Epigenetic View on Interferon γ Signalling in Tumour Cells.

IFN-γ is a pleiotropic cytokine crucial for both innate and adaptive immunity, which also plays a critical role in immunological surveillance of cancer. Genetic defects or gene silencing in the IFN-γ signal transduction pathways as well as in the expression of IFN-γ-regulated genes represent frequent mechanisms by which tumour cells can escape from immune responses. Epigenetic control of the IFN-γ signalling pathway activation associated with epigenetic changes in the corresponding regulatory gene regions, such as chromatin remodelling, histone acetylation and methylation, and DNA demethylation is frequently dysregulated in tumour cells. Epigenetic silencing of the IFN-γ regulatory pathway components, as well as of the IFN-γ-regulated genes crucial for tumour cell recognition or induction of anti-tumour immune responses, has been documented in various cancer models. Expression of both IFN-γ signalling pathway components and selected IFN-γ-regulated genes can be influenced by epigenetic modifiers, namely DNA methyltransferase and histone deacetylase inhibitors. These agents thus can mimic, restore, or boost the immunomodulatory effects of IFN-γ in tumour cells, which can contribute to their anti-tumour therapeutic efficacies and justifies their potential use in combined epigenetic therapy with immunotherapeutic approaches.

IFNγ induces oxidative stress, DNA damage and tumor cell senescence via TGFß/SMAD signaling-dependent induction of Nox4 and suppression of ANT2.

Cellular senescence provides a biological barrier against tumor progression, often associated with oncogene-induced replication and/or oxidative stress, cytokine production and DNA damage response (DDR), leading to persistent cell-cycle arrest. While cytokines such as tumor necrosis factor-alpha (TNFα) and interferon gamma (IFNγ) are important components of senescence-associated secretome and induce senescence in, for example, mouse pancreatic ß-cancer cell model, their downstream signaling pathway(s) and links with oxidative stress and DDR are mechanistically unclear. Using human and mouse normal and cancer cell models, we now show that TNFα and IFNγ induce NADPH oxidases Nox4 and Nox1, reactive oxygen species (ROS), DDR signaling and premature senescence. Unlike mouse tumor cells that required concomitant presence of IFNγ and TNFα, short exposure to IFNγ alone was sufficient to induce Nox4, Nox1 and DDR in human cells. siRNA-mediated knockdown of Nox4 but not Nox1 decreased IFNγ-induced DDR. The expression of Nox4/Nox1 required Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling and the effect was mediated by downstream activation of transforming growth factor-beta (TGFß) secretion and consequent autocrine/paracrine activation of the TGFß/Smad pathway. Furthermore, the expression of adenine nucleotide translocase 2 (ANT2) was suppressed by IFNγ contributing to elevation of ROS and DNA damage. In contrast to mouse B16 cells, inability of TC-1 cells to respond to IFNγ/TNFα by DDR and senescence correlated with the lack of TGFß and Nox4 response, supporting the role of ROS induced by NADPH oxidases in cytokine-induced senescence. Overall, our data reveal differences between cytokine effects in mouse and human cells, and mechanistically implicate the TGFß/SMAD pathway, via induction of NADPH oxidases and suppression of ANT2, as key mediators of IFNγ/TNFα-evoked genotoxicity and cellular senescence.

Radiotherapy-induced plasticity of prostate cancer mobilizes stem-like non-adherent, Erk signaling-dependent cells.

