Cell kinetics of human tumors by in vitro bromodeoxyuridine labeling

We labeled active S-phase cells in primary breast carcinomas with a modified 5-bromo-2'-deoxyuridine (BrdU) procedure using a silver-enhanced colloidal gold visualization step. Separate samples of 29 tumors were labeled with BrdU or tritiated thymidine ([3H]-dThd), and the labeling indices (LI) from the two methods were equivalent (Spearman's correlation coefficient = 0.96). Three breast carcinomas were incubated in various mixes of both BrdU and [3H]-dThd and developed sequentially for each. Paired photomicrographs showed that the same nuclei were labeled by either precursor. The in vitro method yielded LIs similar to those reported after in vivo pulse BrdU labeling for tumors of the central nervous system. The BrdU LI correlated significantly (r = 0.76, p less than 0.001) with % S-phase by DNA flow cytometry in 33 breast carcinomas. The BrdU labeling method is simpler and more rapid than the [3H]-dThd procedure (1-2 days for completion for the former, 7-10 days for the latter), and it provides an equivalent measurement of proliferative index.     

The somatic mutation landscape of the human body

A major challenge in the analysis of DNA methylation (DNAm) data is variability introduced from intra-sample cellular heterogeneity, such as whole blood which is a convolution of DNAm profiles across a unique cell type. When this source of variability is confounded with an outcome of interest, if unaccounted for, false positives ensue. Current methods to estimate the cell type proportions in whole blood DNAm samples are only appropriate for one technology and lead to technology-specific biases if applied to data generated from other technologies. Here, we propose the technology-independent alternative: methylCC, which is available at https://github.com/stephaniehicks/methylCC.     

Iron-supplemented bovine serum as an alternative to fetal bovine serum in the CHO/HGPRT mutation assay

Iron-supplemented bovine calf serum (ICS) was found to be a viable alternative to fetal bovine serum (FBS) in the growth promotion and cloning efficiency of Chinese hamster ovary (CHO) cells that are used in the HGPRT mutation assay. Suspension cultures of CHO cells had an average generation time of 11.5 h in ICS and 13.6 h for cells maintained in FBS. This slight difference was due to lot variability on the part of FBS and could be eliminated by routine quality control measures. The average cloning efficiencies for CHO cells cloned in either ICS or FBS were 107% and 88%, respectively, and these values were not statistically different. No appreciable difference was noted in the spontaneous mutation rates of cells cloned in either ICS or FBS. Furthermore, the use of ICS in mutagenicity studies with genotoxic agents shows the serum to be at least equal or superior to FBS in the detection of both direct-acting mutagens and promutagens. These data suggest that ICS is an appropriate serum to be used in the CHO/HGPRT test system. Since ICS is more readily available and considerably less costly than FBS, a substantial reduction in the cost of the assay can be realized.     

Rapid in vitro bromodeoxyuridine labeling method for monitoring of therapy response in solid human tumors

In vitro bromodeoxyuridine (BrdUrd) incubated single-cell suspensions obtained from solid tumors were fixed on slides for subsequent sample processing. As dispersal of nuclei largely was avoided, only small amounts of cells were needed for examination. The sensitivity of detecting even low BrdUrd incorporation rates could be improved by treatment with intense DNA denaturation conditions. This technique was applied to monitor cytokinetic response to chemotherapy and radiation in oral carcinomas by analysing biopsies taken consecutively in the course of treatment. By combining BrdUrd labeling and DNA flow cytometry, cells arrested in S phase easily could be distinguished from cells showing continuous proliferation.     

The use of distamycin A in human lymphocyte cultures

Summary The effect of the oligopeptide antibiotic distamycin A on human lymphocyte cultures was examined. Distamycin A specifically inhibits the condensation of the Y heterochromatin and induces a fragile site in the chromosome 16 (band q22) in some individuals. The optimal culture conditions under which an undercondensation of the Y heterochromatin and an induction of the fragile site in 16q22 can be achieved by in vitro treatment of lymphocytes were determined. This also permits the use of distamycin A in routine diagnostics of human chromosomes. The use of this technique in the analysis of translocations involving the Y chromosome is presented. The distamycin A-DNA interaction and the different possible explanations for the distamycin A-induced undercondensations of the Y heterochromatin and fragile sites 16q22 are discussed.     

