Genetic transformation of somatic cells. VIII. The effect of the DNA methylation inhibitor 5-azacytidine on the stability of thymidine kinase-negative and -positive phenotypes of transformant clone cells

A study was made of the effect of an DNA methylation inhibitor 5-azacytidine (azaC) on the frequency of reversion to a thymidine kinase-positive (TK+) phenotype in 5-bromodeoxy-uridine (BrdU)-resistant subclones obtained from clones of Chinese hamster cells transformed by thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1). It is shown that in 8 of 15 BrdU-resistant subclones azaC increases 2-1000-fold the frequency of reversion to TK+ phenotype. Variations in the inducibility of reversions to TK+ phenotype indicate that the DNA methylation associated with TK- phenotype affects but differently tk gene of HSV1. Cultivation of TK+ cells of transformant clones in the presence of azaC may lead to stabilization (or decrease in the rate of the loss) of TK+ phenotype, or may not influence the stability of transformant phenotype. The reaction of TK+ cells of transformant clones depends both on genetically determined rate of the loss of TK+ phenotype, and on the structure of transforming DNA introduced to cells. A conclusion is drawn that the TK- phenotype of transformant clone cells arises due to processes which are not associated with methylation of tk gene of HSV1 in spite of the fact that such a methylation may later stabilize significantly the TK- phenotype.     

Genetic transformation of somatic cells. VI. Transfer of the trait of the rate of loss of transformant phenotype by means of DNA from cells containing the thymidine kinase gene of the herpes simplex virus

Using dot-hybridization with thymidine kinase gene (tk gene) of Herpes simplex virus type 1 (HSV 1) of DNA preparations obtained from isolated metaphase chromosomes and lysate fractions of metaphase cells, which presumably contain smaller particles compared to metaphase chromosomes, it has been shown that the tk gene of HSV 1 is localized in chromosomes of cells of transformant clones unstable in TK+-phenotype. The DNA isolated from the metaphase chromosomes from cells of transformant clones is 1.5- or 2-fold more efficient in transforming TK-Chinese hamster cells than is the total high molecular weight DNA from the same cells. Upon transformation of TK- cells by the high molecular weight DNA from the tk gene of HSV 1-containing clones, varying in the rate of the loss of TK+-phenotypes, the character "rate of the loss of transformant phenotype" is transferred together with the tk gene of HSV 1 in 22% of cases. Cells of rerevertant clones, produced from TK- subclones of transformant clones, display the rate of the loss of transformant phenotype characteristic of cells of parental TK+-clones. A comparison of the results allows a conclusion that DNA sequences, determining the character "rate of the loss of transformant phenotype", are linked tightly with the transforming DNA proper containing the tk gene of HSV 1, but are not localized inside such a DNA.     

Genetic transformation of somatic cells. V. Inheritability of the rate of loss of the trait and the stabilization of the transformant phenotype

Subclones were isolated both on selective and nonselective medium from the Chinese hamster cells transformed by thymidine kinase gene (TK-gene) of Herpes simplex virus (HSV-1) and varying in the rate of the loss of transformant phenotype. The study of the stability of thymidine kinase-positive (TK+) phenotype in cell populations the subclones shows that the nonstability and the rate of the loss of transformant phenotype are the characters that are inherited in the cell generations. Durable cultivation on a HAT-selective medium may lead to a complete or partial (expressed as a reduced rate of the loss of the character) stabilization of TK+-phenotype of the cells of transformant clones. The rate of stabilization of TK+-phenotype may differ depending on the structure of transforming DNA introduced into cells of transformant clones.     

Genetic transformation of somatic cells. IX. The loss of the transformant phenotype is accompanied by rearrangements in the plasmid DNA containing the thymidine kinase gene of the herpes virus

