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  • 1
    ISSN: 1432-1335
    Keywords: Cisplatin analogues ; Stability ; MCF-7 cells ; Estrogenic effects ; Growth inhibition
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The platinum(II) complex PtCl2(meso-6), which has the estrogenic ligandmeso-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine (meso-6), has been reported to be an effective antitumor drug for estrogen-receptor(ER)-positive tumors in animal experiments. The goal of this study was to investigate whether the observed biological effects could be ascribed to the intact PtCl2(meso-6). Cultures of the ER-positive human breast cancer cell line MCF-7 were used as the in vitro test system. In culture medium containing 10% fetal calf serum, PtCl2(meso-6) had a half-life of about 2 h, as determined by HPLC analysis, and no PtCl2(meso-6) was detectable after 10 h. The Pt complex bound irreversibly to serum protein. After 30 min, the diamine ligand was found released, with a maximum conversion of about 35% at 24 h. At this time the culture medium still had estrogenic activity, i. e. it induced ER processing in the MCF-7 cells. This indicates that the estrogenic effect was elicited by the released diamine ligand. In contrast, the growth-inhibitory activity of the medium preincubated with PtCl2(meso-6) was lost at a rate similar to the rate of loss of PtCl2(meso-6) from the medium. This accords with the platinum complex being the main cytotoxic entity. When MCF-7 cells were incubated with PtCl2([3H]meso-6), no free Pt complex could be identified in cellular extracts, and most of the cell-associated radioactivity coeluted with meso-6 in HPLC analysis. After 12 h, only 1.4% of the total cellular platinum was bound to DNA, but no tritium label could be detected. In conclusion, diamine ligand is released from the Pt(II) complex and can account for the estrogenic effects so far ascribed to PtCl2(meso-6).
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Journal of cancer research and clinical oncology 120 (1994), S. 286-292 
    ISSN: 1432-1335
    Keywords: Breast cancer ; Interferon ; MCF-7 ; MDA-MB231 ; Platinum complexes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The growth-inhibitory effects of combining interferons (IFN) with platinum(II) complexes were tested with the aim of comparing these in cultures of estrogen-receptor(ER)-negative MDA-MB231 and ER-positive MCF-7 breast cancer cell lines. Another aim was to test whether IFN as a biological response modifier could enhance the effect of the Pt complexes in vitro in an attempt to find an explanation for their more potent antitumor effects in in vivo models. Here it is shown that in both cell lines the combinations of different IFN with all three Pt complexes generally resulted in additive growth inhibition, as calculated by the product of the fraction of surviving cells obtained with each compound alone. Moreover, in MCF-7 cells natural IFNβ (nIFNβ) combined with aqua[meso-1,2-bis-(2,6-dichloro-4-hydroxyphenyl)ethylenediamine]sulfatoplatinum(II) (meso-6-Pt) resulted in synergistic inhibition. This synergy could be attributed to the estrogenic property of meso-6-Pt, since the ligand and estradiol also enhanced the inhibitory effect of nIFNβ. In contrast, the combination of recombinant IFNγ and meso-6-Pt was antagonistic in MDA-MB231 cells. These results show that, in spite of the similar responses of the ER-negative and ER-positive cells to each compound alone, these cells show unexpected differences in their sensitivity to combinations of IFN and the new Pt complex meso-6-Pt.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1432-1335
    Keywords: Cell-cycle arrest ; Cell death ; Micronuclei ; MCF-7 cells ; Tamoxifen ; Cisplatin ; DNA fragmentation ; Endonuclease
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The induction of cell death along with cell-cycle arrest is one of the foremost mechanisms regulating cell growth. In the human breast carcinoma cell line MCF-7 we investigated two chemotherapeutic agents, the antiestrogen tamoxifen and the DNA-damaging drug cisplatin, for the relative contribution of these mechanisms to growth inhibition in culture. Growth kinetics and flow cytometry confirmed that tamoxifen at 1 μM acts mainly by arresting cells in the G0/G1 phase of the cell cycle. Compared to untreated controls, only a few more cells were detached from the monolayer and dead after a 5-day incubation. On the other hand, cisplatin at 1 μM did not induce the well-defined G2/M-arrest reported for other cell types, but resulted in a marked increase in the rate of cell death. A morphological feature observed, especially with cisplatin-treated MCF-7 cells, was the formation of numerous micronuclei (in up to 30% of the cells) and an increase in the number of binucleate cells (up to 20%). In both tamoxifen- and cisplatin-treated cultures, cell death appeared to occur by apoptosis, as indicated morphologically by cellular and nuclear shrinkage accompanied by DNA-condensation and ultimately the formation of DNA containing apoptotic bodies. However, no internucleosomal DNA degradation or endogenous endonuclease activity could be detected in the cells of the monolayer or in the mainly dead and detached cells of the culture supernatant. DNA fragmentation was only observed when isolated MCF-7 nuclei were incubated with exogenous endonucleases. However, as determined by reverse transcriptase/polymerase chain reaction amplification, MCF-7 cells do express the mRNA for DNase I, an endonuclease known to be involved in apoptosis. Thus, apoptosis is part of the growth-inhibitory process and occurs without apparent internucleosomal DNA fragmentation in MCF-7 cell cultures.