Randomized clinical trial on the use of IMAGE1 S LIGHT (SPIES) vs. white light in the prevention of recurrence during transurethral resection of bladder tumors: Analysis after 12-month follow-up.

INTRODUCTION: The improved image resolution of IMAGE1 S technology will increase tumor detection, achieve a greater number of complete resections, and would probably have an impact on the reduction of recurrences. AIM: The primary objective was to compare the recurrence rates of IMAGE1 S vs. white light during transurethral resection of the bladder (TUR); the secondary objective was to compare the complication rates according to Clavien-Dindo (CD) at 12 months of follow-up. METHODS: Prospective, randomized 1:1, blinded clinical trial. Recurrence and complication rates according to CD were analyzed using chi-square/U Mann-Whitney tests and recurrence-free survival (RFS) using Kaplan-Meier curves. The European Association of Urology (EAU) 2021 scoring model was used. RESULTS: The analysis included 103 participants; 49 were assigned to the IMAGE1 S group and 54 to the white light group. Recurrence rates were 12.2% and 25.9%, respectively (P = .080). The low and intermediate risk group had a lower recurrence rate with IMAGE1 S (7.7% vs. 30.8%, P = .003) and a higher RFS with IMAGE1 S (85.2% vs. 62.8% Log Rank: 0.021), with a Hazard Ratio of 0.215 (95% CI: 0.046-0.925). No differences were observed in the high and very high-risk groups. Complications were mostly grade I and rates were similar between both groups (IMAGE1 S 20.4% vs. white light 7.4% P = .083). CONCLUSIONS: There were no differences in the recurrence rates between groups. However, the low and intermediate risk group had a lower recurrence rate with IMAGE1 S. In addition, perioperative complication rates were not higher.

Non-metastatic castration-resistant prostate cancer: management recommendations.

INTRODUCTION AND OBJECTIVE: Survival and quality of life (QoL) of patients with non-metastatic castration-resistant prostate cancer (nmCRPC) deteriorate significantly when they develop metastases. New generation antiandrogens (apalutamide, enzalutamide and darolutamide) can prolong metastasis-free survival (MFS) and overall survival (OS) in these patients, maintaining their QoL. MATERIAL AND METHODS: After the performance of a systematic review of the literature, a scientific committee reached a consensus on simple and practical recommendations to consolidate and improve the management of patients with nmCRPC in urology consultations. RESULTS: Recommendations are made on the frequency of PSA determination and imaging tests in patients with nmCRPC. The importance of co-morbidities in patients with nmCRPC is also highlighted, and recommendations are also made on functional and QoL assessment that can be carried out during urology consultations. The efficacy, safety, and effects on QoL of new generation antiandrogens are reviewed. CONCLUSIONS: To evaluate treatment of patients with nmCRPC, it is necessary to consider co-morbidities and QoL, in addition to age. New generation antiandrogens are a safe and effective treatment option for patients with nmCRPC. The recommendations of this review can be helpful in optimizing the management of nmCRPC patients in urology consultations.

Initial experience with SelectMDx® in the diagnosis of prostate cancer in a real-world evidence clinical practice setting. / Experiencia inicial del uso de SelectMDx® en el diagnóstico de cáncer de próstata en un entorno de práctica clínica habitual «real-world evidence¼.

