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1.
Chin. med. j ; Chin. med. j;(24): 392-395, 2003.
Article in English | WPRIM | ID: wpr-324466

ABSTRACT

<p><b>OBJECTIVE</b>To observe the effects of Dexamethasone (Dex), a synthetic glucocorticoid, on glucocorticoid receptor expression in the human ovarian carcinoma cell line 3AO. The molecular mechanism of glucocorticoid (GC) on 3AO cells was also studied.</p><p><b>METHODS</b>The expression and regulation of the glucocorticoid receptor in 3AO cells was studied by utilizing radioligand binding assay and quantitative RT-PCR.</p><p><b>RESULTS</b>High affinity and low capacity GR existed in 3AO cells, in addition, GR binding activity was down-regulated by Dex in a time-dependent manner, to a level about 28.34% of control following 24 hours treatment, with a concomitant decrease in GR mRNA. The induction of alkaline phosphatase (AKP) activity by Dex was reversed by RU486, a potent glucocorticoid antagonist.</p><p><b>CONCLUSION</b>In 3AO cells, functional GR which can be down-regulated by Dex at the protein and mRNA level, exists suggesting that the regulating effects of Dex on GR occur at least partially at the GR mRNA level, and that the cellular effects of Dex on 3AO cells might be mediated by GR.</p>


Subject(s)
Female , Humans , Dexamethasone , Pharmacology , Dose-Response Relationship, Drug , Mifepristone , Pharmacology , Ovarian Neoplasms , Chemistry , Drug Therapy , RNA, Messenger , Receptors, Glucocorticoid , Genetics , Tumor Cells, Cultured
2.
Article in Chinese | WPRIM | ID: wpr-591192

ABSTRACT

Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two receptor isoforms, termed hGRα and hGRβ. hGRα is a ligand-activated transcription factor which, in the hormone-bound state, modulates the expression of glucocorticoid-responsive genes by binding to specific glucocorticoid response element (GRE) DNA sequences. In contrast, hGRβ dose not bind glucocorticoids and is transcriptionally inactive. We demonstrate here that hGRβ inhibits the hormone-induced, hGRα-mediated stimulations of gene expression, including glucocorticoid-responsive reporter gene (cat) and endogenous p21 gene. We also demonstrate that hGRβ can inhibit hGRα-mediated regulation of proliferation and differentiation of a human osteosarcoma cell line (HOS-8603). Our studies on the expression of hGR mRNA in nephrotic syndrome patients indicate that the hGRα/hGRβ mRNA ratio in peripheral white blood cell of hormone-resistant patients is lower than that of hormone-sensitive patients and health volunteers. These results indicate that hGRβ may be a physiologically and pathophysiologically relevant endogenous inhibitor of hGRα

3.
Article in Chinese | WPRIM | ID: wpr-518247

ABSTRACT

AIM: To explore the possible role of the vitamin D receptor (VDR) in the effect of 1,25-dihydroxyvitamin D 3 and its novel analogues on a human osteosarcoma cell line (HOS-8603) proliferation. METHODS: Detecting the VDR mRNA and protein expression in HOS-8603 cells by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analysis, respectively. Using the method of transient transfection of a reporter gene (DR3-tk-CAT) for VDR to detect its function. The cell VDRas3 which stably expressed VDR antisense mRNA was used to observe the effect of 1,25(OH) 2 D 3 on the proliferation of HOS-8603 cells and the induction of p21 mRNA, one of the VDR target genes, when the VDR in the cells was blocked. RESULTS: HOS-8603 cells expressed the vitamin D receptor which functioned as a hormone-dependent transcriptional factor. When VDR in the HOS-8603 cells decreased, the cells lost the responsiveness to 1,25(OH) 2 D 3 irrespective of either the induction of p21 gene expression or the cell proliferation. CONCLUSION: The effect of 1,25(OH) 2 D 3 on the proliferation of the human osteosarcoma cell line HOS-8603 was mediated by its nuclear receptor VDR.

4.
Article in Chinese | WPRIM | ID: wpr-519858

ABSTRACT

AIM: To investigate the effect of glucocorticoid receptor beta(GR?) on the transcriptional activation function of glucocorticoid receptor alpha(GR?) and elucidate the biological significance of glucocorticoid receptor beta. METHODS: GR? (pRSH-GR?) and GR? responsive reporter gene cat (pMMTV-cat) were cotransfected into human osteosarcoma cell line (HOS-8603). Transient transfectants were treated with dexamethasone and the activity of cat was determined by the method of ELISA. The expression p21 messenger RNA was detected by the method of reverse transcription-polymerase chain reaction. RESULTS: GR? repressed the expression of reporter gene-cat in a dose-dependent manner. The expression of messenger RNA of p21, an endogenous target gene of GR?, was also inhibited by GR?. CONCLUSION: GR? can inhibit the transcriptional activation function of GR?. GR? may be an endogenous inhibitor of GR?.

