- Email: [email protected]
Proliferation and specific functions of neoplastic mast cells in culture
Experimental Cell Research 49 (1971) Y-64
FERATIBN
AND SPECIFIC FUNCTIONS CELLS IN CULTU
9. LAISSUE, W. MARX,l Department
A. GRIEDER
of Pathology,
University
and R. SCHINDLER
of Bern, Switzerland
SUMMARY Cultures in vitro of a murine mastocytoma were incubated in a series of semisynthetic media. In order to produce specific nutritional deficiencies media were used in which one amino acid (L-leucine, L-histidine or L-tryptophan, respectively) was present in optimal and various suboptimal concentrations. Appropriate reduction of the concentration of L-leucine or L-histidine resulted in a decrease of cell multiplication and in an increase of the relative number of dead cells, whereas omission of r,-tryptophan had no appreciable effect on cell proliferation during the observation period of 96 h. After incubation of cells in these media during 4 days, the mean celldar histamine and Shydroxytryptamine contents as well as the incorporation of 35S0, into the protein-bound heparin fraction were determined. The mean cellular content of histamine or 5-hydroxytryptamine was reduced in media containing suboptimal concentrations of the corresponding precursor amino acid (L-histidine or L-tryptophan, respectively). In contrast, appropriate reduction of the kleucine level in the culture medium resulted in an increase of the mean cellular 5hydroxytryptamine content and an increased 35S0, incorporation.
Neoplastic murine mast cells during conroliferation in culture for long tinuous iods of time preserve the capacity to thetize and store histamine, 5-hydroxyamine (5-HT) and heparin [7, 8, 171. confronts us with the opportunity to examine in vitro the question if, in what sense and to what degree synthetic processes resulting in the formation of specific mast elf products are coupled with cell multilication. The present report is concerned with relationships between specialized cell functions and proliferation in neoplastic mast 1 Address: Department of Biochemistry, University of Southern California School of Medicine, Los Angeles, Calif. 90033, USA.
cell populations under a variety of su optimal culture conditions. Pn order the synthesis of histamine or rresponding precursor ammo acids, i.e. rAristidine or r,-tryptophan, were supplied to the culture medium in reduced concentrations, as compared to the levels providing for optimal growth conditions. Conversely, the concentration in the medium of L-leucine, i.e. an amino acid almost exclusively involved in protein synthesis, was modified.
Cell line and culture techniques A culture in vitro of a transplantable murine mastocytoma [41 was kindly provided by Dr G. A. Fischer,
58
J. Laissue et al.
Providence, R. I., USA (cell line HC). Similar to another neoplastic mast cell line [17], the HC cells, except for a minor proportion of their population, do not attach to the surface of culture vessels. From the HC cell population, a clonal subline (termed HCb,) was derived which is characterized by a relatively high cellular content of metachromatic granules. In order to maintain these characteristics, the cells were kept frozen at - 80°C in medium containing 10 % dimethyl sulfoxide. For each experiment, an aliquot of frozen cell suspension was thawed, and the cells were cultured during 2 to 3 weeks in standard medium containing 10 % horse serum. This medium has been previously described as medium I [15]. Subsequently, the celis were centrifuged and resuspended, at a cell density of approx. 1.5 x lo5 cells/ml, in 300 ml of the experimental medium to be tested. Experimental media differed from standard medium as follows: Undialysed serum was substituted by dialysed serum. Dialysis was carried out at 4°C against 6 times 20 vol of deionized water during a total of 24 h. In addition, MgSO, was replaced by an equimolar amount of MgCl,, thus reducing the concentration of inorganic sulfate to 0.1 pmole/ml (added in the form of streptomycin sulfate). This reduction in sulfate concentration had no detectable effect on cell multiplication during observation periods of up to 6 days. In order to produce specific nutritional deficiencies, various experimental media were used in which the concentration of one amino acid (L-leucine, L-histidine or L-tryptophan, respectively) was reduced as compared with that in the standard medium and the “optimal” experimental medium (see figs 2, 4, 5, 6). Concentrations of L-leucine, r.-histidine and L-tryptophan in “optimal” experimental medium were 200 PM, 50 ,uM and 20 PM, respectively. In an additional series of experimental media, different concentrations of dialysed serum were tested (5 %, 10 %, 20 %, 40 x). Unless mentioned otherwise, the cells were incubated in experimental media as spinner cultures during a total of 96 h. Every 24 h, the number of morphologically intact cells per ml of culture was determined, and an appropriate portion of the cell suspension was diluted with the prewarmed experimental medium to be tested in order to obtain again 300 ml of culture containing 1.5 x lo5 morphologically intact cells/ml.
