Le morte du tumour: histological features of tumor destruction in chemo-resistant cancers following intravenous infusions of pathotropic nanoparticles bearing therapeutic genes.

The pathotropic targeting of therapeutic nanoparticles to cancerous lesions is an innovative concept that has recently been reduced to practice in clinical trials for the treatment of metastatic cancer. Previously, we reported that intravenous infusions of Rexin-G, a pathotropic nanoparticle (or vector) bearing a cyto-ablative construct, induced tumor regression, reduced tumor burden, and improved survival, while enhancing the overall quality-of-life of patients with otherwise intractable chemotherapy-resistant cancers. In this report, we describe the major histopathological and radiologic features that are characteristic of solid tumors under the destructive influences of Rexin-G administered as a single therapeutic agent. To further promote tumor eradication and enhance cancer survival, we explored the potential of an auxiliary gene transfer strategy, specifically intended to induce a localized cancer auto-immunization in addition to assisting in acute tumor destruction. This immunization strategy uses Rexin-G in combination with Reximmune-C, a tumor targeted expression vector bearing a granulocyte macrophage-colony stimulating factor (GM-CSF) gene. Intravenous infusions of Rexin-G were given first to induce apoptosis and necrosis in the metastatic tumor nodules, thus exposing tumor neo-antigens, followed by Reximmune-C infusions, intended to recruit immune cells discretely into the same compartments (or lesions). The intent of this two-step approach is to bring a complement of cells involved in humoral and cell-mediated immunity in close proximity to the immunizing tumor antigens in a concerted effort to assist in tumor eradication and to promote a cancer vaccination in situ. Herein, we also describe the distinctive histopathologic and immunocytochemical features of tumors in terminal cancer patients who received Rexin-G infusions in combination with Reximmune-C. In addition to documenting the first histological indications of clinical efficacy achieved by this novel personalized approach to cancer vaccination, we discuss new methods and strategies for advancing its therapeutic utility. Taken together with the clinical data, these histological studies serve as valuable landmarks for medical oncology, and as definitive benchmarks for the emerging field of cancer gene therapy.

Pathotropic nanoparticles for cancer gene therapy Rexin-G IV: three-year clinical experience.

Metastatic cancer is a life-threatening illness with a predictably fatal outcome, thereby representing a major unmet medical need. In 2003, Rexin-G became the world's first targeted injectable vector approved for clinical trials in the treatment of intractable metastatic disease. Uniquely suited, by design, to function within the context of the human circulatory system, Rexin-G is a pathotropic (disease-seeking) gene delivery system bearing a designer killer gene; in essence, a targeted nanoparticle that seeks out and selectively accumulates in metastatic sites upon intravenous infusion. The targeted delivery of the cytocidal gene to primary tumors and metastatic foci, in effective local concentrations, compels both cancer cells and tumor-associated neovasculature to self-destruct, without causing untoward collateral damage to non-target organs. In this study: i) we report the results of three distinctive clinical studies which demonstrate the initial proofs of concept, safety, and efficacy of Rexin-G when used as a single agent for advanced or metastatic cancer, ii) we introduce the quantitative foundations of an innovative personalized treatment regimen, designated the 'Calculus of Parity', based on a patient's calculated tumor burden, iii) we propose a refinement of surrogate end-points commonly used for defining success in cancer therapy, and iv) we map out a strategic plan for the accelerated approval of Rexin-G based on the oncologic Threshold of Credibility paradigm being developed by the Food and Drug Administration.

First clinical experience using a 'pathotropic' injectable retroviral vector (Rexin-G) as intervention for stage IV pancreatic cancer.

Metastatic or non-resectable (stage IV) pancreatic cancer has a rapidly fatal outcome (median survival: 3-6 months), thus making gene therapy a viable therapeutic option. The objectives of the clinical studies are to evaluate the safety/toxicity and potential anti-tumor response/efficacy of intravenous (i.v.) infusions of a 'pathotropic' retroviral vector bearing a cytocidal gene construct (Rexin-G) as a gene transfer intervention for stage IV pancreatic cancer. An intra-patient dose escalation regimen was used wherein increasing doses of Rexin-G were given i.v. daily for 8-10 days. Completion of this regimen was followed by a one-week evaluation period for toxicity, after which, the maximum tolerated dose of Rexin-G was administered for another 8-10 days. In a second protocol, i.v. Rexin-G was administered frontline for 6 days followed by 8 doses of weekly gemcitabine. The NIH Common Toxicity Criteria Vs.2 was used to assess toxicity, and the NCI-RECIST criteria and tumor volume measurements were used to evaluate potential anti-tumor responses. We report the results of the first 3 patients that participated in the studies. Rexin-G arrested tumor growth in 3 of 3 patients without experiencing dose-limiting toxicity. No bone marrow suppression, significant alterations in liver and kidney function, nausea and vomiting, mucositis or hair loss were observed. Two patients are alive with stable disease approximately 5 and 14 months from diagnosis, and 1 patient is alive with progressive disease 20 months from diagnosis. The encouraging results of this first clinical experience will guide the design and planning of phase I/II clinical trials to establish the safety and efficacy of Rexin-G as the first targeted injectable gene therapy vector for stage IV pancreatic cancer.

