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c 3T3 fibroblasts and madin-darby canine kidney epithelial cells
Copyright @ IYXI by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4X27/811120263-08%o?.OO10
Experimental
GROWTH
EFFECTS
FIBROBLASTS
Cell Research 136 (1981) 263-270
OF LITHIUM
CHLORIDE
AND MADIN-DARBY EPITHELIAL
IN BALB/c
CANINE
3T3
KIDNEY
CELLS
SUSANNA M. RYBAK and FRANK E. STOCKDALE Depurtments ofMedicine und Biology, Stunford University. Stunford, CA 94305, USA
SUMMARY The ability of LiCl to initiate DNA synthesis was studied in Madin-Darby canine kidney (MDCK) cells, and mouse BALB/c 3T3 fibroblasts. In a defined culture medium lacking serum, LiCl increased DNA synthesis in BALB/c 3T3 cells lOO-200% over control values. Maximum DNA synthesis was observed with concentrations of LiCl between 10 and 2.5 mM and increases from 40-50% over control were observed with concentrations as low as 1 mM. Exposure of BALB/c 3T3 cultures to LiCl resulted in an increase in the percentage of cells initiating DNA synthesis, total DNA content and cell number. Lithium chloride, in combination with insulin or epidermal growth factor (EGF), had either an additive or synergistic effect upon the growth of BALB/c 3T3 fibroblasts. MDCK cells proved refractory to the growth actions of LiCI, although they responded to EGF and insulin with increased DNA synthesis. Lithium chloride appears to have a direct effect on cell proliferation in some but not all cell types.
The concentration of free divalent cations ported here demonstrate that an epithelial [ 1, 21 as well as monovalent cation fluxes phenotype is not the sole requirement for [3, 41 has been proposed to mediate the LiCl-induced cell proliferation since BALB/ biochemical events which culminate in cell c 3T3 fibroblasts are stimulated to grow by division. Lithium, a monovalent cation this cation, whereas Madin-Darby canine which has a variety of physiological, bio- kidney (MDCK) epithelial cells are not. chemical and biological effects on adult and embryonic tissues [5-71, recently has been shown to stimulate the growth of MATERIALS AND METHODS murine mammary epithelium, both in organ Cell culture [8], and in primary cell culture [9]. These Early passage stock cells were generously supplied studies demonstrate a direct action of LiCl by the following investigators: BALB/c 3T3 cells were on initiation of DNA synthesis in epithelial obtained from Dr Stuart Aaronson at the National Institutes of Health and MDCK cells were obtained cells. In other cells LiCl promotes growth from the laboratory of Dr Dayton Misfeldt at Stanford University. The cell lines were grown over sevonly in the presence of growth factors [lo]. eral passages, aliquots were frozen and new cultures Studies on granulocyte precursors suggest initiated from these frozen stocks approximately every two months. BALB/c 3T3 cells were cultured in Dulthat the mitogenic effects of LiCl may be becco’s Modified Eagle Medium (DME) which conrestricted to epithelial cell types since these tained 5 % fetal bovine serum (FBS) and 1% penicillin-streptomycin and fungizone. MDCK cells were culcells of mesenchymal origin do not directly tured in Waymouth’s medium which contained 10% respond to LiCl [l 11. The experiments re- FBS and 1% penicillin-streptomycin. All cell culture IX-81
1801
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264
Rybak and Stockdale
reagents were obtained from Gibco. The cells were maintained in 75 cm2 flasks and transferred to multiwells or plastic tissue culture plates for the growth experiments. Tissue culture dishes and flasks were from Falcon. In all experiments cells were plated at 2.5~ 10“ cells/cm* in 60 mm dishes (unless otherwise indicated in legends) for 24 h in medium containing the prescribed amount of serum for the particular cell type. At 24 h the cultures were washed hr serumfree medium and incubated an additional 24 h in the appropriate defined medium plus bovine serum albumin (BSA, 5 pg/ml). Lithium chloride or peptide growth factors were then added to fresh defined medium with BSA and the cultures were incubated for 24-48 h before performing an experiment.
Growth assays synthesis. [3H]Thymidine incorporation into acid-soluble or -insoluble material was measured as previously described [9]. The cultures were incubated for 2 h with culture medium containing 2 &i/ml of [methylJH]thymidine (New England Nuclear, sp. act. 20 Cilmmol). Acid-soluble radioactivitv was extracted from’the cells after 2 washes with Hanks’ balanced salt solution (HBSS) by incubating them with 5 % irichloroacetic acid (TCA, w : v). An aliquot of the TCA extract was taken for liquid scintillation counting. The acid-insoluble material was washed with 5% TCA and dieested with 0.5 N NaOH and counted in a liquid sciitillation counter. Data reported as percent stimulation were obtained as follows: (experimental cpmlcontrol cpm - 1.00~ 100). The DNA content of cells was determined by fluorometric assay as described by Hinegardner [ 121.
DNA
Labelling index Cultures were plated into 60 mm dishes and incubated with LiCl or other additions as previously described under “Cell culture”. The cultures were then pulsed for 2 h with [3H]thymidine (2 &i/ml), washed with HBSS, extracted with 5% TCA, fixed with 70% ethanol and air-dried. Fixed cultures were layered with llford liquid emulsion and exposed in the dark for 35 days.
RNA synthesis Lithium chloride-treated and control cell cultures were pulsed with 2 &i/ml [3H]uridine (New England Nuclear, sp. act. 22 Ci/mmol) for 2 h. Uridine incorporation into RNA was then determined by processing the cells as described for [aH]thymidine incorporation into DNA.
Protein synthesis Protein synthesis was determined by pulsing LiCltreated and control cultures for 2 he\h;ith 5 -&X/ml rSHlleucine (SchwarzlMann, sp. act. 59 Cilmmol) in Waymouth’s. medium. Acid-soluble counts were obtained as previously described under “DNA synthesis”. Acid-insoluble counts were obtained by scraping the cells from the dish, precipitating the protein E.rp Cd Rrs 136 (198/j
with hot 5% TCA, and collecting the precipitate in glass fiber filters (Whatman, GF/c). The filters were washed successively with 95% ethanol and acetone, solubilized in NCS solubilizer and counted with a toluene-based scintillant as previously described [ 131.
