Increased Levels of C-C Chemokine RANTES in Asbestos Exposed Workers and in Malignant Mesothelioma Patients from an Hyperendemic Area

Background

Asbestos-induced mesothelial inflammatory processes are thought to be the basic mechanisms underlying Malignant Mesothelioma (MM) development. Detection of MM often occurs at late stage due to the long and unpredictable latent period and the low incidence in asbestos exposed individuals. The aim of this study was to investigate early immunological biomarkers to characterize the prognostic profile of a possible asbestos-induced disease, in subjects from a MM hyperendemic area.

Methods

The Luminex Multiplex Panel Technology was used for the simultaneous measurement of serum levels of a large panel of 47 analytes, including cytokines and growth factors, from workers previously exposed to asbestos (Asb-workers), asbestos-induced MM patients and healthy subjects. In addition, to explore the influence on serum cytokines profile exerted by SV40 infection, a cofactor in MM development, a quantitative real time PCR was performed for sequences detection in the N-terminal and intronic regions of the SV40 Tag gene. Statistical analysis was done by means of the Mann-Whitney test and the Kruskall-Wallis test for variance analysis.

Results

A variety of 25 cytokines linked to pulmonary inflammation and tumor development were found significantly associated with Asb-workers and MM patients compared with healthy controls. A specific pattern of cytokines were found highly expressed in Asb-workers: IFN-alpha (p<0.05), EOTAXIN (p<0.01), RANTES (p<0.001), and in MM patients: IL-12(p40), IL-3, IL-1 alpha, MCP-3, beta-NGF, TNF-beta, RANTES (p<0.001). Notably, the chemokine RANTES measured the highest serum level showing an increased gradient of concentration from healthy subjects to Asb-workers and MM patients (p<0.001), independently of SV40 infection.

Conclusion

This study shows that, in subjects from an hyperendemic area for MM, the C-C chemokine RANTES is associated with the exposure to asbestos fibres. If validated in larger samples, this factor could have the potential to be a critical biomarker for MM prognosis as recently reported for breast tumor.     

Asbestos,chromosomal deletions,and tumor suppressor gene alterations in human malignant mesothelioma

Exposure to the carcinogen asbestos is considered to be a major factor contributing to the development of most malignant mesotheliomas (MMs). We highlight the role of asbestos in MM and summarize cytogenetic and molecular genetic findings in this malignancy. The accumulation of numerous clonal chromosomal deletions in most MMs suggests a multistep process of tumorigenesis, characterized by the loss and/or inactivation of multiple tumor suppressor genes (TSGs). Cytogenetic and loss of heterozygosity (LOH) analyses of MMs have demonstrated frequent deletions of specific sites within chromosome arms 1p, 3p, 6q, 9p, 13q, 15q, and 22q. Furthermore, TSGs within two of these regions, i.e., p16/CDKN2A-p14^ARF at 9p21 and NF2 at 22q12, are frequently altered in MMs. Homozygous deletion appears to be the major mechanism affecting p16/CDKN2A-p14^ARF, whereas inactivating mutations coupled with allelic loss occur at the NF2 locus. Finally, recent studies have demonstrated the presence and expression of simian virus 40 (SV40) in many MMs. SV40 large T antigen has been shown to inactivate the TSG products Rb and p53, suggesting the possibility that asbestos and SV40 could act as cocarcinogens in MM. The frequent occurrence of homozygous deletions of p16/CDKN2A-p14^ARF and the ability of SV40 Tag to bind TSG products suggest that perturbations of both Rb- and p53-dependent growth-regulatory pathways are critically involved in the pathogenesis of MM. J. Cell. Physiol. 180:150–157, 1999. © 1999 Wiley-Liss, Inc.     

