RESUMO
Prostate tumors are initially dependent on androgens for growth, but the majority of patients treated with anti-androgen therapy progress to androgen-independence characterized by resistance to such treatment. This study investigates a novel role for filamin A (FlnA), a 280 kDa cytoskeletal protein (consisting of an actin-binding domain (ABD) followed by 24 sequential repeats), in androgen-independent (AI) growth. Full-length FlnA is cleaved to 170 kDa (ABD+FlnA1-15) and 110 kDa fragments (FlnA16-24); the latter is further cleaved to a 90 kDa fragment (repeats 16-23) capable of nuclear translocation and androgen receptor (AR) binding. Here, we demonstrate that in androgen-dependent LNCaP prostate cancer cells, the cleaved 90 kDa fragment is localized to the nucleus, whereas in its AI subline C4-2, FlnA failed to cleave and remained cytoplasmic. Transfection of FlnA16-24 cDNA in C4-2 cells restored expression and nuclear localization of 90 kDa FlnA. Unlike LNCaP, C4-2 cells proliferate in androgen-reduced medium and in the presence of the AR-antagonist Casodex. They also exhibit increased Akt phosphorylation compared to LNCaP, which may contribute to their AI phenotype. Nuclear expression of 90 kDa FlnA in C4-2 cells decreased Akt phosphorylation, prevented proliferation in androgen-reduced medium and restored Casodex sensitivity. This effect was inhibited by constitutive activation of Akt indicating that FlnA restored Casodex sensitivity in C4-2 cells by decreasing Akt phosphorylation. In addition, FlnA-specific siRNA which depleted FlnA levels, but not control siRNA, induced resistance to Casodex in LNCaP cells. Our results demonstrate that expression of nuclear FlnA is necessary for androgen dependence in these cells.
Assuntos
Antagonistas de Androgênios/farmacologia , Anilidas/farmacologia , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Proteínas Contráteis/farmacologia , Proteínas dos Microfilamentos/farmacologia , Nitrilas/farmacologia , Neoplasias da Próstata/patologia , Compostos de Tosil/farmacologia , Androgênios/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Filaminas , Humanos , Cinética , Masculino , Proteína Oncogênica v-akt/metabolismo , Fragmentos de Peptídeos/farmacocinética , Fragmentos de Peptídeos/farmacologia , Fosforilação , Receptores Androgênicos/efeitos dos fármacos , Receptores Androgênicos/fisiologiaRESUMO
In kidney recipients, the immunosuppressant sirolimus has been associated with a decreased incidence of de novo posttransplant malignancies (including prostate cancer). But the effect of sirolimus on the prostate-specific antigen (PSA) blood level, an important prostate cancer screening tool, remains unknown. We studied male kidney recipients >50 years old (transplanted from January 1994 to December 2006) without clinical evidence for prostate cancer. Pre- and posttransplant PSA levels were analyzed for 97 recipients (n = 19 on sirolimus, n = 78 on tacrolimus [control group]). Pretransplant PSA was similar for sirolimus versus tacrolimus recipients (mean, 1.8 versus 1.7 ng/mL, p = 0.89), but posttransplant PSA was significantly lower for recipients on sirolimus (mean, 0.9 versus 1.9 ng/mL, respectively, p < 0.001). The mean difference between pretransplant and posttransplant PSA was -0.9 ng/mL (50.0%, p = 0.006) for the sirolimus group versus +0.2 ng/mL (+11.8%, p = 0.24) for the tacrolimus group. By multivariate analysis, only pretransplant PSA and immunosuppression with sirolimus independently impacted posttransplant PSA. Our data strongly suggest that sirolimus is associated with a significant PSA decrease in kidney recipients. Future studies must investigate the clinical implications of our findings for the use of PSA for prostate cancer screening in male kidney recipients on sirolimus.
