The enhanced IL-18 production by UVB irradiation requires ROI and AP-1 signaling in human keratinocyte cell line (HaCaT)

Based on our recent observation that enhanced IL-18 expression positively correlates with malignant skin tumors, such as SCC and melanoma, we examined the possible role of UVB, known to be associated with skin cancer development, in the enhancement of IL-18 production using primary human epidermal keratinocytes and human keratinocyte cell line HaCaT. After cells were exposed to UVB irradiation in vitro, IL-18 production was examined by Northern blot analysis and ELISA, and it was found that IL-18 production is enhanced by UVB irradiation in a dose- and time-dependent manner. In addition, we confirmed that it is functionally active form of IL-18 using the inhibitor of caspase-1. The effect of UVB irradiation was blocked by antioxidant, N-acetyl-L-cysteine (NAC), which suggested the involvement of reactive oxygen intermediates (ROI) in the signal transduction of UVB irradiation-enhanced IL-18 synthesis. We also found that UVB irradiation increased AP-1 binding activity by using EMSA with AP-1-specific oligonucleotide. Furthermore, inhibitors of UVB-induced AP-1 activity, such as PD98059, blocked enhanced IL-18 production, indicating that AP-1 activation is required for UVB-induced IL-18 production. Taken together, our results suggest that UVB irradiation-enhanced IL-18 production is selectively mediated through the generation of ROI and the activation of AP-1.     

Human Endogenous Retrovirus Expression Is Inversely Associated with Chronic Immune Activation in HIV-1 Infection

Human endogenous retroviruses (HERV) are remnants of ancestral retroviral infections integrated into the germ line, and constitute approximately 8% of the genome. Several autoimmune disorders, malignancies, and infectious diseases such as HIV-1 are associated with higher HERV expression. The degree to which HERV expression in vivo results in persistent inflammation is not known. We studied the association of immune activation and HERV-K expression in 20 subjects with chronic, untreated progressive HIV-1 infection and 10 HIV-1 negative controls. The mean HERV-K gag and env RNA expression level in the HIV-1 infected cohort was higher than in the control group (p = 0.0003), and was negatively correlated with the frequency of activated CD38+HLA-DR+CD4+ T cells (Rho = −0.61; p = 0.01) and activated CD38+HLA-DR+CD8+ T cells (Rho  = −0.51; p = 0.03). Although HIV-infected persons had higher levels of HERV-K RNA expression (as expected), the level of RNA expression was negatively associated with level of T cell activation. The mechanism for this unexpected association remains to be defined.     

Molecular cloning and phylogenetic analysis of the human endogenous retrovirus HERV-K long terminal repeat elements in various cancer cells

Long terminal repeats (LTRs) of the human endogenous retroviruses K family (HERV-K) have been found to affect expression of genes located nearby. It has been suggested that the HERV-K LTR elements contributed to the structural change in the genome and genetic variation connected to various diseases. We examined the HERV-K LTR elements in human cancer cells. Using genomic DNA from various cancer cells, we performed PCR amplification and identified forty-nine HERV-K LTR elements. Those LTR elements showed a high degree of sequence similarity with human-specific HERV-K LTR elements. A phylogenetic tree, obtained by the neighbor-joining method, revealed that twelve HERV-K LTR elements were closely related to human-specific HERV-K LTR elements. These elements proliferated recently and were detectable in many human cancer cell lines. These results suggest that HERV-K LTR could be implicated in a pathogenic role, although this phenomenon may not directly lead to human cancers. Further studies on the biological function and expression of HERV-K LTR elements in cancer cells are indicated.     

Mapping of positive selection sites in the HIV-1 genome in the context of RNA and protein structural constraints

Background

Viral genomes of the human endogenous retrovirus K (HERV-K) family are integrated into the human chromosome and are transmitted vertically as Mendelian genes. Although viral particles are released by some transformed cells, they have never been shown to be infectious. In general, gammaretroviruses are produced as immature viral particles by accumulation of the Gag polyproteins at the plasma membrane, which subsequently bud from the cell surface. After release from the cell, Gag is further processed by proteolytic cleavage by the viral protease (PR), which results in morphologically mature particles with condensed cores. The HERV-K Gag polyprotein processing and function has not yet been precisely determined.

Results

We generated a recombinant poxvirus, encoding the human endogenous retrovirus K consensus gag-pro-pol genes (MVA-HERV-K_con) and obtained high levels of HERV-K Gag expression. The resulting retroviral particle assembled at the plasma membrane, as is typical for gammaretroviruses; and immature as well as mature retrovirus-like particles (VLPs) were observed around the infected cells. VLPs were purified, concentrated and separated by two-dimensional gel electrophoresis. The HERV-K Gag fragments were identified by mass spectroscopy and N-terminal sequencing which revealed that HERV-K Gag is processed into MA, a short spacer peptide, p15, CA and NC.

