In Vitro Cell. Dev. Biol.--Animal 36:287-289, May 2000 �9 2000 Society for In Vitro Biology 1071-2690/00 $05.00+0.00

Letter to the Edi tor

ISOLEUCINE PREVENTS RAT SALIVARY GLAND EPITHELIAL CELLS FROM APOPTOSIS IN SERUM-FREE CULTURE

Dear Editor: We have studied an amino acid requirement of primary rat sub-

mandibular gland-derived epithelial cells (RSMG) in serum-free cell culture. We have recently reported the establishment of rat submandibular gland epithelial cells designated as RSMG-1 in se- rum-free cell culture to study the mechanisms of morphogenesis in salivary gland regeneration. In glandular organs such as subman- dibular glands (SMG), proper branching during morphogenesis is essential to maintain organ function. We have also revealed hepa- tocyte growth factor-induced branching morphogenesis of RSMG-1 cells in three-dimensional serum-free collagen gel culture (Furue et al., 1999), suggesting that RSMG-1 cell line may be useful for the study of the salivary gland regeneration. During the establish- ment of RSMG-1 cells, we have found that addition of isoleucine in the culture medium promoted the cell proliferation and survival of RSMG-1 cells. Sato et al. (1994) developed a serum-free medium, MCDB 152 supplemented with four factors (4F; insulin, transferrin, 2-mercaptoethanol, 2-aminoethanol) and fibroblast growth factor-1 (FGF-1), for the growth of mouse submaxillary gland epithelial ceils. However, we failed to subculture RSMG cells more than three times, because most of the cells finally died, exhibiting a more rounded and blebed cytoplasmic membrane (Fig. 1A and B). RSMG cells could be maintained in MCDB 153 HAA, which contains increased amounts of several amino acids when compared to the MCDB 153 medium (Shipley and Pittelkow, 1987). Thus, we have speculated that some kind of amino acid contained in MCDB 153 HAA may

be important for maintaining the cell viability and proliferation. Then, we examined a requirement of these amino acids for primary- cultured RSMG cells. Primary explant culture of RSMG cells was carried out as described previously (Fume and Saito, 1997, 1998; Furue et al., 1999). In brief, SMG excised from 10-wk-old Wistar female rats (Japan SLC, Inc., Shizuoka, Japan) were cut, minced, and seeded in 60-mm plastic dishes (Sumitomo Bakelite, Co., To- kyo, Japan) coated with type 1 collagen (Nitta Gelatin Co., Osaka, Japan). The cells were cultured in a humidified atmosphere of 5% C02 at 37 ~ C in MCDB 153 medium (Kyokuto Pharmaceutical Co., Tokyo, Japan) supplemented with 4F and human recombinant FGF- 1 (Upstate Biotechnology, Inc., Lake Placid, NY) at 1 ng/ml. The 4F (Fume et al., 1994) consisted of bovine insulin at 10 ~g/ml, human transferrin at 5 ~g/ml, 10 IxM of 2-mercaptoethanol, and 10 ~M of 2-aminoethanol (all from Sigma Chemical Co., St. Louis, MO). Each factor was prepared as a sterile 100x concentrate and stored at 4 ~ C. We added 0.11 g sodium pyruvate (Sigma) per L, 1.2 g sodium bicarbonate (HEPES) (Wako Chemicals, Tokyo, Japan) per L, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (Wako), 90 mg viccillin (Meiji Seika, Tokyo, Japan) per L, and 90 mg kanamycin (Meiji Seika) per L in MCDB 153 medium. RSMG ceils started to proliferate in MCDB 153 + 4F + FGF-1 between 3 and 10 d after starting the explant cultures of SMG from 10-wk-old female Wistar rats (Fig. 1). The cells became subcon- fluent between 14 and 21 d without fibroblast growth in this serum- free medium. Before reaching confluence, the cells were trypsinized

FIG. 1. Phase contrast photomicrographs of RSMG cells. (A) RSMG ceils at primary culture from the explant specimens on day 7 when cultured in MCDB 153 supplemented with 4F and FGF-1. (B) RSMG cells at the third passage showing rounded and blebed cytoplasmic membrane. (C) RSMG cells cultured in MCDB 153 supplemented with isoleucine (0.75 raM), 4F and FGF-1. Actively proliferating RSMG ceils. Magnification: A, B, C, X40.