INTRODUCTION: The use of biomarkers in the detection of prostate cancer (PC) can decrease overdiagnosis and overtreatment of non-significant PC. We analyze the usefulness and applicability of the SelectMDx® marker in a routine clinical practice setting. MATERIAL AND METHODS: Retrospective study of 48 patients evaluated by the SelectMDx® test between July 2017 and April 2019. Patients were stratified into two groups according to the risk estimated by the clinically significant CP test (CS-PC): <2% or 'very low risk', and >2%. Results were expressed based on previous prostate biopsy (PB) and multi-parametric magnetic resonance imaging (mpMRI) outcomes. RESULTS: Patients with negative PB and normal/doubtful mpMRI had <2% risk in 7/9 cases. Patients without PB and normal/doubtful mpMRI had <2% risk in 12/18 cases, and 2/6 cases with a >2% risk presented CS-PC. Of the 14 patients with no previous PB or mpMRI, 9 had <2% risk, and 2 cases were diagnosed with PC from the group of patients (5) with risk >2%. The number of patients in the remaining subgroups is too small to draw any conclusions. In all cases with pathological digital rectal examination, the test showed a >2% PC risk. CONCLUSION: SelectMDx® is a promising test for detecting patients with a very low risk of CS-PC, especially in patients with suspected PC, with or without negative PB, with normal/doubtful mpMRI. The presence of a pathological digital rectal examination may condition the result of the test.

Evaluation of teleconsultation system in the urological patient during the COVID-19 pandemic. / Evaluación de la teleconsulta en el paciente urológico durante la pandemia COVID-19.

INTRODUCTION: The global pandemic of COVID-19 has led to rapid implementation of telemedicine, but there is little information on patient satisfaction of this system as an alternative to face-to-face care. OBJECTIVE: To evaluate urological patient satisfaction with teleconsultation during the COVID-19 pandemic. MATERIAL AND METHODS: Observational, prospective, cross-sectional, non-interventional study carried out by telephone survey during the period considered as the peak of the pandemic (March-April 2020). A quality survey composed of 11 questions on urological care provided by physicians during the COVID-19 pandemic was conducted, selecting a representative sample of patients attended by teleconsultation. RESULTS: Two hundred patients were contacted by telephone to answer a survey on the quality of teleconsultation. The distribution of patients surveyed among the specialized consultations was homogeneous with the number of consultations cited in the period; 18% of them required assistance from family members. Sixty percent of patients avoided going to a medical center during the pandemic. Of the surveyed patients, 42% had cancelled diagnostic tests, 59% had cancelled medical consultations, 3.5% had cancelled treatments and 1% had cancelled interventions. Ten percent reported a worsening of urological symptoms during confinement. According to physicians, consultations were effectively delivered in 72% of cases, with teleconsultation being carried out by their usual urologist in 81%. Teleconsultation overall satisfaction level was 9 (IQI8-10), and 61.5% of respondents consider teleconsultation as a «health care option¼ after the healthcare crisis. CONCLUSION: Teleconsultation has been evaluated with a high level of satisfaction during the COVID-19 pandemic, offering continuous care to urological patients during the healthcare crisis. The perceived quality offers a field of optional telematic assistance in selected patients, which should be re-evaluated in a period without confinement measures.

Human myoblast fusion requires expression of functional inward rectifier Kir2.1 channels.

Myoblast fusion is essential to skeletal muscle development and repair. We have demonstrated previously that human myoblasts hyperpolarize, before fusion, through the sequential expression of two K+ channels: an ether-à-go-go and an inward rectifier. This hyperpolarization is a prerequisite for fusion, as it sets the resting membrane potential in a range at which Ca2+ can enter myoblasts and thereby trigger fusion via a window current through alpha1H T channels.

Indication for early cystectomy in nonmuscle-invasive bladder cancer. Literature review. / Indicación de cistectomía precoz en el cáncer vesical no músculo infiltrante. Revisión de la literatura.