RESULTS The effects of lithium chloride on [3H]thymidine incorporation in BALBlc 3T3 cells The effects of LiCl on the incorporation of [3H]thymidine into DNA in cultures of BALB/c 3T3 cells is depicted in fig. 1. BALB/c 3T3 cell cultures were deprived of serum for 24 h before LiCl was added to the culture medium. Incorporation of C3H]thymidine was measured 20 h later. In a completely defined medium lacking serum LiCl stimulated DNA synthesis in a concentration-dependent manner over a range of 1-25 mM. The optimal concentration of LiCl is between 10 and 20 mM. Concentrations of LiCl greater than 25 mM result in a vacuolization and a loss of cells from the dishes. The effects of lithium chloride on labelling index, DNA content and cell number in BALBIc 3T3 cells Since changes in [“Hlthymidine incorporation might be secondary to increased transport of the labelled nucleoside rather than cell proliferation, additional parameters of growth were studied. These included measurements of labelling index, DNA content of cultures, and cell number. The labelling index 24 h after LiCl addition was determined by autoradiography (table 1). The results demonstrate that more cells initiate DNA synthesis in the presence of LiCl. To measure changes in DNA content of cell cultures, BALB/c 3T3 cells were grown in the presence of LiCl in serum-free medium for 24 h. The data presented in table 2
Growth effects of lithium chloride
265
Table 2. DNA content of BALB/c 3T3 cell cultures exposed to lithium chloride
0
I, 4
8 LITHIUM
I*
1, 16 CHLORIDE
/ 20
1 24
28
(mM)
Fig. 1. Stimulation of r3H]thymidine incorporation into DNA of BALB/c 3T3 cells by LiCI. Cells were plated as described in Materials and Methods. Incorporation of [3H]thymidine into acid-insoluble material was measured during a 2 h pulse 20 h after addition of LiCI. The mean of triplicate determinations and S.E. is shown. Control mean value was 7613 cpm/well.
demonstrates that a concentration of LiCl as low as 0.5 mM was able to increase total DNA content of BALB/c 3T3 cell cultures. Since it is not possible to sustain cultures of BALB/c 3T3 cells for more than 2 days in the absence of serum, the effects of LiCl on cell number were studied Table 1. Number of labelled nuclei in cultures of BALBlc 3T3 cells in the presence of lithium chloride Additions
% labelled nuclei
None LiCl (0.5 mM) (1.0 mM) (5.0 mM) (10.0 mM) FBS (5%)
27k3.6 25f1.8 32k3.5 47k3.0 39k3.8 64k5.0
BALB/c 3T3 cells were plated in serum-containing medium at a density of lo4 cells/cm2. At 24 h the serum containing medium was removed, the cultures were then starved for 24 h as described in Materials and Methods, and LiCl was added in DME-BSA for an additional 24 h. From the 22nd to the 24th hour [3H]thymidine was added and the labelling index was determined as in Materials and Methods. Nine areas (cm2) were counted on each dish. The results are the mean f S.E.
Additions
DNA content b-dwell)
None LiCl (0.5 mM) (1.OmMj (5.0 mM) (10.0 mM) FBS (5%)
0.38 0.45 (0.43-0.46) 0.53 (0.52-0.54j 0.55 (0.52-0.58) 0.70(0.64-0.76) 1.15 (l.lo-1.20)
BALB/c 3T3 cells were plated at a density of IO1 cells/cm’ and LiCI was added as described in table 1 for 24 h. DNA content was measured as described in Materials and Methods. Data is the mean and the range of duplicate determinations.
over 2 days in serum-free medium. The results presented in table 3 demonstrate that the total number of BALB/c 3T3 cells was greater in the presence of LiCl. Interaction of lithium chloride with insulin and EGF Combinations of insulin and EGF enhance the uptake of Li+ by cultured fibroblasts [14]. Therefore, to determine if insulin and/ or EGF would enhance the growth-promoting effect of Li+, LiCl was combined with maximally effective concentrations of insulin or EGF. Fig. 2 demonstrates that LiCl alone increased [“Hlthymidine incorporation by 90% over control, whereas Table 3. Increase in cell number of BALBIc 3T3 cell cultures exposed to lithium chloride Cell number Additions
24 h
48 h
None LiCl (10 mM)
46 lOO+l 240 58 4OOk2 350
64 lOOk 110 767OO+24OO
Cells were plated and cultured as described in Materials and Methods. Lithium chloride was added in DME-BSA and cells were removed by trypsinization at 24 and 48 h of culture. The cell number shown is the mean of triplicate determinations k S.E. of the mean. Eip Cd Rcs 136 (IYHI)
266
Rybak und Stockdule
I
-
Fig. 2. Effects of mitogens in the presence and ab-
sence of LiCl on C”H]thymidine incorporation into BALB/c 3T3 cells. BALB/c 3T3 cultures were prepared as described in Materials and Methods. Insulin (I pg/ml) and EGF (1 pg/ml) were added alone or in the presence of LiCl (IO mM). Data represents the mean of triplicate determinations. The SE. was within 10% of the mean value. The control mean incorporation was 16961 cpm/well. Fetal bovine serum (5%) stimulated DNA synthesis 379% above control.
insulin and EGF alone increased incorporation 110 and 170% respectively. In each case, incorporation was additive when each mitogen was combined with LiCl. Lithium chloride at a concentration of 0.1 mM had no effect on [3H]thymidine incorporation (fig. 3). In combination with EGF and insulin, LiCl at this suboptimal concentration produced a synergistic effect on DNA synthesis.
Fig. 3. Enhancement of mitogen-stimulated DNA synthesis by a suboptimal concentration of LiCI. BALB/c 3T3 cultures were prepared as described in Materials and Methods. The cells were treated with LiCl (0.1 mM) alone or in combination with insulin (1 pg/ml) and EGF (1 pg/ml) for 24 h. Data represents the mean of triplicate determinations. The SE. was within 10% of the mean value. Control mean value was 10553 cpm/well. Fetal bovine serum (5%) stimulated DNA synthesis 344% above control.
