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.

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.

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.

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.

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.

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.

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.

Potential immunogenicity of oncogene and tumor suppressor gene products.

The immunogenicity of viral oncoproteins has been established beyond doubt. Cytotoxic T lymphocytes directed against viral oncogene products can eradicate large established tumor masses. This stage has not yet been reached for cellular oncogene and tumor suppressor gene products, but T cells have been raised against MHC-binding peptides encoded by both mutant and wild-type alleles of the ras oncogene and the p53 tumor suppressor gene. In addition, T cells specific for joining region peptides of abnormal fusion proteins resulting from chromosome translocation in tumor cells have been generated. Some of these peptides are processed in cells infected with, for example, vaccinia-ras, but direct anti-tumor effects of peptide specific T lymphocytes remain to be demonstrated.

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.

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.

Anti-p53 antibodies in sera of workers occupationally exposed to vinyl chloride.

BACKGROUND: The p53 tumor suppressor gene (also known as TP53) is often mutated in a wide variety of cancers, including angiosarcoma of the liver (ASL). Anti-p53 antibodies have been detected in the sera of patients with leukemia, childhood lymphoma, or cancers such as those of the breast, lung, colon, esophagus, and liver (hepatocellular carcinoma). PURPOSE: The objective of this study was to determine the prevalence and time of appearance of serum anti-p53 antibodies during the pathogenesis of ASL associated with occupational exposure to vinyl chloride. METHODS: Enzyme-linked immunoassay (EIA) was used to detect anti-p53 antibodies in 148 serum samples from 92 individuals occupationally exposed (in France or in Kentucky) to vinyl chloride; 15 of these individuals (six from France and nine from Kentucky) had ASL. A subset of coded EIA-positive and EIA-negative sera was further analyzed for anti-p53 antibodies by immunoblotting and immunoprecipitation. Nucleotide sequence analysis of exons 5-8 of the p53 gene was conducted on ASL DNA from six patients. We tested sera from 31 men who had no occupational exposure to vinyl chloride; they made up the control group. Statistical analyses were done using the Kruskal-Wallis chi-squared approximation and the Wilcoxon two-sample test for normal approximation. All P values result from two-sided tests. RESULTS: Fourteen serum samples (from nine individuals) were positive in the EIA. Five of the 15 individuals with ASL were positive for anti-p53 antibodies by EIA, immunoblotting, and immunoprecipitation: one individual at 11.3 and 10.8 years before diagnosis, another at 4 months before and shortly after diagnosis, and three when diagnosed or shortly thereafter. Four of the 77 vinyl chloride-exposed workers without diagnosed ASL were positive for anti-p53 antibodies; two of the four had symptoms related to vinyl chloride toxicity. Tumors from three of the six vinyl chloride-exposed workers from which sufficient DNA for analysis was obtained had A:T to T:A missense mutations of the p53 gene. Anti-p53 antibodies were detected in two of these individuals. Among the control group, two of 15 serum samples from 15 lung cancer patients and zero of 15 serum samples from control subjects without cancer had anti-p53 antibodies as substantially lower levels than the nine (10%) of 92 vinyl chloride-exposed workers who were positive for anti-p53 antibodies. CONCLUSIONS AND IMPLICATIONS: Serum anti-p53 antibodies can predate clinical diagnosis of certain tumors, such as ASL, and may be useful in identifying individuals at high cancer risk, such as workers with occupational exposure to vinyl chloride.

Mutated p53 gene encodes a nonmutated epitope recognized by HLA-B*4601-restricted and tumor cell-reactive CTLs at tumor site.

Mutations of p53 gene occur in approximately 50% of human cancers, and accumulated p53 protein may be an appropriate target molecule to use for cancer immunotherapy. Indeed, mutated or nonmutated p53-derived peptides can induce HLA class I-restricted and tumor cell-reactive CTLs in vitro. However, to our knowledge, evidence that p53-derived peptides are truly recognized by CTLs at tumor sites has not yet been obtained. This study revealed that a mutated p53 gene encoded a nonmutated nonapeptide recognized by a HLA-B46-restricted and tumor cell-reactive CTL line that was established from T cells infiltrating a colon cancer lesion with the p53 mutation. This p53 peptide, at amino acid positions 99-107, had the ability to induce HLA-B46-restricted and peptide-specific CTLs reactive to tumor cells with the p53 mutation from the peripheral blood mononuclear cells of cancer patients, but not from those of healthy donors. These peptide-induced CTLs did not react to either HLA-B46(+) tumor cells without the p53 mutation or to HLA-B46(+) phytohemagglutinin-blastoid cells. These results provide a scientific basis for the development of p53-directed specific immunotherapy for HLA-B46(+) cancer patients.

