Gene therapy with drug resistance genes.

A major side effect of cancer chemotherapy is myelosuppression. Expression of drug-resistance genes in hematopoietic stem cells (HSC) using gene transfer methodologies holds the promise of overcoming marrow toxicity in cancer chemotherapy. Adequate protection of marrow cells in cancer patients from myelotoxicity in this way would permit the use of escalating doses of chemotherapy for eradicating residual disease. A second use of drug-resistance genes is for coexpression with a therapeutic gene in HSCs to provide a selection advantage to gene-modified cells. In this review, we discuss several drug resistance genes, which are well suited for in vivo selection as well as other newer candidate genes with potential for use in this manner.

Poor expression of MDR1 transgene in HeLa cells by bicistronic Moloney murine leukemia virus-based vector.

Cotransfer of a therapeutic gene together with the human MDR1 gene provides an opportunity to increase the number of transduced marrow cells, expressing the therapeutic gene, by in vivo selection for MDR1. We have used an Lg-MDR1-IRES-neo (LgMIN) retroviral vector, containing MDR1 and neo genes, separated by the EMCV IRES. Human HeLa or canine CTAC cells, transduced with GALV env pseudotyped LgMIN at an MOI of less than 0.01 to ensure 1 proviral copy/genome, were selected with either G418 for neo expression or colchicine for MDR1 expression. The titer determined on HeLa cells with G418 selection was eight-fold higher than that with colchicine selection. In contrast, the same viral supernatant exhibited only a 1.4-fold difference between neo- and MDR1-based viral titer values for CTAC cells. The transduced HeLa cells, with one intact proviral copy per genome, exhibited a 55-fold higher resistance to G418 but only a 4-fold higher resistance to colchicine and a 2-fold higher resistance to Taxol compared with nontransduced cells. About 23% of the transduced cell population did not express vector-derived P-glycoprotein (P-gp) as detected by anti-human P-gp MAb MRK-16. This could explain the difference in viral titers obtained on CTAC cells but not that obtained on HeLa cells. The vector-mediated increase in expression of P-gp was about 20-fold higher in CTAC cells as compared with HeLa cells. These results indicated suppression of expression of vector-derived MDR1 in HeLa cells, in contrast with CTAC cells. To investigate further the possible reasons for this difference, genomic DNA was isolated from the G418-resistant individual colonies of infected cells and analyzed by PCR for full-length proviral MDR1. For transduced CTAC and HeLa cells, selected at a G418 concentration of 1 mg/ml, PCR detected aberrant forms of MDR1 in 17 to 25% of colonies tested. The aberrant forms consisted of MDR1 genes with 2- and 0.7-kb deletions. DNA sequencing across the 2-kb and the 0.7-kb deletion junction suggests cryptic splicing in the producer cell line as the origin of these deletions. The 2-kb deletion corresponds to MDR1 mRNA cryptic splicing via donor (codon 113) and acceptor (codon 773). The 0.7-kb deletion corresponds to splicing via the same donor and a different acceptor (codon 344). When transduced HeLa cells were selected at a higher concentration of G418 (3 mg/ml), the aberrant forms were detected at an increased frequency of about 50% of colonies tested. These results indicate that vector-derived MDR1 is a poor selective marker in HeLa cells but not in CTAC cells and that deletions, which inactivated the MDR1 gene in a bicistronic Mo-MuLV vector, may provide an advantage for expression of the second transgene in HeLa cells.

[Analysis of the genome compartmentalization phenomenon in normal and neoplastic cells]. / Analiz iavleniia kompartmentalizatsii genoma v normal'nykh i opukholevykh kletkakh.

Intact cell nuclei (or whole cell lysates) were immobilized on Celite and extracted gradually with gradients of NaCl, LiCl-urea and temperature. Contrary to the notion of DNA integrity and continuity within chromosomes, a heterogeneous spectrum of DNA fragments of large size was obtained, adhesion of which to the nuclear interior widely varied. Similar chromatographic patterns of DNA were observed in analysis of various origin cells both in normal animal tissues and in malignant cells (Djungarian hamster fibroblasts transformed by SV40). Possible artefacts of apparent genome fragmentation caused by radioactive precursors or hydrodynamic shearing were checked up and ruled out as well as endogenous nucleases appear not to be involved in the phenomenon observed. DNA end-labelling in situ enabled to reveal pre-existence of DNA fragments in isolated nuclei.

Differential dissociation of chromatin digests: a novel approach revealing a hierarchy of DNA-protein interactions within chromatin domains.

