A 33 kDa protein band in enhanced during long‐term adaptation of EUE cells to a hypertonic medium

Giuliani, Attilia; Ferraretto, Anita; Conti, A. M. Fuhrman; Grada, L. De; Fraschini, A; Pellicciari, C.; Romanini, M. G. Manfredi

doi:10.1002/cbf.290090204

Cited by 6 publications

(7 citation statements)

References 20 publications

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“…These findings are in agreement with the biochemical indication that P33 is, for the most part, a soluble protein; in fact, more than 80% of this protein was extracted from cell lysates after exposure to low ionic strength solutions in the absence of detergents. However, a small amount is apparently organelle-bound, being extracted after SDS treatment (7), and this is consistent with our evidence of nuclear immuno-labelling. It is, however, unlikely that P33 plays a similar role in the nucleus to the role hypothesized for the cytoplasm.…”

Section: Discussionsupporting

confidence: 92%

“…Electrophoretic data (7) showed that the relative amount of P33, compared to other protein bands, reached a plateau after 4 d of adaptation. The results of our cytometric study are consistent with the electrophoretic data, since the progressive increase of P33 content per cell (up to 20 d of growth in HT medium) parallels the enlargement of cell volume as observed in a previous work (10).…”

Section: Discussionmentioning

confidence: 99%

“…The level of this protein reverts to the controls when HT-adapted cells were transferred to the isotonic (IT) medium (7), suggesting that there is a direct relationship with resistance to the HT stress (7,10).…”

mentioning

confidence: 98%

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Light and electron microscope immunolabelling and flow cytometric evaluation of a 33kDa stress protein during the cell cycle of eue cells grown in hypertonic medium.

Pellicciari

Viale

Fraschini

et al. 1993

Acta Histochem. Cytochem

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The aim of this work was to look for the stress protein P33, during long-term growth of human EUE cells in a hypertonic medium, with a polyclonal antibody against P33 produced in rabbit, and detectable in cells by both light and electron microscopy, using fluorescein-, peroxidase-anti-peroxidaseand gold-indirect immunolabelling.P33 was found to be mostly located in the cytoplasm, without apparent association with subcellular organelles; a slight, but significant, labelling was also observed in the nucleus. Dual parameter flow cytometric measurements of fluorescein-immunolabelled P33 and DNA content (after propidium iodide staining) showed that the amount of P33 increased progressively with increasing times of growth in a hypertonic medium in all phases of the cycle. Therefore there is no direct relationship between the increase of the P33 and the exit of cells from the cycling compartment that occurs in a hypertonic environment.Recently, we have reported that the synthesis of a 33 kDa protein (P33) is enhanced when EUE (human embryonic epithelium) (13) cells were grown for long periods (4 days onwards) in a hypertonic (HT) medium. The level of this protein reverts to the controls when HT-adapted cells were transferred to the isotonic (IT) medium (7), suggesting that there is a direct relationship with resistance to the HT stress (7, 10).Long term adaptation to an HT medium also entails other characteristics, such as reversible changes in the phenotype of EUE cells: cell volume enlarges (10); there is an increase and a restructuring of cytoskeletal intermediate filaments (4); sulpholipids, gangliosides (2), and enzyme activities linked to ion transport mechanisms (3) also increase. It is of special interest that the growth rate is slowed down under HT stress, and that most cells are blocked in a resting state (12).The aim of this work was first to localize, by both light and electron microscopy, the distribution of P33 proteins in EUE cell populations grown in IT or HT medium. A second aim was to monitor the changes in P33 expression at different times of growth in an HT medium, and to see whether the changes are related to the cell cycle changes.To perform these analyses, a polyclonal antibody against P33 was produced, to be used for fluorescence-, peroxidase-anti-peroxidase (PAP)-, and gold-indirect immunodetection. On suspensions of single cells, dual parameter cytometric measurements of DNA content and P33 were also made, using propidium iodide staining and indirect immunofluorescent labelling. Eagle's medium, supplemented with 10% newborn calf serum, 1 % glutamine, 50 units/ml of streptomycin and penicillin. For the HT medium, the concentration of NaCI was raised from 0.136 M to 0.274 M in the Hank's salt solution used as a component of the culture medium (the final osmolarities were 305 mOsm and 495 mOsm). Cells were grown in a 5% CO2 atmosphere, in either IT or HT media during differentt time periods (1 to 20 d); in addition, 10-d HT-adapted cell samples were transferred to the IT medium, in which they wer...

