RESUMEN
BACKGROUND: Nomograms for biochemical recurrence (BCR) of prostate cancer (PC) after radical prostatectomy can yield very different prognoses for individual patients. Since the nomograms are optimized on different cohorts, the variations may be due to differences in patient risk-factor distributions. In addition, the nomograms assign different relative scores to the same PC risk factors and rarely stratify for tumor growth rate. METHODS: We compared BCR-free probabilities from the GPSM model with a cell kinetics (CK) model that uses the individual's tumor state and growth rate. We first created a cohort of 143 patients that reproduced the GPSM patient distribution in Gleason score, Prostate specific antigen (PSA), Seminal vesicle involvement and Margin status since they form the GPSM score. We then performed 143 CK calculations to determine BCR-free probabilities for comparison with the GPSM results for all scores and with four other prominent nomograms for a high-risk patient. RESULTS: The BCR-free probabilities from the CK model agree within 10% with those from the GPSM study for all scores once the CK model parameters are stratified in terms of the GPSM risk factors and the PSA doubling time (PSADT). However, the probabilities from widely used nomograms vary significantly. CONCLUSIONS: The CK model reproduces the observed GPSM BCR-free probabilities with a broad stratification of model parameters for PC risk factors and can thus be used to describe PC progression for individual patients. The analysis suggests that nomograms should stratify for PSADT to be predictive.
Asunto(s)
Progresión de la Enfermedad , Modelos Biológicos , Recurrencia Local de Neoplasia/epidemiología , Nomogramas , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/patología , Algoritmos , Proliferación Celular , Estudios de Cohortes , Humanos , Masculino , Pronóstico , Antígeno Prostático Específico/sangre , Prostatectomía , Neoplasias de la Próstata/cirugía , Estudios Retrospectivos , Factores de Riesgo , Vesículas Seminales/patologíaRESUMEN
Prostate cancer is the second most prevalent cancer in males in the United States. Standard therapy relies on removing, or blocking the actions of, androgens. In most cases, this therapy results in a regression of the cancer because the prostate and most primary prostate tumors depend on androgens for growth and the avoidance of apoptosis. However, a portion of the cancers eventually relapse, at which point they are termed "androgen refractory" and can no longer be cured by conventional therapy of any type. The precise molecular events that lead from androgen-sensitive prostate cancer to androgen-refractory prostate cancer are, therefore, of great interest. This review seeks to identify specific molecular events that may be linked directly to the progression to androgen-refractory cancer. Some of the mechanisms appear to involve the androgen receptor (AR) directly and include mutations in, or amplification of, the AR gene in a manner that allows the AR to respond to low doses of androgens, other steroids, or antiandrogens. In a less direct manner, coactivators may increase the sensitivity of the AR to androgens and even other nonandrogenic substances through a number of mechanisms. Additional indirect mechanisms that do not result from mutation of the AR may involve activation of the AR by peptide growth factors or cytokines or may involve bypassing the AR entirely via other cellular pathways. Identification of the role of these mechanisms in the progression to androgen-refractory prostate cancer is critical for developing therapies capable of curing this disease.
Asunto(s)
Andrógenos/metabolismo , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Animales , Progresión de la Enfermedad , Sustancias de Crecimiento/metabolismo , Humanos , Masculino , Mutación , Neoplasias Hormono-Dependientes/genética , Próstata/metabolismo , Próstata/patología , Receptores Androgénicos/genética , Transducción de SeñalRESUMEN
Steroid hormones, via their binding to specific receptors, are involved in the development, differentiation, and physiological response of cells to diverse stimuli. Activation by hormonal ligands induces conformational change in the receptor, enabling interaction with the target genes. The steroid receptor superfamily includes androgen, glucocorticoid, mineralocorticoid, progesterone, estrogen, thyroid, vitamin D, retinoic acid, and orphan receptors. This review will focus on the classic steroid receptors, i.e., the androgen, glucocorticoid, progesterone, and estrogen receptors, with emphasis on their transcriptional regulation. Readers are directed to several authoritative reviews for further details of steroid receptors (1-11).