Assuntos
Imunossupressores/farmacologia , Transplante de Rim/fisiologia , Antígeno Prostático Específico/sangue , Sirolimo/farmacologia , Transplante , Estudos de Casos e Controles , Humanos , Imunossupressores/uso terapêutico , Transplante de Rim/imunologia , Masculino , Programas de Rastreamento/métodos , Pessoa de Meia-Idade , Análise Multivariada , Neoplasias da Próstata/sangue , Neoplasias da Próstata/diagnóstico , Estudos Retrospectivos , Sirolimo/uso terapêutico , Tacrolimo/farmacologia , Tacrolimo/uso terapêuticoRESUMO
The antiandrogen bicalutamide is widely used in the treatment of advanced prostate cancer (PCa) in many countries, but its effect on castration-resistant PCa (CRPC) is limited. We previously showed that resistance to bicalutamide results from activation of mechanistic target of rapamycin (mTOR). Interestingly, clinical trials testing combinations of the mTOR inhibitor RAD001 with bicalutamide were effective in bicalutamide-naïve CRPC patients, but not in bicalutamide-pretreated ones. Here we investigate causes for their difference in response. Evaluation of CRPC cell lines identified resistant vs sensitive in vitro models, and revealed that increased eIF4E(S209) phosphorylation is associated with resistance to the combination. We confirmed using a human-derived tumor xenograft mouse model that bicalutamide pre-treatment is associated with an increase in eIF4E(S209) phosphorylation. Thus, AR suppressed eukaryotic initiation factor 4E (eIF4E) phosphorylation, while the use of antiandrogens relieved this suppression, thereby triggering its increase. Additional investigation in human prostatectomy samples showed that increased eIF4E phosphorylation strongly correlated with the cell proliferation marker Ki67. Small interfering RNA-mediated knockdown (k/d) of eIF4E-sensitized CRPC cells to RAD001+bicalutamide, whereas eIF4E overexpression induced resistance. Inhibition of eIF4E phosphorylation by treatment with CGP57380 (an inhibitor of mitogen-activated protein kinase-interacting serine-threonine kinases MAP kinase-interacting kinase 1 (Mnk1/2), the eIF4E upstream kinase) or inhibitors of extracellular signal-regulated kinase 1/2 (ERK1/2), the upstream kinase-regulating Mnk1/2, also sensitized CRPC cells to RAD001+bicalutamide. Examination of downstream targets of eIF4E-mediated translation, including survivin, demonstrated that eIF4E(S209) phosphorylation increased cap-independent translation, whereas its inhibition restored cap-dependent translation, which could be inhibited by mTOR inhibitors. Thus, our results demonstrate that while combinations of AR and mTOR inhibitors were effective in suppressing tumor growth by inhibiting both AR-induced transcription and mTOR-induced cap-dependent translation, pre-treatment with AR antagonists including bicalutamide increased eIF4E phosphorylation that induced resistance to combinations of AR and mTOR inhibitors by inducing cap-independent translation. We conclude that this resistance can be overcome by inhibiting eIF4E phosphorylation with Mnk1/2 or ERK1/2 inhibitors.
Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Anilidas/administração & dosagem , Compostos de Anilina/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Western Blotting , Linhagem Celular Tumoral , Everolimo/administração & dosagem , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos Nus , Nitrilas/administração & dosagem , Fosforilação/efeitos dos fármacos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Purinas/administração & dosagem , Serina/metabolismo , Sirolimo/administração & dosagem , Serina-Treonina Quinases TOR/antagonistas & inibidores , Compostos de Tosil/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Carcinoma of the prostate is the second most common cause of cancer deaths in men. Little is known about the pathogenesis of this disease and the molecular genetic events that contribute to its development. Molecular studies have begun to reveal biologic characteristics of this disease, notably, the loss of genetic material as determined by studies of restriction fragment length polymorphism, oncogene activation, and production and response to growth factors. PURPOSE: Our goal was to characterize p53 gene mutations in human carcinoma of the prostate and to analyze base-pair changes within the coding regions of p53 mRNA (exons 4 through 11). METHODS: Forty-four prostate tissue specimens and four metastatic lesions were obtained from 48 prostate carcinoma patients who had surgical resection. RNA was either immediately extracted or the specimens were snap-frozen in liquid N2 and stored at -70 degrees C until used. Total RNA was extracted from tumor specimens. Expression of p53 was analyzed by polymerase chain reaction (PCR) analysis of mRNA (RNA/PCR). Following confirmation of the RNA/PCR products by Southern blotting, quantitation of message levels was performed by laser densitometry. Absolute area integrations of the curves representing each tissue were then compared after adjustment for the housekeeping gene c-N-ras. Two overlapping regions (exons 4-6 and 6-11) were examined by a nonisotopic PCR single-strand conformation polymorphism (SSCP) analysis system. All specimens displaying SSCP abnormalities were sequenced in both directions to confirm the findings. RESULTS: Of the 48 prostate specimens, three (6%) (two primary and one metastatic) displayed nearly undetectable expression of p53 mRNA (samples PS-70, L113, and PS-95) and 17 (35%) of 48 expressed mutant p53 mRNA encoding amino acid substitutions within exons 4-11 (14 of 17) and/or deletions within the p53 transcripts (three of 17). Overall, the frequency of p53 gene abnormalities that would result in altered protein expression was 20 (42%) of 48 in the tissue samples from prostate carcinoma patients. Nucleotide base-pair transitions of A-->G or T-->C were the most frequent. CONCLUSIONS: p53 mutations are common in prostate cancer. The patterns of p53 gene mutations are dramatically different from data obtained on other cancers and indicate the possible involvement of a carcinogenic agent(s). IMPLICATIONS: Further studies are required to determine the biologic role of p53 gene alterations in the development and progression of this disease and to determine whether p53 mutations can be useful as prognostic markers or for the selection of better treatments for prostate cancer patients.
