding to these activities have been previously identified, namely the stratum corneum chymotryptic enzyme and the mature active form of cathepsin D.
Asunto(s)
Catepsina D/metabolismo , Quimotripsina/metabolismo , Epidermis/metabolismo , Piel/citología , Western Blotting , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Humanos , Insulina/metabolismoRESUMEN
Zn-alpha(2)-glycoprotein (Znalpha(2)gp) is a soluble protein widely distributed in body fluids and glandular epithelia. We have found it to be expressed in stratified epithelia as well. Znalpha(2)gp is clinically correlated with differentiation in various epithelial tumors, including oral and epidermal tumors. We have cloned epidermal Znalpha(2)gp and report the preparation of the recombinant protein in a Baculovirus expression system. Like the native molecule, recombinant Znalpha(2)gp has RNase activity. Znalpha(2)gp functions as a matrix protein for the Tu-138 oral squamous cell carcinoma cell line. Cell attachment to Znalpha(2)gp is comparable to that for fibronectin and is inhibited by the synthetic RGD peptides RGD, RGDV, and RGDS. Attachment is also inhibited by the antibody to integrin alpha(5)beta(1) (the fibronectin receptor), but not by antibodies to integrins alpha(v)beta(3), alpha(3)beta(1), and alpha(2)beta(1). We find that the proliferation of Tu-138 cells is inhibited on a Znalpha(2)gp matrix, as compared with other matrix proteins (fibronectin, vitronectin, laminin, and collagens I and IV) on which growth resembles that on the BSA control. We believe that the role of Znalpha(2)gp in differentiation and its RNase activity are two likely suspects as agents of the inhibition of proliferation.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , División Celular/efectos de los fármacos , Glicoproteínas/metabolismo , Proteínas de Plasma Seminal , Anticuerpos/farmacología , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/farmacología , Clonación Molecular , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/farmacología , Neoplasias Gingivales , Glicoproteínas/farmacología , Humanos , Integrinas/inmunología , Integrinas/metabolismo , Microscopía de Contraste de Fase , Oligopéptidos/química , Oligopéptidos/farmacología , Proteínas Recombinantes/metabolismo , Células Tumorales Cultivadas , Zn-alfa-2-GlicoproteínaRESUMEN
The mRNA levels of keratin K13, involucrin, protein kinase C alpha and epsilon, and interferon-gamma and its receptors were examined in biopsies from human oral squamous cell carcinomas. Expression of all the genes was elevated in the histologically more differentiated tumors, but it was at or below normal (perilesional control) levels in the poorly differentiated ones. For the same set of biopsies, we had previously shown that the well differentiated tumors expressed higher levels of T cell markers. As interferon-gamma stimulates differentiation, its secretion by inflammatory cells at the tumor site may influence the differentiation status of the tumor.
Asunto(s)
Inflamación/patología , Neoplasias de la Boca/patología , Diferenciación Celular , Humanos , Interferón gamma/biosíntesis , Interferón gamma/genética , Queratinas/análisis , Queratinas/genética , Proteína Quinasa C/genética , ARN Mensajero/análisisRESUMEN
Zinc-alpha2-glycoprotein (Znalpha2gp) is a soluble major histocompatibility complex homolog widespread in body fluids and in glandular epithelia; the authors recently demonstrated its presence in stratified epithelia. Znalpha2gp has been associated with tumor differentiation in breast cancers and other carcinomas. We compare here its gene expression in histopathologically graded oral squamous cell carcinomas and in their perilesional normals. Znalpha2gp levels are higher in the controls than in the tumors, and higher in well-differentiated tumors than in poorly differentiated ones. Markers of oral epithelial maturation (keratin K13 and involucrin) are less simply related to tumor histology.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/metabolismo , Glicoproteínas/metabolismo , Neoplasias de la Boca/metabolismo , Proteínas de Plasma Seminal , Carcinoma de Células Escamosas/patología , Diferenciación Celular , Humanos , Neoplasias de la Boca/patología , Zn-alfa-2-GlicoproteínaRESUMEN
Cathepsin D is an ubiquitously expressed lysosomal aspartic proteinase, with well-determined structural and chemical properties but a less clearly defined biological role. In stratified epithelia, the chronology of cathepsin D activation and degradation can be connected with stages of cellular differentiation. We partially purified cathepsin D from human epidermis and from separated stratum corneum by standard biochemical procedures, monitored by SDS-PAGE and Western blotting, and verified its identity as to molecular mass, pH optimum, N-terminal sequencing, reactivity with the specific antibody, inhibition by pepstatin A, and specific enzyme activity. It had hemoglobin-degrading activity over the acid range, with maximum at pH 3. It also degraded bovine serum albumin, human keratins, and stratum corneum extracts at pH 4. We discerned all three isoforms of human cathepsin D (the 52 kDa proenzyme and the active forms at 48 kDa and 33 kDa) in the epidermis; both active forms were also seen in the stratum corneum, but the proenzyme was not. Gene expression of cathepsin D in epidermal keratinocytes resembled that of suprabasal structural proteins (involucrin, keratin K10, transglutaminase) in its response to the calcium switch. An antibody to the 33 kda isoform immunolocalized to the granular layer and the stratum corneum (whereas antibodies to the 48 kDa isoform have been reported to stain mainly the upper spinous and granular layers). A plausible hypothesis to harmonize these results is that cathepsin D is first expressed as the proenzyme in the upper spinous layer, is activated in the lysosomes in the granular layer to the 48 kDa form, and is degraded to the 33 kDa form in the transition zone between the granular layer and the stratum corneum. As the stratum corneum is an acid environment, with an ambient pH of approximately 4.5, cathepsin D is available and suited to contribute to desquamation.