Determination of cell multiplication the proportion of dead cells
and of
Cell multiplication was computed based on morphologically intact cells. Their number, as determined by hemocytometer counts with a phase contrast microscope, corresponded within 5 % to the number of cells remaining unstained for at least a few minutes in isotonic saline containing 0.1 % trypan blue. Similarly, the number of ghost cells, as seen in the phase contrast microscope, agreed within 5 % with that of trypan blue positive cells. Routine counts were, therefore, based on the appearance of cells in the phase contrast microscope. Exptl
Cell Res 69
Determination content
of cellular histamine and 5-HT
For determination of the cellular histamine content, an aliquot of the suspension culture containing 3 to 7 x lo6 cells was centrifuged, and the cells were resuspended in 2 ml of saline, heated to 100°C for 3 min and kept frozen at - 25°C until further processing. The histamine content of these samples was determined in the laboratory of Dr R. Keller, Dermatologische Universitatsklinik, Zurich, by bioassay on the guinea pig ileum as described previously [lo]. For determination of the cellular 5-HT content, a suspension containing approximately 5 x 10’ cells was centrifuged at 750 g for 5 min. The cells in the pellet were resuspended in 2 ml of isotonic saline, and 8.5 ml of 0.1 N HCl solution containing 1 mg ascorbic acid per ml were added, as suggested-by Thompson et al. [19]. Further extraction and fluorimetric determination of 5-HT was carried out according to the method of Bogdanski et al. [l].
Determination of 35S0, incorporation into the protein-bound heparin fraction of mast cells After 4 days incubation in exeerimental media. aliquots of each culture were incubated during 4d with 15 to 50 tiCi/ml of 35S0.,.Subseauentlv. the cell suspensions were chilled by immersion in an ice bath, and all further manipulations were carried out at 0°C. The cells were centrifuged at 750 g for 5 min, the supernatant was withdrawn, and 1.3 ml of cold trichloroacetic acid (TCA) was added to the cell sediment, followed by 1 ml saline containing approx. 10’ non-labeled cells as carrier material, and by 3 ml 5 % TCA. After mixing, the precipitate was sedimented by centrifunation and washed 6 times with 5 ml 5 % TCA andonce with 5 ml absolute ethanol. The final sediment was dissolved in 0.5 ml 0.1 N NaOH for determination of radioactivity in a liquid scintillation counter. It was shown in control experiments that practically all inorganic sulfate, smallmolecular weight sulfate derivatives and sulfolipids were removed by this procedure. The protein-linked glycosaminoglvcans, designated nrotein-bound henaiin fraction,were the major, if not the only components of the TCA-insoluble mast cell fraction which are known to take up sulfate in significant amounts, under the exnerimental conditions described. 3sS0, incorporationL into this fraction was found to be nearly linear during the incubation period of 4 h.
Qualitative morphological examination and size distribution analysis of mast cells A qualitative morphological evaluation of cells with respect to their content of metachromatically staining granules was performed with smears stained with toluidine blue. Cellular size frequency distributions were obtained with a Coulter Counter (model B) using a tube with an aperture of 200 pm diameter.
In a series of preliminary experiments, media containing various concentrations of dialysed serum were compared with respect to their effects on proliferation and amine content of neoplastic mast cells. The increase in cell number during an incubation period of 5 days was most rapid in media containing 10 or % dialysed horse serum and somewhat slower at levels of 5 or 40 %, respectively. No clear differences in mean cellular histamine and 5-HT content were observed in media containing 10, 20 or 40 % dialysed serum: while at the 5 % serum level, the
c
Lmm--
1
2-
3
L
(days); ordinate: (a) log cell multiplication; (b) relative number of morphologicaliy intact cells, 04. Cell multiplication (a) and proportion of morphologically intact cells (b) in neoplastic mast cell cultures, as a function of time of incubation in mediacontaining different concentrations of L-leucine: O-O, 200 ,uM; 3--O, IOOpM; o---o,50/~M; ~...a, 25 LLM. Values for log cell multiplication (log M) were calculated as follows: Fig. 1, Abscissa:
iog M= log
he
Nt
--xD NO
, where Nt is cell number
per ml at the time indicated on the abscissa; N,, cells/ ml at zero time, D, cumulative dilution factor of respective culture.
-0 5/-k-d 25
50
100 200 625 li5
25
50
_.A-_ 0 20
Fig. 2. Abscissa: concentration of amino acid indicated, p”M; ordinate: log ceil multiplication during the 96 h incubation period (A, B) and during the last 24 h of the 96 h incubation period (a, v ).
