Transduction of TAT-HA-beta-galactosidase fusion protein into salivary gland-derived cells and organ cultures of the developing gland, and into rat submandibular gland in vivo.

We have studied the transduction of TAT-HA-beta-galactosidase fusion protein into two cell lines of rat salivary gland origin, A5 and C6-21, into cells of fetal mouse submandibular glands in organ culture, and into rat submandibular gland after retrograde duct injection, using a histochemical method to demonstrate beta-galactosidase activity. Transduction of the fusion protein into A5 and C6-21 cells was concentration- and time-dependent. Therefore, the intensity of the beta-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr. However, the fusion protein was transduced into 100% of both types of cultured cells. When explants of mouse fetuses at 13 days of gestation were exposed to the fusion proteins, both epithelial and mesenchymal cells were stained for the enzyme, with a conspicuous accumulation of the reaction product at perinuclear cytoplasmic regions. The histochemical staining of the mesenchymal cells was more intense compared to that seen in epithelial cells. TAT-HA-beta-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for beta-galactosidase activity of cryostat sections prepared from the injected glands revealed that the transduction of the fusion protein was also time- and dose-dependent. In the glands of rats sacrificed from 10 min to 1 hr after the retrograde injection, essentially all acinar and duct cells showed cytoplasmic staining. The intensity of the staining then declined, and was not seen in the glands of rats killed 24 hr after the injection of the fusion proteins. These results indicate that a full-length, active TAT fusion protein can be targeted to salivary gland cells both in vitro and in vivo to analyze physiological, developmental, and pathophysiological processes.

Passive immunotherapy of mice bearing Ehrlich ascites tumor expressing human, membrane-bound placental alkaline phosphatase.

The objective of our study was to test if a tumor expressing a transgene coding for a membrane-bound protein is amenable to immunotherapy by antibodies to the same protein. To this end, we have established an Ehrlich ascites tumor (EAT) cell line, EAT-DAP, stably expressing human, membrane-bound placental alkaline phosphatase (PLAP) by infecting EAT cells (EATC) with the retroviral vector DAP and selecting neomycin-resistant cells. EATC and EAT-DAP cells grew at similar rates in vitro, and produced ascites tumor in Swiss-Webster mice with similar efficiency. We have treated mice bearing EAT-DAP ascites tumor with a mouse monoclonal antibody to human PLAP or with a monoclonal antibody to human C proteins of the heterogenous ribonucleoprotein complex (hnRNP). The average survival of mice treated with anti-hnRNP was 16.4 +/- 1.1 days (n = 8). Treatment with anti-PLAP prolonged the survival of mice; in 4 mice average survival was 23.3 +/- 5.7 days. Four animals, however, survived for 60 days when they were killed and had no visible signs of tumor. These data support the notion that passive immunotherapy using antibodies against a membrane protein, expressed in tumor cells transduced by a viral vector coding for that protein, may be effective in controlling tumor growth.

Retrovirus-mediated gene transfer into rat salivary gland cells in vitro and in vivo.

A retroviral vector DAP that encodes the human placental alkaline phosphatase (PLAP) and the neomycin-resistant gene was used to transduce the salivary gland-derived 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 beta-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.

Retrovirus-mediated gene transfer into salivary glands in vivo.

In the present report, we show prolonged expression of beta-galactosidase (beta-Gal) in the acinar cells of the submandibular and sublingual glands of rats following retrograde ductal injection of the retroviral vector BAG. To facilitate integration of viral DNA, cell division in the gland was induced by the administration of the beta-adrenergic agonist isoproterenol prior to the delivery of the vector. The frequency of cells stained for beta-Gal was higher if the virus was injected 4-20 hr after the two injections of isoproterenol given 24 hr apart than after the injection of only one dose of the drug. Without stimulation of cell division, no integration of the viral DNA was observed. Expression of the marker enzyme was observed up to 43 days, the limit of the observation period. The data indicate that salivary glands are potential targets of retrovirus-mediated gene transfer for somatic gene therapy.