Assuntos
Carcinoma/genética , Genes p53 , Neoplasias da Próstata/genética , Sequência de Bases , Primers do DNA/química , Humanos , Masculino , Dados de Sequência Molecular , Mutação Puntual , Hiperplasia Prostática/genética , RNA Mensageiro/genética , RNA Neoplásico/genéticaRESUMO
BACKGROUND: The reported frequency of mutation of the p53 tumor suppressor gene (also known as TP53) in human carcinomas of the prostate has varied widely, ranging from 3% to 42%. This variability may be a consequence of tumor heterogeneity and/or the use of different methods of analysis. Since p53 mutation has been associated with clinical outcome for a number of cancer types, determination of its true frequency in primary carcinomas of the prostate is important. PURPOSE: The principal aims of this study were as follows: 1) to validate the utility of detecting p53 gene mutations by means of polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis of complementary DNA (cDNA) (synthesized from prostate tissue RNA and 2) to study the concordance of RNA- and DNA-based PCR-SSCP assays in detecting p53 mutations in individual tumor fragments. METHODS: RNA and genomic DNA were isolated by means of standard techniques from specimens of 19 carcinomas of the prostate, selected on the basis of p53 data obtained in a previous analysis of cDNA (indicating that 14 were mutant and five were wild-type). RNA was converted into cDNA by means of reverse transcription (RT); the cDNA was then amplified by means of nonisotopic (i.e., nonradioactive) PCR, and the PCR products were subjected to SSCP analysis in polyacrylamide gels (RT-PCR-SSCP analysis). Genomic DNA was examined by means of SSCP analysis of isotopically labeled (32PO4) PCR products (DNA-PCR-SSCP analysis). In both approaches, the protein coding region of the p53 gene was divided into multiple, smaller fragments for study. PCR products exhibiting abnormal migration in SSCP gels were subjected to direct nucleotide sequencing or to cloning and sequencing of multiple clones. RESULTS: RT-PCR-SSCP and DNA-PCR-SSCP identified p53 gene abnormalities in 15 of the 19 selected carcinomas, including one previously reported to be wild-type for p53. Overall, PCR-SSCP analysis identified 18 p53 fragments with abnormalities; three carcinomas showed two abnormalities each. Six (33%) of the 18 abnormalities were detected by both RT-PCR-SSCP and DNA-PCR-SSCP, 10 (56%) were detected by RT-PCR-SSCP alone, and two (11%) were detected by DNA-PCR-SSCP alone. The 18 abnormalities were caused by 20 changes in the sequence of the p53 gene; in one carcinoma, double mutations in two individual p53 exons were identified. CONCLUSIONS AND IMPLICATIONS: PCR-SSCP analysis of both RNA and DNA allows the detection of more mutations than the analysis of either alone. Some primary carcinomas of the prostate contain more than one altered p53 gene, consistent with the possibility of intratumoral heterogeneity of mutation of this gene. For comprehensive analysis of p53 mutations in carcinomas of the prostate, and perhaps in other tumor tissues, SSCP analysis of cDNA should be used in combination with SSCP analysis of genomic DNA.
