Hypoxia as a Factor Involved in the Regulation of the apoA-1, ABCA1, and Complement C3 Gene Expression in Human Macrophages.

Hypoxia plays a critical role in progression of atherosclerosis. Local oxygen deficiency in a plaque creates a specific microenvironment that alters the transcriptome of resident cells, particularly of macrophages. Reverse cholesterol transport from plaque to liver is considered a main mechanism for regression of atherosclerosis. Ubiquitously expressed ATP-binding cassette transporter A1 (ABCA1) and liver- and small intestine-derived apolipoprotein A-1 (ApoA-1) are two main actors in this process. We recently reported endogenous apoA-1 expression in human macrophages. While ABCA1 and ApoA-1 have antiatherogenic properties, the role of complement factor C3 is controversial. Plasma C3 level positively correlates with the risk of cardiovascular diseases. On the other hand, C3 gene knockout in a murine atherosclerosis model increases both plaque size and triglycerides level in blood. In the present study, we show for the first time that a hypoxia-mimicking agent, CoCl2, induces the upregulation of the apoA-1 and C3 genes and the accumulation of intracellular and membrane protein ApoA-1 in THP-1 macrophages. The MEK1/2-Erk1/2 and MKK4/7-JNK1/2/3 cascades are involved in upregulation of ABCA1 and C3 via activation of transcription factor NF-κB, which interacts with the HIF-1α subunit of hypoxia-inducible factor 1 (HIF-1). The three major MAP-kinase cascades (Erk1/2, JNK1/2/3, and p38) and the NF-κB transcription factor are involved in the hypoxia-induced expression of the apoA-1 gene in THP-1 macrophages.

[Characterization of Distal and Proximal Alternative Promoters of the Human ApoA-I Gene].

Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoprotein (HDL). ApoA-I constitutes ~75% of the protein content of HDL. The main sites of ApoA-I synthesis in humans are the liver and the small intestine. The mechanisms that govern tissue-specific apoA-I transcription in tissues and organs other than the liver and the small intestine are poorly understood. It is known that the human apoA-I has two additional promoters, the proximal and the distal one. In this work these two alternative apoA-I promoters are characterized, their transcription start sites are mapped and their competition for apoA-Itranscription is demonstrated; the role of the alternative promoters in apoA-I expression in human cells and tissues other than hepatocytes and enterocytes is discussed.

[Non-viral gene therapy approach for regenerative recovery of skin wounds in mammals].

The rate and character of skin tissue regeneration after wounds, burns and other traumas depend on the cell proliferation within damaged area. Acceleration of healing by stimulation of cell proliferation and extracellular matrix synthesis is one of the most important tasks of modern medicine. There are gene therapy approaches to wound treatment consisting in the transfer of genes encoding mitogenic growth factors to wound area. The most important step in the development of gene therapy approaches is the design of gene delivery tools. In spite of high efficacy of viral vectors, the non-viral means have some preferences (low toxicity, low immunogenity, safety and the absence of backside effects). Among non-viral gene delivery tools, molecular conjugates are the most popular because of their efficacy, simplicity, and the capacity to the targeted gene transfer. In the present work we have developed two molecular conjugates--NLS-TSF7 and NLS-TSF12 consisting of the modified signal of nuclear localization of T-antigen of SV40 virus (cationic part) and the peptide ligands of mammalian transferrin receptor (ligand part). These conjugates bind to plasmid DNA with formation of polyelectrolytic complexes and are capable to deliver plasmid DNA into cells expressing transferrin receptors by receptor-mediated endocytosis. Transfer of the expression vector of luciferase gene in the complex with molecular conjugate NLS-TSF7 to murine surface tissues led to about 100 fold increasing of luciferase activity in comparison with the transfer of free expression vector. Treatment of slash wounds in mice with the complexes of expression vector of synthetic human gene encoding insulin-like growth factor 1 with molecular conjugates NLS-TSF7 led to acceleration of healing in comparison with mice treated with free expression vector. The results obtained confirm the high efficiency of the developed regenerative gene therapy approach for the treatment of damaged skin tissues in mammals.

[Transfer of genetic constructions through the transplacental barrier into mice embryos].

