ABSTRACT
PURPOSE: This paper explores the feasibility of a new therapy for the treatment of hypospadias patients. Hypospadias is a very common congenital malformation of male genitals, with very high rate of recurrences after surgery. The field of regenerative medicine, which offers innovative solutions for many pathologies, still does not offer reliable solution for this pathology. Here, we propose quality, safety, and clinical feasibility assessment for an oral mucosa advanced therapy medicinal product (ATMP) grown on a biocompatible scaffold for a clinical study on urethral reconstruction of hypospadias patients. METHODS: Urethral and oral mucosal epithelia from donor biopsies were cultivated between two fibrin layers, under clinical-grade conditions for cell and tissue characterization and comparison, aimed at tissue engineering. In addition, single-clone analyses were performed to analyze gene expression profiles of the two epithelia by microarray technology. RESULTS: Oral mucosa appeared suitable for urethral reconstruction. The resulting ATMP was proven to maintain stem cells and regenerative potency. The preclinical safety studies were performed on human tissues to assess abnormalities and tumorigenicity, and confirmed the safety of the ATMP. Finally, the patient selection and the clinical protocol for the upcoming clinical trial were defined. CONCLUSIONS: Against this backdrop, in this paper, we are proposing a new reproducible and reliable ATMP for the treatment of hypospadias.
Subject(s)
Hypospadias/surgery , Mouth Mucosa/transplantation , Urethra/surgery , Animals , Disease Models, Animal , Feasibility Studies , Humans , In Vitro Techniques , Male , Swine , Tissue Engineering , Tissue Scaffolds , Urologic Surgical Procedures, Male/methodsABSTRACT
Ciliated Bronchial Epithelium (CBE) 1 is a novel gene, which is expressed in ciliated cells. As cilia are important during embryogenesis, the present authors characterised the murine homologue of CBE1 (Cbe1) and compared its temporal expression during murine and human lung development. Cbe1 cDNA was cloned and characterised using sequencing, standard PCR and Western blotting. Mouse and human embryonic/fetal lungs (HELs) were harvested for mRNA analysis and protein localisation in vivo and in vitro using RT-PCR and immunohistochemistry. The Cbe1 amino acid sequence was >75% identical with CBE1 and its alternative splicing and tissue distribution were highly conserved. Pulmonary expression of Cbe1 mRNA was increased at embryonic day (E)16, 1 day later than Foxj1, which is consistent with a role in ciliogenesis. In HELs, CBE1 mRNA was detectable at 8-9 weeks post-conception and increased in explant culture. CBE1 protein expression was weak at 10 weeks post-conception but strong at 12.3 weeks post-conception, in parallel with cilia formation. Additionally, Cbe1 mRNA was expressed at E11 (4-5 weeks post-conception in HELs) in the absence of Foxj1, implying a distinct role in early development. Chronological regulation of CBE1/Cbe1 expression during pulmonary differentiation suggests involvement in ciliogenesis, with an additional role during early lung development.
Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Lung/embryology , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Blotting, Western , Cell Differentiation , Cloning, Molecular , DNA Mutational Analysis , DNA Primers , DNA, Complementary/metabolism , Humans , Immunoenzyme Techniques , Mice , Open Reading Frames , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Statistics, Nonparametric , Time Factors , TransfectionABSTRACT
Hsp60, a mitochondrial chaperonin highly conserved during evolution, has been found elevated in the cytosol of cancer cells, both in vivo and in vitro, but its role in determining apoptosis during oxidative stress (OS) has not yet been fully elucidated. The aim of the present work was to study the effects of OS on Hsp60 levels and its interactions with procaspase- 3 (p-C3) and p53 in tumor cells. NCI-H292 (mucoepidermoid carcinoma) cells were exposed to various concentrations of hydrogen peroxide (H2O2) for 24 hours. Cell viability was determined by Trypan blue and MTT assays. DNA damage was assessed by the Comet assay, and apoptosis was measured by the AnnexinV cytofluorimetric test. Exposure to increasing concentrations of H2O2 resulted in a reduction of cell viability, DNA damage, and early apoptotic phenomena. Hsp60, p-C3, p53, and p21 were assessed by Western blotting and immunocytochemistry before and after OS. Hsp60 and p-C3 were present before and after OS induction. Immunoprecipitation experiments showed an Hsp60/p-C3 complex before OS that persisted after it, while an Hsp60/p53 complex was not detected in either condition. The presence of wild type (wt) p53 was confirmed by RT-PCR, and p21 detection suggested p53 activation after OS. We postulate that, although OS may induce early apoptosis in NCI-H292 cells, Hsp60 exerts an anti-apoptotic effect in these cells and, by extension, it may do so in other cancer cells.
Subject(s)
Carcinoma, Mucoepidermoid/metabolism , Caspase 3/metabolism , Chaperonin 60/metabolism , Lung Neoplasms/metabolism , Oxidative Stress , Apoptosis/drug effects , Blotting, Western , Carcinoma, Mucoepidermoid/drug therapy , Carcinoma, Mucoepidermoid/pathology , Cell Line, Tumor , Cell Survival/drug effects , Comet Assay , DNA/drug effects , DNA Damage , Formazans/metabolism , Gene Expression/drug effects , Humans , Hydrogen Peroxide/pharmacology , Immunohistochemistry , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Tetrazolium Salts/metabolism , Trypan Blue/metabolism , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/geneticsABSTRACT
This review addresses the significance of the expression of proliferating cell nuclear antigen (PCNA), p53 and some heat shock proteins (Hsps) in prostate carcinoma (PC). In fact, PCNA and p53 are two widely discussed tools in PC diagnosis, mainly because of the controversy regarding the significance of their expression during prostate cancer development and progression. At the same time, only few studies have shown the potential role of Hsps in carcinogenesis and their overexpression in pre-neoplastic and neoplastic lesions of the prostate. We briefly describe the physiological roles of Hsps in normal cells, and the significance of their immunohistochemical detection in PC as well as in pre-cancerous lesions of the prostate. We will also discuss the possible functional interactions of these molecules in both dysplastic and neoplastic cells.
