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PubMed2000

Gonadal morphology of female diploid gynogenetic and triploid rainbow trout.

Chromosome sets of fishes can be manipulated; this practice includes the production of triploid and gynogenetic salmonids. Such chromosomal modifications often result in abnormal ovarian development. In rainbow trout (RBT), triploid females have string-like gonads lacking significant developing oocytes and are suggested to be sterile due to the odd set of chromosomes disrupting oogenesis. Aberrant ovarian development is reported to occur in about 30% of gynogenetic females. It has been suggested that gynogenetic fish are more prone to expressing developmental abnormalities due to either increased homozygosity or to incomplete inactivation of the paternal chromatin. This investigation was done to compare the ovarian morphology of female triploid and induced gynogenetic diploid RBT. The objective was to determine whether the presence of supernumerary chromosomal fragments, potentially generated during the process of sperm genome inactivation, would result in abnormal gonadal development in gynogens comparable to that observed in triploid females. Gonadal morphology was observed and karyotypical analysis was completed on 21 gynogenetic fish. In 90% of the fish examined, the presence of chromosomal fragments was positively correlated with irregular ovarian development. The atypical gonadal morphology observed in the gynogens resembled triploid RBT ovarian morphology. The results of this investigation support the hypothesis that disruption of the normal diploid chromosomal complement alters germ cell development in gynogenetic female RBT due to the unbalanced nature of the genome. J. Exp. Zool. 286:505-512, 2000.

Animals↗

PubMed1996

SV40 T antigen increases the expression and activities of p34cdc2, cyclin A, and cyclin B prior to immortalization of human diploid fibroblasts.

SV40 T antigen induces karyotype instability soon after it is expressed in human diploid fibroblasts and ultimately promotes cell immortalization and tumorigenesis. Protein levels and activities of mitotic cell cycle proteins have been shown to be elevated in several immortal cell lines relative to their normal parental cells, suggesting a possible role for the aberrant regulation of mitosis in karyotype instability. We show here that IMR-90 human diploid lung fibroblasts expressing the SV40 tumor antigens display increased protein levels and associated enzymatic activities of cyclin A, cyclin B, and p34cdc2 long before crisis and immortalization. These elevations cannot be explained by faster cell growth or altered cell cycle distributions. Increased protein levels were not totally accounted for by elevated levels of the corresponding mRNA, indicating that T antigen modulates expression at least partially by posttranscriptional mechanisms. These results indicate that perturbation of mitotic regulatory proteins precedes crisis, and imply that altered mitotic control is a direct consequence of T antigen expression rather than an outcome of secondary events associated with immortalization.

Amino Acid Sequence↗

PubMed1999

Malignant transformation of human diploid fibroblasts and suppression of their anchorage independence by introduction of chromosome 13.

Isolation of cell lines that display various degrees of transformed phenotypes may be very useful to clarify multistep mechanisms of oncogenesis, but malignant transformation of human diploid fibroblasts in culture is a very rare event. We attempted to isolate variously transformed cell lines from human diploid fibroblasts (RB) of a patient with hereditary retinoblastoma. The RB cells exhibited normal karyotypes with the exception of one copy of chromosome 13, which contained a large deletion at the q14-22 region, where the RB1 gene is located. By transfection with SV40 early genes and repeated passage, we succeeded in obtaining SV40-transfected mortal, immortalized, anchorage-independent, and tumorigenic RB cell lines. DNA fingerprinting showed that these cell lines were not contaminants, but derivatives of the original RB cells. The remaining RB1 allele may be wild-type even in the malignant cell lines, because the expression and the LT-binding ability were normal. Furthermore, we did not find any homozygous loss in 16 polymorphic markers located in the 13q14-22 region in the transformed cell lines. However, introduction of a copy of a normal chromosome 13 into the anchorage-independent cell line suppressed its anchorage-independent growth ability. All these data, together with the fact that the RB cells containing the deletion progressed to a tumorigenic state spontaneously, but normal fibroblasts did not, raise the possibility that a new tumor suppressor gene, located at 13q14-22, may play a critical role in neoplastic transformation. We conclude that these RB cell lines provide an excellent system for identification of genes involved in malignant transformation of human cells. Genes Chromosomes Cancer 26:47-53, 1999.

