Combination of cabazitaxel and p53 gene therapy abolishes prostate carcinoma tumor growth.

For patients with metastatic prostate cancer, the 5-year survival rate of 31% points to a need for novel therapies and improvement of existing modalities. We propose that p53 gene therapy and chemotherapy, when combined, will provide superior tumor cell killing for the treatment of prostate carcinoma. To this end, we have developed the AdRGD-PGp53 vector which offers autoregulated expression of p53, resulting in enhanced tumor cell killing in vitro and in vivo. Here, we combined AdRGD-PGp53 along with the chemotherapy drugs used in the clinical treatment of prostate carcinoma, mitoxantrone, docetaxel, or cabazitaxel. Our results indicate that all drugs increase phosphorylation of p53, leading to improved induction of p53 targets. In vitro experiments reveal that AdRGD-PGp53 sensitizes prostate cancer cells to each of the drugs tested, conferring increased levels of cell death. In a xenograft mouse model of in situ gene therapy, AdRGD-PGp53 treatment, when combined with cabazitaxel, drastically reduced tumor progression and increased survival rates to 100%. Strikingly, we used a sub-therapeutic dose of cabazitaxel thus avoiding leukopenia, yet still showed potent anti-tumor effects when combined with AdRGD-PGp53 in this mouse model. The AdRGD-PGp53 approach warrants further development for its application in gene therapy of prostate carcinoma.

Cancer-malaria: hidden connections.

Cancer and malaria exemplify two maladies historically assigned to separated research spaces. Cancer, on the one hand, ranks among the top priorities in the research agenda of developed countries. Its rise is mostly explained by the ageing of these populations and linked to environment and lifestyle. Malaria, on the other hand, represents a major health burden for developing countries in the Southern Hemisphere. These two diseases also belong to separate fields of medicine: non-communicable diseases for cancer and communicable diseases for malaria.

Lack of Both Nucleotide-Binding Oligomerization Domain-Containing Proteins 1 and 2 Primes T Cells for Activation-Induced Cell Death.

Nucleotide-binding oligomerization domain (Nod)-containing proteins Nod1 and Nod2 play important roles in the innate immune response to pathogenic microbes, but mounting data suggest these pattern recognition receptors might also play key roles in adaptive immune responses. Targeting Nod1 and Nod2 signaling pathways in T cells is likely to provide a new strategy to modify inflammation in a variety of disease states, particularly those that depend on Ag-induced T cell activation. To better understand how Nod1 and Nod2 proteins contribute to adaptive immunity, this study investigated their role in alloantigen-induced T cell activation and asked whether their absence might impact in vivo alloresponses using a severe acute graft versus host disease model. The study provided several important observations. We found that the simultaneous absence of Nod1 and Nod2 primed T cells for activation-induced cell death. T cells from Nod1 × 2-/- mice rapidly underwent cell death upon exposure to alloantigen. The Nod1 × 2-/- T cells had sustained p53 expression that was associated with downregulation of its negative regulator MDM2. In vivo, mice transplanted with an inoculum containing Nod1 × 2-/- T cells were protected from severe graft versus host disease. The results show that the simultaneous absence of Nod1 and Nod2 is associated with accelerated T cell death upon alloantigen encounter, suggesting these proteins might provide new targets to ameliorate T cell responses in a variety of inflammatory states, including those associated with bone marrow or solid organ transplantation.

Clinicopathological and prognostic significance of programmed cell death ligand-1 expression in lung adenocarcinoma and its relationship with p53 status.

