[Li-Fraumeni syndrome in adult patients with acute lymphoblastic leukemia].

BACKGROUND: LiFraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary disorder that is characterized by an increased risk for certain types of cancer, acute lymphoblastic leukemia (ALL), particularly. Germline TP53 mutations are associated with LFS. Genetic counseling and follow-up is essential for patients with LFS and their relatives. Special therapeutic approaches are needed for treatment of oncological disease in these patients. The article presents a series of clinical cases of patients with ALL and SLF, considers general issues of diagnosis and treatment of adult patients with this hereditary genetic syndrome. AIM: Describe clinical observations of patients with acute lymphoblastic leukemia (ALL) and LFS and consider general issues of diagnosis and treatment of adult patients with LFS and ALL. MATERIALS AND METHODS: TP53 gene mutations were screened using Sanger sequencing in 180 de novo patients with Ph-negative (B- and T-cell) and Ph-positive ALL treated by Russian multicenter protocols (ALL-2009, ALL-2012, ALL-2016) at the National Research Center for Hematology, Moscow, Russia, and at the hematology departments of regional clinics of Russia (multicenter study participants). RESULTS: TP53 gene mutations were found in 7.8% (n=14) of de novo ALL patients. In patients, whose biological material was available TP53 gene mutational status was determined in non-tumor cells (bone marrow and peripheral blood during remission, bone marrow samples after allogeneic hematopoietic stem cells transplantation and in tissue of non-hematopoietic origin) for discriminating germline mutations. The analysis included 5 patients (out of 14 with TP53 mutations), whose non-tumor biological material was available for research. Germline status was confirmed in 4 out of 5 B-cell ALL (n=3), T-cell ALL (n=1) investigated patients. CONCLUSION: Practical value of the research is the observation that the greater part of TP53 gene mutations in patients with Ph-negative B-cell ALL are germinal and associated with LFS.

[Detection of activating mutations in RAS/RAF/MEK/ERK and JAK/STAT signaling pathways].

ISSUE: The study of activating mutations (NRAS,KRAS,FLT3,JAK2,CRLF2genes) of RAS/RAF/MEK/ERK and JAK/STAT signaling pathways in B-cell acute lymphoblastic leukemia (B-ALL) in adult patients which are included in Russian multicenter clinical trials. MATERIALS AND METHODS: Within the multicenter study there were 119 adult patients included withde novoB-ALL. The study was considered as prospective and retrospective. The group withBCR-ABL1-negative B-ALL consisted of up to 93 patients (45 male and 48 female, at the age of 17 to 59, the median age 31), they were treated according to the protocols ALL-2009, ALL-2016. The median follow-up lasted for 19 months (1119). The group withBCR-ABL1-positive B-ALL with up to 26 patients (10 male and 16 female, at the age of 23 to 78, the median age 34 years) was included in the study as well. The treatment was carried out according to the protocols ALL-2009 and ALL-2012 in combination with tyrosine kinase inhibitors. The median follow-up lasted for 23 months (4120). The molecular analysis of activating mutations inNRAS,KRASgenes (RAS/RAF/MEK/ERK signaling pathway) andJAK2,CRLF2genes (JAK/STAT signaling cascade) was performed via Sanger sequencing. The internal tandem duplications (ITDs) inFLT3gene were studied by fragment analysis. The evaluation of CRLF2 expression was fulfilled via flow cytometry. RESULTS: Activating mutations inNRAS,KRAS,FLT3genes were found in 22 (23.6%) patients withBCR-ABL1-negative B-ALL. In total, 23 mutations were revealed in theNRAS(n=9),KRAS(n=12), andFLT3(n=2) genes, according to statistics that was significantly more frequent than withBCR-ABL1-positive B-ALL, these genes mutations were not identified in patients (p=0.007). The frequency of mutations detection inKRASandNRASgenes in patients withBCR-ABL1-negative B-ALL was comparable as 12.9% (12 of 93) to 9.7% (9 of 93), respectively (p=0.488). One patient was simultaneously revealed 2 mutations in theKRASgene (in codons 13 and 61).FLT3-ITD mutations were detected in 3.5% (2 of 57) cases ofBCR-ABL1-negative B-ALL. In patients withBCR-ABL1-positive B-ALLFLT3-ITD mutations were not assessed. Violations in the JAK/STAT signaling cascade were detected in 4 (4.3%) patients withBCR-ABL1-negative B-ALL. They were represented by the missense mutations ofJAK2gene (n=3) and the overexpression of CRLF2 (n=2); in one patient were detected the overexpression of CRLF2 and a mutation inJAK2gene simultaneously. No mutations were found inCRLF2gene. In patients withBCR-ABL1-positive B-ALL noJAK2mutations were detected. As long as analyzing demographic and clinical laboratory parameters between groups of patients with and without mutations, there were no statistically significant differences obtained. In the analyzed groups of patients, long-term therapy results did not differentiate according to the mutations presence inNRAS,KRAS,FLT3,JAK2genes. Also, substantive differences were not shown in the rate of the negative status achievement of the minimum residual disease between patients with and without activating mutations in the control points of the protocol (on the 70th, 133rd and 190th days). CONCLUSION: NRAS,KRAS,FLT3,JAK2activating mutations do not affect the long-term results of the therapy and the rate of the negative status achievement of the minimum residual disease in patients withBCR-ABL1-negative B-ALL treated by the Russian multicenter clinical trials.