Cell multiplication in media containing different concentrations of L-leucine, L-histidine or Ltryptophan, respectively. The symbois represent results obtained in two independent experfments. Values for log cell muhiplication were calculated as described for fig. 1~
mean content of these amines per cell was reduced. In subsequent experiments, media containing 10% dialysed horse serum were used. Fig. 1a illustrates cell pro~~ferat~o~ patterns in a typical experiment in which the effects of media with different concentrations of L-leucine were studied. Reduction of the concentration sf this amino acid from ZOOto 50 pmoles/l resulted in a decrease sn cell multiplication which became apparent cx the second to third day of incubation, while at 25 pmoles/l, the number of morphologically intact cells began to decline 24 to 48 h after incubation of cells under these deficient culture conditions. Concomitant with the decrease in growth rate of the cell population, a decrease :n the relative number of morphologically intact cehs was observed, as seen in fig. 16. Similar results were obtained if the concentration of r=histidine in the culture medium was reduced, whereas omission of z-tryptophan from the medium did not affect cell multiplication to an appreciable extent during
60
J. Laissue et al. I
TRYPTOPHAN.
20
40
60
20 pM
80
100
Fig. 3. Abscissa: cell size (Coulter counter threshold values); ordinate: cells per size class, % of total cell number. Distribution of cell sizes in a culture containing 20 ,uM L-tryptophan (upper), as compared to a culture containing no added tryptophan (lower). Particles with sizes up to 8 were not considered in computing 100 % values.
the incubation period of 96 h. The results obtained with respect to cell multiplication under various culture conditions during the total incubation period of 96 h, as well as during the last 24 h, are summarized in fig. 2. The relative number of ghost cells observed in these experiments may be derived from data presented in figs 4 and 5 (differences between closed and open symbols). Size frequency distribution analysis indicated that after incubation in media containing low concentrations of L-leucine or L-histidine, or lacking L-tryptophan, the mean size of cells was markedly reduced as compared with control cultures in optimal medium. This is illustrated in fig. 3 for a culture without added tryptophan, as compared with a control culture containing 20 pmoles/l of this amino acid. After incubation of cells in experimental media during 4 days, the mean cellular Exptl Cell Res 69
histamine and 5-HT contents, as well as the incorporation of 35S0, into the proteinbound heparin fraction were determined in order to obtain quantitative information on the extent of specific cell functions under these culture conditions. As seen in fig. 4, in media containing suboptimal concentrations of r.-leucine, the mean cellular 5-HT content, as compared with controls (200,~M L-leucine), was increased up to 6 fold if referred to all cells and up to 19 fold if referred to morphologically intact cells. It should be noted that ghost cells also contained metachromatically staining granules. Variation of the concentration of L-histidine in the culture medium had little effect on the mean 5-HT content of cells. On the other hand, the mean 5-HT content was reduced by more than 60% if L-tryptophan was omitted from the medium. The mean histamine content per cell (fig. 5) was not affected to an appreciable extent by variation of the concentration of L-leucine or L-tryptophan in the culture medium; it was, however, profoundly modified by re-
1
Fig. 4. Abscissa: concentration of amino acid indicated, p”M; ordinate: 5-hydroxytryptamine content (logarithmic scale), pg/lOg morphologically intact cells (A, v ) and pg/109 cells including cell ghosts (A, 7). Cellular Shydroxytryptamine content after 4 days’ incubation in media containing different concentrations of L-leucine, L-histidine, or L-tryptophan, respectively. The symbols represent results obtained in two independent experiments.
duction of the concentration of L-histidine: in media containing 12.5 ,umoles/l of this amino acid, cellular histamine content was between 18 and 15 % of control values only. With respect to incorporation of 35S0, into the protein-bound heparin fraction of lastic mast cells (fig. 6), an increase was ved in medium containing L-leucine at a concentration of 25 ,umoles/l. Whereas the difference between controls (200 ,&I Lleucine) and cultures containing 25 ,uM L-leucine, due to the high variation of control values, is not significant, differences between cultures containing this amino acid at 25 and 50 y&I, or 25 and 100 @I, respectively, are significant (P ~0.05) if the t test is used. Modifications of other culture conditions did not result in appreciable changes in uptake of label. Ghost cells obtained by repeated freezing and thawing did not incorporate significant amounts of 35S0,. The results presented in fig. 6 refer, therefore, to morphologically intact cells only.
It has been reported previously that cellular amine levels in neoplastic mast cell cultures
T 20;
Fig. 6. Abscissa: concentration of amino acid dicated, ,&I; ordinate: WQ incorporated into
fnpro-
tein-bound heparin fraction by IO6 intact cells during 4 h, % of total radioactivity. Sulfate incorporation into protein-bound heparin fraction after 4 days’ incubation in media co~ta~~~~~ different concentrations of L-leucine, z-histidine or L-tryptophan, respectively. The symbols represent results obtained in two independent experiments.
