RESUMEN
Chemotherapy, the standard of care treatment for cancer patients with advanced disease, has been increasingly recognized to activate host immune responses to produce durable outcomes. Here, in colorectal adenocarcinoma (CRC) we identify oxaliplatin-induced Thioredoxin-Interacting Protein (TXNIP), a MondoA-dependent tumor suppressor gene, as a negative regulator of Growth/Differentiation Factor 15 (GDF15). GDF15 is a negative prognostic factor in CRC and promotes the differentiation of regulatory T cells (Tregs), which inhibit CD8 T-cell activation. Intriguingly, multiple models including patient-derived tumor organoids demonstrate that the loss of TXNIP and GDF15 responsiveness to oxaliplatin is associated with advanced disease or chemotherapeutic resistance, with transcriptomic or proteomic GDF15/TXNIP ratios showing potential as a prognostic biomarker. These findings illustrate a potentially common pathway where chemotherapy-induced epithelial oxidative stress drives local immune remodeling for patient benefit, with disruption of this pathway seen in refractory or advanced cases.
RESUMEN
Tumor-associated macrophages (TAMs) are a heterogeneous population of cells that facilitate cancer progression. However, our knowledge of the niches of individual TAM subsets and their development and function remain incomplete. Here, we describe a population of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)-expressing TAMs, which form coordinated multi-cellular "nest" structures that are heterogeneously distributed proximal to vasculature in tumors of a spontaneous murine model of breast cancer. We demonstrate that LYVE-1+ TAMs develop in response to IL-6, which induces their expression of the immune-suppressive enzyme heme oxygenase-1 and promotes a CCR5-dependent signaling axis, which guides their nest formation. Blocking the development of LYVE-1+ TAMs or their nest structures, using gene-targeted mice, results in an increase in CD8+ T cell recruitment to the tumor and enhanced response to chemotherapy. This study highlights an unappreciated collaboration of a TAM subset to form a coordinated niche linked to immune exclusion and resistance to anti-cancer therapy.
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Neoplasias , Ratones , Animales , Neoplasias/patología , Macrófagos/metabolismoRESUMEN
PURPOSE: As a promotor of tumor invasion and tumor microenvironment (TME) formation, the protein complex S100A8/S100A9 is associated with poor prognosis. Our aim was to further evaluate its origin and regulatory effects, and to establish an imaging biomarker for TME activity. METHODS: S100A9-/-cells (ko) were created from syngeneic murine breast cancer 4T1 (high malignancy) and 67NR (low malignancy) wildtype (wt) cell lines and implanted into either female BALB/c wildtype or S100A9-/- mice (n = 10 each). Anti-S100A9-Cy5.5-targeted fluorescence reflectance imaging was performed at 0 h and 24 h after injection. Potential early changes of S100A9-presence under immune checkpoint inhibition (anti-PD-L1, n = 7 vs. rat IgG2b as isotype control, n = 3) were evaluated. RESULTS: In S100A9-/-mice contrast-to-noise-ratios were significantly reduced for wt and S100A9-/-tumors. No significant differences were detected for 4T1 ko and 67NR ko cells as compared to wildtype cells. Under anti-PD-L1 treatment S100A9 presence significantly decreased compared with the control group. CONCLUSION: Our results confirm a secretion of S100A8/S100A9 by the TME, while tumor cells do not apparently release the protein. Under immune checkpoint inhibition S100A9-imaging reports an early decrease of TME activity. Therefore, S100A9-specific imaging may serve as an imaging biomarker for TME formation and activity.
