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J T Hsieh

Publications and source records attributed to J T Hsieh.

At least 73 records · Page 4Linked to original sources
PubMed1994

Derivation of androgen-independent human LNCaP prostatic cancer cell sublines: role of bone stromal cells.

A model of human prostate cancer was established to study cellular interaction between prostate cancer and bone stroma in vivo. In this model, subcutaneous co-injection of 2 non-tumorigenic human cell lines--LNCaP, a prostate cancer cell line, and MS, a bone stromal cell-line--into intact adult male mice resulted in formation of carcinomas that secreted prostate-specific antigen (PSA), a clinically useful human serum prostate cancer marker. In castrated hosts, upon cellular interaction with bone fibroblasts, we observed the progression of these tumors from an androgen-dependent (AD) to an androgen-independent state (AI). We derived 4 LNCaP cell sublines from the chimeric LNCaP/MS tumors: the M subline from intact hosts and the C4, C4-2 and C5 sublines from castrated hosts. The LNCaP sublines had chromosomal markers similar to those of the parental LNCaP cells and distinctly different from those of the MS bone stromal cell line. Although the parental and derived cell lines expressed similar steady-state levels of ornithine decarboxylase transcript, the sublines expressed 5- to 10-fold higher basal steady-state levels of PSA transcript than did the parental LNCaP cell line. The LNCaP sublines formed 13- to 26-fold more soft-agar colonies than the parental LNCaP cell line. The sublines became tumorigenic, yielding an incidence of tumors in intact athymic mice of 7-75%. The LNCaP sublines C4 and C5 (but not the parental and M cell line) formed tumors in castrated hosts when co-injected with bone fibroblasts. A second-generation LNCaP subline, C4-2, was derived from a chimeric tumor induced by co-inoculating castrated mouse with C4 cells and MS cells. We found that C4-2 subline was tumorigenic when inoculated into castrated hosts in the absence of inductive fibroblasts. Moreover, C4-2 was the only subline capable of forming soft-agar colonies when cultured in serum-free medium. In comparison with the parental LNCaP cells, the C4-2 subline expressed lower steady-state levels of androgen receptor (AR) protein and mRNA transcript and lost its androgen responsiveness in vitro. Our results suggest that certain genetic traits of prostate cancer cells may be selected or altered through an "adaptive" mechanism that involves cellular interaction with the bone stromal cells.

Animals↗
PubMed1994

Androgen regulation of cell adhesion molecule gene expression in rat prostate during organ degeneration. C-CAM belongs to a class of androgen-repressed genes associated with enriched stem/amplifying cell population after prolonged castration.

Androgen is required for maintaining the differentiated state of prostatic epithelium, and that cell adhesion molecules play active roles in controlling the development of epithelial cells. However, little is known about the regulation of cell adhesion molecules during prostate development. In this study, we demonstrated that the expression of an epithelial cell adhesion molecule, C-CAM, in rat ventral prostatic epithelium are repressed by androgen. A similar regulatory pattern was also observed in the seminal vesicle but not in other androgen-dependent organs (the coagulating gland and the dorsolateral prostate), or other organs (the liver and kidney). These observations suggest that regulation of C-CAM expression by androgen is tissue-specific. Unlike the other androgen-repressed genes associated with apoptosis, which have transient expression patterns, the elevated level of C-CAM in the ventral prostate persisted for more than 30 days postcastration, suggesting that C-CAM belongs to a class of androgen-repressed genes which are not associated with apoptosis. Immunohistochemical study detected C-CAM on the apical surface of secretory epithelium in control rats; castration resulted in an altered localization of C-CAM expression from columnar epithelium to cuboidal basal epithelium. Taken together, the up-regulation and altered expression pattern of C-CAM in ventral prostatic epithelium by androgen deprivation may be associated with the enriched epithelial stem/amplifying cell population in degenerated ventral prostate. These observations suggest that C-CAM may play an active role in the process of prostatic epithelial differentiation.

