Proc. Natl. Acad. Sci. USA Vol. 76, No. 3, pp. 1323-1327, March 1979
Cell Biology
Inducers of mammalian cell differentiation stimulate dome formation in a differentiated kidney epithelial cell line (MDCK) (sodium butyrate/cyclic AMP/cryoprotective solvents/purines/renal transepithelial transport)
JULIA E. LEVER The Salk Institute, P.O. Box 1809, San Diego, California 92112
Communicated by Renato Dulbecco, December 26, 1978
ABSTRACT Cell cultures of a differentiated kidney epithelial cell line, MDCK, spontaneously form fluid-filled domes or hemicysts composed of numbers of cells as a manifestation of specialized epithelial transport phenomena. Addition to MDCK cells of a broad spectrum of compounds that are known as potent inducers of mammalian cell differentiation in cell culture caused a striking increase in the frequency of dome formation. Polar compounds such as NN-dimethylformamide, dimethyl sulfoxide, or hexamethylene bisacetamide stimulated increased dome formation 15-30 hr after addition. Induction of domes by these compounds was prevented either by inhibitors of protein synthesis or by ouabain, cytochalasin B, or vinblastine. Inhibition of DNA synthesis did not block chemical induction of domes. Other inducers were compounds of physiological occurrence such as n-butyrate or adenosine. Furthermore, a variety of conditions expected to elevate intracellular levels of cyclic AMP also stimulated dome formation. These findings suggest the hypothesis that domes are formed in cell culture by a form of cell differentiation that is under positive control by cyclic AMP.
The epithelial cell line MDCK (Madin-Darby canine kidney), established in 1958 from whole normal dog kidney (1), offers considerable promise as a useful model system to study renal transepithelial transport in cell culture (2). Cultures of MDCK cells form a continuous sheet of cells exhibiting the asymmetrical morphological polarity and occluding junctions that are characteristic of renal tubular epithelium (2-4). Furthermore, electrophysiological studies have demonstrated that these cultures retain the transport and permeability properties of the corresponding intact tissue (2). Fluid-filled, multicellular hemicysts known as domes form spontaneously in confluent MDCK cell cultures as a manifestation of transepithelial transport phenomena (3). Domes result from fluid accumulation between the cell layer and the culture dish (3). This property is not unique to MDCK cells but also characterizes epithelial cell cultures from diverse types of fluid-transporting epithelia (5-11). The present study indicates that induction of domes in MDCK cells may represent a novel example of induction of differentiation in a cell culture system. A broad spectrum of compounds known as inducers of differentiation in other cell culture systems, such as Friend erythroleukemia cells (12-15) and neuroblastoma (16, 17), dramatically increased both the size and frequency of dome formation in MDCK cell cultures. Dome formation in MDCK cells was also increased by conditions expected to elevate intracellular levels of cyclic AMP, in confirmation of preliminary findings of Valentich et al. (18). These data suggest the hypothesis that domes arise in cell culture by a process that bears a striking analogy to cell differentiation and is regulated by cyclic AMP.