CONTEXT: High-risk nonmuscle-invasive bladder cancer is a disease that includes a heterogeneous group of patients, for whom close follow-up is recommended due to the risk of progression to a muscle-invasive tumour. The treatment of choice for these tumours is transurethral resection of the bladder tumour followed by a programme of bacillus Calmette-Guerin instillations. There is a subgroup of patients who have a greater risk of progression and who benefit from early radical treatment. OBJECTIVE: To identify which patient group with nonmuscle-invasive bladder cancer will benefit from early radical treatment. SEARCHING THE EVIDENCE: We performed a literature review to identify the risk factors for progression for these patients and thereby recommend a treatment that improves their survival rate. SYNTHESIS OF THE EVIDENCE: We identified the various prognostic factors associated with tumour progression: the persistence of T1 tumour in re-resection of the bladder tumour, the presence of carcinoma in situ, patients refractory to bacillus Calmette-Guerin treatment, patients older than 70 years, tumours larger than 3cm, the substaging of T1 tumours, the presence of lymphovascular invasion and the presence of a tumour in the prostatic urethra. Similarly, we comment on the advantages of radical versus conservative treatment, considering that the performance of an early cystectomy due to a high-risk noninvasive vesical tumour has a better cancer prognosis than those in which the operation is deferred until the progression. CONCLUSIONS: In this disease, it is important to individualise the patients to provide them personalized treatment. For patients with the previously mentioned characteristics, it is recommended that early cystectomy not be delayed.

Methodology of a systematic review. / Metodología de una revisión sistemática.

CONTEXT: The objective of evidence-based medicine is to employ the best scientific information available to apply to clinical practice. Understanding and interpreting the scientific evidence involves understanding the available levels of evidence, where systematic reviews and meta-analyses of clinical trials are at the top of the levels-of-evidence pyramid. ACQUISITION OF EVIDENCE: The review process should be well developed and planned to reduce biases and eliminate irrelevant and low-quality studies. The steps for implementing a systematic review include (i) correctly formulating the clinical question to answer (PICO), (ii) developing a protocol (inclusion and exclusion criteria), (iii) performing a detailed and broad literature search and (iv) screening the abstracts of the studies identified in the search and subsequently of the selected complete texts (PRISMA). SYNTHESIS OF THE EVIDENCE: Once the studies have been selected, we need to (v) extract the necessary data into a form designed in the protocol to summarise the included studies, (vi) assess the biases of each study, identifying the quality of the available evidence, and (vii) develop tables and text that synthesise the evidence. CONCLUSIONS: A systematic review involves a critical and reproducible summary of the results of the available publications on a particular topic or clinical question. To improve scientific writing, the methodology is shown in a structured manner to implement a systematic review.

Cooperation between phosphorylation and acetylation processes in transcriptional control.

We previously reported that the activation of the M promoter of the human choline acetyltransferase (ChAT) gene by butyrate and trapoxin in transfected CHP126 cells is blocked by PD98059, a specific mitogen-activated protein kinase kinase (MEK) inhibitor (E. Espinos and M. J. Weber, Mol. Brain Res. 56:118-124, 1998). We now report that the transcriptional effects of histone deacetylase inhibitors are mediated by an H7-sensitive serine/threonine protein kinase. Activation of the ChAT promoter by butyrate and trapoxin was blocked by 50 microM H7 in both transient- and stable-transfection assays. Overexpression of p300, a coactivator protein endowed with histone acetyltransferase activity, stimulated the ChAT promoter and had a synergistic effect on butyrate treatment. These effects were blocked by H7 and by overexpressed adenovirus E1A 12S protein. Moreover, both H7 and PD98059 suppressed the activation of the Rous sarcoma virus (RSV) and simian virus 40 promoters by butyrate in transfection experiments. Similarly, the induction of the cellular histone H1(0) gene by butyrate in CHP126 cells was blocked by H7 and by PD98059. Previous data (L. Cuisset, L. Tichonicky, P. Jaffray, and M. Delpech, J. Biol. Chem. 272:24148-24153, 1997) showed that the induction of the H1(0) gene by butyrate is blocked by okadaic acid, an inhibitor of protein phosphatases. We now show that the activation of the ChAT and RSV promoters by butyrate in transfected CHP126 cells is also blocked by 200 nM okadaic acid. Western blotting and in vivo metabolic labeling experiments showed that butyrate has a biphasic effect on histone H3 phosphorylation, i.e., depression for up to 16 h followed by stimulation. The data thus strongly suggest that the transcriptional effects of histone deacetylase inhibitors are mediated through the activation of MEK1 and of an H7-sensitive protein kinase in addition to protein phosphatases.