underwent pronounced fluctuation which did not appear to be relat ences in culture conditions [2]. In the studies too, differences in cellular contents and sulfate i~cQr~orati~~ between control cultures of individual experiments were observed, as seenin figs 4 to 6 (controls dium compositions 20 with the sam istidine and 20 PM try leucine, 50 p phan). In every ex~e~~rne~t, therefore, controls were incubated in parallel with c~~~~~r~s containing one amino acid in various reduced
-c
Fig. 5. Abscissa: concentration of amino acid indicated, pi%; ordinate: histamine content, pg/lO’ morpholo-
gically intact cells (A, v ) and ,ug/lO’ cells including Cell ghoStS(A, v >. Cellular histamine content after 4 days’ incubation in media containing different concentrations of L-leucine, L-histidine or L-tryptophan, respectively. The symbols represent results obtained in two independent experiments.
system as previously described [lg]. The possibility can, t not be excluded that amino acid le not entirely constant with time, but underwent fluctuations due to daily addition of fresh medium and subsequ amino acids by the cultured c small amounts of free amin were supplied to the cultu serum. Nevertheless, the results indicate that the observed effects were dependent orn the concentration
of t e amino acid tested. Exptl Cd Res 59
62 J. Laissue et al. A significant decline in cell multiplication concomitant with an increase in the relative number of dead cells was observed on the 2nd to 3rd day of incubation in media deficient in leucine or histidine. Upon omission of tryptophan from the culture medium, cell multiplication continued at a rate comparable with that in control medium during the incubation period of 4 days. In experiments of longer duration, however, omission of tryptophan resulted in a decrease in growth rate beginning approx. 6 days after incubation in the deficient medium. In addition to the effects on cell multiplication, a decrease in mean cell size was observed in cultures containing reduced concentrations of leucine or histidine, or lacking tryptophan. This phenomenon may be attributed to the effects of amino acid depletion on cellular protein metabolism and/or to changes in cell cycle characteristics induced by deficient media [5]. The possibility might be considered that the changes in average cellular amine content and sulfate incorporation, as observed after 4 days’ incubation in amino acid deficient media, have occurred as a result of selection of subpopulations which, under the relatively unfavorable culture conditions, would outgrow the majority of the original cell population. This would imply that the expression of mast cell function, as determined in our studies, is transmitted from parent cell to progeny in the process of cell proliferation. In view of the short duration of the experiments, however, it appears unlikely that the observed modifications in mast cell characteristics are attributable primarily to a selection process. Furthermore, omission of tryptophan or reduction of the histidine concentration to 25 pmoles/l resulted in pronounced changes in average 5-HT or histamine content without a significant decrease in cell multiplication. These findings are in favour of an Exptl Cell Res 69
adaptive change of specific mast cell functions under modified culture conditions. On the other hand, despite the recent clonal origin of the HCb, line, a considerable heterogeneity of the cell population with respect to the amount of metachromatic material per individual cell was observed. This is in agreement with the reported variability in 5-HT contents of individual mastocytoma cells [12, 141. Furthermore, variations in amine content during the cell division cycle, as well as differentiation of “precursor” cells into more mature cell types, cannot be excluded. As long as the relationships between proliferation kinetics and specific functions of mastocytoma cells are not known, it is difficult to draw any conclusions as to the mechanisms underlying the induced changes in average amine content and sulfate incorporation. Cellular amine levels are a function of the synthesis rate and, in addition, depend on the rate of cell multiplication and possibly rates of degradation and/or release of amines into the medium. Mastocytoma cells form 5-HT and histamine from the precursor amino acids, tryptophan and histidine, respectively [7, 91. Free L-tryptophan appears to be the major, if not the sole, precursor of 5-HT [2, 161.It is, therefore, not surprising that reduction of the concentration of tryptophan or histidine in the medium resulted in a decrease of cellular 5-HT or histamine contents, respectively, as seen in figs 4 and 5. On the other hand, n.o increase in cellular amine levels was observed in mastocytoma cells cultured in media containing tryptophan or histidine in concentrations exceeding those providing for optimal growth conditions [2]. In cultures of mastocytoma cells, no catabolites of 5-HT or histamine were detected intracellularly or in the medium [7]. Loss of amines and, to a lesser extent, of heparin from cells into the culture medium has been