Conclusion

The cleavage sites of HERV-K Gag were mapped and found to be highly conserved among HERV-K genomes. The consensus HERV-K gag gene used in this study is known to support viral, infectivity [1], and thus the cleavage sites that were mapped in this study for all the Gag components are relevant for HERV-K infectivity.     

Frequency of Human Endogenous Retroviral Sequences (HERV) K113 and K115 in the Polish Population,and Their Effect on HIV Infection

Background

The human genome contains about 8% of endogenous retroviral sequences originated from germ cell infections by exogenous retroviruses during evolution. Most of those sequences are inactive because of accumulation of mutations but some of them are still capable to be transcribed and translated. The latter are insertionally polymorphic HERV-K113 and HERV-K115. It has been suggested that their presence and expression was connected with several human diseases. It is also believed that they could interfere with the replication cycle of exogenous retroviruses, including HIV.

Results

Prevalence of endogenous retroviral sequences HERV-K113 and HERV-K115 was determined in the Polish population. The frequencies were found as 11.8% for HERV-K113 and 7.92% for HERV-K115. To verify the hypothesis that the presence of these HERVs sequences could affect susceptibility to HIV infection, comparison of a control group (HIV-negative, not exposed to HIV; n = 303) with HIV-positive patients (n = 470) and exposed but uninfected (EU) individuals (n = 121) was performed. Prevalence of HERV-K113 and HERV-K115 in the EU group was 8.26% and 5.71%, respectively. In the HIV(+) group we detected HERV-K113 sequences in 12.98% of the individuals and HERV-K115 sequences in 7.23% of the individuals. There were no statistically significant differences between groups studied.

Conclusion

The frequency of HERV-K113 and HERV-K115 sequences in Poland were found to be higher than usually shown for European populations. No relation between presence of the HERVs and HIV infection was detected.     

Bowman-Birk proteinase inhibitor-mediated radioprotection against UV irradiation is TP53-dependent and associated with stimulation of nucleotide excision repair

The Bowman-Birk proteinase inhibitor (BBI) has previously been described as a radioprotective agent against ionising radiation. It was demonstrated that BBI is able to significantly increase the clonogenic cell survival of normal fibroblasts when applied before exposure to ultraviolet B (UVB) radiation. In transformed TP53-mutated cell lines, however, the BBI-mediated radioprotection was absent. At the molecular level, the radioprotective effect of BBI can be correlated with BBI-mediated stabilisation of TP53 protein prior to irradiation. Following UVB irradiation, the BBI-treated cells present an accelerated removal of cyclobutane pyrimidine dimers. Thus, the cell and molecular biological data presented suggest that BBI is able to protect cells with functional TP53 from UVB-induced DNA damage. This protective effect is most likely achieved via the activation of the TP53 signalling cascade resulting in the activation of nucleotide excision repair. Received: 7 August 2000 / Accepted: 11 January 2001     

Sestrin2 Protein Positively Regulates AKT Enzyme Signaling and Survival in Human Squamous Cell Carcinoma and Melanoma Cells

Skin cancer is the most common cancer in the United States and is mainly caused by environmental UV radiation. Reducing skin cancer incidence is becoming an urgent issue. The stress-inducible protein Sestrin2 (Sesn2) plays an important role in maintaining redox and metabolic homeostasis and their related pathologies. However, the role of Sesn2 in cancer remains unclear. Here we show that UVB radiation induces Sesn2 expression in normal human keratinocytes, mouse skin, normal human melanocytes, and melanoma cells. In addition, Sesn2 promotes AKT activation through a PTEN-dependent mechanism. Sesn2 deletion or knockdown sensitizes squamous cell carcinoma (SCC) cells to 5-fluorouracil-induced apoptosis and melanoma cells to UVB- and vemurafenib-induced apoptosis. In mice Sesn2 knockdown suppresses tumor growth from injected human SCC and melanoma cells. Last, as compared with normal skin, Sesn2 is up-regulated in both human skin SCC and melanoma. Our findings demonstrate that Sesn2 promotes AKT activation and survival in response to UVB stress and chemotherapeutics and suggest that Sesn2 is oncogenic in skin SCC and melanoma.     