Simplification of the CHO/HGPRT mutation assay through the growth of Chinese hamster ovary cells as unattached cultures

A novel technique for the growth of Chinese hamster ovary (CHO) cells as unattached cells on nontissue culture plates was applied to the CHO/HGPRT mutation assay, using EMS and MNNG as mutagens. The subculturing procedures for the unattached cultures involved less time and effort than those for the conventional attached cultures since trypsinization was not required for cell detachment. No significant difference in the maximum mutation frequency was observed for cells grown as unattached or attached cultures during the expression period. The optimum expression time was, however, shorter for the unattached cells (6 days) than for the attached cells (9 days). No selection for or against the mutant population was observed when mutant and wild-type cells were co-cultivated as unattached cultures, indicating that the procedure does not affect the quantitativeness of the mutation assay. The growth of CHO cells as unattached cells during the expression period thus could decrease the cost and effort involved in the use of the CHO/HGPRT mutation assay in the screening of potential mutagens/carcinogens.     

Mutagenicity evaluation of Schistosoma spp. extracts by the umu-test and V79/HGPRT gene mutation assay

Schistosomiasis has been suspected of being a risk factor for various types of cancers for sometime, e.g., bladder cancer, colorectal cancer and hepatic cancer. Among them, the etiological relationship between urinary schistosomiasis and bladder cancer is now widely accepted. However, mechanisms of the carcinogenesis are still unclear. Here, we tested the mutagenicity of the parasite extracts by the umu-test and hypoxanthine guanine phosphoribosyltransferase (HGPRT) gene mutation assay, which both overcome disadvantages of the Ames plate assay. Adult worm extracts and egg extracts of Schistosoma haematobium and Schistosoma mansoni were tested. Under our experimental conditions, neither worm nor egg extracts were shown to have any mutagenicity in both tests even in the presence of S9 mix. Our results suggest that there is very little possibility of immediate gene mutation due to the parasite-derived substances in schistosomiasis-related carcinogenesis.     

The suitability of the human lymphocyte micronucleus assay system for biological dosimetry

Human whole blood was irradiated with 220 keV X-rays at doses of 0-4.0 Gy. After incubation periods of 48, 60, 72, 84 and 96 h, lymphocytes were prepared without colcemid pretreatment according to 2 different methods, and micronuclei were scored. The crucial point of lymphocyte preparation was found to be the osmotic pressure of the hypotonic solution. Only a method that preserves the cytoplasm of lymphoblasts is suitable for a correct association of micronuclei with the main nucleus. Similar as for structural chromosome changes, now their intercellular distribution can be analysed. This is necessary for the derivation of appropriate statistical weights which have to be used for more reliable regression analyses. For 48 h, the data can be described by the linear model, for 84 and 96 h, by the linear-quadratic model. For 60 and 72 h no such definite conclusions can be drawn. For calibration purposes a standardized culture time cannot be recommended. Because the background frequency is high, the lymphocyte micronucleus assay system is not sensitive enough to detect a significant increase in the incidence of micronuclei after exposure to low doses (less than 0.3 Gy).     

A procedure for the CHO/HGPRT mutation assay involving treatment of cells in suspension culture and selection of mutants in soft-agar

A procedure involving treatment of cells in suspension culture and soft-agar cloning was developed for measuring mutation of Chinese hamster ovary (CHO) cells to 6-thioguanine (6TG) resistance. The use of suspension cultures precluded the need for trypsinization and also permitted a 5-fold increase in cell population for compound exposure and mutant selection as compared to former monolayer techniques. Soft-agar cloning reduced the opportunity for metabolic cooperation and permitted the use of non-dialyzed fetal calf serum which resulted in spontaneous mutant frequencies of 6.6 +/- 3.2 X 10(-6) and cloning efficiencies of 91 +/- 18%. Relative total growth values were calculated based on suspension growth and cloning efficiencies such that an assessment of toxicity could be estimated from treatment through cloning. Dose-dependent mutagenic responses were observed in CHO cells following treatment with ethyl methanesulfonate, methyl methanesulfonate, 4-nitroquinoline-1-oxide, methylnitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Clones of 6TG-resistant cells harvested from soft agar maintained 6TG resistance and methotrexate sensitivity and did not incorporate [3H]hypoxanthine into DNA. These preliminary findings indicate that the use of suspension cultures and soft-agar cloning has improved the efficiency and cost effectiveness of the CHO/HGPRT mutation assay.     

