p19Arf sensitizes B16 melanoma cells to interferon-ß delivered via mesenchymal stem cells in vitro.

The immune stimulatory and anti-neoplastic functions of type I interferon have long been applied for the treatment of melanoma. However, the systemic application of high levels of this recombinant protein is often met with toxicity. An approach that provides localized, yet transient, production of type I interferon may overcome this limitation. We propose that the use of mesenchymal stem cells (MSCs) as delivery vehicles for the production of interferon-ß (IFNß) may be beneficial when applied together with our cancer gene therapy approach. In our previous studies, we have shown that adenovirus-mediated gene therapy with IFNß was especially effective in combination with p19Arf gene transfer, resulting in immunogenic cell death. Here we showed that MSCs derived from mouse adipose tissue were susceptible to transduction with adenovirus, expressed the transgene reliably, and yet were not especially sensitive to IFNß production. MSCs used to produce IFNß inhibited B16 mouse melanoma cells in a co-culture assay. Moreover, the presence of p19Arf in the B16 cells sensitizes them to the IFNß produced by the MSCs. These data represent a critical demonstration of the use of MSCs as carriers of adenovirus encoding IFNß and applied as an anti-cancer strategy in combination with p19Arf gene therapy.

Bicistronic transfer of CDKN2A and p53 culminates in collaborative killing of human lung cancer cells in vitro and in vivo.

Cancer therapies that target a single protein or pathway may be limited by their specificity, thus missing key players that control cellular proliferation and contributing to the failure of the treatment. We propose that approaches to cancer therapy that hit multiple targets would limit the chances of escape. To this end, we have developed a bicistronic adenoviral vector encoding both the CDKN2A and p53 tumor suppressor genes. The bicistronic vector, AdCDKN2A-I-p53, supports the translation of both gene products from a single transcript, assuring that all transduced cells will express both proteins. We show that combined, but not single, gene transfer results in markedly reduced proliferation and increased cell death correlated with reduced levels of phosphorylated pRB, induction of CDKN1A and caspase 3 activity, yet avoiding the induction of senescence. Using isogenic cell lines, we show that these effects were not impeded by the presence of mutant p53. In a mouse model of in situ gene therapy, a single intratumoral treatment with the bicistronic vector conferred markedly inhibited tumor progression while the treatment with either CDKN2A or p53 alone only partially controlled tumor growth. Histologic analysis revealed widespread transduction, yet reduced proliferation and increased cell death was associated only with the simultaneous transfer of CDKN2A and p53. We propose that restoration of two of the most frequently altered genes in human cancer, mediated by AdCDKN2A-I-p53, is beneficial since multiple targets are reached, thus increasing the efficacy of the treatment.

p19Arf sensitizes B16 melanoma cells to interferon-β delivered via mesenchymal stem cells in vitro

The immune stimulatory and anti-neoplastic functions of type I interferon have long been applied for the treatment of melanoma. However, the systemic application of high levels of this recombinant protein is often met with toxicity. An approach that provides localized, yet transient, production of type I interferon may overcome this limitation. We propose that the use of mesenchymal stem cells (MSCs) as delivery vehicles for the production of interferon-β (IFNβ) may be beneficial when applied together with our cancer gene therapy approach. In our previous studies, we have shown that adenovirus-mediated gene therapy with IFNβ was especially effective in combination with p19Arf gene transfer, resulting in immunogenic cell death. Here we showed that MSCs derived from mouse adipose tissue were susceptible to transduction with adenovirus, expressed the transgene reliably, and yet were not especially sensitive to IFNβ production. MSCs used to produce IFNβ inhibited B16 mouse melanoma cells in a co-culture assay. Moreover, the presence of p19Arf in the B16 cells sensitizes them to the IFNβ produced by the MSCs. These data represent a critical demonstration of the use of MSCs as carriers of adenovirus encoding IFNβ and applied as an anti-cancer strategy in combination with p19Arf gene therapy.

Molecular targeting of neuroblastoma with a novel p16INK4a transporter system.