Fractionated ionizing radiation combined with surgery or hormone therapy represents the first-choice treatment for medium to high-risk localized prostate carcinoma. One of the main reasons for the failure of radiotherapy in prostate cancer is radioresistance and further dissemination of surviving cells. In this study, exposure of four metastasis-derived human prostate cancer cell lines (DU145, PC-3, LNCaP and 22RV1) to clinically relevant daily fractions of ionizing radiation (35 doses of 2 Gy) resulted in generation of two radiation-surviving populations: adherent senescent-like cells expressing common senescence-associated markers and non-adherent anoikis-resistant stem cell-like cells with active Notch signaling and expression of stem cell markers CD133, Oct-4, Sox2 and Nanog. While a subset of the radiation-surviving adherent cells resumed proliferation shortly after completion of the irradiation regimen, the non-adherent cells started to proliferate only on their reattachment several weeks after the radiation-induced loss of adhesion. Like the parental non-irradiated cells, radiation-surviving re-adherent DU145 cells were tumorigenic in immunocompromised mice. The radiation-induced loss of adhesion was dependent on expression of Snail, as siRNA/shRNA-mediated knockdown of Snail prevented cell detachment. On the other hand, survival of the non-adherent cells required active Erk signaling, as chemical inhibition of Erk1/2 by a MEK-selective inhibitor or Erk1/2 knockdown resulted in anoikis-mediated death in the non-adherent cell fraction. Notably, whereas combined inhibition of Erk and PI3K-Akt signaling triggered cell death in the non-adherent cell fraction and blocked proliferation of the adherent population of the prostate cancer cells, such combined treatment had only marginal if any impact on growth of control normal human diploid cells. These results contribute to better understanding of radiation-induced stress response and heterogeneity of human metastatic prostate cancer cells, document treatment-induced plasticity and phenotypically distinct cell subsets, and suggest the way to exploit their differential sensitivity to radiosensitizing drugs in overcoming radioresistance.

Immunotherapy augments the effect of 5-azacytidine on HPV16-associated tumours with different MHC class I-expression status.

BACKGROUND: Epigenetic mechanisms have important roles in the tumour escape from immune responses, such as in MHC class I downregulation or altered expression of other components involved in antigen presentation. Chemotherapy with DNA methyltransferase inhibitors (DNMTi) can thus influence the tumour cell interactions with the immune system and their sensitivity to immunotherapy. METHODS: We evaluated the therapeutic effects of the DNMTi 5-azacytidine (5AC) against experimental MHC class I-deficient and -positive tumours. The 5AC therapy was combined with immunotherapy, using a murine model for HPV16-associated tumours. RESULTS: We have demonstrated 5AC additive effects against MHC class I-positive and -deficient tumours when combined with unmethylated CpG oligodeoxynucleotides or with IL-12-producing cellular vaccine. The efficacy of the combined chemoimmunotherapy against originally MHC class I-deficient tumours was partially dependent on the CD8(+)-mediated immune responses. Increased cell surface expression of MHC class I cell molecules, associated with upregulation of the antigen-presenting machinery-related genes, as well as of genes encoding selected components of the IFNγ-signalling pathway in tumours explanted from 5AC-treated animals, were observed. CONCLUSION: Our data suggest that chemotherapy of MHC class I-deficient tumours with 5AC combined with immunotherapy is an attractive setting in the treatment of MHC class I-deficient tumours.

Effects of 5-azacytidine and trichostatin A on dendritic cell maturation.

Maturation of dendritic cells (DC) towards functional antigen-presenting cells is a complex process, the regulation of which may also involve epigenetic mechanisms. Thus, it is of interest to investigate how gene expression changes during DC maturation can be influenced with epigenetic agents, such as DNA methyltransferase or histone deacetylase inhibitors. Here, we document the effects of DNA methyltransferase inhibitor 5-azacytidine (5AC) and histone deacetylase inhibitor trichostatin A (TSA) on the murine bone marrow-derived, as well as on the human monocyte-derived DC maturation. The major impact of 5AC and TSA on the DC maturation process consisted in the inhibition of unmethylated CpG oligodeoxynucleotide (CpG ODN) 1826 or LPS-induced activation of pro- and anti-inflammatory cytokine gene expression activation. In the in vitro studies, TSA but not 5AC significantly reduced the capacity of the peptide-pulsed DC to induce total spleen as well as CD8(+) or CD4(+) cell proliferation. IFNγ production by the specific CD4(+) spleen cells co-cultured with TSA- but not with 5AC-treated DC was lower, as compared to the cytokine production after co-cultivation with untreated mature DC. Collectively, these results demonstrate the potential of epigenetic agents, which are under intensive investigation as promising anti-tumour agents, to hamper the immune response induction through their inhibitory effects on DC.

nef gene sequence variation among HIV-1-infected African children.