Asunto(s)
Catepsina D/metabolismo , Diferenciación Celular , Células Epidérmicas , Isoenzimas/metabolismo , Secuencia de Aminoácidos , Catepsina D/genética , Catepsina D/aislamiento & purificación , Células Cultivadas , Epidermis/enzimología , Regulación Enzimológica de la Expresión Génica , Humanos , Inmunohistoquímica , Isoenzimas/genética , Isoenzimas/aislamiento & purificaciónRESUMEN
OBJECTIVES/HYPOTHESIS: This study aimed to ascertain whether cathepsin D expression could be related to the stage of differentiation of oral tumors. STUDY DESIGN: Human oral biopsies of 10 squamous cell carcinomas and of the corresponding perilesional normal tissues were used. The tumors had all been clinically graded as advanced stage but nonmetastatic; five were classified histopathologically as poorly differentiated. METHODS: The gene expression of cathepsin D and keratin K13 in the biopsies was measured by reverse transcription polymerase chain reaction. Ratios of tumor-to-control readings helped compensate for sample variability. RESULTS: Keratin K13, as a suprabasal cell marker, tended to confirm the histological grading of the tumors (but was not otherwise useful in distinguishing tumors from normal tissue). Substantial overexpression of cathepsin D was found in the poorly differentiated tumors. CONCLUSIONS: Cathepsin D overexpression is considered a prognostic indicator of metastasis. In this sample, it was also associated with dedifferentiation. Cathepsin D might serve as a valuable gauge in clinical exploration of the connection between dedifferentiation and metastasis.
Asunto(s)
Carcinoma de Células Escamosas/patología , Catepsina D/análisis , Neoplasias de la Boca/patología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/metabolismo , Transformación Celular Neoplásica/patología , Femenino , Humanos , Queratinas/análisis , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/metabolismo , Reacción en Cadena de la Polimerasa , Pronóstico , ARN Mensajero/análisis , ADN Polimerasa Dirigida por ARNRESUMEN
Normal keratinocytes from epidermis and from buccal mucosa were cultured to confluence in three media with graded differentiation potential and treated with interferon-gamma (IFN-gamma). RT-PCR was used to measure the gene expression of the IFN-gamma receptor (IFNGR-1), as well as the immunomodulator HLA-DR and two enzymes of the 2-5 A pathway. We have previously reported results for a number of structural and regulatory genes in the same system (and include here involucrin for comparison). In epidermal keratinocytes, the induction of IFNGR-1 was upregulated by incubation with IFN-gamma, and this increased with the differentiation potential of the culture medium. A roughly similar pattern occurred for the other genes. In mucosal keratinocytes, in contrast, IFN-gamma failed to induce expression of IFNGR-1 or the other genes. A unique characteristic of HLA-DR was that its induction by IFN-gamma was uniform, for both tissues and all media. The gene expression of the receptor IFNGR-1 appears to be the dominant factor in the sensitivity of other genes to IFN-gamma, although there are substantial disparities among them that presumably reflect functional differences. The difference between the two tissues may be linked to differentiation, as the epidermis has a much more extensive maturation pattern than the buccal mucosa. A clinical implication is a better prognosis for IFN-gamma treatment for more differentiated tumors. Indeed, a previous study has found that the maturation pattern of condylomas responding to interferon treatment resembles that of epidermis, whereas the maturation of nonresponders is more akin to that of buccal mucosa.