Determination of the fraction of S-phase cells in root meristems using bromodeoxyuridine labeling

The use of bromodeoxyuridine (BrdU) and subsequent immunocytochemical visualization for studying cell proliferation in plant meristems was investigated in Allium cepa L. root-tips. We describe the optimization of an indirect immunoperoxidase method for detecting incorporation of this DNA precursor in pulse-labeled cells. The basic object of this study is to quantify the extent to which the fraction of S-phase cells can reliably be estimated in asynchronous populations. A matrix of parallel labeling schedules with tritiated-thymidine or BrdU was developed, and the labeling indices provided by autoradiography or immunocytochemistry were compared. Thus, 0.5 mM BrdU assured saturation S-phase labeling after an exposure time of 30 min, and the mean length of the S-phase determined under such conditions was similar to that previously reported for this plant system. Interestingly, Feulgen staining did not interfere with subsequent detection of the BrdU probe. This allowed comparative evaluations of the nuclear DNA content by Feulgenmicrodensitometry and the position of a given cell in G1, S or G2 compartments. We also explored the possibility of quantifying BrdU-incorporation in single nuclei by densitometry measurement of the peroxidase label.     

Proliferative activity in the cerebellar external granular layer evaluated by bromodeoxyuridine labeling

We have optimised an indirect immunoperoxidase technique demonstrating bromodeoxyuridine (BrdU) incorporation into dividing cells for cerebellar tissue sections of four-day-old rats injected with this marker. This permits confident identification of granule-cell precursors engaged in DNA synthesis in the external granular layer of the developing cerebellum. Preservation of BrdU immunoreactivity is attained using methanol/acetic acid fixation and different pretreatments before immunostaining, while unlabeled nuclei can be recognized clearly after Feulgen or hematoxylin counterstaining. We established conditions to ensure satisfactory BrdU uptake without affecting cell-cycle progression during the postlabeling time period. The dose of BrdU employed provides saturation S-phase labeling from at least 1 h after BrdU delivery. Various kinetic parameters and phase durations have been determined in experiments involving a single injection or cumulative labeling sequences, and the cycle time was calculated based on two models of generative behavior: steady-state and exponential growth. The working hypothesis of steadystate kinetics can be adopted successfully if the existence of neuroblasts with different proliferation rates is taken into account.     

The relationship between transformation and somatic mutation in human and Chinese hamster cells

The frequencies of transformations of primary human and Chinese hamster fibroblasts have been compared with the spontaneous and induced frequencies of mutation for resistance to thioguanine and ouabain, and for ability to use fructose, using the carcinogens benzo (alpha) pyrene and urethane. Whereas the rates and frequencies of mutation were similar in the two cell systems, transformations to morphologically altered cells was observed only in hamster cells. The frequency of this latter transformation event in hamster cells was abour 10(3) greater than the frequencies of mutation in these cells. The morphologically altered cells formed in the above transformation process cannot grow in agar (aga-) and do not produce tumors when injected into animals. The frequency of transition of these latter cells to aga+ cells which produce tumors in animals is similar to the mutation-like events.     

Determination of the fraction of S-phase cells in root meristems using bromodeoxyuridine labeling

The use of bromodeoxyuridine (BrdU) and subsequent immunocytochemical visualization for studying cell proliferation in plant meristems was investigated in Allium cepa L. root-tips. We describe the optimization of an indirect immunoperoxidase method for detecting incorporation of this DNA precursor in pulse-labeled cells. The basic object of this study is to quantify the extent to which the fraction of S-phase cells can reliably be estimated in asynchronous populations. A matrix of parallel labeling schedules with tritiated-thymidine or BrdU was developed, and the labeling indices provided by autoradiography or immunocytochemistry were compared. Thus, 0.5 mM BrdU assured saturation S-phase labeling after an exposure time of 30 min, and the mean length of the S-phase determined under such conditions was similar to that previously reported for this plant system. Interestingly, Feulgen staining did not interfere with subsequent detection of the BrdU probe. This allowed comparative evaluations of the nuclear DNA content by Feulgenmicrodensitometry and the position of a given cell in G1, S or G2 compartments. We also explored the possibility of quantifying BrdU-incorporation in single nuclei by densitometry measurement of the peroxidase label.     