Eflects of lithium chloride on the growth of MDCK cell cultures Lithium chloride initiates growth in mammary epithelial cell cultures [9] and, as shown here, in BALB/c 3T3 fibroblasts. However, LiCl does not stimulate the proliferation of all cell types under these experimental conditions. An epithelial cell line (MDCK), which retains responsiveness to hormones in cell culture [15, 161, was investigated for its responsiveness to LiCl
Table 4. Effects of lithium chloride on DNA synthesis in MDCK cells Acid-soluble radioactivity
Acid-precipitable radioactivity
Additions
[:‘H]Thymidine (cpmlwell)
% increase over control
increase P’HlThWdine % Over control
None FBS (10%) LiCl (0.5 mM) (1.0 mM) (2.5 mM) (5.0 mM) (10.0 mM) (20.0 mM)
447238 767+38 328k 16 377+ I8 439220 484f43 646k.94 754+25
0 72 -
4 212k355 16 316+351 2 933k226 3 284+313 3 120i 54 2 734+333 1 960+235 I 622k 88
8 45 69
(cpmlwell)
0 287 -
MDCK cells were plated, cultured and assayed for [3H]thymidine incorporation into acid-soluble and -insoluble fractions as described in Materials and Methods. The data is the mean of triplicate determinations k S.E. of the mean.
Grou~th yft?cts of’ lithium chloride
267
Table 6. Number of luhelled nlrclei in crrltrrres of MDCK cells in the presence of lithium chloride % labelled nuclei
Fig. 4. Growth of MDCK cells in the presence of mitogens. Cells were plated, cultured and assayed for [:‘H]thymidine incorporation into acid-insoluble material as described in Materials and Methods. The cells were in the presence of the additions for 24 h. The mean control value was 19909 cpmlwell.
and peptide mitogens. Lithium chloride at concentrations sufficient to initiate DNA synthesis (OS-20 mM) in either epithelial or fibroblastic cell types did not increase the incorporation of [3H]thymidine into DNA of MDCK cells, although the acid-soluble [“Hlthymidine pool was increased (table 4). In this experiment MDCK cells responded to FBS with increased DNA synthesis. Insulin, EGF and mammary-stimulating factor (MSF) [ 171also increased the incorporation of [“Hlthymidine into DNA in MDCK cells at concentrations effective in mamTable 5. DNA content of MDCK cell cultures exposed to lithium chloride Additions
DNA content @g/well)
None LiCl (5 mM) (IO mM) FBS (10%)
0.37 (0.34-0.40) 0.39 (0.38-0.40) 0.34 (0.30-0.40) I .33 (1.26-I .40)
MDCK cells were plated in the presence of serum and then starved for 24 h. Lithium chloride was added in a defined medium and DNA content was measured 24 h later as in Materials and Methods. The mean of duplicate determinations and the range is shown.
Additions
6h
24 h
None LiCl (5 mM) FESS(10%)
28.2+4. I 32.2k3.2 43.7k2.8
9.7i I .6” 12.1+1.2” 26.8k3.4
Labelling indices in these experiments were performed as described in table I. (I There was not a significant difference between those two data points (p>O.l (Student’s r-test)). Results are the mean + S.E.
mary epithelial or BALB/c 3T3 cells (fig. 4). The lack of response to LiCl was true whether the cells were grown in DME or Waymouth’s medium. To determine if these observations were caused by an altered intracellular utilization of thymidine, both DNA content and labelling index were measured in MDCK cells exposed to LiCl (tables 5, 6). Neither DNA content nor labelling index were affected by LiCI. The inability of LiCl to increase MDCK cell growth does not appear to be due to toxic effects of this cation, since the cells were unaltered cytologically under phase microscopy after LiCl treatment and the cation stimulated [:‘H]uridine incorporation into RNA by 25% and [:1H]leucine incorporation into protein by 35% above cells grown in the absence of LiCl. DISCUSSION To ascertain whether the difference in responsiveness of granulocyte stem cells (mesenchyme) and mammary cells (epithelium) is due to the expressed cell phenotype, the effects of LiCl on cell lines of epithelial and non-epithelial origin were examined. Lithium chloride initiated DNA synthesis in a concentration-dependent ErpCdl KC\136IIYHI)
268
Rybak and Stockdale
manner in several cell lines in addition to the BALB/c 3T3 cell line which is discussed in this report. The same concentrations of LiCl (l-25 mM) increased DNA synthesis in Swiss 3T3 fibroblast cells as well as in two variants of these cells (NR-2 and NR-5) selected for their lack of response to EGF by Pruss & Herschman [18]. Lithium chloride also initiated DNA synthesis in an established mammary cell line (NAMRU). The BALB/c 3T3 cell line was chosen as a representative fibroblastic cell line for detailed comparison with the MDCK epithelial cell line which proved non-responsive to the growth actions of LiCl. Lithium chloride was able to increase DNA content, initiate DNA synthesis and increase cell number in BALB/c 3T3 cell cultures. Its effects on continuous cell growth remain to be clarified. The additivity of the growth effects of LiCl in combination with maximally effective concentrations of insulin or EGF suggests that this cation and the peptide growth factors elicit DNA synthesis via different mechanisms. Others have shown similar interactions of LiCl with mitogens [lo]. Lithium chloride, at a concentration which by itself was inactive, was combined with insulin and EGF. This resulted in a synergistic enhancement of the DNA synthesis stimulated by the peptide mitogens. Since a combination of EGF and insulin previously was shown to increase Li+ uptake by 3T3 cells [14] our results may indicate that the increased uptake of Li+ at suboptimal concentrations in the presence of insulin and EGF results in an intracellular Li+ concentration sufficient to stimulate DNA synthesis. If this were the case the possibility is raised that the site of action of LiCl in stimulating DNA synthesis may be intracellular.