287

288 FURUE ET AL.

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FIG. 2. Requirement of various amino acids for the RSMG cell growth in serum-free medium. The cells were seeded in a 24-well plate (Sumitonm) coated with type I collagen at the density of 1 • 10 ~ cells/well in MCDB 153 medium supplemented with the 4F and FGF-1 (1 ng/ml), with and with- out addition of each amino acid indicated. (A) Requirement of amino acid; tryptophan (0.06 mM), phenylalanine (0.12 raM), histidine (0.32 mM), iso- leucine (0.75 raM), tyrosine (0.09 raM), or methionine (0.12 mM) were added to each well and cultured for 6 d. The cells were also cultured in MCDB 153 HAA medium + 4F + FGF-1 (1 ng/ml) for 6 d. (B) Effect of various concentrations of isoleucine on RSMG cell growth in serum-free medium. Isoleucine at the final concentration of 0.015, 0.016, 0.02, 0.05, 0.1, 0.75, or 1.5 mM was added to each well and cultured for 6 d. Values are the mean -+ SEM for three measurements.

with 0.05% trypsin (Difco Laboratories, Detroit, MI) in 0.04% eth- ylenediamine-tetraacetic acid (EDTA) (Sigma). The trypsin was then inactivated with 0.1% soybean trypsin inhibitor (Sigma). The cell numbers were counted with a Coulter particle counter (Coulter Elec- tronics, Hialeah, FL). Ceils at the second passages were used in all the experiments. The MCDB 153 HAA (Kyokuto) used for this study contained the following amino acids at the increased amounts of amino acids; tryptophan (0.06 mM), phenylalanine (0.12 mM), his- tidine (0.32 raM), isoleucine (0.75 raM), tyrosine (0.09 mM), or methionine (0.12 mM). The cells were cultured for 6 d; then, the

FIG. 3. DNA analysis by 1% agarose gel electrophoresis of the DNA extracted from RSMG cells. The cells cultured in MCDB 153 + 4F + FGF- 1 or MCDB 153 + 4F + FGF-1 supplemented with isoleucine were harvested and lysed in a lysis buffer (10 mM Tris-HC1, pH 8.0, 10 nrM NaC1, 10 n~/ EDTA, proteinase K [100 Ixg/ml], 1% sodium dodecyl sulfate) and further incubated at 37 ~ C until the mixture became clear. Then, DNA was extracted by phenol/chloroform (1:1) method, and precipitated overnight at -20 ~ C in ethanol containing 0.3 M Na-acetate. After centrifugation, the pellet was resuspended in TE buffer (0.1 M Tris-HC1, pH 8.0, 10 mM EDTA). Ribo- nuclease-treated (Sigma) DNA was reextracted with phenol/chloroform, pre- cipitated with ethanol, and resuspended in TE. DNA samples (0.2 txg) were electrophoretically separated on 1% agarose gel. After electrophoresis, the DNA was stained with ethidium bromide (1 Ixg/ml), and visualized by an ultraviolet transilluminator and photographed with a Polaroid camera. Lane i, DNA extracted from RSMG cells cultured in MCDB 153 + 4F + FGF-1 supplemented with 0.75 mM isoleucine. Lane 2, DNA extracted from RSMG cells cultured in MCDB 153 + 4F + FGF-1. M; DNA molecular weight marker X (Boehringer Mannheim GmbH Biochemiea, Mannheim, Germany).

cell numbers were counted with a Couher particle counter. Addition

of isoleucine to MCDB 153 medium promoted the growth of RSMG ceils suggesting isoleucine is critical for the growth (Fig. 2A). Fur- thermore, the effect of various concentrations of isoleucine on the RSMG cell growth has been studied. The stimulatory effect of iso- leucine was dose-dependent and the concentration of 0.75 mM iso- leucine indicated the maximal cell growth (Fig. 2B). Addition of increased amount of isoleucine prevented RSMG cells from dying (Fig. 1C) and we could serially subculture RSMG cells in the se- rum-free medium. Furthermore, we studied whether addition of iso- leucine in the serum-free medium could prevent deoxyribonucleic acid (DNA) fragmentation in RSMG ceils. We extracted DNA from the RSMG cells cultured in MCDB 153 + 4F + FGF-1 and MCDB 153 + 4F + FGF-1 supplemented with 0.75 mM isoleucine. An agarose gel electrophoresis of DNA extracted from RSMG ceils cul- tured in MCDB 153 + 4F + FGF-1 supplemented with isoleucine showed that there was no DNA fragmentation (Fig. 3, lane 1). On the other hand, DNA from the RSMG cells cultured in MCDB 153 + 4F + FGF-1 showed a nucleosomal ladder, demonstrating that

DNA fragmentations were generated in the cells (Fig. 3, lane 2). These results indicate that RSMG cells cuhured without addition

SERUM-FREE CULTURE OF RAT EPITHELIAL CELLS 289

of isoleucine to MCDB 153 died due to the apoptosis and that isoleucine prevented the cells from apoptosis.

