RESUMO
Genetic prodrug activation therapy (GPAT) is a form of cancer gene therapy that has potential use against tumours such as colorectal malignancy. The characterization of such therapies using laboratory models provides a basis for clinical trials. In this study the gene encoding Herpes Simplex Virus thymidine kinase (HSVtk) was delivered to colorectal tumour cells using an Adenoviral (Ad) vector in vitro. In this way the cells were made susceptible to killing with the prodrug ganciclovir to various degrees depending on cell infectability with Ad. Bystander killing effect appeared minimal both in vitro and when transduced cells were injected in vivo. Mechanisms of cell death, measured in vitro using anti-BrDU (DNA-break labelling) and propidium iodide staining variously showed a combination of apoptosis in the G1 cell cycle phase and late apoptotic or necrotic sub-G1 DNA fragmentation, depending on the tumour cell line. These findings suggest that gene therapy of colorectal cancer by GPAT gives rise to therapeutic forms of direct cell death, but requires improvements in transduction, and possibly immune augmentation.
Assuntos
Morte Celular/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Ganciclovir/metabolismo , Ganciclovir/farmacologia , Terapia Genética , Pró-Fármacos/metabolismo , Adenoviridae/genética , Animais , Ciclo Celular/efeitos dos fármacos , Neoplasias Colorretais/terapia , Fragmentação do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Citometria de Fluxo , Ganciclovir/uso terapêutico , Vetores Genéticos/genética , Humanos , Camundongos , Transplante de Neoplasias , Fosforilação , Simplexvirus/genética , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Timidina Quinase/genética , Timidina Quinase/metabolismo , Transdução Genética , Células Tumorais CultivadasRESUMO
Transduction of 11 pancreatic cancer cell lines with a replication-deficient adenovirus 5 expressing enhanced green fluorescent protein (Ad5EGFP) was analyzed and variable EGFP levels were observed, ranging from <1% to â¼40% of cells transduced, depending on the cell line. Efficient Ad5EGFP transduction was associated mainly with higher levels of cell surface Coxsackie and adenovirus receptor (CAR) but not with expression of α(v)ß(3) and α(v)ß(5) integrins and was fiber dependent. Reduction of CAR by RNA interference resulted in a corresponding decrease in Ad5EGFP transduction. Pre-treatment of Ad5EGFP with blood coagulation Factor X increased virus entry even in the presence of low CAR levels generated by RNA interference, suggesting a potential alternative route of Ad5 entry into pancreatic cancer cells. Immunohistochemistry carried out on 188 pancreatic ductal adenocarcinomas and 68 matched controls showed that CAR was absent in 102 (54%) of adenocarcinomas, whereas moderate and strong staining was observed in 58 (31%) and 28 (15%) cases, respectively. Weak or absent CAR immunolabeling correlated with poor histological differentiation of pancreatic cancer. In normal tissue, strong immunolabeling was detected in islet cells and in the majority of inter- and intralobular pancreatic ducts.
Assuntos
Adenoviridae/genética , Fator X/farmacologia , Neoplasias Pancreáticas/metabolismo , Adenoviridae/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Integrina alfaVbeta3/metabolismo , Pessoa de Meia-Idade , Interferência de RNA , Receptores Virais/genética , Receptores Virais/metabolismo , Receptores de Vitronectina/metabolismo , Transdução GenéticaRESUMO
High-risk human papillomavirus (HPV) is a major causative agent of cervical cancer and the E6 and E7 genes encode the major HPV oncoproteins. The E7 protein from high-risk HPV types alters cell cycle progression and represses genes encoding components of the antigen-presentation pathway, suggesting a role for E7 in tumour immune evasion. We show that knockdown of E7 expression in HPV16- and HPV18-transformed cervical carcinoma cells by RNA interference increased expression of major histocompatibility complex (MHC) class I at the cell surface and reduced susceptibility of these cells to natural killer (NK) cells. Tetracycline-regulated induction of HPV16 E7 resulted in reduced expression of cell surface MHC class I molecules and increased NK cell killing. Our results suggest that, for HPV-associated malignancies, reduced MHC class I expression is the result of an active immune evasion strategy that has evolved to assist viral replication.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 18/imunologia , Células Matadoras Naturais/imunologia , Proteínas Oncogênicas Virais/biossíntese , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/imunologia , Células Cultivadas , Feminino , Células HeLa , Papillomavirus Humano 16/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Proteínas Oncogênicas Virais/fisiologia , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus/metabolismo , Evasão Tumoral/imunologia , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologiaRESUMO
We show that dimerization of major histocompatibility complex (MHC) class I on a human monocytic cell line, THP-1, induces nitric oxide (NO) synthesis. Cells cultured in the presence of a human MHC class I-specific monoclonal antibody produced significant amounts of NO after 72 hr. Reverse transcription-polymerase chain reaction and flow cytometry analysis revealed that the cells synthesized detectable levels of inducible NO synthase mRNA and protein. These effects were not seen after treatment with monovalent Fab fragments or Fc fragments of the same antibody, or after treatment with a control antibody. These data show a link between innate and acquired immune mechanisms mediated by NO and MHC class I.