Ploidy variation in multinucleate cells changes under stress

Ploidy variation is found in contexts as diverse as solid tumors, drug resistance in fungal infection, and normal development. Altering chromosome or genome copy number supports adaptation to fluctuating environments but is also associated with fitness defects attributed to protein imbalances. Both aneuploidy and polyploidy can arise from multinucleate states after failed cytokinesis or cell fusion. The consequences of ploidy variation in syncytia are difficult to predict because protein imbalances are theoretically buffered by a common cytoplasm. We examined ploidy in a naturally multinucleate fungus, Ashbya gossypii. Using integrated lac operator arrays, we found that chromosome number varies substantially among nuclei sharing a common cytoplasm. Populations of nuclei range from 1N to >4N, with different polyploidies in the same cell and low levels of aneuploidy. The degree of ploidy variation increases as cells age. In response to cellular stress, polyploid nuclei diminish and haploid nuclei predominate. These data suggest that mixed ploidy is tolerated in these syncytia; however, there may be costs associated with variation as stress homogenizes the genome content of nuclei. Furthermore, the results suggest that sharing of gene products is limited, and thus there is incomplete buffering of ploidy variation despite a common cytosol.     

A cytophotometric comparison of the age-related changes in the DNA and protein content in human atrial cardiomyocytes in heart diseases

A study was performed on the human auricle muscle cells which were isolated from biopsies obtained in clinics during operation on heart. The nuclear DNA and the total protein in the cytoplasm were revealed by means of the two consecutive tests: the Feulgen and naphthol yellow S staining. DNA and protein contents were determined by two wave-length scanning cytophotometry. It is ascertained that under mitral defects the investigated parameters exhibit a tendency to increase with age: the nuclear ploidy and total protein content in the cytoplasm rise simultaneously with the increase in nuclear and cellular volumes, the polyploidy reaching higher levels than in myocardium of people of the same age without heart disease. If a patient suffered from ischemia, the nuclear polyploidy increased more slowly than cell hypertrophy to reach the level near the natural age limits.     

Micronuclei formation and aneuploidy induced by Vpr, an accessory gene of human immunodeficiency virus type 1.

Vpr, an accessory gene of HIV-1, induces cell cycle abnormality with accumulation at G2/M phase and increased ploidy. Since abnormality of mitotic checkpoint control provides a molecular basis of genomic instability, we studied the effects of Vpr on genetic integrity using a stable clone, named MIT-23, in which Vpr expression is controlled by the tetracycline-responsive promoter. Treatment of MIT-23 cells with doxycycline (DOX) induced Vpr expression with a giant multinuclear cell formation. Increased micronuclei (MIN) formation was also detected in these cells. Abolishment of Vpr expression by DOX removal induced numerous asynchronous cytokinesis in the multinuclear cells with leaving MIN in cytoplasm, suggesting that the transient Vpr expression could cause genetic unbalance. Consistent with this expectation, MIT-23 cells, originally pseudodiploid cells, became aneuploid after repeated expression of Vpr. Experiments using deletion mutants of Vpr revealed that the domain inducing MIN formation as well as multinucleation was located in the carboxy-terminal region of Vpr protein. These results suggest that Vpr induces genomic instability, implicating the possible role in the development of AIDS-related malignancies.     

Nucleus : cell volume ratio directs the timing of the increase in blastomere adhesiveness in starfish embryos

Blastomeres of starfish embryos begin to increase in adhesiveness after the eighth cleavage and form a monolayered hollow blastula. To investigate factors that affect the timing of the adhesiveness increase, we changed the volume of the cytoplasm or the ploidy of embryos and examined the morphologic changes in the descendent blastomeres during early cleavage stages. In parthenogenetic embryos, in which the ploidy is doubled, the timing of the increase in adhesiveness was accelerated by one cell cycle. In contrast, the timing was delayed by approximately one cell cycle in a large-sized embryo formed by the fusion of an egg and a non-nucleate egg fragment. These two sets of observations are in accord with the expectation from the classical concept that the DNA: cytoplasmic ratio may direct the timing of events in early development. However, observations of small-sized embryos with a reduced amount of cytoplasm were contradictory to the expectation based on the DNA: cytoplasmic ratio; the timing of the increase in adhesiveness in half-sized embryos was almost the same as in control embryos and the timing was delayed by only one cell cycle in quarter-sized embryos. Measurement of the diameters of nuclei showed that the size of nuclei was variable, depending on the stage of development, the volume of cytoplasm and ploidy. We calculated a volume ratio of nucleus to cytoplasm (N: C volume ratio) for tetraploid, large-, half- and quarter-sized embryos. We found that the embryonic cells begin to adhere always when their N: C volume ratio reaches 0.06. A plausible model for the cellular timing mechanism of cell contact is proposed.     

