Abstract:

BACKGROUND: The relation between circadian dysregulation and cancer incidence and progression has become a topic of major interest over the last decade. Also, circadian timing has gained attention regarding the use of chronopharmacology-based therapeutics. Given its lack of functional T lymphocytes, due to a failure in thymus development, mice carrying the Foxn1(Δ/Δ) mutation (nude mice) have been traditionally used in studies including implantation of xenogeneic tumors. Since the immune system is able to modulate the circadian clock, we investigated if there were alterations in the circadian system of the athymic mutant mice. METHODS: General activity circadian rhythms in 2-4 month-old Foxn1(Δ/Δ) mice (from Swiss Webster background) and their corresponding wild type (WT) controls was recorded. The response of the circadian system to different manipulations (constant darkness, light pulses and shifts in the light-dark schedule) was analyzed. RESULTS: Free-running periods of athymic mice and their wild type counterpart were 23.86 ± 0.03 and 23.88 ± 0.05 hours, respectively. Both strains showed similar phase delays in response to 10 or 120 minutes light pulses applied in the early subjective night and did not differ in the number of c-Fos-expressing cells in the suprachiasmatic nuclei, after a light pulse at circadian time (CT) 15. Similarly, the two groups showed no significant difference in the time needed for resynchronization after 6-hour delays or advances in the light-dark schedule. The proportion of diurnal activity, phase-angle with the zeitgeber, subjective night duration and other activity patterns were similar between the groups. CONCLUSIONS: Since athymic Foxn1(Δ/Δ) mice presented no differences with the WT controls in the response of the circadian system to the experimental manipulations performed in this work, we conclude that they represent a good model in studies that combine xenograft implants with either alteration of the circadian schedules or chronopharmacological approaches to therapeutics.

Author affiliation: Paladino, Natalia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Duhart, José Manuel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Mul Fedele, Malena Lis. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Golombek, Diego Andrés. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Abstract:

El melanoma es una neoplasia que surge como consecuencia de la proliferación descontrolada de los melanocitos. El comportamiento de este tipo tumoral es impredecible dado que puede variar desde la regresión espontánea hasta una rápida progresión y muerte del portador del tumor. El aumento de la incidencia de esta enfermedad se halla entre las más altas, y hasta el momento no se han desarrollado terapias que sean realmente efectivas. Varios estudios, han demostrado que el desarrollo del melanoma de piel es más desfavorable en pacientes hombres que en mujeres, y que la supervivencia de las mujeres es considerablemente mayor. Ha sido teóricamente propuesto que los andrógenos tienen un papel clave en el peor pronóstico masculino, y que una terapia con antiandrógenos podría tenerse en consideración en pacientes hombres con melanoma. En este trabajo de Tesis se investigó, en primer lugar, la presencia de receptores androgénicos (RA) en el melanoma humano, se procedió luego a la caracterización bioquímica y biológica de los mismos y por último se analizó la influencia de la acción antiandrogénica en el curso clinico de esta enfermedad. En los estudios realizados se utilizaron tres lineas derivadas de melanoma humano obtenidas en el laboratorio: IIB-MEL-J, IIB-MEL-LES e IIB-MEL-LAN. Los análisis de Scatchard de las tres lineas estudiadas, utilizando la unión del andrógeno sintético marcado R1881, revelaron la presencia de un solo tipo de receptor con una constante de disociación aparente (Kd) de 11 nM a 37 °C y una capacidad de binding de 326 fmol/mg de proteina, 58,4 fmol/10 6 células o de 35.000 sitios por célula para IIB-MEL-J, una Kd de 15 nM a 37 °C y una capacidad de binding de 280,6 fmol/mg de proteína, 30,8 fmol/10 (6)células o de 18.500 sitios por célula en el caso de IIB-MEL-LES y Kd de 14 nM a 37 °C, capacidad de binding de 206,4 fmol/mg de proteína, 25,59 fmol/10 (6) células o de 15.400 sitios por célula en el caso de IIB-MEL-IAN. Los estudios sobre la especificidad de la unión del ligando radiactivo a estos receptores, revelaron un comportamiento atípico de los mismos en las tres líneas analizadas. No sólo los andrógenos testosterona (T), dihidrotestosterona (DHT), R1881 y el antiandrógeno hidroxi-flutamida (OH-Flu) compitieron con [3H]R188l, sino que estradiol (E2), progesterona (Pg) y cortisol (F), a 500 X de exceso, también fueron capaces de desplazar la unión del esteroide sintético al RA. A nivel inmunocitoquímico, no existieron evidencias de la presencia de receptores estrogénicos o de progesterona en las tres líneas de melanoma, mientras la inmuno- reactividad para RA fue positiva en las tres líneas estudiadas. La tinción nuclear fue vigorosa, y la intensidad marcada, por encima del 70% de las células analizadas. La presencia del RA fue analizada, además, en biopsias correspondientes a metástasis de melanomas provenientes de distintos pacientes. En todos los casos la reactividad obtenida con el anticuerpo monoclonal (AMC) ANI-IS fue positiva y la intensidad marcada. Estudios in vitro sobre la acción de distintas hormonas y antihormonas revelaron que DHT, T, E2, o Pg estimularon la proliferación celular de IIB-MEL-J, IIB-MEL- LES e IIB-MEL-IAN mientras OH-Flu, casodex (Cas) o Tamoxifeno (Tam) la inhibieron significativamente. La acción inhibitoria del crecimiento celular ejercida por los antiandrógenos también se manifestó in vivo cuando se utilizó flutamida (Flu) en ratones atímicos portadores de tumores. Estos animales mostraron una disminución significativa del crecimiento tumoral y un marcado aumento de la sobrevida respecto de animales controles o sometidos a distintas terapias. Los resultados indican la existencia de RA en melanoma humano y un claro efecto protector por parte del antiandrógeno Flu.

