Autocrine growth factor in defined serum-free medium of human salivary gland adenocarcinoma cell line HSG.

Human salivary gland adenocarcinoma cell line HSG secretes an epidermal growth factor (EGF)-like molecule and contains EGF receptors. Growth of HSG cells is inhibited by glucocorticoid. We have identified that the growth inhibition by glucocorticoid is induced by the reduced secretion of the EGF-like molecule and that addition of anti-human EGF antibody to the culture specifically inhibits the growth of HSG cells, suggesting that autocrine secretion is involved in the growth of HSG cells. To prove that autocrine secretion functions in glucocorticoid-regulated growth of the HSG cell line, we purified the EGF-like molecule from serum-free, defined medium conditioned by the HSG cells and examined the growth-stimulatory effect of the purified molecule. The cultivation of HSG cells in serum-free defined medium, which contains insulin (10 micrograms/ml) and transferrin (10 micrograms/ml) only as proteinaceous components, resulted in establishment of a new cell line (HSG-SF) which had different morphological features from the parental HSG cell line. HSG-SF cells were found to have basically the same responsiveness to glucocorticoid as parental HSG cells. Parental HSG cells secreted high molecular weight EGF-like molecules (Mr 46,000 and 57,000), which were recognized by specific antibody to low molecular weight human EGF (Mr 6,201). From conditioned, serum-free medium of HSG-SF cells, an EGF-like molecule (Mr 46,000) was purified by using an anti-human EGF antibody-coupled Sepharose CL-4B column. This EGF-like molecule induced a maximal increase (36%) in incorporation of [3H]thymidine into DNA of parental HSG cells as well as low molecular weight human EGF. These observations demonstrate that growth of the HSG cell line is regulated by autocrine secretion.

Retinoic acid receptor in subclone of human salivary gland adenocarcinoma cell line HSG and effect of retinoic acid on cellular growth.

Retinoic acid (RA) binding has been detected in the nuclei of a subclone (CL-1) of human submandibular adenocarcinoma cell line HSG conditioned to grow in a serum-free defined medium. Competition assay confirmed the specificity of the RA binding. Scatchard analysis showed the binding molecule to have a high affinity and low capacity. From the analyses by gel-filtration and glycerol density gradient centrifugation, the nuclear binding molecule appears to be distinct from cellular RA binding protein (CRABP) in terms of molecular weight. Furthermore, immunoblotting analysis revealed a band (Mr 47,000) reactive with specific antibody to RA receptor (RAR) alpha in the gel containing the nuclear fraction of CL-1 cells. Northern blotting analysis with specific cDNA probes revealed the expression of RAR alpha and RAR gamma in CL-1 cells. These results indicate that CL-1 cells express two types of RAR subtype, suggesting that these receptor molecules may mediate biological effects of RA. Treatment of CL-1 cells with RA resulted in an increase in the incorporation of [3H]thymidine into TCA-insoluble materials. The maximal increase was observed at 10(-6) M around 48 h. Previously, we demonstrated the autocrine growth of HSG cells mediated by epidermal growth factor (EGF) receptors and EGF-like molecules (Kurokawa et al. (1989) Cancer Res. 49, 5136-5142) and showed that RA had no significant effect on the secretion of the EGF-like molecule. RA induced an increase in [125I]EGF binding to CL-1 cells. The increase in the EGF binding was maximal at 24 h at 10(-6) M RA. RA also increased the amount of [3H]leucine-labeled EGF receptor dose-dependently. No significant change was observed in total protein synthesis of CL-1 cells by treatment with RA. These results suggest that RA stimulates the growth of CL-1 cells by increasing EGF receptor levels.

Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG.

Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor between treated and untreated HSG cells. These results demonstrate that the triamcinolone acetonide-induced increase in [125I]EGF binding capacity is due to the increased synthesis of EGF receptor protein in HSG cells.

Characterization of nontransformed and transformed androgen receptor from rat submandibular gland.

