The Antitumor Efficacy of a Complex Based on Two-Vector System for Coexpression of the Suicide Gene Fcu1 and Cre Recombinase.

In this study, we evaluated the antitumor activity of a gene therapy complex in which the tumor-specific control of the expression of the effector suicide gene FCU1 was performed using a two-vector system based on the site-specific Cre-LoxP recombinase system. The complex of interest showed a high therapeutic potential in a mouse colon adenocarcinoma model.

[The cause of cancer mutations: Improvable bad life or inevitable stochastic replication errors?]

Despite substantial progress in understanding the mechanisms of carcinogenesis and fighting oncology diseases, cancer mortality remains rather high. Therefore, there is a striving to reduce this mortality to the level determined by endogenous biological factors. The review analyzes the mutations that lead to cell malignant transformation and describes the contribution that self-renewal of adult tissues makes to tumorigenesis. Cancer progression is considered as a development of a complicated system where cells mutate, evolve, and are subject to selection. Cancer paradoxes are described in conclusion.

Expression of Therapeutic Gene FCU1 Sensitizes Pancreatic Cancer Cells to 5-Fluorocytosine and Enhances the Cytotoxic Effect of 5-Fluorouracil.

Hybrid therapeutic gene FCU1 gene was cloned into a lentiviral expression vector and the therapeutic effect of its expression was studied in three pancreatic cancer cell lines. Expression of FCU1 gene sensitized cells of two of three studied pancreatic cancer cell lines to 5-fluorocytosine. In addition, uracil phosphoribosyl transferase activity of the hybrid FCU1 protein increased sensitivity of transfected cells of all three studied pancreatic cancer cell lines to 5-fluorouracil, a standard chemotherapeutic agent.

Antitumor efficacy of combined gene and radiotherapy in animals.

Antitumor efficacy of the combined suicide gene therapy and radiotherapy was studied on the model of CT26 murine colon adenocarcinoma. CMV-FCU1-IRES-mGM-CSF-pGL3 construct with PEG-PEI-TAT (FCU1-mGM/5-FC) block copolymer as a vector was used for intratumoral administration. Tumors were irradiated with a single 5 Gy dose. The efficacy was evaluated according to the grade of tumor growth inhibition (T/C) and lifespan of the animals. Pronounced antitumor activity of the combined use of FCU1-mGM/5-FC system with radiotherapy on the background of prolonged lifespan and the synergism of the applied methods was revealed.

Identification of some human genes oppositely regulated during esophageal squamous cell carcinoma formation and human embryonic esophagus development.

Here we directly compared gene expression profiles in human esophageal squamous cell carcinomas and in human fetal esophagus development. We used the suppression subtractive hybridization technique to subtract cDNAs prepared from tumor and normal human esophageal samples. cDNA sequencing and reverse transcription polymerase chain reaction (RT-PCR) analysis of RNAs from human tumor and the normal esophagus revealed 10 differentially transcribed genes: CSTA, CRNN, CEACAM1, MAL, EMP1, ECRG2, and SPRR downregulated, and PLAUR, SFRP4, and secreted protein that is acidic and rich in cysteine upregulated in tumor tissue as compared with surrounding normal tissue. In turn, genes up- and downregulated in tumor tissue were down- and upregulated, respectively, during development from the fetal to adult esophagus. Thus, we demonstrated that, as reported for other tumors, gene transcriptional activation and/or suppression events in esophageal tumor progression were opposite to those observed during development from the fetal to adult esophagus. This tumor 'embryonization' supports the idea that stem or progenitor cells are implicated in esophageal cancer emergence.

Identification of recognition sites for myc/max/mxd network proteins by a whole human chromosome 19 selection strategy.

