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1.
Cancer Res ; 75(24): 5260-72, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26494122

RESUMO

Cancer immunotherapy shows great promise but many patients fail to show objective responses, including in cancers that can respond well, such as melanoma and renal adenocarcinoma. The proteasome inhibitor bortezomib sensitizes solid tumors to apoptosis in response to TNF-family death ligands. Because T cells provide multiple death ligands at the tumor site, we investigated the effects of bortezomib on T-cell responses in immunotherapy models involving low-avidity antigens. Bortezomib did not affect lymphocyte or tissue-resident CD11c(+)CD8(+) dendritic cell counts in tumor-bearing mice, did not inhibit dendritic cell expression of costimulatory molecules, and did not decrease MHC class I/II-associated antigen presentation to cognate T cells. Rather, bortezomib activated NF-κB p65 in CD8(+) T cells, stabilizing expression of T-cell receptor CD3ζ and IL2 receptor-α, while maintaining IFNγ secretion to improve FasL-mediated tumor lysis. Notably, bortezomib increased tumor cell surface expression of Fas in mice as well as human melanoma tissue from a responsive patient. In renal tumor-bearing immunodeficient Rag2(-/-) mice, bortezomib treatment after adoptive T-cell immunotherapy reduced lung metastases and enhanced host survival. Our findings highlight the potential of proteasome inhibitors to enhance antitumor T-cell function in the context of cancer immunotherapy.


Assuntos
Antineoplásicos/administração & dosagem , Bortezomib/administração & dosagem , Proteína Ligante Fas/imunologia , Imunoterapia Adotiva/métodos , Linfócitos T/transplante , Animais , Terapia Combinada , Citotoxicidade Imunológica/imunologia , Citometria de Fluxo , Humanos , Immunoblotting , Imuno-Histoquímica , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Melanoma/imunologia , Melanoma/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout
2.
J Biomol Screen ; 17(9): 1231-42, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22927676

RESUMO

Tumor cell subpopulations called cancer stem cells (CSCs) or tumor-initiating cells (TICs) have self-renewal potential and are thought to drive metastasis and tumor formation. Data suggest that these cells are resistant to current chemotherapy and radiation therapy treatments, leading to cancer recurrence. Therefore, finding new drugs and/or drug combinations that cause death of both the differentiated tumor cells as well as CSC populations is a critical unmet medical need. Here, we describe how cancer-derived CSCs are generated from cancer cell lines using stem cell growth media and nonadherent conditions in quantities that enable high-throughput screening (HTS). A cell growth assay in a 1536-well microplate format was developed with these CSCs and used to screen a focused collection of oncology drugs and clinical candidates to find compounds that are cytotoxic against these highly aggressive cells. A hit selection process that included potency and efficacy measurements during the primary screen allowed us to efficiently identify compounds with potent cytotoxic effects against spheroid-derived CSCs. Overall, this research demonstrates one of the first miniaturized HTS assays using CSCs. The procedures described here should enable further testing of the effect of compounds on CSCs and help determine which pathways need to be targeted to kill them.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Células-Tronco Neoplásicas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Proteína Homeobox Nanog , Neoplasias/tratamento farmacológico
3.
Cancer Res ; 71(7): 2718-27, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21324921

RESUMO

Obesity is a risk factor for development of certain cancers but the basis for this risk is unclear. In this study, we developed a novel mouse model that demonstrates directly how lipogenic phenotypes commonly associated with diet-induced metabolic syndromes can influence hepatic cancer development. Activated AKT and ß-catenin (AKT/CAT) genes were hydrodynamically codelivered using the Sleeping Beauty transposon to initiate liver tumorigenesis. AKT/CAT and MET/CAT combination induced microscopic tumor foci by 4 weeks, whereas no tumorigenesis resulted from delivery of AKT, MET, or CAT alone. Primary AKT/CAT tumor cells were steatotic (fatty) hepatocellular adenomas which progressed to hepatocellular carcinomas (HCC) upon in vivo passage, whereas primary MET/CAT tumors emerged directly as frank HCC. Conversion of AKT/CAT tumor cells to frank HCC during passage was associated with induction of the human HCC marker α-fetoprotein and the stem cell marker CD133. Using hierarchical clustering and gene set enrichment analysis, we compared the primary murine AKT/CAT and MET/CAT tumors to a panel of 53 human HCCs and determined that these two mouse models could be stratified as distinct subtypes associated in humans with poor clinical prognosis. The chief molecular networks identified in primary and passaged AKT/CAT tumors were steatosis and lipid metabolic pathways, respectively. Our findings show how coactivation of the AKT and CAT pathways in hepatocytes can efficiently model development of a lipogenic tumor phenotype. Furthermore, we believe that our approach could speed the dissection of microenvironmental factors responsible for driving steatotic-neoplastic transformation to frank carcinoma, through genetic modification of existing immunodefined transgenic models.


