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

Abstract

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

Passive Immunotherapy of Mice Bearing Ehrlich Ascites Tumor Expressing Human, Membrane-Bound Placental Alkaline Phosphatase

“…Previously, we have transduced A5 cells, a cell line of rat submandibular gland origin, with DAP, and documented the membrane localization of PLAP in A5-DAP cells. The enzyme could be released from A5-DAP cells by treatment with bromelain or phosphatidylinositol-specific phospholipase C. Histochemical staining for heat-resistant AP also indicated the membrane localization of the enzyme [12]. Although in our experiments we have used the DAP vector, it is conceivable to design any number of vectors coding for a membrane protein.…”

“…Producer "2 DAP cells were cultured as described [12]. The virus stock was concentrated by centrifugation [13], and the virus particles were suspended in complete culture medium.…”

“…The histochemical staining was performed as described [12]. For heat treatment, the slides, after fixation, were placed in PBS at 65°C for 20 min.…”

Effect of sodium butyrate on the expression of genes transduced by retroviral vectors

“…A5-DAP cell line was established from A5 cells infected with the retroviral vector DAP and selecting the neomycin (G418) resistant cells. A5-DAP cells express heat-resistant, membrane-bound PLAP [Barka and van der Noen, 1997]. A5 cells also reveal low levels of alkaline phosphatase activity which is heat-sensitive, tissue nonspecific type.…”

“…The role of histone deacetylase in gene regulation has been further confirmed using specific inhibitors, trichostatin A and trapoxin, of the enzyme [Yoshida et al, 1995]. Previously, we have described the transduction of A5 cells, a rat submandibular glandderived cell line, by the retroviral vectors BAG [Barka and van der Noen, 1996] and DAP [Barka and van der Noen, 1997]. These vectors code for Escherichia coli ␤-galactosidase (␤-gal) [Cepko et al, 1984] and human placental alkaline phosphatase (PLAP) [Fields-Berry et al, 1992], respectively, under the control of Moloney (Mo) MuLV long terminal repeat (LTR) enhancer-promoter.…”