In contrast to BALB/c 3T3 fibroblasts, MDCK cells did not initiate DNA synthesis in response to LiCl. MDCK cells ze epithelial cells active in ion transport. If the failure to see a growth effect of LiCl simply was due to increased transport of Li+ by MDCK cells resulting in toxic intracellular levels of this cation, then concentrations found to be suboptimal in other cell types should have produced a stimulation in MDCK cells. In the range of OS-20 mM LiCl does not increase growth in MDCK cells. The length of exposure to LiCl did not alter the responsiveness of these cells. Negative results were also obtained when the length of exposure to LiCl was varied from 6 to 48 h. A 24 h exposure time is optimal for stimulating DNA synthesis in other cell types [9]. Though LiCl did not initiate DNA synthesis in MDCK cells it did promote the transport of [3H]thymidine into the acid-soluble pool and the incorporation of precursors into RNA and protein. These results suggest that MDCK cells may lack specific physiological conditions necessary for LiCl to express its mitogenic potential, or that LiCl antagonizes events leading to DNA synthesis in these cells. Our data support the latter interpretation since LiCl inhibited 44% of the DNA synthesis in MDCK cells stimulated by fetal bovine serum (data not shown). There are several parameters of ion flux which correlate with the initiation of DNA synthesis in a number of cell systems and these may explain the effects of Li+ on growth initiation. In some cell types serum and hormone-stimulated growth responses have been attributed to changes in monovalent cation concentrations induced by these mitogenic agents [4, 191. Since Li+ is in part transported into cells by mechanisms that transport Na+ [14], it is conceivable that the mitogenic action of LiCl
Growth effects of lithium chloride is a consequence of increased intracellular monovalent cation concentrations. However, the growth effects induced by Li+ differ from those mediated by Na+ fluxes. The growth effects caused by increased intracellular Na+ are dependent upon high cell density and the presence of serum [20]. Lithium chloride, on the other hand, stimulates [“Hlthymidine incorporation in subconfluent cultures of 3T3 tibroblasts in the absence of serum. It has been proposed that both Ca’+ [l] and Mg’+ [2] may also regulate cell proliferation, and Rozengurt & Mendoza [4] have suggested that Na+ uptake may modulate intracellular levels of these divalent cations. Lithium may also regulate intracellular levels of divalent cations by indirectly mimicking the effect of Na+ or by directly perturbing Mg2+ or Ca2+ homeostasis because of its physiochemical similarity to these cations [21]. In support of this hypothesis, LiCl has been shown to enhance Ca’+ uptake in isolated fat cells [22] and block Ca’+ uptake in the endoplasmic reticulum [23]. CAMP has been shown to be necessary for a Caz+-regulatory step involved in the proliferative responses of some cell types [I] and in several systems Li+ was able to inhibit adenylate cyclase activation and interfere in a variety of cyclic nucleotidedependent events [24, 251. CAMP has been implicated as a positive modulator of cell growth in MDCK cells [16] and a negative regulator of tibroblastic cell growth [26]. Therefore, it is possible that Li+ interference with the adenylate cyclase system in BALB/c 3T3 and MDCK cells may relate to its effectiveness in promoting cell proliferation. We have established that the responsiveness of cells to the direct actions of LiCl on growth do not necessarily pertain to
269
cellular phenotype since LiCl promotes growth of both epithelial and non-epithelial cells. In this report we also have demonstrated that not all epithelial cells respond to this cation with an increase in DNA synthesis under the experimental conditions described. A comparison of physiological parameters affected by LiCl in BALB/c 3T3 and MDCK cells may help illuminate the mechanism(s) by which this cation initiates DNA synthesis. We wish to thank Sandra Conlon and Gary Schwartz for excellent technical assistance and Shirley Coles for preparation of this manuscript. Grant support is acknowledged from NIH grant HD12083 and NIH Postdoctoral Fellowship HD05809.
REFERENCES I. Whitfield, J, Boynton, A, Macmanus, J, Sikorska, M & Tsang, B, Mol cell biochem 27 (1979) 15. 2. Rubin, H, Proc natl acad sci US 72 (1975) 3551. 3. Koch, K & Leffert, H, Cell 18 (1979) 153. 4. Rozengurt, E & Mendoza, S, Ann NY acad sci 339 (1980) 175. 5. Barth. L&Barth. L. Dev biol 13 (1966) 95. 6. Johnson, F, Lithium research and‘therapy, p. 353. Academic Press, New York (1975). 7. Shou, M, Ann rev pharmacol toxicol 16(1976) 231. 8. Hori. C & Oka. T, Proc natl acad sci US 76 (1979) 2823. 9. Ptashne. K. Stockdale. F & Conlon. S, J cell Iphysiol 103 (1980) 41. 10. Hart, D, Exp cell res 119 (1979) 47. 11. Morley, D & Galbraith, P, Can med assoc j 118 f 1978)288. 12. Hinegardner, R, Ann biochem 39 (1971) 197. 13. Inawall. J. Weiner. C. Morales. M. Davis. E & Stgckdale, F, J cell biol 63 (1974) 145. 14. Smith, J & Rozengurt, E, J cell physiol 97 (1978) 441. 15. Rindler, M, Chuman, L, Shaffer, L & Saier, M, J cell biol 81 (1979) 635. 16. Taub, M, Chuman, L, Saier, M & Sato, G, Proc natl acad sci US 76 (1979) 3338. 17. Ptashne. K. Hsueh. H & Stockdale. F. Biochemistry 18 (1979) 3533. 18. Pruss. R & Herschman. H. Proc natl acad sci US 74 (1977) 3918. 19 Frantz, C, Nathan, D & Scher, C, J cell biol 88 (1981) 51. 20. Toback, F, Proc natl acad sci US 77 (1980) 6654. 21. Birch, N, Biol psych 7 (1973) 269. 22. Clausen, T, Elbrink, J & Martin, B, Acta endocrinol77, suppl. 191 (1974) 137. 23. DeMeis, L, J biol them 246 (1971) 4764. 24. Gelfand, E, Dosch, H, Hastings, D & Shore, A. Science 203 (1979) 365.
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25. Dousa, T & Hechter, 0, Life sci 9 (1970) 765. 26. Hollenberg, M & Cuatrecasas, P, Proc natl acad sci US 70 (1973) 2964.