Rat submandibular gland cytosol contained androgen receptor which had a single class of specific binding and an apparent dissociation constant of (1.1-1.2) X 10(-9) M. The process of transformation was investigated by a slightly modified minicolumn method in which the transformed receptor complexes were separated from the nontransformed receptor and meroreceptor. 10 mM ATP or pyrophosphate at 0 degrees C induced transformation of androgen receptor as did heat or salt treatment. 20 mM of sodium molybdate completely inhibited transformation that resulted from ATP, heat or salt treatment. The nontransformed androgen receptor complexes sedimented at 8 S and eluted at 250-260 mM KCl from DEAE-Sephacel, and its molecular weight was found to be 220 000 on Sephacryl S300 gel chromatography. On the other hand, the transformed androgen receptor complexes sedimented at 4.1-4.3 S (ATP or KCl treatment) or 3.5-3.8 S (heat treatment) and eluted at 60-80 mM KCl from DEAE-Sephacel. The molecular weight of the transformed androgen receptor complexes was 80 000-85 000 (ATP or KCl treatment) or 70 000-80 000 (heat treatment). These results suggest that the transformation of androgen-receptor complexes from rat submandibular gland was induced by the subunit dissociation and that salt bridges may be involved in the subunit interaction.

Binding of [3H]methyltrienolone to androgen receptor in rat liver.

The synthetic androgen methyltrienolone is superior to testosterone and androstenedione for the measurement of androgen receptor in tissues where the native ligands are metabolized into inactive derivatives. [3H]Methyltrienolone binds with a high affinity to androgen receptor in cytosol prepared from male rat livers, as the Scatchard analysis revealed that the Kd value was 3.3 X 10(-8) M and the number of binding sites was 35.5 fmol/mg protein. Since methyltrienolone also binds glucocorticoid receptor which exists in rat liver, the apparent binding of androgen receptor is faulty when measured in the presence of glucocorticoid receptor. The binding of methyltrienolone to glucocorticoid receptor can be blocked by the presence of a 100-fold molar excess of unlabeled synthetic glucocorticoid, triamcinolone acetonide, without interfering in its binding to androgen receptor, because triamcinolone does not bind to androgen receptor. Triamcinolone-blocked cytosol exhibited that the Kd value was 2.5 X 10(-8) M and the number of binding sites was 26.3 fmol/mg protein, indicating a reduction to 3/4 of that in the untreated cytosol. The profile of glycerol gradient centrifugation indicated that [3H]methyltrienolone-bound receptor migrated in the 8-9 S region in both untreated and triamcinolone-blocked cytosols, but the 8-9 S peak in triamcinolone-blocked cytosol was reduced to about 3/4 of that of untreated cytosol.

Mechanism of replenishment of androgen receptors in cytosol of mouse submandibular gland.

Female mice were used to examine the process of depletion and replenishment of cytosolic androgen receptors in submandibular glands, and to investigate the effects of cycloheximide and actinomycin D on these processes. The dose-dependence of receptor depletion and replenishment in the cytosolic fraction, and that of receptor accumulation in the nuclear fraction were investigated. Almost 100% depletion was revealed 1 h after the injection of testosterone propionate at a dose of 500 or 50 micrograms testosterone/100 g body weight. With a 5 micrograms dose, depletion of cytosolic receptors was not complete and replenishment proceeded rapidly compared with that which occurred with the 50 or 500 micrograms dose. The nuclear receptor level increased 1 h after injection of testosterone, and the raised level was gradually reduced to the pretreatment level with all doses. However, the time required for this return to pretreatment level was dependent on the dose of testosterone. The change in the levels of cytosolic and nuclear androgen receptors following injection of testosterone was parallel to the level of circulating androgen. To determine the requirements for transcriptional and translational events in the replenishment process, cycloheximide and actinomycin D were given in vivo. The process of replenishment of cytosolic receptors was inhibited by the injection of cycloheximide. However, actinomycin D exerted no inhibitory effect on receptor replenishment. Neither cycloheximide nor actinomycin D had any effect on the nuclear receptor level until 6 h after the injection of testosterone. Cycloheximide or actinomycin D alone had no effect on the cytosolic or nuclear receptor level. These results suggest that receptor replenishment involves protein synthesis.

Glucocorticoid regulates secretion of epidermal growth factor in the human salivary gland adenocarcinoma cell line.

The treatment of a human submandibular gland adenocarcinoma cell line (HSG cell line) for 48 h with triamcinolone acetonide (TA; 1-100 nmol/l) reduced the secretion of epidermal growth factor (EGF) in a closely related manner to a maximum of 66%. The reduction in the level of EGF secreted resulted in the suppression of DNA synthesis in the HSG cells to a similar extent. When the cells were incubated with TA and exogenous human EGF (hEGF), DNA synthesis was 1.7-fold higher than that without added hEGF. The removal of EGF by the addition of hEGF antibody reduced DNA synthesis in HSG cell cultures to the same extent as did TA. These results suggest that the growth inhibition of HSG cells by TA is due to the reduction in the amount of EGF secreted.