BACKGROUND: There are few data on African children infected with nonclade B HIV-1 in endemic settings, which limits generalizations about pathogenesis and progression. Genotypic and phenotypic variations in host immunogenetics and HIV-1 negative factor (nef) accessory protein may influence disease progression and have frequently been characterized in subjects infected with clade B HIV-1. METHODS: In this descriptive study, we report nef gene sequence variation and host genetic polymorphisms in 32 Kenyan children, including 12 slow progressors. RESULTS: Phylogenetic analysis identified HIV-1 clades A, C and D and a recombinant A/D subtype. Grossly defective nef genes or significant changes from relevant clade reference sequences were not identified in children with delayed disease progression. CONCLUSIONS: nef sequence variations may not be common in perinatally infected African children. Further studies are warranted in HIV-1-infected subjects in settings where infection is endemic.

Long-term analysis of the resistance development in HIV-1 positive patients treated with protease and reverse transcriptase inhibitors: correlation of the genotype and disease progression.

In this study, 27 HIV-1-positive patients on long-term highly active antiretroviral therapy (HAART) in the Czech Republic were followed for a period of up to 7 years. Variability of the HIV-1 protease (PR) sequence common in the Czech Republic was observed. Under the pressure of inhibitors of protease (PRIs) and reverse transcriptase (RTIs) mutations in PR were detected. Development of resistance to PRIs was followed by a decrease in CD4 count and increase in viral load. The dynamics of viral load closely corresponded to the accumulation of specific primary mutations in PR and RT. Out of 27 patients 18 developed resistance to PRIs and the prolonged therapy led to the accumulation of a higher number of amino acid changes associated with the resistance and, consequently, cross-resistance to several PRIs was observed. These multi-resistant variants of HIV-1 with mutations in PR could not be inhibited sufficiently with PRIs that are currently available in clinical practice. Efficient yet temporary suppression of viral replication was achieved by a lopinavir (LPV) treatment.

Immunogenicity of dendritic cell-based HPV16 E6/E7 peptide vaccines: CTL activation and protective effects.

We have investigated the capacity of cellular vaccines based on dendritic cells loaded with human HPV16 E6/E7 oncoprotein-derived peptides to induce immune responses in vitro and to elicit protective immunity in a murine experimental model mimicking human HPV16-associated carcinomas. Dendritic cells loaded with the HPV16 E6/E7 peptides exhibiting CTL or Th epitopes (E6(41-50), E6(81-90), E6(98-107), E6(130-137), E7(49-57), and E7(44-62)) were able to stimulate in vitro DNA synthesis in syngeneic H-2b spleen cells. The priming capacity of peptide-loaded BMDC and peptide-loaded dendritic cell lines DC2.4 and JAWS II was compared. It has been found that both peptide-loaded BMDC and established dendritic cell lines can activate the syngeneic responder cells, but the priming capacity of BMDC was substantially higher. In the second set of experiments, we have examined the cytolytic activity of syngeneic spleen cells after repeated activation in vitro with BMDC loaded with HPV16 synthetic peptides containing CTL epitopes. Significant cytotoxic responses against HPV16 E6/E7 antigen-expressing TC-1 targets have been found after repeated in vitro activation with all peptides containing the CTL epitopes. In contrast, peptide E7(44-62) harbouring both Th and CTL epitopes induced significant cytotoxic responses already after single in vitro activation. This cytotoxic effect could be enhanced with admixture of the E7(49-57) peptide. Experiments with MHC class I proficient (TC-1, MK16-IFNgamma) and deficient (MK16) target cells revealed that the dendritic cells loaded with the E6/E7 HPV16 peptides activated CTLs in vitro, but not the other cytolytic effector mechanisms. The effectiveness of the peptide-loaded BMDC-based cellular vaccines was also investigated in vivo. C57BL/6 (H-2b) mice were immunized with various peptide-loaded BMDC and subsequently challenged with TC-1 cells. The strongest protective effect was achieved with the BMDC loaded with the peptide E7(44-62) harbouring both CTL and Th epitopes. Mice immunized with the E7(44-62) peptide remained tumour-free after s.c. transplantation of the TC-1 cells and exhibited long-lasting protective immunity, whereas the mice immunized with E6(81-90) and E7(49-57) peptides did not remain tumour-free and exhibited only partial inhibition of tumour growth detectable as depression of the tumour growth curves.

