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PubMed2002

Automated identification of diploid reference cells in cervical smears using image analysis.

BACKGROUND: Acquisition of DNA ploidy histograms by image analysis may yield important information regarding the behavior of premalignant cervical lesions. Accurate selection of nuclei for DNA measurement is an important prerequisite for obtaining reliable data. Traditionally, manual selection of nuclei of diagnostic and reference cells is performed by an experienced cytotechnologist. In the present study, a method for the fully automated identification of nuclei of diploid epithelial reference cells in Feulgen- restained Papanicolaou (PAP) smears is described. METHODS: The automated procedure consists of a decision tree implemented on the measurement device, containing nodes with feature threshold values and multivariate discriminant functions. Nodes were constructed to recognize debris and inflammatory cells, as well as diploid and nondiploid epithelial cells of the uterine cervix. Evaluation of the classifier was performed by comparing resulting diploid integrated optical densities with those from manually selected reference cells. RESULTS AND CONCLUSION: On average, automatically acquired values deviated 2.4% from manually acquired values, indicating that the method described in this paper may be useful in cytometric practice.

Algorithms↗

PubMed1986

The fate of the primary diploid population during spontaneous transformation of growth factor-supplemented murine cell cultures.

Primary cultures derived from lung and renal tissue of the newborn harvest mouse (Micromys minutus) were serially passaged in media supplemented with epidermal growth factor, hydrocortisone, transferrin, insulin, and triiodothyronine. Although these growth factor supplements eliminated the growth crisis commonly encountered during the initial stages of murine primary cultures, the original diploid cell fraction clearly underwent such a "crisis"; the truly diploid cells invariably disappeared as these cultures reached 20 to 40 population doublings. They were replaced, either gradually or precipitously, by various heteroploid cell fractions. In three of four independent cultures, these "established" cells were hypotetraploid and appeared to be derived from a small number of progenitors already present during the very early (precrisis) culture stages. In contrast to rather frequent DNA changes displayed by clones and subclones derived from the various heteroploid cell lineages, the predominant components of the established mass cultures displayed a highly constant DNA fluorescence pattern. Our results suggest that primary murine cell cultures develop heteroploid cell lineages even if the initial growth crisis is mitigated by growth factor supplements. These heteroploid cells appear to respond more efficiently to stimulation by various growth factors than the primary diploid cell population.

Animals↗

PubMed1994

Simultaneous cytokinetic measurement of aneuploid tumors and associated diploid cells following continuous labelling with chlorodeoxyuridine.

Cells from a murine tumor, MCa-K, were continuously labelled with the thymidine analogue chlorodeoxyuridine (CldUrd) and analyzed by bivariate flow cytometry in order to measure the growth fraction (GF) and potential doubling time (Tpot) of both the DNA-aneuploid tumor cells and the associated DNA-diploid cells. MCa-K has a DNA index of 1.7, rendering two, partially overlapping, populations observable with labelled and unlabelled cells in each population. The data from these tumors may be divided into three regions of differing DNA content, with one region containing a pure DNA-diploid population, a second region with both cell types, and a third region including only DNA-aneuploid cells. Equations are presented to characterize the fractions of labelled cells in each region as a function of labelling time and cell type, thereby permitting estimation of the proliferative properties of the populations. These equations include the possibility that DNA-aneuploid cells cease cycling both in G1 and in S phase to account for the observed numbers of unlabelled cells with S phase contents. The estimated value of Tpot of the DNA-diploid cells is 126.0 h with a GF of 42%, while that of the DNA-aneuploid cells is 36.9 h with a GF of 69%. It is also estimated that between 2% and 6% of all DNA-aneuploid cells starting DNA synthesis cease cycling, leading to 25% of the cells having an S-phase DNA content being noncycling.

Aneuploidy↗

PubMed2002

Quantitative FISH analysis on interphase nuclei may improve diagnosis of DNA diploid breast cancers.