Adenosine Triphosphatases↗
PubMed1994

Calcium mobilization induced by endothelins and sarafotoxin in cultured canine tracheal smooth muscle cells.

Endothelins (ETs)- and sarafotoxin (S6b)-induced rises in intracellular Ca2+ concentration ([Ca2+]i) were monitored in cultured canine tracheal smooth muscle cells by using a fluorescent Ca2+ indicator fura-2. ET-1, ET-2, ET-3 and S6b elicited an initial transient peak and followed by a sustained elevation of [Ca2+]i, with half-maximal effect (EC50) of 18, 20, 38 and 21 nM, respectively. BQ-123, an ETA receptor antagonist, had a high affinity to block the rise in [Ca2+]i response to ET-1, ET-2, and S6b, as well as a low affinity for ET-3. Removal of external Ca2+ by addition of EGTA during the sustained phase, caused a rapid decline in [Ca2+]i to the resting level. In the absence of external Ca2+, only an initial transient peak of [Ca2+]i was seen, the sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+. Ca2+ influx was required for the changes of [Ca2+]i, since the Ca(2+)-channel blockers, diltiazem, verapamil, and Ni2+, decreased both the initial and sustained elevation of [Ca2+]i response to these peptides. ETs exhibited homologous desensitization of the Ca2+ response, but partial heterologous desensitization of the Ca2+ response mediated by carbachol to different extents. In contrast, ETs did not desensitize the Ca2+ response induced by ATP or vice versa. These data demonstrate that the initial detectable increase in [Ca2+]i stimulated by these peptides is due to the activation of ETA receptors and subsequently the release of Ca2+ from internal stores, whereas the contribution of external Ca2+ follows and partially involves a diltiazem- and verapamil-sensitive process.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Effect of phorbol ester on phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells.

In cultured canine tracheal smooth muscle cells (TSMCs), muscarinic receptor stimulation led to phosphoinositide (PI) hydrolysis, formation of inositol phosphates (IPs), and mobilization of intracellular Ca2+. Desensitization of IPs accumulation and Ca2+ mobilization evoked by carbachol was investigated using [3H]inositol labelling and Ca(2+)-sensitive dye fura-2. Treatment of TSMCs with phorbol 12-myristate 13-acetate (PMA) for 30 min blocked the carbachol-stimulated formation of IPs and mobilization of Ca2+. The concentrations of PMA that gave half-maximal and maximal inhibition of carbachol-induced IPs accumulation were 70 nM and 1 microM, respectively. The inhibitory effect of PMA on carbachol-induced responses was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. Treatment of TSMCs with PMA for 24 h, the cells remained the ability to response to carbachol-induced IPs accumulation and Ca2+ mobilization with the same extent as that observed in the control group. Inactive phorbol ester, 4 alpha-phorbol 12, 13-didecanoate at 1 microM, did not inhibit the responses. The KD and Bmax of the muscarinic receptor for [3H]N-methyl scopolamine binding were not significantly changed by PMA treatment for either 30 min or 24 h. The locus of this inhibition was further investigated by examining the effect of PMA on AlF4(-)-stimulated IPs accumulation in canine TSMCs. AlF4(-)-induced response was inhibited by PMA treatment, supporting that G protein(s) can be directly activated by AlF4- which was uncoupled to phospholipase C (PLC) by PMA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Bradykinin-stimulated calcium mobilization in cultured canine tracheal smooth muscle cells.