Exp Cdl Res 136 (1981)
Received February 27, 1981 Revised version received June 24, 1981 Accepted June 29, 1981
Prmted
in Sweden
Experimental
GROWTH
EFFECTS
FIBROBLASTS
Cell Research 136 (1981) 263-270
OF LITHIUM
CHLORIDE
AND MADIN-DARBY EPITHELIAL
IN BALB/c
CANINE
3T3
KIDNEY
CELLS
SUSANNA M. RYBAK and FRANK E. STOCKDALE Depurtments ofMedicine und Biology, Stunford University. Stunford, CA 94305, USA
SUMMARY The ability of LiCl to initiate DNA synthesis was studied in Madin-Darby canine kidney (MDCK) cells, and mouse BALB/c 3T3 fibroblasts. In a defined culture medium lacking serum, LiCl increased DNA synthesis in BALB/c 3T3 cells lOO-200% over control values. Maximum DNA synthesis was observed with concentrations of LiCl between 10 and 2.5 mM and increases from 40-50% over control were observed with concentrations as low as 1 mM. Exposure of BALB/c 3T3 cultures to LiCl resulted in an increase in the percentage of cells initiating DNA synthesis, total DNA content and cell number. Lithium chloride, in combination with insulin or epidermal growth factor (EGF), had either an additive or synergistic effect upon the growth of BALB/c 3T3 fibroblasts. MDCK cells proved refractory to the growth actions of LiCI, although they responded to EGF and insulin with increased DNA synthesis. Lithium chloride appears to have a direct effect on cell proliferation in some but not all cell types.
The concentration of free divalent cations ported here demonstrate that an epithelial [ 1, 21 as well as monovalent cation fluxes phenotype is not the sole requirement for [3, 41 has been proposed to mediate the LiCl-induced cell proliferation since BALB/ biochemical events which culminate in cell c 3T3 fibroblasts are stimulated to grow by division. Lithium, a monovalent cation this cation, whereas Madin-Darby canine which has a variety of physiological, bio- kidney (MDCK) epithelial cells are not. chemical and biological effects on adult and embryonic tissues [5-71, recently has been shown to stimulate the growth of MATERIALS AND METHODS murine mammary epithelium, both in organ Cell culture [8], and in primary cell culture [9]. These Early passage stock cells were generously supplied studies demonstrate a direct action of LiCl by the following investigators: BALB/c 3T3 cells were on initiation of DNA synthesis in epithelial obtained from Dr Stuart Aaronson at the National Institutes of Health and MDCK cells were obtained cells. In other cells LiCl promotes growth from the laboratory of Dr Dayton Misfeldt at Stanford University. The cell lines were grown over sevonly in the presence of growth factors [lo]. eral passages, aliquots were frozen and new cultures Studies on granulocyte precursors suggest initiated from these frozen stocks approximately every two months. BALB/c 3T3 cells were cultured in Dulthat the mitogenic effects of LiCl may be becco’s Modified Eagle Medium (DME) which conrestricted to epithelial cell types since these tained 5 % fetal bovine serum (FBS) and 1% penicillin-streptomycin and fungizone. MDCK cells were culcells of mesenchymal origin do not directly tured in Waymouth’s medium which contained 10% respond to LiCl [l 11. The experiments re- FBS and 1% penicillin-streptomycin. All cell culture IX-81
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264
Rybak and Stockdale
reagents were obtained from Gibco. The cells were maintained in 75 cm2 flasks and transferred to multiwells or plastic tissue culture plates for the growth experiments. Tissue culture dishes and flasks were from Falcon. In all experiments cells were plated at 2.5~ 10“ cells/cm* in 60 mm dishes (unless otherwise indicated in legends) for 24 h in medium containing the prescribed amount of serum for the particular cell type. At 24 h the cultures were washed hr serumfree medium and incubated an additional 24 h in the appropriate defined medium plus bovine serum albumin (BSA, 5 pg/ml). Lithium chloride or peptide growth factors were then added to fresh defined medium with BSA and the cultures were incubated for 24-48 h before performing an experiment.
Growth assays synthesis. [3H]Thymidine incorporation into acid-soluble or -insoluble material was measured as previously described [9]. The cultures were incubated for 2 h with culture medium containing 2 &i/ml of [methylJH]thymidine (New England Nuclear, sp. act. 20 Cilmmol). Acid-soluble radioactivitv was extracted from’the cells after 2 washes with Hanks’ balanced salt solution (HBSS) by incubating them with 5 % irichloroacetic acid (TCA, w : v). An aliquot of the TCA extract was taken for liquid scintillation counting. The acid-insoluble material was washed with 5% TCA and dieested with 0.5 N NaOH and counted in a liquid sciitillation counter. Data reported as percent stimulation were obtained as follows: (experimental cpmlcontrol cpm - 1.00~ 100). The DNA content of cells was determined by fluorometric assay as described by Hinegardner [ 121.
DNA
Labelling index Cultures were plated into 60 mm dishes and incubated with LiCl or other additions as previously described under “Cell culture”. The cultures were then pulsed for 2 h with [3H]thymidine (2 &i/ml), washed with HBSS, extracted with 5% TCA, fixed with 70% ethanol and air-dried. Fixed cultures were layered with llford liquid emulsion and exposed in the dark for 35 days.
RNA synthesis Lithium chloride-treated and control cell cultures were pulsed with 2 &i/ml [3H]uridine (New England Nuclear, sp. act. 22 Ci/mmol) for 2 h. Uridine incorporation into RNA was then determined by processing the cells as described for [aH]thymidine incorporation into DNA.
Protein synthesis Protein synthesis was determined by pulsing LiCltreated and control cultures for 2 he\h;ith 5 -&X/ml rSHlleucine (SchwarzlMann, sp. act. 59 Cilmmol) in Waymouth’s. medium. Acid-soluble counts were obtained as previously described under “DNA synthesis”. Acid-insoluble counts were obtained by scraping the cells from the dish, precipitating the protein E.rp Cd Rrs 136 (198/j
with hot 5% TCA, and collecting the precipitate in glass fiber filters (Whatman, GF/c). The filters were washed successively with 95% ethanol and acetone, solubilized in NCS solubilizer and counted with a toluene-based scintillant as previously described [ 131.
