Differential in vitro growth properties of cells transformed by DNA and RNA tumor viruses

Mammalian cells transformed by DNA and RNA tumor viruses are shown to display consistently different growth properties. All SV40, adenovirus type 7 and polyoma virus (DNA viruses) transformed cells propagated to high densities. The same cells transformed instead by RNA viruses: MSV strain Kirsten (MSV-Ki) or MSV strain Maloney (MSV-M) grew to densities which were consistently lower than DNA virus-transformed cells but greater than that of untransformed cells. The capacity to synthesize DNA at increasing densities also differentiated the RNA and DNA virus-transformed cells. As growing cultures of untransformed cells neared saturation density, the fraction of cells synthesizing DNA was minimal. The RNA virus-transformed cells were also contact-inhibited but at a significantly higher density. In contrast the DNA virus-transformed cells propagated to still greater densities and continued DNA synthesis at a high rate even at very high densities. Therefore the DNA virus-transformed cells truly are not contact inhibited. It is suggested that the capacity to continue DNA synthesis at high densities explains the attainment of much greater densities by DNA virus-transformed cells. There were no clear-cut differences in the ability to form colonies in agar, although a few of the RNA virus-transformed lines could not be propagated in semi-solid medium. These results may be explained as a persistence of the capacity of DNA tumor viruses to stimulate host cell DNA synthesis.     

Large DNA palindromes as a common form of structural chromosome aberrations in human cancers

Breakage-fusion-bridge cycles contribute to chromosome aberrations and generate large DNA palindromes that facilitate oncogene amplification in cancer cells. At the molecular level, large DNA palindrome formation is initiated by chromosome breaks, and genomic architecture such as short inverted repeat sequences facilitates this process in mammalian cells. However, the prevalence of DNA palindromes in cancer cells is currently unknown. To determine the prevalence of DNA palindromes in human cancer cells, we have developed a new microarray-based approach called Genome-wide Analysis of Palindrome Formation (GAPF, Tanaka et al., Nat Genet 2005; 37: 320-7). This approach is based on a relatively simple and efficient method to purify "snap-back DNA" from large DNA palindromes by intramolecular base-pairing, followed by elimination of single-stranded DNA by nuclease S1. Comparison of Genome-wide Analysis of Palindrome Formation profiles between cancer and normal cells using microarray can identify genome-wide distributions of somatic palindromes. Using a human cDNA microarray, we have shown that DNA palindromes occur frequently in human cancer cell lines and primary medulloblastomas. Significant overlap of the loci containing DNA palindromes between Colo320DM and MCF7 cancer cell lines suggests regions in the genome susceptible to chromosome breaks and palindrome formation. A subset of loci containing palindromes is associated with gene amplification in Colo320DM, indicating that the location of palindromes in the cancer genome serves as a structural platform that supports subsequent gene amplification.     

DNA ligase activity in human cell lines from normal donors and Bloom''s syndrome patients.

DNA ligase activity was studied in several untransformed or virus-transformed human cell lines from normal donors and from Bloom's syndrome (BS) patients. This proneness genetic disease is characterized by several cytological abnormalities and cancer proneness and, recently, some transformed cell lines from these patients were described to present a reduced activity of DNA ligase I. Results presented in this work indicate that: (i) the total DNA ligase activity in crude extract from untransformed or transformed cell lines from several BS patients was significantly higher than in control cells; (ii) the partial purification of the enzyme after gel filtration on fast protein liquid chromatography of crude extracts from lymphoblastoid BS cells showed that the enzyme activity was eluted in a major 180 kDa form in which activity was higher than in control cells; (iii) the activity gel analysis of these enzyme fractions revealed that DNA ligase of human cells was correlated to a major 130 kDa polypeptide and, in BS cells, the extent of the activity of this band was equal or higher than that in control untransformed or transformed cells.     