Bradykinin (BDK)-induced increases in intracellular Ca2+ concentration ([Ca2+]i) were monitored in cultured canine tracheal smooth muscle cells (TSMCs) using a fluorescent Ca2+ indicator, Fura-2. BDK and kallidin caused an initial transient peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner, with half-maximal stimulation (log EC50) obtained at -8.10 M and -8.04 M, respectively. The BDK-induced rise in [Ca2+]i was not affected by the BDK B1 receptor antagonist, des-Arg9[Leu8]-BDK (10 microM). However, the BDK B2 receptor antagonists des-Arg[Hyp3, Thi5,8, D-Phe7]-BDK and Hoe 140 had high affinity in antagonizing BDK with pKB values of 7.5 +/- 0.3 and 8.7 +/- 0.3, respectively. The sustained phase of the rise in [Ca2+]i was dependent on the presence of external Ca2+, as evidenced by a decline to the resting level on addition of EGTA. In the absence of external Ca2+, only an initial transient peak was seen which then declined to the resting level; a sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+ in the continued presence of BDK. Ca2+ influx was required for the changes in [Ca2+]i, since Ca(2+)-channel blockers, diltiazem, verapamil, and Ni2+, decreased both the initial and sustained elevation of [Ca2+]i in response to BDK. In conclusion, these findings indicate that the initial increase in [Ca2+]i stimulated by BDK acting on BDK B2 receptors is due to the release of Ca2+ from internal stores, followed by the influx of external Ca2+ into the cells. The influx of extracellular Ca2+ partially involves a diltiazem- and verapamil-sensitive Ca2+ channel.

Animals↗
PubMed1994

5-Hydroxytryptamine-stimulated calcium mobilization in cultured canine tracheal smooth muscle cells.

5-Hydroxytryptamine (5-HT)-induced increase of intracellular Ca2+ concentration ([Ca2+]i) was monitored in cultured canine tracheal smooth muscle cells (TSMCs) using a fluorescent Ca2+ indicator Fura-2. Stimulation of TSMCs by 5-HT produced an initial transient peak followed by a sustained, concentration-dependent elevation of [Ca2+]i. The log (EC50) values of 5-HT for the peak and sustained plateau responses were -7.43 and -7.60 M, respectively. 5-HT1A and 5-HT3 receptor antagonists, NAN-190 and metoclopramide, inhibited the 5-HT-stimulated increase in [Ca2+]i with pKB values of 6.3 and 6.2, respectively, indicating that the 5-HT receptors mediating Ca2+ signal had low affinity for these receptor antagonists. In contrast, 5-HT2A receptor antagonists, ketanserin and mianserin, had high affinity in antagonizing the changes in [Ca2+]i response to 5-HT with pKB values of 8.3 and 8.3, respectively. The sustained elevation of [Ca2+]i was dependent on the presence of extracellular Ca2+. Removal of extracellular Ca2+ by addition of 2 mM EGTA during the sustained phase caused a rapid decline in [Ca2+]i to the resting level. In the absence of extracellular Ca2+, only an initial peak was observed which then declined to the resting level; the sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+ in the continued presence of 5-HT. Ca2+ influx was required for the changes of [Ca2+]i, since the Ca(2+)-channel blockers, diltiazem, verapamil, and Ni2+, decreased both the initial and sustained elevation of [Ca2+]i in response to 5-HT. These Ca(2+)-channel blockers also decreased the sustained elevation of [Ca2+]i when applied during the plateau phase. In conclusion, these findings indicate that the initial increase in [Ca2+]i stimulated by 5-HT acting on 5-HT2A receptors is due to the release of Ca2+ from internal stores, followed by the influx of external Ca2+ into the cells. The influx of extracellular Ca2+ partially involves a diltiazem and verapamil sensitive Ca2+ channel.

Animals↗
PubMed1994

Endothelin- and sarafotoxin-induced phosphoinositide hydrolysis in cultured canine tracheal smooth muscle cells.