Proliferation reported 13, 6]; no data on the extent of cell death were, however, presented, although media containmg suboptimal concentrations of tryptophan or istidine were used. The pronounced increase in cellular 5-HT content without concomitant increase in histamine content in leucine-deficient media is of special interest. On the basis of the available data, release of amines by dead cells and their uptake by surviving cells cannot be excluded. The amounts of 5-HT that would thus become available are, however, barely sufficient, under the most favorable assumptions, to fully account for the observed increases in cellular 5-HT content. It appears likely, therefore, that under these suboptimal culture conditions, 5-HT metabolism by the neoplastic mast cells is characterized by an enhanced rate of synthesis and/or a reduced rate of release. A similar increase in cellular 5-HT content has been observed in mastocytoma cell cultures following incubation during 24 h in the presence of actinomycin D at an appropriate growthinhibitory concentration [l I]. Treatment of mice bearing a transplantable mastocytoma with doses of cyclophosphamide causing n also resulted in a markedly content of the surviving tumor cells [13]. Labeled sulfate has been shown to be incorporated into heparin by cultured mastocytoma cells [S]. Since inorganic sulfate, small-molecular weight sulfate derivatives and sulfolipids were removed during the extraction procedure, the radioactivity in the TCA-insoluble fraction may be considered to represent label incorporated into the protein-bound heparin fraction, and, therefore, to give an indication of the rate of heparin synthesis during the incubation period of 4 h. A marked increase in 35S0, incorporation was observed in media containing Eeucine at 25 ,umoles/l, i.e. under
and specific functkw conditions
resulting
of m~stQcytom~ cells
53
also in an increase in
In conclusion, the results obtained indicate that appropriate modifications of cell cdture conditions may affect ~ro~~ferat~v~ activity as well as specific functions of neoplastic mast cells. CelEular S-MT or histamine contents were preferentially affected In media containing the corresponding precm-sor amino acid at reduced concentrations: a decrease in amine content was already detectable at a precursor amino acid level which still provided for unimpaired cell proliferation. Reduction of the concentration of an amino acid not directly involved in amine synthesis, such as leucine, resulted in an inhibition of cell multiplication and an increase in ce?lular 5-HT content and sulfate incorporation into the protein-bound heparln fraction. Thus, the variations in the expression of specialized cell functions, as observed under modified culture conditions, do not necessarily paraM the changes in cell proliferation. In addition, 5-HT content, histamine content, and sulfate incorporation into the protein-bound begarm fraction, which are representative of three different functions of the same cell, may be modified independently from each other, This work was supported by the Swiss National Science Foundation -a& by research grant 5 ROT HE 01595 from the National Heart and Lung Institute, USPHS. The authors express their gra&ude tcp Professor H. Cottier for his encouragement and continued interest, and to Dr R. Keller,bermatoloaische Un~versitatsklinik. Zurich. for his kind offer to have the histamine dioassays’ carried out jn h,Es laboratory. One of ns (J. L.) is indebted to Dr W. Burkard and Dr F. Gey, Hoffmann-&a Roche & Co., Base], for being introduced into the technique of’ 5-HT determination. The expert technical assistance of Miss C. Hiirni is gratefully acknowledged.
1. Bogdanski, F, Pletscher, A, Brodie, B Udenfriend, S, 3 pharmacol expel ther I3 7 (I 956) 82. 2. Day, M & Green, J P, J physiol 164 (1962) 220. 3. - Biochem pharmaco: II (1962) 1043.
64
J. Laissue et al.
4. Furth, J, Hagen, P & Hirsch, E I, Proc sot exptl biol med 95 (1957) 824. 5. Gautschi, J R & Schindler, R, Experientia 26 (1970) 693. 6. Green, J P, Biochem pharmacoll2 (1963) 577. 7. - Europ j pharmacol 3 (1968) 68. 8. Green, J P & Day, M, Biochem pharmacol 3 (1960) 190. 9. Hagen, P & Lee, F L, J physiol 143 (1958) 7P. 10. $eller, R & Beeger, I, Int arch allergy 22 (1963) 11. Mannaioni, P F & Giarman, N J, Pharmacol res commun 1 (1969) 218. 12. Mengel, C E, Ann NY acad sci 103 (1963) 225. 13. Mengel, C E & Kelly, M G, Cancer res 21 (1961) 1545.
Exptl
Cell Res 69
14. Orden, L S van, Vugman, I, Bensch, K G & Giarman, N J, J pharmacol exptl ther 158 (1967) 195. 15. Schaer, J C & Schindler, R, Biochim biophys acta 147 (1967) 154. 16. Schindler, R, Biochem pharmacol 1 (1958) 323. 17. Schindler, R, Day, M & Fischer, G A, Cancer res 19 (1959) 47. 18. Schindler, R, Ramseier, L, Schaer, J C & Grieder, A, Exptl cell res 59 (1970) 90. 19. Thompson, J H, Spezia, C A & Angulo, M, Anal biochem 31 (1969) 321. Received March 24, 1971 Revised version received June 14, 1971
FERATIBN
AND SPECIFIC FUNCTIONS CELLS IN CULTU
9. LAISSUE, W. MARX,l Department
A. GRIEDER
of Pathology,
University
and R. SCHINDLER
of Bern, Switzerland
SUMMARY Cultures in vitro of a murine mastocytoma were incubated in a series of semisynthetic media. In order to produce specific nutritional deficiencies media were used in which one amino acid (L-leucine, L-histidine or L-tryptophan, respectively) was present in optimal and various suboptimal concentrations. Appropriate reduction of the concentration of L-leucine or L-histidine resulted in a decrease of cell multiplication and in an increase of the relative number of dead cells, whereas omission of r,-tryptophan had no appreciable effect on cell proliferation during the observation period of 96 h. After incubation of cells in these media during 4 days, the mean celldar histamine and Shydroxytryptamine contents as well as the incorporation of 35S0, into the protein-bound heparin fraction were determined. The mean cellular content of histamine or 5-hydroxytryptamine was reduced in media containing suboptimal concentrations of the corresponding precursor amino acid (L-histidine or L-tryptophan, respectively). In contrast, appropriate reduction of the kleucine level in the culture medium resulted in an increase of the mean cellular 5hydroxytryptamine content and an increased 35S0, incorporation.