Polyomaviruses of Nonhuman Primates: Implications for Research

Polyomaviruses are a family of small nonenveloped DNA viruses that infect birds and mammals. At least 7 nonhuman primate polyomaviruses that occur in macaques, African green monkeys, marmosets baboons, and chimpanzees have been described, as well as 4 polyomaviruses that occur in humans. Simian virus 40 (SV40), which infects macaques, was the first nonhuman primate polyomavirus identified as a contaminant of early polio vaccines. Primate polyomaviruses cause inapparent primary infections but persist in the host and can cause severe disease in situations of immunocompromise. This review describes the primate polyomaviruses, and the diseases associated with the viruses of macaques. In macaques, the greatest current concerns are the potential confounding of study results by polyomavirus infections and the zoonotic potential of SV40.Abbreviations: PML, progressive multifocal leukoencephalopathy; SV40, Simian virus 40Polyomaviruses were previously members of the family Papovaviridae, which included (and derived its name from) rabbit papilloma virus (pa), mouse polyoma virus (po), and simian vacuolating virus (va). Papovaviruses are nonenveloped viruses, with double-stranded circular DNA and an icosahedral capsule. Since the 1980s, studies of Simian virus 40 (SV40) and mouse polyomavirus have demonstrated that these viruses have smaller capsids (45 nm versus 50 nm), smaller genomes (5 kb versus 8 kb), and a different genomic organization than those of papillomaviruses. SV40 and mouse polyomavirus now form an independent family, Polyomaviridae.¹⁸More than 13 members of Polyomaviridae infect mammals and birds. The first polyomavirus was discovered in 1953 in mice²⁸ and was so named because it caused tumors at multiple sites in neonatal mice. Indeed oncogenicity is a common feature of polyomaviruses, particularly tumor production in non-native hosts. Various members of the group transform cell lines and immortalize primary cell cultures as well as induce tumors in susceptible animals. SV40 was identified in 1960 in primary macaque kidney cell cultures, as a contaminant of polio vaccines.⁶⁸ In 1971, the human polyomaviruses BKV²³ and JCV⁵⁴ were identified (both are named after the initials of the patients in which they were first recognized). JCV was discovered in the brain of a patient with progressive multifocal leukoencephalopathy, and BKV was found in the urine of a renal transplant patient. Recently, 2 additional polyomaviruses of the nasopharynx of humans, KIV and WUV, have been identified^2,25 through the use of molecular techniques. KIV was found in nasopharyngeal samples from patients with respiratory disease, and WUV initially was detected in a child with pneumonia. KIV and WUV are closely related genetically and may form a new subfamily of polyomaviruses: their early coding regions (T antigens) are similar to those of other primate polyomaviruses, but their late regions (structural proteins) differ.^7,25 Both KIV and WUV appear to be geographically widespread.The capsids of the polyomaviruses contain 3 structural proteins: VP1, the major capsid protein, and VP2 and VP3, which enclose a single molecule of viral DNA. The viruses also encode regulatory proteins, the T (tumor) antigens. SV40 and other primate polyomaviruses encode 2 T antigens, large T and small t, whereas mouse polyomavirus and some of the other family members have a third, middle T antigen. The T antigens of SV40, BKV, and JCV have about 75% amino-acid homology.⁵⁸ The T antigen of SV40 is essential for initiation of viral DNA replication and promotes transformation and immortalization of host cells, partially through binding to and inhibiting tumor suppressor proteins p53, p107, p130 (pRb2), and pRb (reviewed in reference 10).     

Microbial Regulation of p53 Tumor Suppressor

p53 tumor suppressor has been identified as a protein interacting with the large T antigen produced by simian vacuolating virus 40 (SV40). Subsequent research on p53 inhibition by SV40 and other tumor viruses has not only helped to gain a better understanding of viral biology, but also shaped our knowledge of human tumorigenesis. Recent studies have found, however, that inhibition of p53 is not strictly in the realm of viruses. Some bacterial pathogens also actively inhibit p53 protein and induce its degradation, resulting in alteration of cellular stress responses. This phenomenon was initially characterized in gastric epithelial cells infected with Helicobacter pylori, a bacterial pathogen that commonly infects the human stomach and is strongly linked to gastric cancer. Besides H. pylori, a number of other bacterial species were recently discovered to inhibit p53. These findings provide novel insights into host–bacteria interactions and tumorigenesis associated with bacterial infections.     