Sex difference in the cytosolic and nuclear distribution of androgen receptor in mouse submandibular gland.

Cytosol and nuclear androgen receptors in submandibular glands of male and female mice were measured by an exchange assay at 0 degree C. The binding of [3H]methyltrienolone to cytosol receptors in females was mostly saturated within a short period of incubation (3 h), whereas the saturation was much slower in males; suggesting that almost all of the cytosol receptors were unoccupied in females and the receptors were partially occupied in males. Nuclear receptors were extracted with pyridoxal 5'-phosphate (5 mmol/l) from nuclear fractions with 93-95% efficiency. The exchange of the bound steroids occurred by 24-48 h at 0 degree C, suggesting that most of the nuclear androgen receptor was occupied. The binding was low at higher temperatures, probably due to inactivation of the receptor. Scatchard analysis showed that the apparent dissociation constants of cytosol and nuclear receptors were similar (0.8 and 0.9 nmol/l respectively) in both sexes. On the other hand, the number of androgen-binding sites in the nucleus was much higher in males than in females (1052 fmol/mg DNA and 32 fmol/mg DNA respectively), while the number in the cytosol was higher in females than in males (512 fmol/mg DNA and 368 fmol/mg DNA respectively). These observations show that androgen receptors exist mainly (74%) in the nuclei of males, while they exist mostly (94%) in the cytosol of females.

[Proliferation and differentiation of human salivary gland adenocarcinoma cell line HSG].

The adenocarcinoma cell line derived from an intercalated ductal epithelium of a human salivary gland (HSG) proliferates autonomously mediated by an epidermal growth factor-(EGF)-like molecule with a molecular weight of 46 kDa and an EGF receptor (EGFR). The c-erbB2 protein, a member of EGFR family was also expressed in HSG cells and was involved in the growth signal pathway of HSG cells as well as EGFR. The autocrine growth is regulated by glucocorticoid and retinoic acid (RA) via their receptors. Retinoic acid receptor (RAR) of HSG cells revealed a transcriptional activity in vivo, and the heterodimerization between RAR and 9-cis retinoic acid receptor (RXR) is requisite for the binding with a specific DNA element termed RA response element in vitro. RXR alpha and RXR beta were cloned from HSG cells, and these RXRs, together with RAR, seemed to play a physiological role in RA signaling in vivo.

Co-expression of epidermal growth factor-receptor and c-erb B-2 proto-oncogene product in human salivary-gland adenocarcinoma cell line HSG and the implications for HSG cell autocrine growth.

The autonomous proliferation of HSG cells is mediated by an autocrine growth factor, a 46K epidermal growth factor (EGF)-like molecule. The receptor for this molecule was investigated. Immunoprecipitation and immunoblotting revealed the expression of two possible receptor molecules, EGF-R and p185erbB-2, in HSG cells. Northern blotting also revealed the co-expression of 5.6-kb EGF-R mRNA and 4.6-kb c-erb B-2 mRNA. When the purified EGF-like molecule was added to the cultures, EGF-R but not p185erbB-2 was autophosphorylated. These results suggest that, although both EGF-R and p185erbB-2 are co-expressed in HSG cells, the EGF-R is the genuine receptor for the EGF-like molecule. However, there is a possibility that p185erB-2 is involved in the signal transduction system. This possibility was examined by using specific antibodies to human EGF-R (hEGF-R), p185erbB-2, and EGF to inhibit the functions of these molecules. Addition of these three antibodies to the cultures inhibited the growth of HSG cells. The antibodies to EGF-R and p185erbB-2 also caused morphological changes such as disturbances of the plasma membrane, and some cell death. Surprisingly, the effect of the anti-p185erbB-2 antibody on growth inhibition and morphology was stronger than that of the anti-hEGF-R antibody. Thus, p185erB-2 expressed in HSG cells has an important function in the signal transduction of HSG cell growth.

Expression of cAMP response element binding protein (CREB)-binding protein (CBP) and the implication in retinoic acid-inducible transcription activation in human salivary gland adenocarcinoma cell line HSG.