Genetic modification of mammalian embryos is an important way to model various changes in human development; also, it is an instrument for studying the functions of certain genes in mammals. Using our own experience in developing modes of delivery of genetic constructions to mammals in a nonviral way, we present here data on the delivery of a eukaryotic expression vector to mice embryos through the transplacental barrier with the use of hydrodynamic intravenous injections of DNA-hybrid peptide complexes to pregnant females. The peptide has a cationic part for interaction with DNA and includes a ligand structure towards receptors of the releasing factor of luteinizing hormone (RFLH, luliberin). Advantages of the suggested method are simplicity, economy, nonimmunogenicity for females, and the ability to multiply repeat the procedure. On the basis of the method, systemic gene delivery into tissues of mammalian embryos may be developed.

[The role of ZF5 and CGGBP-20 transcription factors in expression regulation of human FMR1 gene responsible for X-fragile syndrome].

The human FMR1 gene encodes an RNA-binding protein taking part in translation regulation. The 5'-untranslated region of FMR1 gene contains a large number of tandem repeats of GCC triplets (5-50) which increasing (more then 200) is responsible for X-fragile syndrome (human congenital anomaly). As it has been shown earlier, al least two transcription factors (ZF5 and CGGBP-20) are capable of interacting specifically with GCC-repeats in regulatory regions of some genes. In this work, their roles in FMR1 gene expression regulation were studied. It was demonstrated by electrophoretic mobility shift assay that ZF5 recombinant protein specifically bound with GCC-triplet repeats (GCC9). Tissue-specific distributions of ZF5 and FMR1 proteins are very overlapped in mammalian. Inhibition of ZF5 expression in HepG2 cells (by RNA interference) leads to at least 1.5 times stimulations of FMR1 gene expression in these cells. To estimate the contribution of GCC-triplet repeats in FMR1 gene expression regulation we used two alternative variants of genetic construction: containing luciferase reporter gene under 5'-regulatory region fragment devoid of GCC-triplet repeats or including the GCC9 nucleotide sequence. HepG2 cells were co-transfected by these constructions and expressions vectors of ZF5 or (and) CGGBP-20 respectively. It was found that ZF5 downregulated the activity of 5'-regulatory region of FMR1 gene in both cases (acting probably through canonic 5'-GCGCGC3' sites). The presence of GCC-triplet repeats in the construction weakens this ZF5 effect. CGGBP-20 downregulates the activity of 5'-region of FMR1 gene in the presence of GCC-triplets only. The data obtained evidently indicate differently directed ZF5 effects on FMR1 gene expression and suggest the mechanism to explain the earlier demonstrated phenomenon about increasing of mRNA level in permutation FMR1 allele carries.

[Ap1-like cis-elements in 5'-regulatory region of human apolipoprotein A-I gene].

Several Ap1-like cis-acting elements were found within 5'-regulatory region (-2497...+173 versus transcription start point) of human apolipoprotein A-I gene (5'-apoA-I). Those elements are capable to interact with transcription factors belonging to Ap1 and CREB/ATF families. Those elements are localized outside of the hepatic enhancer (-220...-110) and the sequence responsible for apoA-I gene transcription in Caco2 cells (-595...-192). One of Ap1-like sites (5'-TGAGGTCT-3, Cre/jun2/apo) is present within 5'-apoA-I in two copies - distal (-1798 ...-1791) and proximal (+99...+106) ones. This and other Ap1-like sites - 5'-TGACTCT-3' (-1798...-1791, PF1) and 5'-TGACATCA-3' (-1171...-1163, Cre/jun1) were characterized by EMSA. It was shown by using the specific antibodies to c-Jun and ATF2 transcription factors in EMSA supershift experiments, that the DNA-protein complexes formed by Cre/jun2/apo, Cre/jun1 elements with nuclear proteins of human hepatoma HepG2 cells contain ATF2. The functional role of 5'-apoA-I regions containing Ap1-like sites was studied in cotransfection experiments of HepG2 cells (synthesize endogenous ApoA-I), human duodenum adenocarcinoma Hutu80 cells (do not synthesize endogenous ApoA-I), human neuroblastoma SK-N-SH cells (do not synthesize endogenous A-I) with expression vectors of c-jun and mekk1 genes. It was shown, that those Ap1-like sites appears to be responsible (the proximal Cre/jun2/apo is more efficient) for tissue-specific regulation of human apoA-I gene expression.

[Transcription factor ZF5 regulates expression of mammalian gene containing GCC-triplet repeats in 5'-regulatory region in human hepatoma HepG2 cells].