Asunto(s)
Antivirales/farmacología , Células Epiteliales/efectos de los fármacos , Interferón gamma/farmacología , Receptores de Interferón/efectos de los fármacos , 2',5'-Oligoadenilato Sintetasa/efectos de los fármacos , 2',5'-Oligoadenilato Sintetasa/genética , Calcio/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Medios de Cultivo/farmacología , Relación Dosis-Respuesta a Droga , Endorribonucleasas/efectos de los fármacos , Endorribonucleasas/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Genes/efectos de los fármacos , Genes/genética , Antígenos HLA-DR/efectos de los fármacos , Antígenos HLA-DR/genética , Humanos , Masculino , Mucosa Bucal/citología , Mucosa Bucal/efectos de los fármacos , Mucosa Bucal/metabolismo , Precursores de Proteínas/efectos de los fármacos , Precursores de Proteínas/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interferón/genética , Receptor de Interferón gammaRESUMEN
Zinc-alpha 2-glycoprotein (Zn alpha 2gp) is widely distributed in body fluids and in various epithelia; its gene has been completely sequenced, but its function has long remained elusive. We have found that Zn alpha 2gp has RNase activity, comparable to onconase but two orders of magnitude less than RNase A. The RNase activity of Zn alpha 2gp is characterized by maxima in pH at 7.5, in ionic strength at 50 mM NaCl, and in temperature at 60 degreesC. It is strongly inhibited by ZnCl2, but unaffected by MgCl2. It is partially inactivated (down to 20%) by the placental RNase inhibitor. On synthetic polyribonucleotide substrates, the RNase activity of Zn alpha 2gp is specific for pyrimidine residues [poly(C) and poly(U) equally] and cleaves only single-stranded RNA. For onconase, it has been demonstrated that the RNase activity depends on pyroglutamic acid (pyr 1) as the N-terminus; Zn alpha 2gp also has pyr 1, while RNase A does not. Alignment of the amino acid sequences of Zn alpha 2gp and onconase or RNase A reveals only modest matches. Despite the more substantial overall structural homology of Zn alpha 2gp to class I major histocompatibility complex proteins, Zn alpha 2gp has not been proven to be associated with the immune response and, conversely, we could not detect RNase activity in six class I HLA heavy chains.
Asunto(s)
Glicoproteínas/metabolismo , Ribonucleasas/metabolismo , Proteínas de Plasma Seminal , Activación Enzimática/efectos de los fármacos , Epidermis/enzimología , Glicoproteínas/antagonistas & inhibidores , Humanos , Concentración de Iones de Hidrógeno , Masculino , Concentración Osmolar , Hormonas Placentarias/farmacología , Polirribonucleótidos/metabolismo , Próstata/enzimología , ARN de Hongos/metabolismo , Ribonucleasas/antagonistas & inhibidores , Temperatura , Zn-alfa-2-GlicoproteínaRESUMEN
The proteasome is a cytoplasmic high-molecular-weight structure composed of several smaller protein and RNA subunits. It has been associated with non-lysosomal pathways of intracellular degradation, expressing multicatalytic proteinase activities and specific RNase activity. By standard methods, we have isolated andpartially purified proteasomes from human epidermis. We obtained the expected multiple 24-32 kDa subunits by SDS-PAGE, and evidence of RNA. Proteasomes degraded casein, as well as chromogens for t-PA and trypsin but not for chymotrypsin, these proteolytic activities overlap, but do not coincide with those observed in other organs. We found that human epidermal 28 S and 18 S rRNAs were degraded, but yeast RNA was not. By means of zymography, we demonstrated, for the first time, that RNase activity persists after dissociation of the proteasome on the gel and that it co-localizes to the same range of molecular weight subunits as the proteinase activity.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Epidermis/enzimología , Complejos Multienzimáticos/metabolismo , Ribonucleasas/metabolismo , Cromatografía Líquida de Alta Presión , Cisteína Endopeptidasas/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Humanos , Complejos Multienzimáticos/aislamiento & purificación , Complejo de la Endopetidasa Proteasomal , ARN Ribosómico/metabolismo , Dodecil Sulfato de Sodio , Fracciones Subcelulares/enzimología , Especificidad por SustratoRESUMEN
Desquamin is a glycoprotein that we have isolated from the upper granular layer and the stratum corneum of human epidermis; it is not ordinarily expressed in submerged cultures, whose terminal differentiation stops short of formation of these layers. The exogenous addition of desquamin to human cultured keratinocytes extended their maturation, and hematoxylin staining indicated a loss of cell nuclei. For confirmation, cultured cells were lysed in situ, and the nuclei were incubated with desquamin for several days, then stained with hematoxylin. Damage to the nuclei was evident: the nuclear inclusions remained intact, while the surrounding basophilic nuclear matrix was degraded. Desquamin was then tested directly for nuclease activity. Ribonuclease activity was determined by incubating desquamin with human epidermal total RNA and monitoring the dose-dependent disappearance of the 28S and 18S ribosomal RNA bands in an agarose/formaldehyde gel. On RNA-containing zymogels, we confirmed the RNase activity to be specific to desquamin. Using synthetic RNA homopolymers, we found the active RNase domains to be limited to cytosine residues. On the contrary, DNA was not degraded by an analogous procedure, even after strand-separation by denaturation.