MATERIALS AND METHODS Cell Culture. The MDCK cell line, isolated from whole normal adult dog kidney (1), the BSC-1 kidney epithelial line (19), and the BALB/c 3T3 nontransformed mouse fibroblast line (20) were obtained through the courtesy of Robert Holley, Salk Institute. A cell line designated MDCK clone 4, which exhibited enhanced spontaneous dome-forming capacity, was isolated from the MDCK cell line by single-cell plating and colony purification. Frozen aliquots of cells suspended in complete medium plus 20% serum with 10% (vol/vol) dimethylsulfoxide were stored in liquid N2. MDCK cells were propagated in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, nonessential amino acids, hypoxarlthine, putrescine, biotin, lipoic acid, vitamin B12, ascorbic acid, glutathione, p-aminobenzoic acid, trace metals, and linoleic acid (21) in an atmosphere of 10% CO2 in air at 37°C. Growth medium of 3T3 cells and BSC-1 cells was Dulbecco's modified Eagle's medium plus 10% calf serum. Compounds tested as inducers of dome formation were added with medium change to confluent cell cultures on 35-mm dishes. Quantitation of Domes. Cultures were fixed and stained under conditions that preserved domes. The medium was removed by aspiration and a cold solution of 5% glutaraldehyde in phosphate-buffered saline was added to the culture dish for 15 min at room temperature. This was removed, and the cells were stained with Giemsa. Stained dishes were examined with a stereo dissection microscope (Zeiss). The number of domes was counted on duplicate dishes by using 8-20 fields each of 0.07 cm2 area. Larger numbers of fields were examined for cultures with an average of less than 70 domes per cm2. Results are expressed as the mean number of domes per microscope field ± SD. Materials. Prostaglandin El was a generous gift from John Pike (Upjohn), and hexamethylene bisacetamide was kindly donated by P. Marks (Columbia University). Cholera toxin was purchased from Schwarz/Mann. Spectroquality-grade dimethyl sulfoxide was from Mallinckrodt, 1-methyl-2-pyrrolidinone was from Eastman, and N-methylacetamide and cytochalasin B were from Aldrich. Sodium butyrate was prepared by neutralizing n-butyric acid (Fisher). All other compounds were from Sigma. RESULTS Spontaneous Incidence of Domes. Confluent polygonal monolayer cultures of MDCK cells exhibited a low (averaging less than 10 domes/cm2) spontaneous incidence of domes (Fig. 1A). Domes were composed of numbers of cells and varied in size from 70 to 500 Am in diameter. Domes were not randomly dispersed over the cell layer but usually occurred in definite patches. Subconfluent MDCK cell cultures did not form domes.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate
this fact.
1323
~ ~ ~*I
'f'~~~~~~~
Proc. Natl. Acad. Sci. USA 76 (1979)
Cell Biology: Lever
1324
'4' / 4' ~~~~~~~~~~4
O,-.4
r
esi s
S Sb.< 11
t
at
p
'' 44vgr.,
aS S A -4 4s4 sS w ,. d2 -,,>s.,
' ' Ja ;' #
qe*S>
< }4S
^EfsI
r R i $/~~ j > ) C
3,ss pA*,
.t
;
< t
1wo t ; (8
4
o w
i
g
Cell Biology
Inducers of mammalian cell differentiation stimulate dome formation in a differentiated kidney epithelial cell line (MDCK) (sodium butyrate/cyclic AMP/cryoprotective solvents/purines/renal transepithelial transport)
JULIA E. LEVER The Salk Institute, P.O. Box 1809, San Diego, California 92112
Communicated by Renato Dulbecco, December 26, 1978
ABSTRACT Cell cultures of a differentiated kidney epithelial cell line, MDCK, spontaneously form fluid-filled domes or hemicysts composed of numbers of cells as a manifestation of specialized epithelial transport phenomena. Addition to MDCK cells of a broad spectrum of compounds that are known as potent inducers of mammalian cell differentiation in cell culture caused a striking increase in the frequency of dome formation. Polar compounds such as NN-dimethylformamide, dimethyl sulfoxide, or hexamethylene bisacetamide stimulated increased dome formation 15-30 hr after addition. Induction of domes by these compounds was prevented either by inhibitors of protein synthesis or by ouabain, cytochalasin B, or vinblastine. Inhibition of DNA synthesis did not block chemical induction of domes. Other inducers were compounds of physiological occurrence such as n-butyrate or adenosine. Furthermore, a variety of conditions expected to elevate intracellular levels of cyclic AMP also stimulated dome formation. These findings suggest the hypothesis that domes are formed in cell culture by a form of cell differentiation that is under positive control by cyclic AMP.
