Investigations of the mechanism of decreased tumorigenicity of cells grown in BrdU

Transplantable SV40-transformed hamster cells cultivated in the presence of low concentrations of BrdU for prolonged periods of time and cells made deficient in the enzyme thymidine kinase (dTK) by continued exposure to BrdU became less tumorigenic. In both instances, when grown in BrdU the cells contained analog substituted DNA. The tumorigenicity of dTK⁺ cells exposed to low concentrations of BrdU, but not the dTK^? cells, returned to control values when the cells were grown in medium devoid of BrdU. A tumorigenic mouse cell line made dTK deficient also had diminished oncogenicity. However, transformed hamster cells made deficient in another salvage pathway enzyme, hypoxanthineguanine phosphoribosyl-transferase by growth in eight azaguanine, retained their tumorigenicity. Two of five revertant cell lines, in which thymidine kinase activity was restored, transplanted more readily to hamsters than the dTK^? cells from which they were derived. It is concluded that there is a relative loss of tumorigenicity when BrdU is incorporated into the DNA of tumorigenic cell lines, or when there is a genetic modification of thymidine kinase activity.     

Sister chromatid differentiation after in situ detection of ultraviolet-induced DNA breaks under electron microscopy

Summary— Chinese hamster DON cells with 5-bromodeoxyuridine (BrdU)-substituted chromosomes were ultraviolet (UV)-exposed and processed for in situ detection of induced DNA breaks under electron microscopy. For this purpose, UV-induced breaks were amplified by an exonuclease III digestion to obtain single stranded DNA motifs which could hybridize with oligonucleotides of random sequences. These reannealed motifs could be used as primers which were extended by the Klenow polymerase, incorporating biotinylated-dUTP that was detected by a gold-tagged streptavidin. After processing, the chromatid whose DNA was BrdU-substituted in one strand showed a higher electron density than the chromatid substituted in both strands. In contrast, the unifilarly substituted chromatid showed about twice the labelling of DNA breaks as the bifilarly substituted one. This result could be the consequence of a greater loss of chromatin tracts in the bifilarly substituted chromatid, as implied by an X-ray microanalysis which showed that the amount of phosphorous lost by the bifilarly substituted chromatid was higher than that of the unifilarly substituted chromatid.     

Reverse differential staining of sister chromatids induced by Hoechst plus black light and endonuclease

A differential Giemsa staining between sister chromatids was obtained by treating chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) with Hoechst 33258 plus black light at 55 degrees C (HB pretreatment) and deoxyribonuclease (DNase) I, II, or micrococcal nuclease. In this staining pattern the BrdU bifilarly substituted chromatids were darkly and the unifilarly substituted chromatids lightly stained. This staining pattern was obtained only by staining the HB-DNase I-treated chromosomes with Giemsa and methylene blue, not by several other dyes tested. Relatively more DNA labelling was removed from the non-BrdU-substituted than the BrdU-substituted chromosomes, when the HB-pretreated chromosomes were digested with DNase I. But the protein labelling was not removed appreciably in the same treatment. The differential DNase I sensitivity between the non-BrdU-substituted and BrdU-substituted chromosomes disappeared when the HB-pretreated chromosomes were incubated with proteinase K before The DNase I digestion. Moreover, no differential DNase I sensitivity was found between the HB-pretreated isolated DNA containing and not containing BrdU. We propose that during the HB pretreatment, more DNA-protein cross-linkings are induced in BrdU bifilarly substituted than the unifilarly substituted chromatids. This structure protects the chromosomal DNA against the DNase I digestion. Thus, a reverse differential Giemsa staining between sister chromatids is obtained by the HB-DNase I treatment.     

Proteins of BrdU-Dependent Hamster Cell Lines as Characterized by Two-Dimensional Gel Electrophoresis

Cell lines which exhibit the BrdU-dependent phenotype (B4 and HAB) were studied with respect to BrdU-induced alterations in genetic expression by two-dimensional gel electrophoresis. A comparison of the proteins from the HAB cells, in which the DNA is 100% substituted by BrdU, to those of the unsubstituted parent line (3460) showed 55 protein alterations; the synthesis of 15 increased while that of the other 40 decreased. When 3460 cells were grown in BrdU such that their DNA was > 50% substituted, 27 protein changes could be detected; of these, the synthesis of 10 increased while that of 17 decreased. A comparison of all these changes in the various cell lines showed six which were common to the BrdU-substituted cell lines. The proteins from another Syrian hamster cell line, BHK.-21 (C-13) and those of HAB cells grown in thymidine or BrdC were also examined on two-dimensional gels.
Although BrdU has a dramatic effect on many cellular functions, relatively few changes in the pattern of protein synthesis could be detected in these cell lines, perhaps reflecting the specialized action of this analogue on particular cellular functions.     