In the process of retinoic acid (RA) signaling, retinoic acid receptor interacts with a coactivator complex composed of various transcription cofactors such as CREB-binding protein (CBP)/p300 and p160 family member proteins represented by steroid receptor coactivator-1 (SRC-1)/NCoA1 and p300/CBP cointegrator protein (p/CIP)/ACTR. In order to investigate the relationship of CBP to the RA signaling in a human salivary gland (HSG) adenocarcinoma cell line, we examined the expression of CBP in the cells. Immunoprecipitation and immunoblotting of the nuclear extract of HSG cells with anti-human CBP antibody showed a specific 270-kDa band, indicating the expression of CBP in HSG cells. The immunocytochemical analysis confirmed the nuclear localization of CBP. The transfection of HSG cells with a luciferase reporter plasmid harboring an RA-response element at the 5'-upstream region of the reporter gene increased RA-dependent luciferase activity approximately 3-fold. Co-transfection with a CBP-expression plasmid and the luciferase reporter gene enhanced the RA-dependent transcription activation approximately 10-fold. The immunoprecipitates obtained with anti-CBP antibody exhibited a histone acetyl-transferase (HAT) activity 2-fold higher than that obtained with the control antibody, whereas the HAT activity of the immunoprecipitates with anti-SRC-1 and anti-p/CIP, which were used as comparisons, were only a little increased. The RA treatment had no effect on the level of HAT activity except in the case of using the immunoprecipitate obtained with anti-RARalpha, in which case it increased the activity. These findings indicate that CBP expressed in HSG cells mediates the RA-inducible growth and differentiation-regulating transcription activation in concert with the retinoic acid receptors.

Demonstration and characterization of cytosol androgen receptor in rat exorbital lacrimal gland.

The presence of a macromolecule which binds androgen with a high affinity and a low capacity was demonstrated in the cytosol of the lacrimal glands of male and female rats. Evidence was found that this macromolecule was a protein by treatment with protease, trypsin or heat. A specific 8-8.5 S peak was obtained in both sexes by glycerol gradient centrifugation in low salt condition, whereas a specific 5.2 S peak was found in high salt condition. This protein could bind to DNA-cellulose after treatment of androgen-cytosol complexes by warming (25 degrees C 15 min) or exposure under high salt (0.4 M KCl). These results suggested that this protein was an androgen receptor.

Effect of castration and administration of testosterone on cytosol and nuclear androgen receptor in mouse submandibular gland.

The effect of castration or administration of testosterone propionate on the subcellular distribution of androgen receptor in mouse submandibular gland was investigated. Within 10 h after castration of male mice, most of the androgen receptor in nuclei was significantly reduced, the androgen receptor in cytosol increased and the increased cytosol receptor retained for at least 40 h. A single injection of testosterone propionate to female mice resulted in the translocation of cytosol androgen receptor to the nuclei by 30 min. The nuclear receptor level remained for at least 24 h and the cytosol receptor was replenished by 24-72 h. These results reveal that the endocrine manipulations such as castration and testosterone injection cause the change in the subcellular distribution of androgen receptor from mouse submandibular gland in both sexes.

Cancer cells responsible for humoral hypercalcemia express mRNA encoding a secreted form of ODF/TRANCE that induces osteoclast formation.

A novel cDNA encoding a secreted form of osteoclast differentiation factor/tumor necrosis factor-related activation-induced cytokine (sODF/TRANCE, GenBank Accession No. AB037599) was sequenced from 5' RACE cDNA clones of squamous cell carcinoma cell lines, SCC-4 and T3M-1 Cl.2, of which parental malignant tissues had caused severe humoral hypercalcemia. The sODF/TRANCE cDNA was composed of unknown 5' end sequence followed by the 100% identical sequence of the ODF/TRANCE extracellular domain-coding region. The longest open reading frame (ORF) of the novel cDNA completely matched the 3' end of the ORF of the ODF/TRANCE cDNA encoding C-terminal amino acid residues (74-318) in the extracellular region. The corresponding protein that reacted with the antibody specific for the extracellular domain of ODF/TRANCE was detected in the culture media conditioned by the cancer cells. Furthermore, human promyeloblastic leukemia cells, HL60, differentiated into osteoclast-like cells (OCLs) when cultured in the media conditioned by SCC-4 and T3M-1 Cl. 2 cells. The differentiation of HL60 cells into OCLs was inhibited by the anti-ODF/TRANCE antibody. These results strongly suggest that sODF/TRANCE plays an important role in enhanced bone-resorption in humoral hypercalcemia of malignancy.