Asunto(s)
Moléculas de Adhesión Celular/farmacología , Células Epidérmicas , Epidermis/enzimología , Ribonucleasas , Serina Endopeptidasas/farmacología , Moléculas de Adhesión Celular/aislamiento & purificación , Medios de Cultivo Condicionados , Desoxirribonucleasas/análisis , Humanos , Queratinocitos/citología , ARN/efectos de los fármacos , Ribonucleasas/análisis , Serina Endopeptidasas/aislamiento & purificaciónRESUMEN
Zinc-alpha 2-glycoprotein has been detected in most body fluids, and its antibody labels the corresponding glandular epithelia. We have also detected it in human stratified epithelia (epidermis and buccal mucosa). In this study, the mRNA levels of zinc-alpha 2-glycoprotein were found to be about twice as high in epithelial cells of mucosal origin (whether normal primaries or neoplastic cell lines) as in epidermoid cells (normal epidermal primary cultures, an immortalized but non-tumorigenic epidermal cell line, and neoplastic vulvar and cervical cell lines). Interferon-gamma strongly upregulated gene expression, but substantially less in mucosal than epidermoid cells. To compare responses as a clue to the function of zinc-alpha 2-glycoprotein, we ran parallel experiments with three markers of distinct properties, all known to be induced by interferon-gamma. There was the least resemblance for involucrin, a qualitative similarity for HLA-DR, and a rather better match for 2'-5' oligoadenylate synthetase.
Asunto(s)
Células Epiteliales/metabolismo , Glicoproteínas/genética , Interferón gamma/farmacología , Proteínas de Plasma Seminal , 2',5'-Oligoadenilato Sintetasa/metabolismo , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Antígenos HLA-DR/metabolismo , Humanos , Queratinocitos/metabolismo , Precursores de Proteínas/metabolismo , ARN Mensajero/genética , Zn-alfa-2-GlicoproteínaRESUMEN
Zinc-alpha 2-glycoprotein (Zn alpha 2gp) is almost ubiquitous in body fluids, and its antibody labels the corresponding secretory epithelia. We have found that Zn alpha 2gp is also expressed in human epidermis. We cloned the Zn alpha 2gp cDNA by screening our cDNA library, derived from epidermal keratinocytes, with a probe for prostate Zn alpha 2gp. It had complete nucleic acid sequence homology with that from prostate, including the signal peptide. Just as Zn alpha 2gp expression is higher in more differentiated breast tumors, so in skin tumors the highest mRNA levels occurred in the normal controls, the lowest in basal cell carcinomas (the least differentiated epidermal tumor type), and intermediate levels in squamous cell carcinomas and Merkel cell carcinomas. A similar increase in Zn alpha 2gp gene expression with differentiation was observed when epidermal keratinocytes were cultured in media that varied in cellular maturation potential.
Asunto(s)
Clonación Molecular , ADN Complementario/análisis , Expresión Génica , Glicoproteínas/genética , Queratinocitos/metabolismo , Proteínas de Plasma Seminal , Neoplasias Cutáneas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Humanos , Masculino , Datos de Secuencia Molecular , ARN Mensajero/análisis , Zn-alfa-2-GlicoproteínaRESUMEN
Zinc-alpha 2-glycoprotein (Zn alpha 2gp) is almost ubiquitous in body fluids. We have found it to be also present in stratified epithelia. We compare its mRNA expression in cells from human epidermis and buccal mucosa cultured in media of graded differentiation potential (attained by varying calcium ion concentration and adding serum). The Zn alpha 2gp gene is upregulated in both epithelia with differentiation and further with exposure to interferon gamma or transforming growth factor beta 1. The upregulation by these agents increases with differentiation in epidermal cells, but peaks in the low-differentiation medium in buccal epithelia. We compared gene expression levels of Zn alpha 2gp with those of characteristic cytokeratins of stratified epithelia (k5 for basal cells, K10 for epidermal suprabasal cells, and K13 for mucosal suprabasal cells). This pattern correlation associates Zn alpha 2gp cell-type dependently with late differentiation, i.e. with keratin K10 in epidermis and with K13 in buccal epithelium.