The asymmetric methylation of CG palindromic dinucleotides increases sister-chromatid exchanges

Treatment with the base analogue, 5azaC, increases SCEs in CHO but not in mosquito cells. On the other hand, both types of cells show equivalent increases in exchanges when treated with other compounds, such as mitomycin C. Vertebrate DNA is heavily methylated while diptera DNA is heavily demethylated. The sequence of events leading to an increase in SCEs in CHO cells is as follows: first of all, Cs are replaced by 5azaC; in the next cell cycle, CG palindromic dinucleotides exhibit an asymmetric configuration, the Cs in the parental DNA strand being methylated and the Cs in the daughter DNA strand demethylated; after one more cycle, half of the chromosomes show symmetric methylation and the other half symmetric demethylation of both Cs in CG palindromes. The increase of SCEs occurs in the second cell cycle when the hemimethylated DNA enters replication. DNA hemimethylation is believed to be an intermediate stage in the process of demethylation that accompanies gene expression. If so, gene demethylation would be a cause of SCE increase in normal vertebrate cells.     

Histone and nonhistone proteins from normal and virus-transformed rat thyroid epithelial cells

The histone and nonhistone nuclear proteins extracted by different methods from nuclei of normal and virus-transformed rat thyroid epithelial cells (FRTL5 cell line) were studied by polyacrylamide gel electrophoresis in acetic acid/urea and in SDS and by autoradiography. The results have shown that some of these proteins have an higher level of phosphorylation in virus-transformed than in normal cells; moreover, an higher amount of three proteins (C, D, and E), which in normal cells are not detectable at least as Coomassie staining, was found. These proteins, extractable with perchloric acid, are suggested to belong to the High Mobility Group (HMG proteins) and to play some regulatory role.     

The synthesis of surface coat materials in normal and transformed cells

The rate of synthesis and thickness of the surface coat material in a range of virus-transformed and chemically-transformed cell lines were measured by ellipsometry. Cell lines transformed by polyoma virus, SV 40 virus, Rous sarcoma virus and murine sarcoma virus had a significantly thicker coat than the normal parent cells. An increase in the thickness of the cell coat was not a consistent feature of the transformed cell state since this change was not detected in cell lines transformed by methylcholanthrene. The rate of synthesis of the surface coat was significantly faster in transformed cells than in normal cells. Coat synthesis in normal and transformed cells was inhibited rapidly by treatment with cycloheximide. Inhibition of cellular RNA synthesis by actinomycin D produced rapid inhibition of coat synthesis in normal and chemically-transformed cell, but in certain virus-transformed cell lines coat synthesis continued for up to h. The significance of these changes in the pattern of coat synthesis in transformed cells in relation to their altered surface properties is discussed.     

DNA methylation in mouse cells in culture as measured by restriction enzymes

Methylation of DNA in normal mouse cultured 3T3 cells and in their virally or chemically transformed derivatives was studied. DNA methylation was studied by restriction with HpaII, MspI, or HpaII plus MspI. DNA from the chemically transformed cells was cleaved about twice as often with HpaII than was the DNA of normal and virally transformed cells. Digests with MspI and HpaII plus MspI were identical in all cell lines studied. Densitometry of the restriction patterns allowed an estimate of total DNA methylation from the weight average lengths. The chemically transformed cell line showed 25% reduction in methylation compared to the other cell lines. Southern blot hybridization using satellite DNA showed that these sequences followed a pattern of modification similar to that of total DNA.     

Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G(0)/G(1) to S phase transition in normal and tumor cells

DNA methylation is essential for mammalian development, X-chromosome inactivation, and imprinting yet aberrant methylation patterns are one of the most common features of transformed cells. One of the proposed causes for these defects in the methylation machinery is overexpression of one or more of the three known catalytically active DNA methyltransferases (DNMTs) 1, 3a and 3b, yet there are clearly examples in which overexpression is minimal or non-existent but global methylation anomalies persist. An alternative mechanism which could give rise to global methylation errors is the improper expression of one or more of the DNMTs during the cell cycle. To begin to study the latter possibility we examined the expression of the mRNAs for DNMT1, 3a and 3b during the cell cycle of normal and transformed cells. We found that DNMT1 and 3b levels were significantly downregulated in G₀/G₁ while DNMT3a mRNA levels were less sensitive to cell cycle alterations and were maintained at a slightly higher level in tumor lines compared to normal cell strains. Enzymatic activity assays revealed a similar decrease in the overall methylation capacity of the cells during G₀/G₁ arrest and again revealed that a tumor cell line maintained a higher methylation capacity during arrest than a normal cell strain. These results reveal a new level of control exerted over the cellular DNA methylation machinery, the loss of which provides an alternative mechanism for the genesis of the aberrant methylation patterns observed in tumor cells.     