Simultaneous photometry of the DNA and silver-grain count in liver cells labelled with 3H-thymidine or 3H-leucine

A modified method was proposed for reflected light simultaneous measurement of DNA content and of the silver grain number in the nucleus or cytoplasm of the same cell. Specimens-smears of isolated liver cells incorporated 3H-thymidine and 3H-leucine were prepared on coverslips and after processing were mounted on the slide glasses with smeared side facing downwards to avoid the influence of grains on DNA content measurements. To decrease the background, label measurements were carried out in polarized light. It was shown that the intensity of 3H-leucine incorporation in hepatocytes increases proportionally with cell ploidy degree.     

Effect of a food ration with a low-protein content on the processes of liver parenchyma recovery in rats after tetrachloromethane damage

Male Wistar rats weighing 190-200 g were fed a low protein diet. Atrophy of cytoplasm was observed after a 3-week malnutrition. In early periods of liver regeneration (6 hr after CCl4 poisoning), an increase in DNA synthesis was accompanied by an increase in summary concentration of organelle membranes. Though their ploidy was higher than in the control, volume of hepatocytes and summary area of organelles surface membrane was smaller than in standard diet. These data show a reduced capacity for realization of genetic program during the regeneration period in protein-deficient rats.     

Isoform-Specific Redistribution of Calcineurin Aα and Aβ in the Hippocampal CA1 Region of Gerbils After Transient Ischemia

Abstract: To investigate isoform-specific roles of Ca²⁺/calmodulin-dependent phosphatase [calcineurin (CaN)] in ischemia-induced cell death, we raised antibodies specific to CaN Aα and CaN Aβ and localized the CaN isoforms in the hippocampal CA1 region of Mongolian gerbils subjected to a 5-min occlusion of carotid arteries. In the nonischemic gerbil, immunoreactions of both isoforms were highly enriched in CA1 regions, especially in the cytoplasm and apical dendrites of CA1 pyramidal neurons. At 4–7 days after the induced ischemia, immunoreactivities of the CaN Aα isoform in CA1 pyramidal cells were markedly reduced, whereas they were enhanced in the CA1 radiatum and oriens layers. In contrast, CaN Aβ immunoreactivities were reduced in all layers of the ischemic CA1 region, whereas they were enhanced in activated astrocytes, colocalizing with glial fibrillary acidic protein. These findings suggest that up-regulation of CaN Aα in afferent fibers in CA1 and up-regulation of CaN Aβ in reactive astrocytes may be involved in neuronal reorganization after ischemic injury.     

Growth patterns of rat hepatocytes during postnatal development.

During postnatal growth in the liver of the rat, a characteristic shift towards binuclear cells and cells of higher ploidy class occurs. When the protein content of individual isolated hepatocytes of different ploidy classes is analysed cytophotometrically using the specific protein stain Naphthol Yellow S, it appears that the growth in mass in the period 30-99 days is due mainly to increase of protein content of binuclear diploid (BD) and mononuclear tetraploid (MT) cells. The mononuclear diploid (MD) cells play a quickly diminishing role in the parenchymal population after the initial growth phase and cells of highest ploidy degree remain unimportant quantitatively. The quickly growing BD and MT cells only reach a Naphthol Yellow S protein value twice that of MD cells after a certain period of growth, whereas changes in protein content are slight or absent from 99 days onwards in all cell types investigated.     

Effect of low temperatures on the level of nucleic acids and protein in the nuclei of early bird embryos (cytophometric study)]

The content of DNA and protein in the nuclei was determined cytophotometrically as well as DNA and RNA in nucleoli of chicken embryos of different gastrullation stages after 3 days of storage at +6 degrees, +2 degrees and - 2 degrees C. It was established that the response of embryos to cold at the cell level became apparent in inhibiting the protein synthesizing system (under effects of suboptimal and extreme temperature). inhibiting the migration processes of nuclear RNA into the nucleoplasm and nuclear proteins into the cytoplasm and a certain alteration in the nucleus ploidy (under extreme conditions). The reaction of each stage under study and different cellular layers of the same differentiation stage were shown to have certain specificity.     