Asunto(s)
Regulación de la Expresión Génica/fisiología , Receptores de Esteroides/genética , Animales , Humanos , Transcripción Genética/fisiologíaRESUMEN
In vitro studies were conducted to determine whether conditioned medium from rat fetal urogenital sinus explants would affect phenotypic characteristics of NBT-II urinary bladder carcinoma cells in culture. NBT-II cells were exposed to medium (30%, v/v) conditioned for 48 h by intact urogenital sinus explants derived from 18-day fetal rats. Upon exposure for 23 h the [3H]thymidine incorporation by NBT-II cells was decreased by 40.3% relative to control cultures. This effect was paralleled by a similar decrease in proliferation. NBT-II cultures decreased in cell number by 32.1 and 45.8% on days 2 and 4, respectively, after exposure to conditioned medium. Although cell proliferation was inhibited, conditioned medium acted to induce an increase in protein secretion. An increase of 18.6% was observed in the incorporation of [35S]methionine into newly synthesized, secreted proteins by NBT-II cells exposed to conditioned medium for 23 h. Morphologically the NBT-II cells exposed to conditioned medium were larger, more spread out, and exhibited a greater array of lamellipodia and filopodia, although [35S]methionine incorporation into cellular proteins was decreased by 11.1%. These results suggest that diffusable factors produced by fetal urogenital sinus explants can induce changes in proliferation, protein synthesis, protein secretion, and phenotypic morphology of NBT-II carcinoma cells in culture.
Asunto(s)
Neoplasias de la Vejiga Urinaria/patología , Sistema Urogenital/embriología , Animales , División Celular , Células Cultivadas , Medios de Cultivo , ADN/biosíntesis , Sustancias de Crecimiento/fisiología , Biosíntesis de Proteínas , RatasRESUMEN
Proliferating cell nuclear antigen (PCNA) expression is required for DNA replication. Because androgens are critical for prostate cell proliferation, we investigated the effects of androgen on PCNA expression in the prostatic cancer cell line LNCaP. Flow cytometric analysis was used to measure cellular DNA content with dual labeling of PCNA. Semiconfluent LNCaP cells were grown in serum-free medium containing varying concentrations of the synthetic androgen mibolerone and processed for either fluorescence-activated cell sorting or Western analysis. Supplementation of serum-free medium with androgens resulted in dose-dependent changes in PCNA immunoreactivity, with maximum stimulation (2-fold) being achieved at 48 h with 10(-9)M mibolerone. Non-androgenic steroids did not change PCNA immunoreactivity compared with untreated controls, and the antiandrogen, casodex, inhibited the mibolerone-stimulated increase in PCNA immunoreactivity, suggesting that the androgenic induction of PCNA is mediated through the androgen receptor. The presence of a non-consensus androgen response element in the promoter region of the PCNA gene led us to investigate wether androgen responsiveness of the PCNA gene in LNCaP cells might be mediated at the transcriptional level. No change in steady-state mRNA for PCNA with androgen administration was observed. However, an investigation of the androgenic regulation of PCNA protein stability indicated that androgen treatment increased the half-life of 35S-labeled PCNA protein. In addition, polysome run-off translation assays demonstrated an increase in PCNA protein after a 6-h stimulation of LNCaP cells with 10(-9)M mibolerone. These data suggest that androgen induction of prostate cell proliferation may be mediated, at least in part, through PCNA at the posttranscriptional level.
Asunto(s)
Andrógenos/farmacología , Antígeno Nuclear de Célula en Proliferación/análisis , Neoplasias de la Próstata/química , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/patología , Biosíntesis de Proteínas , ARN Mensajero/análisis , Células Tumorales CultivadasRESUMEN
Prostate cancer (PCa) progression is aided by abnormal autocrine growth factor loops. We screened for small cell-permeable inhibitors of receptor tyrosine kinases that could block their signaling and trigger cell death in PCa cell lines. We found that the human epidermal growth factor receptor (HER)-2/neu inhibitor tyrphostin AG825 is preferentially toxic to PCa cells that are phenotypically androgen independent. These effects were dose and time dependent in the human LNCaP, C4, and C4-2 cell line models of progression and correlated with the inhibition of HER-2/neu phosphoactivation and its down-regulation. In addition, we show that the inhibition of HER-2/neu signaling with AG825 triggers an imbalance between extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase activation, which leads to p38-dependent apoptosis. Inhibition of HER-1 with Compound 56 had no effect. These findings suggest that the androgen-independent C4 and C4-2 cells can be killed by selectively inhibiting their HER-2/neu signaling pathway and provide insights into the mechanism of action of AG825 in PCa cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas , Tirfostinos/farmacología , Andrógenos/fisiología , Apoptosis/fisiología , Benzotiazoles , Activación Enzimática , Receptores ErbB/antagonistas & inhibidores , Humanos , Masculino , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Neoplasias Hormono-Dependientes/enzimología , Neoplasias Hormono-Dependientes/patología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/enzimología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptor ErbB-2/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Prostate-specific antigen (PSA) is the most sensitive marker available for monitoring the progression of prostate cancer and response to therapy. In a previous study, we demonstrated tissue-specific expression of PSA glycoprotein and mRNA and its regulation through the androgen receptor. In this study, we examine the effects of protein kinase A (PKA) and protein kinase C (PKC) on the androgen regulation of PSA in a human adenocarcinoma cell line, LNCaP. Northern blot analysis demonstrated that forskolin, an activator of PKA, had no effect on the androgen regulation of PSA. However, the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a direct activator of PKC, showed a time- and dose-dependent repression of the androgen regulation of PSA glycoprotein and mRNA. The biologically inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate, had no effect. Staurosporine, a PKC inhibitor, blocked the TPA-mediated repression of the androgenic stimulation of PSA glycoprotein. In addition, the calcium ionophore, A23187, was able to simulate the actions of TPA, presumably through activation of PKC via calcium mobilization. In summary, the androgenic regulation of PSA protein and mRNA is repressed by tumor-promoting phorbol esters through the PKC pathway. This indicates that the effects of TPA may be secondary to repressed gene transcription or altered mRNA stability. In addition, this study emphasizes that the androgenic regulation of PSA is complex and may involve other extracellular transduction signals.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/metabolismo , Colforsina/farmacología , Nandrolona/análogos & derivados , Neoplasias de la Próstata/metabolismo , ARN Mensajero/metabolismo , Congéneres de la Testosterona/farmacología , Acetato de Tetradecanoilforbol/farmacología , Alcaloides/farmacología , Calcimicina/farmacología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Humanos , Masculino , Nandrolona/antagonistas & inhibidores , Nandrolona/farmacología , Forboles/farmacología , Antígeno Prostático Específico , Estaurosporina , Congéneres de la Testosterona/antagonistas & inhibidores , Células Tumorales CultivadasRESUMEN
p73, a first p53 relative, has recently been identified and demonstrated to be monoallelically expressed. This protein shows significant amino acid sequence and functional similarities to p53. However, it is unclear whether this protein functions as a tumor suppressor. To elucidate the role of p73 in tumor development, we investigated the expression of the p73 gene in lung cancer. In a comparison between normal lung and tumor tissues, p73 was more highly expressed in tumors. Moreover, using a C/T polymorphism in exon 2 for allele-specific expression analysis in 21 pairs of lung tumors and matched normal tissues, we found that five heterozygous samples exclusively expressed both alleles in tumors while showing monoallelic expression in matched normal tissues. This result was confirmed by single-nucleotide primer extension analysis. Mutation analysis of all 13 coding exons of the gene in 21 lung tumor DNAs revealed several polymorphisms, but no tumor-specific mutations were detected. These findings strongly suggest that p73 may play an important role in lung tumorigenesis through activation of a silent allele and overexpression of wild-type p73 rather than as a tumor suppressor.
Asunto(s)
Alelos , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Proteínas Nucleares/genética , Apoptosis , Proteínas de Unión al ADN/biosíntesis , Genes Supresores de Tumor , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/biosíntesis , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Células Tumorales Cultivadas , Proteína Tumoral p73 , Proteínas Supresoras de TumorRESUMEN
Early growth-response (EGR) genes are nuclear transcription factors that are implicated in regulating cell proliferation. Because these genes show divergent expression in various human tumors, we sought to determine their expression in nonmalignant and malignant prostate tissues. Total RNA extracted from prostate tissues was probed with EGR-1, EGR-2, and EGR-alpha cDNA for Northern blots and digoxigenin-labeled cRNA for in situ hybridization. Both Northern blot and in situ hybridization analyses demonstrated increased EGR-1, but not EGR-2 or EGR-alpha expression, in malignant prostate tissue as compared with weak expression in nonmalignant tissue. EGR-1 mRNA was quantified in 96 prostate specimens (86 adenocarcinomas representing different Gleason scores and 10 benign tissues showing no histological manifestation of benign prostatic hypertrophy) using in situ hybridization with an 35S-labeled cRNA probe. EGR-1 mRNA was expressed at significantly higher levels in cancer than in normal prostate (P < 0.001). In cancer with Gleason scores 8-10, the expression of EGR-1 was higher compared with those of lower Gleason scores (P < 0.005). Immunohistochemical staining showed predominately basal cell nuclear EGR-1 protein in prostatic acini. Nuclear staining was weak in nonmalignant tissues, more intense in moderately differentiated carcinoma, and most intense in poorly differentiated carcinoma. These results show that EGR-1 is overexpressed in prostate cancer and suggest a role for EGR-1 in prostate cancer growth.