Mutation of B-Raf in human choroidal melanoma cells mediates cell proliferation and transformation through the MEK/ERK pathway

The BRAF gene, encoding a mitogen-activated protein kinase kinase kinase, is mutated in several human cancers, with the highest incidence occurring in cutaneous melanoma. The activating V599E mutation accounted for 80% of all mutations detected in cutaneous melanoma cell lines. Reconstitution experiments have shown that this mutation increases ectopically expressed B-Raf kinase activity and induces NIH3T3 cell transformation. Here we used tumor-derived cell lines to characterize the activity of endogenous mutated B-Raf protein and assess its specific role in transformation. We show that three cell lines (OCM-1, MKT-BR, and SP-6.5) derived from human choroidal melanoma, the most frequent primary ocular neoplasm in humans, express B-Raf containing the V599E mutation. These melanoma cells showed a 10-fold increase in endogenous B-RafV599E kinase activity and a constitutive activation of the MEK/ERK pathway that is independent of Ras. This, as well as melanoma cell proliferation, was strongly diminished by siRNA-mediated depletion of the mutant B-Raf protein. Moreover, blocking B-RafV599E-induced ERK activation by different experimental approaches significantly reduced cell proliferation and anchorage-independent growth of melanoma cells. Finally, quantitative immunoblot analysis allowed us to identify signaling and cell cycle proteins that are differentially expressed between normal melanocytes and melanoma cells. Although the expression of signaling molecules was not sensitive to U0126 in melanoma cells, the expression of a cluster of cell cycle proteins remained regulated by the B-RafV599E/MEK/ERK pathway. Our results pinpoint this pathway as an important component in choroidal melanoma cell lines.     

Effects of Ultraviolet Irradiation on the Cell Cycle

Cultured mouse Cloudman melanoma cells, EMT6 breast carcinoma cells, and 3T3 fibroblasts all accumulated in the G2/M phase of the cell cycle when exposed to UVB radiation. The effects of UVB were maximal at 20–30 mJ/cm² for all three cell lines, and could be observed by flow cytometry as early as 12 hr post irradiation. It has been known since the mid-1970s that MSH receptor binding activity is highest on Cloudman melanoma cells when they are in the G2/M phase of their cycle. Here we show that either UVB irradiation or synchronization of Cloudman cells with colchicine results in a stimulation of MSH binding within 24 hr following treatment, a time when both treatments have resulted in accumulation of cells in the G2/M phase of the cycle. Furthermore, the two treatments performed together on the melanoma cells stimulated MSH receptor activity to the same extent as either treatment performed separately, suggesting that each may be influencing MSH receptor activity solely through a G2/M accumulation of cells. Together, these results raise the possibility that an increase in the number of cells in the G2 phase of the cell cycle is a generalized cellular response to injury, such as UV irradiation. However, in the case of pigment cells this response includes a mechanism for increasing melanin formation, i.e., increased MSH receptor activity. Should this be the case, similar G2/M “injury responses” of other cell types might be expected, consistent with their differentiated phenotypes.     

Fas Antigen Modulates Ultraviolet B-Induced Apoptosis of SVHK Cells: Sequential Activation of Caspases 8, 3, and 1 in the Apoptotic Process

Interferon-gamma (IFN-gamma) induces various apoptosis-related proteins, including Fas antigen (Fas) in keratinocytes. Ultraviolet B (UVB) irradiation produces "sunburn cells," a specific type of apoptosis. Previously, we reported that IFN-gamma augments Fas-dependent apoptosis of SV40-transformed human keratinocytes (SVHK cells). Caspases are a new class of cysteine proteinases that play an important role in apoptosis. We investigated the mechanism of UVB-induced apoptosis by examining activation of the caspase cascade. UVB irradiation of SVHK cells increased the activities of caspases 1, 3, and 8, which were detected at 3 h, and peak activities occurred at 6 h. Pretreatment of SVHK cells with IFN-gamma significantly increased the activity of caspases 1, 3, and 8. UVB-induced caspase 8 stimulation was significantly suppressed only by caspase 8 inhibitor, while inhibitors of caspases 1, 3, and 8 significantly suppressed UVB-induced caspase 1 stimulation. Caspase 3 and 8 inhibitors, but not caspase 1 inhibitor, significantly suppressed UVB-induced caspase 3 activity, suggesting sequential activation of caspases 8, 3, and 1 in UVB-irradiated SVHK cells. Cross-linking and immunoprecipitation analyses showed multimerization of Fas antigen following UVB irradiation of SVHK cells. Pretreatment of SVHK cells with IFN-gamma significantly augmented UVB-induced apoptosis that was accompanied by increased Fas expression. The susceptibility to UVB-induced apoptosis was also increased in Fas-transfected SVHK cells (F2 cells). Neutralizing anti-Fas antibody significantly suppressed caspase activation and Fas-dependent apoptosis of SVHK cells and F2 cells. In contrast, UVB-induced caspase activation and apoptosis were not inhibited by neutralizing anti-Fas antibody in both cell lines. Our results suggest that UVB directly activates Fas and subsequent caspase cascade resulting in apoptosis of SVHK cells. Furthermore, the expression level of Fas antigen in keratinocytes influenced their susceptibility to UVB-induced apoptosis.