Asunto(s)
Epidermis/fisiología , Glicoproteínas/genética , Mucosa Bucal/fisiología , Proteínas de Plasma Seminal , Células Cultivadas , Medios de Cultivo/farmacología , Células Epidérmicas , Epidermis/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Humanos , Interferón gamma/farmacología , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/fisiología , Mucosa Bucal/citología , Mucosa Bucal/efectos de los fármacos , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Zn-alfa-2-GlicoproteínaRESUMEN
Differences in the effects on confluent epidermal keratinocytes of treatment with interferons (IFNs) alpha, beta, and gamma were observed in their modulation of the mRNA levels of representative structural and functional cellular proteins. Comparisons of the responses in culture media with varying cellular maturation potential indicated the dependence of the modulation on the stage of differentiation. Differentiation was correlated with upregulation of all the genes by interferon gamma, but this effect was not seen with the other interferons. Even though IFNs alpha and beta share the same cell surface receptor, their effects on gene expression were clearly distinguishable and varied with the culture medium. These findings might have relevance in the treatment of skin lesions with varying degrees of differentiation.
Asunto(s)
Proteínas Portadoras , Proteínas de Unión al ADN , Interferones/farmacología , Queratinocitos/fisiología , Proteína Quinasa CDC2/genética , Proteínas de Ciclo Celular/genética , Células Cultivadas , Factores de Transcripción E2F , Expresión Génica/efectos de los fármacos , Humanos , Interferón-alfa/farmacología , Interferón beta/farmacología , Interferón gamma/farmacología , Queratinocitos/química , Queratinocitos/citología , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/metabolismo , Proteína de Retinoblastoma/genética , Proteína 1 de Unión a Retinoblastoma , Piel/citología , Factor de Transcripción DP1 , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Cell lines are useful as models if they retain the relevant characteristics of the tissue of origin. We compared two human squamous carcinoma cell lines derived from tumors of the tongue that vary in their extent of differentiation, with human biopsies of carcinomas of the tongue that were either poorly or well-differentiated. The mRNA levels of suprabasal cell proteins (keratin K13, involucrin, transglutaminase) and of protein kinase C (PKC) isozymes were measured by RT-PCR. Apart from PKC beta and PKC delta (mostly expressed by Langerhans cells and missing in culture), qualitatively similar patterns were found in vitro and in vivo. The more differentiated cells had expression levels moderately lower to higher than the normal controls. The poorly differentiated cells generally had substantially lower levels.
Asunto(s)
Neoplasias de la Boca/patología , Neoplasias de la Lengua/patología , Transcripción Genética , Biomarcadores , Diferenciación Celular , Línea Celular , Humanos , Isoenzimas/análisis , Isoenzimas/biosíntesis , Queratinas/análisis , Queratinas/biosíntesis , Neoplasias de la Boca/metabolismo , Reacción en Cadena de la Polimerasa , Proteína Quinasa C/análisis , Proteína Quinasa C/biosíntesis , Precursores de Proteínas/análisis , Precursores de Proteínas/biosíntesis , ARN Mensajero/biosíntesis , Neoplasias de la Lengua/metabolismo , Transglutaminasas/análisis , Transglutaminasas/biosíntesis , Células Tumorales CultivadasRESUMEN
Induction of inducible nitric oxide synthase (iNOS) mRNA by interferon-gamma (IFNgamma) is well established in various cell types. We observed earlier that this induction is differentiation-dependent in human keratinocytes. Since IFNgamma-mediated growth arrest and differentiation are separable but interrelated processes in keratinocytes, iNOS might play a role in their regulation. We conducted a series of in vitro experiments on normal and transformed epithelial cells with different tissue origins. We found that immortalization and transformation can influence the IFNgamma-mediated iNOS inducibility, but this process is tissue-specific as the induction correlates with the ability of the cells to differentiate.