Assuntos
DNA Complementar/análise , DNA de Neoplasias/análise , Genes p53/genética , Mutação , Polimorfismo Conformacional de Fita Simples , Neoplasias da Próstata/genética , RNA Neoplásico/análise , Genoma Humano , Humanos , Masculino , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
Although oncogenically activated ras alleles are common in some types of human cancers, the frequency in human carcinoma of the prostate (CaP) has not previously been addressed. In this paper, we report a comprehensive screening of 19 CaPs and 3 CaP cell lines for activating point mutations in the sequences of the 12th and 61st codons of c-Ha-ras-1 and the c-Ki-ras-2 genes, as well as the 12th, 13th, and 61st codons of the c-N-ras gene. The 19 CaPs were chosen to represent a wide range of stages (B through D), Gleason scores (3 through 10), and DNA ploidy (diploid with low proliferation to nontetraploid-aneuploid). Fifteen of the tumors had been untreated prior to resection and 4 were obtained postradiation therapy. The polymerase chain reaction was used to amplify genomic DNA sequences and transcribed mRNA sequences of the ras genes prior to allele-specific oligonucleotide probe hybridization. We have detected a mutant allele with a point mutation in the second nucleotide of the 61st codon of the c-Ha-ras-1 gene in one specimen. Thus, we conclude that the activation of ras alleles is not significant in the development or progression of most CaPs.
Assuntos
Adenocarcinoma/genética , Alelos , Regulação da Expressão Gênica , Genes ras , Neoplasias da Próstata/genética , Sequência de Bases , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Metástase NeoplásicaRESUMO
Radioimmunotherapy (RIT) in breast cancer patients using I-131-chimeric L6 (ChL6) and in human breast cancer xenografts in nude mice using Y-90-1,4,7,10-tetraazacylododecant N,N',N",N"'-tetraacetic acid-peptide ChL6 (Y-90-ChL6) has shown promise. Tumor cell response to low-dose rate (5-25 rads/h) irradiation from Y-90-ChL6 RIT, therefore, was correlated with levels of tumor cell mRNA for selected genes linked to programmed cell death (apoptosis). Three groups of 10-16 mice with 1-2 HBT 3477 xenograft tumors were treated with 100, 150, or 250 microCi Y-90-ChL6. Three tumors were taken before and two tumors each were taken 3, 6, and 24 h after injection of 150 microCi Y-90-ChL6. Tumor expression of mRNA was amplified by PCR for p53, PIC1, c-myc, and transforming growth factor-beta 1; quantitated; and standardized to N-ras. Tumors received radiation doses of 2000, 3000, and 5000 rads, respectively, for the groups of mice that received 100, 150, and 250 microCi Y-90-ChL6, and tumor regression occurred in each group, with mean tumor volumes decreased by 10, 50, and 95% at nadir after Y-90-ChL6 injection. At the highest dose level, 30% of mice had complete remissions, and no treatment deaths occurred, although tumors subsequently recurred. Continuous up-regulation of transforming growth factor-beta 1 and c-myc mRNA expression was observed from 3 to 24 h after treatment. Expression of p53 and PIC1 increased at 3 h and subsequently decreased to the untreated control levels. These observations are consistent with previous observations of early responses of p53 and PIC1 to cellular DNA damage and subsequent G1 cell cycle arrest or apoptosis. Apoptosis-associated gene expression patterns observed in this tumor model provide evidence that changes are initiated in the first 24 h of RIT associated with radiation doses of 100-700 rads. These preliminary data suggest that insight into the molecular basis of RIT-induced tumor regression may be gained by further studies using different radiation doses.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Apoptose/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/radioterapia , RNA Mensageiro/biossíntese , Radioimunoterapia , Radioisótopos de Ítrio/uso terapêutico , Animais , Sequência de Bases , Neoplasias da Mama/genética , Feminino , Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Transplante de Neoplasias , RNA Mensageiro/genética , Transplante HeterólogoRESUMO
CDKN2 (p16(INK4A)/MTS1) is found to be mutated in a variety of human tumor types. To explore the involvement of CDKN2 in prostate carcinogenesis, alterations of CDKN2 were examined in 116 human prostate tissues and cell lines and xenografts. Markedly reduced expression of CDKN2 mRNA was found in 43% (26 of 60) of untreated primary carcinomas, whereas no alteration was observed in 10 benign prostatic hyperplasias. In 17 matched sets from individual patients, 41% of cancerous tissues in contrast to 6% of noncancerous tissues expressed low levels of CDKN2 mRNA, supporting the role of CDKN2 as a tumor suppressor in prostate cancer. Alteration of CDKN2 was observed in each prostate tumor cell line, including one with a missense mutation, and in one of three xenograft tumor tissues derived from primary carcinomas. Two cell lines (PC-3 and TSU-Pr1) expressed only CDKN2 E1beta transcripts, indicating that the expression of CDKN2 E1alpha and E1beta are under separate control in the prostate. A high level of CDKN2 expression was related to abnormal RB1 in one primary tumor and in the DU145 cell line, which expressed the mutated CDKN2 allele. Analysis of genomic DNA indicated that altered CDKN2 expression in primary carcinomas of the prostate was more frequently due to down-regulation of transcription (five of seven) than deletion of the gene (two of seven). Additionally, CDKN2 mRNA was induced in nonexpressor cell lines by treatment with 5-aza-2'-deoxycytidine. This study demonstrates that alteration of CDKN2 is one of the most frequent genetic abnormalities in prostate cancer and may contribute to prostate carcinogenesis.