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Neoplasias de la Mama , Inhibidores de Puntos de Control Inmunológico , Animales , Biomarcadores , Neoplasias de la Mama/metabolismo , Calgranulina A/genética , Calgranulina A/metabolismo , Calgranulina B/genética , Femenino , Humanos , Ratones , Ratas , Microambiente TumoralRESUMEN
In Lake Malawi cichlids, each tooth is replaced in one-for-one fashion every â¼20 to 50 d, and taste buds (TBs) are continuously renewed as in mammals. These structures are colocalized in the fish mouth and throat, from the point of initiation through adulthood. Here, we found that replacement teeth (RT) share a continuous band of epithelium with adjacent TBs and that both organs coexpress stem cell factors in subsets of label-retaining cells. We used RNA-seq to characterize transcriptomes of RT germs and TB-bearing oral epithelium. Analysis revealed differential usage of developmental pathways in RT compared to TB oral epithelia, as well as a repertoire of genome paralogues expressed complimentarily in each organ. Notably, BMP ligands were expressed in RT but excluded from TBs. Morphant fishes bathed in a BMP chemical antagonist exhibited RT with abrogated shh expression in the inner dental epithelium (IDE) and ectopic expression of calb2 (a TB marker) in these very cells. In the mouse, teeth are located on the jaw margin while TBs and other oral papillae are located on the tongue. Previous study reported that tongue intermolar eminence (IE) oral papillae of Follistatin (a BMP antagonist) mouse mutants exhibited dysmorphic invagination. We used these mutants to demonstrate altered transcriptomes and ectopic expression of dental markers in tongue IE. Our results suggest that vertebrate oral epithelium retains inherent plasticity to form tooth and taste-like cell types, mediated by BMP specification of progenitor cells. These findings indicate underappreciated epithelial cell populations with promising potential in bioengineering and dental therapeutics.
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Diferenciación Celular , Plasticidad de la Célula , Células Madre/citología , Células Madre/metabolismo , Papilas Gustativas/citología , Papilas Gustativas/metabolismo , Animales , Biomarcadores , Autorrenovación de las Células/genética , Epitelio/metabolismo , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Regeneración , Diente/citologíaRESUMEN
Innate lymphoid cells (ILCs) are largely tissue resident and respond rapidly toward the environmental signals from surrounding tissues and other immune cells. The pleiotropic function of ILCs in diverse contexts underpins its importance in the innate arm of immune system in human health and disease. ILCs derive from common lymphoid progenitors but lack adaptive antigen receptors and functionally act as the innate counterpart to T-cell subsets. The classification of different subtypes is based on their distinct transcription factor requirement for development as well as signature cytokines that they produce. The discovery and subsequent characterization of ILCs over the past decade have mainly focused on the regulation of inflammation, tissue remodeling, and homeostasis, whereas the understanding of the multiple roles and mechanisms of ILCs in cancer is still limited. Emerging evidence of the potent immunomodulatory properties of ILCs in early host defense signifies a major advance in the use of ILCs as promising targets in cancer immunotherapy. In this review, we will decipher the non-exclusive roles of ILCs associated with both protumor and antitumor activities. We will also dissect the heterogeneity, plasticity, genetic evidence, and dysregulation in different cancer contexts, providing a comprehensive understanding of the complexity and diversity. These will have implications for the therapeutic targeting in cancer.
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Susceptibilidad a Enfermedades/inmunología , Inmunidad Innata , Inmunomodulación , Linfocitos/inmunología , Linfocitos/metabolismo , Neoplasias/etiología , Neoplasias/metabolismo , Animales , Biomarcadores , Linaje de la Célula/inmunología , Citocinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Linfocitos/patología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Neoplasias/patología , Transducción de Señal , Microambiente TumoralRESUMEN
In adult tissues and organs with high turnover rates, the generation of transit-amplifying cell (TAC) populations from self-renewing stem cells drives cell replacement. The role of stem cells is to provide a renewable source of cells that give rise to TACs to provide the cell numbers that are necessary for cell differentiation. Regulation of the formation of TACs is thus fundamental to controlling cell replacement. Here, we analyze the properties of a population of mesenchymal TACs in the continuously growing mouse incisor to identify key components of the molecular regulation that drives proliferation. We show that the polycomb repressive complex 1 acts as a global regulator of the TAC phenotype by its direct action on the expression of key cell-cycle regulatory genes and by regulating Wnt/ß-catenin-signaling activity. We also identify an essential requirement for TACs in maintaining mesenchymal stem cells, which is indicative of a positive feedback mechanism.