Culture media for a number of individual types of cells have been improved through the optimization of the compositions of basal nu- trient media and replacement of serum with purified proteins and nonprotein supplements. Ham and his colleagues (Tsao et al., 1982; Boyce and Ham, 1983) studied to refine further the culture condi- tion originally reported by Rheinwald and Green (1975) for the clonal growth of keratinocytes by optimizing nutrient and growth factor concentrations and adding trace elements such that feeder cells were eliminated and serum was replaced by bovine pituitary extract. They reported that this improved basal medium containing higher concentrations of nutrients, MCDB 153, were useful in main- taining a higher density culture of keratinocytes. In this study, we have studied the effect of six kinds of amino acids, individually, on the growth of RSMG cells in MCDB 153 medium supplemented with the 4F and FGF-1. It was revealed that isoleucine was the most important among the amino acids studied, and its optimal concentration was 0.75 mM. Sato et al. (1994) reported that the epithelial cells derived from the mouse submaxillary gland (SMG) were serially maintained and subcuhured in serum-free MCDB 152 medium containing 0.015 mM isoleucine supplemented with the 4F and FGF-1. These findings suggested that primary rat SMG-derived epithelial ceils required relatively high amounts of isoleucine for their optimal growth compared to mouse SMG-derived epithelial cells. Oku et al. (1994) reported that the optimal concentration of isoleucine was 0.05 nrM in the culture medium for rat epidermal keratinocyte, indicating that the optimal concentration of isoleucine in the culture medium may be cell-type specific. In this study, we have found that the addition of isoleucine prevented the RSMG cells from apoptosis. It has been reported that nutrient-deficient medium causes cell cycle arrest in various cell types, including human ke- ratinocytes (Pittelkow et al., 1986; Kobayashi et al., 1998). Human keratinocytes were synchronized by transferring the cells to MCDB 153 medium without isoleucine. These findings suggest that isoleu- cine might be a key factor for the cell growth.

In conclusion, with the addition of isoleucine, at the optimal concentration, to MCDB 153, RSMG ceils could be serially sub- cultured. This culture method may be useful to study the growth and differentiation of rat salivary glands.

REFERENCES

Boyce, S.; Ham, R. G. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and se- rmn free serial culture. J. Investig. Dermatol 81:33s-40s; 1983.

Furue, M.; Okamoto, T.; Hayashi, H., et al. Effects of hepatocyte growth factor (HGF) and activin A on the morphogenesis of rat submandibular gland-derived epithelial cells in serum-free collagen gel culture. In Vitro Cell. Dev. Biol. 35:131-135; 1999.

Furue, M.; Okamoto, T.; Ikeda, M., et al. Primitive neuroectodermal tumor cell lines derived from a metastatic pediatric tumor. In Vitro Cell. Dev. Biol. 30:813-816; 1994.

Furue, M.; Saito, S. Synergistic effect of hepatocyte growth factor and fibro- blast growth facotr-1 on the branching morphogenesis of rat subman- dibular gland epithelial cells. Tissue Culture Res. Commun. 16:189- 194; 1997.

Furue, M.; Saito, S. Hepatocyte growth factor regulates activin [~A mRNA in submandibular gland. In Vitro Cell. Dev. Biol. 34:520-523; 1998.

Kobayashi, T.; Okumura, H.; Hashimoto, K., et al. Synchronization of normal human keratinocyte in culture: its application to the analysis of 1,25- dihydroxyvitamin D3 effects on cell cycle. J. Dermatol. Sci. 17:108- 114; 1998.

Oku, H.; Kumamoto, C.; Miyagi, T., et al. Serum-free culture of rat kerati- nocytes. In Vitro Cell. Dev. Biol. 30A:496--503; 1994.

Pittelkow, M. R.; Wille, J. J.; Scott, R. E. Two functionally distinct classes of growth arrest states in human prokeratinocytes that regulate clon- ogenic potential. J.Investig. Dermatol. 86:410417; 1986.

Rheinwald, J. G.; Green, H. Serial cultivation of strains of human epidermal keratinocytes: the formation of kcratinizing colonies from single cells. Cell 6:331-343; 1975.

Sato, J. D.; Hayashi, I.; Hayashi, J., et al. Specific cell types and their re- quirements. In: Davis, J. M., ed. Basic cell culture, a practical ap- proach. Oxford: Oxford University Press; 1994:181-222.

Shipley, G. D.; Pittelkow, M. R. Control of growth and differentiation in vitro of human prokeratinocytes cultured in serum-free medium. Arch. Dermatol. 123:1541-1544; 1987.

Tsao, M. C.; Wahhall, B. J.; Ham, R. G. Clonal growth of normal human epidermal keratinocytes in a defined medium. J. Cell Physiol. 110: 219-229; 1982.

Miho Furue 1 Shinri Koshika Tetsuji Okamoto Makoto Asashima

Department of Biochemistry Kanagawa Dental College

82 Inaoka-cho Yokosuka City, Kanagawa 238-8580

Japan (M. F., S. K.)

Department of Oral and Maxillofacial Surgery I Hiroshima University School of Dentistry

1-2-3 Kasumi, Hiroshima 734-0037 Japan (T. O.)

Department of Life Sciences (Biology) Graduate School of Arts and Sciences

The University of Tokyo 3-8-1 Komaba

Meguro-ku, Tokyo 153-0041 Japan (M. A.)

(Received 21 October 1999)

1To whom correspondence should be addressed at E-mail: mihofuru @kdcnet.ac.jp