Journal of Histochemistry and Cytochemistry, Vol. 45, 1533-1546, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

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

Tibor Barkaa and Hendrika M. van der Noena
a Department of Cell Biology and Anatomy, Mount Sinai School of Medicine of The City University of New York, New York, New York

Correspondence to: Tibor Barka, Mount Sinai School of Medicine, Box 1007, New York, NY 10029.


  Summary
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Summary
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Materials and Methods
Results
Discussion
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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 ß-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. (J Histochem Cytochem 45:1533-1545, 1997)

Key Words: gene transfer, DAP retroviral vector, submandibular gland, placental alkaline phosphatase, tissue culture, isoproterenol, membrane enzyme


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The major salivary glands could be useful targets for gene transfer for somatic gene therapy and for analysis of regulation of cell-specific gene expression in the glands. One of the major advantages of salivary glands for gene transfer is their accessibility. Genes can be transferred to duct and/or acinar cells by noninvasive retrograde ductal injection via the major excretory ducts that open into the oral cavity. Stable expression of transduced genes could provide for the delivery of gene products into the saliva, and the digestive system in general, for diverse therapeutic purposes. Vectors could also be developed to target delivery of gene products to basolateral cell membranes for endocrine-like secretion. Finally, salivary gland tumors may also be amenable to gene transfer for therapeutic purpose.

Although the potential advantages of salivary glands as targets of gene transfer have been recognized, thus far there are only two publications describing gene transfer to rat salivary glands in vivo. Mastrangeli and co-workers (1994) have shown that adenovirus vectors can effectively transfer genes into the major salivary glands. However, the expression of the transferred genes coding for the Escherichia coli lacZ ß-galactosidase and human {alpha}1-anti-trypsin was restricted to a relatively short period of time. We have previously described (Barka and van der Noen 1996 ) transfer of genes into rat submandibular glands (SG) by retrograde ductal injections of the replication-defective retrovirus BAG encoding the E. coli ß-galactosidase gene and the neomycin resistance gene (Cepko et al. 1984 ). Before injection of the vector, division of acinar cells in the gland, a requirement for integration of retroviral DNA, was induced by administration of the ß-adrenergic agonist isoproterenol (IPR) (Barka and van der Noen 1976 , and references therein). Long-term expression of the transduced gene ß-galactosidase (ß-gal) was observed, but the number of cells expressing the transduced gene was small.

Using the same paradigm, we have carried out experiments aiming at increasing the frequency of cells expressing the transduced gene and coding for a protein that is potentially secreted into the saliva. To this end, we have retrogradely injected into the SG of IPR- and pilocarpine-treated rats the retroviral vector DAP. This vector, structurally similar to the BAG vector, codes for human placental alkaline phosphatase (PLAP) instead of ß-gal (Fields-Berry et al. 1992 ). We surmised that because PLAP is a membrane-bound enzyme it may be released by the acinar cells expressing the transduced PLAP gene. After retrograde injection of the DAP viral vector we observed a high frequency of acinar cells stained for heat-resistant placental alkaline phosphatase and obtained evidence suggesting the secretion of such an enzyme into the saliva.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Retroviral Vector
The {psi}2 DAP cell line (American Type Culture Collection #CRL-1949; Rockville, MD) was produced from the {psi}2 cell line (Mann et al. 1983 ) by the insertion of the recombinant retroviral genome (DAP) into the genome of the {psi}2 cells. These cells produce recombinant retroviruses encoding the human placental AP and the neomycin resistance gene utilizing the same promoters as the BAG vector. Once infected with the DAP virus, the host cell and its progeny express histochemically detectable AP activity (Fields-Berry et al. 1992 ).

Producer cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated calf serum and antibiotics. Supernatant containing retroviral particles was harvested from confluent cultures of producing cells. The viral stock was concentrated by centrifugation (Cepko 1989 ) and the viral particles were suspended in complete culture medium. The titer of the concentrated viral preparation, assayed using NIH-3T3 cells (Miller et al. 1993 ) and used in these experiments, was 1.1 x 107 cfu/ml. The viral stock, containing 10 µg/ml polybrene, was stored at -80°C and was thawed only once.

Cell Culture and In Vitro Infection
The epithelial duct cell line of rat SG origin, A5, was a gift of Dr. B.J. Baum. A5 cells were grown in DMEM supplemented with 10% fetal bovine serum and antibiotics. Cultures grown in 10-cm dishes to approximately 70% confluence were rinsed with Hanks' solution and exposed to 3 ml of medium harvested from confluent cultures of {psi}2-DAP cells. The medium was supplemented with 8 µg/ml of polybrene. To reduce the concentration of polybrene, 4 hr later 10 ml complete medium was added to the cultures. The medium was changed 24 hr later and included 0.75 mg/ml (active component) of G418. The cells were cultured in the selective medium for 2 weeks and frozen. Thawed cultures were again exposed to G418 for several days. The G418-resistant cell line was designated as A5-DAP.

Cloning and Alkaline Phosphatase Activity of Clones of A5 and A5-DAP Cells
Cloning was performed by a limiting dilution technique. The clones were expanded and used for enzyme assays. For determination of AP activity, the cells were collected by trypsinization, and 2 x 105 cells were used for enzyme assay. The cells were sedimented by centrifugation, washed with PBS, resuspended in 0.5 ml of alkaline buffer (2-amino-2-methyl-1-propanol, 1.5 mol/liter, pH 10.3, at 25C) (Sigma Diagnostics 221; Sigma Chemical, St Louis, MO) and were sonicated briefly at 0C. Alternatively, the cells were lysed in alkaline buffer containing 0.5% Nonidet P-40. After addition of 0.5 ml of substrate solution (p-nitrophenyl phosphate disodium salt, final concentration 5 mM), incubation was for 15-60 min at 37C. After addition of 5 ml of 0.05 N NaOH, the absorbance at 400 nm was measured. Enzyme activity, determined in triplicate, is expressed as nmol/min/106 cells of substrate hydrolyzed.

Growth of Clones of A5-DAP Cell Lines
Cells of selected clones of A5-DAP cell lines were seeded in 35-mm dishes (5 x 104 cells/dish) and cultured in DMEM with 10% FBS. Growth was assessed by measuring DNA in triplicate dishes after 1-4 days in culture using a spectrofluorometric method (Hinegardner 1971 ). As standard, deoxyadenosine was used, assuming that 1 mg DNA = 0.385 mg of deoxyadenosine. Doubling time was calculated as described by Hayflick (Hayflick 1973 ).