In this study, we have identified 20 human sequences containing Myc network binding sites in a library from the whole human chromosome 19. We demonstrated binding of the Max protein to these sequences both in vitro and in vivo. The majority of the identified sequences contained one or several CACGTG or CATGTG E-boxes. Several of these sites were located within introns or in their vicinity and the corresponding genes were found to be up- or down-regulated in differentiating HL-60 cells. Our data show the proof of principle for using this strategy in identification of Max target genes, and this method can also be applied for other transcription factors.

Enhanced Vector Design for Cancer Gene Therapy with Hierarchical Enhancement of Therapeutic Transgene Expression.

A set of vectors for Cre recombinase-dependent expression of the hybrid suicidal FCU1 transgene was constructed, including a two-plasmid system wherein the FCU1 and Cre transgenes reside in separate vectors, and single-plasmid variants in which a single plasmid bears both transgenes. To improve the safety profile and specificity in cancer gene therapy applications, as well as to ensure stable propagation of plasmids in bacterial cells, the Cre/LoxP system components were optimized. A bicistronic vector with the Cre expression cassette placed between the LoxP sites unidirectionally with FCU1 cDNA resulted in higher therapeutic efficiency compared with the double-plasmid system in an enzyme-prodrug suicide cancer gene therapy scheme. Therefore, the feasibility of a single-plasmid approach in the development of cancer gene therapy with hierarchical enhancement of therapeutic transgene expression has been demonstrated.

[Tissue specificity of alternative splicing products of mouse mRNA encoding new protein hampin homologous to the Drosophila MSL-1 protein].

A number of mammalian genomes have one gene copy encoding the protein that we named hampin. A search in a number of databases revealed a distant homologue, the well-known Drosophila protein MSL-1 (male-specific lethal 1). An alternative splicing of mRNA led to a significant diversity of structural hampin variants with different domain compositions. We analyzed the tissue-specific expression of five mouse hampin variants using RT-PCR. Two variants encoding hampin proteins with truncated N termini were shown to have a restricted tissue specificity: they are exclusively expressed in the testes. The mRNAs of other hampin variants were detected in all the tested tissues at comparable levels. We obtained polyclonal antibodies to the recombinant hampin and used them to demonstrate that at least one of the variants is predominantly localized in the nucleus. The specific features of the hampin primary structure and its possible functions as a member of the hampin/MSL-1 family of proteins are discussed.

Cloning and characterization of the entire cDNA encoded by ATP1AL1--a member of the human Na,K/H,K-ATPase gene family.

The cDNA for ATP1AL1--the fifth member of the human Na,K-/H,K-ATPase gene family--was cloned and sequenced. The deduced primary ATP1AL1 translation product is 1,039 amino acids in length and has Mr of 114,543. The encoded protein has all of the structural features common to known catalytic subunits of P-type membrane ion-transporting ATPases and is equally distant (63-64% of identity) from the Na,K-ATPase isoforms and the gastric H,K-ATPase. The ATP1AL1 encoded protein was proposed to represent a new separate group within the family of human potassium-dependent ion pumps.

Family of human Na+, K+-ATPase genes. Structure of the gene for the catalytic subunit (alpha III-form) and its relationship with structural features of the protein.

The primary structure of a gene of the Na+, K+-ATPase multigenic family in the human genome has been determined. The gene corresponds to a hypothetical alpha III-form of the enzyme catalytic subunit. The gene comprises over 25,000 bp, and its protein coding region includes 23 exons and 22 introns. Possible correlation between structural features of the protein and location of introns in the gene are discussed.

The family of human Na,K-ATPase genes. ATP1AL1 gene is transcriptionally competent and probably encodes the related ion transport ATPase.

The multigene family of human Na,K-ATPase is composed of 5 alpha-subunit genes, 3 of which were shown to encode the functionally active alpha 1, alpha 2 and alpha 3 isoforms of the catalytic subunits. This report describes the isolation, mapping and partial sequencing of the fourth gene (ATP1AL1) that was demonstrated here to be functionally active and expressed in human brain and kidney. Limited DNA sequencing of the ATP1AL1 exons allowed one to suggest that the gene probably encodes a new ion transport ATPase rather than an isoform of the Na,K-ATPase or the closely related H,K-ATPase.