INTRODUCTION: PD-L1 expression is a predictive biomarker for response to anti-programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) immune checkpoint inhibitors and can be evaluated by immunohistochemistry. Results of the clinicopathologic characteristics of PD-L1-positive lung adenocarcinoma have been inconsistent in previous studies, and there are no reports on the relationship between PD-L1 expression and p53 status in lung adenocarcinoma. METHODS: We examined PD-L1 and p53 expression in a total of 323 surgically resected lung adenocarcinoma cases using anti-PD-L1 (clone SP142) and anti-p53 (clone DO-7) antibodies, and analyzed the clinicopathologic characteristics of PD-L1-positive cases and their relationship with p53 status. RESULTS: PD-L1 expression in tumor cells was positive in 60 of 323 cases (18.6%). Higher PD-L1 expression (≥50%) was more prevalent in former or current smokers (p=0.026) and was associated with more pack-years (p=0.016). PD-L1-positive tumors were significantly associated with solid predominant type (p<0.001), p53 aberrant expression (p<0.001), and PD-L1 expression in tumor-infiltrating immune cells (p<0.001). Patients with stage I to III tumors harboring PD-L1-positive tumor cells showed poor recurrence-free survival (p<0.001) and overall survival (p<0.001) on univariate analysis. CONCLUSIONS: PD-L1 expression in tumor cells, solid predominant histology, p53 aberrant expression, and PD-L1 expression in tumor-infiltrating immune cells are closely related. These variables should be considered when analyzing the clinical outcomes of patients with lung adenocarcinomas treated with anti-PD1/PD-L1 immune checkpoint inhibitors.

Inmunoexpresión p53, sangre oculta en heces de pacientes colecistectomizados y colelitiasis con adenomas colónicos / P53 Immunoexpression, Faecal Occult Blood from Cholecystectomized Patients and Cholelithiasis with Colonic Adenomas

Introducción: la secuencia adenoma- adenocarcinoma, es resultado de fallos genéticos en las células intestinales heredados o adquiridos. Objetivo: determinar la posible relación entre la inmunoexpresión de la p53 y la positividad de la sangre oculta en heces en los adenomas de colon con alto grado de displasia diagnosticados en pacientes colecistectomizados o con colelitiasis. Métodos: se realizó un estudio descriptivo, de corte transversal, en el Instituto de Gastroenterología, en el período de mayo de 2013 hasta junio de 2015. Se realizaron pruebas estadísticas descriptiva y de chi Cuadrado y probabilidad exacta de Fisher. Resultados: la proporción de adenomas con alto grado de displasia fue similar en pacientes colecistectomizados y con colelitiasis (50 por ciento) respectivamente. Una alta proporción se diagnosticó en colecistectomizados femeninas (35 por ciento), con 60 y más años de edad (53 por ciento) y 11 y más años de colecistectomizados (60 por ciento), mientras que en las colelitiasis fueron masculinos (30 por ciento). Conclusiones: una alta proporción de adenomas con alto grado de displasia presentan inmunoexpresión de la p53 y sangre en heces positiva en pacientes colecistectomizados y con colelitiasis, que se reporta por vez primera(AU)

Introduction: The adenoma - adenocarcinoma sequence is a result of inherited or acquired genetic failures in the intestinal cells. Objective: To determine the immunohistochemical expression of p53 and the positivity of the fecal occult blood test in colon adenomas with high degree of diagnosed dysplasia in cholecystectomized patients or with cholelithiasis. Methods: Descriptive, cross-sectional study conducted in the Institute of Gastroenterology in the period of May, 2013 to June, 2015. Statistical tests were statistics testing, exact Chi Square and Fisher's probability tests. Results: The proportion of adenomas with high degree of dysplasia was similar in cholecystectomized patientsand with cholelithiasis (50 percent) respectively. A high proportion diagnosed in colecistectomizados women (35 percent), 60 and more years of age (53 percent) and 11 and more years of performed cholecystectomy (60 percent), whereas cholelithiasis prevailed in males (30 percent). Conclusions: High proportion of adenomas with high degree of dysplasia present p 53 immunoexpression and positive fecal occult blood test in cholecystectomized patients and patients with cholelithiasis that is reported for the first time(AU)

Inmunoexpresión p53, sangre oculta en heces de pacientes colecistectomizados y colelitiasis con adenomas colónicos / P53 Immunoexpression, Faecal Occult Blood from Cholecystectomized Patients and Cholelithiasis with Colonic Adenomas