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Inmediatas-Precoces/genética , Neoplasias de la Próstata/genética , Factores de Transcripción/genética , Dedos de Zinc , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/fisiología , Proteína 1 de la Respuesta de Crecimiento Precoz , Proteína 2 de la Respuesta de Crecimiento Precoz , Factores de Transcripción de la Respuesta de Crecimiento Precoz , Humanos , Proteínas Inmediatas-Precoces/biosíntesis , Hibridación in Situ , Factores de Transcripción de Tipo Kruppel , Masculino , Pronóstico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Mensajero/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/fisiologíaRESUMEN
Prostate-specific antigen (PSA) is a member of the kallikrein gene family and is expressed exclusively in human prostatic epithelial cells. PSA protein has been an important biological marker for prostate cancers. Until now, very little was known about the regulation of PSA expression in prostatic cells. In this study, we have developed a specific oligonucleotide probe which recognizes PSA but not the human glandular kallikrein. This is crucial because both PSA and human glandular kallikrein are expressed in the prostate at relatively high levels and have high nucleotide sequence homology (greater than 82%). Utilizing a S-labeled PSA-specific probe, PSA mRNA was localized within the glandular epithelium of the prostate. Northern blot analysis detected a single 1.6-kilobase transcript in LNCaP cells, a cell line derived from a human prostate adenocarcinoma metastasis. Therefore, LNCaP cells were used to study the androgenic effects on PSA mRNA expression. A time course study demonstrated that PSA mRNA was induced by mibolerone (a nonmetabolizable synthetic androgen) and reached maximal levels after 9 h. The induction of PSA mRNA required as little as 0.3 nM mibolerone. In addition to mibolerone, PSA mRNA could be induced by the natural androgen, dihydrotestosterone, but not by the synthetic glucocorticoid, dexamethasone, or the synthetic estrogen, diethylstilbestrol. Moreover, in the presence of dihydrotestosterone, PSA mRNA was depressed by hydroxyflutamide (an antiandrogen). These results suggest strongly that the androgenic effects on PSA mRNA in LNCaP cells may be via the function of the androgen receptor.
Asunto(s)
Adenocarcinoma/inmunología , Andrógenos/farmacología , Antígenos de Neoplasias/genética , Regulación de la Expresión Génica/efectos de los fármacos , Neoplasias de la Próstata/inmunología , ARN Mensajero/análisis , Adenocarcinoma/genética , Secuencia de Bases , Cicloheximida/farmacología , ADN/análisis , Hormonas/farmacología , Humanos , Masculino , Datos de Secuencia Molecular , Antígeno Prostático Específico , Neoplasias de la Próstata/genética , Células Tumorales CultivadasRESUMEN
We developed a highly sensitive splice variant-specific reverse transcriptase-PCR (RT-PCR) assay for human glandular kallikrein (hK2) mRNA and tested its ability to detect metastatic disease in men with clinically localized prostate cancer. An RT-PCR assay using primers spanning intron IV and including a significant portion of the 3' untranslated region of the hKLK2 gene, with maximum nonhomology to both hK1 and hK3, was developed. The limit of detection of the assay was five copies of hK2 cDNA and one LNCaP cell in 10(9) lymphoblasts. RT-PCR-hK2 was performed on preoperative peripheral blood specimens from 228 consecutive radical prostatectomy patients as well as 7 metastatic prostate cancer patients and 14 healthy men without prostate cancer. This new RT-PCR-hK2 assay amplifies two distinct fragments. The larger fragment (hK2-U) is approximately 680 bp in length and corresponds to the amplified product of a previously reported splice variant in the splice donor site of intron IV in the hKLK2 gene. The smaller fragment (hK2-L) is approximately 643 bp in length and corresponds to the amplified product of the native hK2 mRNA. Whereas the RT-PCR-hK2-L assay was positive in 71% of our patients with metastatic prostate cancer, 14% of healthy control men also tested positive. By univariate (P = 0.028) and multivariate (P = 0.0269) analysis, which controlled for preoperative PSA, clinical stage, and biopsy Gleason score, RT-PCR-hK2-L status added prognostic information to the prediction of lymph node-positive disease. We have developed a new RT-PCR assay which demonstrates a high sensitivity for detecting hK2 mRNA. Preoperative RT-PCR-hK2-L status helps predict pathological lymph node positivity in patients with clinically localized prostate cancer.