Serpin A1 is overexpressed in ALK+ anaplastic large cell lymphoma and its expression correlates with extranodal dissemination.

Anaplastic large cell lymphoma (ALCL) is a distinct subtype of non-Hodgkin's lymphoma. Most of ALCLs (85%) carry a chromosomal translocation involving different partners in the 5' portion, and the anaplastic lymphoma kinase (ALK) receptor kinase domain in the 3' portion. These translocations induce the ectopic expression of X-ALK proteins, thought to be involved in lymphomagenesis, through the dysregulation of cell proliferation and apoptotic pathways. In the present study, based on several ALK+ and ALK- ALCL cell lines and biopsy specimens, we showed that serpin A1, a secretory glycoprotein, was overexpressed in ALK+ ALCL cell lines and ALK+ tumors at both the transcriptional and translational levels. The crucial role of NPM-ALK in the regulation of serpin A1 expression was further demonstrated by using both ectopic expression and downregulation, by RNA interference, of the NPM-ALK oncogene. In addition, in ALK+ tumors, serpin A1 expression appeared to be correlated with the clinical status of the patients as the serpin A1 mRNA level was higher in patients presenting with extranodal dissemination. These data, together with the pattern of expression of serpin A1 we observed in ALK+ tumors, suggest that serpin A1 has an invasion-promoting effect in ALK+ ALCL.

Cáncer de próstata resistente a la castración no metastásico: recomendaciones de manejo / Non-metastatic castration-resistant prostate cancer: management recommendations

Introducción y objetivo: La supervivencia y calidad de vida (QoL) de los pacientes con cáncer de próstata resistente a la castración no metastásico (CPRCnm) se deteriora de forma muy significativa cuando llegan a desarrollar metástasis. Los antiandrógenos de nueva generación (apalutamida, enzalutamida y darolutamida) pueden prolongar la supervivencia libre de metástasis (SLM) y la supervivencia global (SG) en estos pacientes, manteniendo su QoL.Material y método: Tras una revisión sistemática de la literatura, un comité científico alcanzó un consenso sobre recomendaciones sencillas y prácticas que unifiquen y mejoren el manejo de los pacientes con CPRCnm en las consultas de urología.Resultados: Se dan recomendaciones sobre la frecuencia de determinación de antígeno prostático específico (PSA) y pruebas de imagen en pacientes con CPRCnm. También se destaca la importancia de las comorbilidades en el paciente con CPRCnm y se ofrecen recomendaciones sobre la valoración funcional y de la QoL que se pueden llevar a cabo en la consulta de urología. Se revisa la eficacia, seguridad y efectos sobre la QoL de los antiandrógenos de nueva generación.Conclusiones: Para la evaluación del tratamiento de pacientes con CPRCnm, es necesario tener en cuenta no solo la edad, sino también las comorbilidades y la QoL. Los antiandrógenos de nueva generación son una opción de tratamiento segura y eficaz en los pacientes con CPRCnm. Las recomendaciones de trabajo pueden servir de ayuda para optimizar su manejo de los pacientes con CPRCnm en las consultas de urología. (AU)

Introduction and objective: Survival and quality of life (QoL) of patients with non-metastatic castration-resistant prostate cancer (nmCRPC) deteriorate significantly when they develop metastases. New generation antiandrogens (apalutamide, enzalutamide and darolutamide) can prolong metastasis-free survival (MFS) and overall survival (OS) in these patients, maintaining their QoL.Material and methods: After the performance of a systematic review of the literature, a scientific committee reached a consensus on simple and practical recommendations to consolidate and improve the management of patients with nmCRPC in urology consultations.Results: Recommendations are made on the frequency of PSA determination and imaging tests in patients with nmCRPC. The importance of co-morbidities in patients with nmCRPC is also highlighted, and recommendations are also made on functional and QoL assessment that can be carried out during urology consultations. The efficacy, safety, and effects on QoL of new generation antiandrogens are reviewed.Conclusions: To evaluate treatment of patients with nmCRPC, it is necessary to consider co-morbidities and QoL, in addition to age. New generation antiandrogens are a safe and effective treatment option for patients with nmCRPC. The recommendations of this review can be helpful in optimizing the management of nmCRPC patients in urology consultations. (AU)

[Localized prostate cancer Focal Therapy: "A la carte" Model]. / Tratamiento Focal del cáncer de próstata localizado: Modelo "À la carte".