Animals, Treatments, and Retrograde Ductal Injection of the Retroviral Suspension
Female Sprague-Dawley rats weighing about 200 g were kept in air-conditioned quarters and had free access to food and water. The schedule of treatments was as follows. The rats received two injections of 160 mg/kg of isoproterenol-HCl (Sigma) IP. The first injection was given at 1600 hr, followed by the second injection 26 hr later at 1800 hr. IPR was dissolved in 0.1% sodium metabisulfite in saline.

The viral vector suspension (0.1 ml) was injected retrogradely into the SG by cannulating the main excretory duct (Omnell and Qwarnström 1983 ) 15-16 hr after the second dose of IPR. The rats were anesthetized (Ketamine, 60 mg/kg and xylazine, 8 mg/kg, IM) and received 6 mg/kg pilocarpine-HCl 45-60 min before cannulation to deplete the gland of its secretory products. After insertion of the cannula, 1 mg/kg atropine was injected SC to reduce residual saliva flow. For these experiments, 23 rats were used. In carrying out the animal experiments, institutional guidelines were observed.

Collection of Saliva and Assays of Proteins and Alkaline Phosphatase Activity
Saliva was collected between 0900 and 1100 hr from anesthetized rats by cannulating the main excretory ducts of the SG. Although technically difficult, from some rats saliva was obtained from both lobes of the gland by cannulating the two independent secretory ducts. Saliva secretion was induced by administration of 6 mg/kg of pilocarpine-HCl SC. Saliva was collected for 12-15 min from the time of the administration of the sialogogue. Saliva secretion starts 2-3 min after the injection of pilocarpine. Immediately after collection, the saliva samples were centrifuged (5000 rpm, 3 min) and the sediment was discarded. The saliva samples were kept at -20C before assays.

Proteins in saliva samples were determined using the BCA Protein Assay Reagent Kit (Pierce; Rockford, IL). Alkaline phosphatase activity of saliva samples was measured in triplicate using a THERMOmax Microplate Reader (Molecular Devices; Menlo Park, CA). The reaction mixture contained 30 µl saliva, 50 µl alkaline buffer, 50 µl of the substrate solution (4 mg/ml) and H2O to 150 µl. For kinetic assays, the plate was placed in the chamber of the plate reader with the temperature set for 37C. After a 4-min lag period, the optical density at 405 nm minus nonspecific background at 650 nm of each well was determined every 2 min over a period of 90 min. Blank wells contained H2O instead of saliva. The maximum rate of reaction, Vmax, calculated using the software (SOFTmax) of the plate reader, is given in mOD/min (millioptical density units per minute). This was converted into mOD/min/ml saliva, or, by using appropriate p-nitrophenol standards, into nmol/min/ml saliva. In all instances, the correlation coefficient of the kinetic plot was <0.9.

Enzyme-linked Immunoabsorbent Assay (ELISA) for PLAP in Saliva
ELISA was performed using routine methods. Briefly, 96-well plates (Dynatech Immulon plates) were coated for 1 hr at room temperature (RT) with 50 µl of serial dilutions of human placental AP (Sigma; Type XXIV) as standard (0.006-3.125 µg/well), or with saliva (10-20 µl) obtained from untreated rats or from rats infected with the DAP vector 11 days earlier. Dilutions were with bicarbonate buffer: 0.015 M Na2CO3, 0.035 M NaHCO3, pH 9.6. The wells were washed three times with 0.05% Tween-20 in Tris-buffered saline (TBS) and blocked for 10 min with 1% bovine serum albumin in TBS. To each well, 50 µl of the primary antibody, rabbit anti-human placental AP (DAKO; Glostrup, Denmark) (1:500 in TBS containing 1% BSA, 1 hr at RT) was added. After appropriate washing, the second antibody, peroxidase-labeled anti-rabbit IgG (1:500 dilution, 1 hr at RT) (ICN ImmunoBiologicals; Lisle, IL) was added to the wells. After washing, the peroxidase activity was assayed using 2,2'-azinobis-(3-ethylbenzthiazoline sulfonic acid) (ABTS) as chromogene (1 mg/ml in 0.02 M phosphate buffer, pH 6.8) and H2O2 (0.003%) as substrate. The assay was performed using the THERMOmax Microplate Reader in a kinetic assay mode (2-min lag period, 20-min incubation at 37C, readings at 1-min intervals, at 405 nM). The Vmax and the analyses of the data were obtained using log-log curve fit programs of the SOFTmax. The correlation coefficient of the log-log standard curve, used to calculate the relative concentrations of PLAP in the saliva samples, was 0.988.

Histochemical Methods
For histochemical staining of cultures, the cells were grown in Lab-Tek tissue culture chamber/slides. The cultures were rinsed in PBS, fixed in 0.5% glutaraldehyde in PBS for 20 min at RT, washed three times for 3 min in PBS, and incubated for demonstration of enzyme activity. The same fixation protocol was used for cryostat sections (8 µm) cut from SGs. AP activity was demonstrated by an azo dye method using naphthol AS-MX phosphate as substrate and Fast Red TR salt as the coupler, yielding a red azo dye reaction product, or by an indoxyl method using 5-bromo-4-chloro-3-indolyl phosphate as substrate and nitroblue tetrazolium as the chromogen (Fields-Berry et al. 1992 ). For the azo dye method the incubating medium was prepared as follows. To 9 ml of Buffer 3 (Boehringer-Mannheim Genius Kit; 100 mM Tris-HCl, 10 mM NaCl, 50 mM MgCl2, pH 9.5), (Boeh-ringer; Mannheim, Germany), 0.1 ml of naphthol AS-MX phosphate stock solution (50 mg/ml of dimethylformamide), and 10 mg of Fast Red TR salt were added. (If a visible red precipitate is formed the medium should be filtered before use). If incubation longer than 60 min was required, the cells were rinsed in Buffer 3 and incubated further in a freshly prepared medium. Incubation was usually for 60 min at 37C. After incubation, the slides were rinsed in water, postfixed in formalin, washed, and mounted in Plasdon C.

For demonstration of AP activity of acinar cells expressing the transduced PLAP gene, the cryostat sections, after fixation and rinsing, were treated with heat. Exposing the sections (in PBS) to 65C for 10-60 min inhibited AP activity in the SGs without noticeable inhibition of the enzyme activity in transduced cells. Routinely, the sections were heat-treated for 20 min.