Abnormal processing of transfected plasmid DNA in cells from patients with ataxia telangiectasia.

In order to assess spontaneous mutability and accuracy of DNA joining in ataxia telangiectasia, a disorder with spontaneous chromosome breakage, the replicating shuttle vector plasmid, pZ189, was transfected into SV40 virus-transformed fibroblasts from ataxia telangiectasia patients. The ataxia telangiectasia fibroblasts showed elevated frequency of micronuclei, a measure of chromosome breakage. The spontaneous mutation frequency was normal with circular plasmids passed through the ataxia telangiectasia line. These results were compared to those with transformed fibroblasts from a patient with xeroderma pigmentosum, and from a normal donor. Mutation analysis revealed spontaneous point mutations and deletions in the plasmids with all 3 cell lines, however, insertions or complex mutations were only detectable with the ataxia telangiectasia line. To assess DNA-joining ability, linear plasmids which require joining of the DNA ends by host cell enzymes for survival, were transfected into the cells. We found a 2.4-fold less efficient DNA joining in ataxia telangiectasia fibroblasts (p = 0.04) and a 2.0-fold higher mutation frequency (p less than 0.01) in the recircularized plasmids than with the normal line. Plasmid DNA joining and mutation frequency were normal with the xeroderma pigmentosum fibroblasts. These findings with the ataxia telangiectasia fibroblasts of abnormal types of spontaneous mutations in the transfected plasmid and inefficient, error-prone DNA joining may be related to the increased chromosome breakage in these cells. In contrast, an EB virus-transformed ataxia telangiectasia lymphoblast line with normal frequency of micronuclei showed normal types of spontaneous mutations in the transfected plasmid and normal frequency of DNA joining which was error-prone. These data indicate that mechanisms that produce chromosome breakage in ataxia telangiectasia cells can be reflected in processing of plasmid vectors.     

Biosynthesis rates and content of thymosin beta 4 in cell lines

The content and relative biosynthetic rates of thymosin beta 4 have been determined in 28 different cell lines. The highest content of thymosin beta 4 as well as the highest rate of biosynthesis was observed in Epstein-Barr virus-transformed human B-cell lines. The levels observed in these cells are 1 pg thymosin beta 4 per cell, which is three times higher than that in rat peritoneal macrophages. Thus, these B-cell lines have the highest content of thymosin beta 4 of any cell type yet described. Since all of the Epstein-Barr virus-transformed B-cells described here grow in suspension, it is unlikely that the presence of thymosin beta 4 is related to anchorage in these cells. Thymosin beta 4 is not secreted by viable Epstein-Barr virus-transformed B cells in culture, suggesting some intracellular function of the peptide. These results indicate that these B-cell lines may be suitable for the study of thymosin beta 4 function.     

DNA polymerase alpha from the nuclear matrix of cells infected with simian virus 40.

The nuclear matrix prepared from normal, simian virus 40 (SV40)-infected, and SV40-transformed cells contained DNA polymerase activities. Approximately 12% of the total DNA polymerase activities in isolated nuclei remained with the nuclear matrix. alpha-polymerase was the major matrix DNA polymerase activity as judged by sensitivity to various inhibitors: aphidicolin, dideoxy-TTP, and N-ethylmaleimide. Approximately 2-4 fold higher DNA polymerase activity was detected in matrices obtained from lytically infected and virus-transformed cells than that found in normal cells. In lytically infected cells, 30-50% of the matrix-bound DNA polymerase activity solubilized by sonication co-sedimented with majority of the matrix T-antigen, and was co-precipitated with anti-T sera. The results suggest that alpha-polymerase and viral T-antigen may form a functional complex in the matrix.     