Focal therapy has settled as an alternative to radical treatment in selected cases of localized prostate cancer. The selection of patients who are candidates for focal therapy is based on imaging diagnosis relying on multiparametric MRI and image fusion techniques. Thanks to the oncological results and safety profiles of initial series, various energy sources have been developed over the last years. The availability of multiple types of energy sources for focal therapy, commits us to evaluate what type of energy would be the optimal depending on patient's profile and type of lesion. A unique energy for focal therapy would be ideal, but facing the research of the various types of energy we must identify which one is recommended for each lesion. With the experience of our center in different approaches of focal therapy we propose the "A LA CARTE" MODEL based on localization of the lesion. We present the criteria the "a la carte" model is based on, supported by the published evidence on the use of different ablative therapies for the treatment of localized prostate cancer. Lesion localization, technical characteristics of each type of energy, patient's profile and secondary effects must be considered in every choice of focal therapy.

High intensity focused ultrasound with Focal-One® device: Prostate-specific antigen impact and morbidity evaluation during the initial experience. / Ultrasonido focalizado de alta intensidad con el dispositivo Focal-One®: impacto sobre el antígeno prostático específico y evaluación de la morbilidad durante la experiencia inicial.

OBJECTIVE: We report our initial experience in the treatment of prostate cancer (PCa) with high-intensity focused ultrasound (HIFU) using the Focal-One® device. MATERIAL AND METHODS: Retrospective review of the prospectively populated database. Between June 2014 to October 2015, 85 patients underwent HIFU (focal/whole-gland) treatment for localized PCa. Preoperative cancer localization was done with multiparametric magnetic resonance imaging (mpMRI) and transperineal mapping biopsies. Treatment was carried out using the Focal-One® device under general anesthesia. Oncological follow-up: PSA measurement and control biopsy with mpMRI according to protocol. Questionnaire-based functional outcome assessment was done. Complications were reported using Clavien classification. RESULTS: The median PSA was 7.79ng/ml (IQR 6.32-9.16), with a median prostate volume of 38cc (IQR: 33-49.75). Focal and whole-gland therapy was performed in 64 and 21 patients respectively. Ten patients received salvage HIFU. Complications were encountered in 15% of cases, all Clavien 2 graded. Mean hospital stay was 1.8 days (0-7) and bladder catheter was removed on day 2 (1-6). Mean percentage reduction of PSA was 54%. Median follow-up was 3 months (IQR: 2-8). Functional outcomes: All patients were continents at 3 months and potency was maintained in 83% of the preoperatively potent. CONCLUSIONS: Focal-One® HIFU treatment appears to be a safe procedure with few complications. Functional outcomes proved no urinary incontinence and sexual function were maintained in 83%.

Establishment of a novel anaplastic large-cell lymphoma-cell line (COST) from a 'small-cell variant' of ALCL.

Anaplastic large-cell lymphoma (ALCL) is a distinct biological and cytogenetic entity with a broad spectrum of morphological features (common type, small-cell variant and lymphohistiocytic variant). Few cell lines of ALCL are available and they all originate from primary tumors demonstrating the common type morphology (ie large-sized lymphoma cells). We established a new ALCL cell line (COST) from the peripheral blood of a patient with a small-cell variant of ALCL, at diagnosis. Cells growing in vitro and in SCID mice consisted of two populations, that is, small- and large-sized cells as seen in the patient's tumor. Both large and small malignant cells were positive for CD43/MT1 T-cell associated antigen, perforin, granzyme B and TIA-1, but negative for CD2, CD3, CD5, CD7, CD4 and CD8 antigens. Standard cytogenetic studies as well as multiplex FISH confirmed the presence of the canonical t(2;5)(p23;q35) translocation, but also revealed additional numerical and structural abnormalities. The COST cell line is the first ALCL small-cell variant cell line, and thus provides a potentially useful tool for further functional and molecular studies that should improve our understanding of the small-cell variant of ALCL, which is more frequently complicated by a leukemic phase.