Genetic subtypes of HIV type 1 viruses circulating in the Czech Republic.

We analyzed the genetic diversity of HIV-1 strains circulating in the Czech Republic. Phylogenetic analysis of the env and gag gene sequence fragments from 39 isolates revealed that the majority of these strains (32 of 39, 82%) were of subtype B; other genetic subtypes identified were A, C, F, and recombinant circulating form CRF01_AE. The isolates that did not cluster with subtype B originated almost exclusively from a heterosexual route of transmission. The molecular epidemiological data are suggestive of multiple entry of HIV-1 infection into the Czech Republic and show that the genetic pattern of the HIV-1 strains circulating in this country corresponds to that found in other European countries.

Therapy of HPV 16-associated carcinoma with dendritic cell-based vaccines: in vitro priming of the effector cell responses by DC pulsed with tumour lysates and synthetic RAHYNIVTF peptide.

Murine carcinoma induced by MK 16 cells expressing HPV 16 E6/E7 oncogenes was utilized to examine the therapeutic effect of dendritic cell-based tumour vaccines. Mice carrying 5-day MK 16 tumours were injected peritumorally with either dendritic cells (DC) or DC pulsed with MK 16 tumour lysate. Both the unpulsed and MK 16 lysate-pulsed DC vaccines inhibited growth of the MK 16 transplants, the pulsed DC being more efficient than the unpulsed vaccines. In vitro priming of the effector cell-mediated anti-MK 16 responses by DC pulsed with MK 16 tumour lysate and a synthetic HPV 16 E7(49-57) peptide RAHYNIVTF was compared. The priming activity of the lysate was substantially higher than that of the HPV 16 E7(49-57) peptide; the priming activity was similar to that of a standard moderately immunogenic chemically-induced sarcoma. Taken collectively, these results suggest that DC vaccines pulsed with HPV 16-associated tumour lysates represent a prospective modality for treatment of HPV 16-associated carcinomas.

Human immunodeficiency virus 1 strains resistant to nucleoside inhibitors of reverse transcriptase in isolates from the Czech Republic as monitored by line probe assay and nucleotide sequencing.

The genetic resistance to nucleoside inhibitors of the reverse transcriptase (RT) of human immunodeficiency virus I (HIV-1) isolates in the Czech Republic was examined by a line probe assay (LiPA) and nucleotide sequencing. The results of LiPA analysis of 294 blood specimens obtained from 156 patients revealed a high incidence of mutations in the RT gene related to resistance to various drugs (67.3%) in various combinations. Mutations in RT gene (M41L, K70R and T215Y/F) conferring the resistance to zidovudine (ZDV) were most frequent (62.6%), that (M184V) responsible for the resistance to lamivudine (3TC) was less frequent (33.7%), while those linked to the resistance to dideoxyinosine (ddl) and dideoxyinosine together with dideoxycytidine (ddl/ddC) were rather rare (6.5% and 5.1%, respectively). LiPA gave a high rate of uninterpretable results due to codon hybridization failure, especially in HIV-1 isolates of non-B subtype. Thirty-two specimens were analyzed also by direct sequencing of a part of RT gene. The results obtained by LiPA and the sequencing were highly concordant for codons successfully analyzed by both methods, but the sequencing provided information also about the codons that could not be analyzed by LiPA. A high prevalence of resistant strains in the Czech Republic and their heterogeneity justifies a regular HIV-1 resistance testing. LiPA turned out as a fast, powerful and most reliable tool for such a purpose. However, due to an increasing diversity of HIV-1 strains circulating in the Czech Republic, LiPA cannot replace the nucleotide sequence analysis.

Peritumoral administration of antigen-unstimulated bone marrow-derived dendritic cells inhibits tumour growth.