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

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Light and electron microscope immunolabelling and flow cytometric evaluation of a 33kDa stress protein during the cell cycle of eue cells grown in hypertonic medium.

Pellicciari

Viale

Fraschini

et al. 1993

Acta Histochem. Cytochem

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“…2 1 We also investigated, by polyacrylamide gel electrophoresis, the expression of a 35 kDa protein band, whose amount was observed to increase in EUE cells grown in an HT medium. 13 This protein has been recently found to share sequence homology with an aldose r e d~c t a s e , '~ which may be involved in increasing in the cellular osmolytes, which is necessary to cope with the higher extracellular tonicity of the HT medium.22…”

Section: Introductionmentioning

confidence: 99%

Cell cycle effects of hypertonic stress on various human cells in culture

Pellicciari¹,

Filippini²,

Grada³

et al. 1995

Cell Biochemistry & Function

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Long-term exposure to hypertonic (HT) culture media has been found to perturb the cell cycle and change gene expression in various animal cell types. A lower growth rate, with exit of cells from the cycling compartment has been observed previously in human transformed EUE cells. The aim of this study was to investigate if the kinetic changes after long-term HT stress, were typical of transformed cells or could be also found in primary cultures of normal cells. Human transformed cells from normal and neoplastic tissues, and normal human cells of epithelial and connective origin have been studied. After the incorporation of bromodeoxyuridine (BrdUrd), the frequency of S-phase cells was estimated by dual-parameter flow cytometry of DNA content versus BrdUrd immunolabelling; the total growth fraction was also estimated, after immunolabelling with an anti-PCNA antibody. We also investigated, by polyacrylamide gel electrophoresis, changes in the amount of a 35 kDa protein band, which increased in EUE cells grown in an HT medium, and which may be directly involved in cell resistance to hypertonicity. Lower BrdUrd labelling indices and higher frequencies of cells in the G0/1 range of DNA content were common features of all the cells in HT media, irrespective of their tissue of origin; other cycle phases may also be involved, depending on the cell type considered. The mechanisms by which cells cope with the HT environment could however differ, since only some cell types showed an increase of the 35 kDa stress protein found originally in HT EUE cells.

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“…In their physiological environment, such cells have to cope with major osmolarity changes, since the osmolarity of the medulla increases considerably during periods of antidiuresis (Some et al, 1993;Yancey and Burg, 1989). An aldose reductase is then induced, which is responsible for the production of sorbitol, a major osmolyte accumulated during the osmotic adjustment of these cells (Bagnasco et Giuliani et al, 1991) focused on human embryonic epithelial (EUE) cells. This material is derived, like ours, from cells which never have to experience major changes in osmolarity under physiological conditions.…”

Section: Partial Purification Of Aldose Reductasementioning

confidence: 99%

Protein patterns, osmolytes, and aldose reductase of L-929 cells exposed to hyperosmotic media

Libioulle¹,

Llabrès²,

Gilles³

1996

J. Cell. Physiol.

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L-929 cells acclimated to media made hyperosmotic (600 mosmol/kgH2O) by addition of NaCl, sorbitol, or mannitol show, on SDS-polyacrylamide gels, a markedly enhanced protein band at 40 kDa, most likely corresponding to the enzyme aldose reductase. The effect was not observed in cells acclimated to a medium rendered hyperosmotic by addition of proline. The major organic osmolyte accumulated is sorbitol in cells acclimated to high-sorbitol or high-NaCl medium, proline in cells acclimated to high-proline medium. Cells acclimated to any of these hyperosmotic media display unaltered Na+ levels and similarly increased K+ levels and decreased Cl-levels. These results are interpreted in terms of the mechanisms involved in aldose reductase induction and in regulation of the enzyme activity in long-term acclimation to hyperosmotic media.

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A 33 kDa protein band in enhanced during long‐term adaptation of EUE cells to a hypertonic medium

Cited by 6 publications

References 20 publications

Light and electron microscope immunolabelling and flow cytometric evaluation of a 33kDa stress protein during the cell cycle of eue cells grown in hypertonic medium.

Light and electron microscope immunolabelling and flow cytometric evaluation of a 33kDa stress protein during the cell cycle of eue cells grown in hypertonic medium.

Cell cycle effects of hypertonic stress on various human cells in culture

Protein patterns, osmolytes, and aldose reductase of L-929 cells exposed to hyperosmotic media

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