Cell Adhesion↗

PubMed1990

Diploid predominance in hereditary nonpolyposis colorectal carcinoma evaluated by flow cytometry.

Fifty-nine colorectal carcinomas of patients with verified cancer family syndrome (CFS) were analyzed for DNA ploidy using flow cytometry. Sixty-eight percent of the tumors were diploid, and 32% were aneuploid. The aneuploid tumors had a median DNA index of 1.24 (range, 1.12-1.97). In 90% of all tumors the DNA index was less than 1.27. This predominance of diploid/near-diploid tumors was seen both in primary and in metachronous carcinomas. In 21 cases a cell cycle analysis was possible. Tumors with the S-phase fraction (SPF) greater than or equal to 9.8% had a worse prognosis than tumors with the SPF of less than 9.8%. These findings suggest that the predominance of diploid/near diploid DNA values is one of the characteristics of colorectal carcinomas in CFS. This might signify the existence of two or more pathogenetically different subgroups of colorectal carcinoma and explain the proposed better prognosis of colorectal carcinoma in CFS compared with other colorectal carcinomas.

Adult↗

PubMed1986

Diploid-triploid mosaicism: report of necropsy findings.

This is the first report of necropsy findings associated with diploid-triploid mosaicism. The important pathological findings are presented and compared to those of pure triploidy and those noted in noninvasive studies of diploid-triploid mosaics. The clinical findings in this patient are compared with those of other reported cases.

Adrenal Glands↗

PubMed1983

Isolation by flow sorting of cytokinetic and morphological heterogeneity in late-passage cultures of human diploid fibroblasts (IMR-90).

Considerable structural, metabolic, and proliferative heterogeneity develops in populations of cultured diploid cells which have reached advanced levels of population doubling. Isolation of noncycling cells from late-passage cultures would permit more definitive investigation of the structure and behavior of individual senescent cells. In this paper, we report the viable sorting of late-passage cultures of human diploid fibroblasts (IMR-90) into two subpopulations of cells with different proliferative potentials. Sorting is based on cellular light-scattering properties and autofluorescence. Structural and behavioral features of the subpopulation exhibiting increased forward-angle light scatter are more characteristic of senescent cells than the subpopulation sorted by decreased forward-angle light scatter.

Autoradiography↗

PubMed1991

Use of diploid and triploid trout erythrocytes as internal standards in flow cytometry.

DNA content determination requires the use of standards. Vindelov has shown the need to use two standards. Chicken and trout erythrocytes are commonly used, but they are not ideal standards. On the one hand, their DNA contents rarely frame the studied sample DNA content, and, on the other hand, as their base compositions are different in terms of A + T/G + C, their relative indices change according to the stains used. Use of triploid trout erythrocytes instead of chicken erythrocytes allows elimination of these two drawbacks; however, diploid trout must be differentiated from triploid trout. The present paper shows that an anatomic malformation is found with the triploid trout and so justifies the use of paired diploid and triploid trout as standards to measure nuclear DNA content.

Animals↗

PubMed1995

A haploid and a diploid cell coexist in an in vitro immortalized spermatogenic cell line.

We have recently established a conditionally immortalized germ cell line [GC-2spd(ts)] that, at the permissive temperatures of 37 degrees C and 32 degrees C, is able to undergo meiosis in vitro and form round spermatids [Hofmann et al., (1994): Proc Natl Acad Sci USA 91:5533-5537]. In this report, we provide data that indicate that the GC-2spd(ts) cell line consists of two cell populations undergoing a haploid (n/2n) cell cycle and a diploid (2n/4n) cell cycle, respectively. The cells containing 2n DNA, when sorted by fluorescence-activated cell sorting, are able to reconstitute the full population of n/2n/4n DNA cells, indicating that they are able to commit to the reductive meiotic division and form haploid spermatids or to continue self-replication through a diploid cell cycle. This GC-2spd(ts) cell line provides a valuable tool to study the molecular mechanisms involved in the cellular decision between self-renewal by mitosis and commitment to meiosis.