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Incisivo/citología , Incisivo/crecimiento & desarrollo , Células Madre Mesenquimatosas/citología , Animales , Ciclo Celular/genética , Regulación de la Expresión Génica , Genoma , Código de Histonas , Células Madre Mesenquimatosas/metabolismo , Ratones , Complejo Represivo Polycomb 1/metabolismo , Nicho de Células Madre/genética , Vía de Señalización Wnt/genéticaRESUMEN
The extent to which heterogeneity within mesenchymal stem cell (MSC) populations is related to function is not understood. Using the archetypal MSC in vitro surface marker, CD90/Thy1, here we show that 30% of the MSCs in the continuously growing mouse incisor express CD90/Thy1 and these cells give rise to 30% of the differentiated cell progeny during postnatal development. In adulthood, when growth rate homeostasis is established, the CD90/Thy1+ MSCs decrease dramatically in number. When adult incisors are cut, the growth rate increases to rapidly re-establish tooth length and homeostasis. This accelerated growth rate correlates with the re-appearance of CD90/Thy+ MSCs and re-establishment of their contribution to cell differentiation. A population of Celsr1+ quiescent cells becomes mitotic following clipping and replenishes the CD90/Thy1 population. A sub-population of MSCs thus exists in the mouse incisor, distinguished by expression of CD90/Thy1 that plays a specific role only during periods of increased growth rate.
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Linaje de la Célula/genética , Incisivo/citología , Células Madre Mesenquimatosas/citología , Osteogénesis/genética , Antígenos Thy-1/genética , Animales , Biomarcadores/metabolismo , Recuento de Células , Diferenciación Celular , Proliferación Celular , Citometría de Flujo , Expresión Génica , Incisivo/crecimiento & desarrollo , Incisivo/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Transgénicos , Mitosis , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Antígenos Thy-1/metabolismoRESUMEN
Caveolin-1 is a scaffolding protein involved in the formation of cholesterol-rich caveolae lipid rafts within the plasma membrane and is capable of collecting signaling molecules into the caveolae and regulating their activity, including extracellular matrix metalloproteinase inducer (EMMPRIN). However, detailed expression patterns of caveolin-1 and EMMPRIN in the developing dental germ are largely unknown. The present study investigated the expression patterns of caveolin-1 and EMMPRIN in the developing mouse tooth germ by immunohistochemistry and real-time polymerase chain reaction. At the bud stage, caveolin-1 expression was initiated in the epithelium bud and mesenchymal cells, while EMMPRIN was weakly expressed at this stage. At the cap stage, caveolin-1 protein was located in the lingual part of the tooth germ; however, EMMPRIN protein was located in the labial part. From the bell stage to 2 days postnatal, caveolin-1 expression was detected in the ameloblasts and cervical loop area; with EMMPRIN expression in the ameloblasts and odontoblasts. Real-time polymerase chain reaction results showed that both caveolin-1 and EMMPRIN mRNA levels increased gradually with progression of developmental stages, and peaked at day two postnatal. The current finding suggests that both caveolin-1 and EMMPRIN take part in mouse tooth development, especially in the differentiation and organization of odontogenic tissues.
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Basigina/genética , Caveolina 1/genética , Regulación del Desarrollo de la Expresión Génica , Odontogénesis/genética , Diente/embriología , Diente/metabolismo , Animales , Basigina/metabolismo , Caveolina 1/metabolismo , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Germen Dentario/embriología , Germen Dentario/metabolismoRESUMEN
Human teeth contain stem cells in all their mesenchymal-derived tissues, which include the pulp, periodontal ligament, and developing roots, in addition to the support tissues such as the alveolar bone. The precise roles of these cells remain poorly understood and most likely involve tissue repair mechanisms but their relative ease of harvesting makes teeth a valuable potential source of mesenchymal stem cells (MSCs) for therapeutic use. These dental MSC populations all appear to have the same developmental origins, being derived from cranial neural crest cells, a population of embryonic stem cells with multipotential properties. In rodents, the incisor teeth grow continuously throughout life, a feature that requires populations of continuously active mesenchymal and epithelial stem cells. The discrete locations of these stem cells in the incisor have rendered them amenable for study and much is being learnt about the general properties of these stem cells for the incisor as a model system. The incisor MSCs appear to be a heterogeneous population consisting of cells from different neural crest-derived tissues. The epithelial stem cells can be traced directly back in development to a Sox10(+) population present at the time of tooth initiation. In this review, we describe the basic biology of dental stem cells, their functions, and potential clinical uses.