The epithelial cell line MDCK (Madin-Darby canine kidney), established in 1958 from whole normal dog kidney (1), offers considerable promise as a useful model system to study renal transepithelial transport in cell culture (2). Cultures of MDCK cells form a continuous sheet of cells exhibiting the asymmetrical morphological polarity and occluding junctions that are characteristic of renal tubular epithelium (2-4). Furthermore, electrophysiological studies have demonstrated that these cultures retain the transport and permeability properties of the corresponding intact tissue (2). Fluid-filled, multicellular hemicysts known as domes form spontaneously in confluent MDCK cell cultures as a manifestation of transepithelial transport phenomena (3). Domes result from fluid accumulation between the cell layer and the culture dish (3). This property is not unique to MDCK cells but also characterizes epithelial cell cultures from diverse types of fluid-transporting epithelia (5-11). The present study indicates that induction of domes in MDCK cells may represent a novel example of induction of differentiation in a cell culture system. A broad spectrum of compounds known as inducers of differentiation in other cell culture systems, such as Friend erythroleukemia cells (12-15) and neuroblastoma (16, 17), dramatically increased both the size and frequency of dome formation in MDCK cell cultures. Dome formation in MDCK cells was also increased by conditions expected to elevate intracellular levels of cyclic AMP, in confirmation of preliminary findings of Valentich et al. (18). These data suggest the hypothesis that domes arise in cell culture by a process that bears a striking analogy to cell differentiation and is regulated by cyclic AMP.
MATERIALS AND METHODS Cell Culture. The MDCK cell line, isolated from whole normal adult dog kidney (1), the BSC-1 kidney epithelial line (19), and the BALB/c 3T3 nontransformed mouse fibroblast line (20) were obtained through the courtesy of Robert Holley, Salk Institute. A cell line designated MDCK clone 4, which exhibited enhanced spontaneous dome-forming capacity, was isolated from the MDCK cell line by single-cell plating and colony purification. Frozen aliquots of cells suspended in complete medium plus 20% serum with 10% (vol/vol) dimethylsulfoxide were stored in liquid N2. MDCK cells were propagated in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, nonessential amino acids, hypoxarlthine, putrescine, biotin, lipoic acid, vitamin B12, ascorbic acid, glutathione, p-aminobenzoic acid, trace metals, and linoleic acid (21) in an atmosphere of 10% CO2 in air at 37°C. Growth medium of 3T3 cells and BSC-1 cells was Dulbecco's modified Eagle's medium plus 10% calf serum. Compounds tested as inducers of dome formation were added with medium change to confluent cell cultures on 35-mm dishes. Quantitation of Domes. Cultures were fixed and stained under conditions that preserved domes. The medium was removed by aspiration and a cold solution of 5% glutaraldehyde in phosphate-buffered saline was added to the culture dish for 15 min at room temperature. This was removed, and the cells were stained with Giemsa. Stained dishes were examined with a stereo dissection microscope (Zeiss). The number of domes was counted on duplicate dishes by using 8-20 fields each of 0.07 cm2 area. Larger numbers of fields were examined for cultures with an average of less than 70 domes per cm2. Results are expressed as the mean number of domes per microscope field ± SD. Materials. Prostaglandin El was a generous gift from John Pike (Upjohn), and hexamethylene bisacetamide was kindly donated by P. Marks (Columbia University). Cholera toxin was purchased from Schwarz/Mann. Spectroquality-grade dimethyl sulfoxide was from Mallinckrodt, 1-methyl-2-pyrrolidinone was from Eastman, and N-methylacetamide and cytochalasin B were from Aldrich. Sodium butyrate was prepared by neutralizing n-butyric acid (Fisher). All other compounds were from Sigma. RESULTS Spontaneous Incidence of Domes. Confluent polygonal monolayer cultures of MDCK cells exhibited a low (averaging less than 10 domes/cm2) spontaneous incidence of domes (Fig. 1A). Domes were composed of numbers of cells and varied in size from 70 to 500 Am in diameter. Domes were not randomly dispersed over the cell layer but usually occurred in definite patches. Subconfluent MDCK cell cultures did not form domes.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate
this fact.
1323
~ ~ ~*I
'f'~~~~~~~
Proc. Natl. Acad. Sci. USA 76 (1979)
Cell Biology: Lever
1324
'4' / 4' ~~~~~~~~~~4
O,-.4
r
esi s
S Sb.< 11
t
at
p
'' 44vgr.,
aS S A -4 4s4 sS w ,. d2 -,,>s.,
' ' Ja ;' #
qe*S>
< }4S
^EfsI
r R i $/~~ j > ) C
3,ss pA*,
.t
;
< t
1wo t ; (8
4
o w
i
g