As demonstrated by dot-hybridization, the cells of HT-subclones isolated from the cells of transformant clones cultured on a non-selective medium differ significantly in the number of copies of thymidine kinase gene (tk-gene) of Herpes simplex virus (HSV1). Since the cells of transformant clones lose thymidine kinase-positive (TK+) phenotype during cultivation, this data are indicative of high frequence rearrangements in the region of transforming DNA as responsible for the transformant phenotype nonstability. These rearrangements, among other things, induce alterations in the number of copies of tk gene of HSV1. The analysis of cells of subclones isolated on a medium containing 5-bromodeoxyuridine (BrdU) shows that the number of copies of tk gene of HSV1 decreases as compared to the cells of parental clones. The decrease in the number of copies of tk gene of HSV1 in a row of BrdU-resistant subclones is accompanied by simultaneous increase in the number of sequences of pBR325 plasmide DNA to which tk gene of HSV1 is linked covalently in the pST826 plasmide introduced into cells of transformant clones. This evidence implies a most complex nature of transforming DNA rearrangements reducing the number of copies of tk gene of HSV1 due possibly to a genetic correction. The analysis of results permits a hypothesis that instability of cells in transformant phenotype may be determined by the genetic instability of insertion type. The rate of the loss of transformant phenotype depends on the frequency of rearrangements in the transforming DNA locus.     

Genetic transformation of somatic cells. IV. The rate of loss of transformant phenotype depends on the structure of the transforming DNA and changes when the cells are treated with the tumor promotor 12-o-tetradecanoyl-phorbol-13-acetate

Analysis was made of the phenotype stability of some clones of thymidine kinase deficient (TK-) Chinese hamster cells transformed by thymidine kinase gene (TK-gene) of Herpes simplex virus type (HSV 1). The presence of a fragment of human satellite DNA III in the plasmid DNA carrying the TK-gene of HSV 1 reduced notably the rate of the loss of TK+-phenotype, and the treatment of the cells with a tumour promoter--12-o-tetradecanoyl-phorbol-13-acetate--immediately after transformation destabilizes TK+-phenotype of transformant clones. Removal of the eukaryotic carrier DNA for the plasmid DNA without the TK-gene of HSV 1 destabilizes the clone transformant phenotype. Changes in the structure of the plasmid DNA containing no TK-gene of HSV 1 and introduced into cells simultaneously with TK-gene containing plasmids affects the rate of the loss of TK+-phenotype transformed cells.     

Genetic transformation of somatic cells. II. An analysis of the status of the plasmid nucleotide sequences in chromosomal DNA and the thymidine kinase activity in transformant clone cells

Chinese hamster A238 TK- -cells were transformed with plasmids (derivatives of pBR325) containing thymidine kinase (TK) gene of Herpes simplex virus type 1 (HSV1). The results of dot- and blot-hybridization indicate the presence of pBR325 sequences in the chromosomal fractions of DNA in the transformant clones. These sequences are probably tandemly arranged, and each cluster contains 25--50 copies. SV40 sequences cloned in pBR325 were introduced into the Chinese hamster cells by co-transformation with TK-gene of HSV1-containing plasmid DNA, and all the co-transformant clones selected for TK+-phenotype were shown by hydridization to contain 3V40 DNA fragments. Isoelectrofocusing in polyacrylamide gel shows that thymidine kinase from TK+-transformant clones is of viral type (isoelectric point 7), in contrast to the cellular enzyme (coded by chromosomal gene) having alkaline isoelectric point (pH 9). The results suggest that the true TK+-transformant cells are selected by the procedure used in this study.     

Thymidine kinase activity and trifluorothymidine resistance of spontaneous and mutagen-induced L5178Y cells in RPMI 1640 medium

L5178Y/TK+/- cells were treated with 3-methylcholanthrene (3MC) in order to obtain thymidine-kinase-deficient mutants (TK-/-) which were resistant to trifluorothymidine (TFTr). Clones of TK-/- cells were harvested from soft agar and adapted to growth in suspension culture. The phenotype of the TK-/- and TK+/- clones was confirmed by measuring thymidine kinase activity. These studies were undertaken with cells from 16 3MC-induced large colony clones (lambda TK-/-), 21 3MC-induced small colony clones (sigma TK-/-), and 51 spontaneous sigma TK-/- clones. Thymidine kinase activity was absent in all of the lambda TK-/- and sigma TK-/- 3MC-induced clones and also in 49 of 51 sigma TK-/- spontaneous clones. After at least 50 generations in suspension culture, TFTr was retained by 80% of the 3MC-induced lambda TK-/- cells, by 75% of the 3MC-induced sigma TK-/- cells, and by 89% of the spontaneous sigma TK-/- cells. The collective results showed that 86 of the 88 TFTr colonies examined lacked thymidine kinase activity and indicate that at least 98% of all TFTr colonies seen in the L5178Y assay are true TK-/- mutants.     