Animals↗

PubMed1985

Effects of some DNA-ligands and of some mutagenic compounds on the induction of meiotic disomic or diploid yeast products.

Data were collected following the administration of various compounds to meiotic yeast cultures to investigate their effects on disomic or diploid induction. Epi chlorohydrin, actinomycin D, and 9-aminoacridine were unable to induce either diploidy or disomy. Ethyl methanesulphonate was active in inducing diploids, while hycanthone induced disomics. beta-Propiolactone, N-methyl-N'-nitro-N-nitro-soguanidine, and beta-naphtylamine were able to increase both disomy and diploidy.

DNA↗

PubMed2005

Characterizing allelic associations from unphased diploid data by graphical modeling.

A method for estimating a graphical model to describe allelic associations between genetic loci is extended to use diploid genotypes rather than haploid data. It also provides haplotype frequency estimates, estimates of phase for sampled individuals, allows for missing or partial information, genotyping errors, and arbitrary penetrance functions. A data set of 688 unrelated individuals genotyped on 25 genetic loci is used to illustrate haplotype frequency estimation. The frequencies obtained are shown to be similar to those obtained by the PHASE program. We also illustrate how putative loci for traits can be included in the analysis in order to detect allele-phenotype associations. Haplotype reconstruction is illustrated on a standard set of 40 male X-chromosome haplotypes, randomly paired to simulate diploid genotype data. The novel method is shown to be comparable to most existing ones, though the PHASE method does consistently better on this problem. The graphical model approach is shown to have some advantages in terms of tractability and can be used to select informative subsets of loci and to map loci influencing phenotypes.

Alleles↗

PubMed1979

Growth of diploid, Epstein-Barr virus-carrying human lymphoblastoid cell lines heterotransplanted into nude mice under immunologically privileged conditions.

Human Epstein-Barr virus-carrying lymphoid cell lines which have been classified on the basis of studies on clonality and morphological, chromosomal and functional parameters as lymphoblastoid cell lines (LCL) of presumed non-neoplastic origin were inoculated intracerebrally into nude mice. All eighteen of them grew, killing the host mice within 7 to 25 days, except for 2 which grew more slowly. At autopsy, the brain of the nudes was found to be invaded by infiltrating lymphomas. Sixteen of these lymphomas, when recultured in vitro, gave rise to cell lines with growth properties and morphology indistinguishable from those of the inoculated LCL. Chromosomal examinations showed that 3/7 cell lines injected, which grew as lymphomas in the brain, were still normal diploid on reexplantation whereas the remaining four had become aneuploid. Four lines derived from intracerebral lymphomas (2 diploid, 1 aneuploid and 1 untested) were inoculated subcutaneously into adult nude mice. None of them grew. When the corresponding four original LCL lines were inoculated subcutaneously into newborn nude mice, they grew rapidly, but failed to do so in newborn normal mice or intracerebrally in adult normal mice. One such line, U-1450, was treated with anti-lymphocyte serum (ALS). Small nodules developed at the site of inoculation. From one nodule a cell line was cultured, 1450 ALSAD. It was morphologically indistinguishable from the line of origin. The lines obtained from nude mice inoculated with polyclonal LCL seem to have a restricted clonal representation, but were not monoclonal, as evidenced by analyses of their pattern of immunoglobulin synthesis.

Aneuploidy↗

PubMed1985

Stabilized non-complementing diploids (Ncd) from fused protoplast products of B. subtilis.