Asunto(s)
Calicreínas/análisis , Calicreínas/metabolismo , Neoplasias de la Próstata/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto , Anciano , Empalme Alternativo , Secuencia de Bases , Estudios de Casos y Controles , Cartilla de ADN/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Intrones , Metástasis Linfática/diagnóstico , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Análisis Multivariante , Pronóstico , Neoplasias de la Próstata/sangre , ARN Mensajero/metabolismo , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
Causing prostate cancer cells to express functionally active sodium iodide symporter (NIS) by targeted NIS gene transfer might offer the possibility of radioiodine therapy of prostate cancer. Therefore, we investigated radioiodine accumulation and therapeutic effectiveness of 131I in NIS-transfected prostate cancer cells in vitro and in vivo. The human prostatic adenocarcinoma cell line LNCaP was stably transfected with NIS cDNA under the control of the prostate-specific antigen promoter. The stably transfected LNCaP cell line NP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake in vitro that resulted in selective killing of these cells by 131I in an in vitro clonogenic assay. Xenografts were established in athymic nude mice and imaged using a gamma camera after i.p. injection of 500 microCi of 123I. In contrast to the NIS-negative control tumors (P-1) which showed no in vivo uptake of 123I, NP-1 tumors accumulated 25-30% of the total 123I administered with a biological half-life of 45 h. In addition, NIS protein expression in LNCaP cell xenografts was confirmed by Western blot analysis and immunohistochemistry. After a single i.p. application of a therapeutic 131I dose (3 mCi), significant tumor reduction was achieved in NP-1 tumors in the therapy group compared with P-1 tumors and tumors in the control group. In conclusion, a therapeutic effect of 131I has been demonstrated in prostate cancer cells after induction of tissue-specific iodide uptake activity by prostate-specific antigen promoter-directed NIS expression in vitro and in vivo. This study demonstrates the potential of NIS as a novel therapeutic gene for nonthyroidal cancers, in particular prostate cancer.
Asunto(s)
Adenocarcinoma/radioterapia , Proteínas Portadoras/genética , Radioisótopos de Yodo/uso terapéutico , Proteínas de la Membrana/genética , Neoplasias de la Próstata/radioterapia , Simportadores , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Western Blotting , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/metabolismo , Terapia Genética , Humanos , Inmunohistoquímica , Radioisótopos de Yodo/farmacocinética , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Especificidad de Órganos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Transfección , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Human glandular kallikrein (hK2) protein, like prostate-specific antigen (PSA), is produced mainly in prostatic epithelium. It may be useful as a new diagnostic indicator for prostate cancer. Recently, a number of hK2-specific monoclonal antibodies have been developed that enable us to detect hK2 protein in human prostate tissue, seminal fluid, and sera. Whether hK2 can be expressed, like PSA, in nonprostatic cells is not known. In this study, we have characterized the presence of hK2 in an androgen-responsive breast cancer cell line T47-D at both the protein and mRNA levels with an immunoassay, Western blot analysis, Northern blot analysis, and the reverse transcription-PCR. Using a sensitive immunoassay with monoclonal antibodies to hK2, we found that T47-D cells could be induced with androgens, mineralocorticoids, glucocorticoids, and progestins to produce significantly more hK2 than PSA. Estrogens failed to mimic the effect of the other steroids, blocking instead the stimulatory effect of androgens. Androgen induction of hK2 in T47-D cells was dose dependent. More interestingly, we found that the hK2 in androgen-induced T47-D cell spent media appears to be the pro-form of hK2 rather than mature hK2. Our study demonstrates that hK2, a serine protease thought to be found only in prostate-related tissues and fluids, is also produced in a breast cancer cell line T47-D after steroid stimulation. This finding suggests that hK2 may have a potential role in breast cancer as well as prostatic cancer and will be the impetus for further studies of hK2 distribution and function.