The detection of DNA aneuploid cells using flow cytometry is an indication for the presence of tumor cells, but when DNA diploid cells are found in 25-33% of the cases, the diagnostic and prognostic significance of DNA ploidy is more limited. We analyzed interphase nuclei after in situ hybridization and using image cytometry on 50 breast tumors with diploid DNA content to investigate whether early chromosome rearrangements were detectable and if their occurrence was clinically significant. Imbalances between the two arms of chromosome 1 were found in 55% of the cases and values ranged from 1.5-3.0. Comparison with histological data showed that Grade I tumors mainly have imbalances (67%) and that Grade III tumors were mainly without the imbalance (67%), whereas Grade II tumors were intermediate (50% imbalance). These data suggest that the diagnosis of DNA diploid cases may be improved by using interphase FISH. In addition, the data also indicates that early breast tumors may have different genetic origins, which is important in the comprehension of tumor malignancy in early stages, especially for preinvasive lesions.

Adult↗

PubMed1982

Glycopeptide hormone production by cultured human diploid fibroblasts.

Human diploid fibroblasts in culture were examined for production of glycopeptide hormones. Forty-one percent of the strains produced human chorionic gonadotropin (hCG) under normal growth conditions. Constitutive hCG synthesis was apparently unrelated to donor age, length of time in culture, or number of passages. Follicle stimulating hormone (FSH) was not found in any strain investigated. Only one cell strain produced free alpha-chains of glycopeptide hormones. Hydroxyurea (HU) at a concentration of 1 mM mediated a small, statistically significant increase in hCG production (p less than 0.01) in all constitutive strains, but had no effect on non-hCG-producing fibroblast strains. Sodium butyrate (Bu) was effective in increasing hCG synthesis in only one constitutive strain, derived from a newborn foreskin. HU treatment had no apparent effect on cell structure. All Bu-treated strains, both those producing hCG and the nonproducers, showed morphological alterations; cells were flattened and they contained ordered arrays of refractile granules. It is suggested that hCG synthesis in cultured human diploid fibroblasts may result from a localized chromosomal event in which the loci responsible for this hormone are activated. Human diploid fibroblasts in culture are shown to be amenable to the study of gene expression and its modulation.

Butyrates↗

PubMed1986

Changes of serum-induced ornithine decarboxylase activity and putrescine content during aging of IMR-90 human diploid fibroblasts.

The roles of ornithine decarboxylase (ODC, EC 4.1.1.17) and polyamines in cellular aging were investigated by examining serum-induced changes of these parameters in quiescent IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL) and in human progeria fibroblasts. Serum stimulation caused increases of ODC and DNA synthesis in IMR-90 human diploid fibroblasts, with maximal values occurring, respectively, 10 hr and 22 hr after serum stimulation. Both serum-induced ODC activity and DNA synthesis in IMR-90 cells were found to be inversely related to their PDL. Maximal ODC activity and DNA synthesis in young cells (PDL = approximately 18-22) were, respectively, five-fold and six-fold greater than that in old cells (PDL = approximately 50-55), which in turn were comparable or slightly higher than that in progeria fibroblasts. Polyamine contents (putrescine, spermidine, and spermine) in quiescent IMR-90 cells did not show significant PDL-dependency. The putrescine and spermine contents in quiescent progeria cells were comparable to those in quiescent IMR-90 cells. The spermidine content in quiescent progeria cells, however, was extremely low, less than half of that in quiescent IMR-90 cells. Serum stimulation caused a marked increase in putrescine content in young cells but not in old cells or in progeria cells. The spermidine and the spermine content in IMR-90 cells, either young or old, and in progeria cells did not change significantly after serum stimulation. Our study indicated that aging of IMR-90 human diploid fibroblasts was accompanied by specific changes of polyamine metabolism, namely, the serum-induced ODC activity and putrescine accumulation. These changes were also observed in progeria fibroblasts derived from patients with Hutchinson-Gilford syndrome.

Blood↗

PubMed1987

Establishment and some mutational characteristics of 3T3-like near-diploid mouse cell line.

A stable and nonmalignant near-diploid cell line, designated m5S, has been established by serial in vitro transfers of embryonic skin cells of ICR mouse. The cells are stable in karyotype and growth, display high sensitivity to contact inhibition of cell division, are nontumorigenic in nude mice, and the transformation and mutation may be reproducibly tested. Unlike most of the mouse cell lines, the m5S underwent a unique karyotypic change during establishment and showed high stability in a diploid range, making amenable to the study of mutagenesis. The m5S is nearly unique; it represents the second near-diploid cell line sensitive to postconfluence inhibition of cell division derived from mouse embryo cells to be reported and characterized. These cells are being successfully used for qualitative and quantitative studies of oncogenic transformation as well as mutation to drug resistance by radiation and chemicals.