Inhibition of retinoic acid-inducible transcription by COUP-TFI in human salivary gland adenocarcinoma cell line HSG.

Human salivary gland adenocarcinoma cells (HSG) express nuclear receptors, all-trans-retinoic acid (at-RA) receptors (RARs), and retinoid X/9-cis-retinoic acid (9-c-RA) receptors (RXRs). In order to investigate whether the endogenous RARs or RXRs of HSG cells can induce transcription activation, the thymidine kinase promoter (TK)-driven luciferase reporter gene containing the retinoic acid response element (RARE), of RARbeta, betaRARE2-TK-Luc, was transfected into HSG cells and ligand-dependent transcription activation was examined. Luciferase activity of cell lysate increased by the treatment with either at-RA or 9-c-RA. Co-transfection of RARalpha and (or) RXRalpha-expression plasmids with the reporter gene enhanced the luciferase activity, suggesting that endogenous RARs and RXRs work as ligand-dependent transfactors in HSG cells. Reverse transcriptase - polymerase chain reaction analysis revealed that HSG cells express chicken ovalbumin upstream promoter - transcription factor I (COUP-TFI). Co-transfection of COUP-TFI-expression plasmid suppressed the at-RA-induced transcription activation of the reporter gene. Similar results were shown using a chromatin-integrated reporter gene system, using a stably transfected beta-RARE2-TK-beta-galactosidase (beta-Gal) reporter gene. The at-RA-dependent increase in the beta-Gal expression was completely inhibited by COUP-TFI. The transfection of antisense oligonucleotide of COUP-TFI squelched the RA-dependent growth inhibition induced by RAR-RXR heterodimers. Conclusively, RARs and RXRs of HSG cells are functional and play roles as transactivators in at-RA-sensitive processes such as the proliferation or differentiation of cells. COUP-TFI very likely regulates these processes by repressing the functions of these transactivators.

Interaction of hepatic chromatin with androgen-receptor complex.

When the chromatin prepared from male rat livers interacted with [3H] testosterone-receptor complex prepared from male rat livers, the binding was much higher than when interacted with free [3H] testosterone, indicating that the formation of testosterone-receptor complex is prerequisite for interaction with chromatin. When the [3H] testosterone-receptor complex was interacted with the chromatin and the bound chromatin proteins were extracted into three fractions based on their extractability or solubility in salt solutions, higher radioactivities were found in the 0.35 M NaCl-extract and 2.0 M NaCl residual fraction. Chromatin was fractionated into 0.35 M NaCl-soluble, 2.0 M NaCl-soluble and insoluble fractions, and interaction of free [3H] testosterone or [3H] testosterone-receptor complex with chromosomal fractions was studied. The binding of the chromosomal fractions with free [3H] testosterone was negligible compared with that of [3H] testosterone-receptor complex. The binding of 0.35 M NaCl-soluble fraction with [3H] testosterone-receptor complex was higher than that of 2.0 M NaCl-soluble fraction. The binding ability of 2.0 M NaCl-insoluble fraction with [3H] testosterone-receptor complex was twice as much as that of the intact chromatin. This observation indicated that salt-soluble fractions of chromatin contributes only partly to the binding of chromatin with [3H] testosterone-receptor complex and that other components in 2.0 M NaCl-insoluble fraction may participate in the binding.

Expression of bone morphogenetic protein in human adenocarcinoma cell line.

A subclone termed as HSG-S8 was previously isolated from human salivary adenocarcinoma cell line, HSG, and proliferated in a serum-free culture. When HSG-S8 and parental HSG cells were transplanted into nude mice i.m. or s.c., both cells reproducibly induced adenocarcinoma. Furthermore, injection of HSG-S8 consistently induced formation of cartilage i.m. or bone s.c. in tumors, but did not parental HSG cells. The cytoplasm of HSG-S8 cells was specifically immunostained by anti-bone morphogenetic protein (BMP)-2 antibody. The conditioned medium of HSG-S8 cells contained a protein of M(r) 18,000 which specifically reacted with anti-BMP-2 protein antibody. Northern blot analysis also revealed that HSG-S8 cells expressed transcripts for BMP-2. On the other hand, parental HSG cells gave a very slightly positive signal only in Northern blot analysis. These results indicate that HSG-S8 cells synthesize and secrete BMP-2 protein, which is probably involved in the formation of cartilage and bone in the tumor tissues in nude mice.