Asunto(s)
Neoplasias de la Mama/metabolismo , Calicreínas/metabolismo , Northern Blotting , Western Blotting , Relación Dosis-Respuesta a Droga , Estradiol/farmacología , Femenino , Humanos , Inmunoensayo , Masculino , Antígeno Prostático Específico/metabolismo , Esteroides/farmacología , Calicreínas de Tejido , Células Tumorales CultivadasRESUMEN
Currently, no curative therapy for metastatic prostate cancer exists. Causing prostate cancer cells to express functionally active sodium iodide symporter (NIS) would enable those cells to concentrate iodide from plasma and might offer the ability to treat prostate cancer with radioiodine. Therefore, the aim of our study was to achieve tissue-specific expression of full-length human NIS (hNIS) cDNA in the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP and in subcell lines C4, C4-2, and C4-2b in vitro. For this purpose, an expression vector was generated in which full-length hNIS cDNA coupled to the prostate-specific antigen (PSA) promoter has been ligated into the pEGFP-1 vector (NIS/PSA-pEGFP-1). The PSA promoter is responsible for androgen-dependent expression of PSA in benign and malignant prostate cells and was therefore used to mediate androgen-dependent prostate-specific expression of NIS. In addition, two control vectors were designed, which consist of the pEGFP-1 vector containing the PSA promoter without NIS cDNA (PSA-pEGFP-1) and NIS cDNA without the PSA promoter (NIS-pEGFP-1). Prostate cancer cells were transiently transfected with each of the above-described expression vectors, incubated with or without androgen (mibolerone) for 48 h, and monitored for iodide uptake activity. In addition, stably transfected LNCaP cell lines were established for each vector. Prostate cells transfected with NIS/PSA-pEGFP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake in a range comparable to that observed in control cell lines transfected with hNIS cDNA. Perchlorate-sensitive iodide uptake was not observed in cells transfected with NIS/PSA-pEGFP-1 and treated without androgen or in cells transfected with the control vectors. In addition, prostate cancer cell lines without PSA expression (PC-3 and DU-145) did not show iodide uptake activity when transfected with NIS/PSA-pEGFP-1. Western blotting of LNCaP and C4-2b cell membranes transfected with NIS/PSA-pEGFP-1 using a monoclonal antibody that recognizes the COOH-terminus of hNIS revealed a band with a molecular weight of 90,000 that was not detected in androgen-deprived cells or in cells transfected with the control vectors, as well as a minor band at Mr 150,000 in transiently transfected LNCaP cell membranes. In conclusion, tissue-specific androgen-dependent iodide uptake activity has been induced in prostate cancer cells by PSA promoter-directed NIS expression. This study represents an initial step toward therapy of prostate cancer with radioiodine.
Asunto(s)
Adenocarcinoma/patología , Andrógenos , Proteínas Portadoras/biosíntesis , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Yoduros/farmacocinética , Proteínas de la Membrana/biosíntesis , Nandrolona/análogos & derivados , Neoplasias Hormono-Dependientes/patología , Antígeno Prostático Específico/genética , Neoplasias de la Próstata/patología , Proteínas Recombinantes de Fusión/biosíntesis , Simportadores , Adenocarcinoma/metabolismo , Proteínas Portadoras/genética , Membrana Celular/química , ADN Complementario/genética , Vectores Genéticos/genética , Humanos , Masculino , Proteínas de la Membrana/genética , Nandrolona/farmacología , Neoplasias Hormono-Dependientes/metabolismo , Especificidad de Órganos , Neoplasias de la Próstata/metabolismo , Proteínas Recombinantes de Fusión/genética , Transfección , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Human kallikrein (hK) 2 is an arginine-selective serine protease expressed predominantly in the prostate that has an 80% sequence identity with prostate-specific antigen. Expression of hK2 is elevated in the tumor epithelium compared to benign prostate tissue. We have purified, sequenced, and identified a novel hK2 complex in prostate tissue consisting of hK2 and a serine protease inhibitor known as protease inhibitor-6 (PI-6). This 64-kDa SDS-PAGE stable complex is elevated in the tumor and is approximately 10% of total hK2. No comparable complex of prostate-specific antigen was detected. PI-6, also known as cytoplasmic antiprotease, has been characterized as an intracellular inhibitor of trypsin and chymotrypsin-like proteases, which has high homology to plasminogen activator inhibitor 1 and 2. The physiological role of PI-6 in the prostate and its relationship to hK2 and prostate cancer are under investigation.
