[Maturation and Antigen Loading Protocols Influence Activity of Anticancer Dendritic Cells].

The practical use of dendritic cell-based vaccines in anticancer therapy is limited by a lack of standards for dendritic cell (DC) generation, as well as standard procedures for controlling their activation and the technique of DC loading with nucleic acids encoding tumor antigens. Analyzing the currently available data, the most promising cocktails for DC maturation were selected and a comparative study of the cocktails and time of maturation on the capacity of DC to activate T-cell immune response has been performed. A study of the expression of surface markers and the production of IL-12, IL-6, and IL-10 cytokines, as well as the efficacy of T-cell activation showed that the use of the standard 7-day maturation protocol is preferable to the 4-day maturation protocol. Cocktails composed of TNF-α, IL-lß, IFN-α, IFN-γ, and poly(I:C), as well as TNF-α, IL-lß, IFN-γ, R848, and PGE2 were shown to be the most efficient activators of DCs. A comparison of the efficacy of different methods of DNA transfection into DCs and RNA delivery using alphavirus vectors demonstrated the superiority of magnet-assisted transfection (MATra) to other protocols.

Design of Polyepitope DNA Vaccine against Breast Carcinoma Cells and Analysis of Its Expression in Dendritic Cells.

Polyepitope DNA vaccine inducing T-cell-mediated immune response against cancer-specific antigens is a promising tool for selective elimination of tumor cells. Breast cancer-specific polyepitope DNA vaccine was designed using TEpredict and PolyCTLDesigner software on the basis of immunogenic peptides of HER2 and Mammaglobin-1 (Mam) tumor antigens. LPS-free preparations of plasmid DNA encoding polyepitope T-cell antigen and full-length copies of HER2 and Mam antigens were obtained. TaqMan-PCR systems for evaluation of the expression of immunogens in cells were created. The protocol of vaccine DNA delivery into dendritic cells was optimized. Expression of the target immunogens in dendritic cells derived from human peripheral blood mononuclear fraction after transfection with plasmid DNA preparations is demonstrated.

[Preparation of dendritic cells for cancer immunotherapy].

Development of new effective method for cancer therapy is one of the most important trends in the modern medicine. Along with surgery, chemotherapy and radiotherapy, induction of an immune response against the tumor cells is a promising approach for therapy of cancer, particularly metastatic, slowly dividing tumors and cancer stem cells. Induction of the antitumor T-cell immune response involves activation of antigen-presenting cells, which can efficiently present the cancer antigens and activate T-lymphocytes. The immune response may be activated by dendritic cells (DC) loaded with tumor antigens, such as tumor-specific proteins, tumor cell lysates, apoptotic or necrotic tumor cells, as well as nucleic acids encoding tumor antigens. Regardless of the selected source of the tumor antigen, preparation of mature DC is a principal step in the development of anticancer vaccines aimed at the induction of the cytotoxic T-cell immune response. Recently, various research groups have proposed several strategies for producing mature DC, differed by the set of agents used. It has been shown that the maturation strategy influences both their phenotype and the ability to induce the immune response. In this review we have analyzed the results of studies on the various strategies of preparation of mature DCs.

[Flap endonuclease-1 and its role in the processes of DNA metabolism in eucaryotic cells].

Flap endonuclease-1 (FEN1) is a structure specific endonuclease. The natural substrates of FEN1 are 5'-flap structures formed by three DNA chains one of them has unannealed flapped 5'-end (flap). Flap structures are the intermediates of different processes of DNA metabolism, such as DNA recombination, Okazaki fragment maturation during replication of lagging strand, as well as strand displacement DNA synthesis in base excision repair. FEN1 also possesses 5'-exonuclease activity and newly discovered gap endonuclease activity. FEN1 is known to interact physically and functionally with a number of DNA replication and repair proteins such as the proliferating cell nuclear antigen, helicase/nuclease Dna2, WRN and BLM proteins, replication protein A, apurinic/apyrimidinic endonuclease 1, DNA polymerase beta, poly(ADP-riboso) polymerase 1, high mobility group protein 1, integrase of human immunodeficiency virus, transcription coactivator p300, chromatin proteins, cyclin-dependent kinases (Cdk1, Cdk2, Cyclin A). FEN1 activity is significant for maintaining the integrity of repeat sequences in genome. Recent data suppose the correlation between the abnormality of hFEN1 activity and arising/progression of neurodegenerative and cancer diseases. FEN1 has the dramatic effect on cell growth and development thereby attracting the interest to this enzyme.

Study of interaction of XRCC1 with DNA and proteins of base excision repair by photoaffinity labeling technique.