Introducción: la secuencia adenoma- adenocarcinoma, es resultado de fallos genéticos en las células intestinales heredados o adquiridos.Objetivo: determinar la posible relación entre la inmunoexpresión de la p53 y la positividad de la sangre oculta en heces en los adenomas de colon con alto grado de displasia diagnosticados en pacientes colecistectomizados o con colelitiasis.Métodos: se realizó un estudio descriptivo, de corte transversal, en el Instituto de Gastroenterología, en el período de mayo de 2013 hasta junio de 2015. Se realizaron pruebas estadísticas descriptiva y de chi Cuadrado y probabilidad exacta de Fisher.Resultados: la proporción de adenomas con alto grado de displasia fue similar en pacientes colecistectomizados y con colelitiasis (50 por ciento) respectivamente. Una alta proporción se diagnosticó en colecistectomizados femeninas (35 por ciento), con 60 y más años de edad (53 por ciento) y 11 y más años de colecistectomizados (60 por ciento), mientras que en las colelitiasis fueron masculinos (30 por ciento).Conclusiones: una alta proporción de adenomas con alto grado de displasia presentan inmunoexpresión de la p53 y sangre en heces positiva en pacientes colecistectomizados y con colelitiasis, que se reporta por vez primera(AU)

Introduction: The adenoma - adenocarcinoma sequence is a result of inherited or acquired genetic failures in the intestinal cellsObjective: To determine the immunohistochemical expression of p53 and the positivity of the fecal occult blood test in colon adenomas with high degree of diagnosed dysplasia in cholecystectomized patients or with cholelithiasis.Methods: Descriptive, cross-sectional study conducted in the Institute of Gastroenterology in the period of May, 2013 to June, 2015. Statistical tests were statistics testing, exact Chi Square and Fisher's probability tests.Results: The proportion of adenomas with high degree of dysplasia was similar in cholecystectomized patientsand with cholelithiasis (50 percent) respectively. A high proportion diagnosed in colecistectomizados women (35 percent), 60 and more years of age (53 percent) and 11 and more years of performed cholecystectomy (60 percent), whereas cholelithiasis prevailed in males (30 percent).Conclusions: High proportion of adenomas with high degree of dysplasia present p 53 immunoexpression and positive fecal occult blood test in cholecystectomized patients and patients with cholelithiasis that is reported for the first time(AU)

Promising systemic immunotherapies in head and neck squamous cell carcinoma.

Patients with head and neck squamous cell carcinoma (HNSCC) demonstrate poor survival and significant treatment morbidity with standard therapy. The immune profile in HNSCC, whether caused by carcinogen exposure or human papillomavirus (HPV), is notably immunosuppressive. Early clinical trials of immunotherapy in HNSCC were troubled by systemic toxicity or difficulties in local administration. Now, interest in immunotherapy has been revitalized by mechanistic insights into immune evasion by HNSCC, coupled to ongoing development of novel immunotherapies. This review will summarize immune escape mechanisms in HNSCC, namely downregulation of tumor antigen (TA) presentation, aberrant regulation of the signal transducer and activator of transcription (STAT) family, the immunosuppressive cytokine milieu, and dysregulation of immune effector cells. Therapeutic strategies hypothesized to specifically counter HNSCC immunosuppression will then be discussed. We will survey TA- targeted monoclonal antibodies (mAb), including the prototype cetuximab, as well as adjunctive strategies to enhance antibody-dependent cell-mediated cytotoxicity. We will review immunomodulation to restore STAT1/STAT3 activation balance. Examples of mAb therapy to block immunosuppressive cytokines, such as interleukin-6 or VEGF, will be provided. mAbs which release co-inhibitory T cell receptors such as CTLA-4 and PD-1, overexpressed in HNSCC, also hold therapeutic promise. Finally, we will describe principles for therapeutic vaccination in HPV-associated HNSCC, where non-host TAs such as viral oncoproteins represent ideal targets, and HPV-negative HNSCC, where p53 is a promising target. Insights into immunosuppression in HNSCC have elucidated mechanistic targets for immunotherapy. Rational clinical investigation may lead to effective stand alone or combinatorial treatment approaches.