Carrier DNA affects the integration of HSV-1 TK gene in the genome of L929TK- cells.

L929TK- cells were cotransfected with DNA mixtures containing tk gene of HSV-1, plasmids carrying LTR of MoMLV or RSV and carrier DNA of salmon sperm or chromosomal DNA of recipient cells. Selection of TK+ transformants was conducted in DMEM supplemented with HAT. Plasmids carrying LTR sequences of MoMLV or RSV retroviruses showed enhancing effect on the frequency of TK+ transformation. Southern blot analysis of chromosomal DNA of TK+ transformants demonstrated in clones deriving from cotransfections of tk gene and carrier DNA of L929TK- cells multiple copies of tk gene integrated into several genomic sites of host. Single copies of tk gene integrated into different sites of host genome occurred in chromosomal DNA of TK+ clones deriving from cotransfections of tk gene and carrier DNA of salmon sperm. Cells cotransfected with tk gene and plasmids carrying LTR sequences of MoMLV or RSV formed three dimensional colonies in semisolid agar medium. No effect of carrier DNA on the morphology of TK+ transformant clones was noticed.     

Transformation of the gene for hypoxanthine phosphoribosyltransferase.

Purified DNA from wild-type Chinese ovary (CHO) cells has been used to transform three hypoxanthine phosphoribosyltransferase (HPRT) deficient murine cell mutants to the enzyme positive state. Transformants appeared at an overall frequency of 5 x 10(-8) colonies/treated cell and expressed CHO HPRT activity as determined by electrophoresis. One gene recipient, B21, was a newly isolated mutant of LMTK- deficient in both HPRT and thymidine kinase (TK) activities. Transformation of B21 to HPRT+ occurred at 1/5 the frequency of transformation to TK+; the latter was, in turn, an order of magnitude lower than that found in the parental LMTK- cells, 3 x 10(-6). Thus both clonal and marker-specific factors play a role in determining transformability. The specific activity of HPRT in transformant extracts ranged from 0.5 to 5 times the CHO level. The rate of loss of the transformant HPRT+ phenotype, as measured by fluctuation analysis, was 10(-4)/cell/generation. While this value indicates stability compared to many gene transferents, it is much greater than the spontaneous mutation rate at the indigenous locus. The ability to transfer the gene for HPRT into cultured mammalian cells may prove useful for mutational and genetic mapping studies in this well-studied system.     

Stability of the transformants obtained by phage particle-mediated gene transfer

Recombinant lambda phage DNA, encapsulated in phage particles and coprecipitated with calcium phosphate, efficiently transforms cultured mammalian cells without a requirement for carrier DNA. The present paper analyzes the stability of the transformants obtained by the phage transfer method. lambda phage particles containing recombinant DNA that includes the thymidine kinase (TK) gene of herpes simplex virus type 1 as a selective marker were introduced into Ltk- cells deficient in TK activity, and TK+ transformants were selected in HAT medium. To test the stability of the TK+ phenotype of the transformants, seven individual transformant clones were isolated, cultured in HAT selective medium and then in non-selective medium for various lengths of time. After such culture, transformants were allowed to develop colonies in both selective and non-selective medium. For all seven transformant clones, the numbers of colonies obtained in the two types of medium were almost identical, irrespective of whether or not each transformant clone had been previously cultured for 15 to 50 days in non-selective medium. This result suggests that most transformants obtained by the phage transfer method maintain the TK+ phenotype stably, for at least 50 days, when grown in non-selective medium.     

Utilization of exogenous thymidine by Chlamydia psittaci growing in the thymidine kinase-containing and thymidine kinase-deficient L cells.

