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PubMed1988

Three-dimensional light microscopy of diploid Drosophila chromosomes.

Fluorescence microscopy, uniquely, provides the ability to examine specific components within intact, even living, cells. Unfortunately, high-resolution conventional fluorescence microscopy is intrinsically a two-dimensional technique and performs poorly with specimens thicker than about 0.5 micron. Probing the spatial organization of components within cells has required the development of new methods optimized for three-dimensional data collection, processing, display, and interpretation. Our interest in understanding the relationship between chromosome structure and function has led us to develop the necessary methodology for exploring cell structures in three dimensions. It is now possible to determine directly the three-dimensional spatial organization of diploid chromosomes within intact nuclei throughout most of the mitotic the cell cycle.

Animals↗

PubMed2002

Flow cytometry measurement of the DNA contents of G0/G1 diploid cells from three different teleost fish species.

BACKGROUND: Although there is a lot information in the literature about genome size in fish, a high variability among data for the same species is reported, being mainly related to methodological aspects. Flow cytometry-based fluorescence measurements of intercalating dyes is the most attractive approach due to its precision, objectivity, high speed, and relative simplicity. METHODS: We analyze the DNA content of G0/G1 diploid nuclei of three teleost species (Carassius auratus, Tinca tinca, and Danio rerio) using flow cytometry. Forty-three animals were used and up to 50,000 retinal cells were analyzed per sample. Propidium iodide-associated fluorescence was assessed using a FACSCalibur flow cytometer. Standard human leukocytes were used as a reference. RESULTS: Our results show that C. auratus (3.584 +/- 0.058 pg per nucleus) and D. rerio (3.357 +/- 0.074 pg per nucleus) showed similar DNA contents per cell, whereas it was significantly lower (2.398 +/- 0.038 pg per nucleus) in T. tinca. Interestingly, a low intraspecies variability was observed, the coefficient of variation being 1.608%, 2.198%, and 1.573% for C. auratus, D. rerio, and T. tinca, respectively. CONCLUSIONS: The methodology used in this study provides an accurate and easy measurement of the genome size of a species.

Animals↗

PubMed1989

Correlation of DNA content, p53, T antigen, and V antigen in simian virus 40-infected human diploid cells.

Human diploid fibroblasts (HDF) have a finite life span in cell culture which can be extended when transformed with simian virus 40 (SV40). Flow cytometric analysis of SV40-HDF transformation allowed DNA content changes to be correlated with the appearance, quantity, and distribution of T antigen, p53, and V antigen, three proteins associated with this process. These studies demonstrated a shift in the DNA content to tetraploidy, which was correlated with the age of the SV40-HDF but not the time of infection. A significant increase of the epitope recognized by PAb122 to host p53 and the epitope PAb101 to SV40 T antigen occurred at the same time the tetraploid population appeared. However, an antigen reactive with SV40 V antibody was present at high levels in most of the population early after infection, but the levels declined with time. The percentage of PAb101-T antigen-positive cells increased more rapidly in cells infected at a late passage, and this was concomitant with the shift in DNA content to tetraploid. Analysis of the mean fluorescence of total, gated populations (G1, G2, and greater than G2) demonstrated that a threshold level of p53 and T antigen was reached in each compartment of the cell cycle. As the transformed phenotype appeared, a population of cells was continually released into the supernatant, and although these cells had a DNA pattern similar to the monolayer cells, the T antigen and p53 levels were 3-5 times higher in the tetraploid G2 cells. These studies correlated the expression of proteins associated with viral transformation in HDF which vary with time and shift in DNA content.

Antigens, Viral↗

PubMed1983

Immunoglobulin expression in diploid and polyploid interspecies hybrid of Xenopus: evidence for allelic exclusion.

The expression of species-specific surface IgM was studied in various Xenopus species hybrids. Whether the hybrids are diploid or polyploid, a lymphocyte does not produce all of the immunoglobulins (Ig) encloded by the Ig genes present in its genome. This suggests that the selection pressure to make a lymphocyte synthesize only one antibody is high and that allelic exclusion already existed in the ancestors of amphibians which appeared 300 million years ago. The apparent lack of cells producing multiple Ig in polyploid hybrids having more than one pair of functional Ig genes suggests that if a stochastic model for allelic exclusion is correct, the frequency of multiple successful rearrangements has to be very low, resulting in a huge waste of lymphocyte precursors. Given the particularity of frog development it is argued that this is not likely to be the case.