The endothelins (ETs) and sarafotoxin are two structurally related classes of potently contractile peptides. To understand the mechanism of action of ETs, we have examined the effect of ETs and sarafotoxin on phosphoinositide (PI) hydrolysis in cultured canine tracheal smooth muscle cells (TSMCs). ET-1, ET-2, ET-3, and sarafotoxin caused dose-dependent accumulation of inositol phosphatase (IPs) and tracheal smooth muscle contraction. BQ-123, an ETA receptor antagonist, had a high affinity to block the ET-1-induced IP accumulation and tracheal smooth muscle contraction with pKB values of 7.3 and 7.4, respectively. Pretreatment of TSMCs with cholera toxin impaired the ability of ET-1 and ET-2 to stimulate IP formation, whereas there was no effect by treatment with pertussis toxin. Stimulation of PI turnover by these peptides required the presence of extracellular Ca2+ and was blocked by treatment with EGTA. The addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs directly stimulated IP accumulation. A further Ca(2+)-dependent increase in IP formation was obtained by inclusion of either GTPrS or ET-1. The combined presence of GTPrS and ET-1 elicited an additive effect on IP formation. Short-term exposure to phorbol 12-myristate 13-acetate (PMA, 1 microM) abolished the stimulation of PI hydrolysis induced by these peptides. The inhibitory effect of PMA on ET-induced response was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Prolonged incubation of TSMCs with PMA resulted in a recovery of receptor responsiveness that may be due to downregulation of PKC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

4-Aminopyridine-sensitive neurologic deficits in patients with spinal cord injury.

4-Aminopyridine (4-AP) is a potassium channel blocking agent with the ability to restore conduction in demyelinated internodes of axons of the spinal cord. The present investigation sought to obtain electrophysiologic evidence of the effect of 4-AP in ameliorating central conduction deficits in a group of patients (n = 6) with spinal cord injury (SCI). The group was selected on the basis of having temperature-dependent central conduction deficits. 4-AP (24-25 mg total dose) was delivered intravenously at 6 mgh-1 or 15 mgh-1 while somatosensory evoked potentials (SEPs) and motor evoked potentials (MEPs) were recorded as indices of central conduction. Two patients exhibited marked increases in the amplitude of cortical SEPs, and in one of these, 4-AP brought about a reduced central conduction time from L1 to cortex. Four patients revealed increased amplitude MEPs with concomitant reduction in latency indicative of enhanced conduction in corticospinal or corticobulbospinal pathways. Two of these patients demonstrated increased voluntary motor unit recruitment following 4-AP. Clinical examination revealed reduced spasticity (n = 2), reduced pain (n = 1), increased sensation (n = 1), improved leg movement (n = 3), and restored voluntary control of bowel (n = 1). These results support the hypothesis that 4-AP induces neurologic benefits in some patients with SCI. They are also consistent with the emerging concept that pharmaceutical amelioration of central conduction deficits caused by focal demyelination may contribute to the management of a select group of patients with compressive or contusive SCI.

4-Aminopyridine↗
PubMed1994

The effect of cyclic AMP elevating agents on bradykinin- and carbachol-induced signal transduction in canine cultured tracheal smooth muscle cells.

1. The effects of cholera toxin (CTX), forskolin and dibutyryl cyclic AMP on bradykinin (BK)- and carbachol-induced accumulation of inositol phosphates (IPs) and Ca2+ mobilization were investigated in canine cultured tracheal smooth muscle cells (TSMCs). The BK-induced responses were mediated via a G protein which was not inhibited by CTX or pertussis toxin treatment. 2. BK-stimulated IPs accumulation and Ca2+ mobilization were potentiated by CTX (10 micrograms ml-1) pretreatment which was time-dependent. Maximal increase of these responses occurred after 24 h treatment with CTX. The concentration-effect relationship of BK-induced responses were shifted to the left and BK was substantially more effective in CTX-treated cells than in the control cells. This enhancing effect of CTX did not occur with carbachol. 3. Short-term (< 4 h) treatment with forskolin (10 microM) or dibutyryl cyclic AMP (1 mM) failed to accentuate BK-induced responses, but long-term (> 4 h) treatment of TSMCs with these agents mimicked the enhancing effect of CTX, suggesting that CTX-induced enhancement of BK responsiveness might be due to a rise in cyclic AMP. 4. Prolonged treatment of TSMCs with these agents was accompanied by an increase in cell surface [3H]-BK binding sites, which was inhibited by concurrent incubation with cycloheximide, an inhibitor of protein biosynthesis. Cycloheximide also abolished the potentiating actions of CTX, forskolin, and dibutyryl cyclic AMP on BK-induced IPs formation and Ca2+ mobilization. 5. The locus of this enhancement was further investigated by examining the effects of CTX, forskolin and dibutyryl cyclic AMP on A1F4(-)-induced IPs accumulation in canine TSMCs. AIF4-induced IPs accumulation was not affected by CTX, forskolin, or dibutyryl cyclic AMP treatment, supporting the contention that this stimulatory effect is located at the BK receptor level.6. These results demonstrate that the augmentation of BK-induced IPs accumulation and Ca2+mobilization produced by CTX, forskolin and dibutyryl cyclic AMP involves a cyclic AMP-dependent mechanism which is induced by a sustained increase in the level of intracellular cyclic AMP. CTX and forskolin may promote an increase of the synthesis of BK receptors, and thereby enhance BK-induced responses.