Photoperiod regulates neuronal bromodeoxyuridine labeling in the brain of a seasonally breeding mammal

Seasonal changes in vertebrate brain function are pervasive, but annual cycles in the rates of neuronal incorporation are established only in songbirds. Although cell division continues in the subependymal and hippocampal subgranular zones of adult rodents, there exists no parallel evidence that seasonal plasticity in mammals extends to changes in neuronal or glial number. We examined the effect of photoperiod on incorporation of new neurons in the brain of the adult golden hamster, a long-day breeder. We administered the cell birth marker 5′-bromode-oxyuridine (BrdU) to males which had either been maintained in long days, transferred to short days for 10 weeks, or moved acutely from long to short or short to long days. The number of cells in specific brain regions immunoreactive (ir) for this thymidine analog was determined 7 weeks later. The number of BrdU-ir cells in the dentate gyrus and subependymal zone increased twofold in short days. Transfer between photoperiods 10 days before the BrdU injections produced intermediate numbers of BrdU-labeled cells in the dentate gyrus, but was as effective as long-term photoperiodic exposure in the subependymal zone. Photoperiod also had similar effects in the hypothalamus and cingulate/retrosplenial cortex, but not in the central gray or preoptic area. Double-label immunocytochemistry indicated that very few of the BrdU-ir cells were glia, but that a majority had neuronal phenotype. In the subependymal zone, short days significantly increased the number of BrdU-labeled neurons. We did not detect significant effects of photoperiod on the volume of either the granule cell layer of the hippocampus or the dentate gyrus as a whole. We conclude that short day lengths increase neuronal birth and/or survival in several brain regions of adult hamsters. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 410–420, 1998     

Transgenerational genomic instability as revealed by a somatic mutation assay using the medaka fish

We previously established a somatic mutation assay of the medaka wl (white leucophores) locus based on visual inspection, and showed that somatic mutations at paternally derived alleles frequently arise during the development of F1 embryos fertilized by sperm/late spermatids that had been exposed to gamma-rays. To further study such delayed mutations, we determined the frequency of mutant embryos obtained from three different crosses between irradiated males and non-irradiated females. When sperm and late spermatids were irradiated, the mutant frequency within non-irradiated maternally derived alleles was approximately 3 times higher than in the control group. In the F2 generation, however, no increase in mutant frequency was observed. Similarly, there was no significant increase in the F1 mutant frequency when stem spermatogonia were irradiated. These data suggest that irradiation of sperm and late spermatids can induce indirect mutations in F1 somatic cells, supporting the idea that genomic instability arises during F1 embryonic development. Moreover, such instability apparently arises most frequently when eggs are fertilized just after the sperm are irradiated.     

Detection of alkylation damage in human lymphocyte DNA with the comet assay

The enzyme 3-methyladenine DNA glycosylase II (AlkA) is a bacterial repair enzyme that acts preferentially at 3-methyladenine residues in DNA, releasing the damaged base. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay (single cell gel electrophoresis) they appear as DNA strand breaks. AlkA is no t lesion-specific, but has a low activity even w ith undamagedbases. We have tested the enzyme at different concentrations to find conditions that maximise detection of alkylated bases with minimal attack on normal, undamaged DNA. AlkA detects damage in the DNA of cells treated with low concentrations of methyl methanesulphonate. We also find low background levels of alkylated bases in normal human lymphocytes.     

Changes in bromodeoxyuridine labeling index during radiation treatment of an experimental tumor

Cell proliferation kinetics in a spontaneous mouse fibrosarcoma (FSaII) growing in C3H mice has been studied by in vivo pulse labeling of cells synthesizing DNA with bromodeoxyuridine (BrdUrd). A monoclonal antibody to BrdUrd and flow cytometry were used to quantify these cells. Labeling indices (LI) were measured before and after radiation. Unirradiated 10-mm tumors had a mean LI of 17.5%. After a single dose of 20 Gy there was depression of LI after 1 day followed by a rapid increase to greater than control values after 5 days. Analysis performed after five fractions showed that LI was dependent on the dose per fraction and interval between fractions. After 5 and 7 Gy/fraction LI remained similar to control values during daily fractionation but was significantly depressed after twice daily fractionation. With doses greater than 10 Gy/fraction there was marked depression of LI using both fractionation schedules. These changes in LI correlated well with changes in tumor volume after radiation. Tumors were also biopsied after 5 fractions of a 20-fraction course to see if LI would predict for tumor control. LIs of greater than or equal to 10% were associated with lack of tumor control at 90 days while all controlled tumors had a significant depression of LI. Changes in LI after radiation were a reasonable indication of the amount of repopulation occurring and might be useful in selecting patients for altered fractionation schedules.