Some nuclear proteins of human HeLa and HepG2 cells are capable of binding to GCC-triplet repeats--(GCC)n > 3 in 5'-regulatory regions of a number of mammalian genes--G-C-elements. According to our previous data, nucleotide sequence (GCC)4 in promoter of mouse ribosomal protein L32 gene (rpL32) between 17 and 6 bp upstream of transcription start site interacts to nuclear proteins from HepG2 cells, and may be considered as a GCC-element. We suggest that one of those proteins, with molecular weight about 52 kDa, which may interact with rpL32 GCC-element, is a known conservative mammalian transcription factor ZF5. DNA-binding domain of ZF5 contains a few Kruppel-like Zn-fingers (Cys2His2-type) interacting with the GC-rich nucleotide sequences in 5'-regulatory regions of a number of mammalian genes. Our results (obtained by EMSA) showed that recombinant GST-ZF5 fused protein containing ZF5 DNA-binding domain specifically binds a few GS-rich sequences: (GCC)g-9riplet repeats, 5'-GCGCGC-3' (known ZF5 consensus binding site) and (more preferable) the fragment (-24...+1 bp) of rpL32 promoter. The high affinity of ZF5 DNA-domain binding with the latter may be explained by the presence in this fragment of two overlapped subsequences, each being capable of binding to ZF5: (GCC)4 and 5'-GCGCGC- 3'. Zf5 cDNA was cloned from HepG2 cells by RT-PCR method, and then used for construction of the gene expression vector. It has been shown that Zf5 cDNA expression vector specifically down-regulates (in luciferase assays) the activity of rpL32 promoter (-155...+159) including the above mentioned GC-rich subsequences by cotransfection of HepG2 cells. Therefore, our results enable us to consider GCC-elements as a novel class of ZF5 targets in 5'-regulatory regions of mammalian genes.

Complexes of DNA with cationic peptides: conditions of formation and factors effecting internalization by mammalian cells.

This work was devoted to the study of conditions of the formation of DNA/K8 complex and analysis of factors effecting the entry of DNA/K8 complex into mammalian cells in comparison with DNA complexes with arginine-rich fragment (47-57) of human immunodeficiency virus (type 1) transcription factor Tat (Tat peptide). The stoichiometry of positively charged DNA/K8 complexes has been studied for the first time. Non-cooperative character of DNA-K8 interaction was revealed. It has been shown that along with the positive charge of such complexes, the presence of an excess of free K8 peptide in the culture medium is a necessary condition for maximal efficiency of cell transfection with DNA/K8 complexes. A stimulatory effect of free K8 peptide on the efficiency of mammalian cell transfection by DNA/K8 complexes is likely to be mediated by the interactions of cationic peptide K8 with negatively charged proteoglycans on the cell surface, which leads to protection of DNA/K8 complexes from disruption by cellular heparan sulfates. However, the protective role of free cationic peptides depends not only on their positive charge, but also on the primary structure of the peptide. In contrast with the results obtained for DNA complexes with molecular conjugates based on poly-L-lysine, the aggregation of DNA/K8 complexes leads to a significant increase in the expression of transferred gene.

[Hydrodynamics-based transfer of human apolipoprotein A-I gene into mice: study of factors involving an efficacy and duration of the transferred gene expression in animals' liver]. / Perenos gena apolipoproteina A-I cheloveka mysham metodom gidrodinamicheskikh in''ektsii: faktory, vliiaiushchie na éffektivnost' i prodolzhitel'nost' ékspressii gena v pecheni zhivotnogo.