Assuntos
Carcinoma Hepatocelular/metabolismo , Transformação Celular Neoplásica/metabolismo , Neoplasias Hepáticas/metabolismo , Proteína Oncogênica v-akt/metabolismo , beta Catenina/metabolismo , Adenoma/genética , Adenoma/metabolismo , Adenoma/patologia , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Elementos de DNA Transponíveis , Ativação Enzimática , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Oncogenes , Proteínas Proto-Oncogênicas c-met/metabolismo
4.
J Invest Dermatol ; 130(7): 1841-8, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20237491

RESUMO

The immune system surveys the skin for keratinocytes (KCs) infected by viruses or with acquired genetic damage. The mechanism by which T cells mediate KC elimination is however undefined. In this study we show that antigen-specific CD8 T cells can eliminate antigen-bearing KCs in vivo and inhibit their clonogenic potential in vitro, independently of the effector molecules perforin and Fas-ligand (Fas-L). In contrast, IFN-gamma receptor expression on KCs and T cells producing IFN-gamma are each necessary and sufficient for in vitro inhibition of KC clonogenic potential. Thus, antigen-specific cytotoxic T lymphocytes (CTLs) may mediate destruction of epithelium expressing non-self antigen by eliminating KCs with potential for self-renewal through an IFN-gamma-dependent mechanism.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Interferon gama/genética , Interferon gama/imunologia , Queratinócitos/imunologia , Pele/citologia , Pele/imunologia , Animais , Apoptose/imunologia , Linfócitos T CD8-Positivos/citologia , Comunicação Celular/imunologia , Divisão Celular/imunologia , Linhagem Celular , Células Clonais/citologia , Células Clonais/imunologia , Proteína Ligante Fas/imunologia , Feminino , Rejeição de Enxerto/imunologia , Queratinócitos/citologia , Complexo Principal de Histocompatibilidade/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Citotóxicas Formadoras de Poros/imunologia , Transplante de Pele/imunologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia
6.
Nucleic Acids Res ; 35(14): 4820-32, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17621583

RESUMO

Recently we reported that gene codon composition determines differentiation-dependent expression of the PV L1 genes in mouse primary keratinocytes (KCs) in vitro and in vivo (Zhao et al. 2005, Mol. Cell Biol. 25:8643-8655). Here, we investigated whether generalized substitution of isoencoding codons affects the duration of expression of PV L1 genes in mouse and human KCs in day 1 culture transiently transfected with native (Nat) and codon modified (Mod) L1 genes. Following transient transfection, KC continuously transcribed both Nat and Mod PV L1 genes for at least 12 days, with the levels of L1 mRNAs from the Mod L1 genes significantly higher than those from the Nat L1 genes. However, continuous L1 protein expression at day 9 post-transfection was observed for both mouse and human KCs transfected with the Nat L1 genes only. Further, aa-tRNAs prepared from D8 KC cultures enhanced translation of two PV Nat L1 DNAs in RRL lysate and PV Nat L1 mRNAs in D0 cell-free lysate, whereas aa-tRNAs from D0 KCs enhanced translation of PV Mod L1 mRNAs in D8 cell-free lysate. It appears that aa-tRNAs in less-differentiated and differentiated KCs differentially match the PV Nat and Mod L1 mRNAs to regulate their translations in vitro.