Clonal Composition of Human Multipotent Mesenchymal Stromal Cells: Application of Genetic Barcodes in Research.

Clonal composition of human multipotent mesenchymal stromal cells (MMSCs) labeled with lentiviral vectors carrying genetic barcodes was studied. MMSCs were transduced with a cloned library of self-inactivating lentiviral vectors carrying 667 unique barcodes. At each cell culture passage, 120 cells were plated one cell per well in 96-well plates. The efficiency of cloning and labeling of the clonogenic cells was determined. DNA was extracted from the cell-derived colonies, and the barcodes were identified by Sanger sequencing. Also, DNA was extracted from the total MMSC population at each passage to analyze the diversity and representation of barcodes by deep sequencing using the Illumina platform. It was shown that the portion of MMSCs labeled with the lentiviral vectors remained stable in the passaged cells. Because of the high multiplicity of infection, the labeling procedure could decrease the proliferative potential of MMSCs. Identification of barcodes in individual cell clones confirmed the polyclonal character of the MMSC population. Clonal composition of MMSCs changed significantly with the passages due to the depletion of proliferative potential of most cells. Large clones were found at the first passage; at later passages, many small clones with a limited proliferative potential were detected in the population. The results of deep sequencing confirmed changes in the clonal composition of MMSCs. The polyclonal MMSC population contained only a small number of cells with a high proliferative potential, some of which could be stem cells. MMSCs with a high proliferative potential were detected more often in the earliest passages. In this regard, we would recommend to use MMSCs of early passages for regenerative medicine applications based on cell proliferation.

Coinheritance of HbD-Punjab/ß+-thalassemia (IVSI+5 G-C) in patient with Gilbert's syndrome.

Thalassemia and qualitative hemoglobinopathy are hereditary disorders of Hb synthesis that lead to change in the Hb conformation or a decrease in the synthesis of structurally normal Hb, and consequently, to erythron pathology. Many variants of Hb are unstable or have altered affinity for oxygen, and, in heterozygous form can be associated with clinical and hematological manifestations (hemolytic anemia, hypochromic microcytic anemia, erythrocytosis). HbD-Punjab [ß121 (GH4) Glu → Gln; HBB: C.364G> C] is variant of Hb carrying the amino acid substitution in the 121 position of ß-globin chain. In all cases reported so far, patients with HbD-Punjab/ß+-thalassemia (IVSI+5 G-C) combination experienced typical thalassemia with hypochromic microcytosis. HbD-Punjab was detected by electrophoresis from 37 to 94% of total Hb. The article describes rare clinical case of the cohabitation of HbD-Punjab/ß+-thalassemia (IVSI+5 G-C) in a patient with homozygous variant of Gilbert's syndrome observed in AS Loginov Moscow Clinical Scientific Center.