Cutaneous malignant melanoma is becoming increasingly important in western countries due to its rising incidence, especially on the white population. A significant difference in survival between male and female patients has been reported, and the proportion of females cured was much greater than that of males. It has been theoretically proposed that an androgen dependency of melanoma could explain the longer survival time in women patients irrespective of the hormonal status of the female host. To evaluate the presence of androgen receptors in human melanoma cell lines, Scatchard analysis was performed in whole cells with [3H]-Rl881. IIB-MEL-J cells in culture revealed a single binding component with an apparent dissociation constant (KD) at 37°C of 11 nM and a binding capacity of 326 fmol/mg protein, 58,4 fmol/10(6) cells or 35.000 binding sites/cell. For IIB-MELLES cell, the KDobtained was 15 nM at 37°C, with a binding capacity of 280,6 fmol/mg total cell protein, 30,8 fmol/10(6) cells, or 18.500 binding sites/cell. IIB-MEL- IAN cells showed a KD of 14 nM at 37°C with a binding capacity of 206,4 fmol/mg total cell protein, 25,59 fmol/10(6) cells, or 15.400 binding sites/cell. Competition analysis revealed an atypical relaxation of specificity, since not only androgen testosterone (T), dihydrotestosterone (DHT), R188] and antiandrogcn hydroxy-flutamide (OH-Flu) competed for [3H]-R1881 binding, but also estradiol (E2), progesterone (Pg), and cortiso] (F) at 500-fold excess concentration. Immunocytochemistry of human melanoma cell lines IIB-MEL-J, IIB-MEL-LES and IIB-MEL-IAN was performed with monoclonal antibody anti-AR ANI-IS. Cells lines were deeply stained,'the intensity of the staining being high in the majority of specimen. When the same cells were tested with monoclonal antibodies raised against estrogen and progesterone receptors, no staining was found. All of the specimens of melanoma metastases that were tested for the presence of AR were deeply stained, the intensity of the staining being high in the majority of the specimens. Several hormones and antihormones were tested for their ability to stimulate cell proliferation on both cell lines with similar results. DHT, T, E2 or Pg stimulate significantly cell proliferation, this increase being reversed by OH-Flu, casodex (Cas) or tamoxifen (Tam). In addition, male and nude mice transplanted with IIB-MEL-J tumors were treated with flutamide (Flu) when tumors were palpable. Flu was effective in diminishing tumor growth and increasing survival rate of animals. As a conclusion, the presence of functional androgen receptors in these cells has been demonstrated by growth inhibition in vitro and in vivo with antiandrogens. In this study, we demonstrate a clear stimulation of cell proliferation by an androgen in human melanoma cell lines. The pure antiandrogen Flu showed an inhibitory effect on the cell growth in nude mice and a remarkable increase in survival rates of nude mice. Binding studies by Scatchard plot and competition studies showed the presence of androgen receptor with atypical sensitivity to different steroids. An interesting consequence of the present results is the possibility of clinical application of antiandrogen treatment to melanoma patients with androgen receptor.