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Células Madre Embrionarias/fisiología , Células Madre Mesenquimatosas/fisiología , Regeneración/fisiología , Diente/fisiología , Animales , Diferenciación Celular/genética , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/metabolismo , Cresta Neural/citología , Cresta Neural/embriología , Cresta Neural/crecimiento & desarrollo , Organogénesis/genética , Regeneración/genética , Diente/embriología , Diente/crecimiento & desarrolloRESUMEN
Mesenchymal stem cells occupy niches in stromal tissues where they provide sources of cells for specialized mesenchymal derivatives during growth and repair. The origins of mesenchymal stem cells have been the subject of considerable discussion, and current consensus holds that perivascular cells form mesenchymal stem cells in most tissues. The continuously growing mouse incisor tooth offers an excellent model to address the origin of mesenchymal stem cells. These stem cells dwell in a niche at the tooth apex where they produce a variety of differentiated derivatives. Cells constituting the tooth are mostly derived from two embryonic sources: neural crest ectomesenchyme and ectodermal epithelium. It has been thought for decades that the dental mesenchymal stem cells giving rise to pulp cells and odontoblasts derive from neural crest cells after their migration in the early head and formation of ectomesenchymal tissue. Here we show that a significant population of mesenchymal stem cells during development, self-renewal and repair of a tooth are derived from peripheral nerve-associated glia. Glial cells generate multipotent mesenchymal stem cells that produce pulp cells and odontoblasts. By combining a clonal colour-coding technique with tracing of peripheral glia, we provide new insights into the dynamics of tooth organogenesis and growth.
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Diferenciación Celular , Linaje de la Célula , Incisivo/citología , Células Madre Mesenquimatosas/citología , Neuroglía/citología , Animales , Rastreo Celular , Células Clonales/citología , Pulpa Dental/citología , Femenino , Incisivo/embriología , Masculino , Ratones , Modelos Biológicos , Cresta Neural/citología , Odontoblastos/citología , Regeneración , Células de Schwann/citologíaRESUMEN
Rodent incisors are capable of growing continuously and the renewal of dental epithelium giving rise to enamel-forming ameloblasts and dental mesenchyme giving rise to dentin-forming odontoblasts and pulp cells is achieved by stem cells residing at their proximal ends. Although the dental epithelial stem cell niche (cervical loop) is well characterized, little is known about the dental mesenchymal stem cell niche. Ring1a/b are the core Polycomb repressive complex1 (PRC1) components that have recently also been found in a protein complex with BcoR (Bcl-6 interacting corepressor) and Fbxl10. During mouse incisor development, we found that genes encoding members of the PRC1 complex are strongly expressed in the incisor apical mesenchyme in an area that contains the cells with the highest proliferation rate in the tooth pulp, consistent with a location for transit amplifying cells. Analysis of Ring1a(-/-);Ring1b(cko/cko) mice showed that loss of Ring1a/b postnatally results in defective cervical loops and disturbances of enamel and dentin formation in continuously growing incisors. To further characterize the defect found in Ring1a(-/-);Ring1b(cko/cko) mice, we demonstrated that cell proliferation is dramatically reduced in the apical mesenchyme and cervical loop epithelium of Ring1a(-/-);Ring1b(cko/cko) incisors in comparison to Ring1a(-/-);Ring1b(fl/fl)cre- incisors. Fgf signaling and downstream targets that have been previously shown to be important in the maintenance of the dental epithelial stem cell compartment in the cervical loop are downregulated in Ring1a(-/-);Ring1b(cko/cko) incisors. In addition, expression of other genes of the PRC1 complex is also altered. We also identified an essential postnatal requirement for Ring1 proteins in molar root formation. These results show that the PRC1 complex regulates the transit amplifying cell compartment of the dental mesenchymal stem cell niche and cell differentiation in developing mouse incisors and is required for molar root formation.