Ouabain-sensitive H,K-ATPase: tissue-specific expression of the mammalian genes encoding the catalytic alpha subunit.

Human ATP1AL1 and corresponding genes of other mammals encode the catalytic alpha subunit of a non-gastric ouabain-sensitive H,K-ATPases, the ion pump presumably involved in maintenance of potassium homeostasis. The tissue specificity of the expression of these genes in different species has not been analyzed in detail. Here we report comparative RT-PCR screening of mouse, rat, rabbit, human, and dog tissues. Significant expression levels were observed in the skin, kidney and distal colon of all species (with the exception of the human colon). Analysis of rat urogenital organs also revealed strong expression in coagulating and preputial glands. Relatively lower expression levels were detected in many other tissues including brain, placenta and lung. In rabbit brain the expression was found to be specific to choroid plexus and cortex. Prominent similarity of tissue-specific expression patterns indicates that animal and human non-gastric H,K-ATPases are indeed products of homologous genes. This is also consistent with the high sequence similarity of non-gastric H,K-ATPases (including partial sequences of hitherto unknown cDNAs for mouse and dog proteins).

[Study of expression of Na+,K+-ATPase subunit genes in the human brain using cDNA libraries]. / Izuchenie ékspressii genov alpha-sub''edinitsy Na+,K+-ATP-azy v mozge cheloveka metodom analiza kDNK-bankov.

Human brain cDNA libraries were screened with cDNA inserts corresponding to the mRNA for the Na+,K(+)-ATPase alpha-subunit from pig kidney. The results obtained demonstrate the existence of two highly homologous mRNAs encoding the alpha- and alpha III-isoforms of the Na+,K(+)-ATPase catalytic subunit.

[Phage mimotopes of monoclonal antibodies against plasma membrane Ca2+-ATPase]. / Fagovye mimotopy monoklonal'nykh antitel k Ca2+-ATPaze plazmaticheskikh membran.

Libraries of random phage-displayed pentadeca- and hexapeptides were screened with the use of four monoclonal antibodies against the human plasma membrane Ca2(+)-ATPase. Bacteriophages specifically binding the antibodies were selected, and the amino acid sequences of the expressed peptides (mimotopes) were determined. Mimotopes for three antibodies (8B8, 2D8, F9) did not correspond to the Ca2(+)-ATPase sequence. Pentadecapeptides for the 7C8 antibodies displayed similarity to the fragment Glu1097-Arg1113 of the Ca2(+)-ATPase calmodulin-binding site. However, these antibodies failed to bind recombinant fragment Leu1069-Leu1220; therefore, the structure of this epitope remains obscure. This work opens a series of studies of the plasma membrane Ca2(+)-ATPase structure by means of monoclonal antibodies and the phage display method.

[Study of the transmembrane organization of the C-terminal domain of the alpha subunit of Na+,K+-ATPase using polyclonal antibodies to its perimembrane fragments]. / Izuchenie transmembrannoi organizatsii C-kontsevogo domena alpha- edinitsy Na+,K+-ATP-azy s pomoshch'iu poliklonal'nykh antitel k ee primembrannym fragmentam.

The fragments 825-842, 868-886, 928-945, 946-967, 962-978 of the alpha-subunit of the porcine Na+,K(+)-ATPase were expressed in E. coli as fusion proteins with human tumour necrosis factor. Polyclonal antibodies were obtained to these fragments. The location of the chosen fragments according to the plasma membrane was determined with the polyclonal antibodies by ELISA on intact, lysed or solubilized pig kidney embryo cells. The fragments 825-842, 928-945, and 962-978 contained intracellular epitopes and did not contain extracellular ones; the fragments 868-886 and 946-967 did not contain any exposed epitopes.