Assuntos
Adenocarcinoma/genética , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Regulação Neoplásica da Expressão Gênica , Genes p16 , Proteínas de Neoplasias/biossíntese , Neoplasias da Próstata/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Western Blotting , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/genética , Decitabina , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes do Retinoblastoma , Humanos , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Proteína do Retinoblastoma/biossíntese , Proteína do Retinoblastoma/genética , Transcrição Gênica/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
An androgen receptor (AR) gene mutation identified in the androgen-dependent human prostate cancer xenograft, CWR22, changed codon 874 in the ligand-binding domain (exon H) from CAT for histidine to TAT for tyrosine and abolished a restriction site for the endonuclease SfaNI. SfaNI digestion of AR exon H DNA from normal but not from prostate cancer tissue indicated H874Y is a somatic mutation that occurred before the initial tumor transplant. CWR22, an epithelial cell tumor, expresses a 9.6-kb AR mRNA similar in size to the AR mRNA in human benign prostatic hyperplasia. AR protein is present in cell nuclei by immunostaining as in other androgen-responsive tissues. Transcriptional activity of recombinant H874Y transiently expressed in CV1 cells in the presence of testosterone or dihydrotestosterone was similar to that of wild type AR. With dihydrotestosterone at a near physiological concentration (0.01 nM), H874Y and wild type AR induced 2-fold greater luciferase activity than did the LNCaP mutant AR T877A. The adrenal androgen, dehydroepiandrosterone (10 and 100 nM) with H874Y stimulated a 3- to 8-fold greater response than with wild type AR and at 100 nM the response was similar with the LNCaP mutant. H874Y, like the LNCaP cell mutant, was more responsive to estradiol and progesterone than was wild type AR. The antiandrogen hydroxyflutamide (10 nM) had greater agonist activity (4- to 7-fold) with both mutant ARs than with wild type AR. AR mutations that alter ligand specificity may influence tumor progression subsequent to androgen withdrawal by making the AR more responsive to adrenal androgens or antiandrogens.