Murine BM cells from B6 mice were grown in vitro in medium supplemented with GM-CSF and IL-4 to differentiate DC from DC precursors. After 10 days of culture, approximately 20% of the cell population exhibited the characteristic morphology of BMDC. In cytofluorometric analysis the morphological changes of cells were accompanied by upregulation of the expression of the MHC class II, CD11c, CD80, and CD86 molecules. The BMDC were pulsed with a lysate of syngeneic MK16 carcinoma cells and used for in vitro activation of SC. Co-cultivation of the carcinoma lysate-pulsed BMDC with SC induced a proliferative response of the syngeneic SC. Priming of the proliferative responses was more efficient when the BMDC were grown in the presence of GM-CSF and IL-4 for 10 days than for 7 days. The in vivo effect of mature, tumour lysate-unstimulated BMDC was examined in mice carrying syngeneic MK16 carcinoma transplants. It has been found that local pretreatment with BMDC inhibits growth of a subsequent challenge inoculum of the MK16 cells. Similarly, treatment of mice carrying small MK16 tumours and of those with MK16 surgical minimal residual disease performed with BMDC significantly inhibited tumour growth. It can be concluded from these results that local concentration of mature BMDC at the tumour site can control the development and growth of the transplanted tumour inoculum.

Irradiation of genetically modified plasmacytoma vaccines results in upregulation of CD80 molecule expression, IL-2 production and higher therapeutic efficacy of the vaccines.

It has been found previously that irradiated, IL-2 gene-modified plasmacytoma (X63-m-IL-2) vaccines are more efficient in the therapy of the parental (X63-Ag8.653) plasmacytoma than live plasmacytoma vaccines. In this communication, we have demonstrated that irradiation of murine IL-2-producing plasmacytoma vaccines resulted in upregulation of CD80 molecule expression and IL-2 production. The expression of MHC class I antigens was not altered. The upregulation of the CD80 membrane molecule expression in X63-m-IL-2 cells was higher after irradiation with 150 Gy than after irradiation with 50 Gy. Comparable upregulation of the CD80 molecule expression has also been demonstrated after irradiation of the parental murine X63-Ag8.653 plasmacytoma cells. The results indicate that upregulation of the CD80 molecule expression and enhanced IL-2 production in irradiated X63-m-IL-2 cells was responsible for the higher therapeutic effectiveness of the irradiated plasmacytoma vaccine.

Interleukin 2 gene therapy of surgical minimal residual tumour disease: characterization of cytolytic effector cells from tumour progressors and regressors.

Experiments were designed to characterize cytolytic effector cells from mice with SMRTD treated with IL-2 gene therapy. Mice were inoculated with syngeneic murine MK16 carcinoma cells. When the tumours reached 8-12 mm in diameter, they were excised and the operated mice were randomized into two groups. The first group without any further treatment was designated as operated-only; the second group, vaccinated 3 days after the operation with IL-2-producing tumour vaccine, is referred to as operated-vaccinated. Tumour recurrence rate in the operated-only mice was 90 percent; in the operated-vaccinated group the recurrence rate was 38.5 percent (progressors). The remaining 61.5 percent of mice were permanently protected (regressors). On day 53, the tumour progressors, regressors and healthy controls were sacrificed, and their spleen cells were used for 51Cr microcytotoxicity assay. Splenocytes from any group of mice were not cytolytic when allowed to react with MK16, YAC-1 (NK sensitive) and C1498 (NK resistant) targets. However, when grown for 3 days in IL-2-containing medium, the splenocytes from all groups of mice could develop cytolytic activity. The cytolytic activity of splenocytes from tumour progressors and regressors was substantially lower then that of splenocytes from healthy controls. In addition, significantly lower cytolytic activity was observed with IL-2-activated splenocytes from tumour progressors as compared to that of tumour regressors. Depletion of NK1.1+ cells or CD4+ plus CD8+ cells prevented the induction of significant IL-2-stimulated cytotoxicity directed against MK16 and C1498 targets in spleen cell cultures from tumour progressors, regressors, and healthy control mice, indicating that both, NK1.1+ and CD4+ plus CD8+, cells participate in the antitumour effect of IL-2 gene therapy. This was further supported by the finding that after depletion of CD4+ plus CD8+ cells, a residual cytolytic activity directed exclusively against NK-sensitive YAC-1 cells was observed.