Human apolipoprotein A-I gene (apoA-I) plasmid expression vectors were transferred into mice by hydrodynamic injections into tail vein. Two types of expression vectors were used. First one -pCMVcapoAI contains cDNA of apo A-I driven by human cytomegalovirus early gene promoter (CMV). Second one--pAlg contains genomic locus of intron-containing apo A-I under control of own extended 5'-regulatory region (APOAI). Hydrodynamic intravenous injections of both expression vectors led to appearance of human apo A-I mRNA in the liver and human Apo A-I protein in the serum of injected mice. Dynamics of human Apo A-I content in the serum of mice injected by pCMVcapoAI and pAlg were different. When pCMVcapoAI was used, maximal concentration of human Apo A-I protein in the mouse serum was detected one day after injection with following decline to zero level during next two weeks. Under the same conditions injections of pAlg led to maximal level of human Apo A-I concentration in the mouse serum (up to 20 mkg/ml in some animals) on the 5th-7th day of experiment with following graduate decline during several months (human Apo A-I concentration in the serum of oldest analyzed mouse (6 months after injection) was about 25% of its maximal level in the same animal). Levels of human Apo A-I concentration in the mouse serum were compatible after injections of both expression vectors, in spite of much more strong activity of CMV promoter in comparison with APOAI in cultured human hepatoma cells HepG2. We ascribe the revealed difference in dynamics of human Apo A-I expression to delay of apo A-I transcription from pAlg vector, that was confirmed by nested RT-PCR. Significant level and long-term persistence of human Apo A-I in the serum of mice injected by pAlg could be explained by properties of APOAI or (and) exon-intron structure of genomic apo A-I gene. To test the role of APOAI in long-term expression of human Apo A-I in the mice we performed hydrodynamic injections of plasmid vectors containing cDNA of reporter gene encoding luciferase driven by variants of APOAI. No long-term expression of luciferase was found in the livers of injected mice. Therefore, our data suggest the role of exon-intron structure in maintaining of efficient and long-term expression of transferred human apo A-I.

Transcription regulatory cis-element (GCC)8 in the 5'-untranslated region of the gene for human very-low-density lipoprotein receptors.

The 5'-untranslated region of very-low-density lipoprotein (VLDL) receptor gene includes 2 groups of triplet repeats (GCC)n. Four repeats are localized near the promoter region in position 15.23 from the transcription initiation site. Eight repeats were detected in position 573.597. Sequence (GCC)8 in VLDL receptor gene forms specific complexes with nuclear proteins of HepG2 cells, the formation of these complexes depended on Zn(2+). Superexpression of the CGGBP-20 protein interacting with long sequences (GCC)n suppressed transcriptional activity of VLDL receptor gene. Removal of fragment (397.616) containing cis-element (GCC)8 from the 5'-untranslated region of VLDL receptor gene led to activation of the linked marker gene cat in Hutu80 cells, but did not abolish the repressor effect of CGGBP-20 protein. Our results suggest that (GCC)n-binding proteins differing from CGGBP-20 regulate activity of the VLDL receptor gene via cis-element (GCC)8.

[Delivery of "suicide" thymidine kinase gene of herpes virus in the complex with cationic peptide into human hepatoma cells in vitro]. / Dostavka "suitsidnogo" gena timidinkinazy virusa gerpesa v komplekse s kationnym peptidom v kletki gepatomy cheloveka in vitro.

The delivery of "suicide" herpes simplex virus type-1 thymidine kinase gene (tk) into tumor cells, followed by treatment with synthetic nucleotide analogues (gancyclovir, acyclovir), is a perspective approach to cancer therapy. Serious limitations in employment of the existing means of gene delivery into target cells constitute the main obstacle for cancer gene therapy development. In the present work a possibility to use a nonviral gene delivery system is shown based on the employment of lysine rich peptide K8 and amphipathic peptide JTS-1 for transferring tk gene into human hepatoma HepG2 cells. Cationic peptide K8 forms compact complexes with plasmid DNA, and JTS-1 acts as a pH-dependent endosomal releasing agent. Transfection of HepG2 cells by tk expression vector coupled with K8/JTS-1 peptides, followed by acyclovir administration (50-100 micrograms/ml) for 24 h leads to cell cycle arrest in the G1/S checkpoint of some cells, which eventually die through apoptosis. Treatment of HepG2 cells with higher acyclovir concentration (200 micrograms/ml) additionally results in a nonspecific toxic effect. The above results demonstrate the efficacy of K8/JTS-1 delivery system for the "suicide" cancer gene therapy, and may be regarded as a basis for further elaboration of "suicide" cancer approaches in vivo.

Receptor-mediated transfer of DNA--galactosylated poly-L-lysine complexes into mammalian cells in vitro and in vivo.