Assuntos
Proteínas do Capsídeo/genética , Códon/química , Regulação Viral da Expressão Gênica , Queratinócitos/virologia , Proteínas Oncogênicas Virais/genética , Biossíntese de Proteínas , Animais , Proteínas do Capsídeo/biossíntese , Diferenciação Celular , Sistema Livre de Células , Células Cultivadas , Humanos , Queratinócitos/citologia , Cinética , Camundongos , Proteínas Oncogênicas Virais/biossíntese , RNA de Transferência/metabolismo , Transcrição Gênica , Transfecção
7.
J Natl Cancer Inst ; 96(21): 1611-9, 2004 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-15523090

RESUMO

BACKGROUND: Although immunization with tumor antigens can eliminate many transplantable tumors in animal models, immune effector mechanisms associated with successful immunotherapy of epithelial cancers remain undefined. METHODS: Skin from transgenic mice expressing the cervical cancer-associated tumor antigen human papillomavirus type 16 (HPV16) E6 or E7 proteins from a keratin 14 promoter was grafted onto syngeneic, non-transgenic mice. Skin graft rejection was measured after active immunization with HPV16 E7 and adoptive transfer of antigen-specific T cells. Cytokine secretion of lymphocytes from mice receiving skin grafts and immunotherapy was detected by enzyme-linked immunosorbent assay, and HPV16 E7-specific memory CD8+ T cells were detected by flow cytometry and ELISPOT. RESULTS: Skin grafts containing HPV16 E6-or E7-expressing keratinocytes were not rejected spontaneously or following immunization with E7 protein and adjuvant. Adoptive transfer of E7-specific T-cell receptor transgenic CD8+ T cells combined with immunization resulted in induction of antigen-specific interferon gamma-secreting CD8+ T cells and rejection of HPV16 E7-expressing grafts. Specific memory CD8+ T cells were generated by immunotherapy. However, a further HPV16 E7 graft was rejected from animals with memory T cells only after a second E7 immunization. CONCLUSIONS: Antigen-specific CD8+ T cells can destroy epithelium expressing HPV16 E7 tumor antigen, but presentation of E7 antigen from skin is insufficient to reactivate memory CD8+ T cells induced by immunotherapy. Thus, effective cancer immunotherapy in humans may need to invoke sufficient effector as well as memory T cells.


Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Carcinoma/imunologia , Carcinoma/terapia , Rejeição de Enxerto/imunologia , Imunização Passiva , Imunoterapia Ativa , Proteínas Oncogênicas Virais/imunologia , Transplante de Pele/imunologia , Adjuvantes Imunológicos/farmacologia , Transferência Adotiva , Animais , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Interferon gama/metabolismo , Queratina-14 , Queratinócitos/imunologia , Queratinas/imunologia , Camundongos , Camundongos Transgênicos , Proteínas E7 de Papillomavirus , Proteínas Repressoras/imunologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/terapia
8.
Immunol Cell Biol ; 80(5): 415-24, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12225377

RESUMO

Keratinocytes expressing the human papillomavirus (HPV) type 16 E7 protein, as a transgene driven by the K14 promoter, form a murine model of HPV-mediated epithelial cancers in humans. Our previous studies have shown that K14E7 transgenic skin grafts onto syngeneic mice are not susceptible to immune destruction despite the demonstrated presence of a strong, systemic CTL response directed against the E7 protein. Consistent with this finding, we now show that cultured, E7 transgenic keratinocytes (KC) express comparable endogenous levels of E7 protein to a range of CTL-sensitive E7-expressing cell lines but are not susceptible to CTL-mediated lysis in vitro. E7 transgenic and non-transgenic KC are susceptible to conventional mechanisms of CTL-mediated lysis, including perforin and Fas/FasL interaction when an excess of exogenous peptide is provided. The concentration of exogenous peptide required to render a cell susceptible to lysis was similar between KC and other conventional CTL targets (e.g. EL-4), despite large differences in H-2Db expression at the cell surface. Furthermore, exposure of KC to IFN-gamma increased H-2Db expression, but did not substantially alter the exogenous peptide concentration required to sensitize cells for half maximal lysis. In contrast, the lytic sensitivity of transgenic KC expressing endogenous E7 is modestly improved by exposure to IFN-gamma. Thus, failure of CTL to eliminate KC expressing endogenous E7, and by inference squamous tumours expressing E7, may reflect the need for a sustained, local inflammatory environment during the immune effector phase.


Assuntos
Interferon gama/farmacologia , Queratinócitos/efeitos dos fármacos , Proteínas Oncogênicas Virais/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Animais , Apresentação de Antígeno , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/virologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/imunologia , Citotoxicidade Imunológica , Modelos Animais de Doenças , Proteína Ligante Fas , Antígenos H-2/imunologia , Antígeno de Histocompatibilidade H-2D , Queratinócitos/imunologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus , Perforina , Proteínas Citotóxicas Formadoras de Poros , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/virologia , Linfócitos T Citotóxicos/imunologia , Receptor fas/fisiologia
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