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 107 (1981), S. 155-163 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Confluent, quiescent Swiss 3T3 cells in culture can be stimulated to initiate DNA synthesis and divide by addition of growth factors to the culture medium. Here we show that hydrocortisone and other steroids which have glucocorticoid activity inhibit the stimulation of these cells by epidermal growth factor (EGF) in contrast to their reported enhancement of stimulation by fibroblast growth factor (FGF). Binding studies using [3H]-triamcinolone acetonide show that Swiss 3T3 cells contain a single class of glucocortioid receptor of uniform affinity (KD = 2.0 nM), and about 34,000 receptor sites per cell. Those steroids which displace bound [3H]-triamcinolone acetonide are also effective in inhibiting the stimulation of DNA synthesis by EGF in the presence or absence of insulin, and the concentration of triamcinolone acetonide required for one-half maximal biological effect is in the same range as the KD. A similar concentration is required for one-half maximal enhancement of the effect of FGF. These results suggest that both the inhibitory and stimulatory effects of glucocorticoids may be mediated via these receptors, the different effects thus being due to differences in the intracellular events triggered by each growth factor.
    Additional Material: 5 Ill.
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  • 5
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Fibroblastic growth factor (FGF) and prostaglandin F2α (PGF2α) are two different growth factors in Swiss 3T3 cells: each stimulates the initiation of DNA synthesis after a lag phase of 14-15 hr. Colchicine, colcemid, and nocodazole have no mitogenic effect on their own, but each has a synergistic effect on the rate of initiation of DNA synthesis stimulated by either FGF or PGF2α. Independent of the growth factor, the microtubule-disrupting drug had to be added before 8 hr of the lag phase to enhance the stimulation. Colchicine could be removed at 5 hr of the lag phase with no impairment of the synergistic effect. However, both colcemid and nocodazole had to remain present up to 10 hr of the lag phase to achieve the full synergy. This result can be accounted for by the fact that colchicine, in contrast to colcemid and nocodazole, binds almost irreversibly to microtubules, and it strengthens the interpretation that the state of microtubular organization plays a role in regulating the rate of entry into S phase. Preincubating quiescent Swiss 3T3 cells with colchicine, colcemid, or nocodazole before stimulation by FGF or PGF2α shortened the lag phase by about 3 hr. When the microtubule-disrupting drug was removed prior to stimulation, the lag phase was also shortened, but there was no enhancement of the rate of initiation of DNA synthesis, except upon preincubation with colchicine. This suggests that microtubule disruption can independently affect the length of the lag phase and the rate of initiation of DNA synthesis, and that these two phenomena can be uncoupled.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Prostaglandin F2α(PGF2α); which stimulates DNA synthesis in resting 3T3 cells, also stimulates the incorporation of [32P]PO4 into phosphatidylinositol. The effect is selective for PGF2α when compared with PGE1, PGE2, and PGF2α. Epidermal growth factor (EGF) also stimulates DNA synthesis but does not affect phosphatidylinositol turnover. PGE1, which acts synergistically with PGF2α to enhance DNA synthesis, does not affect the ability of PGF2α to enhance the incorporation of [32P]PO4 into phosphatidylinositol. PGF2α also causes a small increase in the cellular content of 1,2-diacylglycerol. This effect is not shared by EGF or PGE1. Stimulation of phosphatidylinositol metabolism resulting in an increase in the cellular content of 1,2-diacylglycerol may thus constitute an event in the pathway leading to the initiation of DNA synthesis in which PGF2α differs in its action from EGF.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Epidermal growth factor (EGF) stimulates the initiation of DNA synthesis in Swiss 3T3 cells after a constant prereplicative period of 14-15 hours. The final rate of initiation follows apparent first-order kinetics and can thus be quantified by a rate constant k. The value of k can be changed by later additions during the prereplicative period: When cells stimulated by a very low concentration of EGF, alone or with insulin, which results in a relatively low value of k, receive a saturating amount of EGF at 15 hours, then k is markedly increased after 4-6 hours. Insulin alone (up to 200 ng/ml) is unable to set the lag phase, but does have a synergistic effect on the value of k given by EGF. When added at 15 hours, insulin also increases k, but after a delay of 4-6 hours. In contrast, both hydrocortisone and prostaglandin E1 (PGE1) inhibit the stimulation of DNA synthesis by EGF only during the first 8 hours of the prereplicative period of decreasing the value of k.Prostaglandin F2α (PGF2α), which stimulates DNA synthesis in a similar mode as EGF, when added with EGF has a synergistic effect on DNA synthesis. This suggests that EGF and PGF2α, nevertheless, act through different regulatory events.