[Molecular biology and serologic analysis in an epidemic of viral hepatitis B in vaccinated patients on hemodialysis]. / Molekulárne-biologická a sérologická analýza epidemie virové hepatitidy B u vakcinovaných hemodialyzovaných pacientu.

The authors analyzed an epidemic of viral hepatitis B during which 43% patients of the hemodialyzation centre in K were infected. Using the method of sequence analysis of the DNA of hepatitis B virus they identified the source of infection. They monitored serologically the course of infection in all infected subjects and the state of specific antibody immunity in patients and staff. 22% infections were manifested clinically and 75% infected HBsAg positive patients developed chronic hepatitis. Although 38 of 42 hemodialyzed patients were vaccinated against hepatitis B, 20 patients lacked anti-HBs antibodies before the epidemic. Only one of the patients without anti-HBs was not infected. In 10 immune patients the rise of antibody levels confirmed that they were also exposed to HBV. The effectiveness of vaccination against VHB in hemodialyzed patients is markedly lower. Vaccination of these patients is an important but only supplementary provision in VHB prevention. Sequence analysis of HBV DNA may be a useful tool of epidemiological analysis.

CD38 functions are regulated through an internalization step.

The endocytosis of the human CD38 molecule has been investigated in normal lymphocytes and in a number of leukemia- and lymphoma-derived cell lines. CD38 internalization was followed using radioiodinated Abs in an acidic elution endocytosis assay to monitor the effects of cross-linking on internalization processes and to quantify the ratio of the internalized molecule. Second, conventional, confocal, and electron microscopies were used to evaluate the morphologic effects induced by ligation of the molecule with Abs mimicking the natural ligand(s). The results demonstrated that internalization is a reproducible phenomenon following CD38 ligation with both agonistic and nonagonistic specific Abs and involving only a fraction of the entire amount of the surface molecule. It is independent from signal transduction as can be inferred by the observation that 1) both agonistic and non agonistic Abs are effective and 2) the dynamic of internalization is much slower than that of cellular signaling. Morphologic studies demonstrated that endocytosis induced as a result of CD38 ligation presents a very specific pathway consisting of subcellular organelles fundamental to the processing of the complex. Our data indicate that down-regulation by endocytosis may be, in parallel with shedding, a regulatory element in activation and adhesion processes mediated by CD38. However, internalization seems not to be a key step in triggering intracellular signaling; more likely, it is a negative feedback control mechanism which interrupts signal transduction or cell-cell cross-talks mediated by membrane CD38.

Functional associations of CD38 with CD3 on the T-cell membrane.

CD38 is a multifunctional membrane surface glycoprotein expressed by different cells and tissues, including T cells at certain stages of their development. Besides its involvement in transmembrane signaling, CD38 play a role in cell adhesion processes. Structurally, membrane CD38 was reported as presenting lateral associations with molecules involved in recognition and signaling, namely with the TCR/CD3 complex in T cells. Here we report that ligation of CD38 by agonistic and non-agonistic monoclonal antibodies exerts different effects on T cells, the former inducing down-modulation of the associated molecules, probably through a protein kinase C-dependent mechanism. This observation supports the view that the reduced expression of TCR/CD3 is secondary to interplay with CD38-mediated signaling, which partially overlaps with the CD3-mediated pathway. CD3 ligation by monoclonal antibodies leads not only to the expected internalization of the TCR/CD3 complex but also to down-modulation of surface CD38. The results obtained indicate that CD38 is closely associated with the CD3/TCR complex and that co-modulation of CD38 with TCR/CD3 is a critical step in signaling processes on T lymphocytes.

Receptor-mediated transport of oligodeoxynucleotides into hepatic cells.

Receptor-mediated endocytosis was employed for a highly efficient transport of oligodeoxynucleotides into hepatoma cell line PLC/PRF/5. The oligodeoxynucleotides were bound to the asialofetuin-poly-L-lysine conjugate and this complex was internalized by the cells via asialoglycoprotein receptor, an endocytic receptor unique for hepatocytes. Binding of the oligodeoxynucleotides to the complex dramatically increased their cellular uptake more than 20-fold. Chloroquine, a lysosomatropic agent, further increased the transport of the complex but not of the free oligodeoxynucleotides.