Immunization with wild-type p53 gene sequences coadministered with Flt3 ligand induces an antigen-specific type 1 T-cell response.

We examined the ability of immunization with sequential adenovirus/plasmid DNA vectors expressing human wild-type p53 to stimulate a type 1 T-cell response and induce protection against challenge from a metastatic tumor that expresses mutated murine p53. We found that tumor protection and an antigen (Ag)-specific immune response were enhanced by prior injection of Flt3 ligand (Flt3L) at a dose and schedule that significantly increased dendritic cell (DC) number and frequency. Preliminary studies using enzyme-linked immunospot and Winn assays suggested that Ag-specific CD8 cells, with their significant increase in IFN-gamma-secreting activity (Tc1 cells), were responsible for the tumor protection. The delayed-type hypersensitivity response to p53 was increased in mice immunized with p53 alone or p53 and Flt3L compared with a negative control. In contrast, spleen cells from mice immunized with p53 and Flt3L exhibited a higher Ag-specific proliferative response than mice immunized with p53 alone. The frequencies of Ag-specific IFN-gamma and interleukin (IL)-4-secreting cells were determined using an enzyme-linked immunospot assay, which demonstrated that the frequency of IFN-gamma-secreting cells was significantly higher in mice immunized with p53 and Flt3L than in mice receiving Flt3L, excipient, or p53 treatment alone. In contrast, the frequency of IL-4-secreting cells did not differ significantly among these groups. We also observed an increased frequency of IL-12 and IFN-gamma-secreting cells (but not IL-4 or IL-10) in the spleens of mice immediately after 10 days of Flt3L treatment, which was also the day of p53 priming. This observation supports the likelihood that there are multiple mechanisms of Flt3L adjuvant activity, including expansion of DC and type 1 T-cell number. Overall, these results suggest that immunization with p53 genetic sequences after in vivo expansion of DC, using Flt3L, provides a useful strategy to induce p53-specific, and protective, type 1 T-cell responses.

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.

Evidence for a second cell cycle block at G2/M by p53.

Wild type p53 can induce cell cycle arrest at specific points in the cell cycle, in particular G1/S, an ability lost by most p53 mutants. We have previously reported that p53 mutant genes can rescue REF52 cells from ras-induced growth arrest and that over expression of wild type p53 inhibits cell growth in these cells. In this paper we examined whether p53 can also induce cell cycle arrest at the G2/M boundary of the cell cycle. To accomplish this we used the REF52 cell line and the temperature sensitive p53val135 mutant allele. Cells were enriched in the late G1 and early S phases before the temperature shift. REF52 cells expressing mutant-p53val135 alone with an activated H-ras gene arrest primarily at the G1/S and G2/M parts of the cell cycle at the restrictive temperature, as determined by flow cytometry analysis. These results suggest that the anti-proliferative activity of p53 may be involved in regulation of the cell cycle at the G2/M restriction point as well as transit through G1/S and initiation of DNA synthesis.

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.

p53 antibodies in patients with various types of cancer: assay, identification, and characterization.

Alteration of the p53 gene is the most frequent genetic alteration in human cancer and leads to the accumulation of mutant p53 in the nucleus of tumor cells. In addition, it has been shown that patients with various types of neoplasia have p53 antibodies in their sera which could be used as an indirect diagnostic procedure for p53 alteration. Using a new ELISA, we have analyzed the sera from more than 1000 patients with various types of cancer and from healthy blood donors. We demonstrate that p53 antibodies are detected mainly in cancer patients and are strictly proportional to the occurrence of p53 mutations. Using various immunological approaches, these antibodies were unambiguously demonstrated to be directed toward the human p53 protein. Isotyping analysis of these antibodies strongly suggested that they correspond to a humoral response to the p53 protein which accumulates in the tumor cell. This finding suggests that serological analysis, combined with histochemistry, is suitable for assessing the integrity of the p53 gene in cancer patients.