Alleles↗

PubMed1984

Binding and uptake of rabbit IgG complexes by diploid fibroblasts via plasma membrane Fc receptors.

The existence of IgG receptors on the plasma membrane of diploid human fibroblasts is demonstrated. The receptors specifically bind heat-aggregated rabbit IgG as well as rabbit IgG within antigen-antibody complexes. Monomeric rabbit IgG were only poor ligands of the receptor. Competition experiments with Fab and Fc fragments of IgG revealed that the receptor specifically recognizes the Fc domain of the IgG molecule. Heat-aggregated IgG or antigen-antibody IgG complexes are specifically bound to the receptors, endocytosed and subsequently degraded. The receptors do not seem to be recycled because protein synthesis is a prerequisite for further cycles of endocytosis.

Biological Transport↗

PubMed1998

Diploid Musa acuminata genetic diversity assayed with sequence-tagged microsatellite sites.

The sequence-tagged microsatellite site (STMS) discrimination potential was explored using nine microsatellite primer pairs. STMS polymorphism was assayed by nonradioactive urea-polyacrylamide gel electrophoresis. Genetic relationships were examined among 59 genotypes of wild or cultivated accessions of diploid Musa acuminata. The organization of the subspecies was confirmed and some clone relationships were clarified.

DNA, Plant↗

PubMed1988

Induction of mitotic chromosome loss in the diploid yeast Saccharomyces cerevisiae D61.M by genotoxic carcinogens and tumor promoters.

Three genotoxic carcinogens and eight tumor promoters were tested for induction of aneuploidy, specifically chromosome loss, in Saccharomyces cerevisiae D61.M. This is a heterozygous diploid yeast strain that permits the scoring of segregants expressing three linked recessive markers (cyhR2, ade6, and leu1), two of which (ade6 and leu1) are located close to the centromere on opposite arms of chromosome VII. The centromere marker leu was routinely checked, and a positive control (bavistan) was run with every experiment. The three genotoxic carcinogens aflatoxin B1, benzo(a)pyrene, and 7,12-dimethylbenz(a)anthracene did not induce aneuploidy, independent of the presence or absence of an exogenous metabolic activation system (rat liver homogenate; S9). Four of the eight tumor promoters tested induced chromosome loss but not mitotic recombination or mutation: cholic acid, lithocholic acid, phenobarbital, and saccharin. Diethylstilbestrol (DES) led to positive as well as to negative results in several independent experiments. In the case of the positive experiment, DES also induced putative recombinants. Three tumor promoters induced neither chromosome loss nor mitotic recombination: anthralin, 4,4'-dichloro-diphenyl-ethane (DDT) and gamma-hexachlorcyclohexane (lindane). From our experiments it can be concluded that the hypothesis put forward by Parry et al. [Nature; 294:263-265], according to which tumor promoters induce chromosome loss in yeast, is not correct in a general sense. In our set of eight tumor promoters, only one half distinctly induced chromosome loss.

Aneuploidy↗

PubMed1992

Induction of 6-thioguanine-resistant mutants in human diploid fibroblasts in vitro with ethylene oxide.

Human diploid fibroblasts (strain VH-10) were exposed to the direct-acting alkylating agent, ethylene oxide (EtO), in vitro, and the frequency of HPRT mutants was evaluated by selection in medium containing 6-thioguanine. A dose-dependent increase of the mutant frequency was found in the dose range of 2.5-10 mMh of EtO. The EtO-induced mutant frequency increased 5-19 times the background frequency at low or moderately toxic doses, which indicates that EtO is a strong mutagen in human fibroblasts in vitro. The mutagenic potency was 9.8 x 10(-6) per mMh.

Alkylating Agents↗

PubMed1992

Effects of Captan on DNA and DNA metabolic processes in human diploid fibroblasts.

The fungicide Captan has been examined for its effects on DNA and DNA processing in order to better understand the genotoxicity associated with this agent. Captan treatment resulted in production of DNA single strand breaks and DNA-protein cross-links and elicited an excision repair response in human diploid fibroblasts. Captan was also shown to inhibit cellular DNA synthesis and to form stable adducts in herring sperm and human cellular DNA. Misincorporation of nucleotides into Captan-treated synthetic DNA templates was significantly elevated in an in vitro assay using E. coli DNA polymerase I, suggesting that DNA adduct formation by Captan could have mutagenic consequences. In sum, these studies demonstrate that Captan is capable of interacting with DNA at a number of levels and that these interactions could provide the basis for Captan's genotoxicity. The extreme cytotoxicity of this fungicide, however, could be due to other cellular effects since at the IC50 for cell killing, approximately 0.8 microM, none of the above genotoxic events could be detected by the methods employed.