Potential molecular targets in neuroblastoma include ALK mutations, p16 deletion and CDK2A mutations; however, targeted therapeutics have not been developed for these factors. We developed Wr-T, a new system for intracellular peptide and protein delivery with a 30-residue sequence that mediates molecule entrapment and intracellular permeability. Wr-T was used to introduce the p16INK4a functional peptide to restore the tumor suppressor function of p16INK4a. Introduction of Wr-T into rats with subcutaneous grafts of neuroblastoma produced an astonishing 75.6% tumor suppression (p<0.0005). Thus, the p16INK4a functional peptide can be introduced in low doses and, because it exists in vivo, it should produce fewer side-effects than standard chemotherapy. We suggest this system could be used for molecular-targeted peptides other than p16INK4a and should be pursued for further development.

Systemic transduction of p16INK4A antitumor peptide inhibits the growth of MBT-2 mouse bladder tumor cell line grafts.

p16(INK4a) (p16), a key molecule in bladder tumor development, inhibits the activities of cyclin-dependent kinases (CDKs) and maintains the retinoblastoma protein (pRb) in its active hypophosphorylated state. Following the finding that the p16 antitumor peptide dramatically inhibits the growth of aggressive leukemia/lymphoma through the restoration of p16 function using the Wr-T peptide transporter system, in this study, we developed a systemic therapy using mouse­p16 peptide (m­p16) in subcutaneous p16­null mouse bladder tumors. In vitro analysis showed that the growth of p16­null bladder tumor cells and the hyperphosphorylation of their pRbs were inhibited by p16 transduction in a concentration­dependent manner. In an animal model, p16­null MBT­2 cells were injected subcutaneously into KSN/SKC nude mice. The systemic delivery of the m­p16 peptide using Wr­T by cardiac injection significantly inhibited the growth of solid MBT­2 tumors compared with the control phosphate­buffered saline (PBS) injection. Histological examination by TUNEL staining revealed that apoptosis was increased and pRb phosphorylation was inhibited. Thus, the systemic peptide delivery of p16 restores the hypophosphorylation of pRb and may be a useful tool for the treatment of bladder tumors.

Papel de p16 en lesiones preinvasivas e invasivas de cáncer de cuello uterino. Aplicación de la técnica a citología convencional / Role of p16 expression in preinvasive and invasive cervical lesions: application of conventional cytology

Introducción: La detección de la sobreexpresión de la proteína p16 mediante técnicas inmunohistoquímicas e inmunocitoquímicas es un valor seguro en la identificación de lesiones epiteliales cervicales de alto grado, sirviendo al mismo tiempo para detectar aquellas lesiones de bajo grado cito o histológico con integración viral, firmes candidatas a la progresión a lesión de alto grado. Material y métodos: Hemos evaluado la eficacia de la detección inmunohistoquímica e inmunocitoquímica de la sobreexpresión de la proteína p16 sobre muestras de cérvix uterino. Para ello hemos recurrido a 58 casos de biopsias de cérvix y 53 citologías con diagnóstico de positividad para la infección por el virus del papiloma humano. Resultados: Hemos observado cómo el 100% de las lesiones de alto grado eran teñidas mientras que so ́lo un porcentaje de las de bajo grado hacían lo propio. Los resultados obtenidos en citología fueron extrapolados a las citologías (correspondientes a las mismas pacientes que las biopsias) obteniendo similares resultados. Conclusiones: La identificación de la sobreexpresión de la proteína p16 por métodos inmunocitoquímicos sobre citologías cervicovaginales convencionales muestra resultados superponibles a los obtenidos sobre muestras histológicas (AU)

Introduction: Detection of p16 expression by immunohistochemistry and immunocytochemistry is a good standard for the identification of high-grade cervical epithelial lesions and low-grade lesions with DNA HPV viral integration (with a tendency for progression). Material and methods: We evaluated p16 expression in 58 HPV-positive cervical biopsies and 53 conventional cytological samples that tested HPV-positive with immunohistochemical and immunocytochemical techniques. Results: All high-grade lesions were positive for p16 while only some of the low-grade lesions were positive. The results obtained in histological samples could be extrapolated to cytological samples from the same patients. Conclusions: p16 expression in conventional cytology provides similar results to those in histological samples (AU)