Expression of the cysteine proteinase inhibitor cystatin C mRNA in rat eye.

BACKGROUND: Cystatin C, a naturally occurring inhibitor of cysteine proteinases, belongs to family 2 of the cystatin superfamily. While cystatins in general, and cystatin C specifically, are expressed in various cell types and found in biological fluids, cystatins in ocular structures have not been investigated. In the present study, the expression of cystatin C mRNA in the eye of the rat was studied. METHODS: Total RNA was extracted from eyes as well as from pooled corneae, retinas, lenses, sclerae, and corneae of adult rats. Cystatin C mRNA was detected in the RNA samples by reverse transcriptase--polymerase chain reaction and Northern blot hybridization. In addition, in situ hybridizations of formalin-fixed cryostat sections were carried out using a digoxigenin-labeled cystatin C probe. RESULTS: Cystatin C mRNA was demonstrated in total RNAs extracted from the eye, sclera, and retina, but not in RNAs isolated from the cornea and lens. In situ hybridizations revealed cystatin C mRNA in most of the stromal cells of the sclera. In the retina, a strong signal was localized in the outer nuclear layer. The distribution of the reaction product suggested that in the retina Müller cells and rod cells are the primary sites of expression of cystatin C. In addition, some glial cells in the inner nuclear and ganglion cell layers were stained. No specific signal for cystatin C mRNA was detected in the cornea, lens, iris, ciliary body, and choroid. CONCLUSIONS: In the eye of the rat, significant levels of cystatin C mRNA are detected in the sclera and retina. In the sclera cystatin C may play a role in modulating the activities of cysteine proteinases, mostly cathepsins, involved in the turnover and remodeling of the stroma. In the retina, cystatins synthesized and presumably released by Müller cells and rod cells may have a protective function against the harmful effects of cysteine proteinases released under physiologic and pathologic conditions.

Expressions of the genes for cysteine proteinase inhibitors cystatin C and cystatin S in rat submandibular salivary gland.

Rat cystatin S and rat cystatin C are members of family 2 (cystatin) of the cystatin superfamily. All members of the cystatin family inhibit cysteine proteinases to varying degree. The expression of these two inhibitors, which have a 48% similarity at the nucleotide level, was studied in the submandibular gland using reverse transcriptase-polymerase chain reaction (RT-PCR). Northern blot hybridization and in situ hybridization with digoxigenin-labelled DNA probes. Both inhibitors were expressed in the serous acinar cells of the submandibular gland. In accord with previous findings, cystatin S mRNA was induced by the beta-adrenergic agonist isoproterenol. The level of cystatin S mRNA, which was very low in the glands of untreated rats and was demonstrable by RT-PCR but not by Northern blot hybridization, was not altered by acute inflammation produced by turpentine. Neither the administration of isoproterenol nor acute inflammation had any effect on the level of cystatin C mRNA, indicating beta-adrenoreceptors are not involved in the regulation of the cystatin C gene(s) in the submandibular gland. The data indicate that these two closely related genes, expressed in the same cells, are differently regulated. The consequence of this difference in gene regulation on the physiological and pathological roles of these inhibitors remains to be established.

Lack of expression of T-kininogen gene in the hearts of untreated and turpentine-injected rats.

Cystatins, inhibitors of cysteine proteinases, are present in rat heart. However, the controls of genes coding for various cystatins in the heart, and the cellular sites of expression of these genes are not known. With a sensitive reverse transcriptase--polymerase chain reaction T-kininogen mRNA was readily detected in the submandibular glands and livers, but not in the hearts, of control or turpentine-injected rats. Immunocytochemical observations employing a monoclonal antibody to bradykinin, which reacts with kininogens in general, revealed no specific staining in cardiac structures, but a weak staining was apparent in blood vessels and on the surface of endothelial cells of both control and turpentine-injected rats. The monoclonal antibody revealed the presence of kininogens in the acinar cells of the submandibular gland, and, in acute inflammation, in the hepatocytes. These findings suggest that the T-kininogen gene is not expressed in the heart, and the T-kininogen demonstrable in heart extracts derives from the blood. Circulating kininogens are likely bound to endothelial cells, and may be a local source of kinins. In addition, kininogens, as potent inhibitors of cysteine proteinases, may play a role in pathologic conditions of the heart by controlling the deleterious effects of cathepsins released from lysosomes or secreted by macrophages.