Captan↗

PubMed1994

Induction of DNA strand breaks by ethylene oxide in human diploid fibroblasts.

In vitro exposure of normal human diploid fibroblasts (strain VH-10) to ethylene oxide (EtO) induced DNA strand breaks in the dose range of 2.5-30 mMh of EtO. Alkaline DNA unwinding (ADU), neutral filter elution (NFE), pulsed field gel electrophoresis (PFGE), and the comet assay were used to measure DNA single (SSBs) and double strand breaks (DSBs). Different induction rates of SSBs and DSBs, depending on applied method and also on treatment conditions (cells in monolayer or in suspension were used), were found. A dose-dependent increase of DNA strand breaks was found by the ADU method in the dose range of 2.5-20 mMh of EtO when treatment was performed in monolayer and in suspension. DSBs were detected by NFE only when the cells were treated with EtO in suspension (doses 10-30 mMh). The highest induction rate of DSBs (about 4 DSBs per 100 Mbp per 1 mMh of EtO) was detected in suspension with PFGE applied. We have shown that heat-labile sites are formed by EtO. Presumably, the different DSB levels detected by PFGE and NFE result from the conversion of these sites to DSBs during cell lysis at elevated temperature in the PFGE method. The results of the comet assay confirmed that apoptotic processes are not involved in the formation of DSBs in our experimental conditions (less than 1% of apoptotic cells were observed at all doses studied). Possible mechanisms for the induction of DNA strand breaks by EtO-treatment are discussed. The capacity to repair DSBs in EtO-exposed (5-7.5 mMh) cells was studied, and it was found that a considerable part of the damage (about 50%) could be repaired during 18 hr of incubation.

Cells, Cultured↗

PubMed1981

Induction of sister chromatid exchanges in human diploid fibroblasts by mutagens with and without rat liver microsomal activation.

The frequency of sister chromatid exchange (SCE) in WI-38 cells was estimated by the 5-bromodeoxyuridine (BrdUrd)-dye technique after one hour's exposure to cyclophosphamide (CY), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline-1-oxide (4NQO), and maleic hydrazide (MH) with and without the addition of rat liver microsomal suspension (S-9) fraction with cofactors (S-9 mix). CY at concentrations from 1 x 10(-5) M to 1 x 10(-3) M with S-9 mix increased the number of SCEs per cell in a dose-dependent manner. Without S-9 mix, CY at concentrations below 1 x 10(-3) M failed to produce more SCEs than the controls. MNNG at 1 x 10(-8) M and 4NQO at 1 x 10(-7) M without S-9 produced significant increases in SCEs per cell. Addition of S-9 during treatment slightly decreased the effects of MNNG and 4NQO in the formation of SCEs. MH was tested at pH 6.4 and pH 7.6. At pH 7.6, MH at 1 x 10(-3)M without S-9 mix inhibited cell multiplication, but did not cause a significant increase of SCEs per cell. There were no interactions between MH (2 x 10(-4) M) and S-9 mix nor between MH and the pH levels tested. These results indicate that in the presence of metabolic activation, SCE formation in human diploid fibroblasts in vitro may be used as a potential assay for mutagenicity.

Biotransformation↗

PubMed1982

Sister chromatid exchange response of human diploid fibroblasts and Chinese hamster ovary cells to dimethylnitrosamine and benzo(a)pyrene.

In the search for relevant assays for mutagenicity testing, considerable attention has been given to the use of mammalian cells in vitro and the incorporation of metabolic activation in the protocol. Chinese hamster ovary (CHO) cells are commonly chosen as the target cells for cytogenetic tests because of their excellent growth characteristics and long lifespan in culture. However, there may be cellular factors affecting the uptake, metabolism, and repair of damage which are not the same in all cell lines. The response of CHO cells and three human diploid fibroblast strains (IMR-90, WI-38, S-3299) to benzo(a) pyrene (BP) and dimethylnitrosamine (DMN) were compared using sister chromatid exchange (SCE) analysis as a measure of genetic damage. For both BP and DMN the human cells and the CHO cells showed dose-response slopes that were significantly different from zero, except CHO cells treated with BP for 1 hr and S-3299 cells treated with DMN. Whereas human and CHO cells showed similar dose-responses to BP and the three human cell strains had similar dose-responses to BP and DMN, the dose-response of the human cells to DMN was statistically less significant than that of CHO cells. Reducing the duration of chemical treatment in CHO cells had no effect on the slope of the dose-response curves for BP or DMN. The observed differences between human and CHO cells may reflect differences in the fate of metabolic intermediates of DMN.