Animals↗
PubMed1994

Bradykinin-stimulated phosphoinositide metabolism in cultured canine tracheal smooth muscle cells.

1. Stimulation of bradykinin (BK) receptors coupled to phosphoinositide (PI) hydrolysis was investigated in canine cultured tracheal smooth muscle cells (TSMCs). BK, kallidin, and des-Arg9-BK, stimulated [3H]-inositol phosphates (IPs) accumulation in a dose-dependent manner with half-maximal responses (EC50) at 20 +/- 5, 13 +/- 4, and 2.3 +/- 0.7 nM, (n = 5), respectively. 2. D-Arg[Hyp3, D-Phe7]-BK and D-Arg[Hyp3, Thi5,8, D-Phe7]-BK, B2 receptor antagonists, were equipotent in blocking the BK-induced IPs accumulation with pKB = 7.1 and 7.3, respectively. 3. Short-term exposure of TSMCs to phorbol 12-myristate 13-acetate (PMA, 1 microM) attenuated BK-stimulated IPs accumulation. The concentrations of PMA that gave half-maximal and maximal inhibition of BK-induced IPs accumulation were 15 +/- 4 nM and 1 microM, n = 3, respectively. The inhibitory effect of PMA on BK-induced response was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. 4. Prolonged incubation of TSMCs with PMA for 24 h, resulted in a recovery of receptor responsiveness which may be due to down-regulation of PKC. The inactive phorbol ester, 4 alpha-phorbol 12, 13-didecanoate at 1 microM, did not inhibit this response. 5. The site of this inhibition was further investigated by examining the effect of PMA on AlF(4-)-induced IPs accumulation in canine TSMCs. AlF(4-)-stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the G protein(s) can be directly activated by AlF4-, which is uncoupled from phospholipase C by PMA treatment. 6. Incubation of TSMCs in the absence of external Ca2+ or upon removal of Ca2+ by addition of EGTA, caused a decrease in IPs accumulation without changing the basal levels. Addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs stimulated IPs accumulation was obtained by inclusion of either guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or BK. The combination of GTP gamma S and BK caused an additive effect on IPs accumulation.7. Pretreatment of TSMCs with cholera toxin enhanced BK-stimulated IPs accumulation, whereas there was no effect with pertussis toxin.8. These data suggest that BK-stimulated PI metabolism is mediated by the activation of BK B2 receptors coupling to a G protein which is not blocked by cholera toxin or pertussis toxin treatment and dependent on external Ca2+. The transduction mechanism of BK coupled to PI hydrolysis is sensitive to feedback regulation by PKC.

Animals↗
PubMed1994

5-Hydroxytryptamine receptor-mediated phosphoinositide hydrolysis in canine cultured tracheal smooth muscle cells.