Assuntos
Desidroepiandrosterona/farmacologia , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genética , Antagonistas de Androgênios/farmacologia , Animais , Mapeamento Cromossômico , Epitélio/metabolismo , Estradiol/farmacologia , Flutamida/análogos & derivados , Flutamida/farmacologia , Haplorrinos , Humanos , Ligantes , Masculino , Mutação , Progesterona/farmacologia , Receptores Androgênicos/metabolismo , Transcrição Gênica , Ativação Transcricional , Transfecção , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
PURPOSE: To assess the acute toxicity of three-dimensional conformal radiotherapy (3D-CRT) in prostate cancer patients eligible for implant monotherapy. METHODS AND MATERIALS: Between December 1991 and June 1998, 198 prostate cancer patients were treated with 3D-CRT at the University of California Davis Medical Center. Fifty-two of these patients had a prostate-specific antigen (PSA) level = 10.0 ng/ml, Gleason score = 6, and a 1997 AJCC clinical stage T1bN0-T2bN0. Eleven (21%) patients received radiotherapy to the prostate and seminal vesicles; the remaining patients were treated to the prostate only. The 3D-CRT treatment planning guidelines in Radiation Therapy Oncology Group (RTOG) 9406 were followed after 1994 (similar treatment planning was used before the protocol became available). Typically, 4 oblique and 2 lateral fields were treated. All patients were seen at least weekly while under treatment, 1 month postirradiation and then every 3 months. Total radiation doses ranged from 66.0-79.2 Gy, with a median dose of 73.8 Gy in 41 fractions over 8 weeks. Acute toxicity is described according to the RTOG acute toxicity scoring system. RESULTS: Overall, 3D-CRT was well-tolerated: 29% of patients experienced RTOG Grade 1 and 27% experienced Grade 2 acute lower gastrointestinal (GI) toxicity. Forty percent and 33% of patients experienced Grade 1 and 2 acute genitourinary (GU) toxicity, respectively. As expected, more acute morbidity, especially GI, was observed with a larger clinical target volume (prostate and seminal vesicles versus prostate only; p = 0. 05). Neoadjuvant hormonal therapy did not increase the incidence or severity of radiation-induced side effects. No acute toxicity >/= Grade 3, e.g., hourly nocturia, gross hematuria, diarrhea requiring parenteral support, narcotics for pain control, or catheterization for acute urinary retention, was observed. CONCLUSION: Although relatively high doses of radiation are delivered to prostate cancers with 3D-CRT compared with conventional radiotherapy, 3D-CRT is surprisingly well-tolerated. No patients in the cohort eligible for implant monotherapy experienced acute toxicity >/= Grade 3.
Assuntos
Braquiterapia/métodos , Sistema Digestório/efeitos da radiação , Neoplasias da Próstata/radioterapia , Radioterapia Conformacional/efeitos adversos , Transtornos Urinários/etiologia , Doença Aguda , Humanos , Masculino , Dosagem Radioterapêutica , Estudos RetrospectivosRESUMO
Clinical staging and histologic grading do not have sufficient predictive value to determine the response to therapy of any given prostate cancer. A review of the findings from the largest prospective study of patients with localized and locally advanced prostate cancer and from retrospective flow cytometric studies of specific disease stages suggests that DNA flow cytometry offers additional prognostic information for this disease. However, for the individual patient, this added information may have limited value, since approximately 15% of those with diploid disease will experience disease progression within 5 years as compared with half of those with nondiploid disease. We have found that ploidy does not predict length of survival once prostate cancer becomes disseminated, nor does it predict those who will benefit from receiving definitive radiation therapy for localized prostate cancer. On the other hand, for those who have persistent tumor, we have frequently found increased ploidy abnormalities in the tumor sampled after radiation therapy and are currently correlating this finding with clinical outcome. We have also found that DNA flow cytometry can be used to predict tumor volume. For larger, grade-matched diploid tumors, there are significant increases in the proliferation of both the tumor and the adjacent benign tissue, which we take to be evidence of "field effects" in this disease. An even more obvious manifestation of the same phenomenon is seen in the occurrence of aneuploidy in benign tissue near high-grade, large-volume prostate cancer. It is concluded that DNA flow cytometry has much to tell us about the natural history and biologic behavior of prostate cancer.
Assuntos
DNA de Neoplasias/análise , Citometria de Fluxo , Neoplasias da Próstata/patologia , Previsões , Humanos , Masculino , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/terapiaRESUMO
We describe the detection and characterization of an activated c-N-ras allele from a gamma-radiation-induced canine acute nonlymphocytic leukemia (ANLL). The activated allele was detected by use of the NIH3T3 transfection/transformation assay. The leukemia DNA had a transforming activity of 0.0125 foci/microgram. By the use of a double anti-ras antibody enzyme-linked immunoblot assay, we have dissected the lesion within the activated c-N-ras allele. Aspartic acid has been substituted for the normal glycine at position 12 in the activated p21c-N-ras. The expression of the mutant p21ras has also been detected in an in vivo passage of the radiation-induced canine ANLL from which the activated c-N-ras allele was isolated. We have demonstrated sufficient homology between canine c-N-ras genes and the human cDNA c-N-ras clone, p6a1, that allows this probe to be used in Southern blotting of canine tissues. In addition, anti-ras antibodies generated against both murine and human ras antigens are capable of detecting canine p21ras species.