Demonstration of AP activity in unfixed cells was carried out by a modification of the azo dye method. The cultures were first rinsed in PBS and then briefly in 0.1 M Tris-HCl buffer, pH 8.6. They were incubated at 37C in a medium consisting of 0.5 mg/ml (1.35 mM) of naphthol AS-MX-phosphate, 1 mM MgCl2, 1 mg/ml Fast Red TR salt, 0.1 M Tris-HCl buffer, pH 8.6. The medium was filtered through a 22-µm MILLEXC-GS filter unit (Millipore; Bedford, MA). The progression of staining was monitored microscopically. In general, it was for 10-20 min. The stained cells were photographed, and if permanent preparations were needed they were rinsed in PBS and fixed in formalin or glutaraldehyde (1%)-formalin (4%) mixture. Cells grown on coverslips were treated similarly and mounted in Plasdon.

Semiquantitation of acinar cells expressing PLAP was carried out by counting the number of acinar cells stained for PLAP in 80-100 microscopic fields using x 10 ocular and x 10 objective lenses (area 0.16 cm2).

Release of Membrane-bound AP from A5-DAP Cells by Bromelain Treatment
Bromelain treatment was carried out as described by Kottel and Hanford (Kottel and Hanford 1980 ). HeLa (JW-36) (2 x 105) cells and A5-DAP (5 x 105) cells were collected by trypsinization, washed with PBS, resuspended in 0.5 ml of 50 mM phosphate buffer, pH 6.8, containing 0-1 mg/ml bromelain (7.5 U/mg protein; Sigma B-5144), and incubated for 60 min at 37C. After incubation, the cells were sedimented by centrifugation (14,000 rpm, 5 min). The supernatant was removed for assay of AP activity. The sediment was resuspended in 0.5 ml alkaline buffer + 0.15 ml of phosphate buffer, sonicated, and used for AP assay. The results are shown as percent of total activity released by the bromelain treatment. Total activities (OD400) were HeLa cells 0.192 ± 0.02, A5-DAP cells 0.121 ± 0.01. Activities of cells kept on ice for 60 min (the time of bromelain treatment) were HeLa 0.193, A5-DAP 0.123, indicating that no activity was lost during the bromelain treatment and sonication. Incubation for 60 min at 37C without bromelain also resulted in no loss of enzyme activity.

Release of Membrane-bound AP from HeLa and A5-DAP Cells by Phosphatidylinositol-specific Phospholipase C
HeLa (JW-36) and A5-DAP cells, each 2 x 105, were collected by trypsinization, washed with PBS, and resuspended in 80 µl of release buffer (25 mM Tris-HCl, pH 5, 0.25 M sucrose, 10 mM glucose) (Berger et al. 1987 ) to which 20 µl enzyme solution containing 1 unit of phosphatidylinositol-specific phospholipase C was added (Sigma P5542). According to the supplier, this enzyme was purified from Bacillus cereus by the method of Sundler et al. (1978). The suspension was incubated for 90 min at 37C. Control cells were incubated in 100 µl of release buffer without the enzyme or were kept at 0C. After incubation, the cells were sedimented by centrifugation (14,000 rpm, 5 min), and the supernatant was used for assay of AP activity. The sedimented cells were resuspended in 0.5 ml of alkaline buffer, sonicated, and enzyme activity was measured.

Secretion of AP by A5-DAP Cells
A5-DAP-Cl-4 cells (4 x 105), which express high levels of AP activity, were seeded in 35-mm dishes. Next day, the cultures were rinsed with Dulbecco's PBS and incubated at 37C for 30, 60, or 90 min in 0.5 ml of RPMI 1640 medium (GIBCO; Grand Island, NY). There were two dishes for each time point. After incubation, the spent medium was removed and centrifuged at 14,000 rpm for 2 min to sediment cells that may have become loose during the incubation. AP activity was assayed in duplicate using 0.2 ml of medium. The results are expressed as units of AP secreted per dish per 106 cells, and per 100 µg DNA. One unit = nmol/min of substrate hydrolyzed.


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Retrovirus-mediated Gene Transfer and Expression of Transduced Genes in Submandibular Gland-derived A5 Cells In Vitro
Expression of PLAP in A5 Cells Transduced by the DAP Retroviral Vector. Before gene transfer to the SG in vivo, we performed in vitro experiments using the retroviral vector DAP and the A5 cell line. This cell line, established from the submandibular glands of weanling Fischer 344 rats (Brown et al. 1989 ), has been used in previous gene transfer experiments (Mastrangeli et al. 1994 ; Barka and van der Noen 1996 ). The objectives were to establish that (a) salivary gland-derived cells can be infected by the DAP vector, (b) the transduced cells express PLAP encoded by the viral DNA, (c) the PLAP is membrane-bound, and (d) the transduced cells release AP into the culture medium.

A5 cells exposed to the spent medium of virus-producing 2 DAP cells were readily infected with the viral vector DAP, and the transduced cells, A5-DAP, selected by their resistance to G418, revealed AP activity when stained by indoxyl or azo dye methods.

There was a cell-to-cell variation in the intensity of staining, but after longer incubation time and using the more sensitive indoxyl method, every cell revealed some AP activity. In contrast, by histochemical staining, A5 cells showed no or minimal alkaline phosphatase (AP) activity (Figures 1 and 2).



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Figures 1-2. Alkaline phosphatase activity in A5-DAP and A5 cells. A5 cells transduced by the viral vector DAP and selected by resistance to G418, A5-DAP cells revealed staining for alkaline phosphatase (Figure 1). The parent A5 cells revealed no or minimal staining for the enzyme (Figure 2). A5-DAP and A5 cells grown on uncoated coverslips were fixed with 0.5% glutaraldehyde in PBS for 20 min at RT. After washing in PBS, the cells were stained for the demonstration of AP activity using an azo dye method as described in Materials and Methods. Incubation time was 40 min at 37C. Bar shown in Figure 1 = 10 µm (valid for 1-4).

Figure 3. Alkaline phosphatase activity in A5-DAP cells. With relatively short incubation times, enzyme activity is localized predominantly at the peripheries of the cells, suggesting that the enzyme, PLAP, is membrane-bound. Cells were fixed and stained for the demonstration of AP activity using an indoxyl method as described in Materials and Methods. Incubation time was 20 min at 37C. Bar = 10 µm.

Figure 4. Alkaline phosphatase activity in A5-DAP cells. Deposits of the reaction product of the histochemical staining are seen mostly on the cell membrane. These deposits are frequently in clusters (arrows), suggesting an uneven distribution of the enzyme in the plasma membrane. Cells were grown on coverslips and stained for demonstration of AP activity without fixation, using the azo dye method. Incubation time was 10 min at 37C. Bar = 10 µm.