Literature review and clinical observation of acquired idiopathic hemophilia with a new missense mutation in the factor VIII gene (His2026Arg).

The article provides review of possible mechanisms of inhibitor coagulopathies, in particular of acquired hemophilia A. This pathology is an extremely rare disease occurring in 1-2 cases in 1 million per year. In the present study we provide data for two clinical cases of hemophilia A in women. These cases had different development mechanisms, although both women have a newly discovered missense mutation His2026Arg in the VIII factor gene. The matter of main interest is the description of the disease development in the patient with an acquired idiopathic hemophilia A with a possible disease occurrence due to an asymmetric X-chromosome inactivation (lyonization). In this particular case lyonization led to the late manifestation of the hemophilia A carrier's state and development of severe form of the inhibitor-associated acquired hemophilia A. We also discuss therapeutic approaches to these forms of the disease, considering there are no concise protocols for case management due to an extreme rarity of the pathology. Acquainting the clinical personnel working it the different areas of medicine with suchlike inhibitor coagulopathies has a major practical importance.

Investigation of the Mesenchymal Stem Cell Compartment by Means of a Lentiviral Barcode Library.

The hematopoietic bone marrow microenvironment is formed by proliferation and differentiation of mesenchymal stem cells (MSCs). The MSC compartment has been less studied than the hematopoietic stem cell compartment. To characterize the structure of the MSC compartment, it is necessary to trace the fate of distinct mesenchymal cells. To do so, mesenchymal progenitors need to be marked at the single-cell level. A method for individual marking of normal and cancer stem cells based on genetic "barcodes" has been developed for the last 10 years. Such approach has not yet been applied to MSCs. The aim of this study was to evaluate the possibility of using such barcoding strategy to mark MSCs and their descendants, colony-forming units of fibroblasts (CFU-Fs). Adherent cell layers (ACLs) of murine long-term bone marrow cultures (LTBMCs) were transduced with a lentiviral library with barcodes consisting of 32 + 3 degenerate nucleotides. Infected ACLs were suspended, and CFU-F derived clones were obtained. DNA was isolated from each individual colony, and barcodes were analyzed in marked CFU-F-derived colonies by means of conventional polymerase chain reaction and Sanger sequencing. Barcodes were identified in 154 marked colonies. All barcodes appeared to be unique: there were no two distinct colonies bearing the same barcode. It was shown that ACLs included CFU-Fs with different proliferative potential. MSCs are located higher in the hierarchy of mesenchymal progenitors than CFU-Fs, so the presented data indicate that MSCs proliferate rarely in LTBMCs. A method of stable individual marking and comparing the markers in mesenchymal progenitor cells has been developed in this work. We show for the first time that a barcoded library of lentiviruses is an effective tool for studying stromal progenitor cells.

[Mutational Analysis of Hemophilia B in Russia: Molecular-Genetic Study].

Hemophilia B is a hereditary X-linked coagulation disorder. This pathology is caused by various defects in the factor IX gene, which is, being about 34 kb long and consisting of eight exons, localized in the Xq27 locus of the. X-chromosome long arm. Mutations were revealed in 56 unrelated patients with hemophilia B in this study by using direct sequencing of factor IX gene functionally important fragments. Forty-six mutations were found with prevailing missense mutations (n = 30). The rest of the mutations were nonsense (n = 4) and splicing (n = 4) mutations, large deletions (n = 3), microdeletions (n = 2), microinsertions (n = 2), and promoter mutations (n = 1). Eleven of 46 mutations were previously unknown for human populations.

[Molecular serological characteristics of weak D antigen types of the Rhesus system]. / Molekulyarno-serologicheskie kharakteristiki tipov slabogo antigena D sistemy rezus.