5.
Article in Chinese | WPRIM | ID: wpr-520542

ABSTRACT

AIM: To explore the possible role of p21 wafl gene in the effect of 1,25-dihydroxyvitamin D 3 on a human osteosarcoma cell line (HOS-8603) proliferation and differentiation. METHODS: The effect of 1,25(OH) 2D 3 on the expression of p21 wafl mRNA was determined by quantitative reverse transcription-polymerase chain reaction. The human osteosarcoma cell line stably expressing the p21 wafl mRNA, which named HOS-p21 wafl , was constructed by lipofectamine transfection, and the expression of p21 wafl protein was detected by Western blot. Then the growth curve of the HOS-p21 wafl cells and the expression of alkaline phosphatase (AKP) in the HOS-p21 wafl cells were investigated. RESULTS: The expression of endogenous p21 wafl mRNA in HOS-8603 cells was induced by 1,25(OH) 2D 3 in a time-dependent manner, reaching the maximum at 4 h, and remaining the inducible action for up to 24 h over basal levels. The HOS-p21 wafl cells grew much slower than the control cells, only reaching 50% of the control ones when cultured for up to 6 days. Histochemical analysis showed that the expression of alkaline phosphatase (AKP) in the HOS-p21 wafl cells increased remarkably.CONCLUSION: These results suggest that the p21 wafl mRNA expression induced by 1,25(OH) 2D 3 is one of important mechanisms responsible for the cellular effects of the hormone on HOS-8603 cells.

6.
Article in Chinese | WPRIM | ID: wpr-518848

ABSTRACT

AIM: To clarify the effects of glucocorticoids on the messenger RNA expression of glucocorticoid receptor (GR? and GR?). METHODS: messenger RNA of GR ? and GR? were determined by quantitive RT-PCR. RESULTS: Besides GR? mRNA, GR? mRNA is also expressed in human osteosarcoma cell line HOS-8603, human ovarian carcinoma cell line 3AO and human peripheral white blood cells. After administration of dexamethasone, both GR? mRNA and GR? mRNA were down-regulated in a time-dependent and dose-dependent manner. CONCLUSION: GR? expresses in various kinds of human tissue cells. The expression of GR? is regulated by glucocorticoids.

7.
Article in Chinese | WPRIM | ID: wpr-550321

ABSTRACT

The effect of glucocorticoids on the down-regulation of glucocorticoid receptor (GR) mRNA was studied in intact rats. GR mRNA was characterized by Northern blot hybridization and quantitated by dot blot hybridization using a human GR cDNA fragment as a probe. Administration of hydrocortisone in polyvinyl alcohol resulted in a rapid increase in plasma glucocorticoids which maintained at stress levels (20 to 40 ?g/dl) for about 3 d. Hepatic GR mRNA decreased significantly to 73.5?63% of control values 6 h following hydrocortisone treatment, after which the decline of GR mRNA was gradual, reaching a minimum of 44.0?5.0% of control levels 3 d after the treatment The effect of hydrocortisone on the down-regulation of hepatic GR mRNA lasted up to 11 d. In contrast, hydrocortisone treatment had no effect on GR mRNA in rat brain. These results are consistent with the changes in GR in rats as reported previously, except that even though the hepatic cytosol GR decreased markedly, no significant changes in hepatic GR mRNA were found 1 h after hydrocortisone treatment, strongly suggesting that the down-regulation of GR by its ligands in vivo occurs at both transcriptional and posttranscriptional levels and is of tissue-specific fashion.

8.
Article in Chinese | WPRIM | ID: wpr-550955

ABSTRACT

The effects of retinoic acid (RA) on the proliferation and differentiation of a newly established human osteosarcoma cell line, HOS-8603, were studied. The results showed that RA could inhibit the proliferation of HOS-8603 cells at different concentrations ranging from 10-6 to 10-10 mol/L, and the inhibitory effect of RA was in a dose-dependent manner. In addition, RA (10-8 mol/L) could also significantly suppress the clonal proliferation of HOS-8603 cells. The effects of RA on the alkaline phosphatase activity in HOS-8603 cells were also determined. It was found that the alkaline phosphatase activity could be induced 48 h after treatment with RA (10-7 mol/L). The induction of alkaline phosphatase activity and morphological changes in HOS-8603 cells by RA suggested that RA might play roles in the differentiating processes. The interactions between retinoic acid and dexmathasone (Dex), a synthetic glucocorticoid, in the control of HOS-8603 cell proliferation were also investigated, and it was found that RA at low concentrations (10-8 to 10-10 mol/L) had a synergistic effect, while high concentrations of RA (10-6 and 10-7 mol/L) had a antagonistic effect with Dex on the proliferation of HOS-8603 cells.