Interactions between the tumor suppressor p53 and immune responses.

PURPOSE OF REVIEW: The p53 tumor suppressor is a master regulator of antitumor defenses through its control of growth arrest, senescence and apoptosis. In recent years, p53 regulation was found to extend to a variety of biological processes including autophagy, fertility, metabolism and immune responses. Here, we focus on the role of p53 in the immune system. We explore the relationship between p53 and the innate immune response with particular emphasis on the Toll-like receptor (TLR) pathway and implications for cancer therapy. RECENT FINDINGS: Numerous studies have shown that the immune system, especially innate immunity, has a critical role in tumor development. It appears that p53 can influence innate immune responses as part of its tumor suppressor activities and recent work suggests that the complete set of innate immune TLR genes are responsive to chromosomal stress and the transcriptional network regulated by p53. Activation of p53 by common antitumor agents results in p53 dependent regulation of expression of most TLR genes in human primary and cancer cell lines, resulting in modulation of TLR downstream responses to cognate ligands. In addition several tumor-associated p53 mutants can also affect TLR gene expression. These observations together with the discovery of other immune-related p53 target genes provide new insights into the relationship between p53 and immunity and suggest approaches that might be useful in cancer therapies. SUMMARY: The tumor suppressor p53 can modulate innate immune gene responses in response to factors that can activate p53. This is expected to provide new opportunities in cancer diagnosis and in chemotherapeutic strategies that employ specific TLR agonists or antagonists that target the TLR pathway.

p53-dependent senescence delays Emu-myc-induced B-cell lymphomagenesis.

The effect of p53-dependent cell-cycle arrest and senescence on Emu-myc-induced B-cell lymphoma development remains controversial. To address this question, we crossed Emu-myc mice with the p53(515C) mutant mouse, encoding the mutant p53R172P protein that retains the ability to activate the cell-cycle inhibitor and senescence activator p21. Importantly, this mutant lacks the ability to activate p53-dependent apoptotic genes. Hence, Emu-myc mice that harbor two p53(515C) alleles are completely defective for p53-dependent apoptosis. Both Emu-myc::p53(515C/515C) and Emu-myc::p53(515C/+) mice survive significantly longer than Emu-myc::p53(+/-) mice, indicating the importance of the p53-dependent non-apoptotic pathways in B-cell lymphomagenesis. In addition, the p53(515C) allele is deleted in several Emu-myc::p53(515C/+) lymphomas, further emphasizing the functionality of p53R172P in tumor inhibition. Lymphomas from both Emu-myc::p53(515C/515C) and Emu-myc::p53(515C/+) mice retain the ability to upregulate p21, resulting in cellular senescence. Senescence-associated beta-galactosidase (SA beta-gal) activity was observed in lymphomas from Emu-myc::p53(+/+), Emu-myc::p53(515C/515C) and Emu-myc::p53(515C /+) mice but not in lymphomas isolated from Emu-myc::p53(+/-) mice. Thus, in the absence of p53-dependent apoptosis, the ability of p53R172P to induce senescence leads to a significant delay in B-cell lymphoma development.

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Closing the manufacturing process of dendritic cell vaccines transduced with adenovirus vectors.