Expression of the cysteine proteinase inhibitor cystatin C gene in rat heart: use of digoxigenin-labeled probes generated by polymerase chain reaction directly for in situ and northern blot hybridizations.

Cystatins represent a widely distributed superfamily of cysteine proteinase inhibitory proteins. We investigated the expression of the cystatin C gene, belonging to the family 2 of cystatins, in the hearts of female rats. Using a highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) we have detected cystatin C mRNA in the ventricule and atrium, as well as in liver and submandibular gland. A digoxigenin-labeled cystatin C probe, generated by PCR, hybridized to a single mRNA species of about 700 nucleotides on Northern blots. Northern blot hybridizations established that neither an acute inflammation produced by injection of turpentine nor administration of the beta-adrenergic agonist isoproterenol had an effect on the level of cystatin C mRNA in the heart. In situ hybridizations with digoxigenin-labeled probe localized the expression of the cystatin C gene to cardiac muscle fibers but not to other cardiac cellular elements. Cystatin C may be released by cardiac muscle fibers under physiological and pathological conditions and may modify inflammatory and necrobiotic processes.

Cysteine proteinase inhibitor in cultured human medullary thyroid carcinoma cells.

The TT cell line of human medullary thyroid carcinoma, that retains some of the differentiated functions of thyroid C cells including the synthesis and secretion of calcitonin, was found to contain and release into the culture medium cysteine proteinase inhibitor(s), cystatin(s). The major inhibitor, which is similar to, if not identical with, cystatin C, is constitutively released, or secreted, by TT cells. The rate of secretion of cystatin, quantified by titration of inhibition of papain, was stimulated by dibutyryladenosine 3':5'-cyclic monophosphate, forskolin, the calcium ionophore A 23187, and by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Neither forskolin nor TPA had, however, an effect on the level of the inhibitor in TT cells. Treatment with n-butyrate strongly inhibited the proliferation of TT cells, and led, in 4 to 7 days, to a doubling of the intracellular concentration of cystatins. Northern blot hybridizations to a 32P-labeled riboprobe complementary to human cystatin C cDNA indicated that cAMP, forskolin, and TPA had no effect on the steady-state levels of cystatin C mRNA. These data indicate that release of cystatin(s) from TT cells is regulated by cAMP-calcium-protein kinase C mechanisms that appear to be similar to those that regulate the secretion of calcitonin from these cells. However, in contrast to the calcitonin gene, the expression of the cystatin C gene in these cells is not regulated by cAMP or TPA. By a combination of acetone fractionation, affinity chromatography on Cm-papain-Sepharose, and gel exclusion chromatography a protein of approximately 14 kilodaltons was isolated from TT cells that reacted with antibodies against human cystatin C, and strongly inhibited papain. Cystain secreted by TT cells also had a molecular weight of 14 kilodaltons, and reacted with anti-human cystatin C antibodies. The physiologic and pathologic roles of cystatins in different cell types remain to be established. The TT cells provide a suitable cell type to study the regulation of the expression of the cystatin gene and the mechanism of cystatin release.

Cystatins in human tear fluid.