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Proteínas de Unión al ADN/metabolismo , Incisivo/citología , Incisivo/metabolismo , Células Madre Mesenquimatosas/metabolismo , Proteínas Represoras/metabolismo , Nicho de Células Madre/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Esmalte Dental/citología , Esmalte Dental/crecimiento & desarrollo , Esmalte Dental/metabolismo , Dentina/citología , Dentina/crecimiento & desarrollo , Dentina/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Incisivo/anomalías , Incisivo/crecimiento & desarrollo , Células Madre Mesenquimatosas/citología , Ratones , Ratones Noqueados , Ratones Transgénicos , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Transducción de Señal , Nicho de Células Madre/genética , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Resistance to anticancer drugs is often observed in prostate cancer therapy. Kindlin-2 was recently found overexpressed during cancer progression. In this study, we examined the functional role of Kindlin-2 in cisplatin-induced prostate cancer cell death. Kindlin-2 was highly expressed in the androgen-insensitive (PC-3 and DU-145), but not in the androgen-sensitive cell lines (e.g., LNCaP). Overexpression of Kindlin-2 in LNCaP protected the cells from cisplatin-induced death, while Kindlin-2 knock-down in PC-3 cells enhanced cisplatin sensitivity. Mechanistically, Kindlin-2 regulation of the anti-apoptotic Bcl-xL may explain the increased cell death in the absence of Kindlin-2. Taken together, Kindlin-2 appears to play a functional role in prostate cancer cell sensitivity to cisplatin. Targeting Kindlin-2 may therefore improve drug efficacy and reduce drug doses, and would likely be beneficial for the treatment of prostate cancer.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Cisplatino/farmacología , Proteínas de la Membrana/fisiología , Proteínas de Neoplasias/fisiología , Neoplasias de la Próstata/tratamiento farmacológico , Línea Celular Tumoral , Humanos , Masculino , Proteínas de la Membrana/análisis , Proteínas de Neoplasias/análisis , Neoplasias de la Próstata/química , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Interferencia de ARNRESUMEN
Kindlin-2 is a novel integrin-interacting focal adhesion protein that belongs to the Kindlin family. Focal adhesion proteins control cytoskeleton dynamics and promote cancer cell growth, survival, migration and metastasis. Little is known, however, about expression of Kindlin-2 in association with human cancer. We now reveal high Kindlin-2 expression in malignant mesothelioma (MM) cell lines using an affinity-purified anti-Kindlin-2 antibody. Furthermore, we show by immunohistochemistry that Kindlin-2 is highly expressed in 92 of 102 (90%) MMs with epitheliod; sarcomatoid, biphasic and poorly differentiated morphologies. In addition, Kindlin-2 expression correlates to cell proliferation, suggesting a role for Kindlin-2 in tumor growth. We also detect increased expression of Kindlin-2 at the invasion front of tumors concurrent with increased expression of integrin-linked kinase, a Kindlin-binding protein. Besides the high expression of Kindlin-2 in pleural MMs, pleural metastases of lung adenocarcinoma also express large amounts of Kindlin-2, but not Kindlin-1. Notably, in vitro, when endogenous Kindlin-2 was knocked down with RNAi in MM cells, this impaired cell spreading, adhesion and migration. Overall, our study suggests that heightened expression of Kindlin-2 might contribute to tumor progression in MM.
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Neoplasias Pulmonares/metabolismo , Proteínas de la Membrana/metabolismo , Mesotelioma/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pleurales/metabolismo , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Humanos , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
P21-activated kinase 5 (PAK5) is the recently identified member of the group B p21-activated kinase (PAK) family which are effectors of the small GTPase Cdc42 and Rac1, known to regulate cell motility and activate cell-survival signaling pathways. However, overexpression of PAK5 has not been associated with any cancers so far. Interestingly, we found that PAK5 was overexpressed in a variety of colorectal carcinoma (CRC) cell lines in a Western-blotting examination. Therefore, in this study, we aim to examine the PAK5 expression during CRC progression and to answer if PAK5 is involved in malignant progression of CRC. By immunohistochemistry, our results showed that PAK5 expression was increased with CRC progression through the adenoma to carcinoma sequence, with the most significant increases in invasive and metastatic CRCs (p < 0.0001). Furthermore, increased PAK5 expression was also found with the development of CRC from lower Duke's grades to higher ones (p < 0.01). Moreover, PAK5 was also increased from well to poorly differentiated CRCs (p < 0.01). Using gain and loss of function experiments, we found that PAK5 reduced CRC cell adhesion but promoted their migration on collagen type I. Taken together, our study demonstrated that PAK5 expression increased significantly with malignant progression of CRC and that PAK5 might promote CRC metastasis by regulating CRC cell adhesion and migration.
