RESUMEN
Uncontrolled cell proliferation and inhibition of apoptosis are considered to be vital for cancer initiation, maintenance, infiltration, metastasis and recurrence after anti-cancer therapy. Here we report the generation of a novel cell line by reprogramming child foreskin fibroblast with the full length apoptosis inhibitor gene PIWIL2. The fibroblasts transfected with PIWIL2 expressed the stem cell markers OCT-4, NANOG, SOX-2, KLF-4 and C-MYC; endoderm marker AFP and GATA6; mesoderm markers ACTA2 and BRACHYURY; and ectoderm markers NESTIN and TUBB3. The karyotype was found to be hyperdiploid. The PIWIL2 transfected fibroblast cells grew into tumorous masses within 5 weeks of subcutaneous injection into adult nude mice. Although the injected cell expressed markers for all three germlines, ectoderm, mesoderm, and endoderm, they did not form teratomas in vivo. This study indicates that the PIWIL2 gene could play a key role in cancer induction and maintenance. This method for generating induced tumorigenic cells (ITGC) provides a new research tool to study oncogenesis that in turn may lead to a better understanding of cancer etiology and the development of novel anti-cancer therapies.
Asunto(s)
Proteínas Argonautas/genética , Fibroblastos/metabolismo , Expresión Génica , Células Madre Neoplásicas/metabolismo , Animales , Proteínas Argonautas/metabolismo , Línea Celular Transformada , Transformación Celular Neoplásica/genética , Niño , Fibroblastos/citología , Prepucio/citología , Humanos , Inmunohistoquímica , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones Desnudos , Microscopía Fluorescente , Proteína Homeótica Nanog/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Proteínas Proto-Oncogénicas c-myc/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción SOXB1/genética , Factores de Tiempo , Transfección , Trasplante HeterólogoRESUMEN
To compare KnockOut serum replacement (KSR) and fetal bovine serum (FBS) for the development of germ cells. Testicular tissues from Sprague-Dawley rats were cultured for 4 weeks in culture media supplemented with FBS or KSR. Tissue area was measured at the beginning and end of the culturing period. Testicular histology, development of the germ cells, and the diameter of seminiferous tubules were analyzed by hematoxylin and eosin staining. After 4 weeks in culture, apoptosis and expression of the stage-specific spermatogenesis marker genes Kit, Sycp3, and Crisp1 were assayed. Tissues cultured in KSR-supplemented media were able to sustain growth and gradually increase seminiferous tubule diameter during the culture period. In addition, spermatogonia, primary spermatocytes, secondary spermatocytes, and round spermatids were observed after 4 weeks in culture, and reverse transcription-PCR confirmed expression of the marker genes. In comparison, tissues cultured in FBS-supplemented media showed dwindling testicular organization, necrotic seminiferous tubules, and expression of Kit, but inconsistent expression of Sycp3 and Crisp1 KnockOut serum replacement outperforms FBS as a growth media supplements for culturing immature spermatogonial tissue culture.
Asunto(s)
Medios de Cultivo , Suero , Espermatozoides/crecimiento & desarrollo , Testículo/citología , Técnicas de Cultivo de Tejidos , Animales , Apoptosis , Bovinos , Sangre Fetal , Expresión Génica , Masculino , Ratas , Ratas Sprague-Dawley , Túbulos Seminíferos/anatomía & histología , Espermátides/citología , Espermatocitos/citología , Espermatogénesis/genética , Espermatogonias/citología , Testículo/crecimiento & desarrolloRESUMEN
This study explored the expression of connexin 43 (Cx43) in the testes of prepubertal Sprague-Dawley (SD) rats following in utero flutamide (Flu) exposure. Connexins constitute the major protein type in gap junctions. Connexin 43, the most prominent connexin family member expressed by testes, is localized at the base of seminiferous tubules in humans and rodents, and may be involved in fertility. Flutamide was injected subcutaneously into pregnant SD rats on gestational days 12-21 (25 mg/kg/day). Immunohistochemistry, Western blotting, and real-time PCR was used to investigate the distribution and the expression of Cx43 protein and mRNA in the testis on postnatal day 20 (PD20). Following Flu-exposure, Cx43 was observed between Sertoli cells in the seminiferous tubules. On PD20, no Cx43 protein was expressed by the spermatogonial cell layer of the seminiferous tubules in the controls, but was observed in the Flu-exposed group. Western blotting showed that Cx43 was expressed at significantly lower levels in Flu-exposed testes than controls on PD20 (p < 0.001). On PD20, levels of Cx43 mRNA in undescended Flu-exposed testes were significantly lower than in controls (p < 0.05) and descended Flu-exposed testes (p < 0.01). After Flu-exposure in the rat embryonic period, Cx43 mRNA and protein expression were downregulated, and its distribution in the seminiferous tubules was abnormal.