The activities of cysteine proteinases which include several lysosomal cathepsins are controlled by naturally occurring inhibitory proteins termed cystatins. Cystatins occur both intracellularly and extracellularly in various tissue fluids including tears. Tears were collected by the Schirmer paper strip method from healthy volunteers who had no history or signs of external ocular disease. The tear components were extracted from the filter papers, and used to determine the apparent free cystatin activity and cystatin levels of tears, and for immunoblots. Tears were also collected using capillary tubes for the measurements of cystatins. By titrating papain, a cysteine proteinase, of known specific activity with tear fluid, relatively high levels of apparent free cystatin activity were demonstrated in tears: 28.8 +/- 3.47 (S.E.M.) pmols papain inhibited per mg tear protein (n = 9). The concentrations of cystatins in tear samples were measured by an indirect enzyme-linked immunosorbent assay (ELISA) using antibodies against human salivary cystatin S and purified cystatin S as standard. The ELISAS revealed that tears contain high levels of cystatin-like immunoreactive material, amounting to about 10% of tear proteins. In microgram cystatin S/mg protein the values were: right eye: 94.7 +/- 9.9; left eye: 115.5 +/- 14.8; n = 12. Cystatin levels of tears collected using capillary tubes were comparable: 120.7 +/- 19 micrograms/mg protein (n = 10). Immunoblots of tear fluids revealed a protein of about 14,000 molecular weight which reacted with antihuman cystatin SN monoclonal antibodies. Protein(s) of similar molecular weight were visualized using antibodies against human cystatins S and C. Less abundant additional cystatin-like immuno-reactive proteins were detected by using the two latter antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic regulation of c-fos expression in cultured BC3H1 muscle cells.

Administration of adrenergic agonists induced c-fos mRNA in the salivary glands of the mouse and in the heart of the mouse, rat, and hamster (Barka et al., 1986, Mol. Cell Biol. 6, 2984-2989; 1987; Oncogene 1, 439-443). To further analyze transcriptional and post-transcriptional control of c-fos expression by adrenergic receptors and the putative role of fos in replication and differentiation pathways, we have examined c-fos expression in BC3H1 cells, a tumor-derived nonfusing muscle cell line. BC3H1 cells possess alpha 1- and beta 2-adrenergic receptors as well as receptors for histamine and acetylcholine. Furthermore, rapidly proliferating BC3H1 cells undergo differentiation toward muscle phenotype when exposed to low serum-containing culture media. Both alpha- and beta-adrenergic agonists and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate caused a rapid, transient increase in the steady-state level of c-fos mRNA. This induction was essentially independent of whether the cells were in the proliferative, relatively quiescent, or differentiated state. Protein synthesis inhibitors cycloheximide and anisomycin also increased markedly the concentration of c-fos mRNA, and in the presence of anisomycin c-fos mRNA was superinduced by the alpha-adrenergic agonist norepinephrine. Run-on transcription assays indicated that the c-fos gene is expressed in both proliferating and differentiated cells, although the steady-state levels of c-fos mRNA were low, or even undetectable, in such cells. The adrenergic agonists and the tumor promoter stimulated the transcription of the c-fos gene in both proliferating and differentiated cells. This stimulation, however, was modest, two- to three-fold compared to controls, in contrast to the marked elevation of the level of c-fos mRNA they caused. Neither the proliferation nor the expression of muscle type creatine kinase activity was influenced by adrenergic agonists. It is suggested that activation of the c-fos gene is a consequence of adrenoreceptor stimulation in diverse cell types, and thus it is involved in pleiotropic cellular responses to adrenergic agonists. Catecholamines may be one of the physiologic regulators of the c-fos gene.

Proto-oncogene fos (c-fos) expression in the heart.

Administration of the beta-adrenergic agonist isoproterenol led to a marked rapid increase in the steady-state level of c-fos mRNA in the heart of mice, rats, and Syrian hamsters. Stimulation of c-fos expression by isoproterenol was inhibited by the beta-adrenergic antagonist propranolol. An increase in Ca2+ influx through voltage-dependent calcium channels is probably not required for the activation of the c-fos gene by isoproterenol since the calcium channel blockers verapamil, nifedipine, and diltiazem had no effect on the induction of c-fos by the drug. In the heart of the rat, c-fos expression was also stimulated by the alpha-adrenergic agonist phenylephrine, histamine, and prostaglandin E1. The histamine-induced expression of the c-fos gene was blocked by the histamine H1-receptor antagonist pyrilamine but not by H2-receptor antagonists ranitidine and cimetidine. It is concluded that in the heart, hormones which increase cAMP and cytosolic Ca2+, such as beta-adrenergic agonists and prostaglandin E1, and/or stimulate the turnover of inositol phospholipids, such as alpha-adrenergic agonists and histamine H1-receptor agonists, regulate c-fos gene expression. The fos protein is likely to play a role in the mechanisms of neurotransmitters and hormones that modulate the functioning of the heart and of cardiac hypertrophy, degeneration and necrosis.