Asunto(s)
Calicreínas/metabolismo , Neoplasias de la Próstata/metabolismo , Serpinas/metabolismo , Biomarcadores de Tumor , Citoplasma/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/patología , Unión Proteica , Inhibidores de Serina Proteinasa/metabolismo , Calicreínas de TejidoRESUMEN
p73, a protein that has substantial structural and functional similarity to p53, has recently been identified. It was found to be monoallelically expressed in all cell lines and normal individuals tested. To elucidate its role in cancer development and as a potential imprinted tumor suppressor, we investigated the allele-specific expression of the human p73 gene in 28 cases of renal cell carcinoma and its imprinting status in fetal pancreatic and thymic tissues. Of 12 informative pairs of renal cell carcinoma and matched normal tissues identified by StyI restriction fragment length polymorphism (RFLP) in exon 2, p73 showed monoallelic expression in 11 out of 12 normal tissues but biallelic expression in 8/12 and switched allele expression in 2/12 of the matched corresponding cancers. An imprinting study of the p73 gene in two families using a newly identified exonic BanI RFLP indicated that expression of p73 was limited to the maternal allele in RNA from fetal pancreas and thymus, demonstrating that p73 is imprinted in at least these two tissues. These findings strongly suggest that loss of imprinting or switching of allelic expression of the p73 gene is associated with the development of renal cell carcinoma.
Asunto(s)
Alelos , Carcinoma de Células Renales/genética , Proteínas de Unión al ADN/genética , Genes Supresores de Tumor , Impresión Genómica , Neoplasias Renales/genética , Proteínas Nucleares/genética , Femenino , Humanos , Masculino , Linaje , Proteína Tumoral p73 , Proteínas Supresoras de TumorRESUMEN
p73, a first p53 relative, was recently identified and shown to be monoallelically expressed in a number of different human tissues. To determine the potential role of this gene in human bladder cancer, we investigated p73 expression levels, allelic expression patterns, and analysed p73 mutations in 23 unselected primary invasive bladder cancers with matched normal tissues and in seven bladder cancer cell lines. In a comparison between normal and tumor tissues using quantitative RT-PCR analysis, we found that p73 was overexpressed in 22/23 bladder cancers, sometimes as great as 20-fold. Allelic expression analysis using a C/T polymorphism in exon 2 and a newly identified T/C polymorphism in exon 5 revealed that p73 was biallelically expressed in both normal bladder and cancer tissues, suggesting that p73 is not imprinted in bladder tissue. Mutation screening of the p73 gene in bladder cancer DNAs using denaturing high-performance liquid chromatography analysis and DNA sequencing revealed no tumor-specific mutations in any coding exons of the p73 gene. These data suggest that the p73 is unlikely to be a tumor suppressor gene, but that overexpression of p73 may contribute to tumorigenesis in bladder cancer.
Asunto(s)
Carcinoma de Células Transicionales/genética , Proteínas de Unión al ADN/biosíntesis , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Proteínas Nucleares/biosíntesis , Neoplasias de la Vejiga Urinaria/genética , Alelos , Carcinoma de Células Transicionales/metabolismo , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Proteínas de Unión al ADN/genética , Exones/genética , Genes Supresores de Tumor , Humanos , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de Restricción , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Tumoral p73 , Proteínas Supresoras de Tumor , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
A putative tumor suppressor, PTEN/MMAC1 gene at 10q23 was recently identified and found to be mutated in many different human tumors. To determine the role of the PTEN/MMAC1 gene in lung cancer, we screened 34 small cell lung cancer (SCLC) cell lines, 10 SCLC tumors, 13 non-small cell lung cancer (NSCLC) cell lines and 10 NSCLC tumors using Denaturing HPLC (DHPLC) and direct sequencing methods. In SCLC, six (18%) of the cell lines and one of the primary tumor samples (10%) showed alterations of the PTEN/MMAC1 gene including point mutations, small fragment deletions, and homozygous deletions. All of the point mutations and small fragment deletions were observed in hemizygously deleted cell lines. In contrast to SCLC, none of the NSCLC tumors or cell lines had mutations in the PTEN/MMAC1 gene. These data indicate that PTEN/MMAC1 mutations contribute to the pathogenesis and neoplastic evolution in SCLC but not in NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Pequeñas/genética , Genes Supresores de Tumor , Neoplasias Pulmonares/genética , Mutación , Monoéster Fosfórico Hidrolasas , Proteínas Tirosina Fosfatasas/genética , Proteínas Supresoras de Tumor , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Pequeñas/patología , Exones , Humanos , Neoplasias Pulmonares/patología , Fosfohidrolasa PTEN , Empalme del ARN , Células Tumorales CultivadasRESUMEN