RESULTS The effects of lithium chloride on [3H]thymidine incorporation in BALBlc 3T3 cells The effects of LiCl on the incorporation of [3H]thymidine into DNA in cultures of BALB/c 3T3 cells is depicted in fig. 1. BALB/c 3T3 cell cultures were deprived of serum for 24 h before LiCl was added to the culture medium. Incorporation of C3H]thymidine was measured 20 h later. In a completely defined medium lacking serum LiCl stimulated DNA synthesis in a concentration-dependent manner over a range of 1-25 mM. The optimal concentration of LiCl is between 10 and 20 mM. Concentrations of LiCl greater than 25 mM result in a vacuolization and a loss of cells from the dishes. The effects of lithium chloride on labelling index, DNA content and cell number in BALBIc 3T3 cells Since changes in [“Hlthymidine incorporation might be secondary to increased transport of the labelled nucleoside rather than cell proliferation, additional parameters of growth were studied. These included measurements of labelling index, DNA content of cultures, and cell number. The labelling index 24 h after LiCl addition was determined by autoradiography (table 1). The results demonstrate that more cells initiate DNA synthesis in the presence of LiCl. To measure changes in DNA content of cell cultures, BALB/c 3T3 cells were grown in the presence of LiCl in serum-free medium for 24 h. The data presented in table 2
Growth effects of lithium chloride
265
Table 2. DNA content of BALB/c 3T3 cell cultures exposed to lithium chloride
0
I, 4
8 LITHIUM
I*
1, 16 CHLORIDE
/ 20
1 24
28
(mM)
Fig. 1. Stimulation of r3H]thymidine incorporation into DNA of BALB/c 3T3 cells by LiCI. Cells were plated as described in Materials and Methods. Incorporation of [3H]thymidine into acid-insoluble material was measured during a 2 h pulse 20 h after addition of LiCI. The mean of triplicate determinations and S.E. is shown. Control mean value was 7613 cpm/well.
demonstrates that a concentration of LiCl as low as 0.5 mM was able to increase total DNA content of BALB/c 3T3 cell cultures. Since it is not possible to sustain cultures of BALB/c 3T3 cells for more than 2 days in the absence of serum, the effects of LiCl on cell number were studied Table 1. Number of labelled nuclei in cultures of BALBlc 3T3 cells in the presence of lithium chloride Additions
% labelled nuclei
None LiCl (0.5 mM) (1.0 mM) (5.0 mM) (10.0 mM) FBS (5%)
27k3.6 25f1.8 32k3.5 47k3.0 39k3.8 64k5.0
BALB/c 3T3 cells were plated in serum-containing medium at a density of lo4 cells/cm2. At 24 h the serum containing medium was removed, the cultures were then starved for 24 h as described in Materials and Methods, and LiCl was added in DME-BSA for an additional 24 h. From the 22nd to the 24th hour [3H]thymidine was added and the labelling index was determined as in Materials and Methods. Nine areas (cm2) were counted on each dish. The results are the mean f S.E.
Additions
DNA content b-dwell)
None LiCl (0.5 mM) (1.OmMj (5.0 mM) (10.0 mM) FBS (5%)
0.38 0.45 (0.43-0.46) 0.53 (0.52-0.54j 0.55 (0.52-0.58) 0.70(0.64-0.76) 1.15 (l.lo-1.20)
BALB/c 3T3 cells were plated at a density of IO1 cells/cm’ and LiCI was added as described in table 1 for 24 h. DNA content was measured as described in Materials and Methods. Data is the mean and the range of duplicate determinations.
over 2 days in serum-free medium. The results presented in table 3 demonstrate that the total number of BALB/c 3T3 cells was greater in the presence of LiCl. Interaction of lithium chloride with insulin and EGF Combinations of insulin and EGF enhance the uptake of Li+ by cultured fibroblasts [14]. Therefore, to determine if insulin and/ or EGF would enhance the growth-promoting effect of Li+, LiCl was combined with maximally effective concentrations of insulin or EGF. Fig. 2 demonstrates that LiCl alone increased [“Hlthymidine incorporation by 90% over control, whereas Table 3. Increase in cell number of BALBIc 3T3 cell cultures exposed to lithium chloride Cell number Additions
24 h
48 h
None LiCl (10 mM)
46 lOO+l 240 58 4OOk2 350
64 lOOk 110 767OO+24OO
Cells were plated and cultured as described in Materials and Methods. Lithium chloride was added in DME-BSA and cells were removed by trypsinization at 24 and 48 h of culture. The cell number shown is the mean of triplicate determinations k S.E. of the mean. Eip Cd Rcs 136 (IYHI)
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I
-
Fig. 2. Effects of mitogens in the presence and ab-
sence of LiCl on C”H]thymidine incorporation into BALB/c 3T3 cells. BALB/c 3T3 cultures were prepared as described in Materials and Methods. Insulin (I pg/ml) and EGF (1 pg/ml) were added alone or in the presence of LiCl (IO mM). Data represents the mean of triplicate determinations. The SE. was within 10% of the mean value. The control mean incorporation was 16961 cpm/well. Fetal bovine serum (5%) stimulated DNA synthesis 379% above control.
insulin and EGF alone increased incorporation 110 and 170% respectively. In each case, incorporation was additive when each mitogen was combined with LiCl. Lithium chloride at a concentration of 0.1 mM had no effect on [3H]thymidine incorporation (fig. 3). In combination with EGF and insulin, LiCl at this suboptimal concentration produced a synergistic effect on DNA synthesis.
Fig. 3. Enhancement of mitogen-stimulated DNA synthesis by a suboptimal concentration of LiCI. BALB/c 3T3 cultures were prepared as described in Materials and Methods. The cells were treated with LiCl (0.1 mM) alone or in combination with insulin (1 pg/ml) and EGF (1 pg/ml) for 24 h. Data represents the mean of triplicate determinations. The SE. was within 10% of the mean value. Control mean value was 10553 cpm/well. Fetal bovine serum (5%) stimulated DNA synthesis 344% above control.