Anticancer immunotherapy using dendritic cell (DC) based vaccines provides an adjuvant therapeutic strategy that is not cross reactive with conventional therapeutics. However, manufacturing of DC vaccines requires stringent adherence to Good Manufacturing Practice (GMP) methods and rigorous standardization. Optimally this includes a closed system for monocyte isolation, in combination with closed culture and washing systems and an effective vector transduction strategy. In this study, we used the Gambro Elutra to enrich monocytes from non-mobilized leukapheresis products collected from healthy donors. This approach enriched monocytes from an average frequency of 13.6+3.2% (mean+SEM), to an average frequency of 79.5+4.3% following enrichment with a yield of 79 to 100%. The monocytes were then cultured in a closed system using gas permeable Vuelife fluoroethylene propylene (FEP) bags and X-vivo-15 media containing 10 ng/ml granulocyte-macrophage colony-stimulation factor (GM-CSF) and 5 ng/ml Interleukin (IL) 4. The cultures were re-fed on days two and four, with a 25% media volume and cytokines. Following culture for seven days, the cells were harvested using a Cobe-2991 and concentrated using a bench centrifuge retrofitted with blocks to allow centrifugation of 72 ml bags and supernatant removed using a plasma extractor. This approach reduced the media volume to an average of 17.4 ml and an average DC concentration of 6.3+1.0x10(7) cells/ml, a viability of 93.8+2.2%, a purity of 88.9+3.3% and a total yield of 8.5+1.4x10(8) DCs. Based on the identification of DR+ cells as DCs we had an average yield of 46+8% using a calculation based on the number of monocytes in the apheresis product and the resulting DCs differentiated from monocytes. The use of DCs as a vaccine, required transduction with an adenovirus (Adv) vector with the tumor suppressor, p53 transgene (Adv5CMV-p53) as the antigen at a DC concentration of 9x10(6) DCs/ml at an Ad5CMV-p53: DC ratio of 20,000:1, and a 2 or 3 hour co-culture, followed by a 1:10 dilution with media and an additional 16-22 hour incubation. Following incubation, the DCs were washed twice and the supernatants removed using a plasma extractor. The average viability after infection with Ad5CMV-p53 was 87.9+/-2.6% with an average of 20.3+5.4% of the DCs expressing p53. The calculated yield of DCs following Ad5CMV-p53 transduction, based on the number of monocytes in the apheresis products, averaged 12.4+3.8%. We conclude that it is possible to efficiently manufacture Adv transduced DCs using a functionally closed system.

Overexpression of VEGF is associated with positive p53 immunostaining in hepatocellular carcinoma (HCC) and adverse outcome of HCC patients.

BACKGROUND AND OBJECTIVES: To elucidate the clinicopathological correlations among vascular endothelial growth factor (VEGF), microvessel density (MVD) and tumor suppressor gene p53 in hepatocellular carcinomas (HCCs), we adopted a new definition of "VEGF overexpression." METHODS: The expressions of VEGF, MVD, and p53 in 113 HCC specimens were analyzed by immunohistochemistry. RESULTS: VEGF expression in surrounding liver tended to be stronger (VEGF overexpression, 31%) than, or similar to (57%) that in HCCs (P = 0.001). P53 positivity was noted in 42 cases (37.1%). MVD ranged from 22 to 201 microvessels/field determined for 5 high-power fields. VEGF expression in HCCs was positively correlated with MVD (P = 0.001). VEGF overexpression is positively correlated with young age (P = 0.008), male gender (P = 0.01), hepatitis B viremia (P = 0.013), high alpha-fetoprotein levels (P < 0.001), p53 (+) (P = 0.036), advanced-stage HCC (P = 0.015), and HCC dedifferentiation (P = 0.004). Survival analyses indicated that VEGF overexpression, high MVD, and advanced-stage HCC were independent poor prognostic factors for disease-free and overall survival. CONCLUSION: This study provides evidence of a positive association between parameters reflective of angiogenesis, and p53 expression in HCCs. VEGF overexpression exhibited a significant correlation with viremia and survival.