Effects of copper deficiency on pollen fertility and nucleic acids in the durum wheat anther

Summary The effects of continuous copper deficiency and of temporary deficiencies initiated prior to (initial deficiency) or after (terminal deficiency) the end of tillering (a stage preceding the meiotic cycle) on pollen formation were investigated in durum wheat grown on a nutrient solution. The effects of the treatments on pollen viability (FCR test) and on the proline content of the pollen grains suggested that copper deficiency induced a nearly complete sterility of the pollen formed and inhibited all grain production. Temporary copper deficiencies significantly reduced the viability rate and the number of proline-rich pollen grains without affecting pollengrain production. Cytophotometric measurements showed that initial copper deficiency induced a significant increase in the proportion of polyploid microspore mother cells (MMCs), whereas terminal copper deficiency blocked endomitotic DNA syntheses. Protein metabolism was markedly altered by the treatments. The RNA content of the cytoplasm of tapetum cells was decreased by 34%–48%, depending on the treatment. The autoradiographic study showed that the stress caused by copper deficiency enhanced [³H]uridine incorporation into microspore cytoplasm RNA and also into the tapetum cells in the case of temporary deficiencies. The incidence of the treatments on the ploidy of the mother cells and on the disturbance of protein metabolism, particularly in tapetum cells, is discussed.     

Importance of protein kinase C targeting for the phosphorylation of its substrate, myristoylated alanine-rich C-kinase substrate

We visualized the translocation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in living Chinese hamster ovary-K1 cells using MARCKS tagged to green fluorescent protein (MARCKS-GFP). MARCKS-GFP was rapidly translocated from the plasma membrane to the cytoplasm after the treatment with phorbol ester, which translocates protein kinase C (PKC) to the plasma membrane. In contrast, PKC activation by hydrogen peroxide, which was not accompanied by PKC translocation, did not alter the intracellular localization of MARCKS-GFP. Non-myristoylated mutant of MARCKS-GFP was distributed throughout the cytoplasm, including the nucleoplasm, and was not translocated by phorbol ester or by hydrogen peroxide. Phosphorylation of wild-type MARCKS-GFP was observed in cells treated with phorbol ester but not with hydrogen peroxide, whereas non-myristoylated mutant of MARCKS-GFP was phosphorylated in cells treated with hydrogen peroxide but not with phorbol ester. Phosphorylation of both MARCKS-GFPs reduced the amount of F-actin. These findings revealed that PKC targeting to the plasma membrane is required for the phosphorylation of membrane-associated MARCKS and that a mutant MARCKS existing in the cytoplasm can be phosphorylated by PKC activated in the cytoplasm without translocation but not by PKC targeted to the membrane.     

Escherichia coli alkaline phosphatase localized to the cytoplasm slowly acquires enzymatic activity in cells whose growth has been suspended: a caution for gene fusion studies.

Alkaline phosphatase is normally localized to the periplasm of Escherichia coli and is unable to fold into its native conformation if retained in the cytoplasm of growing cells. The alkaline phosphatase activity of E. coli expressing a version of the protein without a signal sequence was nonetheless found to increase gradually when the growth of cells was suspended. At least 30% of the protein was activated over the course of several hours when freshly grown exponential-phase cells were held on ice. Similar behavior was observed with cells expressing certain other mutant versions of alkaline phosphatase that are retained in the cytoplasm. The activation resulted not from the passage of the alkaline phosphatase into the periplasm but from the slow folding of alkaline phosphatase into its native conformation in the cytoplasm. These findings indicate that the mechanism by which proteins are normally kept reduced in the cytoplasm fails to function if cells are not growing. It was found that the addition of the sulfhydryl-alkylating agent iodoacetamide to cells after growth blocks this activation completely. This treatment can therefore diminish the likelihood of spurious enzyme activity measurements in studies that make use of alkaline phosphatase fusion proteins.     