Neoplastic murine mast cells during conroliferation in culture for long tinuous iods of time preserve the capacity to thetize and store histamine, 5-hydroxyamine (5-HT) and heparin [7, 8, 171. confronts us with the opportunity to examine in vitro the question if, in what sense and to what degree synthetic processes resulting in the formation of specific mast elf products are coupled with cell multilication. The present report is concerned with relationships between specialized cell functions and proliferation in neoplastic mast 1 Address: Department of Biochemistry, University of Southern California School of Medicine, Los Angeles, Calif. 90033, USA.
cell populations under a variety of su optimal culture conditions. Pn order the synthesis of histamine or rresponding precursor ammo acids, i.e. rAristidine or r,-tryptophan, were supplied to the culture medium in reduced concentrations, as compared to the levels providing for optimal growth conditions. Conversely, the concentration in the medium of L-leucine, i.e. an amino acid almost exclusively involved in protein synthesis, was modified.
Cell line and culture techniques A culture in vitro of a transplantable murine mastocytoma [41 was kindly provided by Dr G. A. Fischer,
58
J. Laissue et al.
Providence, R. I., USA (cell line HC). Similar to another neoplastic mast cell line [17], the HC cells, except for a minor proportion of their population, do not attach to the surface of culture vessels. From the HC cell population, a clonal subline (termed HCb,) was derived which is characterized by a relatively high cellular content of metachromatic granules. In order to maintain these characteristics, the cells were kept frozen at - 80°C in medium containing 10 % dimethyl sulfoxide. For each experiment, an aliquot of frozen cell suspension was thawed, and the cells were cultured during 2 to 3 weeks in standard medium containing 10 % horse serum. This medium has been previously described as medium I [15]. Subsequently, the celis were centrifuged and resuspended, at a cell density of approx. 1.5 x lo5 cells/ml, in 300 ml of the experimental medium to be tested. Experimental media differed from standard medium as follows: Undialysed serum was substituted by dialysed serum. Dialysis was carried out at 4°C against 6 times 20 vol of deionized water during a total of 24 h. In addition, MgSO, was replaced by an equimolar amount of MgCl,, thus reducing the concentration of inorganic sulfate to 0.1 pmole/ml (added in the form of streptomycin sulfate). This reduction in sulfate concentration had no detectable effect on cell multiplication during observation periods of up to 6 days. In order to produce specific nutritional deficiencies, various experimental media were used in which the concentration of one amino acid (L-leucine, L-histidine or L-tryptophan, respectively) was reduced as compared with that in the standard medium and the “optimal” experimental medium (see figs 2, 4, 5, 6). Concentrations of L-leucine, r.-histidine and L-tryptophan in “optimal” experimental medium were 200 PM, 50 ,uM and 20 PM, respectively. In an additional series of experimental media, different concentrations of dialysed serum were tested (5 %, 10 %, 20 %, 40 x). Unless mentioned otherwise, the cells were incubated in experimental media as spinner cultures during a total of 96 h. Every 24 h, the number of morphologically intact cells per ml of culture was determined, and an appropriate portion of the cell suspension was diluted with the prewarmed experimental medium to be tested in order to obtain again 300 ml of culture containing 1.5 x lo5 morphologically intact cells/ml.
Determination of cell multiplication the proportion of dead cells
and of
Cell multiplication was computed based on morphologically intact cells. Their number, as determined by hemocytometer counts with a phase contrast microscope, corresponded within 5 % to the number of cells remaining unstained for at least a few minutes in isotonic saline containing 0.1 % trypan blue. Similarly, the number of ghost cells, as seen in the phase contrast microscope, agreed within 5 % with that of trypan blue positive cells. Routine counts were, therefore, based on the appearance of cells in the phase contrast microscope. Exptl
Cell Res 69
Determination content
of cellular histamine and 5-HT
For determination of the cellular histamine content, an aliquot of the suspension culture containing 3 to 7 x lo6 cells was centrifuged, and the cells were resuspended in 2 ml of saline, heated to 100°C for 3 min and kept frozen at - 25°C until further processing. The histamine content of these samples was determined in the laboratory of Dr R. Keller, Dermatologische Universitatsklinik, Zurich, by bioassay on the guinea pig ileum as described previously [lo]. For determination of the cellular 5-HT content, a suspension containing approximately 5 x 10’ cells was centrifuged at 750 g for 5 min. The cells in the pellet were resuspended in 2 ml of isotonic saline, and 8.5 ml of 0.1 N HCl solution containing 1 mg ascorbic acid per ml were added, as suggested-by Thompson et al. [19]. Further extraction and fluorimetric determination of 5-HT was carried out according to the method of Bogdanski et al. [l].