Membrane Localization of AP Activity in A5-DAP Cells
Histochemical staining of glutaraldehyde-fixed cells suggested that the enzyme is localized predominantly in the plasma membrane (Figure 3). AP activity could also be demonstrated in unfixed cells, providing a staining pattern consistent with plasma membrane localization. This was particularly conspicuous after short (5-10 min) incubation. Frequently, the distribution of the reaction product was patchy, suggesting an uneven distribution of the enzyme in the plasma membrane (Figure 4). Because the phosphate ester substrate is not expected to penetrate the plasma membranes of intact, unfixed cells, the membrane staining indicated that the enzyme is an ectoenzyme with active sites exposed at the cell surface.

The plasma membrane localization of the PLAP was further substantiated by its release on treatment with bromelain or phosphatidylinositol-specific phospholipase C.

Treatment of A5-DAP cells with 0.05 mg/ml or 0.25 mg/ml bromelain for 60 min at 37C released 83% or 85% of total AP activity, respectively. In the case of HeLa cells, 55% of total AP activity was released by digestion with 0.05 mg/ml of bromelain. Treatment with bromelain for 60 min at 37C had no effect on the total AP activity of A5-DAP cells.

Treatment of A5-DAP or HeLa cells for 90 min at 37C with phosphatidylinositol-specific phospholipase C released 77% or 52%, respectively, of total AP activity. Cells incubated without the enzyme for the same period of time released 1.5% or 2.2%, respectively, of total activity (Figure 5).



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Figure 5. Release of alkaline phosphatase activity from A5-DAP and HeLa cells by treatment with bromelain or phospholipase C. Treatment with bromelain or with phospholipase C released 83% or 79%, respectively, of membrane-bound AP activity from A5-DAP cells. PL, Phospholipase C. A5-DAP and HeLa cells were incubated with indicated concentrations of bromelain or phospholipase C for 60 or 90 min, respectively, at 37C and the released AP activity was determined in triplicate as described in Materials and Methods.

Clonal Variation in the Expression of AP Activity in A5-DAP Cells
Histochemical staining of A5-DAP cells indicated a heterogeneity with respect to the level of AP activity. To analyze this phenomenon, A5-DAP cells were cloned by a limiting dilution technique and the enzyme activities of 20 randomly selected clones were determined. For the assays, clones in logarithmic growth phase were used. There was a great variation in AP activities of clones, ranging from 0 to 574 nmol/min/106 cells (Figure 6). When unfixed clones of A5-DAP cells were stained for AP activity using the azo dye method, even within a given colony the intensity of staining varied greatly from cell-to-cell (Figure 7). Hence, the level of AP activity in clones of A5-DAP cells measured chemically is proportional to the number of cells expressing the transduced gene and to the level of PLAP expression in individual cells.



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Figure 6. Alkaline phosphatase activity in clones of A5-DAP cells. Clones of A5-DAP cells revealed a great variation in AP activity. A5-DAP cells were cloned by a limiting dilution technique, and AP activity was determined, in triplicate, of randomly selected clones as described in Materials and Methods. The triplicate values agreed within 5%.



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Figure 7. Alkaline phosphatase activity in a colony of A5-DAP cells. This photomicrograph illustrates a group of cells within a colony of A5-DAP cells. Two of these cells show high enzyme activity. Cells were grown on coverslips and stained for the demonstration of AP activity without fixation, using the azo dye method. Incubation time was 30 min at 37C. Bar = 10 µm.

Compared to A5-DAP cells, the alkaline phosphatase activity of A5 cells was low, 5-20 nmol/min/106 cells. Clones of A5 cells also differed in the level of AP activity (Figure 8).



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Figure 8. Alkaline phosphatase activity in clones of A5 cells. Clones of A5 cells had a lower AP activity compared to that of A5-DAP cells (Figure 6). The enzyme activity of individual clones, however, varied. A5 cells were cloned by a limiting dilution technique and AP activity was determined, in triplicate, of randomly selected clones as described in Materials and Methods. The triplicate values agreed within 5%.

Effects of Specific Inhibitors and Heat on AP Activity of A5 and A5-DAP Cells
AP in A5 cells was sensitive to L-homoarginine, less sensitive to L-leucine and resistant to L-phenylalanine. In contrast, AP in A5-DAP cells was resistant to L-homoarginine but sensitive to L-phenylalanine. These data (Table 1) indicate that most of the AP expressed by A5-DAP cells is PLAP whereas the enzyme in A5 cells is of a tissue-nonspecific type.


 
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Table 1. The effect of inhibitors on alkaline phosphatase activity of A5-DAP and A5 cellsa

As expected, AP in A5-DAP cells was relatively heat resistant; 30-min exposure to 56C had no effect on enzyme activity, and more than 80% of activity remained after exposure to 65C for 10-30 min. In contrast, AP in A5 cells was heat-sensitive; about 80% of the activity was destroyed by heat treatment for 10-30 min at 56C (Figure 9).



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Figure 9. Effect of heat on the alkaline phosphatase activity of A5 and A5-DAP cells. AP in A5-DAP cells is heat-resistant; 30-min exposure to 56C had no effect on its activity. AP in the parent A5 cells is heat-sensitive. About 80% of activity was lost after 30-min exposure to 56C. A5-DAP and A5 cells were suspended in PBS and heated for 10-30 min. The remaining enzyme activity was determined in triplicate and compared to that of cells kept at 0C (=100%).

Cell Growth Rate and Expression of PLAP in A5-DAP Clones
Because it has been reported that in HeLaS3 cells PLAP activity is inversely related to cell growth rate (Telfer and Green 1993 ), we investigated whether such a negative correlation exists in clones of A5-DAP cells. To this end, we measured the doubling time, in terms of DNA, of selected clones expressing different levels of AP activity. We found no correlation between the doubling time and level of AP activity (Table 2).


 
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Table 2. Doubling times and alkaline phosphatase activity of A5 cells and selected clones of A5-DAP cellsa

Release of AP by A5-DAP Cells
The membrane localization of PLAP in A5-DAP cells suggested that these cells may release or secrete AP. Indeed, when A5-DAP-Cl-4 cells, which express high level of AP activity, were incubated in a serum-free medium there was a time-dependent release of AP (Figure 10).



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Figure 10. Release of AP A5-DAP cells. A5-DAP cells incubated in a serum-free culture medium released AP in a time-dependent manner. A5-DAP (clone 4) cells were incubated for 30-90 min in a serum-free medium and the AP activity released into the medium was determined in duplicate as detailed in Materials and Methods.

