Simian Virus 40-Induced T and Tumor Antigens

Antigen extracts from simian virus 40 (SV40) transplanted hamster tumors were studied by rate-zonal centrifugation. Three species or molecular forms of antigen were demonstrated. The major antigen component corresponded to a molecular weight of 65,000 to 75,000, and two larger species were detectable in smaller quantities. Similar studies were carried out on SV40 virus-induced T antigen from BSC-1 cells. Three antigen components were again detected. Quantitative differences in the expression of "T" and tumor antigen species were reproducibly found.     

T Antigens of Bovine and Canine Adenoviruses Demonstrated by Immunofluorescence

The intracellular development in acutely infected cells of bovine and canine adenovirus T antigens was followed by immunofluorescent staining. With both species of adenovirus, antigen was first detected as intranuclear pin-points at 18 hr postinfection and coalescence into large lobular masses was noticed by 24 hr. Cross-reactions between bovine 1 (nononcogenic) and bovine 3 (oncogenic) T antigens were not observed by the direct technique although the more sensitive indirect procedure did reveal cross-reactivity. Extensive cross-reactions were observed between the T antigens of the oncogenic canine hepatitis virus and the "nononcogenic" Toronto strain of canine adenovirus. The magnitude of these reactions places the two canine strains in the same T antigen subgroup. The canine and bovine T antigens were not stained by tumor antisera against any of the known human or simian T antigen subgroups. Antigen synthesis was not prevented by inhibitors of deoxyribonucleic acid synthesis although the appearance was altered markedly.     

Inhibition of the Synthesis of Simian Virus 40 Antigens in Cells Preinfected with Yaba Tumor Virus

The synthesis of tumor and viral antigens after infection of an established line of cynomolgus monkey kidney cells with simian virus 40 (SV40) was compared in cells previously infected with Yaba virus and in cells not preinfected. SV40 failed to induce synthesis of tumor or viral antigens in cells preinfected with Yaba virus. The inhibitory state in preinfected cells was shown to develop sequentially. Increase in the rate of deoxyribonucleic acid synthesis in the nuclei of preinfected cells occurred after infection with SV40. This rate of increase was significantly lower than that which occurred in SV40-infected cells which had not been preinfected. Cytosine arabinoside did not exert significant effect on the development of the inhibitory effect against SV40 in Yaba virus-infected cells.     

Serological Studies of the Antigenic Similarity between Typhus Group Rickettsiae and Weil-Felix Test Antigens

The sera from two patients with murine typhus reacted with whole cells of Rickettsia prowazekii, R. typhi, and Proteus vulgaris OX19, and with lipopolysaccharides (LPS) from the spotted fever group rickettsia strain TT-118 and P. vulgaris OX19 in the enzyme-linked immunosorbent assay. Sera from these patients reacted with ladder-like bands of LPS from R. prowazekii and R. typhi in the immunoblot, whereas the reactivity of these sera with LPS from P. vulgaris OX19 differed from each other. These results indicate that LPS from the typhus group rickettsiae and P. vulgaris OX19 contain similar epitopes.     

Replication and Complementation of Human Adenoviruses and Simian Papovavirus at an Elevated Temperature

The simian papovavirus SV40 replicated as well in simian cells incubated at 41 C as in cells incubated at 37 C, although the latent period was shortened at the elevated temperature. Human adenoviruses differed in their responses to the elevated temperature. Some serotypes, such as 3, 4, 5, 7, 8, 16, and 21, replicated as well, or almost as efficiently, in human cells incubated at 41 C as in cells incubated at 37 C, whereas with other serotypes, such as 1, 2, 6, 12, and 14, maximal yields in cultures incubated at 41 C were much lower than the yields from companion cultures incubated at 37 C. This difference was also detected in simian cells co-infected with SV40 and a human adenovirus; maximal complementation occurred with some serotypes at the elevated temperature but not with other serotypes. The degree of complementation observed in the simian cells at 41 C was directly correlated with the ability of the adenovirus to replicate at 41 C in human cells. Therefore, the capacity of SV40 to serve as a helper virus is not affected by the elevated temperature, showing that the complementation event supplied by the simian virus is heat-stable between 37 and 41 C. Maximal complementation appeared to depend upon a characteristic present in the adenovirus genome.     