Eflects of lithium chloride on the growth of MDCK cell cultures Lithium chloride initiates growth in mammary epithelial cell cultures [9] and, as shown here, in BALB/c 3T3 fibroblasts. However, LiCl does not stimulate the proliferation of all cell types under these experimental conditions. An epithelial cell line (MDCK), which retains responsiveness to hormones in cell culture [15, 161, was investigated for its responsiveness to LiCl
Table 4. Effects of lithium chloride on DNA synthesis in MDCK cells Acid-soluble radioactivity
Acid-precipitable radioactivity
Additions
[:‘H]Thymidine (cpmlwell)
% increase over control
increase P’HlThWdine % Over control
None FBS (10%) LiCl (0.5 mM) (1.0 mM) (2.5 mM) (5.0 mM) (10.0 mM) (20.0 mM)
447238 767+38 328k 16 377+ I8 439220 484f43 646k.94 754+25
0 72 -
4 212k355 16 316+351 2 933k226 3 284+313 3 120i 54 2 734+333 1 960+235 I 622k 88
8 45 69
(cpmlwell)
0 287 -
MDCK cells were plated, cultured and assayed for [3H]thymidine incorporation into acid-soluble and -insoluble fractions as described in Materials and Methods. The data is the mean of triplicate determinations k S.E. of the mean.
Grou~th yft?cts of’ lithium chloride
267
Table 6. Number of luhelled nlrclei in crrltrrres of MDCK cells in the presence of lithium chloride % labelled nuclei
Fig. 4. Growth of MDCK cells in the presence of mitogens. Cells were plated, cultured and assayed for [:‘H]thymidine incorporation into acid-insoluble material as described in Materials and Methods. The cells were in the presence of the additions for 24 h. The mean control value was 19909 cpmlwell.
and peptide mitogens. Lithium chloride at concentrations sufficient to initiate DNA synthesis (OS-20 mM) in either epithelial or fibroblastic cell types did not increase the incorporation of [3H]thymidine into DNA of MDCK cells, although the acid-soluble [“Hlthymidine pool was increased (table 4). In this experiment MDCK cells responded to FBS with increased DNA synthesis. Insulin, EGF and mammary-stimulating factor (MSF) [ 171also increased the incorporation of [“Hlthymidine into DNA in MDCK cells at concentrations effective in mamTable 5. DNA content of MDCK cell cultures exposed to lithium chloride Additions
DNA content @g/well)
None LiCl (5 mM) (IO mM) FBS (10%)
0.37 (0.34-0.40) 0.39 (0.38-0.40) 0.34 (0.30-0.40) I .33 (1.26-I .40)
MDCK cells were plated in the presence of serum and then starved for 24 h. Lithium chloride was added in a defined medium and DNA content was measured 24 h later as in Materials and Methods. The mean of duplicate determinations and the range is shown.
Additions
6h
24 h
None LiCl (5 mM) FESS(10%)
28.2+4. I 32.2k3.2 43.7k2.8
9.7i I .6” 12.1+1.2” 26.8k3.4
Labelling indices in these experiments were performed as described in table I. (I There was not a significant difference between those two data points (p>O.l (Student’s r-test)). Results are the mean + S.E.
mary epithelial or BALB/c 3T3 cells (fig. 4). The lack of response to LiCl was true whether the cells were grown in DME or Waymouth’s medium. To determine if these observations were caused by an altered intracellular utilization of thymidine, both DNA content and labelling index were measured in MDCK cells exposed to LiCl (tables 5, 6). Neither DNA content nor labelling index were affected by LiCI. The inability of LiCl to increase MDCK cell growth does not appear to be due to toxic effects of this cation, since the cells were unaltered cytologically under phase microscopy after LiCl treatment and the cation stimulated [:‘H]uridine incorporation into RNA by 25% and [:1H]leucine incorporation into protein by 35% above cells grown in the absence of LiCl. DISCUSSION To ascertain whether the difference in responsiveness of granulocyte stem cells (mesenchyme) and mammary cells (epithelium) is due to the expressed cell phenotype, the effects of LiCl on cell lines of epithelial and non-epithelial origin were examined. Lithium chloride initiated DNA synthesis in a concentration-dependent ErpCdl KC\136IIYHI)
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Rybak and Stockdale
manner in several cell lines in addition to the BALB/c 3T3 cell line which is discussed in this report. The same concentrations of LiCl (l-25 mM) increased DNA synthesis in Swiss 3T3 fibroblast cells as well as in two variants of these cells (NR-2 and NR-5) selected for their lack of response to EGF by Pruss & Herschman [18]. Lithium chloride also initiated DNA synthesis in an established mammary cell line (NAMRU). The BALB/c 3T3 cell line was chosen as a representative fibroblastic cell line for detailed comparison with the MDCK epithelial cell line which proved non-responsive to the growth actions of LiCl. Lithium chloride was able to increase DNA content, initiate DNA synthesis and increase cell number in BALB/c 3T3 cell cultures. Its effects on continuous cell growth remain to be clarified. The additivity of the growth effects of LiCl in combination with maximally effective concentrations of insulin or EGF suggests that this cation and the peptide growth factors elicit DNA synthesis via different mechanisms. Others have shown similar interactions of LiCl with mitogens [lo]. Lithium chloride, at a concentration which by itself was inactive, was combined with insulin and EGF. This resulted in a synergistic enhancement of the DNA synthesis stimulated by the peptide mitogens. Since a combination of EGF and insulin previously was shown to increase Li+ uptake by 3T3 cells [14] our results may indicate that the increased uptake of Li+ at suboptimal concentrations in the presence of insulin and EGF results in an intracellular Li+ concentration sufficient to stimulate DNA synthesis. If this were the case the possibility is raised that the site of action of LiCl in stimulating DNA synthesis may be intracellular.