AIM: to estimate the spread of weak D antigen types of the Rhesus system in the citizens of the Russian Federation and a possibility of serologically identifying these types. SUBJECTS AND METHODS: The red blood cells and DNA of people with weakened expression of D antigen were investigated using erythrocyte agglutination reaction in salt medium (2 methods); agglutination reaction in the gel columns containing IgM + IgG anti-D antibodies, indirect antiglobulin test with IgG anti-D antibodies (2 methods); polymerase chain reaction to establish the type of weak D. RESULTS: A rhesus phenotype was determined in 5100 people in 2014-2015. The weakened agglutinable properties of red blood cells were detected in 102 (2%) examinees. 63 examinees underwent genotyping to identify the variants of the weak D antigen, which identified 6 weak D types. There were the most common weak D types 3 (n=31 (49.2%)) and weak D type 1 (n=18 (28.6%)), including weak D type 1.1 in one (1.6%) case. The other 4 weak D antigen types were as follows: weak D type 2 (14.3% (n=9)), weak D type 15 (4.8% (n=3)), weak D type 4.2 (DAR) (1.6% (n=1)) and weak D type 6 (1.6% (n=1)). The antiglobulin test in the gel column containing antiglobulin serum was the most sensitive serological assay to identify the weak D antigen. Only a molecular test could establish weak D type 15 in 2 samples of red blood cells with Ccdee and ccdEe phenotypes. CONCLUSION: The weak D antigen could be serologically identified in 96.8% of cases. When testing for weak D, particular attention should be given to people with the D-negative phenotype who had the C or E antigens. Our investigations conducted for the first time in Russia will be able to improve the immunological safety of red blood cell-containing medium transfusions for patients.

[Hereditary afibrinogenemia: A literature review and clinical observations]. / Nasledstvennaia afibrinogenemiia: obzor literatury i klinicheskie nabliudeniia.

Afibrinogenemia is a rare congenital coagulopathy that leads to life-threatening bleeding. In afibrinogenemia, plasma fibrinogen levels are less than 0.1 g/L. The clinical manifestations of the disease can be both bleeding and thromboses of different localizations, which is determined by the multifunctional role of fibrinogen in hemostasis. The described cases demonstrate different clinical phenotypes of the disease. In both cases the diagnosis was confirmed by genetic examinations that revealed homozygous mutations in the fibrinogen A genes. The nature of the mutations assumes consanguineous marriages, as confirmed by the results of a genealogical analysis. Fibrinogen preparations are promising in treating afibrinogenemia in Russia.

[Molecular genetic study of acute intermittent porphyria in Russia: mutation analysis and functional polymorphism search in porphobilinogen deaminase gene].

Acute intermittent porphyria (AIP) is an autosomal dominant hereditary disease, caused by partial deficiency of porphobilinogen deaminase (PBGD), one of the key enzymes ofheme biosynthesis. This study describes molecular genetics of AIP in Russia. Mutation analysis of PBGD gene in 70 unrelated patients revealed 47 various genetic defects, 28 of which had not been described previously. Mutations 53delT and Argl 73 Trp (recorded 8 times, in total 23%) proved to be the most common in Russia. Microdeletion 53delThas monophyletic origin and was found only in Russia. Molecular genetic examination of 132 relatives of AIP patients from 40 families revealed 52 latent carriers of the disease. Low (about 10%) AIP penetrance indicates that a mutation in the PBGD gene is an important but not sufficient prerequisite for clinical manifestation of the disease. Modulation of penetrance in erythropoietic protoporphyria by coinheritance of a mutant allele and a functionally defective wild type allele of ferrochetalase gene has been shown previously. We hypothesized that similar mechanism works in AIP. Sequencing of the full length PBGD genes from unrelated AIP patients as well as SN P analysis, and the analysis of abnormal PBGD mRNA splicing showed that in case ofAIP, this hypothesis is not true and some other factors are responsible for the penetrance of this disease.

[Maintaining of the morphological specificity and genetic introgression in populations of the great tit Parus major and the Japanese tit P. minor in the middle Amur region].