[Epitope mapping of monoclonal antibodies to human plasma membrane Ca2-ATPase]. / Kartirovanie épitopov monoklonal'nykh antitel k Ca2-ATPase plazmati cheskikh membran cheloveka.

Overlapping fragments of the fourth isoform of human plasma membrane Ca(2+)-ATPase (hPMCA4) and several fragments of hPMCA1 were expressed in bacterial cells and purified by metal affinity chromatography. Enzyme immunoassays of the fragments helped map epitopes for 4 monoclonal antibodies (2D8, 8B8, 7C8 and 5E6). The epitope for 2D8 was localized within the 222-249 site (i.e., in the putative transduction domain), the epitopes for 8B8 and 7C8 were localized within the 330-353 site, in which phospholipids are presumably bound, and the 5E6 epitope was found within the 791-843 site, where the putative hinge region is situated. 2D8 recognizes hPMCA1 and hPMCA4 isoforms, while 8B8 and 7C8 are specific for hPMCA4. The amino acid sequences of these epitopes and phage-displayed mimotopes were compared.

[Primary structure of rhodopsin. I. Cyanogen bromide degradation peptides]. / Pervichnaia struktura rodopsina. I. Pepidy bromtsianovogo rasshchepleniia.

Carboxymethylated bovine rhodopsin was subjected to cyanogen bromide cleavage at methionine residues. The resultant products were fractionated into the two groups according to the solubility of peptides in 2 M guanidine hydrochloride. Gel-filtration on Bio-Gel P-30 in 80% formic acid of each group followed by rechromatography and high performance liquid chromatography resulted in 15 peptides embracing the whole polypeptide chain of rhodopsin. Amino acid sequence of these peptides was determined.

[Primary structure of rhodopsin. II. Chymotrypsin hydrolysis peptides]. / Pervichnaia struktura rodopsina. II. Peptidy khimotripticheskogo gidroliza.

Apomembranes prepared from the photoreceptor disks were subjected to chymotryptic hydrolysis. The insoluble material, containing the membrane-bound peptides was removed by centrifugation, and the water-soluble peptides of the supernatant were separated by ion-exchange chromatography on AG 50W X 4 followed by high performance liquid chromatography. The insoluble peptides were separated by gel-filtration on Bio-Gel P-30 in 80% formic acid. Amino acid sequence of peptides containing in total 296 amino acid residues and comprising 85% of the rhodopsin polypeptide chain was determined.

[Study of the molecular organization of visual rhodopsin in photoreceptor membranes by limited proteolysis]. / Issledovanie molekuliarnoi organizatsii zritel'nogo rodopsina v fotoretseptornykh membranakh metodom ogranichennogo proteinoliza.

Proteolysis of rhodopsin in disc membranes of right-side out orientation by thermolysin, papain and St. aureus V8 protease allowed to identify two highly exposed regions of polypeptide chain located on the cytoplasmic membrane surface: carboxyl terminal sequence 321-348 and the fragment 236-241. Incubation with chymotrypsin reveals the third site on the cytoplasmic surface, 146-147, accessible to proteolytic enzymes. Frozen-thawed membranes comprise a mixture of vesicles with normal and inverted orientation. Both thermolytic and chymotryptic digests of rhodopsin in these membranes contain the polypeptide which represents the amino terminal sequence lacking the first 30 amino acid residues. Thus at least 30 amino acids from the N-terminus must protrude into the intradiscal space. One additional site was located on the intradiscal surface: papain digests rhodopsin in the inverted membranes at the position 186-187. Localization of the proteolytic cleavage sites allowed to propose a model for rhodopsin topography in disc membrane: the polypeptide chain traverses the bilayer thickness seven times; each of seven transmembrane segments containing approximately 40 amino acid residues includes a sequence of approximately 30 hydrophobic amino acids; which are probably in close contact with hydrocarbon matrix of the membrane. Hydrophobic sequences are terminated with fragments containing clusters of hydrophilic amino acids, possibly interacting with lipid polar head groups and orienting each segment in the bilayer.