Proteins of BrdU-dependent hamster cell lines as characterized by two-dimensional gel electrophoresis.

Cell lines which exhibit the BrdU-dependent phenotype (B4 and HAB) were studied with respect to BrdU-induced alterations in genetic expression by two-dimensional gel electrophoresis. A comparison of the proteins from the HAB cells, in which the DNA is 100% substituted by BrdU, to those of the unsubstituted parent line (3460) showed 55 protein alterations; the synthesis of 15 increased while that of the other 40 decreased. When 3460 cells were grown in BrdU such that their DNA was greater than 50% substituted, 27 protein changes could be detected; of these, the synthesis of 10 increased while that of 17 decreased. A comparison of all these changes in the various cell lines showed six which were common to the BrdU-substituted cell lines. The proteins from another Syrian hamster cell line, BHK-21 (C-13) and those of HAB cells grown in thymidine or BrdC were also examined on two-dimensional gels. Although BrdU has a dramatic effect on many cellular functions, relatively few changes in the pattern of protein synthesis could be detected in these cell lines, perhaps reflecting the specialized action of this analogue on particular cellular functions.     

Opposite staining effect of two silver-staining techniques on sister chromatids

Opposite differential staining between sister chromatids was obtained by two silver-staining techniques on chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) and pretreated with Hoechst plus black light. Both silver-nitrate and silver-carbonate staining were affected by chemical extraction and enzyme digestion of chromosomal proteins. Prestaining of silver nitrate or silver carbonate also blocked the fluorescences of protein dyes. However, removal of chromosomal DNA affected the silver-carbonate but not the silver-nitrate staining; the fluorescences of DNA dyes were blocked by the prestaining of silver carbonate but not silver nitrate. Chromosomal protein labelling was released only slightly and its relative amount between BrdU bifilarly substituted and unifilarly substituted chromatids was unchanged during pretreatment of Hoechst plus black light. We speculate that chromosomal non-histones are the targets for silver-nitrate stain, and DNA-non-histone complexes for silver-carbonate stain.     

Sister-chromatid exchanges and gene mutations are induced by the replication of 5-bromo- and 5-chloro-deoxyuridine substituted DNA

The thymidine (dT) analogue 5-chlorodeoxyuridine (CldU) induces 7–8-fold more sister-chromatid exchanges (SCE) than does 5-bromodeoxyuridine (BrdU) at equal substitution for dT in Chinese hamster ovary cells in culture. This difference facilitates study of the mechanism of induction of SCE by these analoques. Cultures were incubated with either BrdU or CldU for one cell cycle, followed by incubation in the presence of dT alone or BrdU or CldU for the second cell cycle and the SCE frequency determined in M2 cells. The results suggest that the induction of SCE is dependent only on the replication of the analogue-substituted DNA during the second cell cycle. Additional studies employed cultures grown in the presence of BrdU or CldU for 7 days to obtain mainly bifilarly substituted DNA, followed by 2 rounds of replication in the presence of dT alone. The SCE frequencies were approximately twice those found in cultures which had undergone the usual 2 rounds in the presence of the analogue; this is consistent with the replication of twice the amount of analogue-substituted DNA. Furthermore, such long-term growth in the presence of BrdU or CldU also results in concentration-dependent increases in the frequency of 6-thioguanine-resistant mutants, suggesting that gene mutations also result from the replication of analogue-substituted DNA.     

Label-retaining epithelial cells in mouse mammary gland divide asymmetrically and retain their template DNA strands

It has been postulated that the stem cells of somatic tissues protect themselves from mutation and cancer risk by selective segregation of their template DNA strands. Self-renewing mammary epithelial stem cells that were originated during allometric growth of the mammary ducts in pubertal females were labeled using [3H]-thymidine (3HTdR). After a prolonged chase during which much of the branching duct morphogenesis was completed, 3HTdR-label retaining epithelial cells (LREC) were detected among the epithelium of the maturing glands. Labeling newly synthesized DNA in these glands with a different marker, 5-bromodeoxyuridine (5BrdU), resulted in the appearance of doubly labeled nuclei in a large percentage of the LREC. By contrast, label-retaining cells within the stroma did not incorporate 5BrdU during the pulse, indicating that they were not traversing the cell cycle. Upon chase, the second label (5BrdU) was distributed from the double-labeled LREC to unlabeled mammary cells while 3HTdR was retained. These results demonstrate that mammary LREC selectively retain their 3HTdR-labeled template DNA strands and pass newly synthesized 5BrdU-labeled DNA to their progeny during asymmetric divisions. Similar results were obtained in mammary transplants containing self-renewing, lacZ-positive epithelial cells suggesting that cells capable of expansive self-renewal may repopulate new mammary stem cell niches during the allometric growth of new mammary ducts.     