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 119 (1984), S. 155-162 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Quiescent Swiss mouse 3T3 cells react to a heat treatment at 46°C for 20 min by changing their flat, well-extended morphology to a round appearance with retracted cytoplasmic processes during the subsequent 2 h at 37°C. The percentage of morphologically changed cells was used to quantify changes in heat sensitivity, or resistance, in response to mitogenic stimulation. Stimulating quiescent cells with serum or with the specific growth factors epidermal growth factor (EGF) and prostaglandin F2α (PGF2α) markedly increased the heat resistance to a 46°C treatment, but only when the heat treatment, but only when the heat treatment was applied within 2-3 h after the addition. When insulin (which is not mitogenic, but synergistic with EGF and PGF2α in these cells) was added alone or in combination with either EGF or PGF2α, it had no effect on the development of heat resistance. Neither did cycloheximide nor tunicamycin inhibit heat resistance induced by EGF, and cycloheximide even enhanced it after 2-4 h. However, adding colcemid before or at the beginning of the heat treatment abolished the increased heat resistance. The results indicate that the resistance to a single heat treatment at 46°C may be related to changes in the metabolic state after mitogenic stimulation, even though these changes need not be reflected in the rate of entry into S phase. Furthermore, the cytoskeletal organization appears to be a crucial component in heat resistance of Swiss 3T3 cells.
    Additional Material: 7 Ill.
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  • 9
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: An approach to the investigation how growth factors and hormones regulate mammalian cell proliferation is to study the activity of enzymes involved in DNA replication. Quiescent cultures of Swiss mouse 3T3 cells were stimulated with prostaglandin F2α insulin, and/or hydrocortisone for a time at which less than 50% of the cells had initiated DNA synthesis. Such cells were lysed with a Ca+ +-containing hypotonic buffer and incubated with a nucleotide mixture including [3H]thymidine-triphosphate for 1 hr at 37°C. The amount of radioactive label incorporated into the trichloroacetic acid (TCA)-precipitate and the percentage of labeled nuclei correlated with the in vivo stimulation. Analysis of radioactively and density-labeled DNA in sucrose and CsC gradients indicated that the incorporation of label reflected semiconservative replication. DNA polymerase activities were assayed in supernatants from whole-cell lysates prepared with a hypotonic buffer not containing Ca+ +. Using various templates, it was shown that the increase in activity of DNA polymerase α correlated with the percentage of cells in S phase upon the different stimulation, while DNA polymerase β activity after various times of stimulation showed that this activity increased only when cells began to enter S phase, regardless of the combination of growth factor and hormones.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 13 (1980), S. 281-294 
    ISSN: 0091-7419
    Keywords: glycosaminoglycans ; cell cycle ; biosynthesis ; fibroblasts ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cultures of embryonic fibroblasts from Balb/c or CBA/J mice were given 12-h pulses of 14C-galactose, or were double-labelled with 3H-galactose and 35H-sulfate. The time course of the rates of labelling of glycosaminoglycans - galactose label was found in the uronic acid moiety - was studied in synchronously and asynchronously growing cultures. Partial synchrony was achieved by trypsinising quiescent, confluent cells and subsequent transfer of cells to new cultures with fresh medium. Synchrony was monitored by measurement of thymidine uptake in parallel cultures. The distribution of label in the hyaluronic acid, chondroitin sulfate, and heparan sulfate fractions from cells and culture media was determined at each time point.Peaks of DNA synthesis were accompanied by or followed 12 h later by a maximal rate of labelling with galactose of secreted glycosaminoglycans, and - with the exception of hyaluronic acid - also of cellular glycosaminoglycans. The rate of labelling with galactose of glycosphingolipids in parallel cultures followed a different time course. In double-label experiments the rates of labelling of glycosaminoglycan sulfates with 3H-galactose and 35S-sulfate did not go parallel. In older, quiescent cultures the labelling rate with galactose decreased while the sulfation rate increased.It is discussed that the labelling rate with galactose is indicative of the biosynthetic rate of the glycosaminoglycans. The conclusion is reached that glycosaminoglycans are preferentially synthesized and secreted after the S phase of the cell cycle.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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