In Vivo Experiments
Expression of PLAP in Acinar Cells in the SG after Retrograde Duct Injection of the DAP Vector. To evaluate the feasibility of in vivo gene transfer to the salivary glands by the retroviral vector DAP, we performed retrograde duct injections of the vector suspension containing polybrene. Before injection of the vector, division of acinar cells, a prerequisite for integration of viral DNA (Springett et al. 1989 ; Miller et al. 1990 ; Schuitemaker et al. 1994 ), was induced by administration of the ß-adrenergic agonist IPR. Furthermore, the acinar cells were depleted of most of their secretory products by administration of pilocarpine before delivery of the vector. Expression of the transduced gene coding for human PLAP was ascertained by histochemical staining for heat-resistant AP.

Several cell types in rat SG contain AP demonstrable histochemically in cryostat sections fixed in glutaraldehyde (Figure 11). The AP in the gland, however, is heat-sensitive, and no AP activity could be demonstrated by either of the histochemical stainings used in any cell in sections prepared from the glands of untreated rats and exposed to 65C for 20 min. In contrast, human PLAP is relatively heat-resistant (Neale et al. 1965 ; Stigbrand 1984 ), and exposure to 65C for up to 40-50 min had no obvious effect on AP activity of cells expressing PLAP (Figure 12). This difference in heat sensitivity allowed the detection and semi-quantitation of cells transduced by DAP and expressing PLAP.



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Figures 11-12. Alkaline phosphatase activity in the submandibular gland of a rat injected retrogradely with the viral vector DAP 6 days earlier. These figures represent serial sections prepared from the gland of a rat injected with the viral vector. The sections were stained simultaneously but the slide illustrated in Figure 12 was exposed to 65C for 20 min in PBS after fixation but before staining. AP activity is seen in several cell types in the gland (Figure 11). This endogenous AP is heat-sensitive. After heat treatment, only cells expressing the heat-stable PLAP are stained (arrows). Indoxyl method; incubation time 2 hr at 37C.

Figures 13-15. Histochemical demonstration of heat-stable AP activity in the submandibular gland of a rat 25 days after retrograde duct injection of the viral vector DAP. Many acinar cells stained for PLAP are seen in the vector-injected lobe (Figures 13 and 14) but not in the contralateral lobe (Figure 16). No stained cells were observed in the sublingual gland (Figure 14, SL). Enzyme activity was demonstrated by the indoxyl method as given in Materials and Methods. Bar shown in Figure 15 = 10 µm (valid for 11-16).

Figure 16. Composite figure illustrating membrane localization of AP activity in acinar cells transduced by the retroviral vector DAP. Deposits of the reaction product of the histochemical staining are seen on the apical cell membranes of acinar cells forming acini. This pattern of staining was observed after short incubation times using the azo dye or the indoxyl method in the glands of rats injected with the vector 6-25 days earlier.

Rats were sacrificed 6-25 days after retrograde injection of the viral vector, and cryostat sections prepared from the SGs were stained histochemically for the demonstration of heat-resistant AP activity. Similarly, SGs of untreated rats and lobes contralateral to the injected lobes were also examined.

PLAP was detected only in the acinar cells in the SGs of rats retrogradely injected with the viral vector DAP (Figures 13 and 14). There was no staining for the enzyme in any other cell types or in cells in the sublingual gland (Figure 14), which is adjacent to the SG but has an independent excretory duct. No staining was observed in heat-treated sections prepared from the glands of untreated rats, or from lobes contralateral to the injected lobes (Figure 15) of rats that were injected retrogradely into one of the two lobes. After a relatively short incubation time, deposits of the histochemical reaction were seen in many acinar cells on the apical cell membrane (Figure 16). With longer incubation, such as that used to quantify the frequency of cells expressing PLAP, this membrane staining was obscured.

Semiquantitation of the number of stained cells in the glands of rats injected 6-25 days earlier with the vector revealed a relatively high frequency of cells expressing PLAP (Table 3). Of 14 rats injected with the vector, no stained cells were seen in the glands of three rats sacrificed 11 days later.


 
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Table 3. Frequency of acinar cells stained for heat-resistant alkaline phosphatase in submandibular glands of rats after retrograde injection of DAPa

None of the glands injected with the viral vector showed any signs of inflammation or structural changes on routine H&E preparations.

AP Activity in Submandibular Saliva of Untreated and DAP-injected Rats
PLAP is a membrane enzyme, and, as described above, A5 cells transduced by the DAP vector (A5-DAP cells) release the enzyme into the culture medium. This finding and the localization of the enzyme in the apical cell membrane of the acinar cells suggested that acinar cells transduced by DAP in vivo and expressing the PLAP gene may secrete AP into the saliva. To test this possibility, we collected saliva by cannulating the main excretory duct from untreated rats and from rats 11 days after injection of the retroviral vector DAP. The saliva samples were assayed for AP activity, for PLAP by ELISA using a polyclonal antibody against human placental AP, and for protein concentration. Saliva was collected, in general, for 10-15 min after administration of pilocarpine given as a secretogogue. The flow rate was calculated from the time of the injection of pilocarpine. Because the flow of saliva starts about 2-3 min after the SC injection of pilocarpine, the average actual flow rates were higher than those given in Table 4.


 
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Table 4. Submandibular saliva of control and DAP-injected ratsa

The flow rates and protein concentrations of saliva samples obtained from control and virus-injected rats were quite variable but were not statistically different. However, on average, the AP activity of saliva from rats injected with the viral vector was threefold higher than that of untreated rats. This difference was statistically significant, p = 0.046 (Table 4).

From seven rats, saliva was obtained from both lobes, i.e., virus-injected lobe and contralateral lobe, 7-25 days after injection of the vector. One rat (#5) was cannulated twice, 7 and 20 days after the injection of the vector. In all but one instance, the saliva secreted by cells of the vector-injected lobe revealed higher AP activity compared to the saliva from the contralateral lobe (Figure 17). However, the difference was statistically not significant by the paired (non-independent samples) t-test.



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Figure 17. Comparison of AP activities of submandibular saliva samples obtained from the virus-injected lobe and from the contralateral lobe of the same rat. Rats received the viral vector DAP via retrograde duct injection. Saliva was collected by cannulating both excretory ducts, i.e., the duct of the lobe into which the vector was injected 7-25 days earlier and the duct of the contralateral lobe. AP activity in saliva samples was determined in triplicates as described in Materials and Methods. The triplicate values agreed within 5%. The numbers over the columns indicate days after injection of the vector. One rat (5) was cannulated twice.

