Enumeration of 6-thioguanine-resistant tumour cells using flow cytometry and comparison with a microtitration cloning assay

Measurement of mutant frequency in tumour specimens has been hampered by low cloning efficiency in soft agar. A method was developed to detect cell proliferation using the thymidine analogue 5-bromo-2'-deoxyuridine (BrUdR). BrUdR incorporation was monitored by immunofluorescent staining of fixed cells using a monoclonal antibody highly specific for this nucleoside analogue. The 6-thioguanine (6TG) exposure conditions which inhibited DNA synthesis, as measured by BrUdR incorporation, in wild-type cells while allowing proliferation of spontaneous hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutants were investigated using tumour cell lines. It was shown that exposure to 10(-5) M BrUdR for the equivalent of 1 cell cycle time did not affect growth of wild-type cells, nor did it affect the growth of HPRT- mutants in the presence of 6TG. Methods for rapid flow cytometric enumeration of BrUdR-labelled 6TG-resistant cells were developed using fluorescent microspheres as an internal standard. To validate the BrUdR mutation assay, the 6TG mutant frequency (MF) was measured in L1210 R/S, a mouse leukaemic cell line (BrUdR 6TG MF = 7.0 X 10(-5] and the results directly compared with those from a microtitration cloning assay (MF = 4.6 X 10(-5]. The results were similar and within the range reported for HPRT MF in mammalian cells.     

Immunohistochemical detection of proliferating cells in vivo

Incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine (BrUdR) into newly synthesized DNA provides the basis of a simple technique for identifying proliferating cells. BrUdR was administered to C57BL/6 mice by continuous infusion for 1-7 days, or by intraperitoneal injection for shorter intervals. Various tissue types, including gut, kidney, and liver, were excised, fixed in neutral buffered formalin, and paraffin-embedded for sectioning. De-paraffinized 4-micron tissue sections and bone marrow samples were incubated with an anti-BrUdR antibody and cells that had traversed S-phase during the BrUdR exposure period were identified immunohistochemically. Proliferation and migration of intestinal epithelial cells were identified by antibody staining after continuous in vivo exposure to BrUdR for 1-4 days, and BrUdR incorporation into proliferating marrow cells was detected within 30 min. Tissues such as normal liver, known to have low levels of proliferation, remained unstained after 3 days' exposure to BrUdR. After we established that normal proliferating cells could be identified using this technique, BrUdR was administered to mice bearing B16 melanomas. Again, proliferating tumor cells were clearly identified in histological sections. The nuclei from these paraffin-embedded tumors were also collected for flow cytometric analysis after de-waxing, rehydration, and pepsin treatment. This combination of techniques made possible the comparison in adjacent tissue sections of labeling index, obtained from stained sections, with percentage S-phase, measured using DNA flow cytometry. The % S-phase was consistently higher than the labeling index obtained with immunocytochemistry, and two-parameter DNA vs BrUdR flow cytometry showed that this difference could be accounted for by a population of unlabeled cells with an S-phase DNA content.     

Effect of estradiol on the early incorporation of (3H) thymidine into uterine DNA on immature mouse.

The incorporation of [3H]thymidine into uterine DNA was markedly depressed within 10 to 30 minutes after intraperitoneal administration of 17beta-estradiol to immature mouse. Maximum inhibition occurred about 6 hours after the hormone was administered. Uterine DNA content and the amount of [3H]thymidine incorporated into the acid-soluble fraction was not affected during the period of hormone-induced inhibition. Moreover, the in vitro incorporation of [3H]thymidine by isolated estradiol-treated mouse uterus was blocked. In contrast to the uterus, 17beta-estradiol did not influence the incorporation of thymidine into mouse liver DNA. Evidence is presented to show that the incorporation of thymidine into uterine DNA was blocked initially by 17beta-estradiol.     

Uptake and fate of bacteriophage phi W-14 DNA in competent Bacillus subtilis.