The cDNA cloning and immunological characterization of hamster p53.

We have cloned and sequenced the p53-encoding cDNA of Syrian hamster. The encoded product is 78% and 75% homologous to human and mouse p53, respectively. Immunoprecipitations of the cDNA-encoded protein by monoclonal antibodies specific for mammalian p53 confirmed the identity of the protein.

The relationship between angiogenesis and the immune response in carcinogenesis and the progression of malignant disease.

Recent studies have demonstrated that angiogenesis and suppressed cell-mediated immunity (CMI) play a central role in the pathogenesis of malignant disease facilitating tumour growth, invasion and metastasis. In the majority of tumours, the malignant process is preceded by a pathological condition or exposure to an irritant which itself is associated with the induction of angiogenesis and/or suppressed CMI. These include: cigarette smoking, chronic bronchitis and lung cancer; chronic oesophagitis and oesophageal cancer; chronic viral infections such as human papilloma virus and ano-genital cancers, chronic hepatitis B and C and hepatocellular carcinoma, and Epstein-Barr virus (EBV) and lymphomas; chronic inflammatory conditions such as Crohn's disease and ulcerative colitis and colorectal cancer; asbestos exposure and mesothelioma and excessive sunlight exposure/sunburn and malignant melanoma. Chronic exposure to growth factors (insulin-like growth factor-I in acromegaly), mutations in tumour suppressor genes (TP53 in Li Fraumeni syndrome) and long-term exposure to immunosuppressive agents (cyclosporin A) may also give rise to similar environments and are associated with the development of a range of solid tumours. The increased blood supply would facilitate the development and proliferation of an abnormal clone or clones of cells arising as the result of: (a) an inherited genetic abnormality; and/or (b) acquired somatic mutations, the latter due to local production and/or enhanced delivery of carcinogens and mutagenic growth factors. With progressive detrimental mutations and growth-induced tumour hypoxia, the transformed cell, to a lesser or greater extent, may amplify the angiogenic process and CMI suppression, thereby facilitating further tumour growth and metastasis. There is accumulating evidence that long-term treatment with cyclo-oxygenase inhibitors (aspirin and indomethacin), cytokines such as interferon-alpha, anti-oestrogens (tamoxifen and raloxifene) and captopril significantly reduces the incidence of solid tumours such as breast and colorectal cancer. These agents are anti-angiogenic and, in the case of aspirin, indomethacin and interferon-alpha have proven immunomodulatory effects. Collectively these observations indicate that angiogenesis and suppressed CMI play a central role in the development and progression of malignant disease.

Ionizing radiation enhances immunogenicity of cells expressing a tumor-specific T-cell epitope.

BACKGROUND: p53 point mutations represent potential tumor-specific cytolytic T lymphocyte (CTL) epitopes. Whether ionizing radiation (IR) alters the immunological properties of cells expressing mutant p53 in respect of the CTL epitope generated by a defined point mutation has not been evaluated. METHODS: Mutant p53-expressing syngeneic, nontumor forming BALB/c 3T3 fibroblasts, tumor forming ras-transfected BALB/c 3T3 sarcomas, and DBA/2-derived P815 mastocytoma cells, which differ at the level of minor histocompatibility antigens, were used as cellular vaccines. Cells were either injected with or without prior IR into naive BALB/c mice. Cellular cytotoxicity was assessed after secondary restimulation of effector spleen cells in vitro. RESULTS: Injection of P815 mastocytoma cells expressing the mutant p53 induced mutation-specific CTL in BALB/c mice irrespective of prior irradiation. However, syngeneic fibroblasts or fibrosarcomas endogenously expressing mutant p53 were able to induce significant mutation-specific CTL only when irradiated prior to injection into BALB/c mice. IR of fibroblasts did not detectably alter the expression of cell surface molecules involved in immune response induction, nor did it alter the short-term in vitro viability of the fibroblasts. Interestingly, radioactively-labeled fibroblasts injected into mice after irradiation showed altered organ distribution, suggesting that the in vivo fate of these cells may play a crucial role in their immunogenicity. CONCLUSIONS: These findings indicate that IR can alter the immunogenicity of syngeneic normal as well as tumor forming fibroblasts in vivo, and support the view that ionizing radiation enhances immunogenicity of cellular tumor vaccines.