PIP: Most antiandrogens appear to act by binding to the androgen receptor and competitively inhibiting the binding of testosterone and cihydrotestosterone to the receptor. Focusing on those compounds which appear to inhibit androgen receptor mediated responses, this review discusses the chemistry of those antiandrogens which have been studied to the extent that their mechanism of action is at least partially understood, outlines the mechanism of androgen action as it is currently understood and suggests how antiandrogens might fit in with this mechanism, indicates the major metabolites of several important antiandrogens, and discusses the clinical applications of several antiandrogens. Cyproterone acetate has been studied extensively as a potential male contraceptive. Although it was recognized that 100 mg of cyproterone acetate per day inhibited spermatogenesis, that dose also reduced libido and potency. Following the administration of 10 or 20 mg of cyproterone acetate per day to 15 males for 26 weeks, the following observations were made: the number of motile sperm was reduced; the quality of their motion was impaired; and the ability of the sperm to penetrate cervical mucus was decreased. Sperm density was also suppressed, but neither it nor sperm motility were inhibited to the extent necessary for contraception. Antiandrogens have been demonstrated to be beneficial in treating 5 clinical syndromes or diseases: acne, seborrhea, hirsutism with or without menstrual abnormalities; precocious puberty; benign prostatic hypertrophy; cancer of the prostate; and sexual deviates. Since 3 of these conditions are very common, effective and safe treatment would have a large market. At this time, antiandrogens are widely used in Europe for treatment of seborrhea, acne, and hirsutism and a large Veterans Administration Cooperative Study in the US was approved but has not yet been funded to compare antiandrogens with other treatments for cancer of the prostate. Studies to assess antiandrogen interaction with other hormones or drugs have been limited. Side effects in the female have been best evaluated when cyproterone acetate was administered in combination with ethinyl estradiol. In 46 women followed over 317 cycles, side effects were similar to those reported with estrogen-progestin contraceptives. Administration of 10-20 mg of cyrproterone acetate per day to males caused no significant side effects, but 100 mg or more/day has caused loss of libido, impotence, gynecomastia, tiredness, weakness, decreased efficiency, weight gain, drying and desquamation of skin over the legs, and loss of hair on the trunk and pubic area.^ieng
Asunto(s)
Antagonistas de Andrógenos , Receptores Androgénicos/metabolismo , Receptores de Esteroides/metabolismo , Esteroides , Anomalías Inducidas por Medicamentos , Acné Vulgar/tratamiento farmacológico , Antagonistas de Andrógenos/análisis , Antagonistas de Andrógenos/metabolismo , Antagonistas de Andrógenos/uso terapéutico , Antagonistas de Andrógenos/toxicidad , Animales , Unión Competitiva , Fenómenos Químicos , Química , Anticonceptivos Masculinos , Femenino , Hirsutismo/tratamiento farmacológico , Humanos , Masculino , Hiperplasia Prostática/tratamiento farmacológico , Neoplasias de la Próstata/tratamiento farmacológico , Pubertad Precoz/tratamiento farmacológico , Receptores Androgénicos/análisis , Disfunciones Sexuales Fisiológicas/tratamiento farmacológico , Esteroides/análisis , Esteroides/metabolismo , Esteroides/uso terapéutico , Esteroides/toxicidadRESUMEN
The androgen receptor (AR) mediates the biological functions of androgens and is essential for normal growth and differentiation of urogenital organs as well as initiation and maintenance of spermatogenesis. Withdrawal of androgens by castration or other methods has been shown to cause a marked, although often temporary, regression of many prostate cancers. In order to gain a better understanding of the transcriptional regulation of the AR, a series of truncation mutants derived from the 5'-region of the mouse AR (mAR) were inserted into the promoter-less plasmid pBLCAT3 and transiently expressed in the mouse alpha T3-1 and GT1-7 cell lines. The results of these experiments indicate the presence of a negative regulatory element in the 5'-untranslated region of the gene, which is able to reduce chloramphenicol acetyltransferase (CAT) activity by 77-89%. We have named this element the mAR suppressor (mARS). DNase-I protection assays of the 5'-untranslated region disclosed a protected domain. Gel mobility assays using the mARS revealed the presence of three protein-DNA complexes that could specifically bind to this protected domain. Insertion of the mARS into the thymidine kinase promoter containing pBLCAT2 vector resulted in a 2- to 10-fold decrease in CAT activity, but only if the insert was 3' to the start of transcription initiation. Finally, point mutations within the mARS were able to increase transcription of the AR promoter by 2.3-fold. The results of these experiments indicate that the mAR 5'-untranslated region contains a suppressor element.(ABSTRACT TRUNCATED AT 250 WORDS)