Phage phi W-14 DNA (in which one-half of the thymine residues are replaced by alpha-putrescinyl thymine) was taken up by competent Bacillus subtilis cells at a rate threefold higher than the rate of homologous DNA uptake. In contrast to other types of heterologous DNA, the amount of phi W-14 DNA taken up in 15 min exceeded the amount of homologous DNA taken up by a factor of two to three, as measured in terms of acid-precipitable material. The amount of phi W-14 DNA taken up was even greater than this analysis indicated if allowance was made for the fact that phi W-14 DNA was degraded more rapidly after uptake than homologous DNA. Competition experiments showed that the affinity of phi W-14 DNA for homologous DNA receptors was lower than the affinity of homologous DNA and was similar to the affinities of other types of heterologous DNA. The more rapid and more extensive uptake of phi W-14 DNA appeared to occur via receptors other than the receptors for homologous DNA, and these receptors (like those for homologous DNA) were an intrinsic property of competent cells. Uptake of phi W-14 DNA was affected by temperature, azide, EDTA, and chloramphenicol, as was uptake of homologous DNA. This was consistent with entry of both DNAs by means of active transport. After uptake, undegraded phi W-14 [3H]DNA was found in the cells in a single-stranded form, whereas a portion of the label was associated with recipient DNA, presumably as a result of incorporation of monomers resulting from degradation. Acetylation of the amino groups of the putrescine side chains in phi W-14 DNA decreased the affinity of this DNA for its receptors without affecting its ability to compete with homologous DNA.     

Effects of thymidine analogues on murine and human cells.

Three mouse tumour cell lines grew continuously in 3 micro M 5-bromodeoxyuridine (BUdR). One line (MC-2) produced a retrovirus and altered in morphology in the presence of BUdR or 5-iododeoxyuridine (IUdR). These effects, which could be reversed by growth in normal medium were similar to those reported for the B-16 mouse melanoma line. The B-16 line used in this study, however, as well as a variety of human cells (six melanoma lines and three fibroblast strains), were much more sensitive to BUdR, 0.03-0.1 micro M being the maximum tolerated levels for continuous growth. No virus production or changes in morphology were induced in these cells by BUdR, deoxyuridine (UdR), 5-fluorodeoxyuridine (FUdR) or thymidine (TdR). The results of cell labelling and growth studies showed a correlation of incorporation of BUdR into DNA with toxicity. Compared on a competitive basis with 1 micro M TdR, the order of incorporation of 1 micro M nucleosides by two human cell lines was TdR = BUdR = IUdR greater than UdR greater than FUdR. In contrast to previous reports that FUdR is incorporated into RNA but not into DNA, half of the FUdR label was found in alkalistable, DNase-sensitive material. Over 90% of the other compounds was incorporated into DNA. All of the UdR and 60% of the IUdR label was incorporated as thymidine; this conversion could be inhibited by labelling in the presence of FUdR.     

Interaction of non-histone proteins with DNA and chromatin from Drosophila and mouse cells. Specificity, isolation and analysis of complexes.

The specificity of the binding of purified non-histone proteins to DNA has been investigated through two types of experiments. Using a nitrocellulose filter assay at a low protein/DNA ratio, the binding of mouse non-histone proteins to mouse DNA was twice as great as the binding of mouse non histone protein to Drosophila DNA. The reverse experiment using Drosophila non-histone protein confirmed the interpretation that some protein . DNA complexes were specific. Protein . DNA complexes isolated by gel filtration chromatography indicated that 20% or 10% of the non-histone protein was bound to homologous or heterologous DNA respectively. Purified non-histone proteins bound with lower efficiency (15%) than unpurified but with higher specificity to soluble chromatin than to naked DNA. This binding did not result from an exchange between chromatin non-histone proteins and purified non-histone proteins added in excess. DNA-bound and chromatin-bound proteins were analysed on polyacrylamide gels. Whereas no major qualitative differences were observed with DNA-bound proteins, some proteins bound to homologous mouse chromatin were different from those bound to heterologous Drosophila chromatin. These results suggest a possible role of DNA-bound non-histone proteins in the regulation of gene expression.     

Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA.     

Homologous recombination of polyoma virus DNA in mouse cells

Summary We have produced nonviable deletion mutants of polyoma virus in order to study homologous recombination after DNA transfection into mouse cells. The frequency of recombination was determined by the formation of infectious virus. It was dependent on the amount of DNA transfected and the size of the region of homology between the mutations. Recombination frequencies were highest when both mutated genomes were transfected in closed circular form rather than after linearization of one or both of the recombination partners. The system described may be useful for a more detailed analysis of physiological and genetic conditions influencing the frequency of homologous recombination in mouse cells as well as to study enzymes involved and intermediates produced in this process.     

Optimal conditions for uptake of exogenous DNA by Chinese hamster lung cells deficient in hypoxanthine-guanine phosphoribosyltransferase.

