ABSTRACT
Malignant melanoma is a highly aggressive cancer that retains functional p53 and p73, and drug unresponsiveness largely depends on defects in death pathways after epigenetic gene silencing in conjunction with an imbalanced p73/DNp73 ratio. We constructed oncolytic viruses armed with an inhibitor of deacetylation and/or p73 to specifically target metastatic cancer. Arming of the viruses is aimed at lifting epigenetic blockage and re-opening apoptotic programs in a staggered manner enabling both, efficient virus replication and balanced destruction of target cells through apoptosis. Our results showed that cooperative expression of shHDAC1 and p73 efficiently enhances apoptosis induction and autophagy of infected cells which reinforces progeny production. In vitro analyses revealed 100% cytotoxicity after infecting cells with OV.shHDAC1.p73 at a lower virus dose compared to control viruses. Intriguingly, OV.shHDAC1.p73 acts as a potent inhibitor of highly metastatic xenograft tumors in vivo. Tumor expansion was significantly reduced after intratumoral injection of 3 x 108 PFU of either OV.shHDAC1 or OV.p73 and, most important, complete regression could be achieved in 100 % of tumors treated with OV.shHDAC1.p73. Our results point out that the combination of high replication capacity and simultaneous restoration of cell death routes significantly enhance antitumor activity.
Subject(s)
Adenoviridae/genetics , DNA-Binding Proteins/biosynthesis , Histone Deacetylase 1/antagonists & inhibitors , Histone Deacetylase 1/genetics , Melanoma/therapy , Nuclear Proteins/biosynthesis , Oncolytic Virotherapy/methods , Oncolytic Viruses/genetics , RNA, Small Interfering/administration & dosage , Tumor Suppressor Proteins/biosynthesis , Cell Line, Tumor , DNA-Binding Proteins/genetics , Gene Knockdown Techniques , HEK293 Cells , Humans , Melanoma/genetics , Melanoma/virology , Nuclear Proteins/genetics , RNA, Small Interfering/genetics , Transgenes , Tumor Protein p73 , Tumor Suppressor Proteins/geneticsABSTRACT
Eighteen congenital melanocytic naevi (CMN) from 17 patients and 18 dysplastic melanocytic naevi (DMN) from 18 patients were screened for mutations in the BRAF oncogene (present study) and the N-ras oncogene (in the course of two foregoing studies) by single-strand conformational polymorphism (SSCP)/sequencing analysis. BRAF mutations were demonstrated in both types of lesion. As a whole, 17 of 18 CMN (94.4%) and five of 18 DMN (27.7%) harboured either BRAF or N-ras mutations. As the BRAF oncogene is frequently found to be mutated in human cutaneous melanomas, it may constitute a risk factor for melanoma formation within CMN and DMN.
Subject(s)
Nevus, Pigmented/congenital , Nevus, Pigmented/genetics , Proto-Oncogene Proteins B-raf/genetics , Skin Neoplasms/congenital , Skin Neoplasms/genetics , Adolescent , Adult , Aged , Child , Child, Preschool , DNA Mutational Analysis , Female , Gene Expression Regulation, Neoplastic , Genes, ras/genetics , Humans , Male , Middle Aged , Polymerase Chain Reaction , Polymorphism, Single-Stranded ConformationalABSTRACT
Seven tumour suppressor genes (Chk1, Chk2, Apaf1, Rb1, p53, p16(INK4a) and p14(ARF)) and two oncogenes (N-ras and BRAF) were screened in nine human malignant melanoma (HMM) cell lines for point mutations or small deletions/insertions by DGGE, TGGE and SCCP analysis. For the first time in human mesothelioma, Chk1 gene mutations were detected in two of the nine investigated HMM cell lines. P53 gene mutations were found in three cell lines and p16(INK4a) mutations in 5. Mutation of the Chk1 gene implies a novel disruption mechanism of the p53 pathway in HMM, without affecting p53 itself. According to our knowledge, this is the first mutation screening of Chk1, Chk2, Apaf1 and Rb1 in human malignant mesothelioma.
Subject(s)
Mesothelioma/genetics , Mutation , Oncogene Proteins/genetics , Tumor Suppressor Proteins/genetics , Base Sequence , Cell Line, Tumor , Checkpoint Kinase 1 , Cyclin-Dependent Kinase Inhibitor p16/genetics , DNA Mutational Analysis , Humans , Mesothelioma/pathology , Protein Kinases/genetics , Sequence Homology, Nucleic Acid , Tumor Suppressor Protein p53/geneticsABSTRACT
Fourteen primary human malignant mesothelioma (HMM) samples obtained from 14 patients were screened for point mutations and microdeletions/microinsertions in exons 1-16 of the chromosome 22q-located tumour suppressor gene neurofibromin 2 (nf2) by single strand conformation polymorphism (SSCP) analysis. In one tumour (7%) a 10 basepair microdeletion of exon 10 was detected by SSCP and subsequently characterised in detail by sequencing. Deletion of the second nf2 allele in laser-microdissected regions of the 10 bp mutation-harbouring tumour was demonstrated by denaturing gradient gel electrophoresis (DGGE) analysis. Simultaneous comparative genomic hybridisation (CGH) analysis also showed losses at chromosome 22q. Our data indicate that functional loss of the NF2 protein may be involved in the formation of a subset of HMMs.
