RESUMO
Early genetic events in the development of high-grade serous ovarian cancer (HGSOC) may define the molecular basis of the profound structural and numerical instability of chromosomes in this disease. To discover candidate genetic changes we sequentially passaged cells from a karyotypically normal hTERT immortalised human ovarian surface epithelial line (IOSE25) resulting in the spontaneous formation of colonies in soft agar. Cell lines transformed ovarian surface epithelium 1 and 4 (TOSE 1 and 4) established from these colonies had an abnormal karyotype and altered morphology, but were not tumourigenic in immunodeficient mice. TOSE cells showed loss of heterozygosity (LOH) at TP53, increased nuclear p53 immunoreactivity and altered expression profile of p53 target genes. The parental IOSE25 cells contained a missense, heterozygous R175H mutation in TP53, whereas TOSE cells had LOH at the TP53 locus with a new R273H mutation at the previous wild-type TP53 allele. Cytogenetic and array CGH analysis of TOSE cells also revealed a focal genomic amplification of CXCR4, a chemokine receptor commonly expressed by HGSOC cells. TOSE cells had increased functional CXCR4 protein and its abrogation reduced epidermal growth factor receptor (EGFR) expression, as well as colony size and number. The CXCR4 ligand, CXCL12, was epigenetically silenced in TOSE cells and its forced expression increased TOSE colony size. TOSE cells had other cytogenetic changes typical of those seen in HGSOC ovarian cancer cell lines and biopsies. In addition, enrichment of CXCR4 pathway in expression profiles from HGSOC correlated with enrichment of a mutated TP53 gene expression signature and of EGFR pathway genes. Our data suggest that mutations in TP53 and amplification of the CXCR4 gene locus may be early events in the development of HGSOC, and associated with chromosomal instability.
Assuntos
Transformação Celular Neoplásica/genética , Ovário/citologia , Receptores CXCR4/genética , Proteína Supressora de Tumor p53/genética , Animais , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Cariotipagem , Perda de Heterozigosidade , Camundongos , Ovário/metabolismo , RNA MensageiroRESUMO
BACKGROUND: Dendritic cells (DC) as antigen presenting cells play an important role in immunotherapy of cancer. Mucin, encoded by the gene MUC1, is a human tumor antigen expressed in breast, pancreatic and ovarian cancers. Therefore, MUC1-transfected DC would be an attractive tool in constructing cancer vaccines. MATERIALS AND METHODS: Using two different cationic liposome preparations and, for comparison, a recombinant adenovirus expressing mucin, we tested the efficiency of mucin gene transfer into DC by flow cytometry. We investigated if these transfected DC were able to specifically stimulate autologous peripheral blood lymphocytes (PBL) from healthy donors. RESULTS: Flow cytometry revealed that 5-20% of DC transfected with liposomes Lipofectin and 20-40% of DC transduced with adenovirus expressed the relevant mucin epitopes. The expression of mucin on DC was similar to the expression of mucin found on carcinoma cells. After antigen uptake, DC specifically stimulated autologous PBL. CONCLUSION: We have shown that cationic liposomal gene transfer into human DC was feasible. We could obtain antigen specific stimulation of PBL at a similar rate as with adenoviral MUC1-transduced DC.
Assuntos
Células Dendríticas/fisiologia , Mucina-1/genética , Transfecção/métodos , Adenoviridae/genética , Antígenos CD/biossíntese , Antígenos CD1/biossíntese , Antígeno B7-1/biossíntese , Antígeno B7-2 , Resinas de Troca de Cátion , Cátions , DNA Complementar/administração & dosagem , DNA Complementar/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Citometria de Fluxo , Antígenos HLA-DR/biossíntese , Humanos , Lipídeos , Lipossomos , Ativação Linfocitária/imunologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Glicoproteínas de Membrana/biossíntese , Mucina-1/imunologia , Fosfatidiletanolaminas , Fito-Hemaglutininas/farmacologiaRESUMO
Naked DNA is an attractive tool for vaccination studies. We have studied naked DNA vaccination against the human tumor antigen mucin, encoded by the gene MUC1. C57/BL6 mice were immunized twice, on day 1 and day 10. with plasmid pCI-MUC1, intramuscularly. Five days after the last immunization tumor challenge experiments were performed using the tumor cell line MC38, expressing human MUC1. In 85% of mice immunized with the mucin plasmid tumor growth inhibition was observed, whereas control mice developed tumors. Re-tumor challenge after three months revealed no tumor growth in mice immunized with the mucin plasmid. These encouraging results, showing long-term protection against tumor growth, indicate the potential usefulness of naked DNA vaccination for clinical immunotherapy.