The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in base excision repair (BER) interacting with and modulating activity of key BER proteins. To estimate the influence of XRCC1 on interactions of BER proteins poly(ADP-ribose) polymerase 1 (PARP1), apurinic/apyrimidinic endonuclease 1 (APE1), flap endonuclease 1 (FEN1), and DNA polymerase beta (Pol beta) with DNA intermediates, photoaffinity labeling using different photoreactive DNA was carried out in the presence or absence of XRCC1. XRCC1 competes with APE1, FEN1, and PARP1 for DNA binding, while Pol beta increases the efficiency of XRCC1 modification. To study the interactions of XRCC1 with DNA and proteins at the initial stages of BER, DNA duplexes containing a photoreactive group in the template strand opposite the damage were designed. DNA duplexes with 8-oxoguanine or dihydrothymine opposite the photoreactive group were recognized and cleaved by specific DNA glycosylases (OGG1 or NTH1, correspondingly), although the rate of oxidized base excision in the photoreactive structures was lower than in normal substrates. XRCC1 does not display any specificity in recognition of DNA duplexes with damaged bases compared to regular DNA. A photoreactive group opposite a synthetic apurinic/apyrimidinic (AP) site (3-hydroxy-2-hydroxymethyltetrahydrofuran) weakly influences the incision efficiency of AP site analog by APE1. In the absence of magnesium ions, i.e. when incision of AP sites cannot occur, APE1 and XRCC1 compete for DNA binding when present together. However, in the presence of magnesium ions the level of XRCC1 modification increased upon APE1 addition, since APE1 creates nicked DNA duplex, which interacts with XRCC1 more efficiently.

Use of modified flap structures for study of base excision repair proteins.

To investigate interactions between proteins participating in the long-patch pathway of base excision repair (BER), DNA duplexes with flap strand containing modifications in sugar phosphate backbone within the flap-forming oligonucleotides were designed. When the flap-forming oligonucleotide consisted of two sequences bridged by a decanediol linker located in the flap strand near the branch point, the efficiency and position of cleavage by flap endonuclease 1 (FEN1) differed from those for natural flap. The cleavage rate of chimeric structure by FEN1 was lower than that of a normal substrate. When we introduced the second modification in the flap-forming oligonucleotide, the cleavage rate decreased significantly. To estimate efficiency of recognition and processing of the chimeric structures by BER proteins, we studied the rate of DNA synthesis by DNA polymerase beta (Pol beta) and the rate of nucleotide excision at the 3'-end of the initiating primer by apurinic/apyrimidinic endonuclease 1 (APE1) compared with those for the natural DNA duplexes. Efficiency of strand-displacement DNA synthesis catalyzed by Pol beta was shown to be higher for flap structures containing non-nucleotide linkers. The chimeric structures were processed by the 3'-exonuclease activity of APE1 with efficiency lower than that for a normal flap structure. Thus, DNA duplexes with modifications in sugar phosphate backbone can be used to mimic intermediates of the long-patch pathway of BER in reconstituted systems containing FEN1. Based on chimeric and natural oligonucleotides, photoreactive DNA structures were designed. The photoreactive dCMP moiety was introduced into the 3'-end of DNA primer via the activity of Pol beta. The photoreactive DNA duplexes--3'-recessed DNA, nicked DNA, and flap structures containing natural and chimeric oligonucleotides--were used for photoaffinity labeling of BER proteins.

[Interaction of replication protein A and flap endonuclease 1 with DNA duplexes containing a nick or flap]. / Issledovanie vzaimodeistviia replikativnogo belka A i flép-éndonukleazy-1 s DNK-dupleksami, soderzhashchimi odnotsepochechnye razryvy i flép-struktury.

Nicks and flaps are intermediates in various processes of DNA metabolism, including replication and repair. Photoaffinity modification was employed in studying the interaction of the replication protein A (RPA) and flap endonuclease 1 (FEN-1) with DNA duplexes similar to structures arising during long-patch base excision repair. The proteins were also tested for effect on DNA polymerase beta (Pol beta) interaction with DNA. Using Pol beta, a photoreactive dTTP analog was added to the 3' end of an oligonucleotide flanking a nick or a flap in DNA intermediates. The character and intensity of protein labeling depended on the type of intermediates and on the presence of the phosphate or tetrahydrofuran at the 5' end of a nick or a flap. Photoaffinity labeling of Pol beta substantially (up to three times) increased in the presence of RPA or FEN-1. Various DNA substrates were used to study the effects of RPA and FEN-1 on Pol beta-mediated DNA synthesis with displacement of a downstream primer. In contrast to FEN-1, RPA had no effect on DNA repair synthesis by Pol beta during long-patch base excision repair.