Accumulation of c-fos mRNA in slices of mouse submandibular gland incubated in vitro.

Incubation of slices or isolated lobules of murine submandibular gland at 37 degrees C in physiologic solutions or in tissue culture media, or dissociation of the cells by collagenase-hyaluronidase treatment, increased the steady-state level of c-fos mRNA without any additional stimulus. This activation of c-fos expression required the presence of Na+ and K+ but not extracellular Ca2+. It was augmented by depolarizing concentrations of K+ and by veratridine, and inhibited by high concentrations of amiloride. Alterations in membrane permeability and in ion fluxes and/or perturbation in membrane phospholipids may play a role in this transitional activation of the c-fos gene expression in incubated tissue slices in which the cells are not viable and undergo a necrobiotic process.

Beta-adrenergic stimulation of c-fos gene expression in the mouse submandibular gland.

Isoproterenol (IPR), a beta-adrenergic agonist, induces division of acinar cells in the parotid and submandibular glands of adult rodents and produces hyperplastic and hypertrophic enlargements of these organs. We analyzed the effects of IPR on thymidine incorporation, c-fos mRNA levels, and the immunocytochemical localization of c-fos protein in the submandibular glands of adult and of 5- and 14-day-old mice. In the glands of untreated mice c-fos transcripts were not detectable. In all experimental groups, administration of IPR led to a rapid, transient increase in the c-fos mRNA level. Propranolol blocked the IPR effect, while treatment with IPR and cycloheximide led to superinduction. We observed no correlation between the effect of IPR on cell replication or organ growth and stimulation of c-fos expression, and conclude that the latter is the result of beta-adrenergic receptor-IPR interaction. The c-fos protein was localized immunocytochemically in both the cytoplasm and the nuclei of acinar cells and in the nuclei of duct cells.

Induction of the synthesis of a specific protein in rat submandibular gland by isoproterenol.

Prolonged treatment of rats with the beta-adrenergic agonist, isoproterenol (IPR), produces hypertrophy and hyperplasia of the parotid and submandibular glands. The drug induces the synthesis of several secretory proteins that are absent or occur at very low concentrations in the gland or saliva of the untreated rat. We have measured the relative concentrations of one of these proteins (termed "large mobile" (LM) protein, Menaker et al. (1974) Lab. Invest. 30, 341-349) by using a solid phase enzyme-linked immunoabsorption assay. LM protein was not measurable in gland extracts of 20-day-old fetuses or 2-day-old rats. Its concentration was very low in the glands of 6- and 13-day-old and adult rats. Administration of IPR for 4 to 7 days to adult or 6-day old rats increased the levels of the LM protein by 20 to 25-fold. The LM protein was localized immunocytochemically primarily in the acinar cells in the glands of control and IPR-treated adult rats. In vitro translation studies using a mRNA-dependent reticulocyte lysate system and labeling with [35S]methionine showed: little synthesis of pro-LM from poly(A+) RNA from glands of adult rats and none from 13-day-old animals, and that, in comparison, poly(A+) RNA from glands of adult or 13-day-old IPR-treated rats directed the synthesis of a much greater concentration of pro-LM. The in vitro precursor of the LM protein migrated electrophoretically as a single band in anti-LM immunoprecipitates, and had a molecular weight of 14,000. The LM protein, which appears to be a single, unique polypeptide induced by IPR in the submandibular glands of developing and adult rats, will be useful in studies examining the effects of catecholamine beta-agonists on gene expression in an exocrine cell.