Animals↗

PubMed2006

Premeiotic endomitosis produces diploid eggs in the natural clone loach, Misgurnus anguillicaudatus (Teleostei: Cobitidae).

The natural clone loach produces unreduced eggs genetically identical to somatic cells of the mother fish and such diploid eggs normally develop as a clone without genetic contribution of sperm. Following the identification of clonal nature and diploidy of eggs, we conducted cytological studies to determine the mechanisms responsible for this unusual oogenesis. Cytolological observation of full-grown oocytes cultured in vitro revealed that oocytes of both the clone and the control loach underwent two successive meiotic divisions: formation of a bipolar spindle and metaphase in meiosis I and equal segregation of chromosomes, extrusion of the first polar body and the appearance of metaphase of meiosis II. However, spindle size of the clone was larger than that of the control. Bivalent chromosome number of germinal vesicle of oocytes was 25 in the control diploid, whereas 50 in the clone. The results suggest that chromosomes are duplicated by mitosis without cytokinesis before meiosis, i.e. premeiotic endomitosis and then oocytes differentiated from tetraploid oogonia undergo a quasinormal meiosis followed by two successive divisions to produce diploid eggs.

Animals↗

PubMed1994

Diploid expression and translational regulation of rat acrosin gene.

Acrosin, a sperm acrosomal serine protease has been implicated in the recognition, binding and penetration of the zona pellucida of the ovum. Biosynthesis of acrosin was found to start in early round spermatids which are haploid germ cells. Here, we report that acrosin gene transcription occurs as early as at day 19 of rat spermatogenesis which contains diploid but not haploid spermatogenic cells. Translational control of the acrosin gene may be due to cytoplasmic protein factors which through RNA-bandshift experiments were found to bind to the 5'UTR of the acrosin mRNA. In order to differentiate between diploid and haploid spermatogenic cells at the molecular level, transcription of the protamine 2 gene during rat testicular development was evaluated. Protamine 2 transcripts could be demonstrated for the first time in 25-day-old testes which contain diploid as well as haploid spermatogenic cells.

Acrosin↗

PubMed1990

Mitochondrial genome transmission in Chlamydomonas diploids obtained by sexual crosses and artificial fusions: role of the mating type and of a 1 kb intron.

The linear mitochondrial DNAs of the two infertile algal species Chlamydomonas smithii and C. reinhardtii are co-linear with the exception of a 1 kb intron (alpha intron) located in the cytochrome b gene of C. smithii. C. smithii also possesses an additional HpaI restriction site (H marker) located in the COXI gene, about 5 kb from the intron. In reciprocal crosses, C. smithii (H+ alpha +) x C. reinhardtii (H- alpha -), the alpha intron is transmitted to all diploid progeny, whereas the H marker is frequently transmitted either biparentally or paternally depending on whether the C. smithii parent is maternal (mt+) or paternal (mt-). In diploids resulting from artificial fusion between vegetative cells, the absolute transmission of alpha is accompanied by the frequent transmission of the H+ marker, irrespective of the mating type of the parental strains. Finally, in reciprocal crosses between C. smithii (H+ alpha +) and recombinant H- alpha + clones, the transmission of the H marker is predominantly paternal or biparental. These results allow us to conclude that (1) the alpha intron behaves as a group I intron whose unidirectional conversion influences the transmission of the H marker; and (2) the mt- paternal mitochondrial genome is transmitted more often than the mt+. The mating type has no effect in diploids obtained by artificial fusion.

Chlamydomonas↗

PubMed1976

Arg-7 mutant X wild-type crosses in Chlamydomonas reinhardi: study of the enzyme produced in diploid strains.