Non-complementing diploids (Ncd) displaying the parental phenotype can be selected from polyethylene glycol (PEG)-treated fused polyauxotrophic protoplasts of Bacillus subtilis. These bacteria carry the two parental genomes, but only one of them is phenotypically expressed, the other being replicated but not expressed. Cellular cloning and DNA-DNA in situ hybridization led to the discovery of non-complementing diploid cells which at first sight could have been considered as parental haploids. The new class of stabilized Ncd (10(-7) segregants) can be obtained either directly after the primary fusion event or from segregating Ncd after further growth. The totally inactive chromosome of a stable Ncd can be activated after PEG-induced self fusion. DNA-mediated transformation studies using crude stable Ncd lysates as DNA donors show low frequencies for the genetic markers from the 'silent' chromosome. Contrary to the unstable Ncd situation, however, these frequencies remain low even with purified donor DNA. The differences in the transformation properties of the non-expressed markers are correlated to Ncd clone stability. These facts suggest that chromosome inactivation in PEG-induced fusion involves at least a two-stage process. The first would be reversible and the second irreversible, thus preserving the inactive chromosome state.

Bacillus subtilis↗

PubMed1994

Expression of cell growth and bone phenotypic genes during the cell cycle of normal diploid osteoblasts and osteosarcoma cells.

Establishing regulatory mechanisms that mediate proliferation of osteoblasts while restricting expression of genes associated with mature bone cell phenotypic properties to post-proliferative cells is fundamental to understanding skeletal development. To gain insight into relationships between growth control and the developmental expression of genes during osteoblast differentiation, we have examined expression of three classes of genes during the cell cycle of normal diploid rat calvarial-derived osteoblasts and rat osteosarcoma cells (ROS 17/2.8): cell cycle and growth-related genes (e.g., histone), genes that encode major structural proteins (e.g., actin and vimentin), and genes related to the biosynthesis, organization, and mineralization of the bone extracellular matrix (e.g., alkaline phosphatase, collagen I, osteocalcin, and osteopontin). In normal diploid osteoblasts as well as in osteosarcoma cells we found that histone genes, required for cell progression, are selectively expressed during S phase. All other genes studied were constitutively expressed both at the transcriptional and posttranscriptional levels. Alkaline phosphatase, an integral membrane protein in both osteoblasts and osteosarcoma cells, exhibited only minimal changes in activity during the osteoblast and osteosarcoma cell cycles. Our findings clearly indicate that despite the loss of normal proliferation-differentiation interrelationships in osteosarcoma cells, cell cycle regulation or constitutive expression of growth and phenotypic genes is maintained.

Actins↗

PubMed1975

Studies on the regulation of the three enzymes of the Leloir pathway in cultured mammalian cells. I. Effect of substitution of galactose for glucose as the sole hexose in the medium in human diploid cell strains and in a rat hepatoma line.

In human diploid cell strains, the substitution of galactose for glucose as the sole hexose in the medium had no measurable effect on the specific activity of the cell protein for any of the three enzymes of the Leloir pathway. These enzymes are galactokinase, alpha-D-galactose-1-phosphate: UDP glucose uridyl transferase and UDP galactose 4-epimerase. A cell strain from a patient with galactosemia had no detectable activity for the transferase. The substitution of galactose for glucose in the medium of these cells (which has been shown to cause the cells to accumulate galactose-1-phosphate) also failed to affect cellular activity for the three enzymes. Similarly, the three activities failed to respond to the substitution of galactose for glucose in cultures of a rat hepatoma line. Cells of this line have been shown by others to perform a number of the tissue-specific functions of liver. The failure of galactose to stimulate increasd cellular activity for the three enzymes represents a striking difference between the behavior of these enzymes in human diploid cell strains and their behavior in E. coli.

Bucladesine↗

PubMed1976

Regulation of lipid synthesis from acetate in diploid fibroblast cultures -- variation with passage level and stage of cell growth.