The tumour suppressor gene p53 modulates the severity of antigen-induced arthritis and the systemic immune response.

p53 is a transcription factor with a well-described role in the induction of apoptosis and cell cycle arrest as part of a protective response to a variety of stressful stimuli. Expansion of inflamed tissue in rheumatoid arthritis has been related to the loss of functioning p53, and the severity of collagen-induced arthritis is increased in p53-/- mice. Our objective was to assess the role of p53 in a model of adaptive immunity, antigen-induced arthritis (AIA). AIA was induced in p53-/- and wild-type mice by priming with methylated bovine serum albumin followed by intra-articular challenge. Severity of arthritis was assessed using a standardized scoring system and synovial apoptosis was detected by TdT-mediated biotin-dUTP nick-end labelling. Splenocyte proliferation was measured by [H(3)] incorporation and interferon (IFN)-gamma release. Splenocyte viability was assessed using Titreglow. Splenic T cell activation status was assessed by flow cytometry. Serum cytokines were measured using enzyme-linked immunosorbent assay. Increased severity of AIA in p53-/- mice was associated with decreased synovial apoptosis and with increased delayed-type hypersensitivity response, increased mitogen and antigen-induced splenocyte proliferation and increased IFN-gamma release in p53-/- mice compared with wild-type mice. Antigen-specific immunoglobulin responses were equivalent in both groups. Splenocyte viability was increased in p53-/- mice but T cell apoptosis was equivalent. T cell activation markers were increased in p53-/- mice compared with wild-type mice. Lipopolysaccharide-induced tumour necrosis factor release was increased in p53-/- mice with a trend to increased interleukin-6 in p53-/- mice compared with littermates. p53 is involved in the modulation of adaptive and innate immune responses relevant to arthritis models and is also involved in the modulation of severity of AIA by both cell-cycle dependent and cell-cycle-independent mechanisms.

Antiproliferative activity of cisplatin detected by CFSE in p53-proficient and p53-deficient cells.

We have previously developed experimental and data analysis procedures to measure the antiproliferative activity of drugs in continuously proliferating cancer cell lines using carboxyfluorescein diacetate succinimidyl ester (CFSE). The method was applied here to analyze the role of p53 in the effect of the anticancer drug cisplatin, distinguishing events occurring in the first generation of cells from those in the second and subsequent generations. A CFSE-loaded colon carcinoma cell line expressing functional wild-type p53 was treated for 1 with cisplatin in parallel with its p53-deficient counterpart, collecting frequency distributions of DNA and CFSE content up to 72 h after treatment. At a sublethal cisplatin concentration proliferation was temporarily inhibited but then the block was overcome and most cells were able to divide several times. The initial block was stronger in HCTp53-/- cells, resulting in a larger proportion of undivided cells at 24 h. This was confirmed and amplified at a higher, lethal concentration, where undivided G(2)M-blocked p53-deficient cells eventually died by non-apoptotic mechanisms, while p53-proficient cells avoided this with a less stringent block. This gave p53-proficient cells more time to repair and eventually decide on survival or apoptotic death before traversing the cycle into their second generation.

Genetically modified myeloma cell vaccine inducing antitumor immune response in vivo.

This study was aimed to evaluate the in vivo antitumor effect of genetically modified myeloma cell vaccine on human myeloma xenografts implanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Human immune system was established in NOD/SCID mice by intraperitoneal injection of human peripheral blood lymphocytes (PBLs). After being inoculated subcutaneously with irradiated myeloma cell line sko-007, adenovirally transferred with GFP or p53, granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7-1 genes, huPBL-NOD/SCID mice were challenged by subcutaneous injection of non-transferred sko-007 cells. The results indicated that Ad-p53/GM-CSF/B7-1-infected sko-007 cell vaccination significantly reduced local tumor growth compared with controls. Histopathological and immunohistochemical analysis showed that tumor tissues increasingly displayed diffuse necrosis, mainly caused by apoptosis, accompanied with significant fibroplasias and blood vessel hyperplasia, and human T cells infiltrated into the tumor tissues. It is concluded that transgenic p53, GM-CSF and B7-1 expression produces an immune response against myeloma cells and may be of therapeutic value for multiple myeloma in human being.