The arg-7 locus is the structural gene for the argininosuccinate lyase (ASL). Interallelic complementation was previously found to occur between several mutants of the locus: this is indicative for the homomultimeric nature of ASL. Two complementing (arg-7-5 and arg-7-7) and two non-complementing (arg-7-1 and arg-7-6) mutants of the arg-7 locus were crossed to the pab-2 strain (which is wild-type for the arg-7 locus). In each cross, heterozygote phenotypically wild-type strains were isolated; their diploid pattern was demonstrated by various criteria: mating type, cell volume, nuclear size. The four heterozygotes were compared to the haploid wild-type and in some experiments, to the diploid strain arg-1 X pab-2 homozygous for the arg-7 locus. No difference was found in growth rate and in the Michaelis constant values for ASL. The specific activity of the enzyme produced in the heterozygotes was about 50 percent of the activity found in haploid or diploid wild-type. The heat sensitivity of ASL was also investigated in the different strains: two (containing the complementing mutations arg-7-5 and arg-7-7) of the four heterozygotes produce ASL varieties different from the wild-type enzyme as far as the thermolability is concerned. These results suggest that hybrid ASL can be formed by interaction between the products of wild-type and mutant genes. A clear dominance of the wild-type allele is expected only when the mutant allele has no product of the gene: this could be the case for arg-7-1 and arg-7-6.

Argininosuccinate Lyase↗

PubMed1980

Quantitation of in situ hybridization of ribosomal ribonucleic acids to human diploid cells.

The hybridization of 5S and 28S ribosomal RNAs to human fibroblast and leukocyte cells was used as a model system to quantitative the technique of in situ hybridization for human diploid cell types. Quantitation consisted of counting (scoring) the number of grains formed over both interphase nuclei and metaphase chromosomes on slides after various hybridization procedures. The average number of grains/nucleus per slide was then used to determine hybridization percentages. As with nitrocellulose filter hybridizations the kinetics of in situ hybridizations can be fit with a single first-order rate-constant. However, the in situ hybridization rate was approximately 10 times slower than the corresponding filter hybridization rate. The efficiency of in situ hybridization was found to range between 5 and 15% for both leukocyte and fibroblast cell types and for both metaphase and interphase nuclei. Determination of the parameters of the in situ hybridization reaction of ribosomal RNAs to diploid chromosomes define the experimental conditions needed for the localization of single copy genes to diploid chromosomes.

Cells, Cultured↗

PubMed1988

Restriction fragment length polymorphisms in diploid and allotetraploid Gossypium: assigning the late embryogenesis-abundant (Lea) alloalleles in G. hirsutum.

We have determined the copy number of 21 genes in an allotetraploid and several diploid species of cotton by gel and dot blot hybridization with cloned cDNAs. The legumin A, legumin B, and all 18 unique Lea (late embryogenesis-abundant) cDNA sequences isolated from the AD allotetraploid Gossypium hirsutum are present in one copy in A, D, E, and F diploid species and in two copies in G. hirsutum. Gel blot analysis of DNAs digested with EcoRI or BamHI usually detects different sized fragments in A and D diploids. Conservation of these restriction fragment length polymorphisms in G. hirsutum allows most of these fragments to be assigned to their respective subgenomes. Furthermore, both subgenomes in G. hirsutum can be distinguished from those in the interfertile allotetraploid G. barbadense. These results show that physical mapping of both sets of chromosomes in an allotetraploid should be possible by segregation analysis.

Alleles↗

PubMed1986

High-resolution 2-dimensional protein mapping of "diploid" and "aneuploid" mammary adenocarcinomas.

Two-dimensional-electrophoretic analysis has been applied to non-neoplastic mammary epithelium from eight healthy women, tumor tissue from eight "diploid" mammary adenocarcinomas, and tumor tissue from eight "aneuploid" mammary adenocarcinomas. Compared with non-neoplastic mammary epithelium, a slight numerical net non-neoplastic mammary epithelium, a slight numerical net increase of the protein spots was detected in "diploid" tumors and a marked increase in "aneuploid" tumors. Two prominent spots were present in all 16 malignant tissues examined and absent in all eight non-neoplastic tissues (silver staining method). The results suggest that a difference in the composition of cellular proteins exists both between non-neoplastic mammary cells and malignant tumor cells, and between "diploid" and "aneuploid" tumors.

Adenocarcinoma↗

PubMed1980

Biochemical properties and level of expression of alcohol dehydrogenases in the allotetraploid plant Tragopogon miscellus and its diploid progenitors.