Author affiliation: Morvillo, Verónica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Abstract:

Thymulin is a thymic hormone exclusively produced by the epithelial cells of the thymus. After its discovery and initial characterization in the ‘70s, it was demonstrated that the production and secretion of thymulin are strongly influenced by the neuro-endocrine system. Conversely, a growing body of evidence, to be reviewed here, suggests that thymulin is a hypophysiotropic peptide. Additionally, a substantial body of information pointing to thymulin and a synthetic analog as anti-inflammatory and analgesic peptides in the central nervous system brain and other organs will be also reviewed. In recent years, a synthetic DNA sequence encoding a biologically active analog of thymulin, metFTS, was constructed and cloned in a number of adenovectors. These include bidirectional regulatable Tet-Off vector systems that simultaneously express metFTS and green fluorescent protein and that can be down-regulated reversibly by the addition of the antibiotic doxycycline. A number of recent studies indicate that gene therapy for thymulin may be an effective therapeutic strategy to prevent some of the hormonal and reproductive abnormalities that typically appear in congenitally athymic (nude) mice, used as a suitable model of neuroendocrine and reproductive aging. Summing up, this article briefly reviews the publications on the physiology of the thymulin-neuroendocrine axis and the anti-inflammatory properties of the molecule and its analog. The availability of novel biotechnological tools should boost basic studies on the molecular biology of thymulin and should also allow an assessment of the potential of gene therapy to restore circulating thymulin levels in thymodeficient animal models and eventually, in humans

Author affiliation: Reggiani, Paula Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina

Author affiliation: Schwerdt, José Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina

Author affiliation: Console de Avegliano, Gloria Miriam. Universidad Nacional de La Plata; Argentina

Author affiliation: Roggero, Eduardo Angel. Universidad Abierta Interamericana; Argentina

Author affiliation: Dardenne, Mireille. Université Paris Descartes. Paris; Francia

Author affiliation: Goya, Rodolfo Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina

Abstract:

Objectives: There is clear evidence on the existence of a thymus-pituitary axis which seems to be particularly important during perinatal life. In particular, the thymic peptide thymulin has been shown to be a relevant player in thymuspituitary communication. Our goal was to explore the effect of thymulin on circulating prolactin (PRL) levels in different animal models. To this end we undertook a series of experiments in rats and mice, implementing adult thymectomy, thymulin immunoneutralization in normal C57BL/6 mice and neonatal thymulin gene therapy in nude mice. Methods: We assessed the impact of the above manipulations on PRL secretion and lactotrope morphology by measuring serum PRL by radioimmunoassay and by performing morphometric analysis of the lactotropic cell population in the anterior pituitary gland. Results: Adult thymectomy in female rats slightly increased serum PRL, an effect that was partially reversed by thymulin gene therapy. In mice, thymulin immunoneutralization from birth to age 32 days reduced serum PRL both in males and females. Thymulin immunoneutralization induced a significant (p < 0.01) decrease in lactotrope cell density (CD) and volume density (VD) without changes in cell size (CS). Neonatal thymulin gene therapy markedly increased serum thymulin (p < 0.01) and lactotrope CD, CS and VD in nude mice of both sexes. Conclusions: Our findings suggest a modulatory effect of thymulin on the lactotrope cell population and on serum PRL, particularly during early life.

Author affiliation: Martines, Eliana V.. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; Argentina. Universidad Adventista del Plata; Argentina

Author affiliation: Reggiani, Paula Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; Argentina

Author affiliation: Camihort, Gisela A.. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; Argentina

Author affiliation: Luna, Georgina. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; Argentina

Author affiliation: Zappa Villar, María Florencia. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; Argentina

Author affiliation: Brown, Oscar Alfredo. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; Argentina

Author affiliation: Goya, Rodolfo Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; Argentina

Author affiliation: Console, Gloria M.. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; Argentina