Two germ line polymorphisms of the tumour suppressor gene p53 may influence the biology of chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is characterised by the accumulation of mature B lymphocytes. Defects in the tumour suppressor gene p53 pathway are known to be important in CLL and p53 inactivation is associated with a particularly aggressive form of CLL. A single nucleotide polymorphism (SNP) in codon 72 of TP53 leads to a single amino acid change leading to a change in apoptotic potential and alters prognosis in squamous carcinomas. A polymorphism within intron 6 of TP53 has been postulated to alter the susceptibility to lung cancer. Our study looked at the influence of these two polymorphisms in a cohort of approximately 200 CLL patients. The codon 72 polymorphism A2/A2 genotype (homozygous arginine) was associated with an increased susceptibility to CLL and CD38 negativity but did not appear to influence other biological behaviour or clinical response. The intron 6 polymorphism A2/A2 genotype was strongly associated with early stage disease, CD38 negativity and a longer time to first treatment. The effect on time to treatment did not retain significance in multivariate analysis and the polymorphism did not predict for overall survival (OS). Detailed investigation of the complete TP53 genotype is warranted to further characterise the role of SNPs in p53 and their influence on CLL.

Adenoviral-mediated transfer of human wild-type p53, GM-CSF and B7-1 genes results in growth suppression and autologous anti-tumor cytotoxicity of multiple myeloma cells in vitro.

Multiple myeloma (MM) remains incurable despite the use of high-dose chemotherapy and stem cell transplantation. However, immunotherapy is expected to offer long-term disease control, or even possibly a cure. We have previously demonstrated the suppressive effect of a recombinant adenovirus carrying human wild-type p53, granulocyte-macrophage colony-stimulating factor, and B7-1 genes (Ad-p53/GM-CSF/B7-1) on the growth of laryngeal cancer cells. In the present study, we evaluated the effects of an Ad-p53/GM-CSF/B7-1-modified myeloma cell vaccine strategy aimed to induce apoptosis and to augment the immunogenicity of MM cells. Both MM cell lines and purified primary myeloma cells were infected with Ad-p53/GM-CSF/B7-1. High expression levels of these three genes were confirmed separately by Western blot, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. When wild-type p53, GM-CSF and B7-1 genes were introduced, the growth of MM cells was inhibited via enhanced apoptosis and the immunogenicity of tumor cells was augmented. The combinatorial effect of these three genes on inducing cytotoxic T lymphocytes (CTLs) was more evident than that of p53 individually or any combinations of two (p53 plus GM-CSF or p53 plus B7-1). Furthermore, significant proliferation of autologous peripheral blood lymphocytes (PBLs) and specific cytotoxicity against autologous primary MM cells were induced in vitro. These results suggest that myeloma cell vaccination co-transferred with p53, GM-CSF and B7-1 genes may be a promising immunotherapeutic approach against MM.

Resistance against Friend leukemia virus-induced leukemogenesis in DNA-dependent protein kinase (DNA-PK)-deficient scid mice associated with defective viral integration at the Spi-1 and Fli-1 site.

Retroviral DNA integration is mediated by the viral protein integrase. However, elements of the host DNA repair machinery such as the phosphatidylinositol 3-kinase (PI-3K)-related protein kinase family system would play a role in the integration of viral DNA into the host DNA. Here, we show that a host PI-3K-related protein kinase, DNA-dependent protein kinase (DNA-PK), plays a role in the specific integration of retroviral DNA and induction of retroviral diseases in vivo. DNA-PK-deficient scid mice inoculated with Friend leukemia virus (FLV) exhibited a random integration into their genomic DNA and expressed the viral envelope protein gp70. However, the specific integration of FLV at Spi-1 or Fli-1 sites did not occur in association with the significant resistance of scid mice to FLV-induced leukemogenesis. In contrast, the knockout of another member of the PI-3K-related protein kinase family, encoded by the ataxia telangiectasia mutated (ATM) gene, resulted in mice as sensitive to FLV-induced leukemogenesis as the wild type mice. FLV was specifically integrated into the DNA at Spi-1 and Fli-1 sites with significant expression of these transcription factors. These findings indicated that DNA-PK would be essential for controlling the in vivo integration of FLV at specific sites as well as the susceptibility to FLV-induced leukemogenesis.