Increased Transformation Efficiency of Simian Virus 40 in Rat Embryo Cells infected with Rauscher Leukaemia Virus

SIMIAN virus 40 (SV40) is oncogenic for hamsters¹ and Mastomys². In vitro studies have shown that SV40 is capable of transforming cells derived from hamster³, mouse⁴, rabbit⁴, pig⁴, cow⁵, monkey⁶, human⁷, guinea-pig⁸ and rat⁹. We have established and studied continuous lines of uninfected and Rauscher leukaemia virus (RLV) infected rat embryo (RE) cells¹⁰. Rat embryo cells exposed to RLV have produced infectious virus and complement-fixing (CF) antigen characteristic of the murine leukaemia-sarcoma virus complex for more than 18 months. This communication describes increased transformation efficiency of SV40 in RLV infected RE cells (RLV-RE) compared with uninfected RE cells.     

Characterization of the simian virus 40-specific messenger RNAs isolated from HeLa cells infected with the non-defective adenovirus 2-simian virus 40 hybrid viruses Ad2+ND2 and Ad2+ND4

Previous work has shown that cells infected with the non-defective adenovirus 2-simian virus 40 hybrid viruses, Ad2⁺ND2 and Ad2⁺ND4 synthesize more than one SV40⁴ large T antigen-related protein. These proteins overlap in amino acid sequence and have their carboxy-terminal sequences in common (Mann et al., 1977). We have characterized the messenger RNAs coding for these SV40-specific proteins. By translating in vitro SV40-specific mRNA isolated from cells infected with these viruses we have shown that each SV40-specific protein can incorporate ³⁵S-labeled formyl methionine at its N-terminus donated by [³⁵S]-fmet-tRNA_f^met, demonstrating that each protein results from a de novo initiation event. Furthermore, analysis of the N-terminal tryptic peptides of these proteins indicates that each protein has a unique N-terminal peptide and therefore a unique initiation site for protein synthesis, with the possible exception of the 74,000 and 95,000 molecular weight proteins, which may have the same N-terminal sequence. Therefore, these proteins cannot be derived by proteolytic cleavage of a large precursor protein.The messenger activities for many of the hybrid virus proteins can be resolved by gel electrophoresis, demonstrating the presence of multiple SV40-specific mRNA species. This result is consistent with the possibility that each SV40-specific protein is coded by a distinct species of RNA.     

Intermediate in SV40 DNA Chain Growth

PREVIOUS studies of the DNA replication of simian virus 40 (SV40), an oncogenic member of the papoyavirus group, have been concerned with separation and characterization of replicative intermediates^1–4. Circular replicating intermediates have been identified for SV40^1–3, as well as for the similar replication system of polyoma viral DNA5,6. The replicative intermediates of SV40 DNA have been observed by electron microscopy to contain two forks, three branches and no free ends^1–3 as is the case for the circular replicating molecules of polyoma, bacteriophage λ⁷, Escherichia coli⁸ and colicin E1 in mini-cells^9,10. An important property of replicative intermediates of SV40 DNA that has also been observed in replicating molecules of colicin E1¹⁰ is that most molecules contain a superhelical region in the unreplicated portion of the molecule¹.     

Tumor Immunity against a Simian Virus 40 Oncoprotein Requires CD8+ T Lymphocytes in the Effector Immune Phase