This study demonstrates that homoeologous genes in two diploid plant species that specify different amounts of an enzyme maintain the same relative level of expression in an allotetraploid derivative. The three predominant alcohol dehydrogenase (ADH) isozymes (DD, DP, PP) in seeds of the recently evolved allotetraploid plant Tragopogon miscellus (Compositae) are dimers specified by Adh3-D and Adh3-P genes derived from its diploid progenitors T. dubius and T. pratensis. Seeds of T. pratensis contain twice as much ADH activity as those of T. dubius, while T. miscellus is intermediate. The three isozymes were similar in a number of catalytic properties; the densitometric ratio of the isozymes purified from T. miscellus was 1 DD : 4DP : 4PP for both ADH activity and protein; and dissociation-reassociation of the DP enzyme gave a 1:2:1 ratio of the three isozymes. Therefore, the enzymes were similar in specific activity, but twice as many P as D subunits were present in active enzymes in T. miscellus, precisely the difference in activity between the parents. In T. miscellus, the specific activity of ADH and its activity per mg tissue are intermediate to those of the diploids, because relative expression of the Adh gene in each genome is not influenced by the presence of the other genome.

Alcohol Oxidoreductases↗

PubMed1975

Microphotometry of rat liver nucleoproteins during the cell cycle, and comparison of diploid nuclei in the G2 period with tetraploid nuclei.

The following facts were established with a microphotometric investigation of isolated nuclei from rat liver in different stages of the cell cycle. During the mitotic wave occurring in the liver of newborn animals after injection of casein it was found that the naphtol yellow S (NYS) protein content of the nuclei increases about 30% during the G1-period. A second increase of around 70% was established during the S-phase whereas no increase could be observed during the G2-phase. An indication for the existence of a "critical protein mass" of the nuclei before the onset of the S-phase could be observed. The protein content of diploid nuclei in the G1-phase of adult animals is about 50% higher than in newborns. This makes it probable that there is no significant difference in the NYS-protein content of diploid nuclei in the G2-period and tetraploid nuclei of adult rats. No differences were observed between diploid nuclei in the G2-period of newborn rats and tetraploid nuclei of adult rats in their Fastgreen histon, RNA and SH plus SS content. The only criterion to distinguish between G2 nuclei and tetraploid nuclei seems to be the number of nucleoli, but this is rather unreliable.

Aging↗

PubMed1983

Hidden breaks in ribosomal RNA of phylogenetically tetraploid fish and their possible role in the diploidization process.

Hidden breaks occur in the ribosomal RNA of tetraploid Cyprinid fish such that the large ribosomal RNA (28 S) yields upon denaturation two RNA fragments of 8.7 X 10(5) and 5.0 X 10(5) daltons, whereas the small rRNA (18 S) yields fragments of 3.2 X 10(5) to 5.0 X 10(4) daltons. In tetraploid Cyprinids hidden breaks occur only in the rRNA of somatic tissue and not in oocytes and sperm cells. Hidden breaks can be detected only slightly in diploid Cyprinid species. Ribosomes purified from somatic tissue of tetraploid Cyprinids show a reduced efficiency in protein synthesis in vitro. The ribosomal proteins from diploid and tetraploid Cyprinid fish show considerable electrophoretic differences. This is discussed in light of a possible functional role of hidden breaks in rRNA in the process of diploidization of gene expression in tetraploid Cyprinid species.

Animals↗

PubMedDate not supplied

Association of near-diploid DNA content and N-myc amplification in neuroblastomas.

Seventeen neuroblastomas at different clinical stages were analysed for their N-myc copy number and flow cytometrically determined DNA content. Aneuploidy was found in 11 patients (65 per cent), whereas the remaining were near-diploid. N-myc amplification was found significantly (P less than 0.05) confined to near-diploid tumors (3 out of 6 cases). This finding indicates a very selective mechanism of oncogene amplification which is independent of gross chromosomal imbalance and limited to specific loci in the human genome. Association of near-diploidy and age at diagnosis older than 24 months was also demonstrated (P less than 0.05). Thus, flow cytometric analysis of DNA content together with N-myc gene dosage allowed us to distinguish two different subsets of neuroblastoma tumors: the first one aneuploid, with single-copy N-myc, usually observed in patients younger than 24 months with localized or IV-S clinical stages; the second one near-diploid, with frequent N-myc amplification, usually observed in patients older than 24 months with advanced clinical stages.

Child↗