With the goal of developing non-viral techniques for exogenous gene delivery into mammalian cells, we have studied receptor-mediated gene transfer using complexes of plasmid DNA and galactosylated poly-L-lysine, poly(L-Lys)Gal. To evaluate the optimal parameters for efficient gene transfer into human hepatoma HepG2 cells by the DNA-poly(L-Lys)Gal complexes, the bacterial reporter genes lacZ and cat were used. Examination of the reporter gene expression level showed that the efficiency of DNA delivery into the cells depends on the structure of DNA--poly(L-Lys)Gal complexes formed at various ionic strength values. The efficiency of DNA transfer into the cells also depends on DNA/poly(L-Lys)Gal molar ratio in the complexes. Plasmid vector carrying human apolipoprotein A-I (apoA-I) gene was injected as its complex with poly(L-Lys)Gal into rat tail vein. Some level of ApoA-I was detected in the serum of the injected rats. Also, the human apoA-I-containing plasmid was found to be captured specifically by the rat liver cells and transported into the cell nuclei, where it can persist as an episome-like structure for at least a week. After repeated injections of DNA--poly(L-Lys)Gal complexes, the level of human ApoA-I in rat serum increases, probably, due to accumulation of functional human apoA-I gene in the liver cell nuclei. The data seem to be useful for the development of non-viral approaches to gene therapy of cardiovascular diseases.

DNA-protein interactions between mammalian nuclear proteins and a GCC-element included in a composite cis-acting element of mouse ribosomal protein L32 promoter.

DNA-protein complex formation between the sequence GC(GCC)4 (GCC-element) of mouse ribosomal protein L32 (rpL32) promoter and nuclear proteins of mouse and human cells has been studied using gel retardation and South-Western blotting methods. The rpL32 promoter fragment (-24.+11) was able to form specific complexes with mouse and human nuclear proteins mainly due to the presence of the GCC-element (-19.-6). DNA-protein complex patterns exhibited marked tissue-specificity. Three nuclear polypeptides of approximately 18, 28, and 50 kD that bind to the rpL32 promoter region (-24.+11) have been detected in HeLa cells by ligand blotting. At least one of them (18 kD) interacted with the GCC-element directly. The same fragment of the promoter interacted only with one nuclear polypeptide (28-31 kD) from human fibroblasts. DNA-protein complex formation between the investigated rpL32 promoter fragment containing the GCC-element and human fibroblast nuclear proteins is Zn2+-dependent. The method of functional titration (in vivo competition in the CAT-test) revealed that the GCC-element within the rpL32 promoter functions as a positive cis-acting transcriptional element in NIH 3T3 cells. Thus, our data characterize the sequence GC(GCC)4 as a functionally active cis-element included as a component in the more complex (composite) cis-element of mouse rpL32 promoter exhibiting tissue-specific properties. In various mammalian cell types the GCC-element can interact with various nuclear proteins, and the mode of these interactions can be determined by its relative position to other cis-elements in the regulatory sites of the genome.

[Study of the effect of the cDNA for the human apolipoprotein A-I gene in transgenic rabbits: modeling the neurological syndrome of human Tangier disease]. / Issledovanie na transgennykh krolikakh deistviia kDNK gena apolipoproteid A-I cheloveka: modelirovanie neirologicheskogo sindroma tenzhirskoi bolezni cheloveka.

Two transgenic rabbits which carried human apolipoprotein A-1 (apo A-1) cDNA under mouse ribosomal protein L/32 promoter were obtained. The effectiveness of transgenosis was confirmed by DNA dot/blot and Southern blot hybridizations. Both transgenic animals had paralyses of fore or fore and high limbs. Electron microscopy demonstrated distinct degradative changes of those parts of spinal cord which were responsible for leg skeletal muscle innervation. RNA dot/blot hybridization showed transgene expression in liver and brain but not in kidney of adult transgenic animal. However, analysis of blood serum lipids and immunochemical determinations gave no indications of the presence of human apo A-1 in adult transgenic rabbit. The data obtained allow us to suggest that the observed pathology was due to interference of native and foreign protein products of apo A-1 gene expression in CNS in the course of embryo development. This suggestion was supported by results of in situ hybridization of 5- and 9-week human embryo sections with apo A-1 cDNA, showing effective expression of apo A-1 gene in neural cells of CNS. Results of transgenosis may be viewed as modeling of the neurological syndrome of human Tangier disease.

[Isoelectric focusing of 3-hydroxy-3-methylglutaryl-CoA-reductase from the postmicrosomal fraction of rat liver]. / Izoélektrofokusirovanie 3-gidroksi-3-metilglutaril-CoA-redutazy postmikrosomnoi fraktsii pecheni krysy.

Using isoelectrofocusing, the existence of multiple forms of 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) in the postmitochondrial fraction of rat liver has been demonstrated for the first time. The isoelectric points for the enzyme isoforms are 6.25, 7.5 and 8.25.