Monoclonal antibodies against rat saliva and salivary gland antigens.

Hybridomas were produced by the fusion of NS1 myeloma cells with spleen cells of a BALB/c mouse immunized with rat submandibular saliva. Growth of hybridomas was evident in 60/96 wells, and colonies secreting antibodies against saliva components were identified in 20 wells by using a solid phase enzyme-linked immunoassay. Cloning of cells from 12 wells yielded originally 43 hybridoma cell lines secreting anti-saliva antibodies. After recloning, one hybridoma (4Cl3) was selected for further studies. The hybridoma (4Cl3) cells were grown as ascites tumors, and the antibodies were purified from the ascitic fluid by diethylaminoethyl Affi-gel Blue chromatography. The purified antibody (MA4), immunoglobulin G1, immunoprecipitated a 39K dalton protein from submandibular saliva, and also reacted with a protein of the same electrophoretic mobility on immunoblots. From extracts of submandibular gland slices, incubated with [3H]leucine, the antibody again immunoprecipitated a 39K protein, indicating that this protein is synthesized in the gland. MA4 was used for immunocytochemical stainings of submandibular glands of rats of different ages. In general, immunostaining was seen only in acinar cells. Thus, there was no staining in the glands of 1-day-old rats that lack differentiated acinar cells. In the glands of 1- to 4-week-old rats the number of immunoreactive cells and the extent of immunostaining paralleled the differentiation of the acinar cells. In the glands of adult rats a uniform staining of the secretory granules of the acinar cells was observed. The immunoreactive 39K protein seemed to be restricted to the acinar cells in the submandibular gland; there was no immunostaining in the parotid, sublingual, or lingual salivary glands, or in the pancreas, colon, and duodenum. Stimulation of saliva secretion by isoproterenol resulted in a virtual depletion of the antigen from the acinar cells. These results indicate the feasibility of producing mouse hybridomas that secrete antibodies against rat saliva components. The monoclonal antibody at hand will be useful in analyzing the differentiation of the acinar cells, and the factors that influence this differentiation process.

Transport of 125I-EGF into milk and effect of sialoadenectomy on milk EGF in mice.

Epidermal growth factor (EGF) (urogastrone) is found in high concentrations in mouse and human milk. The origin of milk EGF is unknown. Milk samples were collected from lactating mice 2-6 h after the intravenous administration of a tracer dose of 125I-labeled EGF. The milk contained relatively high levels of radioactivity of which 35-46% was precipitated by trichloroacetic acid-phosphotungstic acid (TCA-PTA) and 26% by a specific antimouse EGF antiserum. Part of the radioactivity in milk was eluted from a Bio-Gel P-10 column at the point at which pure standard 125I-EGF was eluted. These data indicate that the mammary gland of the lactating mouse is capable of sequestering and transporting 125I-EGF into milk. Administration of a thousand-fold excess of unlabeled EGF caused no reduction in TCA-PTA-precipitable radioactivity in milk samples of mice given 125I-EGF. When mice were given 10 micrograms of unlabeled EGF and milk was collected 4 h later, compared with controls the EGF level in milk was doubled. Administration of EGF had no effect on lactose and protein concentrations in the milk, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed no qualitative or quantitative changes in the major milk proteins. Milk collected from lactating mice that were sialoadenectomized 6 mo earlier contained about 50% less EGF compared with controls. After the administration of 125I-EGF high concentrations of radioactivity were also found in the mammary and submandibular glands and in the stomach. In the latter organs, however, 95-96% of the radioactivity was in the acid-soluble fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Culture of A-431 human epidermoid carcinoma cells in serum-free medium: effect of culture conditions on the binding of [125I]-epidermal growth factor.