The ranges of the great tit Parus major and the Japanese tit P. minor overlap in the middle Amur region, where hybridization of these two species occur. These species have contacted for nearly a century on the western slope of the Malyi Khingan Ridge (the central part of the sympatry zone), but the great tit has colonized territories to the east of the ridge only in the last two decades. The percentage of the P. minor's allele of intron 2 of the mioglobin gene has significantly increased from 8.9% in the west to 27.8% in the east in phenotypically major's populations. Thus, the percentage of foreign mtDNA in P. major populations did not change significantly from west (6.2%, n = 120) to east (3.2%, n = 61). Simultaneous use of two genetic markers (one nuclear and the other mitochondrial) supports our conclusion on strong introgression in the populations of both species, which nevertheless maintain their morphological specificity in the contact zone.

[Analysis of the AluI polymorphism in intron 1 of the human coagulation factor VIII gene: a new marker for the hemophilia A carrier detection].

Frequencies of the CIT SNP alleles at position 2403 of the human coagulation factor VIII gene intron 1, containing the AluI restriction endonuclease recognition site, were examined. Genomic DNA samples for the analysis were obtained from the consulted women and their relatives from the families with hemophilia A. A total of 221 unrelated X chromosomes were studied. The two allelic variants were found with similar frequencies of T(Alu+), 0.53 and C(Alu-), 0.47. The heterozygosity index evaluated as equal to 0.50 was correlated with the experimental heterozygote number. The absence of a tight linkage between the AluI SNP and the widely used in the hemophilia A gene diagnostics HindIII polymorphism (CIT SNP at position 103 of intron 19) was demonstrated. Summarized informativity of these two markers for obligate carriers and for those detected in this study constituted 68% (32 out of 47). At the same time using one of the markers, only 40% (HindIII) and 51% (AluI) of the consulted women were informative. The new marker was used in 13 prenatal DNA diagnostics of hemophilia A. A new deletion polymorphism (del TGA, position 2281-2283 of intron 1) was described in close proximity of the AluI SNP with the frequency of about 0.05. among the five other SNP of the factor VIII gene examined (Bme 18I, intron 1; HpaII, intron 13; MnlI, exon 14; Bst4CI, exon 25; and MseI, exon 26) no effective diagnostic markers were found. Only the MnlI polymorphism could be recommended for limited usage.

[New efficient extragenic microsatellite markers for hemophilia A carrier state diagnostics].

In search of new efficient markers for genetic diagnostics of hemophilia A, two tri-nucleotide microsatellite repeats (STR) at chromosome X loci, which flank coagulation factor VIII gene (F8), namely STR HA472--CTT-repeat, which is localized adjacent to the GAB3 gene 163 bp apart from the 3' end of the F8 gene and STR HA544--repeat (CTT)x(ATT)y located at a distance of 375 bp from the 5' end of the F8 gene were discovered. Detailed analysis using PCR and sequencing has shown that STR HA472 contains two long variable CTT-blocks separated by small spacer CCTCCC. The location of recognition site of restriction endonuclease Mnl1 (CCTC) in the spacer permits to test differentially the polymorphic blocks and thus to increase the analysis informativity. STR HA544 is also represented by two polymorphic blocks (CTT and ATT), for separate amplification of which highly informative PCR amplification assays were elaborated. The study has been done using DNA samples of 212 individuals (125 women) from 48 families with hemophilia A carriers. Our results point to Mendelian inheritance of the markers studied, a high number of allelic variants and high heterozygosity, which was 90% and 100% for HA544 and HA472, respectively. This permitted us to use these data for practical gene diagnostics of the carriers and prenatal diagnostics of hemophilia A. In addition to high informativity STR HA472 and HA544 are highly important for diagnostics as they are located at a shorter distance than other known extragenic polymorphisms of the F8 gene. In contrast to dinucleotide repeats, trinucleotide repeats are readily tested, not requiring high-resolution electrophoretic systems. In addition, they are located on the opposite sites of the F8 gene. This permits to control homologous recombination events in the locus and thus to prevent diagnostic mistakes.