We describe here a novel approach to the dissection of chromatin structure by extracting DNA fragments from digested nuclei irreversibly immobilized (via proteins) on Celite columns. Three successive gradients (NaCl, LiCl-urea, temperature) are used to release three families of DNA fragments: namely, the 'DNA adherence' classes DNA-0, DNA-I and DNA-II, respectively. This 'protein image' DNA chromatography separates DNA fragments in accordance with the tightness of their bonds with proteins in situ. There are at least two DNA-skeleton attachment sites differing from each other by their resistance to the dissociating agents used as well as their susceptibility to DNAase I and S1 nuclease treatments, DNA cross-linking and single-stranded breaks. Several lines of evidence show a specific, topological rather than chemical, DNA-protein linkage at the tight attachment site. A hierarchy of chromatin loops demarcated by these attachment sites was determined. The technique described is generally applicable and can be used both to probe DNA-protein interactions and to map specific DNA sequences within the chromatin domain.

A procedure for DNA and RNA transfer to membrane filters avoiding weight-induced gel flattening.

We describe a technique of rapid (within 1-2 h) transfer of DNA and RNA from agarose gels to nitrocellulose or nylon membrane filters. It is characterized by nearly complete elimination of mechanical action on the gel (a thin layer of liquid is placed over the gel and, filtering through the gel into a stack of paper towels beneath, it transfers nucleic acids onto the filter under the gel). This "descending" transfer, as opposed to the widely used "ascending" Southern transfer, reduces the transfer time (to about 1 h) with equal or higher quality of the hybridization signal. The comparison of transfer kinetics by the both methods shows that (a) the Southern transfer of large size DNA fragments proceeds quicker than it has been thought so far and is almost complete within 4 h; (b) the descending transfer has an advantage over the ascending one in the rate of transfer (1-2 h) and its efficiency; and (c) the time of transfer may become a critical parameter upon using a filter with an apparently low retention capacity (Hybond N, Amersham) that is manifested by a decreased signal at longer than optimal transfer times.

[Changes in gene expression in the brain of rats with Zajdela's ascitic hepatoma]. / Izmeneniia ékspressii nekotorykh genov v golovnom mozge krys s perevivaemoi astsitnoi gepatomoi Zaidela.

Expression of some genes in the brain of ascitic hepatoma of Zajdela bearing rats was compared with that of control animals using Northern blot hybridization technique. The differences revealed were: an increased expression of actin gene and decreased expression of hsp70 gene in the brain of tumor-bearing animals.

[Comparative analysis of subdomain fragments of chromatin]. / Sravnitel'nyi analiz subdomennykh fragmentov khromatina.

Nuclei isolated from Djungarian hamster fibroblasts transformed by SV40 were treated with restriction endonuclease Bsp RI, fixed on Celite columns and underwent successive gradients of dissociating agents, such as NaCl, LiCl-urea, and temperature. This procedure leads to fractionation of DNA fragments in accordance with the tightness of DNA-protein bonds in situ. The fractions obtained were analysed by agarose gel electrophoresis and dot-hybridization technique with the use of various DNA probes. The results received are as follows: a) a DNA fragment size is not a factor determining the chromatographic position, the latter is probably stipulated by DNA-protein interactions; b) an analysis of cells synchronized at the G1/S border shows that the distribution of specific DNA sequences, such as actin, histone, hsp 70, and c-Ha-ras genes as well as reiterated DNA sequences, does not coincide with that of total genomic DNA; the nuclear matrix-attached fragments of those sequences are enriched to various extents. By nick-translation labeling in situ, DNase I-sensitive and hypersensitive regions were tentatively identified among subdomain chromatin fragments.

[Identification of the stable DNA-matrix complex (type II) as a site of replication fork]. / Identifikatsiia prochnogo kompleksa DNK-matriks (tip II) kak mesta nakhozhdeniia replikativnoi vilki.

The two types of DNA-matrix complexes (the weak and tight ones, or type I and type II, respectively) identified in our previous work were studied with respect to their involvement in DNA replication. Nuclei isolated from human fibrosarcoma HT1080 cell line were treated with either restriction endonucleases or ultrasonic desintegrator and afterwards subjected to the triple-gradient Nucleoprotein--Celite chromatography. This permitted fractionation of nuclear DNA into fragments not attached, weakly attached, and tightly attached to the nuclear matrix (DNA 0, DNA I, and DNA II, respectively). It was shown that pulse labelled RNA migrates from DNA II fraction where it resides initially to DNA 0 and further to DNA I during the 2 h chase period. This finding allowed us to consider the tight DNA-matrix complex as the replicative one. The experiments aiming to follow the movements of specific DNA sequences (histone genes) in relation to the DNA-matrix attachment sites were conducted on synchronous HT1080 cells progressing through S phase. The histone sequences appeared to undergo similar movements during the first 30 min of S phase. They reside initially in DNA 0 and DNA I fractions, but as soon as DNA synthesis was restored they migrate consequently to DNA II and DNA 0 fractions. This approach can appear to be a useful tool for studying the schedule of replication of specific genes during S phase.