The incorporation of [3H]thymidine into the deoxyribonucleic acid (DNA) of Chlamydia psittaci (strain 6BC) growing in thymidine kinase (adenosine 5'-triphosphate-thymidine 5'-phosphotransferase, EC 1.7.1.21)-containing L cells, L(TK+), and thymidine kinase-deficient L cells, LM(TK-), was examined by autoradiography. Label was detected over C. psittaci inclusions in L(TK+) but not LM(TK-) cells. No evidence for a chlamydia-specific thymidine kinase activity in either L(TK+) or LM(TK-) cells was obtained. Entry of [3H]thymidine into the DNA of C. psittaci growing in L(TK+) cells was quantitated by measuring label in purified C. psittaci. It was 265 times less efficient than entry into infected host cell DNA. It is concluded that low levels of exogenous thymidine are incorporated into the DNA of C. psittaci and that this incorporation is dependent on a fully competent host thymidine kinase activity. Evidence also is presented that L cells possess at least two thymidine kinase activities, both of which are capable of supplying thymidylate precursors for nuclear DNA synthesis.     

DNA-mediated gene transfer without carrier DNA

DNA-mediated gene transfer is a procedure which uses purified DNA to introduce new genetic elements into cells in culture. The standard DNA-mediated gene transfer procedure involves the use of whole cell DNA as carrier DNA for the transfer. We have modified the standard DNA-mediated gene transfer procedure to transfer the Herpes simplex virus type 1 thymidine kinase gene (TK) into TK- murine recipient cells in the absence of whole cell carrier DNA. The majority (8/10) of carrier-free transformant lines expressed the TK+ phenotype stably, in sharp contrast to our results with carrier-containing DNA-mediated gene transfer. There was a wide range in donor DNA content among independent transformants. Further analysis on one transformant line using DNA restriction digests and in situ hybridization provided evidence that, in the absence of whole cell carrier DNA, multiple donor DNA sequences became integrated at a single chromosomal site.     

Evidence for allelic exclusion in Chinese hamster ovary cells.

Earlier results suggested that the functional hemizygosity of genes in pseudodiploid Chinese hamster ovary (CHO) cells is due to the silencing of one allele by DNA methylation. From this one could make a strong prediction that we have now been able to confirm by genetic experiments, using thymidine kinase (TK) alleles. TK- mutants induced by ethylmethane sulphonate (EMS) were all revertible to TK+ at high frequency by the demethylating agent 5-azacytidine (5-aza-CR). This revertibility was due to reactivation of a silent nonmutant TK allele. Further mutagenesis by EMS yielded TK- derivatives that were no longer revertible by 5-aza-CR; these are assumed to have mutations in both alleles. TK- cells were also transfected with equine herpes virus TK+ DNA, and the TK+ derivatives were shown to be markedly less stable than cells with the normal TK+ gene. CHO cells lack metallothionein activity (sensitive to cadmium), and also require proline for growth, because genes have become silenced during the establishment of the cell line. In both cases 5-aza-CR reactivates these genes to give the cadmium resistant and proline independent phenotypes. Long-term experiments with reactivants in the absence of selection showed that the genes become silent, presumably as a result of de novo methylation. A strain resistant to cytosine arabinoside (araCR) was also resistant to 5-azadeoxycytidine (5-aza-CdR), but not to 5-aza-CR, which would be expected if the araCR strain lacked deoxycytidine kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of thymidine kinase activity in hybrids between human leukemic cells and a TK-mouse cell line.

A series of actively proliferating clones have been isolated after PEG-induced fusion of a thymidine kinase deficient murine line and white blood cells from two leukemic patients. Their hybrid nature was proved both cytologically and biochemically. All the hybrids tested showed levels of TK activity significantly higher than the TK- mouse parental cell line and comparable to those exhibited by replicating TK+ cells.     

Deoxyribonucleic acid-mediated gene transfer in mammalian cells: molecular analysis of unstable transformants and their progression to stability.

To elucidate mechanisms involved in deoxyribonucleic acid-mediated gene transfer, we transferred the herpes simplex virus thymidine kinase gene (TK) into mouse Ltk- cells. Independent TK+ clones (transformants) and derivatives of each were tested for phenotypic expression and the presence and arrangement of TK sequences. Initially, transformants expressed viral TK unstable, with 10% of the cells in each generation losing both the TK+ phenotype and virally derived TK sequences. After a prolonged period in culture, stable subpopulations arose from which the TK+ phenotype and viral sequences were no longer lost at detectable frequency. Analysis of unstable cell populations indicated that individual viral deoxyribonucleic acid molecules were reduced in size, but were linked to other deoxyribonucleic acid to form molecules large enough to be precipitated in a Hirt fractionation. We term these molecules transgenomes. Analysis of independent unstable subclones derived from the primary transformants demonstrated that individual transgenomes could contain multiple copies of the viral TK sequences. Recipient cell lines frequently possessed more than one type of transgenome and possibly multiple copies per cell of each type. Stable derivatives possessed only one of the transgenomes present in the unstable parent, and these sequences were associated with a recipient cell chromosome.     

