Regulation of the expression of alkaline phosphatase in a human breast-cancer cell line

Chang, Tsu‐Chung; Wang, Jehng-Kang; Hung, Mei-Whey; Chiao, Chih-Hung; Tsai, Li-Ping; Chang, Gu‐Gang

doi:10.1042/bj3030199

Cited by 27 publications

(32 citation statements)

References 31 publications

(35 reference statements)

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“…For experiments in which the effect of actinomycin D (Act D) or cycloheximide (CHX) on the induction of AP expression was determined, MCF‐7 cells were incubated with Act D (0.1 µg·mL −1 ) or CHX (2 µg·mL −1 ) simultaneously with Dex (10 −7 m ) or RA (10 −6 m ) for 48 h before being harvested to assay enzyme activities. The effects of Act D and CHX on transcription and translation were assayed as described previously [15]. The concentrations of Act D and CHX used in these experiments were chosen from preliminary results that indicated that higher concentrations of either agent showed toxic effects on the cells after 48 h incubation.…”

Section: Methodsmentioning

confidence: 99%

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Expression and regulation of alkaline phosphatases in human breast cancer MCF‐7 cells

Tsai¹,

Hung²,

Chen

et al. 2000

European Journal of Biochemistry

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The effect of retinoic acid and dexamethasone on alkaline phosphatase (AP) expression was investigated in human breast cancer MCF-7 cells. Cellular AP activity was induced significantly by retinoic acid or dexamethasone in a time-dependent and dose-dependent fashion. A marked synergistic induction of AP activity was observed when the cells were incubated with both agents simultaneously. Two AP isozymes, tissuenonspecific (TNAP) and intestinal (IAP), were shown to be expressed in MCF-7 cells as confirmed by the differential rate of thermal inactivation of these isozymes and RT-PCR. Based on the two-isozyme thermalinactivation model, the specific activities for TNAP and IAP in each sample were analyzed. TNAP activity was induced only by retinoic acid and IAP activity was induced only by dexamethasone. Whereas dexamethasone conferred no significant effect on TNAP activity, retinoic acid was shown to inhibit IAP activity by < 50%. Interestingly, TNAP was found to be the only isozyme activity superinduced when the cells were costimulated with retinoic acid and dexamethasone. Northern blot and RT-PCR analysis were then used to demonstrate that the steady-state TNAP mRNA level was also superinduced, which indicates that the superinduction is regulated at the transcriptional or post-transcriptional levels. In the presence of the glucocorticoid receptor antagonist RU486, the dexamethasone-mediated induction of IAP activity was blocked completely as expected. However, the ability of RU486 to antagonize the action of glucocorticoid was greatly compromised in dexamethasone-mediated superinduction of TNAP activity. Furthermore, in the presence of retinoic acid, RU486 behaved as an agonist, and conferred superinduction of TNAP gene expression in the same way as dexamethasone. Taken together, these observations suggest that the induction of IAP activity by dexamethasone and the superinduction of TNAP by dexamethasone were mediated through distinct regulatory pathways. In addition, retinoic acid plays an essential role in the superinduction of TNAP gene expression by enabling dexamethasone to exert its agonist activity, which otherwise has no effect.

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Section: Methodsmentioning

confidence: 99%

“…TNAP has been shown to be involved in bone mineralization and IAP is thought to play a role in the transport of inorganic phosphates [12,13]. To date, several studies have indicated the involvement of APs in cellular events such as the regulation of protein phosphorylation, cell growth, apoptosis and cellular migration during embryonic development [14–18].…”

mentioning

confidence: 99%

Expression and regulation of alkaline phosphatases in human breast cancer MCF‐7 cells

Tsai¹,

Hung²,

Chen

et al. 2000

European Journal of Biochemistry

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“…[4][5][6] In some cancer cells, the placental isoenzyme is inducible by compounds that arrest cell cycle at G1 phase, i.e., glucocorticoid or butyrate. 7,8 In the HeLaS3 cervical cancer cell, the placental alkaline phosphatase activity is inversely related to cell growth rate and the enzyme may play a crucial role in growth regulatory mechanism. 7 Among the mammalian alkaline phosphatase isoenzymes, the placental enzyme has characteristic thermal and chemical stabilities, which have been utilized as one of the clinical diagnostics in distinguishing between the various alkaline phosphatase isoenzymes in serum.…”

Section: Introductionmentioning

confidence: 99%

Biphasic denaturation of human placental alkaline phosphatase in guanidinium chloride