The growth inhibitory effect of p21 adenovirus on androgen-dependent and -independent human prostate cancer cells.

OBJECTIVE: To assess the potential of p21 as a gene therapy treatment for prostate cancer, by introducing p21 into both androgen-dependent (AD) and -independent (AI) human prostate cancer cell lines via a recombinant adenoviral vector, Ad5CMV-p21, carrying human p21 cDNA. MATERIALS AND METHODS: The LNCaP, DU145 and PC-3 human prostate cancer cell lines were cultured and infected with Ad5CMV-p21. Cell growth, cell-cycle progression and tumorigenicity were then assessed by thymidine incorporation into cellular DNA, and cell number, flow cytometry, and tumour growth after inoculating the cells into nude mice. RESULTS: Growth was inhibited in Ad5CMV-p21 viral-infected AD and AI prostate cancer cells. The effects were dose-dependent, regardless of the androgen status of the cell lines. Flow cytometric analysis showed that Ad5CMV-p21 arrested cell-cycle progression at G1/S with no appreciable effect on the levels of apoptotic cells. The tumorigenicity of cancer cells infected with Ad5CMV-p21 was greatly reduced in athymic mice. CONCLUSIONS: These results suggest that Ad5CMV-p21 may be a new therapeutic agent for human prostate cancer gene therapy.

Long term inhibition of neointima formation in balloon-injured rat arteries by intraluminal instillation of a matrix-targeted retroviral vector bearing a cytocidal mutant cyclin G1 construct.

Restenosis from neointimal proliferation is a frequent complication of intracoronary stenting and catheter-based revascularization procedures. Currently, there is no known therapeutic strategy that has been sufficiently effective to warrant its widespread use. In the present study, the anti-proliferative properties of a matrix (collagen)-targeted retroviral vector bearing a mutant cyclin G1 (DNT 41-249) construct was evaluated in vitro and in vivo. In controlled one-month efficacy studies, the intraluminal instillation of the mutant cyclin G1 vector significantly inhibited neointima lesion formation in balloon-injured rat arteries without neointimal growth, associated necrosis or intense inflammatory reaction. Taken together, these data extend the potential utility of the matrix-targeted mutant cyclin G1 retroviral vector for management of vascular restenosis.

Suppression of arthritis by forced expression of cyclin-dependent kinase inhibitor p21(Cip1) gene into the joints.

Rheumatoid synovial fibroblasts (RSF) express cyclin-dependent kinase (CDK) inhibitors p16(INK4a) and p21(Cip1) when they are growth-inhibited in vitro. The induction of p16(INK4a) is characteristic of RSF and intra-articular p16(INK4a) gene therapy has been shown to suppress adjuvant arthritis (AA) of rats. The other inducible CDK inhibitor, p21(Cip1), has multiple functions depending on the cell type. They include inhibition of CDK as well as promotion of active CDK complex formation and induction of apoptosis. This study is to discern the biological effects of p21(Cip1) gene transfer into RSF and its therapeutic effects on AA. A recombinant adenovirus containing a human p21(Cip1) gene and control adenoviruses were prepared. RSF infected with these viruses were examined for their cell growth. Apoptotic cell death was evaluated by nuclear staining and DNA fragmentation analysis. In vivo gene therapy of rat AA was carried out by intra-articular injection of the viruses. Severity of the arthritis was clinically scored. The treated joints were examined histologically and proliferating cell nuclear antigens (PCNA) were detected immunohistochemically. The adenoviral p21(Cip1) gene transfer inhibited growth of RSF without inducing apoptosis. p21(Cip1) gene therapy suppressed AA clinically and histologically. The effects were comparable to p16(INK4a) gene therapy. PCNA expression was reduced in the p21(Cip1)-treated joints. The adenoviral gene transfer of p21(Cip1) ameliorated rat AA. The effect was attributable to inhibition of proliferation. Because p21(Cip1) is induced more easily by many chemicals than p16(INK4a), it also appears to be a feasible target in developing anti-rheumatic drugs.

In vitro and in vivo transfection of p21 gene enhances cyclosporin A-mediated inhibition of lymphocyte proliferation.