Virus-Specific Deoxyribonucleic Acid in Simian Virus 40-Exposed Hamster Cells: Correlation with S and T Antigens

Several homologous hamster embryonic cell lines, transformed in association with simian virus (SV) 40 infection, were examined for the presence of deoxyribonucleic acid (DNA) complementary to SV40 ribonucleic acid (RNA) made in vitro. The methods employed permitted the detection of 10(-5) mug of viral DNA in 100 mug of cellular DNA, corresponding to one-fifth of an SV40 DNA molecule per cell. Those lines which contained both the SV40 surface (S) and tumor (T) antigens also contained DNA complementary to SV40 RNA synthesized in vitro. In contrast, neither of two lines which contained S, but not T, antigen contained detectable DNA complementary to SV40 RNA. These findings suggest that the production of S antigen does not depend upon the persistence of SV40 DNA in transformed cells.     

Serological Analysis of the Repertoire of Human Cancer Antigens and Autoantigens

The spectrum of human antigens allows a monitoring of various pathological processes such as autoimmune disorders and tumorigenesis. Serological analysis of cDNA expression libraries (SEREX) is now used to search for new cancer-associated antigens, which are potential diagnostic markers or targets for immunotherapy of cancer. The results obtained for several solid tumors are reviewed. Groups of antigens detectable by SEREX, causes of immunogenicity of autoantigens, and prospective implication of antigens in diagnostics and monitoring of cancer are discussed.     

Hypoxia Integration in the Serological Proteome Analysis Unmasks Tumor Antigens and Fosters the Identification of Anti-Phospho-eEF2 Antibodies as Potential Cancer Biomarkers

The expression by tumor cells of proteins with aberrant structure, expression or distribution accounts for the development of a humoral immune response. Autoantibodies (aAb) directed against tumor-associated antigens (TAA) may thus be particularly relevant for early detection of cancer. Serological proteome analysis (SERPA) aims to identify such circulating aAb through the immunoblotting of 2D-separated tumor cell proteins with cancer patient serum and the consecutive MS identification of proteins in reactive spots. This method has the advantage to use post-translationally modified proteins as a source of potential TAA. Here, we applied this strategy by using colorectal tumor cells pre-exposed to hypoxia in order to promote the expression of a pattern of TAA more likely to represent in vivo conditions. We used two human HCT116 and HT29 colorectal cancer cell lines exposed for 48 hours to 1% O₂. Spots positive after immunoblotting of 2D-separated lysates of hypoxic cells with the sera of tumor-bearing mice, were collected and analysed by MS for protein identification. Among the hypoxia-specific immunogenic proteins, we identified a phosphorylated form of eukaryotic translation elongation factor 2 (phospho-Thr56 eEF2). We confirmed the increased phosphorylation of this protein in hypoxic colorectal tumor cells as well as in mouse tumors. Using a specific immunoassay, we could detect the presence of corresponding anti-phospho-Thr56 eEF2 aAb in the serum of tumor-bearing mice (vs healthy mice). We further documented that the detection of these aAb preceded the detection of a palpable tumor mass in mice and validated the presence of anti-phospho-Thr56 eEF2 aAb in the serum of patients with adenomatous polyps and colorectal carcinoma. In conclusion, this study validates a phosphorylated form of eEF2 as a new TAA and more generally, provides evidence that integrating hypoxia upstream of SERPA offers a more relevant repertoire of TAA able to unmask the presence of circulating aAb.     

Serological Specificity of the Lipopolysaccharides, the Major Antigens of Pseudomonas syringae

The nature of major antigens of Pseudomonas syringae was studied on one strain of four pathovars (pvs aptata, mors-prunorum, phaseolicola and tabaci) belonging to four separate serogroups. Bacterial antigens were prepared by 4 procedures: extraction by phenol-water (PW), by citrate-NaCl (CN), by trichloracetic acid (TCA), and precipitation of a glycoproteic extracellular complex (GP). 3-Deoxy-2-octulosonic acid (KDO) revelation in all the extracts showed that the four procedures led to antigens containing similar amounts of lipopolysaccharide (LPS). Twenty polyclonal antisera were raised in rabbits against whole bacteria and the different extracts. Serological reactions were tested by gel double diffusion (DD) and indirect immunofluorescent staining (IF). The anti-whole cell sera were shown to contain mostly anti-LPS antibodies. For each pathovar, whole bacteria used as antigens in DD gave precipitation bands identical to the bands given by the LPS extracts (PW, CN or TCA), identical to the heated bacteria (HB), and identical to LPS sidechain preparations. The GP extract itself was shown to be rich in LPS. To serotype P. syringae, it is advised to raise antisera against either whole bacteria or GP extracts; whereas the reacting antigens for DD would be heated bacteria.     