Immunocytochemical localization of S-100b protein in degenerating and regenerating rat sciatic nerves

We studied the cellular and subcellular distribution of S-100b protein in normal, crushed, and transected rat sciatic nerves by an immunocytochemical procedure. In uninjured nerves, S-100b protein was restricted to the cytoplasm and membranes of Schwann cells, with no reaction product present in the nucleus or in axons. Similar images were seen from the first to the thirtieth day after the crush in activated Schwann cells during the degeneration period, i.e., up to the seventh post-lesion day, and in normal Schwann cells reappearing during the regeneration period, i.e., after the seventh post-lesion day, in the zone of the crush and proximal and distal to it. By the technique employed, there seemed to be no differences in the intensity of the immune reaction product in normal and activated Schwann cells. Also, similar images were seen in the proximal stump of transected nerves. Only a slight S-100b protein immune reaction product could be observed in the rare activated Schwann cells present in the distal stump around the seventh post-lesion day, the majority of cell types being represented by fibroblasts and elongated cells at this stage and thereafter. By immunochemical assays, similar results as those presented here have been reported and interpreted as indicative of the presence of S-100 protein in axons or, alternatively, of axonal control over expression of S-100 protein in Schwann cells. Our immunocytochemical data clearly show that the strong reduction in the S-100 protein content of the distal stump of transected nerves is owing to the paucity of Schwann cells and to the decrease in the S-100 protein content of these cells, rather than to degeneration of axons.     

Comparative Ploidy Studies Using Cytological and Paraffin Section Preparations

The ploidy profiles of benign and malignant tumours can be obtained using image analysis. However, the results of ploidy studies have varied according to the type of specimen used. We compared the ploidy profiles of paraffin embedded thin sections, cytospin preparations of disaggregated cells, and cytological smears from the same specimen as defined by image analysis. Ten benign breast lesions, 10 breast carcinomas and 10 malignant melanomas were investigated in this way. Preparations stained by the Feulgen technique were examined using the MD20 video image analysis densitometry system. Ploidy profiles were obtained by measuring the integrated optical density of at least 200 nuclei. By paying proper attention to the quality of fixation and presence or absence of cytoplasm around cells, comparable results were found for all preparations in each case. We therefore conclude that if careful attention is paid to the technical quality of the material, reliable ploidy results can be obtained by image analysis.     

APC senses cell-cell contacts and moves to the nucleus upon their disruption.

The adenomatous polyposis coli (APC) tumor suppressor protein is involved in the Wnt/wingless pathway, modulating beta-catenin activity. We report the development of a highly specific, chemically synthesized oligobody (oligonucleotide-based synthetic antibody), directed against the N-terminal region of APC. Using this reagent, we found that within 16 h of disrupting HT-29 cell-cell contacts by harvesting cells with trypsin/EDTA treatment and replating, APC was translocated from the cytoplasm to the nucleus. Five days after plating the cells, when the cells had returned to their normal confluent phenotype and cell-cell contacts were reestablished, APC returned to the cytoplasm. These results suggest that APC functions as part of a "sensor" system, and responds to the loss of cell-cell contacts by moving to the nucleus, and returning to the cytoplasm when the contacts are fully restored.     

Fractionation of Rat Hepatocyte Subpopulations with Varying Metabolic Potential, Proliferative Capacity, and Retroviral Gene Transfer Efficiency

The liver contains hepatocytes with varying ploidy and gene expression. To isolate cells on the basis of ploidy for analyzing mechanisms concerning cell proliferation and differentiation, we used Percoll gradients to separate F344 rat hepatocyte subpopulations. Specific fractions were enriched in polyploid (H2 fraction) or diploid (H3 and H4 fractions) hepatocytes containing glycogen and glucose-6-phosphatase. H4 cells were relatively smaller with greater nuclear/cytoplasmic ratios, less complex cytoplasm, and higher serum albumin or ceruloplasmin biosynthetic rates. H2 fraction cells were larger with lesser nuclear/cytoplasmic ratio, more complex cytoplasm, and more cytochrome P450 activity. Phenotypic marking showed that H4 cells originated in zone one and H2 cells in zones two or three of the liver lobule. H4 cells showed much greater mitogenic responsiveness to human hepatocyte growth factor. Retroviral gene transfer, which requires both viral receptors and cellular DNA synthesis, was significantly more efficient in H4 cells. The findings indicated thatsmalldiploid andlargepolyploid hepatocytes show unique biological differences. The ability to isolate hepatocytes of varying maturity is relevant for mechanisms concerning liver growth control and hepatic gene expression.