[Acute porphyrias: problem of primary diagnosis in Russia and CIS countries].

AIM: To analyse manifestations and experience in primary screening diagnosis of acute porphyrias which are rarely encountered and little known by general practitioners. MATERIAL AND METHODS: The data on 100 patients with the diagnosis acute porphyria have been analysed. Porphyrin metabolism in differential diagnosis was estimated according to standard techniques. RESULTS: Analysis of primary diagnosis of acute porphyria hepatica in Russia (region-related prevalence, duration of diagnosis, complications because of late pathogenetic treatment) demonstrates the importance of screening diagnosis of acute porphyria at the level of municipal clinics. CONCLUSION: Early diagnosis prevents severe complications of acute porphyria and reduces cost of examinations in search of accurate diagnosis.

[A search for Y-chromosomal species-specific markers and their use for hybridization analysis in ground squirrels].

In four ground squirrel species from the Volga region-yellow (Spermophilus fulvus), russet (S. major), little (S. pygmaeus), and speckled (S. suslicus)--four hybridization variants (major/fulvus, major/pygmaeus, major/suslicus, and pygmaeus/suslicus) have been reliably described. Earlier we have shown that populations of S. major from the Volga region were characterized by wide introgression of mtDNA from S. fulvus and S. pygmaeus, which probably, resulted from ancient hybridization. In this study, the same populations were used to analyze the introgression of the Y chromosome, which (unlike mtDNA) is paternally inherited. Three genes, ZfY, SRY, and SmcY were tested as Y-chromosomal candidate markers. It was demonstrated that Y chromosome of ground squirrels lacked the ZfY gene, while its homologous structure, ZfY(X), was presumably linked to the X chromosome. The SRY region examined was rather conservative. In particular, the sequences determined in S. major and S. fulvus were identical, while three out of four substitutions found in S. pygmaeus were located in the coding region. The SmcY gene was found to be the most suitable marker, providing distinguishing of all of the four ground squirrel species by nine nucleotide substitutions. Introgression at the Y chromosome was observed only in two cases: in one S. major individual (out of 51 phenotypically pure animals) caught in the major/fulvus sympatry zone, and in four (one litter) out of fourteen S. fulvus individuals caught in close vicinity of the sympatry zone of these two species. Among 28 S. pymaeus and 9 S. suslicus individuals, no foreign SmcY genes were detected. Two colonies of the "hybrid accumulation" type were examined with eight major/suslicus hybrids analyzed in the first and seventeen major/fulvus hybrids in the second colony. The prevalence of the S. major paternal lineages was observed in both colonies (87.5 and 82.4%, respectively). The data obtained suggest that compared to wide mtDNA introgression, introgression of Y chromosome in the Volga region ground squirrels is statistically significantly less frequent event.

Gene therapy model for stromal precursor cells of hematopoietic microenvironment.

Marker bacterial Neor gene was transduced by retroviral gene transfer into stromal precursor cells making up the hematopoietic microenvironment in murine long-term bone marrow cultures (LTBMC). Cultures were infected six times during the first 3 weeks of cultivation. At 4 weeks, the adherent cell layers (ACLs) were implanted under the renal capsule of syngeneic unirradiated and irradiated mice. Cells from newly formed ectopic foci were explanted into secondary LTBMC. ACLs containing the marker gene were detected by polymerase chain reaction. About 74% of stromal cells in ACLs contained Neor gene. The possibility of stable gene transduction into stromal precursor cells competent to transfer the hematopoietic microenvironment was established.

[Nucleotide sequence of the E1B area and the adjacent 3'-terminal segment of the E1A oncogene from monkey adenovirus SA7 (C8)]. / Nukleotidnaia posledovatel'nost' oblasti E1B i primykaiushchego k nei 3'-kontsevogo uchastka oblasti E1A onkogena adenoviursa obez'ian SA7 (C8).