LINE-1 methylation status of endogenous DNA double-strand breaks

DNA methylation and the repair of DNA double-strand breaks (DSBs) are important processes for maintaining genomic integrity. Although DSBs can be produced by numerous agents, they also occur spontaneously as endogenous DSBs (EDSBs). In this study, we evaluated the methylation status of EDSBs to determine if there is a connection between DNA methylation and EDSBs. We utilized interspersed repetitive sequence polymerase chain reaction (PCR), ligation-mediated PCR and combined bisulfite restriction analysis to examine the extent of EDSBs and methylation at long interspersed nuclear element-1 (LINE-1) sequences nearby EDSBs. We tested normal white blood cells and several cell lines derived from epithelial cancers and leukemias. Significant levels of EDSBs were detectable in all cell types. EDSBs were also found in both replicating and non-replicating cells. We found that EDSBs contain higher levels of methylation than the cellular genome. This hypermethylation is replication independent and the methylation was present in the genome at the location prior to the DNA DSB. The differences in methylation levels between EDSBs and the rest of the genome suggests that EDSBs are differentially processed, by production, end-modification, or repair, depending on the DNA methylation status.     

Myc oncogene-induced genomic instability: DNA palindromes in bursal lymphomagenesis

Genetic instability plays a key role in the formation of naturally occurring cancer. The formation of long DNA palindromes is a rate-limiting step in gene amplification, a common form of tumor-associated genetic instability. Genome-wide analysis of palindrome formation (GAPF) has detected both extensive palindrome formation and gene amplification, beginning early in tumorigenesis, in an experimental Myc-induced model tumor system in the chicken bursa of Fabricius. We determined that GAPF-detected palindromes are abundant and distributed nonrandomly throughout the genome of bursal lymphoma cells, frequently at preexisting short inverted repeats. By combining GAPF with chromatin immunoprecipitation (ChIP), we found a significant association between occupancy of gene-proximal Myc binding sites and the formation of palindromes. Numbers of palindromic loci correlate with increases in both levels of Myc over-expression and ChIP-detected occupancy of Myc binding sites in bursal cells. However, clonal analysis of chick DF-1 fibroblasts suggests that palindrome formation is a stochastic process occurring in individual cells at a small number of loci relative to much larger numbers of susceptible loci in the cell population and that the induction of palindromes is not involved in Myc-induced acute fibroblast transformation. GAPF-detected palindromes at the highly oncogenic bic/miR-155 locus in all of our preneoplastic and neoplastic bursal samples, but not in DNA from normal and other transformed cell types. This finding indicates very strong selection during bursal lymphomagenesis. Therefore, in addition to providing a platform for gene copy number change, palindromes may alter microRNA genes in a fashion that can contribute to cancer development.     

Potassium fluxes and ouabain binding in growing,density-inhibited and rous sarcoma virus-transformed chicken embryo cells

Potassium fluxes, ouabain binding, and Na⁺ and K⁺ intracellular concentrations were determined for cultures of growing normal, density-inhibited and Rous sarcoma virus-transformed chicken embryo fibroblasts. No significant differences in K⁺ influx or ouabain binding were detected between growing normal cells and Rous sarcoma virus-transformed cells; however, ouabain binding and ouabain-sensitive K⁺ influx were 1.5- to 1.8-fold lower in density-inhibited cells. Thus, potassium influx in this system can be classified as a growth-related, but not transformation-specific change. As determined by both flame photometry and radioisotopic (⁴²K) equilibration, growing normal and density-inhibited cells had similar potassium contents, whereas transformed cells exhibited 1.4-fold higher potassium levels. Sodium ion levels, as measured by flame photometry, were also 2- to 4.5-fold higher in transformed than normal or density-inhibited cells. Complementary studies of potassium efflux showed a 1.3- to 1.5-fold higher rate (based on the percentage of pool exiting the cell) in growing normal versus density-inhibited or transformed fibroblasts. Because of the larger potassium pool in transformed cells, efflux based on absolute number of potassium ions is similar in normal and transformed chicken embryo fibroblasts.     