Assuntos
Ácido Aspártico/genética , Códon/genética , Genes ras , Leucemia Mieloide Aguda/genética , Leucemia Induzida por Radiação/genética , RNA Mensageiro/genética , Alelos , Animais , Radioisótopos de Cobalto , Cães , Raios gama , Regulação da Expressão Gênica , Leucemia Mieloide Aguda/etiologiaRESUMO
To correlate molecular changes with clinical information in prostate tissue, it is necessary to have accurate methods for screening for mutations in clinically available specimens. We have refined the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis for detection of p53 mutations in routine pathology specimens. These improvements help overcome technical barriers that interfere with SSCP analysis of archival tissues when only small amounts of poorly preserved formalin-fixed DNA are available. Furthermore, prostate samples are heterogeneous in containing tumor, normal tissue, and hyperplastic tissue. To address the first issue, the method included an initial selection of PCR products using exonuclease I, followed by a second-step selection using nested PCR. This step ensures adequate amplification of the target sequence while minimizing artifactual products that could otherwise interfere with mutation analysis. For the second issue, in addition to morphologic selection of appropriate tissue areas, we improved the sensitivity of detection of mutations by using restriction enzyme digestion of products prior to SSCP analysis. Detection of mutations in heterogeneous tissue was evaluated by determining the minimal detectable mutant allele frequencies in exons 4, 5, 6, 7, 8-9, and 10 by using mixtures of known mutant and wild-type cell lines, which were found to be 17.6, 9.1, 12.5, 8.1 14.0, and 14.3%, respectively. To determine the utility of this method when used on heterogeneous clinical samples, we performed study of 19 archival prostate specimens (14 primary prostate cancers, three benign prostatic hyperplasia and two metastases) and detected abnormally migrating products in six of the prostate cancer specimens (four primaries and two metastases). In five of these samples, there was sufficient DNA to perform sequencing, which disclosed single-base change mutations in all five samples.
Assuntos
Análise Mutacional de DNA/métodos , DNA de Neoplasias/análise , Genes p53/genética , Polimorfismo Conformacional de Fita Simples , Neoplasias da Próstata/genética , Enzimas Reparadoras do DNA , Exodesoxirribonucleases , Humanos , Masculino , Mutação , Reação em Cadeia da Polimerase/métodos , Neoplasias da Próstata/patologia , Células Tumorais CultivadasRESUMO
Benign intrascrotal neoplasms of the testicle account for less than 10 per cent of all intrascrotal masses in postpubertal males. Radical inguinal orchiectomy is standard treatment for a firm, intratesticular mass. Of those patients so treated, 93 per cent of patients with this finding will have had the appropriate operation. There are misleading conditions, however, that can cause firm lesions in the testicle. There is a controversy over whether or not our current technology preoperatively can detect these benign lesions accurately. We encountered five different benign testicular masses within a twelve-month period. We present our findings and comments on some of the difficulties in dealing with these.
Assuntos
Doenças Testiculares/patologia , Neoplasias Testiculares/patologia , Adolescente , Adulto , Diagnóstico Diferencial , Cisto Epidérmico/patologia , Hematoma/patologia , Humanos , Malacoplasia/patologia , Masculino , Mesotelioma/patologia , Orquiectomia , UltrassonografiaRESUMO
The ability to detect and monitor the course of transitional cell carcinoma (TCC) of the bladder using DNA histograms obtained from flow cytometry was studied. Voided urine and barbotage specimens were collected from patients with active TCC or a past history of TCC. These specimens were submitted to routine cytologic and flow cytometric analyses. Samples were considered to be positive if they met one or more of three criteria: if they had aneuploid or tetraploid peaks, if the DNA index of the major G1 peak was shifted more than 10 per cent from that of diploid cells, or if 15 per cent or more of the cells fell to the right of the major diploid G1 cell population thereby constituting a significant hyperdiploid cell population. Using these methods for patients with active disease, the detection rate was 91 per cent. In patients with a past history of TCC, positive histograms preceded the appearance of visible tumor in one third of the cases. Flow cytometry proved to be an excellent way of following patients with a past history of TCC or of screening patients suspected of having active disease. Following this protocol, few biologically active tumors go undetected. However, in 112 patients without a history of bladder cancer, the false positive or suspicious rate was 38 per cent. Before flow cytometry can be recommended as a widespread screening method for patients thought to be at risk of TCC of the bladder developing, this suspicious group will have to be eliminated.