Animals↗

PubMed1983

Optimal phenotypic expression times for HPRT mutants induced in foreskin-, skin-, and lung-derived human diploid fibroblasts.

A series of paired lung- and skin-derived fibroblast cultures has been established from human embryonic tissues under carefully controlled, identical conditions, providing the unique opportunity to study differences between normal diploid fibroblast populations from skin and lung without the confusion of genetic differences between donors. To reliably assess differences in the induced mutation frequencies observed among different cell populations, optimal phenotypic expression times in the HPRT mutagenesis system were determined for neonatal foreskin, fetal skin, and fetal lung cultures. Cell populations were mutagenized with several doses of N-methyl-N'-nitro-N-nitrosoguanidine and were replated in 6-thioguanine selective medium at intervals over 14 days. Survivals following MNNG exposure ranged from 1.6% to 45.5%. For all doses and survivals tested a 7-day expression period was the optimal value for cultures from the three different tissue sources in six independent experiments. Mutant frequency data derived from untreated control populations confirmed that spontaneous mutations during the expression period contributed negligibly to the final mutant frequency. Differences between the mutation frequencies obtained using an in situ and a replating protocol were approximately twofold for lung-, skin-, and foreskin-derived cultures.

Cell Line↗

PubMed1984

Assessment of mutagenic damage by monofunctional alkylating agents and gamma radiation in haploid and diploid frogs, Xenopus laevis.

Adult male South African clawed frogs, Xenopus laevis, were mutagenized by 3-day immersion in aqueous solutions of ethyl methanesulfonate (EMS), diethyl nitrosamine (DEN), or ethyl nitrosourea (ENU), or by acute exposure to gamma radiation. They were then spawned repeatedly at 2-week intervals with untreated females, and embryonic survival of the progeny was used to assess genetic damage. Recessive lethal effects were assessed from reduced survival of androgenetic haploid progeny. Neither recessive nor dominant lethal effects were obtained after exposure to 100 mg/liter EMS or 2 g/liter DEN. At 250 mg/liter EMS, peak dominant lethality occurred 3-5 wk after treatment. Most embryos hatched, but many were abnormal and died shortly after hatching. Haploid survival was significantly reduced over a broader period, from 1 to 13 wk after mutagenesis. Treatment with 75 mg/liter ENU produced effects similar to the 250-mg/liter EMS mutagenesis. At 400 mg/liter EMS, the frequency and severity of the effects on both diploid and haploid embryos were increased over the lower dose. Gamma irradiation at 1,500 R produced effects similar to the 400-mg/liter mutagenesis, except that peak dominant lethality extended from 1 to 7 wk.

Alkylating Agents↗

PubMed1985

Autoradiographic detection of diphtheria toxin resistant mutants in human diploid fibroblasts.

An autoradiographic procedure for the detection of diphtheria toxin (DT) resistant (DipR) mutants in human diploid fibroblast (HDF) cells has been developed. The assay is based on the observation that when HDFs from confluent cultures are seeded in medium containing 0.01 flocculating units/ml or higher concentration of DT, protein synthesis in sensitive cells is severely inhibited by 4-6 hr. If at this or later time, a radiolabeled protein precursor (eg, 3H-leucine) is added to the culture, it is almost exclusively incorporated into the resistant cells, which are then readily identified by autoradiography. The DipR cells can also be identified by labeling in the presence of 3H-thymidine, although a higher background is observed in these experiments. Reconstruction experiments using DipS and DipR HDFs show that the frequency of heavily labeled cells that are detected by autoradiography show an excellent correlation with the number of DipR cells added and to the number of DipR cells as detected by conventional colony forming assay. These studies provide strong evidence that the labeled cells identified by autoradiography are bona fide DipR mutants. The detection of DipR cells by autoradiography is apparently not affected by the presence of the sensitive cells in the mixtures. The spontaneous frequency of DipR cells in HDFs has been found to be in the range of 1-5 X 10(-6), and this increases in a dose dependent manner upon treatment with the mutagen ethyl methanesulfonate. These results indicate that the autoradiographic assay could be used for quantitative mutagenesis. Since the autoradiographic assay does not depend on cell division, it may prove useful in estimating the incidence of pre-existing mutations in cell populations that either do not divide or have very limited growth potential (eg, lymphocytes, muscle cells, neurons, senescent fibroblasts, etc).