Determination of 35S0, incorporation into the protein-bound heparin fraction of mast cells After 4 days incubation in exeerimental media. aliquots of each culture were incubated during 4d with 15 to 50 tiCi/ml of 35S0.,.Subseauentlv. the cell suspensions were chilled by immersion in an ice bath, and all further manipulations were carried out at 0°C. The cells were centrifuged at 750 g for 5 min, the supernatant was withdrawn, and 1.3 ml of cold trichloroacetic acid (TCA) was added to the cell sediment, followed by 1 ml saline containing approx. 10’ non-labeled cells as carrier material, and by 3 ml 5 % TCA. After mixing, the precipitate was sedimented by centrifunation and washed 6 times with 5 ml 5 % TCA andonce with 5 ml absolute ethanol. The final sediment was dissolved in 0.5 ml 0.1 N NaOH for determination of radioactivity in a liquid scintillation counter. It was shown in control experiments that practically all inorganic sulfate, smallmolecular weight sulfate derivatives and sulfolipids were removed by this procedure. The protein-linked glycosaminoglvcans, designated nrotein-bound henaiin fraction,were the major, if not the only components of the TCA-insoluble mast cell fraction which are known to take up sulfate in significant amounts, under the exnerimental conditions described. 3sS0, incorporationL into this fraction was found to be nearly linear during the incubation period of 4 h.
Qualitative morphological examination and size distribution analysis of mast cells A qualitative morphological evaluation of cells with respect to their content of metachromatically staining granules was performed with smears stained with toluidine blue. Cellular size frequency distributions were obtained with a Coulter Counter (model B) using a tube with an aperture of 200 pm diameter.
In a series of preliminary experiments, media containing various concentrations of dialysed serum were compared with respect to their effects on proliferation and amine content of neoplastic mast cells. The increase in cell number during an incubation period of 5 days was most rapid in media containing 10 or % dialysed horse serum and somewhat slower at levels of 5 or 40 %, respectively. No clear differences in mean cellular histamine and 5-HT content were observed in media containing 10, 20 or 40 % dialysed serum: while at the 5 % serum level, the
c
Lmm--
1
2-
3
L
(days); ordinate: (a) log cell multiplication; (b) relative number of morphologicaliy intact cells, 04. Cell multiplication (a) and proportion of morphologically intact cells (b) in neoplastic mast cell cultures, as a function of time of incubation in mediacontaining different concentrations of L-leucine: O-O, 200 ,uM; 3--O, IOOpM; o---o,50/~M; ~...a, 25 LLM. Values for log cell multiplication (log M) were calculated as follows: Fig. 1, Abscissa:
iog M= log
he
Nt
--xD NO
, where Nt is cell number
per ml at the time indicated on the abscissa; N,, cells/ ml at zero time, D, cumulative dilution factor of respective culture.
-0 5/-k-d 25
50
100 200 625 li5
25
50
_.A-_ 0 20
Fig. 2. Abscissa: concentration of amino acid indicated, p”M; ordinate: log ceil multiplication during the 96 h incubation period (A, B) and during the last 24 h of the 96 h incubation period (a, v ).
Cell multiplication in media containing different concentrations of L-leucine, L-histidine or Ltryptophan, respectively. The symbois represent results obtained in two independent experfments. Values for log cell muhiplication were calculated as described for fig. 1~
mean content of these amines per cell was reduced. In subsequent experiments, media containing 10% dialysed horse serum were used. Fig. 1a illustrates cell pro~~ferat~o~ patterns in a typical experiment in which the effects of media with different concentrations of L-leucine were studied. Reduction of the concentration sf this amino acid from ZOOto 50 pmoles/l resulted in a decrease sn cell multiplication which became apparent cx the second to third day of incubation, while at 25 pmoles/l, the number of morphologically intact cells began to decline 24 to 48 h after incubation of cells under these deficient culture conditions. Concomitant with the decrease in growth rate of the cell population, a decrease :n the relative number of morphologically intact cehs was observed, as seen in fig. 16. Similar results were obtained if the concentration of r=histidine in the culture medium was reduced, whereas omission of z-tryptophan from the medium did not affect cell multiplication to an appreciable extent during
60
J. Laissue et al. I
TRYPTOPHAN.