1. 5-Hydroxytryptamine (5-HT) has been shown to induce contraction of tracheal smooth muscle. However, the mechanisms of action of 5-HT are not known. We therefore investigated the effects of 5-HT on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and its regulation in canine cultured tracheal smooth muscle cells (TSMCs) labelled with [3H]-inositol. 5-HT-induced inositol phosphates (IPs) accumulation was time- and dose-dependent with a half-maximal response (EC50) and a maximal response at 0.38 +/- 0.05 and 10 microM, respectively. 2. Ketanserin and mianserin (10 and 100 nM), 5-HT2 receptor antagonists, were equipotent in blocking the 5-HT-induced IPs accumulation with pKB values of 8.46 and 8.21, respectively. In contrast, the dose-response curves of 5-HT-induced IPs accumulation were not shifted until the concentrations of NAN-190 and metoclopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased up to 10 microM. 3. Pretreatment of TSMCs with pertussis toxin or cholera toxin did not inhibit the 5-HT-induced IPs accumulation, but partially inhibited the AlF(4-)-induced IPs response. 4. Stimulation of IPs accumulation by 5-HT required the presence of external Ca2+ and was blocked by EGTA. The addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs directly stimulated IPs accumulation. A further Ca(2+)-dependent increase in IPs accumulation was obtained by inclusion of either guanosine 5'-O-(3-thiotriphoshate) (GTP gamma S) or 5-HT. The combination of GTP gamma S and 5-HT elicited an additive effect on IPs accumulation. 5. Treatment with phorbol 12-myristate 13-acetate (PMA, 1 microM, 30 min) abolished the 5-HT-induced IPs accumulation. The concentrations of PMA that gave a half-maximal and maximal inhibition of 5-HT-induced IPs accumulation were 2.2 +/- 0.4 nM and 1 microM, n = 3, respectively. The protein kinase C (PKC) activator, 4 alpha-phorbol 12,13-didecanoate, at 1 microM, did not influence this response. The inhibitory effect of PMA was reversed by staurosporine, a PKC inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 6. The site of this inhibition was further investigated by examining the effect of PMA on AlF(4-)-induced IPs accumulation in canine TSMCs. AlF(4-)-stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the effect of PMA is distal to the 5-HT receptor. 7. Acetylcholine-induced IPs accumulation was completely inhibited by atropine, but not affected by ketanserin or mianserin, suggesting that 5-HT-induced IPs accumulation is not due to release of acetylcholine.8. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis via a pertussis toxin- and cholera toxin-insensitive GTP binding protein in canine TSMCs and that this coupling process is negatively regulated by PKC. 5-HT2 receptors may be predominantly mediating IPs accumulation and presumably IP-induced Ca2+ release may function as the transducing mechanism for 5-HT stimulated contraction of tracheal smooth muscle.

Animals↗
PubMed1994

Sarafotoxin-induced calcium mobilization in cultured dog tracheal smooth muscle cells.

Sarafotoxin b (S6b)-induced changes in intracellular Ca2+ concentration ([Ca2+]i) were monitored in cultured canine tracheal smooth muscle cells (TSMCs) by a fluorescent Ca2+ indicator fura-2. S6b elicited an initial transient peak followed by a sustained elevation of [Ca2+]i. BQ-123, an endothelin-A (ETA) receptor antagonist, had a high affinity to block the rise in [Ca2+]i response to S6b. In the absence of external Ca2+, only an initial transient peak of [Ca2+]i was seen, the sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+. Ca2+ influx was required for the changes of [Ca2+]i, since the Ca(2+)-channel blockers, diltiazem, verapamil, and Ni2+, decreased both the initial and sustained elevation of [Ca2+]i in response to S6b. TSMCs pretreated with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min attenuated Ca2+ mobilization induced by S6b, which was reversed by staurosporine, a protein kinase C (PKC) inhibitor. The change of [Ca2+]i induced by S6b was attenuated by cholera toxin pretreatment, but not by pertussis toxin. These data demonstrate that the initial detectable increase in [Ca2+]i stimulated by S6b is due to the activation of ETA receptors and subsequent release of Ca2+ from internal stores, whereas the contribution of external Ca2+ follows and partially involves a diltiazem- and verapamil-sensitive process. The inhibition of PMA on S6b-induced Ca2+ mobilization was inversely correlated with membraneous PKC activity.