Reduced N-methyl-N′-nitro-N-nitrosoguanidine sister chromatid exchange induction in chinese hamster V79 cells pre-exposed to 5-bromodeoxyuridine

The sequence in which N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and 5-bromodeoxyuridine (BrdU) are added to cell cultures affects the number of sister chromatid exchanges (SCE) induced by MNNG. When V79 Chinese hamster cell monolayer cultures were treated with MNNG for 2 h prior to addition of BrdUrd, approximately a 4–5-fold increase in SCE was observed at the second division metaphases compared to controls exposed to BrdU alone. This effect was independent of whether one or three DNA strands had been substituted as a result of incubating the cells through one or two DNA synthesis periods in the presence of BrdU. This increase in SCE also occurred after MNNG exposure and BrdU incubation was extended for three division cycles. In contrast, when BrdU incorporation preceded MNNG treatment, the average number of SCE/metaphase was reduced 70–80% at the second division cycle and 60% relative to the total number found in three division cycles. SCE induction by MNNG does not involve a caffeine sensitive step since caffeine had no effect on the SCE frequency regardless of the treatment protocol. The conditions in which BrdU preceded MNNG exposure may be responsible for either reducing the number of DNA sites available for interaction with MNNG or preventing the expression of SCE.     

Induction of erythroid differentiation in Friend leukemia cells by bromodeoxyuridine

Treatment of Friend leukemia cells with BrdU, the thymidine analog which interferes with DMSO induced differentiation in these cells as well as the expression of differentiated character in many other cell systems, is capable of inducing erythroid differentiation. Globin mRNA, as assayed by hybridization to globin cDNA, increases 2.5- to 30-fold after appropriate treatment with BrdU. This effect was observed with several different subclones of three independent Friend tumor cell lines. After BrdU treatment, globin mRNA content may reach up to 10-20% of the levels in DMSO induced cultures. The induction of erythroid differentiation is also apparent when accumulated heme content or the appearance of benzidine positive cells is monitored. One Friend cell line (745) we examined was not induced by BrdU although it incorporated an amount of BrdU into its DNA comparable to that incorporated by the other cell lines. In addition, BrdU did interfere with DMSO induction in this cell line. These results suggest that two different mechanisms may be operative in regulating erythroid differentiation in Friend leukemia cells. While BrdU interferes with the mechanism activated by DMSO treatment, this analog could independently activate an alternative mechanism.     

"Trophic" effect of transferrin on amphibian limb regeneration blastemas

In light of the recent demonstration that one "neurotrophic factor" of peripheral nerves is the iron-transport glycoprotein transferrin, we tested the effects of heterologous transferrin on cellular events in cultured newt forelimb blastemas. Addition of transferrin to medium containing 1% fetal bovine serum resulted in DNA labeling and mitotic activity approximately twice as high as that of blastemas cultured in medium with 1% serum alone. Blastemas maintained for 24 hr in medium with 1% serum were stimulated to increased levels of DNA synthesis by the addition of transferrin, and this response was dose-dependent. Varying the concentrations of iron and transferrin in the medium gave results indicating that the glycoprotein's trophic effect is due to its ability to furnish iron to the cells in an appropriate manner. Results of the study are consistent with the hypothesis that blastema cell proliferation is promoted by transferrin or transferrin-like factors released from nerves.     

The diverse effects of 5'-bromodeoxyuridine on enzyme activities in cultured H35 hepatoma cells.

Reuber (H35) hepatoma cells were grown in medium containing 10(-5)M bromodeoxyuridine (BrdU), which was incorporated into their DNA. Cell growth rate was unaffected by BrdU for the first two generations, after which it was reduced by about 50%. The effect of BrdU incorporation on the activities of several enzymes with rapid turnover rates was examined to test the hypothesis that the synthesis of such enzymes will be preferentially inhibited by BrdU. Tyrosine amino-transferase (TAT) activity decreased by 70% within two generations whereas thymidine kinase activity remained at control values. PEP carboxykinase activity was unchanged during the first generation in BrdU-containing medium but, during the second, its activity increased by at least 30%. Ornithine decarboxylase levels decreased by about 50% only after two generations in the presence of BrdU. There appeared to be no simple relationship between turnover rates and the effect of BrdU on enzyme activity. Incorporation of BrdU was found to inhibit the induction of both TAT and PEP carboxykinase by dexamethasone and to enhance the inhibition of cell growth by this steroid. These results are discussed with respect to possible mechanisms of gene expression and development in both normal and neoplastic cells.     