Conditions were characterized for maximizing the uptake of exogenous mammalian cell DNA by hypoxanthine-guanine phosphoribosyltransferase-deficient Chinese hamster lung cells. Recipient cell cultures in an exponential growth phase were found to be more competent in taking up DNA than stationary cultures. Polyornithine enhanced the uptake of exogenous DNA more reproducibly and to a greater extent than did any of the other facilitators tested (DEAE-dextran, CaCl2, latex spheres, spermine, polylysine and polyarginine). Maximal DNA incorporation occurred when polyornithine and DNA were mixed together prior to inoculation. About 25-30% of the DNA inoculum became deoxyribonuclease-resistant in a typical experiment utilizing polyornithine as the facilitator. Both homologous and heterologous exogenous DNAs rapidly became associated with recipient cell nuclei: approximately 95% of the deoxyribonuclease-resistant donor DNA was nuclear-associated 15 min after inoculation.     

Interference of DNA sequence divergence with precise recombinational DNA repair in mammalian cells.

Studies done in prokaryotes and eukaryotes have indicated that DNA sequence divergence decreases the frequency of homologous recombination. To determine which step(s) of homologous recombination is sensitive to DNA sequence divergence in mammalian cells we have used an assay that does not rely on the recovery of functional products. The assay is based on the acquisition by homologous recombination of endogenous LINE-1 sequences by exogenous LINE-1 sequences. In parallel experiments, we introduced into mouse cells two gapped exogenous LINE-1 sequences, one from the mouse, L1Md-A2, and the other from the rat, L1Rn-3. Although L1Rn-3 is on average less than 85% homologous to the LINE-1 elements of the mouse, the frequency of homologous recombination with endogenous LINE-1 elements obtained with L1Rn-3 was the same as the one obtained with L1Md-A2 which is on average 95% homologous to the LINE-1 elements of the mouse. The endogenous LINE-1 sequences rescued by L1Rn-3 were 8-18% divergent from L1Rn-3 sequences, whereas those rescued by L1Md-A2 were 2-5% divergent from L1Md-A2 sequences. The gap which had been introduced into the exogenous LINE-1 sequences had been precisely repaired in 50% of the recombinants obtained with L1Md-A2. None of the L1Rn-3 recombinants showed precise gap repair.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium orthovanadate stimulation of DNA synthesis in Nakano mouse lens epithelial cells in serum-free medium

Quiescent cultured Nakano mouse lens cells incubated for 40 hours with sodium orthovanadate incorporated 3H-thymidine at an accelerated rate; the greatest response occurred at 20 microM vanadate, whereas by 2 microM an incorporation rate equivalent to unstimulated cells was noted. Microscopic examination of the cells revealed that those exposed to concentrations of vanadate greater than 100 microM had lysed by the end of the 40-hour incubation. Reduction in vanadate exposure time to 1 hour caused the cells to incorporate the greatest amount of 3H-thymidine at a vanadate concentration of 200 microM to 500 microM. Half-maximum incorporation of 3H-thymidine (after a 40-hour incubation) was induced by a 2-hour incubation with 20 microM vanadate. Studies with insulin showed that while 20 ng/ml insulin alone did not increase 3H-thymidine incorporation, 20 ng/ml insulin in combination with 20 microM vanadate resulted in a significant increase in 3H-thymidine uptake over cells exposed to only vanadate. Insulin alone will increase cell number and insulin with vanadate are synergistic in the stimulation of DNA synthesis, but the two together show no further increase in cell number over that produced by insulin alone. Thus, vanadate can increase progression from G1/G0 to S-phase, but cannot stimulate cells to divide. Studies designed to detect DNA damage and repair rather than S-phase DNA synthesis demonstrated that vanadate was not causing increased 3H-thymidine uptake by damaging DNA. Cell counts revealed that vanadate, while able to induce DNA synthesis, does not induce mitosis. Autoradiography and equilibrium sedimentation experiments demonstrated that gene amplification was not occurring. A known vanadate exchange inhibitor blocked the ability of vanadate to increase 3H-thymidine incorporation which is consistent with the idea that cellular internalization of vanadate is required for this effect to be seen. 86Rb+ uptake experiments demonstrate that the vanadate concentration inducing 50% inhibition of (Na+, K+)ATPase is nearly two orders of magnitude more concentrated that vanadate concentrations shown capable of inducing 3H-thymidine uptake. This strongly suggests that (Na+, K+)ATPase inhibition is not the central mechanism by which DNA synthesis is stimulated by vanadate.     