Adaptation of a retrovirus as a eucaryotic vector transmitting the herpes simplex virus thymidine kinase gene.

We investigated the feasibility of using retroviruses as vectors for transferring DNA sequences into animal cells. The thymidine kinase (tk) gene of herpes simplex virus was chosen as a convenient model. The internal BamHI fragments of a DNA clone of Moloney leukemia virus (MLV) were replaced with a purified BamHI DNA segment containing the tk gene. Chimeric genomes were created carrying the tk insert in both orientations relative to the MLV sequence. Each was transfected into TK- cells along with MLV helper virus, and TK+ colonies were obtained by selection in the presence of hypoxanthine, aminopterin, and thymidine (HAT). Virus collected from TK+-transformed, MLV producer cells passed the TK+ phenotype to TK- cells. Nonproducer cells were isolated, and TK+ transducing virus was subsequently rescued from them. The chimeric virus showed single-hit kinetics in infections. Virion and cellular RNA and cellular DNA from infected cells were all shown to contain sequences which hybridized to both MLV- and tk-specific probes. The sizes of these sequences were consistent with those predicted for the chimeric virus. In all respects studied, the chimeric MLV-tk virus behaved like known replication-defective retroviruses. These experiments suggest great general applicability of retroviruses as eucaryotic vectors.     

De novo methylation as major event in the inactivation of transfected herpesvirus thymidine kinase genes in human cells

Spontaneous inactivation of integrated thymidine kinase genes was studied in three human cell lines, one with multiple copies and two with a single copy of a transfected shuttle plasmid containing two selectable genes: the HSV tk gene and the Eco gpt gene. Selection for gpt expression prevented the isolation of TK- mutants which are the result of plasmid loss. Under these conditions TK- clones were isolated with a frequency of 5.10(-6) both with the cell line containing 5 or 6 copies of the tk gene and with one of the two cell lines containing one copy of this gene. This inactivity of the tk gene was associated with de novo methylation as the number of HAT-resistant (TK+) clones strongly increased after growth of the TK- derivatives in the presence of the demethylating agent, 5-azacytidine. Digestion with methylation-sensitive restriction enzymes revealed two different patterns of DNA methylation in the genomic DNA of TK- variants. In the TK- derivatives of the cell line containing multiple copies of the tk gene many HpaII restriction sites in the gene copies were insensitive to digestion. These HpaII sites were, however, not methylated in TK- variants of the cell line containing one copy of the plasmid, and methylated CpGs could be detected only with EcoRI which recognizes the cGAATTCg sequence in the tk promoter region. With the other of the two single-copy TK+ cell lines no TK- mutants were obtained, suggesting that the position of a gene in the genome is an important factor in determining the frequency and the extent of de novo methylation. Additionally, we observed that remethylation is an even more efficient process of gene inactivation as TK+ clones reactivated with 5-azacytidine can become TK- again at a 100-fold higher rate than the original TK+ cell line.     

A cytotoxic effect associated with 9-(1,3-dihydroxy-2-propoxymethyl)-guanine is observed during the selection for drug resistant human cells containing a single herpesvirus thymidine kinase gene

A cytotoxic effect associated with 9-(1,3-dihydroxy-2-propoxymethyl)-guanine (DHPG) was discovered while searching for spontaneous mutations in a single copy, integrated HSV-1 thymidine kinase (TK) gene in the human 143 TK- cell line. It was found that spontaneous DHPGR mutations could not be selected while other anti-TK drugs resulted in selectable mutation frequencies of 10(-4) to 10(-3). When 143 TK- cells were mixed with these HSV-1 TK+ cells and subjected to DHPG, a 90% to 100% decrease in recoverable TK- colonies was observed. In addition, the media from the HSV-1 TK+ cells metabolizing DHPG was shown to inhibit the growth of the TK- cells.