The required activities of CD4⁺ T cells and antibody against the virally encoded oncoprotein simian virus 40 (SV40) Tag have previously been demonstrated by our laboratory to be mediators in achieving antitumor responses and tumor protection through antibody-dependent cell-mediated cytotoxicity (ADCC). In this study, we further characterize the necessary immune cell components that lead to systemic tumor immunity within an experimental pulmonary metastatic model as the result of SV40 Tag immunization and antibody production. Immunized animals depleted of CD8⁺ T cells at the onset of experimental tumor cell challenge developed lung tumor foci and had an overall decreased survival due to lung tumor burden, suggesting a role for CD8⁺ T cells in the effector phase of the immune response. Lymphocytes and splenocytes harvested from SV40 Tag-immunized mice experimentally inoculated with tumor cells synthesized increased in vitro levels of the Th1 cytokine gamma interferon (IFN-γ), as assessed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry assays. CD8⁺ T-cell activity was also heightened in SV40 Tag-immunized and tumor cell-challenged mice, based upon intracellular production of perforin, confirming the cytolytic properties of CD8⁺ T cells against tumor cell challenge. Altogether, these data point to the role of recombinant SV40 Tag protein immunization in initiating a cytotoxic T-lymphocyte (CTL) response during tumor cell dissemination and growth. The downstream activity of CD8⁺ T cells within this model is likely initiated from SV40 Tag-specific antibody mediating ADCC tumor cell destruction.Determining the immunologic mechanisms involved in antitumor responses can provide valuable insight into developing and formulating appropriate immunotherapeutic strategies against a range of human cancers (25). Cell-mediated immunity involving CD8⁺ T lymphocytes is generally regarded as the primary response to utilize due to its potent and efficient cytotoxicity against tumor cell targets in vitro and in animal models (10). Indeed, the proof of concept of this approach is best characterized by specialized conditioning protocols that involve autologous transfer of tumor infiltrating lymphocytes (TILs) in metastatic melanoma patients, with objective responses that approximate 70% (8). However, the efficacy of TILs harvested from additional cancer types have been less than effective, and additional strategies, such as genetic modification of peripheral blood mononuclear cells, are being explored to improve and extend the approach of cytotoxic T-lymphocyte (CTL) immunotherapy clinically (33, 46).The roles of immune components such as CD4⁺ T cells and antibody have been given less attention within the context of promoting tumor immunity against a range of tumor antigens. For example, the ability of CD4⁺ T cells to activate humoral immunity can lead to antitumor responses that involve antibody-dependent cell-mediated cytotoxicity (ADCC) (17). In this scenario, antibody binds its targeted antigen and effectors such as natural killer (NK) cells lyse tumorigenic cells through interaction with the Fc region of the bound antibody. The efficacy of ADCC has been realized in scenarios involving breast cancer and non-Hodgkin''s lymphoma, for example, and to date, the only FDA-approved immunologic treatments against these malignancies involve antibody-based therapies (5).The concurrent roles of antibody—with specific emphasis on ADCC—and CD8⁺ T-cell immunity within the context of tumor immunity have not been widely reported. Several recent studies have commented on the ability of antibody-bound tumor cells, particularly as a whole tumor cell-dendritic cell (DC) vaccination approach, to initiate CTL activity by engaging DCs through Fc receptors (9, 19, 34). However, to our knowledge, the mechanistic aspects of ADCC (e.g., NK-mediated lysis) promoting CD8⁺ T-cell activity have been explored in relatively few studies (27, 41). From an immunotherapeutic standpoint, it may be preferable in certain settings to induce both the humoral and cell-mediated arms of the immune system to offset the progression of tumor cell growth and dissemination. Namely, these strategies could include active or passive approaches to first effectively induce ADCC in response to a tumor antigen, which would promote CTL activity against additional tumor targets through cross-presentation.Our laboratory has been involved in determining the immunologic mechanisms of tumor immunity induced by the virally encoded tumor-specific antigen simian virus 40 (SV40) large tumor antigen (Tag). The mechanistic aspects of SV40 Tag-induced tumor immunity have been examined within an experimental murine model of pulmonary metastasis. To date, CD4⁺ T cells and SV40 Tag-specific antibody have been implicated as required immune components within this murine system in order to achieve complete systemic tumor immunity (18). These studies demonstrated that during the course of immunization with SV40 Tag (i.e., the induction-phase response), CD4⁺ T cells were required to induce an SV40 Tag humoral response. The specific role of the antibody response against an experimental tumor cell challenge was observed to involve ADCC-mediated clearance pathways (4, 23).In the present study, we further characterize the immunologic response to SV40 Tag immunization by observing the necessary immune cell components following experimental challenge (i.e., the effector-phase response) with a tumor cell line expressing SV40 Tag. With the development of an SV40 Tag antibody response following SV40 Tag immunization in vivo, CD8⁺ T-cell depletion during the effector phase resulted in the formation of lung tumor foci and decreased survival not observed with the abrogation of CD4⁺ T cells. SV40 Tag-immunized mice also displayed a heightened Th1 response and CD8⁺ CTL activity after experimental tumor cell challenge, as assessed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry assays. In all, these data indicate that CD8⁺ T cells mediate tumor immunity following antibody activation in response to the tumor-specific antigen SV40 Tag. We hypothesize that CD8⁺ T-cell activity is initiated due to cross-presentation mechanisms as a result of ADCC activity against SV40 Tag. We are not aware of another published report that formulates a role for ADCC activity against a viral oncoprotein in this manner in order to engage CD8⁺ T-cell activation.SV40 Tag has been reported to be expressed in a number of human cancers, including malignant pleural mesothelioma and non-Hodgkin''s lymphoma, although a causal link between SV40 infection and tumorigenesis is uncertain (11, 24, 35). The results of this study have direct implications for the construction of an appropriate immunotherapeutic strategy for patients suffering malignancies expressing the SV40 Tag tumor-specific antigen.     