Effect of N-trifluoroacetyladriamycin-14-valerate on [3H]thymidine uptake and DNA synthesis of human lymphoma cells

The effects of N-trifluoroacetyladriamycin-14-valerate on the uptake of [3H]thymidine and its incorporation into DNA of human P3HR-1 lymphoma cells were studied. In the absence of the drug, at 0 degrees C, [3H]thymidine was transported into the cells but not incorporated into DNA, as determined by both the trichloroacetic acid-soluble and -precipitable counts obtained with the cells. At 37 degrees C, [3H]thymidine was readily transported into the cells and incorporated into DNA. In the presence of the drug, both [3H]thymidine uptake (as shown by acid-soluble counts) and the amount of its incorporation into acid-precipitable materials were markedly reduced. However, the uptake of [3H]thymidine at 0 degrees C was found to be equally sensitive to drug inhibition as at 37 degrees C. The incorporation at 37 degrees C of [3H]thymidine into acid-precipitable materials of the cells, which had been prelabeled at 0 degrees C with [3H]thymidine, was found to be insensitive to inhibition by the drug. The in vitro activities of DNA polymerases alpha and beta purified from human P3HR-1 cells were also found not to be susceptible to inhibition. Nuclei purified from cells pretreated with the drug continued to synthesize DNA. The cytofluorograms of the cells treated with the drug indicated that the treated cells accumulated at the G2/M phase, whereas the S phase of the cells was not arrested. These results suggest that N-trifluoroacetyladriamycin-14-valerate inhibits [3H]thymidine uptake but not cellular DNA synthesis in human P3HR-1 lymphoma cells.     

Incorporation of homologous and heterologous proteins into the envelope of Moloney murine leukemia virus.

The efficiencies with which homologous and heterologous proteins are incorporated into the envelope of Moloney murine leukemia virus (M-MuLV) have been analyzed by utilizing a heterologous, Semliki Forest virus-driven M-MuLV assembly system and quantitative pulse-chase assays. Homologous M-MuLV spike protein was found to be efficiently incorporated into extracellular virus particles when expressed at a relatively low density at the plasma membrane. In contrast, efficient incorporation of heterologous proteins (the spike complex of Semliki Forest virus and a cytoplasmically truncated mutant of the human transferrin receptor) was observed only when these proteins were expressed at high densities at the cell surface. These results imply that homologous and heterologous proteins are incorporated into the M-MuLV envelope via two distinct pathways.     

Termination of DNA synthesis by 9-beta-D-arabinofuranosyl-2-fluoroadenine. A mechanism for cytotoxicity.

The action of 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) on DNA synthesis was evaluated both in whole cells and in vitro. 9-beta-D-Arabinofuranosyl-2-fluoroadenine was converted to its 5'-triphosphate 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (F-ara-ATP) in cells and then incorporated into DNA in a self-limiting manner. More than 94% of the analogue was incorporated into DNA at the 3' termini, indicating a chain termination action. In vitro DNA primer extension experiments further revealed that F-ara-ATP compared with dATP for incorporation into the A site of the extending DNA strand. The incorporation of F-ara-AMP into DNA resulted in termination of DNA strand elongation. Human DNA polymerase alpha incorporated more F-ara-AMP into DNA than polymerase epsilon (proliferating cell nuclear antigen-independent DNA polymerase delta) and was more sensitive to inhibition by F-ara-ATP. On the other hand, DNA polymerase epsilon was able to excise the incorporated F-ara-AMP from DNA in vitro. The incorporation of F-ara-AMP into DNA was linearly correlated both with inhibition of DNA synthesis and with loss of clonogenicity; thus it may be the mechanism of cytotoxicity.     

Induction of mutation in mouse FM3A cells by N4-aminocytidine-mediated replicational errors.

To explore the potential use of a nucleoside analog, N4-aminocytidine, in studies of cellular biology, the mechanism of mutation induced by this compound in mouse FM3A cells in culture was studied. On treatment of cells in suspension with N4-aminocytidine, the mutation to ouabain resistance was induced. The major DNA-replicating enzyme in mammalian cells, DNA polymerase alpha, was used to investigate whether the possible cellular metabolite of N4-aminocytidine, N4-aminodeoxycytidine 5'-triphosphate (dCamTP), can be incorporated into the DNA during replication. Using [3H]dCamTP in an in vitro DNA-synthesizing system, we were able to show that this nucleotide analog can be incorporated into newly formed DNA and that it can serve as a substitute for either dCTP or dTTP. dCamTP in the absence of dCTP maintained the activated calf thymus DNA-directed polymerization of deoxynucleoside triphosphates as efficiently as in its presence. Even in the presence of dCTP, dCamTP was incorporated into the polynucleotide. When dCamTP was used as a single substrate in the poly(dA)-oligo(dT)-directed polymerase reaction, it was incorporated into the polynucleotide fraction. The extent of incorporation was 4% of that of dTTP incorporation when dTTP was used as a single substrate. Even in the presence of dTTP, dCamTP incorporation was observed. A copolymer containing N4-aminocytosine residues was shown to incorporate guanine residues opposite the N4-aminocytosines. However, we were unable to observe adenine incorporation opposite N4-aminocytosine in templates. These cell-free experiments show that an AT-to-GC transition can take place in the presence of dCamTP during DNA synthesis, strongly suggesting that the mutation induced in the FM3A cells by N4-aminocytidine is due to replicational errors.     