[A new method of DNA and RNA transfer onto nitrocellulose filters during blot hybridization]. / Novyi sposob perenosa DNK i RNK na nitrotselliuloznye fil'try v metode blot-gibridizatsii.

A quick (1-2 hour) method of DNA and RNA transfer onto nitrocellulose filters for subsequent blot-hybridization was elaborated. The main features of the method proposed are, firstly, almost complete exclusion of the mechanical impact on the gel and, secondly, addition to the transfer medium (20 X SSC) of a chaotropic agent, 0.5 M NaClO4. The latter results in a slight dissolution of the gel matrix and, on the other hand, somewhat increases the binding of the nucleic acid to the nitrocellulose. The method shortens significantly the time of DNA or RNA transfer at equal, or even higher, quality of hybridization.

[Radioactive labeling of DNA with 3H-thymidine: effect of the internal irradiation on cell growth, chromatin structure and gene activity]. / Radioaktivnoe mechenie DNK 3H-timidinom: vliianie vnutrennego oblucheniia na rost kletok, strukturu khromatina i gennuiu aktivnost'.

The growth of djungarian hamster fibroblasts 4/21 is inhibited by 3H-thymidine present in a culture medium in concentrations from 18.5 to 740 KBq/ml. As judged from the gradient elution of DNA from isolated nuclei (the nucleoprotein-celite chromatography), DNA fragmentation increases together with the increase in 3H-thymidine concentration and the decrease in the cell growth rate. DNA fragmentation does not activate the family of heat shock genes (hsp70). On the contrary, the hsp70 gene transcription is somewhat inhibited in both heat shock and non-heat shock conditions even at a concentration of 3H-thymidine of as low as 37 KBq/ml. Hence the 3H labelling of radiosensitive cultured cells can lead to some deviations in cellular processes under study.

[Hydroxyapatite-mediated transfer of DNA on nitrocellulose filters in dot-hybridization experiments]. / Oposredovannyi oksiapatitom perenos DNK na nitrotselliuloznye fil'try v metode gibridizatsii v piatnakh.

The method of hydroxylapatite-mediated rapid and effective transfer of DNA onto nitrocellulose filters for following dot-hybridization was elaborated. The analysed DNA occurred initially in diluted and large volume solutions (from 1 to 10 ml) with various composition (2 M NaCl; 4 M LiCl--8 M urea; 4 M CsCl; 5 and 20% sucrose) was adsorbed on hydroxylapatite and quantitatively transferred onto nitrocellulose after hydroxylapatite solubilization in a small volume of acid (usually, 200 microliters of 10% TCA). As exemplified by the hybridization of total rat liver DNA with the plasmid ph22 DNA containing a cluster of sea urchin histone genes, the method presented appears to be not only simple and useful for handling multiple probes of diluted DNA solutions with high concentrations of salts, sucrose and urea but also more sensitive than some convenient DNA dot-hybridization methods.

[Relation between the structural, metabolic and "adhesive" properties of nuclear and cytoplasmic non-ribosomal RNA]. / Vzaimosviaz' strukturnykh, metabolicheskikh i "adgezivnykh" svoistv iadernykh i tsitoplazmaticheskikh neribosomnykh RNK.

Nuclear RNAs release from nucleoproteins of isolated nuclei absorbed on a celite column in a wide range of dissociating conditions (from 1 M LiCl--2 M urea at 2 degrees C to 4 M LiCl--8 M urea at 70-80 degrees C) was demonstrated. Such a high "adhesive" heterogeneity of nuclear RNAs (i.e., variations in the tightness of RNA-protein bonds) appears to be due to the association of nuclear matrix proteins. A direct correlation was found to exist between the metabolic turnover of RNA and the tightness of its association with the nuclear matrix. Actually, the pulse label which rapidly incorporates into the RNAt greater than 50 degrees, the RNA fraction being most tenaciously bound to the matrix, could be chased later into RNAs weakly bound to it. As the RNA-matrix binding weakens, the metabolic and structural properties of a given RNA change, e.g., sedimentation coefficients decrease, while the poly(A)+-RNA content and stability increase. The "adhesive" heterogeneity was found to be inherent in not only nuclear RNAs but also in cytoplasmic non-ribosomal RNAs, showing the same correlation, i.e., the tighter the RNA--protein complex, the higher the rate of RNA turnover. Cytoplasmic RNAs which differ in their adhesiveness may fulfil various intracellular functions, since polyribosomal mRNPs and informosomal mRNPs appear to be enriched in tightly and weakly bound RNA fractions, respectively. The interrelationships between nuclear and cytoplasmic RNAs are discussed.