Cyclosporine has potent antiproliferative properties, some of which may be via the induction of the cyclin inhibitor p21. In this study, we describe the effects of in vitro and in vivo transfection of p21 in lymphoid and nonlymphoid cells. For in vitro studies, p21 sense plasmid DNA was transfected in A-549 cells (lung adenocarcinoma cell line) and Jurkat cells (human lymphoid cell line). This in vitro transfection of p21 resulted in the inhibition of spontaneous and mitogen-induced cellular proliferation ([3H]thymidine uptake) and also augmented the antiproliferative effects of cyclosporine. In vivo transfection of p21 was accomplished in mice via the i.m. injection of p21 sense plasmid DNA complexed with cationic lipids. As was the case in the cell lines, p21 mRNA was augmented in heart, lung, liver, and spleen 7 days after i.m. injection of p21 sense plasmid DNA. The mitogen (anti-CD3)-induced proliferation of splenocytes from p21-overexpressing mice was significantly decreased, and again this effect was augmented by cotreatment with cyclosporine. These novel findings demonstrate the potential of targeting the cell cycle directly to inhibit alloimmune activation in organ transplantation. This may serve as an alternate strategy to induce immunosuppression, perhaps with less toxicity than that which is seen with conventional immunosuppressive agents.

Bases moleculares del proceso de muerte celular programada: implicaciones de la proteína supresora de tumores p53 y otras proteínas en el control del ciclo celular: mecanismos de acción apoptótica: revisión / Molecular bases of programmed cell death: involvement of tumor suppresor protein p53 and other proteins in the control of the cellular cycle: review

La apoptosis es un mecanismo de daño celular que ocurre en el normal desarrollo y en la regulación de tejidos y órganos en vertebrados. Neuronas bajo apoptosis dependiente o independiente de p53, dependiendo UPON del estímulo que conduce a fragmentación del DNA. Muchas neuronas en el desarrollo del sistema nervioso sufren apoptosis, siendo la ciclina D1 un esencial mediador del daño celular neural. Otras características de apoptosis son la condensación del núcleo, la fragmentación de la cromatina junto a los sitios de ruptura en los nucleosomas, la rotura de la membrana y la formación de cuerpos apoptóticos. Entre los posibles mecanismos moleculares estan: (a) la activación de proteasas, como ICE (II-1 ß converting enzyme); y una pequeña Ribonucleoproteína U1, siendo sustratos para ICE y sus homólogos (tales como ICH y otras proteínas). El gen p53 codifica un factor de transcripción que contribuye a varias diferentes actividades celulares, incluyendo la opoptosis, la respuesta celular a la radiación, y la actividad de proteínas tales como GADD, Bcl-2 (represor de apoptosis) y Bax. p53 ejecuta un papel como un inhibidor de apoptosis por transactivación de la expresión del gen Bax. El gen supresor de tumores p53 limita la proliferación celular por parada del ciclo celular en G1, o por apoptosis, dependiendo del contexto celular. La proteína p21 es un inhibidor de ciclinas dependientes de kinasas, la cual es transactivada por p53. Durante la apoptosis hay una activación de c-myc, y del factor de transcripción NF-kB (factor nuclear kappa ß), el cual es un importante regulador de la apoptosis. Como un ejemplo de señalización apoptosis nosotros hemos seleccionado el problema relatando el sistema Fas/APO en timocitos

Resumption of meiosis is initiated by the accumulation of cyclin B in bovine oocytes.

In mammals, oocytes are maintained at the G2/M border of the first meiotic division, often for years, before being ovulated. The oocytes wait for a signal that will trigger the chromosome segregation necessary to produce a haploid cell, which is required for fertilization. This event, meiotic resumption, is determined by activation of the M phase-promoting factor (MPF), constituted of the p34cdc2 kinase (p34) and cyclin B. MPF activation in oocytes of large mammals requires protein synthesis. The accumulation of an initiator protein to a critical level plays a key role in determining the cell-cycle timing of p34 kinase activation. In this study, we undertook a search to identify the initiator protein that controls meiosis in the cow. Oocytes were cultured in various conditions before being processed for two-dimensional (2D) gels or Western blotting or fixed for nuclear evaluation. Comparison of 2D patterns of the synthesized protein required for meiotic resumption suggest an initiator role for cyclin. Immunodetection of other proteins that may be initiator proteins (p34, cdc25, and microtubule-associated protein kinases) in immature oocytes, and the absence of cyclin in these oocytes, suggest an important role for cyclin B. Human cyclin B1 protein microinjected into cycloheximide-treated bovine oocytes triggered meiotic resumption. We can conclude that the quantity of cyclin present in bovine oocytes is critical to meiotic resumption.