Evaluación de la teleconsulta en el paciente urológico durante la pandemia COVID-19 / Evaluation of teleconsultation system in the urological patient during the COVID-19 pandemic

INTRODUCCIÓN: La pandemia global de COVID-19 ha provocado una rápida implantación de la telemedicina, pero existe escasa información sobre la satisfacción percibida por el paciente como alternativa a la asistencia presencial. OBJETIVO: Se evalúa la satisfacción del paciente urológico con la teleconsulta durante la pandemia COVID-19. MATERIAL Y MÉTODOS: Estudio observacional, prospectivo transversal, no intervencionista, mediante encuesta telefónica durante el periodo considerado pico de pandemia (marzo-abril 2020). Se realiza una encuesta de calidad compuesta por 11 preguntas sobre la atención urológica durante la pandemia COVID-19 por los facultativos, seleccionando una muestra representativa de los pacientes atendidos en el periodo por teleconsulta. RESULTADOS: Doscientos pacientes fueron contactados telefónicamente para responder a una encuesta de calidad sobre teleconsulta. La distribución de pacientes encuestados entre las consultas monográficas fue homogénea entre el número de consultas citadas en el periodo, requiriendo el 18% de ellos ayuda por familiar. El 60% de los pacientes evitaron acudir a un centro médico durante la pandemia. El 42% de los pacientes encuestados tenían cancelada alguna prueba complementaria, el 59% alguna consulta médica, el 3,5% tratamientos y el 1% intervenciones. El 10% apreciaron un empeoramiento de su sintomatología urológica durante el confinamiento. La resolución subjetiva de la consulta por el facultativo fue alcanzada en el 72% de los casos, siendo la teleconsulta por el urólogo habitual en el 81%. El grado de satisfacción global con la teleconsulta fue de 9 (RIQ 8-10), considerando la teleconsulta como una «opción de asistencia sanitaria» pasada la crisis sanitaria por el 61,5% de los encuestados. CONCLUSIÓN: La teleconsulta ha sido valorada con un alto grado de satisfacción durante la pandemia COVID-19, ofreciendo asistencia continuada a los pacientes urológicos durante la crisis sanitaria. La calidad percibida ofrece un campo de asistencia telemática opcional en pacientes seleccionados, que debe reevaluarse fuera de una situación de confinamiento

INTRODUCTION: The global pandemic of COVID-19 has led to rapid implementation of telemedicine, but there is little information on patient satisfaction of this system as an alternative to face-to-face care. OBJECTIVE: To evaluate urological patient satisfaction with teleconsultation during the COVID-19 pandemic. MATERIAL AND METHODS: Observational, prospective, cross-sectional, non-interventional study carried out by telephone survey during the period considered as the peak of the pandemic (March-April 2020). A quality survey composed of 11 questions on urological care provided by physicians during the COVID-19 pandemic was conducted, selecting a representative sample of patients attended by teleconsultation. RESULTS: Two hundred patients were contacted by telephone to answer a survey on the quality of teleconsultation. The distribution of patients surveyed among the specialized consultations was homogeneous with the number of consultations cited in the period; 18% of them required assistance from family members. Sixty percent of patients avoided going to a medical center during the pandemic. Of the surveyed patients, 42% had cancelled diagnostic tests, 59% had cancelled medical consultations, 3.5% had cancelled treatments and 1% had cancelled interventions. Ten percent reported a worsening of urological symptoms during confinement. According to physicians, consultations were effectively delivered in 72% of cases, with teleconsultation being carried out by their usual urologist in 81%. Teleconsultation overall satisfaction level was 9 (IQI8-10), and 61.5% of respondents consider teleconsultation as a «health care option» after the healthcare crisis. CONCLUSION: Teleconsultation has been evaluated with a high level of satisfaction during the COVID-19 pandemic, offering continuous care to urological patients during the healthcare crisis. The perceived quality offers a field of optional telematic assistance in selected patients, which should be re-evaluated in a period without confinement measures