Characteristics of cytosol androgen receptor in rat thymus.

Male and female rat thymic cytosol contained specific androgen receptor. The apparent dissociation constants (Kd) were 2.4 nM in males and 2.5 nM in females, and the number of binding sites (NBS) were 23.7 fmol/mg protein in males and 34.2 fmol/mg protein in females. Transformation of receptor to the DNA binding state was achieved by heat or KCl treatment of [3H]R1881-receptor complex, and the characteristics of transformed and nontransformed receptors were investigated. The nontransformed androgen-receptor complex eluted at 0.20-0.25 M KCl from DEAE-Sephacel and sedimented at 9.1 S and its molecular weight was 255,000 on agarose gel chromatography, while the transformed receptor complex eluted at 0.03-0.15 M KCl with a broad peak and sedimented at 4.5 S and its molecular weight was 80,000-85,000. The minicolumn binding assay revealed that approximately 57% of the total receptor complexes bound to DNA-cellulose following heat treatment (20 degrees C, 1 h). Castration exerted no effect on the physicochemical properties of cytosol androgen receptor, but it increased the number of binding site to the female level.

Proliferative signal transduction by epidermal growth factor (EGF) in the human salivary gland adenocarcinoma (HSG) cell line.

We examined the proliferative signal transduction by EGF in HSG-AZA 3, a subclone of HSG cell line. The treatment of cells with EGF resulted in an increase in [3H]thymidine incorporation into DNA depending upon EGF concentrations. In addition, the nuclear proto-oncogene c-fos was rapidly induced by EGF. Moreover, EGF induced transient expression of EGF receptor mRNA followed by the de novo synthesis of EGF receptor protein. On the other hand, treatment of the cells with EGF occurred phosphorylation by tyrosine kinase comprised in the EGF receptor, autophosphorylation, followed by activation of MAP kinase. These results indicate that the proliferative response to EGF is modulated by the phosphorylation cascade mediating EGF receptor-associated tyrosine kinase and MAP kinase, and transient activation of c-fos protein is implicated in the cell proliferation.

Expression of retinoid X receptors and COUP-TFI in a human salivary gland adenocarcinoma cell line.

The growth of the adenocarcinoma cell line derived from human salivary gland (HSG) is regulated by all-trans-retinoic acid (t-RA), which binds to its specific receptor, retinoic acid receptors (RARs), located in the nucleus, and thereby transactivates target genes. In this study, we examined the binding characteristics of the nuclear extract of HSG cells to the retinoic acid response element (RARE) compared with those of in vitro translated RAR alpha and retinoid X receptor alpha (RXR alpha), a heterodimeric partner of RAR alpha. Gel shift analysis using anti-RAR alpha and anti-RXR alpha antibodies revealed that the translated RAR alpha bound to RARE as a heterodimer with RXR alpha. In contrast, the binding of the nuclear extract of HSG cells to RARE showed a heterogeneous pattern, suggesting the existence of several species of RXRs as well as RARs in the nuclei of HSG cells. We therefore tried to clone these putative RXRs by the polymerase chain reaction using degenerated oligonucleotide primers conserved across the RXR family. The DNA sequencing of the recombinant clones revealed the expression of RXR alpha and RXR beta. In addition, chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI), which is also an RXR family member, was cloned. To evaluate the transcriptional activity of RARs and RXRs endogenously expressed in HSG cells, we performed a transient transfection analysis. When HSG cells were transfected with a luciferase reporter plasmid containing two repeats of either the RARE of the RAR beta gene or that of cellular retinol-binding protein II gene, positioned upstream of a thymidine kinase promoter fused to the luciferase sequence, a 2-3-fold induction of luciferase activity was observed in both cases. These results suggest that RARs and RXRs endogenously expressed in HSG cells were transcriptionally active in vivo. Thus, our findings showed that RXR alpha, RXR beta, and COUP-TFI are expressed in HSG cells and suggest that these molecules function as heterodimeric partners of RARs and (or) competitive repressors for RAREs and are involved in cellular responses mediated by retinoids.