Regulation of lipid synthesis from acetate in human diploid fibroblast cultures has been studied at various passage levels and at different stages of cell growth. When cultures were transferred to lipid free medium, a stimulation of [14C]acetate incorporation into lipid occurred within three to six hours after removal of exogenous lipid. In early passage cultures, this stimulation was observed whether cells were transferred to protein-free medium or medium supplemented with delipidized serum protein. However, in late passage cultures the presence of delipidized serum protein was required for the stimulation of lipid synthesis. When logarithmically dividing and stationary phase cultures were compared, the cultures in log phase showed stimulation of acetate incorporation into lipid in the presence or absence of delipidized serum protein, whereas in the stationary cultures the delipidized serum protein was required. When cultures were partially synchronized by a thymidine block, stimulation of acetate incorporation into lipid in the blocked cells only occurred in the presence of delipidized serum protein; in released cells stimulation occurred in protein free medium. When inhibition of lipid synthesis from acetate was compared in young vs. old or dividing vs. stationary cultures, however, no differences were observed. The data indicate the response of diploid fibroblast cultres to change in exogenous lipid is dependent on passage level and state of growth.

Acetates↗

PubMed1978

Reciprocal regulation of glucose and glutamine utilization by cultured human diploid fibroblasts.

Human diploid fibroblasts utilize both glucose and glutamine as energy sources. The utilization of glutamine by fibroblasts is regulated by glucose, and vice versa. This conclusion is supported by the following observations: (1) essentially identical growth rates were observed in Eagle's minimum essential medium (MEM)3 in which the glucose concentration was either 5.5 mM or was maintained between 25 and 40 micrometer, (2) the total glutamine utilization by fibroblasts increase at least 30% in medium with 25 micrometer to 70 micrometer glucose compared to medium with 5.5 mM glucose, while the rate of glutamine-1 or 5-14C oxidation to CO2 increased 5-fold as the glucose concentration was decreased to zero, (3) 2 mM glutamine inhibited glucose-6-14C oxidation by 88% and stimulated glucose-1-14C by 77% in log phase cells and (4) glutamine oxidation in normal medium contributed approximately 30% of the energy requirement of human diploid fibroblasts.

Cell Division↗

PubMed1979

A new cell surface marker of aging in human diploid fibroblasts.

The relationship of cell surface changes to proliferative decline of human diploid fibroblasts was investigated using the concanavalin A-mediated red blood cell adsorption assay. The amount of the red blood cells adsorbed to human diploid fibroblasts via concanavalin A increased continuously from the early phases of cell passage up through cell senescence, while the amount of 3H-concanavalin A binding did not change to a significant extent. The red blood cell adsorption is not a function of cell cycle phase and time spent in culture. Cocultivation of young cells with old cells also did not affect the adsorption capacity of respective cells. Thus, the concanavalin A-mediated red blood cell adsorption can be expected to serve as a new cell surface marker for aging in vitro. Using this marker, it was revealed that transient cell size or 3H-thymidine incorporating capacity di not have a direct relationship with the division age of a cell. Small rapidly dividing cells in old populations resemble large slowly dividing or nondividing cells of the same populations and differ from small rapidly dividing cells in young populations, in terms of cell surface properties.

Cell Adhesion↗

PubMed1980

Lactate: a major product of glutamine metabolism by human diploid fibroblasts.

Human diploid fibroblasts metabolize up to 13% of the glutamine in tissue culture medium to lactate. Four microCi of glutamine-U-14C were added to media containing 5 mM or 65 microM glucose or medium containing no added glucose, but supplemented with purine and pyrimidine nucleosides (HGTU). Aliquots of the media were taken at daily intervals and were assayed for glucose, lactate, pyruvate, malate, citrate, aspartate, glutamine, and glutamate. The label incorporation into these compounds was determined, except for glutamine and glucose. The distribution of label from glutamine-U14C in 5 mM glucose medium by day 4 was lactate (10.2%), glutamate (15.2%), citrate (1.9%), pyruvate (2.0%), malate (1.1%), and aspartate (< 0.1%). The accumulation of label in lactate and glutamate occurred continuously during the growth cycle. Malate, citrate, and aspartate accumulation occurred primarily in confluent cultures. The label in aspartate was seen only in stationary phase cells or when the glucose concentration was decreased to 65 microM or less; net aspartate accumulation was increased twofold in low glucose media. These data demonstrate an actively functioning pathway for the conversion of 4-carbon TCA-cycle intermediates to 3-carbon glycolytic intermediates in human diploid fibroblasts.

Carboxylic Acids↗