Animals↗
PubMed1994

[Cloning and characterization of a specific cytokeratin-8 cDNA from rat prostatic epithelium].

In this study, we report the cloning and sequencing of a full-length cytokeratin-8 (CK-8) cDNA from a rat prostatic epithelial cDNA library. The effects of androgen on CK-8 expression in rat prostate were also studied. The data indicated that the steady-state level of CK-8 mRNA was elevated by about 5- to 10-fold in both prostate and seminal vesicles in castrated rats, and the elevated levels persisted during a 23-day experimental period. Androgen but not estrogen administration repressed the expression of CK-8 mRNA. This effect could be antagonized by the simultaneous administration of an antiandrogen, flutamide, indicating that CK-8 is a new class of androgen-repressed genes whose regulation is presumably mediated by androgen receptor mechanisms.

Androgens↗
PubMed1994

[Localization and regulation of cytokeratin 8 mRNA expression in rat prostate epithelia].

The effects of androgen on cytokeratin (CK) 8 mRNA expression in rat ventral prostate (VP) were studied by in situ hybridization with an antisense RNA-probe. It was found that 1) the CK 8 antisense RNA-probe was accumulated only within prostatic epithelial cells, indicating that the CK 8 mRNA is a specific and sensitive marker for prostate epithelia. 2) After castration, the signals of CK 8 mRNA in VP sections were significantly increased. Administration of androgen to the castrated male host repressed the elevated expression of CK 8 mRNA in VP. This effect can be antagonized by the simultaneous administration of an antiandrogen. 3) Unlike other androgen-repressed gene, the elevated expression of CK 8 mRNA in VP was persisted even 2 months after the process of glandular involution was completed. 4) During prostate development, the strongest CK 8 mRNA staining was found in the early neonatal prostatic epithelia which were composed mainly of prostatic stem cells. Thereafter, when the levels of serum androgen were gradually increased, the shift of CK 8 mRNA staining to peripheral region and decreased level of overall CK 8 mRNA were noted. These data support the notion that the CK 8 gene of prostate epithelia is a new class of androgen-repressed one. The data also indicated that the expression of CK 8 gene is closely related with the proliferation and differentiation of prostate stem cells, and excessive expression of CK 8 mRNA is a characteristic for prostatic stem cells.

Animals↗
PubMed1994

Androgen repression of cytokeratin gene expression during rat prostate differentiation: evidence for an epithelial stem cell-associated marker.

Cytokeratin (CK) 8 mRNA expression in developing and degenerating rat prostate was studied using in situ hybridization with an antisense RNA-probe. It was found that: 1) the CK 8 antisense probe was accumulated only within prostatic epithelial cells; 2) after castration, CK 8 mRNA signals in ventral prostate (VP) sections were significantly increased, and elevated CK 8 mRNA expression persisted even long after prostate involution was complete; and 3) during prostate development, the strongest CK 8 mRNA staining was found in the early neonatal prostatic epithelia which were composed mainly of prostatic stem cells. Thereafter, a shift of CK 8 mRNA staining to peripheral regions and decreased overall CK 8 mRNA levels were noted. These data indicate that excessive expression of CK 8 mRNA is a characteristic of prostatic stem cells, and CK molecules are excellent markers for determining the hierarchical pathway of cell differentiation in prostate epithelium.

Animals↗
PubMed1993

Epidermal growth factor receptor-mediated autocrine and paracrine stimulation of human transitional cell carcinoma.