Differential Oncogenic Ras Signaling and Senescence in Tumor Cells

Several studies have shown that forced expression of oncogenic H-ras can induce a senescence-like permanent growth arrest in normal cells. Here we report that expression of oncogenic H-ras in human osteosarcoma U2OS cells also resulted in a senescence-like flat and enlarged cell morphology and permanent growth arrest. In contrast to normal human fibroblasts, U2OS cells were arrested independently of the p16 and ARF tumor suppressors. Treatment with a MEK inhibitor or a p38MAPK inhibitor interrupted oncogenic H-ras-induced growth arrest in U2OS cells, suggesting that activation of MAPK pathways is important. To further determine whether this process is unique to oncogenic H-ras signaling, we examined the effect of oncogenic K-ras on normal cells and human osteosarcoma cells. Similar to oncogenic H-ras, oncogenic K-ras also induced senescence in normal fibroblasts, while transforming immortalized mouse fibroblasts. However, in contrast to oncogenic H-ras, oncogenic K-ras failed to induce a permanent growth arrest in osteosarcoma U2OS cells. Additionally, cells transduced with oncogenic K-ras exhibited distinguishable cellular changes compared to those transduced with oncogenic H-ras. In summary, we report for the first time that oncogenic H-ras signaling can trigger a senescence-like growth arrest in tumor cells, independent of the p16 and ARF tumor suppressors. This result suggests that tumor cells may harbor a senescence-like program that can be activated by ras signaling. Moreover, our study uncovered a cell type-dependent differential response to oncogenic K-ras, as compared to oncogenic H-ras.     

Transformation Potentials of the Noninfectious (Defective) Component in Pools of Adenoviruses Type 12 and Simian Adenovirus 7

Pools of adenovirus 12 and simian adenovirus 7 were separated into four or five fractions by density gradient centrifugation in cesium chloride. Each fraction was analyzed for total in vitro infectivity units, total transformation activity, and for total virus particle (VP) content. Two major subpopulations were separated with mean densities of 1.30 +/- 0.02 and 1.34 +/- 0.02 g/ml, respectively. Virions in the 1.34 g/ml range were highly infectious (10(2) to 10(3) VP per infectivity unit) in contrast to virions at 1.30 g/ml density (10(4) to 10(5) VP per infectivity units). Transformation capacity was evenly distributed throughout fractions of both viruses, indicating that genetically incomplete or defective virus particles were not deficient in their ability to induce transformation. The average VP per transformation unit for adenovirus 12 (2.85 x 10(6)) and for simian adenovirus 7 (4.00 x 10(6)) did not vary significantly from fraction to fraction. These values were obtained with optimal input multiplicities of 16 to 64 VP per cell. At higher multiplicities the apparent increase in VP per transformation unit was attributable to the viral cytocidal effect on hamster cells. These studies revealed that quantitation of in vitro transformation based on VP multiplicities was more reliable than on the basis of infectious units. These estimates were independent of method of virus production, extraction, and purification.     

Serological Relationships of Type I Antigens of Group B Streptococci

Some of the complex antigenic relationships of type I group B streptococci from various clinical sources were defined by means of immunodiffusion, absorption, and precipitin tests. Three predominant types are described: Ia, Ib, and Ii. Methods for preparing antisera for differentiating type I strains are presented.     

Chemotherapy Enhances Cross-Presentation of Nuclear Tumor Antigens

Cross-presentation of tumor antigen is essential for efficient priming of naïve CD8⁺ T lymphocytes and induction of effective anti-tumor immunity. We hypothesized that the subcellular location of a tumor antigen could affect the efficiency of cross-presentation, and hence the outcome of anti-tumor responses to that antigen. We compared cross-presentation of a nominal antigen expressed in the nuclear, secretory, or cytoplasmic compartments of B16 melanoma tumors. All tumors expressed similar levels of the antigen. The antigen was cross-presented from all compartments but when the concentration was low, nuclear antigen was less efficiently cross-presented than antigen from other cellular locations. The efficiency of cross-presentation of the nuclear antigen was improved following chemotherapy-induced tumor cell apoptosis and this correlated with an increase in the proportion of effector CTL. These data demonstrate that chemotherapy improves nuclear tumor antigen cross-presentation and could be important for anti-cancer immunotherapies that target nuclear antigens.     

Enhancer-Mediated Oncogenic Function of the Menin Tumor Suppressor in Breast Cancer

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