Saliva samples were also assayed for the relative concentration of PLAP by ELISA, employing a polyclonal antibody to human PLAP and purified human placental AP as standard. The mean antigen concentrations reacting with the antibody to human PLAP were in ng/ml (means ± SE; n = 8 in both groups): saliva of untreated rats 34 ± 25; DAP-infected rats 425 ± 147. This difference, because of the high variation, was statistically not significant (p = 0.058). In five of the eight saliva samples of untreated rats no PLAP was measurable; three of eight saliva samples from DAP-injected rats had no PLAP or the PLAP concentration was below the sensitivity of the method. These data suggested that a fraction of AP in the saliva of virus-injected rats is of the PLAP type.

We sought further evidence that the saliva of rats injected with the viral vector includes PLAP secreted by the transduced acinar cells by studying the effects of heat and specific inhibitors on AP activity. PLAP, in contrast to tissue-nonspecific AP, is relatively heat-stable, resistant to L-homoarginine, sensitive to L-phenylalanine, and less sensitive to L-leucine (Chang et al. 1994 ; Stigbrand 1984 ).

The effects of heat and inhibitors were tested on pooled saliva samples obtained from rats injected with the viral vector 11 days earlier and from untreated rats. In addition, heat sensitivity was tested using individual saliva samples obtained from two rats 6 days after retrograde injection of the vector and from one rat injected 20 days earlier. In addition, heat sensitivity of saliva AP of three untreated rats was measured. AP of saliva from control rats was heat-sensitive; no activity was measurable after exposure to 65C for 20 min in any sample. In contrast, measurable AP activity remained in saliva samples of virus-injected rats after heat treatment (Table 5).


 
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Table 5. Effect of heat on alkaline phosphatase activity of saliva samplesa

The relative sensitivity of saliva AP to the specific inhibitors also suggested that the saliva obtained from virus-injected rats contained PLAP. Thus, the AP of pooled saliva obtained 11 days after the injection of the vector was less sensitive to L-homoarginine and more sensitive to L-phenylalanine than the pooled saliva of control rats.


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

We have shown previously (Barka and van der Noen 1996 ) that rat submandibular gland-derived A5 cells can be infected and transduced by the retroviral vector BAG, which codes for bacterial ß-galactosidase. A5 cells were also transduced by the retroviral vector DAP, which is structurally similar to BAG but codes for, in addition to the neomycin resistance gene, human placental alkaline phosphatase (Fields-Berry et al. 1992 ). Human PLAP is a heat-stable (Neale et al. 1965 ; Posen et al. 1969 ; Jensen et al. 1968 ; Stigbrand 1984 ), membrane-bound enzyme (Jensen et al. 1968 ; Jemmerson et al. 1985 ), anchored to the plasma membrane via its carboxyl terminus by glycosyl-phosphatidylinositol (Low and Zilversmit 1980 ; Abu-Hasan and Sutcliffe 1985 ; Low et al. 1986 ; Low 1989 ). Consequently, the enzyme is released from the membrane not only by bromelain (Kottel and Hanford 1980 ) but also by phosphatidylinositol-specific phospholipase C (Ikezawa et al. 1976 ; Low et al. 1988 ; Fleming et al. 1995 ).

A5 cells transduced by DAP, A5-DAP cells, expressed heat-stable human PLAP, which in these cells was also membrane-bound, as indicated by histochemical staining and by the release of the enzyme by bromelain and phosphatidylinositol-specific phospholipase C. The histochemical staining suggested that the enzyme is in clusters, as has been described for the placenta and different cancer cells (Tokumitsu et al. 1981 ; Jemmerson et al. 1985 ). Simian (COS) cells transfected with a eukaryotic expression vector containing the PLAP cDNA also transiently expressed active, membrane-bound PLAP (Berger et al. 1987 ). In polarized cells (MDCK) stably transfected with PLAP cDNA, the enzyme was expressed at the apical cell surface (Brown et al. 1989 ; Brown and Rose 1992 ; Arreaza and Brown 1995 ). Histochemical staining suggested that in acinar cells transduced by the DAP vector in vivo PLAP is also localized to the apical cell membrane. Similarly, Fields-Berry and co-workers (1992) have observed membrane localization of PLAP in developing murine retina after transduction with the DAP vector.

A5-DAP cells revealed a great clonal variation in the level of AP activity, and even within a single clone expression of PLAP varied from cell to cell. This phenomenon, "mosaic expression," was observed by Cepko 1989 in fibroblasts infected with lacZ-containing viral vectors in which the expression of ß-gal was driven by the LTR promoter. In such cells, the lack of ß-gal expression correlated with the lack of detectable levels of mRNA for lacZ. This phenomenon appeared to be epigenetic and reversible. The cause of mosaicism in A5-DAP cells remains to be investigated.

From the in vitro experiments we concluded that a salivary gland-derived cell line A5 can be infected by the retroviral vector DAP. In the transduced cells the viral promoter LTR is effective, and the cells express heat-stable PLAP localized mostly in the plasma membrane.

Our previous work (Barka and van der Noen 1996 ) has established that retrograde duct injection of a retroviral vector is an effective means of gene transfer to the acinar cells of the SG in vivo, resulting in integration and long-term expression of the genes encoded by the vector. One of the limitations to the use of retroviral vectors, the requirement for cell division for integration of the viral DNA, was overcome by induction of cell division of acinar cells by administration of the ß-adrenergic drug isoproterenol. Injection of one or two doses of IPR stimulates cell replication of the essentially quiescent acinar cells in the glands of adult rats (Selye et al. 1961 ; Barka 1965a , Barka 1965b ; Baserga 1966 ; Schneyer 1969 ; Barka and van der Noen 1976 ). We have also shown that both the schedule of the administration of IPR and the timing of the retrograde injection of the vector were important for effective gene transfer and transduction. Although long-term expression of ß-gal was obtained, the frequency of acinar cells expressing the vector was low, less than one to 2.6 cells per microscopic field defined by a x 10 objective and a x 10 ocular lens.

In the present study, a much greater efficiency of transduction was obtained; staining for heat-resistant alkaline phosphatase revealed 9-59 cells/microscopic field expressing the enzyme. In comparison, using a similar semiquantitative method, Branchereau and co-workers (1994) found a maximum of 35 cells stained for ß-gal in regenerating liver after asanguineous perfusion with the MGF NB vector, which contains the lacZ gene of E. coli. Three factors may account for the higher efficiency observed: use of a different retroviral vector; modification of the injection schedule; or administration of a secretogogue before retrograde injection of the vector.