In contrast to BALB/c 3T3 fibroblasts, MDCK cells did not initiate DNA synthesis in response to LiCl. MDCK cells ze epithelial cells active in ion transport. If the failure to see a growth effect of LiCl simply was due to increased transport of Li+ by MDCK cells resulting in toxic intracellular levels of this cation, then concentrations found to be suboptimal in other cell types should have produced a stimulation in MDCK cells. In the range of OS-20 mM LiCl does not increase growth in MDCK cells. The length of exposure to LiCl did not alter the responsiveness of these cells. Negative results were also obtained when the length of exposure to LiCl was varied from 6 to 48 h. A 24 h exposure time is optimal for stimulating DNA synthesis in other cell types [9]. Though LiCl did not initiate DNA synthesis in MDCK cells it did promote the transport of [3H]thymidine into the acid-soluble pool and the incorporation of precursors into RNA and protein. These results suggest that MDCK cells may lack specific physiological conditions necessary for LiCl to express its mitogenic potential, or that LiCl antagonizes events leading to DNA synthesis in these cells. Our data support the latter interpretation since LiCl inhibited 44% of the DNA synthesis in MDCK cells stimulated by fetal bovine serum (data not shown). There are several parameters of ion flux which correlate with the initiation of DNA synthesis in a number of cell systems and these may explain the effects of Li+ on growth initiation. In some cell types serum and hormone-stimulated growth responses have been attributed to changes in monovalent cation concentrations induced by these mitogenic agents [4, 191. Since Li+ is in part transported into cells by mechanisms that transport Na+ [14], it is conceivable that the mitogenic action of LiCl
Growth effects of lithium chloride is a consequence of increased intracellular monovalent cation concentrations. However, the growth effects induced by Li+ differ from those mediated by Na+ fluxes. The growth effects caused by increased intracellular Na+ are dependent upon high cell density and the presence of serum [20]. Lithium chloride, on the other hand, stimulates [“Hlthymidine incorporation in subconfluent cultures of 3T3 tibroblasts in the absence of serum. It has been proposed that both Ca’+ [l] and Mg’+ [2] may also regulate cell proliferation, and Rozengurt & Mendoza [4] have suggested that Na+ uptake may modulate intracellular levels of these divalent cations. Lithium may also regulate intracellular levels of divalent cations by indirectly mimicking the effect of Na+ or by directly perturbing Mg2+ or Ca2+ homeostasis because of its physiochemical similarity to these cations [21]. In support of this hypothesis, LiCl has been shown to enhance Ca’+ uptake in isolated fat cells [22] and block Ca’+ uptake in the endoplasmic reticulum [23]. CAMP has been shown to be necessary for a Caz+-regulatory step involved in the proliferative responses of some cell types [I] and in several systems Li+ was able to inhibit adenylate cyclase activation and interfere in a variety of cyclic nucleotidedependent events [24, 251. CAMP has been implicated as a positive modulator of cell growth in MDCK cells [16] and a negative regulator of tibroblastic cell growth [26]. Therefore, it is possible that Li+ interference with the adenylate cyclase system in BALB/c 3T3 and MDCK cells may relate to its effectiveness in promoting cell proliferation. We have established that the responsiveness of cells to the direct actions of LiCl on growth do not necessarily pertain to
269
cellular phenotype since LiCl promotes growth of both epithelial and non-epithelial cells. In this report we also have demonstrated that not all epithelial cells respond to this cation with an increase in DNA synthesis under the experimental conditions described. A comparison of physiological parameters affected by LiCl in BALB/c 3T3 and MDCK cells may help illuminate the mechanism(s) by which this cation initiates DNA synthesis. We wish to thank Sandra Conlon and Gary Schwartz for excellent technical assistance and Shirley Coles for preparation of this manuscript. Grant support is acknowledged from NIH grant HD12083 and NIH Postdoctoral Fellowship HD05809.
REFERENCES I. Whitfield, J, Boynton, A, Macmanus, J, Sikorska, M & Tsang, B, Mol cell biochem 27 (1979) 15. 2. Rubin, H, Proc natl acad sci US 72 (1975) 3551. 3. Koch, K & Leffert, H, Cell 18 (1979) 153. 4. Rozengurt, E & Mendoza, S, Ann NY acad sci 339 (1980) 175. 5. Barth. L&Barth. L. Dev biol 13 (1966) 95. 6. Johnson, F, Lithium research and‘therapy, p. 353. Academic Press, New York (1975). 7. Shou, M, Ann rev pharmacol toxicol 16(1976) 231. 8. Hori. C & Oka. T, Proc natl acad sci US 76 (1979) 2823. 9. Ptashne. K. Stockdale. F & Conlon. S, J cell Iphysiol 103 (1980) 41. 10. Hart, D, Exp cell res 119 (1979) 47. 11. Morley, D & Galbraith, P, Can med assoc j 118 f 1978)288. 12. Hinegardner, R, Ann biochem 39 (1971) 197. 13. Inawall. J. Weiner. C. Morales. M. Davis. E & Stgckdale, F, J cell biol 63 (1974) 145. 14. Smith, J & Rozengurt, E, J cell physiol 97 (1978) 441. 15. Rindler, M, Chuman, L, Shaffer, L & Saier, M, J cell biol 81 (1979) 635. 16. Taub, M, Chuman, L, Saier, M & Sato, G, Proc natl acad sci US 76 (1979) 3338. 17. Ptashne. K. Hsueh. H & Stockdale. F. Biochemistry 18 (1979) 3533. 18. Pruss. R & Herschman. H. Proc natl acad sci US 74 (1977) 3918. 19 Frantz, C, Nathan, D & Scher, C, J cell biol 88 (1981) 51. 20. Toback, F, Proc natl acad sci US 77 (1980) 6654. 21. Birch, N, Biol psych 7 (1973) 269. 22. Clausen, T, Elbrink, J & Martin, B, Acta endocrinol77, suppl. 191 (1974) 137. 23. DeMeis, L, J biol them 246 (1971) 4764. 24. Gelfand, E, Dosch, H, Hastings, D & Shore, A. Science 203 (1979) 365.
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25. Dousa, T & Hechter, 0, Life sci 9 (1970) 765. 26. Hollenberg, M & Cuatrecasas, P, Proc natl acad sci US 70 (1973) 2964.
Exp Cdl Res 136 (1981)
Received February 27, 1981 Revised version received June 24, 1981 Accepted June 29, 1981
Prmted
in Sweden