Loss of p53 exacerbates multiple myeloma phenotype by facilitating the reprogramming of hematopoietic stem/progenitor cells to malignant plasma cells by MafB

Multiple myeloma (MM) is a serious, mostly incurable human cancer of malignant plasma cells. Chromosomal translocations affecting MAFB are present in a significant percentage of multiple myeloma patients. Genetically engineered Sca1-MafB mice, in which MafB expression is limited to hematopoietic stem/progenitor cells (HS/P-Cs), display the phenotypic features of MM. Contrary to many other types of cancer, it is not yet known if the p53 gene plays any essential role in the pathogenesis of this disease. Here, we show, taking advantage of the Sca1-MafB MM mouse model, that loss of p53 does not rescue the multiple myeloma disease, but instead accelerates its development and exacerbates the MM phenotype. Therefore, the efficiency of the MafB-induced MM reprogramming of normal HS/P-Cs to terminally differentiated malignant plasma cells is enhanced by p53 deficiency, in analogy to what happens in reprogramming to pluripotency. These results raise caution about interfering with p53 function when treating multiple myeloma.     

Simulation,Fabrication and Characterization of THz Metamaterial Absorbers

Metamaterials (MM), artificial materials engineered to have properties that may not be found in nature, have been widely explored since the first theoretical¹ and experimental demonstration² of their unique properties. MMs can provide a highly controllable electromagnetic response, and to date have been demonstrated in every technologically relevant spectral range including the optical³, near IR⁴, mid IR⁵ , THz⁶ , mm-wave⁷ , microwave⁸ and radio⁹ bands. Applications include perfect lenses¹⁰, sensors¹¹, telecommunications¹², invisibility cloaks¹³ and filters^14,15. We have recently developed single band¹⁶, dual band¹⁷ and broadband¹⁸ THz metamaterial absorber devices capable of greater than 80% absorption at the resonance peak. The concept of a MM absorber is especially important at THz frequencies where it is difficult to find strong frequency selective THz absorbers¹⁹. In our MM absorber the THz radiation is absorbed in a thickness of ~ λ/20, overcoming the thickness limitation of traditional quarter wavelength absorbers. MM absorbers naturally lend themselves to THz detection applications, such as thermal sensors, and if integrated with suitable THz sources (e.g. QCLs), could lead to compact, highly sensitive, low cost, real time THz imaging systems.     

Structure of two adenovirus-simian virus 40 hybrids which contain the entire SV40 genome

Two defective adenovirus-simian virus 40 hybrids which contain the entire SV40 genome (Ad2⁺⁺HEY and Ad2⁺⁺LEY)² have been isolated. Upon infection of cells permissive for SV40 both hybrids give rise to infectious SV40 virions, but with markedly different efficiencies. In the case of Ad2⁺⁺HEY nearly all cells infected with a hybrid particle yield SV40 progeny, whereas in the case of Ad2⁺⁺LEY infectious SV40 is produced in only about one in 10⁴ cells infected with hybrid particles. The structures of the DNA molecules in the Ad2⁺⁺HEY and Ad2⁺⁺LEY populations were examined using electron microscope heteroduplex methods. Both populations were found to be heterogeneous. Ad2⁺⁺HEY contained three hybrids (HEY-I, HEY-II, and HEY-III) whose genomes differed only in their content of SV40 DNA (0.45 ± 0.02, 1.43 ± 0.04, and 2.39 ± 0.09 SV40 genomes, respectively). Ad2⁺⁺LEY contained two hybrids (LEY-I and LEY-II), which also differed only in their content of SV40 DNA (0.03 ± 0.01 and 1.05 ± 0.01 SV40 genomes, respectively). In those hybrids which contained more than one complete SV40 genome (HEY-II, HEY-III, LEY-II) the excess SV40 DNA was shown to be organized as a tandem repetition. These data suggest that the various hybrid genomes within each population are interconvertible by recombination events, which insert or excise an SV40 genome. It is proposed that HEY-II and HEY-III yield infectious SV40 with higher efficiency than LEY-II because their SV40 DNA segments contain longer tandem repetitions; thus, the probability of an intramolecular recombination event which results in excision of an SV40 genome is greater.     