Previously, we have used the BAG vector coding for bacterial ß-gal, whereas in the present study the DAP vector coding for PLAP was employed. Although these vectors are structurally similar and utilize the same promoter, LTR, to drive the genes coding for ß-gal or PLAP, respectively, the host genome may regulate the expression of the transduced genes differently. The timing of the injections of IPR and the retrograde injection of the vector might have been more favorable to achieve infection and integration of the genes encoded by the vector. Finally, we surmised that saliva components in the duct system, primarily mucins and glycoproteins, may interfere with binding and uptake of the vector particles. By injecting a secretogogue, pilocarpine, before retrograde injection of the vectors, the gland was depleted of most of its secretory product. This approach was suggested by Sandberg and co-workers (1994; see also Henning 1995 ) in an attempt to overcome the mucous barrier to gene transfer to the epithelial cells in the intestine.

In A5-DAP cells the PLAP is membrane-bound, and the cells release the enzyme into the culture medium. Acinar cells transduced in vivo by DAP expressed heat-resistant AP in the apical membrane. These findings suggested that such cells may secrete AP into the saliva.

Rat submandibular saliva contains various levels of AP, a finding which is not well documented in the literature. In the few studies we were able to locate (Baer et al. 1967 ; Marchenko et al. 1973 ; Petrovich and Podorozhnaya 1973 ; Levitskii and Barabash 1974 ), enzyme assays were performed on mixed saliva obtained from the oral cavity. It appears that the level of AP in saliva is strain-dependent (Baer et al. 1967 ) and is influenced, among others factors, by the age of the rat (Marchenko et al. 1973 ) and by the diet (Baer et al. 1967 ; Petrovich and Podorozhnaya 1973 ). Our attempt to collect saliva from the SG after overnight fasting of the animals was not successful. Although the presence of AP in the saliva of untreated rats complicated evaluation of the possible secretion of PLAP by cells transduced by the viral vector, several lines of evidence point to such a secretion. The level of AP activity of saliva obtained from rats 11 days after retrograde injection of DAP was threefold higher compared to that of untreated rats. This difference was statistically significant. Higher levels of AP activity were also detected in saliva collected from the lobe of the SG that was injected with the vector compared to that which derived from the contralateral lobe. ELISA using a polyclonal antibody to human PLAP also revealed a higher average level of the enzyme in saliva after injection of DAP compared to that of control. The interpretation of the ELISA data is complicated, however, by lack of data on the crossreactivity of rat saliva AP with antibodies to human PLAP. Finally, although the AP activity of saliva of untreated rats was inactivated by heat (20 min at 65C), some enzyme activity of saliva obtained from virus-injected rats was heat-resistant. In addition, there was a difference in sensitivity to specific inhibitors of AP in saliva samples of control and virus-injected animals, further suggesting the presence of PLAP in the saliva of virus-injected rats. Because there were no obvious morphological changes in the glands of virus-injected rats, alteration in AP level is unlikely to be caused by pathological processes. Collectively, these data strongly suggest that a measurable amount of PLAP is secreted by the acinar cells of the SG gland after retrograde duct injection of DAP coding for the enzyme. Unequivocal proof of this, however, will require the purification and characterization of AP of rat submandibular saliva. Nevertheless, because a relatively large volume of saliva is secreted daily (Ship et al. 1991 ), even low levels of the product of a transduced gene can have physiological or therapeutic effects.

The data presented here further support the notion that retrograde duct injection of a retroviral vector is a feasible means of achieving transduction of acinar cells and long-term expression of the transduced gene in salivary glands.


  Acknowledgments

Support for this research was provided from grant DE11729 from the National Institute of Dental Research.

Received for publication February 12, 1997; accepted June 11, 1997.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Abu-Hasan NS, Sutcliffe RG (1985) Placental alkaline phosphatase integrates via its carboxyl-terminus into the microvillous membrane: its allotypes differ in conformation. Placenta 6:391-404 [Medline]

Arreaza G, Brown DA (1995) Sorting and intracellular trafficking of a glyosylphosphatidylinositol-anchored protein and two hybrid transmembrane proteins with the same ectodomain in Madin-Darby canine kidney epithelial cells. J Biol Chem 270:23641-23647 [Abstract/Free Full Text]

Baer PN, Hawkins GR, Wells H, Mantel N, Zipkin I (1967) Studies on experimental calculus formation in the rat. XI. Relation to diet and selected salivary constituents. J Periodontol 57:323-329

Barka T (1965) Induced cell proliferation: the effect of isoproterenol. Exp Cell Res 37:662-679

Barka T (1965) Stimulation of DNA synthesis by isoproterenol in the salivary glands. Exp Cell Res 39:355-364 [Medline]

Barka T (1970) Further studies on the stimulation of deoxyribonucleic acid synthesis in the submandibular gland by isoproterenol. Lab Invest 22:73-80 [Medline]

Barka T, van der Noen H (1976) Stimulated growth of submandibular gland. Lab Invest 35:507-514 [Medline]

Barka T, van der Noen H (1996) Retrovirus-mediated gene transfer into salivary glands in vivo. Hum Gene Ther 7:613-618 [Medline]

Baserga R (1966) Inhibition of stimulation of DNA synthesis by isoproterenol in submandibular glands of mice. Life Sci 5:2033-2039

Berger J, Howard AD, Gerber L, Cullen BR, Udenfriend S (1987) Expression of active, membrane-bound human placental alkaline phosphatase by transfected simian cells. Proc Natl Acad Sci USA 84:4885-4889 [Abstract]

Branchereau S, Calise D, Ferry N (1994) Factors influencing retroviral-mediated gene transfer into hepatocytes in vivo. Hum Gene Ther 5:803-808 [Medline]

Brown AM, Rusnock EJ, Sciubba JJ, Baum BJ (1989) Establishment and characterization of an epithelial cell line from the rat submandibular gland. J Oral Pathol Med 18:206-213 [Medline]

Brown DA, Crise B, Rose JK (1989) Mechanism of membrane anchoring affects polarized expression of two proteins in MDCK cells. Science 245:1499-1501 [Medline]

Brown DA, Rose JK (1992) Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to apical cell surface. Cell 68:533-544 [Medline]

Cepko C (1989) Lineage analysis in vertebrate nervous system by retrovirus-mediated gene transfer. Methods Neurosci 1:367-392

Cepko CL, Roberts BE, Mulligan RC (1984) Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell 37:1053-1062 [Medline]