Autoradiography↗

PubMed2003

Chromosomes that show partial loss or gain in near-diploid tumors coincide with chromosomes that show whole loss or gain in near-triploid tumors: evidence suggesting the involvement of the same genes in the tumorigenesis of high- and low-risk neuroblastomas.

We performed two-color fluorescence in situ hybridization analysis to detect the numbers of chromosomes 1 and 17, 1p deletion, and 17q gain in 177 neuroblastomas, including 101 tumors that were found by a mass-screening program for infants. Sixty-eight tumors with disomy 1 or tetrasomy 1 were classified as the Dis1 group, and 109 tumors with trisomy 1, pentasomy 1, or a mixed population of cells with trisomy 1 and cells with tetrasomy 1 were classified as the Tris1 group. 17q gain was the most frequent genetic event, followed by 1p deletion, and MYCN amplification in both Dis1 and Tris1 tumors. However, the incidence of all the genetic events was higher in Dis1 tumors than in Tris1 tumors. These findings suggest that Tris1 tumors are more resistant to acquiring the genetic events than are Dis1 tumors. In addition, there was an accumulation of genetic events in more advanced stages, with the exception of a high incidence of 17q gain in the stage IVS Tris1 tumors. Comparative genomic hybridization analysis, which was performed in 59 of the 177 tumors, showed that chromosomes partially lost or gained in Dis1 tumors coincided with chromosomes totally lost or gained in Tris1 tumors. Dis1 and Tris1 tumors were considered to have near-diploid/tetraploid and near-triploid/pentaploid chromosome numbers, respectively. These findings suggest that the same tumor-suppressor genes or oncogenes may be involved in the development and progression of both high- and low-risk neuroblastomas, and that the ploidy state of the tumor plays a fundamental role in the heterogeneous behavior.

Allelic Imbalance↗

PubMed1981

Diploid human lymphoblastoid and Burkitt lymphoma cell lines: susceptibility to murine NK cells and heterotransplantation to nude mice.

Human lymphoid cell lines which had been classified on the basis of studies on clonality and morphological, on the basis of studies on clonality and morphological, chromosomal and functional parameters as lymphoblastoid cell line (LCL) of presumed non-neoplastic origin and Burkitt lymphoma (BL) lines of proven malignant origin, were tested for susceptibility to natural killer (NK) cells obtained from the spleens of athymic nude mice. The 20 lines included normal diploid LCL and aneuploid BL lines. All cells carried the Epstein-Barr virus (EBV) genome. In addition, two EBV-negative BL lines were tested. The pronase-induced release of 14C-DNA from 14C-thymidine-labelled target cells was used to assess the sensitivity of the cell lines to NK activity. When attempts were made to correlate the growth of the EBV-positive LCL and the EBV-positive BL cell lines in the subcutaneous space of adult nude mice with their susceptibility to NK cells, no significant correlation was observed. The EBV-negative BL cell line, Ramos, however, could be transplanted subcutaneously in nude mice and was more resistant to NK activity than was the EBV-negative BL cell line, BJAB, which cannot be transplanted subcutaneously. Growth of heterotransplanted EBV-converted cell lines in the subcutaneous space of adult nude mice may be influenced by immune effectors other than NK cells.

Animals↗

PubMed1984

Isolation of HTLV derived from Japanese adult T-cell leukemia patients in human diploid fibroblast strain IMR90 and the biological characters of the infected cells.

HTLV was isolated in the human diploid fibroblast strain IMR90 directly from the leukemic cells of the Japanese ATL-patients. The infection was checked by the presence of virus-specific p24 and proviral DNA. The isolation was successful in three of four patients. The patient whose virus isolation was unsuccessful contained only a defective viral genome in his leukemic cells. The infected cells produced polykaryons when co-cultured with catS + L-cells. Human leukemia HL60 cells formed clusters adherent to the infected IMR90 cells but not to uninfected IMR90 cells. The former phenomenon was blocked by the ATLL patients' serum.

Cell Nucleus↗