Hung

Chang

1998

Proteins

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Human placental alkaline phosphatase is a membrane-anchored dimeric protein. Unfolding of the enzyme by guanidinium chloride (GdmCl) caused a decrease of the fluorescence intensity and a large red-shifting of the protein fluorescence maximum wavelength from 332 to 346 nm. The fluorescence changes were completely reversible upon dilution. GdmCl induced a clear biphasic fluorescence spectrum change, suggesting that a three-state unfolding mechanism with an intermediate state was involved in the denaturation process. The half unfolding GdmCl concentrations, [GdmCl]0.5, corresponding to the two phases were 1.45 M and 2.50 M, respectively. NaCl did not cause the same effect as GdmCl, indicating that the GdmCl-induced biphasic denaturation is not a salt effect. The decrease in fluorescence intensity was monophasic, corresponding to the first phase of the denaturation process with [GdmCl]0.5 = 1.37 M and reached a minimum at 1.5 M GdmCl, where the enzyme remained completely active. The enzymatic activity lost started at 2.0 M GdmCl and was monophasic but coincided with the second-phase denaturation with [GdmCl]0.5 = 2.46 M. Inorganic phosphate provides substantial protection of the enzyme against GdmCl inactivation. Determining the molecular weight by sucrose-density gradient ultracentrifugation revealed that the enzyme gradually dissociates in both phases. Complete dissociation occurred at [GdmCl] > 3 M. The dissociated monomers reassociated to dimers after dilution of the GdmCl concentration. Refolding kinetics for the first-phase denaturation is first-order but not second-order. The biphasic phenomenon thereby was a mixed dissociation-denaturation process. A completely folded monomer never existed during the GdmCl denaturation. The biphasic denaturation curve thereby clearly demonstrates an enzymatically fully active intermediate state, which could represent an active-site structure intact and other structure domains partially melted intermediate state.

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“…PLAP, in contrast to tissue-nonspecific AP, is relatively heat-stable, resistant to l-homoarginine, sensitive to l-phenylalanine, and less sensitive to l-leucine (Chang et al 1994;Stigbrand 1984). The effects of heat and inhibitors were tested on pooled saliva samples obtained from rats injected with the viral vector 11 days earlier and from untreated rats.…”

Section: Ap Activity In Submandibular Saliva Of Untreated and Dap-injmentioning

confidence: 99%

Retrovirus-mediated Gene Transfer into Rat Salivary Gland Cells In Vitro and In Vivo

Barka

Noen

1997

J Histochem Cytochem.

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SUMMARY A retroviral vector DAP that encodes the human placental alkaline phosphatase (PLAP) and the neomycin-resistant gene was used to transduce the salivary glandderived cell line A5 in vitro and acinar cells in rat submandibular gland in vivo. Expression of the transduced PLAP gene was established by histochemical staining for heat-resistant AP and by determination of enzyme activity. From the in vitro experiments, we concluded that the salivary gland-derived cell line A5 can be infected by the retroviral vector DAP. In the transduced cells the viral long terminal repeat (LTR) promoter was effective, and the cells expressed heat-stable PLAP which was localized mostly in the plasma membrane and could be released by treatment with bromelain or phosphatidyinositol-specific phospholipase C. A5-DAP cells secreted PLAP into the medium. Clones of A5-DAP cells expressed various levels of the enzyme. The level of enzyme activity in different clones was unrelated to growth rate. Retrograde ductal injection of the viral vector into the duct of the submandibular gland of rats resulted in integration and long-term expression of PLAP gene in acinar cells. Expression of PLAP was seen up to 25 days, the limit of the observation period. To facilitate integration of the viral DNA, cell division of acinar cells was induced by administration of the ␤ -adrenergic agonist isoproterenol before administration of the virus. PLAP was secreted into submandibular saliva. The data support the notion that salivary glands are suitable targets for gene transfer in vivo by a retroviral vector.

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Regulation of the expression of alkaline phosphatase in a human breast-cancer cell line

Cited by 27 publications

References 31 publications

Expression and regulation of alkaline phosphatases in human breast cancer MCF‐7 cells

Expression and regulation of alkaline phosphatases in human breast cancer MCF‐7 cells

Biphasic denaturation of human placental alkaline phosphatase in guanidinium chloride

Retrovirus-mediated Gene Transfer into Rat Salivary Gland Cells In Vitro and In Vivo

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