[Analysis of cellular nucleoproteins by the nucleoprotein-celite chromatography method. III. Two types of DNA-nuclear matrix interaction]. / Analiz kletochnykh nukleoproteidov metodom nukleoproteid-tselit-khromatografii. III. Dva tipa vzaimodeistvii DNK s iadernym matriksom.

There are two types of DNA-nuclear matrix interactions in animal cells as revealed by the release of DNA from isolated nuclei by three successive gradients: NaCl, LiCl-urea and temperature. Nuclei were treated with dissociating agents while being adsorbed on the Celite columns. "Weak" DNA-matrix interactions which dissociate in 1.5 M LiCl-3 M urea at 2 degrees appear to be sensitive to ethidium bromide and resistant to exogeneous nucleases (DNAase I, DNAase II and micrococcal nuclease), to DNA-damaging agents, including alkylators and gamma-irradiation, and also to psoralen-induced cross-links. "Strong" DNA-matrix interactions proved to be very different. They dissociate in 4 M LiCl-8 M urea at approximately 90 degrees, are very sensitive to DNAase I and other nucleases, slightly sensitive to chemicals and irradiation at doses stimulating single-stranded DNA breaks, but resistant to ethidium bromide. DNA strand separation seems to be necessary prerequisite for DNA release from its "strong" complex with nuclear matrix. A model for the topological link between DNA and the nuclear matrix involved in DNA replication complex is discussed.

[Analysis of cellular nucleoproteins by nucleoprotein-celite-chromatography. I. Structural rearrangements of the chromosomal apparatus coupled with cellular quiescence-growth transitions]. / Analiz kletochnykh nukleoproteidov metodom nukleoproteid-tselit-khromatografii. I. Izmeneniia struktury khromatina, sopriazhennye s kletochnymi perekhodami pokoi-delenie.

By using the nucleoprotein-celite-chromatography of unfractionated cell lysates it was found that two alternative states of the chromosomal apparatus of eukaryotic cells exist, one of them being characteristic of resting cells (relaxed form), while another of growing cells (stabilized form). This finding evidences for the real occurrence of a special state of cellular quiescence, G0. DNA in form beta is much more tightly associated with proteins than DNA alpha. Upon cellular transitions from resting to growing state and vice versa the coupled rearrangements of chromosomal apparatus were observed, judging from corresponding chromatographic patterns. One state or another characterizes the chromosomal apparatus as a whole, rather than some of its subfractions. The tight DNA-protein interactions were observed in proliferating cells independent of the cell cycle phase.

[Turnover of nuclear RNA. II. Conveyer model of synthesis and maturation of nuclear RNA as an alternative for a stochastic model]. / Obmen iadernykh RNK. II. Konveiernaia model' sinteza i sozrevaniia iadernykh RNK kak al'ternativa stokhasticheskoi modeli.

It is shown that the stochastic model of hnRNA decay is inconsistent with a number of experimental data. The model of ordered in time and space movement of nascent and post-transcriptional RNA molecules "on conveyer" coupled with certain processing steps is put forward. Since degradation of the pre-mRNA molecule proceeds not instantaneously but in several subsequent steps, the notion of the "exiton" is introduced. The exiton is proposed to be a submolecular metabolically indivisible fragment of RNA molecule which decays as a unit. From this point of view, a population of nuclear RNAs, as a whole, can be presented (and easily mathematically treated) as a community of exiton transporters of various life spans moving in parallel. The life-time spectrum of exitons in population of rat liver hnRNA was computed. The share of exitons with life-time from 41.5 to 42.5 min appeared to be most in the population. Exitons with life-times above 1 h and 2 h comprise approximately 50 and 20% of the total population, respectively. The consequences of the model proposed are discussed.

[Nuclear RNA turnover. 1. Metabolic heterogeneity]. / Obmen iadernykh RNK. I. Vyiavlenie metabolicheskoi geterogennosti.

It is usually necessary to compare the kinetics of labelling of nucleoside triphosphates and nuclear RNAs to determine the turnover rate (half-life, T1/2) of nuclear RNAs. It is shown that the widely adopted correction for non-constant specific radioactivity of precursor pool is not correct in general and could be used only for very stable RNAs. The method for T1/2 determination is described which is suitable for any form of UTP labelling kinetics. Besides, the criterion was found for revelation of metabolic heterogeneity of nuclear RNA population. Rat liver nuclear DNA-like RNA appeared to be heterogeneous and consisted of two subpopulations, one rapidly labelled with T1/2 about 30 min and other, three times larger, with no labelling during the experiment.