9.
Article in Chinese | WPRIM | ID: wpr-550987

ABSTRACT

The effects of MC903, a novel 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3] analogue, on proliferation and differentiation of a human megakaryoblastic leukemia cell line (HIMeg) were investigated. MC903 was found to inhibit cell proliferation and induce cell differentiation of HIMeg cells. Colony formation assay showed that MC903 (10-10 to 10-6mol/L) almost completely inhibited the colony-forming ability of HIMeg cells. Cell cycle analysis demonstrated that MC903 altered the cell cycle distribution; the fraction of cells in G0 + G1 increased while those in S and G2 + M decreased after treatment with MC903 for 4d. The effect of MC903 on proliferation and differentiation of HIMeg cells was in a dose- and time-dependent manner and was more active than that of 1, 25(OH)2, D3,. The results of the present study suggested that 1,25(OH)2 D3 as well as its novel analogue MC903 might play an important role in modulating the proliferation and differentiation of megakaryocytic lineage.

10.
Article in Chinese | WPRIM | ID: wpr-551319

ABSTRACT

The cellular effects of glucocorticoids on proliferation and differentiation of a human inegakaryoblastic leukemia cell line (HIMeg) were studied in comparison with sex steroid hormones. In both liquid and methylcellulose culture systems, glucocorticoids suppressed the proliferate of HIMeg cells in a dose-dependent manner. In contrast, sex steroid hormones except progesterone had little effects on the proliferation of HIMeg cells. In liquid culture systems, morphological changes were not evident, and the percentage of cells with multilobular nuclei increased slightly from 2% to 8% after glucocorticoid treatment. Similarly, only 2% of HIMeg cells expressed glycoprotein Ⅱb/Ⅲa (GP Ⅱb/Ⅲa) antigen without hormone, whereas 30% of the cells expressed GP Ⅱb/Ⅲa with the addition of glucocorticoids. These data indicated that glucocorticoids could induce differentiation of HIMeg cells. To clarify the molecular mechanisms, glucocorticoid receptor (GR) expression was examind by Scatchard analysis, and it was found that there was a saturable, high affinity GR in HIMeg cells. Furthermore, the cellular effects of glucocorticoids on HIMeg cells could be reversed by RU486, a potent glucocorticoid antagonist. These observations suggest that the cellular effects of glucocorticoids on HIMeg cells were mediated by GR.

11.
Article in Chinese | WPRIM | ID: wpr-551354

ABSTRACT

The induction of hsp70 mRNA in human osteosarcoma cells (HOS-8603) and intacl rats under heat stress was studied using hsp70 cDNA labeled with a-32P dATP. The results showed that the induction of hsp70 mRNA was evident in liver, lung, spleen and the most evident was found in brain when the core body temperature of rats was brought to 42℃ for 15 min. The induction of hsp70 mRNA in HOS-8603 was also significant when cells were cultured at 42℃ for 30 min. These results indicated that hsp70 mRNA could be induced both in vitro and in vivo under heat stress condition and the induction of hsp70 mRNA in intact rats was of tissue-specific fashion.

12.
Article in Chinese | WPRIM | ID: wpr-551522

ABSTRACT

To explore the potential regulatory effects of 1, 25-dihydroxyvitamin D, L1' 25 (0H),D,j on the expression of vitamln D, receptor (VDR ) mRNA in human megakaryoblastic leukemia(HIMeg) and human osteosarcoma (HOS-86O3) cell lines. Methods: VDR mRNA expression was deter-mined by the utilization of our recently established quantitative reverse-transcripti0n p0lymerase chain re-acti0n (RT~PCR) method. Results: lt was found that VDR mRNA expression in HOS-86O3 cells was notchanged significantly after treatment with 1, 25 (OH),D, for O~ 24 h- On the other hand, treatment 0fHIMeg ce1ls with 1, 25 (OH),D, cou1d significantly induce a d0wn-regulati0n of VDR mRNA expressi0n ina time-dependent manner, reaching a level of about only 15% of control after 24 h treatment. C0ncIusi0n:The regulation of VDR mRNA expressi0n by 1,25 (OH), D, is of tissue-specific fashi0n.

13.
Article in Chinese | WPRIM | ID: wpr-550864

ABSTRACT

Glucocorticoid could significantly inhibit the growth of a human acute leukimia cell line (CEM-C7) in dose- and time-dependent manners. Qucocorticoid had also cytotoxic effect on CEM-C7 cells which was reflected in inducing DNA fragmentation preceding cell death. Glucocorticoid -induced CEM-C7 cell killing belongs to programmed cell death, which could be reversed by RU486, a potent glucocorticoid receptor antagonist These results demonstrated that the effects of glucocorticoids on lymphocytosis were mediated by glucocortioid receptor, and that the programmed cell death induced by glucocorticoids might be the molecular basis of the therapeutic effects of glucocorticoids in the treatment of acute lymphoblastic leukemia

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