Translesional synthesis past acetylaminofluorene-derived DNA adducts catalyzed by human DNA polymerase kappa and Escherichia coli DNA polymerase IV.

Human DNA polymerase kappa (pol kappa) has a sequence significantly homologous with that of Escherichia coli DNA polymerase IV (pol IV). We used a truncated form of human pol kappa (pol kappaDeltaC) and full-length pol IV to explore the miscoding properties of these enzymes. Oligodeoxynucleotides, modified site-specifically with N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-AF), were used as DNA templates in primer extension reactions that included all four dNTPs. Reactions catalyzed by pol kappaDeltaC were partially blocked one base prior to dG-AAF or dG-AF, and also opposite both lesions. At higher enzyme concentrations, a significant fraction of primer was extended. Analysis of the fully extended reaction product revealed incorporation of dTMP opposite dG-AAF, accompanied by much smaller amounts of dCMP, dAMP, and dGMP and some one- and two-base deletions. The product terminating 3' to the adduct site contained AMP misincorporated opposite dC. On templates containing dG-AF, dAMP, dTMP, and dCMP were incorporated opposite the lesion in approximately equal amounts, together with some one-base and two-base deletions. Steady-state kinetics analysis confirmed the results obtained from primer extension reactions catalyzed by pol kappa. In contract, primer extension reactions catalyzed by pol IV were blocked effectively by dG-AAF and dG-AF. At high concentrations of pol IV, full-length products were formed containing primarily one- or two-base deletions with dCMP, the correct base, incorporated opposite dG-AF. The miscoding properties of pol kappa observed in this study are consistent with mutational spectra observed when plasmid vectors containing dG-AAF or dG-AF are introduced into simian kidney cells [Shibutani, S., et al. (2001) Biochemistry 40, 3717-3722], supporting a model in which pol kappa plays a role in translesion synthesis past acetylaminofluorene-derived lesions in mammalian cells.     

Detection of cytosine arabinoside resistant cells at low frequency using the bromodeoxyuridine/DNA assay

This report describes a rapid and sensitive procedure for detection of cytosine arabinoside- (Ara-C) resistant mouse leukemia cells (L1210) in a predominantly Ara-C-sensitive population. L1210 cell lines sensitive or resistant to Ara-C were grown and treated with Ara-C in vitro or in vivo. Ara-C-resistant cells were detected as those cells with S-phase DNA content retaining the ability to incorporate bromodeoxyuridine (BrdUrd) after treatment with Ara-C. The BrdUrd incorporation ability of the S-phase cells was assessed by simultaneous flow cytometric measurement of cellular DNA content and amount of incorporated BrdUrd. The proportion of Ara-C-resistant cells was accurately estimated at frequencies approaching 10(-3).     

Maturation of nascent DNA fragment is disturbed after treatment of cultured mouse FM3A cells with 8-methoxypsoralen plus near-ultraviolet radiation

By the method of sedimentation in 5–20% alkaline sucrose gradient, the process of maturation of the nascent DNA fragment was studied with cultured mouse FM3A cells treated with 8-methoxypsoralen plus near-ultraviolet radiation. This treatment is known to cause crosslinks of the chromosomal DNA strands. The profile of the newly-replicated DNA, labeled for 10 min with [³H]thymidine immediately after treatment, was the same as that of the untreated cells, where the incorporated radioactivity was present in the intermediate DNA fragment (about 50–80 S). But, when the treated cells were labeled after several hours of incubation, the labeled DNA became much shorter due to inhibition of maturation of the initial DNA fragment (the Okazaki fragment) to the intermediate DNA. With the use of aphidicolin, a specific inhibitor of eukaryotic DNA polymerase α, it became apparent that, in addition to formation of the crosslinks, further DNA replication is required to cause this inhibition of DNA maturation. Aphidicolin also suppressed the inhibition of incorporation of [³H]thymidine into cellular DNA after treatment, but inhibition of this incorporation resumed after its removal.