Chang T-C, Wang J-K, Hung M-W, Chiao C-H, Tsai L-C, Chang G-G (1994) Regulation of the expression of alkaline phosphatase in human breast-cancer cell line. Biochem J 303:199-205 [Medline]

Fields-Berry SC, Halliday AL, Cepko CL (1992) A recombinant retrovirus encoding alkaline phosphatase confirms clonal boundary assignment in lineage analysis of murine retina. Proc Natl Acad Sci USA 89:693-697 [Abstract]

Fleming H, Begley M, Campi T, Condon R, Dobyns K, McDonagh J, Wallace S (1995) Induction of heat labile alkaline phosphatase by butyrate in differentiating endometrial cells. J Cell Biochem 58:509-516 [Medline]

Hayflick L (1973) Subculturing human diploid fibroblast cultures. In Kruse PF, Patterson MK, eds. Tissue Culture. Methods and Applications. New York, Academic Press, 220-223

Henning SJ (1995) Gene transfer into the intestinal epithelium. Adv Drug Deliv Rev 17:341-347

Hinegardner RT (1971) An improved fluorometric assay for DNA. Anal Biochem 39:197-201 [Medline]

Ikezawa H, Yamanegi M, Taguchi R, Miyashita T, Ohyabu T (1976) Studies on phosphatidylinositol phosphodiesterase (phospholipase C type) of Bacillus cereus. I. Purification, properties and phosphatase-releasing activity. Biochim Biophys Acta 450:154-164 [Medline]

Jemmerson R, Kiler FG, Fishman WH (1985) Clustered distribution of human placental alkaline phosphatase on the surface of both placental and cancer cells. J Histochem Cytochem 33:1227-1234 [Abstract]

Jensen H, Lyngbye J, Davidsen S (1968) Histochemical investigation of thermostable alkaline phosphatase in the normal full-term placenta. Acta Obstet Gynecol Scand 47:436-442 [Medline]

Kottel RH, Hanford WC (1980) Differential release of membrane-bound alkaline phosphatase isoenzymes from tumor cells by bromelain. J Biochem Biophys Methods 2:325-330 [Medline]

Levitskii AP, Barabash RD (1974) Sexual characteristics of the acid and alkaline phosphatase activity of the saliva and salivary glands of rats. Fiziol Zh 20:251-253 (in Ukrainian) [Medline]

Low MG (1989) Glycosyl-phosphatidylinositol: a versatile anchor for cell surface proteins. FASEB J 3:1600-1608 [Abstract/Free Full Text]

Low MG, Ferguson MAJ, Futerman AH, Silman I (1986) Covalently attached phosphatidylinositol as a hydrophobic anchor for membrane proteins. Trends Biochem Sci 11:212-215

Low MG, Stiernberg J, Waneck GL, Flavell RA, Kincade PW (1988) Cell-specific heterogeneity in sensitivity of phosphatidylinositolanchored membrane antigens to release by phospholipase C. J Immunol Methods 113:101-111 [Medline]

Low MG, Zilversmit DB (1980) Role of phosphatidyinositol in attachment of alkaline phosphatase to membranes. Biochemistry 19:3913-3918 [Medline]

Mann R, Mulligan RC, Baltimore D (1983) Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 33:153-159 [Medline]

Marchenko AI, Levytsky AP, Barabash RD (1973) Age changes in alkaline and acid phosphatase activity of saliva and salivary glands in rats. Ukr Biokhim Zh 45:307-311 (in Ukrainian) [Medline]

Mastrangeli A, O'Connell B, Aladib W, Fox PC, Baum BJ, Crystal RG (1994) Direct in vivo adenovirus-mediated gene transfer to salivary glands. Am J Physiol 266:G1146-G1155 [Abstract/Free Full Text]

Miller AD, Miller DG, Garcia JV, Lynch CM (1993) Use of retroviral vectors for gene transfer and expression. Methods Enzymol 217:581-599 [Medline]

Miller DG, Adam MA, Miller AD (1990) Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 10:4239-4242 [Medline]

Neale FC, Clubb JS, Hotchkis D, Posen S (1965) Heat stability of human placental alkaline phosphatase. J Clin Pathol 18:359-363 [Medline]

Omnell K-A, Qwarnström EE (1983) A technique for intraoral cannulation and infusion of the rat submandibular gland. Dentomaxillofac Radiol 12:13-15 [Medline]

Petrovich YA, Podorozhnaya RP (1973) Influence of a sucrose diet on the aminotransferase and phosphatase activity in the saliva of rats of different age. Voprosy Pitaniia 32:54-56 (in Russian) [Medline]

Posen S, Cornish CJ, Horne M, Sainin PK (1969) Placental alkaline phosphatase and pregnancy. Ann NY Acad Sci 166:733-741 [Medline]

Sandberg JW, Lau C, Jacomino M, Finegold M, Henning SJ (1994) Improving access to intestinal stem cells as a step toward intestinal gene transfer. Hum Gene Ther 5:323-329 [Medline]

Schneyer CA (1969) ß-adrenergic effects by autonomic agents on mitosis and hypertrophy in rat parotid. Proc Soc Exp Biol Med 131:71-75

Schuitemaker H, Kootstra NA, Fouchier RAM, Hooibrink B, Miedema F (1994) Productive HIV-1 infection of macrophages restricted to the cell fraction with proliferative capacity. EMBO J 13:5929-5936 [Abstract]

Selye H, Veilleux R, Cantin M (1961) Excessive stimulation of salivary gland growth by isoproterenol. Science 133:44-45

Ship JA, Fox PC, Baum BJ (1991) How much saliva is enough? "Normal" function defined. J Am Dent Assoc 122:63-69 [Medline]

Springett GM, Moen RC, Anderson S, Blaese RM, Anderson WF (1989) Infection efficiency of T lymphocytes with amphotropic retroviral vectors is cell cycle dependent. J Virol 63:3865-3869 [Medline]

Stigbrand T (1984) Present status and future trends of human alkaline phosphatase. Prog Clin Biol Res 166:3-14 [Medline]

Telfer JF, Green CD (1993) Placental alkaline phosphatase activity is inversely related to cell growth rate in HeLaS3 cervical cancer cells. FEBS Lett 329:238-244 [Medline]

Tokumitsu S-I, Tokumitsu K, Fishman WH (1981) Immunocytochemical demonstration of intracytoplasmic alkaline phosphatase in HeLa TCRC-1 cells. J Histochem Cytochem 29:1080-1087 [Abstract]