Efficient non-viral DNA-mediated gene transfer to human primary myoblasts using electroporation.

Gene transfer of human primary myoblasts with various non-viral methods has been hampered by low yield of transfection. We report here an efficient, simple and reproducible non-viral DNA-mediated gene transfer procedure for transfecting human myoblasts. We found that electroporation promotes a highly efficient DNA uptake by human primary cultures of myogenic cells. Under optimal conditions, 60-70% of human myoblasts transfected with the enhanced green fluorescent gene expressed the enhanced green fluorescent protein. Electroporated myoblasts behaved normally as judged by their ability to synthesize and express developmentally regulated proteins and to undergo terminal differentiation, i.e. to fuse and form myotubes. We showed, in addition, that a subpopulation of cultured human myoblasts with self-renewing properties and equivalent to native muscle satellite cells were as efficiently transfected by electroporation as proliferating myoblasts. Thus, the development of gene therapies based on the engineering and transplantation of human myoblasts may greatly benefit from gene transfer by electroporation.

Activation of the MAP kinase cascade by histone deacetylase inhibitors is required for the stimulation of choline acetyltransferase gene promoter.

We previously described that the major promoter (M) of human choline acetyltransferase (ChAT) gene is activated by three inhibitors of histone deacetylase, butyrate, trichostatin and trapoxin, in transfected CHP126 neuroepithelioma cells. We now show that trapoxin and butyrate triggered a rapid and transient phosphorylation of ERK1/2 kinases, that was suppressed by PD98059, a highly specific inhibitor of MAP kinase kinase MEK1. The stimulation of ChAT promoter activity by trapoxin or butyrate did not require ongoing protein synthesis, and was suppressed by PD98059. The overexpression of dominant negative mutants of H-ras or ERK2 proteins depressed ChAT promoter activation by trapoxin in transient transfection assays. Conversely, the overexpression of constitutively active mutants of H-ras or MEK1 proteins had little or no effect on ChAT promoter activity, but strongly synergized with trapoxin. These data thus suggest that the activation of the MEK/ERK kinase cascade plays a necessary, but not sufficient, role in the regulation of ChAT promoter by inhibitors of histone deacetylase.

Histone hyperacetylating agents stimulate promoter activity of human choline acetyltransferase gene in transfection experiment.

Butyrate (5 mM), Trichostatin A (1 microM) or Trapoxin A (30 nM) increased choline acetyltransferase (ChAT) activity in cultured rat sympathetic neurons 3- to 8-fold in 2 days. On the contrary, the three drugs decreased ChAT activity in human CHP126 cells. Butyrate had little effect on ChAT mRNA level in these cells, suggesting post-transcriptional mechanisms for the decrease in ChAT activity. However, transient transfection experiments using CHP126 cells revealed that the M promoter, but not the R promoter, of human ChAT gene was activated 20- to 130-fold by the three hyperacetylating agents. A butyrate-responsive element was localized in the 1 kbp region upstream of exon M. Constructs containing in addition the genomic segment between exons M and 1 displayed maximal basal activity and inducibility by butyrate, suggesting the presence of butyrate-activated promoter/enhancer elements in this region. The stimulatory effects of butyrate and Trichostatin A were also observed in stably transfected CHP126 clones, suggesting that the chromatin environment was not preventing the induction of the endogenous ChAT gene by butyrate. Rather, the data suggest different chromatin organizations for the stable transgene and the endogenous ChAT gene.