Assuntos
Carcinoma de Células de Transição/diagnóstico , Citometria de Fluxo , Neoplasias da Bexiga Urinária/diagnóstico , HumanosRESUMO
An adult male with acute urinary retention had spontaneous resolution after passage of a proteinaceous clot. He admitted to passing milky urine intermittently over the past twenty years. A history of hydrocele with associated lymphadenitis in a patient who emigrated from Australia was suspicious for a parasitic etiology. Urinalysis, lymphangiography, and positive indirect hemagglutination studies confirmed the diagnosis of filarial chyluria.
Assuntos
Quilo , Filariose/complicações , Transtornos Urinários/etiologia , Adulto , Filariose/fisiopatologia , Humanos , Masculino , UrinaRESUMO
Intravesical mitomycin C is an effective agent for treatment of superficial transitional cell carcinoma of the bladder. A 60 to 70 per cent overall response rate has been reported. Its side effects have been minor. We present a case of severe bladder contracture after intravesical therapy with mitomycin C.
Assuntos
Carcinoma de Células de Transição/tratamento farmacológico , Mitomicinas/efeitos adversos , Doenças da Bexiga Urinária/induzido quimicamente , Neoplasias da Bexiga Urinária/tratamento farmacológico , Administração Intravesical , Contratura/induzido quimicamente , Feminino , Humanos , Pessoa de Meia-Idade , Mitomicina , Mitomicinas/administração & dosagem , Neoplasias da Bexiga Urinária/patologiaRESUMO
Flow cytometry can be performed on testicular aspirates of vasovasostomy candidates preoperatively. On the basis of ploidy ratios and debris components, DNA histograms can be classified as normal or abnormal. Using this method, the likelihood of the presence of sperm may be predicted.
PIP: Of the approximately 10,000 vasectomy reversals performed in the US, 40%-50% of cases do not result in pregnancy. At the time of vasovasostomy, testicular aspirates were taken from 42 men, with a mean age of 38.1 and a mean duration since vasectomy of 10.9 years. DNA histograms were collected from the aspirates by automated flow cytometry. The histograms were classified as normal or abnormal based on the percent of haploid, diploid, and tetraploid cells and on the amount of low-signal debris. 21 (54%) of the 39 men available for analysis had normal ploidies, sperm present at the time of operation, and approximately 5.5% debris. Of the 14 men who had no sperm in the vasal fluid, 11 had abnormal histograms, showing abnormal distribution of ploidies; 5 (35.7%) had high levels of cellular debris. Normal distribution of ploidies, as shown by DNA flow cytometry, and low levels of cellular debris correlate with presence of sperm. Testicular parenchymal aspiration, therefore, provides a rapid, reproducible method for evaluating male fertility status after vasectomy reversal.
Assuntos
DNA/análise , Testículo/análise , Vasovasostomia , Adulto , Autoanticorpos/análise , Líquidos Corporais/análise , Líquidos Corporais/citologia , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Ploidias , Espermatozoides/imunologiaRESUMO
While 80% of transitional cell carcinomas (TCC) present as Ta Tl lesions, they account for only 15% of deaths caused by TCC. We have evaluated the ability of DNA ploidy analysis to predict outcome in 228 patients with Ta Tl TCC. All patients were judged to be at increased risk for tumor recurrence due to having two occurrences of Stage TI tumor within 56 weeks, or three or more tumors presenting simultaneously within 16 weeks of registration. Concurrent carcinoma in situ was acceptable. All patients were treated with either bacillus Calmette Guerin (BCG) immunotherapy or mitomycin-C (MMC) intravesical chemotherapy. Patients with nondiploid tumors had higher hazard rates for both tumor progression and death (p = 0.007 and p = 0.016, respectively); however, the prognostic information of DNA ploidy was not additive to tumor grade.
RESUMO
The overwhelming determining factor in survival for renal cell carcinoma is the total surgical removal of the tumor. Preoperative assessment of tumor stage is critical in determining the surgical approach especially regarding the presence and extent of tumor thrombus. Interoperative techniques are discussed as well as parenchymal sparing procedures and the role of surgery in advanced disease.