The SA7 (C8) simian adenovirus was sequenced from the 1478th to 3194th nucleotide. The region includes the 3'-terminal part of E1A and the major part of the E1B coding region. The sequence obtained was compared with the structure of SA7 (P) DNA previously determined in the region 1-2338, and many differences were found which are nucleotide substitutions, microdeletions and microinsertions. Among point substitutions the most frequent was the C-->T transition in CG pairs known as hot spots of mutations. Differences of our sequence from the previously published one was also revealed.

[Structure of integrated oncogens E1A and E1B in a malignant line of rat SH2 fibroblasts, transformed by monkey adenovirus SA7 (C8) DNA]. / Struktura integrirovannykh onkogenov D1A i E1B v zlokachestvennoi linii fibroblastov krysy SH2, transformirovannykh DNK adenovirusa obez'ian SA7 (C8).

In this investigation the primary structure of E1A and E1B regions of SA7 (C8) simian adenovirus integrated in malignant SH2 rat cell line was studied. Southern blotting revealed at least two copies of the SA7 oncogene integrated in the SH2 genome. PCR analysis of E1A and E1B regions showed heteroduplex structures, proving the different structure of the integrated copies. The heteroduplex molecules with different electrophoretic mobility were separated, and chains corresponding to different copies were sequenced according to the modified Sanger method. We found that two copies differ mainly in microsatellite regions, in E1A between positions 894-902 (GCG)3/(GCG)4, in E1B between positions 2037-2048 (GCA)3/(GCA)4. It is necessary to stress that all deviations found belong to the coding regions of the SA7 oncogene.

[Penetration of oligo/polynucleotides and their polyalkylating derivatives into rat cells transformed by simian adenovirus DNA]. / Proniknovenie oligo/polinukleotidov i ikh poliakriliruiushchikh proizvodnykh v kletki krysy, trnasformirovannye DNK adenovirusa obez'ian.

The transport regularity of the [32P]-oligo/polynucleotides and their polyalkylating derivatives into SH2 rat cells transformed with SA7 adenovirus DNA was investigated. Derivatives penetrate the SH2 cells and their distribution in the subcellular fractions are proportional to the concentration of reagent in the medium. The transport efficiency of the derivatives is inhibited sharply with cell concentration increase and practically does not depend on the action of cell metabolism inhibitors. The data obtained assumes the mechanism of the derivatives transport to be liquid endocytosis. Being distributed in the cell components the polyalkylating derivatives were accumulated by the cell nuclei up to 10(5)-10(7) molecules per nucleus. Transport efficiency is much greater in the anchored cells than in the suspended ones. Though essential dephosphorylation of the utilized substances is observed in the SH2 cells, part of them maintain native chain length and the 5'-phosphate group after 1 h incubation in nucleic acids obtained from the cell nuclei.

[Oncogene-directed mutagenesis in vivo. Polyalkylating derivatives of short single-stranded polynucleotides, complementary E1-adeno-oncogene, in the normalization of adenovirus-transformed rodent cell lines]. / Onkogen-naprovlennyi mutagenez in vivo. Polialkiliruiushchie proizvodnye korotkikh odnotiazhevykh polinukleotidov, komplementarnykh E1- adenoonkogenu, v normalizatsii transformirovannykh adenovirusom kloetochnykh linii gryzunov.

Polyalkylating derivatives of single-stranded polynucleotides (30-200-mers) complementary to the long E1 oncogene sequences of simian adenovirus SA7 cause inherited normalization of SH2 and G11 cells transformed with adenovirus SA7; certain deletions in the integrated proviral E1A oncogene were observed in several cases during this process. The transformed cells are indifferent to reagents noncomplementary to the E1 region. Thus polyalkylating derivatives of single-stranded 30-200-mers act as addressed mutagenes which react in a specific way with the integrated complementary DNA sequences of E1 oncogene in transformed rodent cells and realize oncogene-directed mutagenesis in vivo. During this treatment temporary normalized cells reverting to the initial transformed phenotype are also produced.