ROLE OF MULTIPLE FTSZ RINGS IN CHLOROPLAST DIVISION UNDER OLIGOTROPHIC AND EUTROPHIC CONDITIONS IN THE UNICELLULAR GREEN ALGA NANNOCHLORIS BACILLARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE)1

Chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann cultured under nutrient‐enriched conditions have multiple rings of FtsZ, a prokaryote‐derived chloroplast division protein. We previously reported that synthesis of excess chloroplast DNA and formation of multiple FtsZ rings occur simultaneously. To clarify the role of multiple FtsZ rings in chloroplast division, we investigated chloroplast DNA synthesis and ring formation in cells cultured under various culture conditions. Cells transferred from a nutrient‐enriched medium to an inorganic medium in the light showed a drop in cell division rate, a reduction in chloroplast DNA content, and changes in the shape of chloroplast nucleoids as cells divided. We then examined DNA synthesis by immunodetecting BrdU incorporated into DNA strands using the anti‐BrdU antibody. BrdU‐labeled nuclei were clearly observed in cells 48 h after transfer into the inorganic medium, while only weak punctate signals were visible in the chloroplasts. In parallel, the number of FtsZ rings decreased from 6 to only 1. When the cells were transferred from an inorganic medium to a nutrient‐enriched medium, the number of cells increased only slightly in the first 12 h after transfer; after this time, however, they started to divide more quickly and increased exponentially. Chloroplast nucleoids changed from punctate to rod‐like structures, and active chloroplast DNA synthesis and FtsZ ring formation were observed. On the basis of our results, we conclude that multiple FtsZ ring assembly and chloroplast DNA duplication under nutrient‐rich conditions facilitate chloroplast division after transfer to oligotrophic conditions without further duplication of chloroplast DNA and formation of new FtsZ rings.     

Effects of thymidine analogs on Syrian hamster melanoma cells: phenotypes arising from selection for analog resistance

In order to compare the biological effects of different thymidine (dT) analogs, two unusual cell lines (B-4 and HAB) previously isolated from a Syrian hamster melanoma line by selection with 5-bromodeoxyuridine (BrdU) were analyzed for their response to other analogs. B-4 cells require high concentrations of BrdU for optimal growth, and it was seen that the requirement for BrdU could be satisfied partially by 5-chlorodeoxyuridine (CldU) but not by the other dT analogs tested. HAB cells are able to grow with all the dT residues in nuclear DNA replaced by BrdU, and it was found that they could also grow with essentially all the dT residues in nuclear DNA replaced by CldU but not by other analogs. New cell lines resistant to 100 micrometer concentrations of CldU, 5-iododeoxyuridine (IdU), and 5-hydroxymethyldeoxyuridine (HMdU) were isolated from the melanoma line and tested for cross-resistance to the other dT analogs. A high level of cross-resistance was observed only with BrdU and CldU. The ability of the cell lines resistant to BrdU, CldU, and IdU to incorporate these analogs into nuclear DNA also was determined. BrdU and CldU were incorporated efficiently by all of the lines tested, but the IdU-resistant cells seemed to preferentially exclude IdU.     

Flow cytometric analysis of chromosomes and cells using a modified BrdU-Hoechst method

Chromosomes and interphase cells were harvested from cultures of the Chinese hamster line B14 F28 grown in medium containing BrdU up to four cell cycles and stained with the fluorescent dye 33342 Hoechst for flow cytometry. The newly synthetized BrdU-DNA is not stainable by the Hoechst dye which is highly specific for thymidine. The temporal development of the DNA fluorescence after addition of BrdU to the growth medium has been investigated. The chromosomal fluorescence intensity is reduced one step per generation. The extent of the intensity decrease by BrdU incorporation is proportional to the amount of new DNA and it is realized by repeated measurement following an UV-exposure. This UV-illumination stops the quenching by BrdU of the Hoechst stain induced DNA fluorescence. Therefore, the entire DNA content of these chromosomes now becomes measurable. The obtained intensity gain serves as a measure of the extent of the previous BrdU caused intensity shift. In this way we could establish 3 successive mitoses. Principally, this method is suitable also for measurement of whole cells in order to obtain both the number of generations in the experimental period and the phase distribution of the cell cycle.     