Serum-free culture conditions that permit the continuous growth of A-431 human epidermoid carcinoma cells were developed. In Dulbecco's modified Eagle's synthetic nutritional medium (DME) supplemented with fetuin, insulin, transferrin, biotin, and oleic acid-fatty acid-free bovine serum albumin complex A-431 cells grew at a rate comparable to that observed in the presence of calf or fetal calf serum. Of the factors tested, oleic acid had the most pronounced stimulatory effect on the growth and [3H]-thymidine incorporation of A-431 cells in serum-free medium. A-431 cells have a high number of receptors for epidermal growth factor (EGF); they bind and rapidly internalize EGF. Nevertheless, EGF did not stimulate either the growth or the [3H]-thymidine incorporation of these cells. Analyses of [125I]-EGF binding data indicated that A-431 cells grown in the presence of calf serum had about 3.2-3.9 X 10(6) specific, saturable EGF receptor sites on their surface. Linear Scatchard plots indicated a single class of noninteracting receptors with an apparent equilibrium dissociation constant of about 2.8 X 10(-9) M. The average number of receptors of A-431 cells maintained in DME supplemented with only fetuin, insulin, and transferrin for several months was significantly less, 1.54 X 10(6), than that of A-431 stock cells cultured in the same medium for 2 days only (2.68 X 10(6)). The apparent dissociation constants for the same cell populations were, however, similar, 4.5 X 10(-9) M and 4.1 X 10(-9) M, respectively. Stimulation of growth by oleic acid resulted in about 20% decrease in the average number of receptor sites, with an increase in the apparent equilibrium dissociation constant.

Hormonal regulation of epidermal growth factor and protease in the submandibular gland of the adult mouse.

The structure of the granular convoluted tubules of the mouse submandibular gland is influenced by androgens, adrenal steroids, and thyroid hormones. We wished to investigate the effects of variations in hormonal status on the quantitative and qualitative distribution of two secretory products of these tubules, epidermal growth factor (EGF) and protease. The effects of the thyroid and adrenal glands on EGF content and protease activity of the submandibular glands of adult female mice were studied by RIAs (EGF), enzyme assays (protease), and immunocytochemical methods. In animals rendered chronically hypothyroid by propylthiouracil (4 months) or in animals which were adrenalectomized and ovariectomized (3 weeks), protease activity and EGF levels were reduced by 81-97%. The administration of testosterone induced these polypeptides even in hypothyroid animals. Daily administration of L-T4 (T4; 1 micrograms/g BW) for 7 days increased EGF and protease activity 3.6-fold in intact mice and reversed the effect of hypothyroidism. EGF and protease were also induced by T4 in adrenalectomized and ovariectomized mice, although to a lesser degree than in intact animals. Immunocytochemical stainings of submandibular glands indicated that the number of granular convoluted tubule cells immunoreactive for EGF correlated with the levels of EGF determined by RIAs. With respect to immunostaining for protease, such a correlation was not observed. The data indicate multihormonal regulation of EGF and protease in the mouse submandibular gland.

Hypertrophic and hyperplastic effects of thyroxine on the submandibular gland of the mouse.

The nature of the trophic response of the mouse submandibular gland to thyroxine (T4) was examined. Adult female Swiss-Webster mice were given daily subcutaneous injections of T4 (1 microgram/gm body weight) for two or four days; two injections of tritiated thymidine (3H-TdR) were given 24 and 29 hours after the last injection of hormone, and the mice were killed one hour after the last injection of 3H-TdR. One gland was analyzed chemically for DNA content and for incorporation of 3H-TdR, while the other was used to prepare autoradiograms. The cellular composition of each gland was analyzed by counting 1000 nuclei, and the frequency and labelling index (LI) of six cell types were established. A rise in specific activity of DNA and a fall in its concentration were seen in response to T4. The LI for the entire gland more than doubled. The LIs and frequencies of granular convoluted tubule and granular intercalated duct cells were increased more than those of acinar and nongranular intercalated duct cells; striated and excretory duct cells were not affected. It is concluded that the enlargement of the submandibular gland of the mouse caused by T4 is due to both hyperplastic and hypertrophic effects.