Stromal-epithelial interactions may play a key role in tumor growth and metastasis. We have established a model to study the cellular and molecular basis of this paracrine interaction both in vivo and in vitro using a human transitional cell carcinoma cell line (WH). s.c. coinoculation of 1 x 10(6) WH cells with 1 x 10(6) nontumorigenic fetal rat urogenital sinus mesenchymal (rUGM) cells in athymic mice accelerated carcinoma growth 20 times faster than isolated WH cell inoculations and 4 times faster than coinoculations of the same number of NIH-3T3 or human bladder fibroblasts. Characterization of these chimeric tumors with immunohistochemical and DNA dot-blot analyses documented their predominantly human component. To evaluate the underlying mechanisms involved in this paracrine-mediated in vivo tumor growth acceleration, Northern analyses for growth factors (GFs) and extracellular matrix (ECM) expression in the different cell lines, as well as in vitro mitogenic assays, were performed. Northern analysis revealed basic fibroblast growth factor, transforming growth factor alpha, and epidermal growth factor receptor expression by WH cells but not rUGM cells; ECM components (fibronectin and collagens I and IV) were expressed only in the fibroblast cell lines. Cell type-specific paracrine growth factors are produced by cultured stromal and epithelial cells with a 2-3-fold bidirectional increase in WH and rUGM cell growth when cultured with reciprocal cell-type conditioned medium. An autocrine growth loop was observed for WH but not rUGM cells. WH cell growth is stimulated in vitro by low concentrations of transforming growth factor alpha and epidermal growth factor, while rUGM cell growth is stimulated 3-fold by basic fibroblast growth factor. Antiepidermal growth factor receptor antibodies completely inhibited autocrine and paracrine pathways stimulating WH cell growth, while anti-basic fibroblast growth factor antibodies had no inhibitory effect. These observations suggest that autocrine and paracrine growth factor stimulation of WH bladder carcinoma cell growth is most likely mediated by an epidermal growth factor receptor-related pathway. The predominant expression of ECM by fibroblasts in this model suggests that stromal cell ECM components may modulate tumor cell growth and angiogenesis possibly through mechanisms involving cellular adhesion, chemotaxis, or growth factor action.

3T3 Cells↗
PubMed1993

Autocrine regulation of prostate-specific antigen gene expression in a human prostatic cancer (LNCaP) subline.

Prostate-specific antigen (PSA), a M(r) 34,000 serine protease, is recognized as a useful marker for the detection and prognosis of patients with prostate cancer. Although serum PSA is an excellent prognostic indicator, an increasing number of factors were found to regulate the PSA expression of prostatic cancer cells, which include androgenic steroids, the growth factors (GFs) and the extracellular matrix. The purpose of this study is to define a novel protein factor that may be responsible for regulating PSA expression by androgen-independent (AI) human prostate cancer cells. We have established a LNCaP subline (C4) from a parental LNCaP tumor grown in a castrated host. The C4 subline overexpressed PSA mRNA and protein. Serum-free conditioned medium (CM) isolated from the C4 subline is able to stimulate PSA gene expression in parental LNCaP cells in a concentration-dependent manner. This autocrine PSA-inducing activity was found to be organ specific because CMs from other fibroblast cell lines (such as bone, prostate, kidney, and lung fibroblasts) and the CMs from several prostatic carcinoma cell lines (such as parental LNCaP, PC-3, DU-145) and a bladder transitional carcinoma cell line (WH) fail to exhibit similar activity. The activity of the CM from the C4 subline cannot be substituted by GFs such as TGF-alpha, TGF-beta, bFGF, HGF, KGF, or NGF; neuropeptide (bombesin/GRP); secondary messenger analogue (dibutyryl cAMP); beta 2-adrenergic agonist (isoproterenol); or alpha 1-adrenergic agonist (phenylephrine), indicating that the factor(s) may be a novel prostate-specific autocrine factor (PSAF). Both androgen and PSAF exhibit an additive effect on up-regulating PSA gene expression, suggesting that the signal transduction pathway elicited by PSAF may differ from that mediated by the androgen receptor. Further characterization of PSAF by heat, acid, and trypsin digestion revealed that the PSAF may be a protein factor with a unique amino acid composition. These observations suggest that a novel autocrine pathway mediated by PSAF may be responsible for the overexpression of PSA mRNA and protein in a human prostatic cancer cell line. The potential clinical significance of this factor will be discussed.

Castration↗