Induction of sister chromatid exchanges by 5-fluorodeoxycytidine: correlation with DNA methylation

Chinese hamster ovary cells grown in medium containing 10 M 5-fluorodeoxycytidine for forty-eight hours were found to have up to 5% of the deoxycytidine residues of the DNA substituted by this analog. Cytological studies of these cells showed that the incorporated 5-FdC caused a two-fold increase of sister chromatid exchanges (SCE) over the control level. However, 5-FdC was capable of inducing SCE only after it had been present in the cell for at least two cycles of DNA synthesis. This is in contrast to several other chemicals that we have tested which induced SCE immediately after the first DNA synthesis. We consider the possibility that the delayed effect may be related to hypomethylation of cytosine in the newly replicated DNA.     

Growth and differentiation of epidermal cells from the rainbow trout established as explants and maintained in various media

Growth and a number of differentiated characteristics of cultured epidermal cells from the rainbow trout Oncorhynchus mykiss were compared using two commercially available serum–free media, a dermal substrate/serum free kit and a serum–containing medium which had been previously optimized for epidermal cell culture. Each medium supported short term growth over 15 days. Only the medium supplied for dermal substrate culture supported longer growth periods. This medium was supplied for use with a collagen/stromal substrate but gave good cultures even without the substrate. Differentiation, measured by examining mucous cells, cytokeratins, epidermal growth factor receptor, gap junction status and ultrastructure showed that serum–free media gave quantitatively and qualitatively superior expression and short term retention of differentiation over serum–containing medium. Epithelial cell growth with expression of differentiated characteristics can be maintained in primary culture in serum–free medium for at least as long as in serum–containing medium. This provides a useful technique for use when serum presence in medium is undesirable or proves toxic to the specialized cell type under investigation.     

Altered thymidylate kinase substrate specificity in mammalian cells selected for resistance to iododeoxyuridine

A clone of Syrian hamster melanoma cells was selected for resistance to high levels of the thymidine (dT) analog 5-iododeoxyuridine (IdU). Unlike cell lines previously isolated as IdU resistant (IdU^r), these IdU^r lines had normal levels of thymidine kinase (EC 2.7.1.21) activity, grew in HAT medium, and readily incorporated exogenous dT and 5-bromodeoxyuridine (BrdU) into DNA. However, these IdU^r cells were found to preferentially exclude IdU from their DNA. Analyses of the soluble nucleotide pools of the IdU^r cells showed that they were able to take up and phosphorylate exogenous dT as well as the wild-type cells, and both mutant and wild-type cells accumulated dTTP as the major phosphorylated component. In contrast, while the wild-type cells in the presence of exogenous IdU accumulated significant levels of IdUTP (as well as IdUMP), the IdU^r cells accumulated only IdUMP. Thus, the mutant cells appear to have a markedly decreased ability to phosphorylate IdU beyond the monophosphate level. Assays of thymidylate kinase (EC 2.7.4.9) activity in extracts of the IdU^r cells indicated a marked preference for dTMP as substrate over IdUMP (in comparison to the wild-type enzyme activity). The cell lines described in this study represent a new phenotype arising from selection for resistance to a halogenated dT analog. The resistance appears to involve a change in the substrate specificity of thymidylate kinase, such that the enzyme in the IdU^r cells has an enhanced ability to discriminate between very closely related compounds.     

Possible effect of the cell synchronization method on the pattern of 5-bromodeoxyuridine incorporation into early synthesized DNA

Synchronously and asynchronously growing chick embryo fibroblasts have been used to study the pattern of 5-bromodeoxyuridine (BrdU) incorporation into DNA. In the synchronous cell system, the density of unifilarly substituted DNA is about 0.010 g/ml higher during first half of S phase than during second half of S phase. The density of unifilarly substituted DNA isolated from asynchronously growing cells is similar to that of DNA synthesized during the second half of S phase of synchronously growing cells for a given concentration of analogue in culture medium. Reassociation kinetics experiments have shown the oversubstitution to occur at the level of early synthesized repeated and/or intermediate DNA sequences. It is then assumed that the oversubstitution is due to some metabolic changes caused by the synchronization procedure itself. As BrdU incorporation into early replicating DNA is known to induce alterations of the cell metabolism, the implication of this phenomenon is discussed at the level of the inhibition of transformation which takes place when chick embryo fibroblasts are infected with Rous sarcoma virus during G1 and subsequently treated with BrdU during early S phase.