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Transformación Celular Neoplásica/metabolismo , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Quinasas p21 Activadas/metabolismo , Adenoma/enzimología , Adulto , Anciano , Western Blotting , Adhesión Celular , Movimiento Celular , Separación Celular , Transformación Celular Neoplásica/patología , Pólipos del Colon/enzimología , Pólipos del Colon/patología , Progresión de la Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Hiperplasia , Inmunohistoquímica , Metástasis Linfática , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Plásmidos , Transfección , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
OBJECTIVE: To study the role of PAK6 in prostate cancer by cloning PAK6-N terminal sequence into E.coli and preparing its polyclonal rabbit antibody to detect PAK6 expression in prostate cancer. METHODS: Based on human PAK6 cDNA sequence, we designed a pair of primers to amplify the PAK6-N terminal sequence by PCR. The PCR product was subcloned into the bacterial expression vector pGEX-4T-1 via EcoRI/XhoI sites, and the recombinant plasmids were identified by enzymatic cleavage followed by DNA sequence analysis. By transforming the expression vector into component E.coli BL21 cells, the GST-PAK6-N fusion protein was expressed with IPTG induction. Glutathione-Sepharose beads were used to purify GST- PAK6-N fusion protein. Anti-PAK6 polyclonal antibody was produced by immunizing rabbits with purified GST-PAK6 N-terminal fusion protein. Anti-PAK6 polyclonal antibody was purified by protein A beads and used for detection of PAK6 expression in 3 prostate cancer specimens. RESULTS AND CONCLUSION: We cloned PAK6-N terminal gene fragment successfully, purified GST-PAK6 N-terminal fusion protein, and obtained polyclonal rabbit PAK6 antibody. Immunohistochemistry indicated that PAK6 expressed in the stroma instead of the cancer cells in prostate cancer. All of the 3 prostate cancer specimens showed positive staining in the stroma, suggesting that PAK6 may participate in the stroma-cancer cell interaction in prostate cancer.
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Anticuerpos Monoclonales/inmunología , Neoplasias de la Próstata/enzimología , Quinasas p21 Activadas/inmunología , Quinasas p21 Activadas/metabolismo , Anciano , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Reacción en Cadena de la Polimerasa , Neoplasias de la Próstata/genética , Conejos , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Quinasas p21 Activadas/genéticaRESUMEN
OBJECTIVE: To clone PAK5-N terminal sequence for expression in E. coli to prepare its polyclonal antibody, and examine the role of PAK5 in dental germ cells. METHODS: Based on human PAK5 cDNA sequence, PCR primers were designed to amplify PAK5-N terminal sequence. The PCR product was cloned into the expression vector pGEX-4T-1 EcoRI/XhoI sites, and the recombinant plasmids were identified by agarose gel electrophoresis followed by DNA sequence analysis. The recombinant plasmids were transformed into E. coli BL21 and the expression of GST-fusion protein was induced by IPTG. Glutathione-Sepharose beads were used to purify GST-fusion PAK5-N-terminal fragment. Anti-PAK5 polyclonal antibody was obtained in immunizing rabbits with purified GST-PAK5 N-terminal fusion protein, and the antibodies were purified by protein A beads and used for detection of PAK5 expression in dental germ cells. RESULTS AND CONCLUSIONS: We successfully cloned PAK5-N terminal gene fragment, and achieved protein expression, purification and production of PAK5-NT polyclonal antibody. The results of Western blotting indicated that PAK5 can be highly expressed in the dental germ cells, suggesting that PAK5 may play an important role in biological function of dental germ cells.