DNA methylation status in somatic and placenta cells of cloned cats

We recently produced 11 cloned kittens by somatic cell nuclear transfer (SCNT) using fibroblasts from a feline fetus (donor A, three kittens), an adult domestic cat (donor B, one kitten), and a deaf adult Turkish Angora cat (donor C, seven kittens). Two kittens were stillborn and three died a month after birth. The donor C-derived kittens did not share their donor's eye color or deafness. To test whether this and the low cloning success rate are due to epigenetic modifications, we compared the methylation of somatic and placental cells from the cloned cats and domestic normal cats by bisulfite mutagenesis sequencing analysis. The DNA methylation of somatic cells from the cloned kittens ranged from 78.0% to 88.9%, and did not differ significantly depending on whether they were stillborn, died early after birth, or were healthy. Donors B and C showed similar levels of methylation (77.0% and 79.1%, respectively), as did somatic cells from normal domestic and Turkish Angora cats (range, 75.7-88.0%). However, donor A showed less methylation (70.6%) than the somatic cells from the kittens derived from it (range, 82.2-88.9%). Moreover, placental cells from three donor C-derived kittens showed significantly higher DNA methylation (range, 76.7-80.5%) than placental cells from normal domestic cats (range, 64.2-74.9%). Thus, methylation of satellite regions in somatic cells may not be responsible for the stillbirth, early death, or different eye and hearing attributes of cloned cats. However, hypermethylation in the placenta of cloned cats may be responsible for low success rates in cloning cats.     

Cell shape and hexose transport in normal and virus-transformed cells in culture

The rate of hexose transport was compared in normal and virus-transformed cells on a monolayer and in suspension. It was shown that: (1) Both trypsin-removed cells and those suspended for an additional day in methyl cellulose had decreased rates of transport and lower available water space when compared with cells on a monolayer. Thus, cell shape affects the overall rate of hexose transport, especially at higher sugar concentrations. (2) Even in suspension, the initial transport rates remained higher in transformed cells with reference to normal cells. Scanning electron micrographs of normal and transformed chick cells revealed morphological differences only in the flat state. This indicates that the increased rate of hexose transport after transformation is not due to a difference in the shape of these cells on a monolayer.     

Hypomethylation of HeLa cell DNA and the absence of 5-methylcytosine in SV40 and adenovirus (type 2) DNA: analysis by HPLC

Methylation of the purified virion DNA of both SV40 and adenovirus (type 2) was measured by high performance liquid chromatography (HPLC) and compared to their hosts, African green monkey kidney cells and Hela cells, respectively. Essentially, no 5-methylcytosine was detected in either viral DNA. Implications for viral gene regulation by methylation are discussed. In comparison with normal human cell DNA methylation levels, HeLa cell DNA methylation is reduced significantly.     

Inhibition of friend murine leukemia virus production by low-ionic-strength medium.

The effect of medium of low ionic strength on the release of virus from Friend leukemia cells has been studied. The release of infectious Friend leukemia virus is almost completely inhibited in medium of low ionic strength, as measured by a focus-forming assay (XC assay), by endogenous RNA-dependent DNA polymerase activity of released virus particles, and by electron microscope studies of the production of C-type particles. Friend leukemia virus-transformed proerythroblasts undergo extensive morphological changes in low-ionic-strength medium. The cells are viable in this medium, but they can no longer be stimulated with dimethyl sulfoxide to produce hemoglobin and increase virus production. Infectious virus is released between 30 and 120 min of resuspension of inhibited cells in normal medium. The rate of virus release after reversal of the inhibition is much greater than the rate of virus release during normal cell growth. The morphological changes occurring after dimethyl sulfoxide stimulation of Friend leukemia cells are compared with those resulting from resuspension in normal medium of cells inhibited by low ionic strength.