Iodine Uptake and Prostate Cancer in the TRAMP Mouse Model

Iodine supplementation exerts antitumor effects in several types of cancer. Iodide (I⁻) and iodine (I₂) reduce cell proliferation and induce apoptosis in human prostate cancer cells (LNCaP and DU-145). Both chemical species decrease tumor growth in athymic mice xenografted with DU-145 cells. The aim of this study was to analyze the uptake and effects of iodine in a preclinical model of prostate cancer (transgenic adenocarcinoma of the mouse prostate [TRAMP] mice/SV40-TAG antigens), which develops cancer by 12 wks of age. ¹²⁵I⁻ and ¹²⁵I₂ uptake was analyzed in prostates from wild-type and TRAMP mice of 12 and 24 wks in the presence of perchlorate (inhibitor of the Na⁺/I⁻ symporter [NIS]). NIS expression was quantified by quantitative polymerase chain reaction (qPCR). Mice (6 wks old) were supplemented with 0.125 mg I⁻ plus 0.062 mg I₂/mouse/day for 12 or 24 wks. The weight of the genitourinary tract (GUT), the number of acini with lesions, cell proliferation (levels of proliferating cell nuclear antigen [PCNA] by immunohistochemistry), p53 and p21 expression (by qPCR) and apoptosis (relative amount of nucleosomes by enzyme-linked immunosorbent assay) were evaluated. In both age-groups, normal and tumoral prostates take up both forms of iodine, but only I⁻ uptake was blocked by perchlorate. Iodine supplementation prevented the overexpression of NIS in the TRAMP mice, but had no effect on the GUT weight, cell phenotype, proliferation or apoptosis. In TRAMP mice, iodine increased p53 expression but had no effect on p21 (a p53-dependent gene). Our data corroborate NIS involvement in I⁻ uptake and support the notion that another transporter mediates I₂ uptake. Iodine did not prevent cancer progression. This result could be explained by a strong inactivation of the p53 pathway by TAG antigens.     

Poldip2 Knockout Results in Perinatal Lethality,Reduced Cellular Growth and Increased Autophagy of Mouse Embryonic Fibroblasts

Polymerase-δ interacting protein 2 (Poldip2) is an understudied protein, originally described as a binding partner of polymerase delta and proliferating cell nuclear antigen (PCNA). Numerous roles for Poldip2 have been proposed, including mitochondrial elongation, DNA replication/repair and ROS production via Nox4. In this study, we have identified a novel role for Poldip2 in regulating the cell cycle. We used a Poldip2 gene-trap mouse and found that homozygous animals die around the time of birth. Poldip2−/− embryos are significantly smaller than wild type or heterozygous embryos. We found that Poldip2−/− mouse embryonic fibroblasts (MEFs) exhibit reduced growth as measured by population doubling and growth curves. This effect is not due to apoptosis or senescence; however, Poldip2−/− MEFs have higher levels of the autophagy marker LC3b. Measurement of DNA content by flow cytometry revealed an increase in the percentage of Poldip2−/− cells in the G1 and G2/M phases of the cell cycle, accompanied by a decrease in the percentage of S-phase cells. Increases in p53 S20 and Sirt1 were observed in passage 2 Poldip2−/− MEFs. In passage 4/5 MEFs, Cdk1 and CyclinA2 are downregulated in Poldip2−/− cells, and these changes are reversed by transfection with SV40 large T-antigen, suggesting that Poldip2 may target the E2F pathway. In contrast, p21^CIP1 is increased in passage 4/5 Poldip2−/− MEFs and its expression is unaffected by SV40 transfection. Overall, these results reveal that Poldip2 is an essential protein in development, and underline its importance in cell viability and proliferation. Because it affects the cell cycle, Poldip2 is a potential novel target for treating proliferative conditions such as cancer, atherosclerosis and restenosis.