20
40
60
20 pM
80
100
Fig. 3. Abscissa: cell size (Coulter counter threshold values); ordinate: cells per size class, % of total cell number. Distribution of cell sizes in a culture containing 20 ,uM L-tryptophan (upper), as compared to a culture containing no added tryptophan (lower). Particles with sizes up to 8 were not considered in computing 100 % values.
the incubation period of 96 h. The results obtained with respect to cell multiplication under various culture conditions during the total incubation period of 96 h, as well as during the last 24 h, are summarized in fig. 2. The relative number of ghost cells observed in these experiments may be derived from data presented in figs 4 and 5 (differences between closed and open symbols). Size frequency distribution analysis indicated that after incubation in media containing low concentrations of L-leucine or L-histidine, or lacking L-tryptophan, the mean size of cells was markedly reduced as compared with control cultures in optimal medium. This is illustrated in fig. 3 for a culture without added tryptophan, as compared with a control culture containing 20 pmoles/l of this amino acid. After incubation of cells in experimental media during 4 days, the mean cellular Exptl Cell Res 69
histamine and 5-HT contents, as well as the incorporation of 35S0, into the proteinbound heparin fraction were determined in order to obtain quantitative information on the extent of specific cell functions under these culture conditions. As seen in fig. 4, in media containing suboptimal concentrations of r.-leucine, the mean cellular 5-HT content, as compared with controls (200,~M L-leucine), was increased up to 6 fold if referred to all cells and up to 19 fold if referred to morphologically intact cells. It should be noted that ghost cells also contained metachromatically staining granules. Variation of the concentration of L-histidine in the culture medium had little effect on the mean 5-HT content of cells. On the other hand, the mean 5-HT content was reduced by more than 60% if L-tryptophan was omitted from the medium. The mean histamine content per cell (fig. 5) was not affected to an appreciable extent by variation of the concentration of L-leucine or L-tryptophan in the culture medium; it was, however, profoundly modified by re-
1
Fig. 4. Abscissa: concentration of amino acid indicated, p”M; ordinate: 5-hydroxytryptamine content (logarithmic scale), pg/lOg morphologically intact cells (A, v ) and pg/109 cells including cell ghosts (A, 7). Cellular Shydroxytryptamine content after 4 days’ incubation in media containing different concentrations of L-leucine, L-histidine, or L-tryptophan, respectively. The symbols represent results obtained in two independent experiments.
duction of the concentration of L-histidine: in media containing 12.5 ,umoles/l of this amino acid, cellular histamine content was between 18 and 15 % of control values only. With respect to incorporation of 35S0, into the protein-bound heparin fraction of lastic mast cells (fig. 6), an increase was ved in medium containing L-leucine at a concentration of 25 ,umoles/l. Whereas the difference between controls (200 ,&I Lleucine) and cultures containing 25 ,uM L-leucine, due to the high variation of control values, is not significant, differences between cultures containing this amino acid at 25 and 50 y&I, or 25 and 100 @I, respectively, are significant (P ~0.05) if the t test is used. Modifications of other culture conditions did not result in appreciable changes in uptake of label. Ghost cells obtained by repeated freezing and thawing did not incorporate significant amounts of 35S0,. The results presented in fig. 6 refer, therefore, to morphologically intact cells only.
It has been reported previously that cellular amine levels in neoplastic mast cell cultures
T 20;
Fig. 6. Abscissa: concentration of amino acid dicated, ,&I; ordinate: WQ incorporated into
fnpro-
tein-bound heparin fraction by IO6 intact cells during 4 h, % of total radioactivity. Sulfate incorporation into protein-bound heparin fraction after 4 days’ incubation in media co~ta~~~~~ different concentrations of L-leucine, z-histidine or L-tryptophan, respectively. The symbols represent results obtained in two independent experiments.
underwent pronounced fluctuation which did not appear to be relat ences in culture conditions [2]. In the studies too, differences in cellular contents and sulfate i~cQr~orati~~ between control cultures of individual experiments were observed, as seenin figs 4 to 6 (controls dium compositions 20 with the sam istidine and 20 PM try leucine, 50 p phan). In every ex~e~~rne~t, therefore, controls were incubated in parallel with c~~~~~r~s containing one amino acid in various reduced
-c
Fig. 5. Abscissa: concentration of amino acid indicated, pi%; ordinate: histamine content, pg/lO’ morpholo-
gically intact cells (A, v ) and ,ug/lO’ cells including Cell ghoStS(A, v >. Cellular histamine content after 4 days’ incubation in media containing different concentrations of L-leucine, L-histidine or L-tryptophan, respectively. The symbols represent results obtained in two independent experiments.
system as previously described [lg]. The possibility can, t not be excluded that amino acid le not entirely constant with time, but underwent fluctuations due to daily addition of fresh medium and subsequ amino acids by the cultured c small amounts of free amin were supplied to the cultu serum. Nevertheless, the results indicate that the observed effects were dependent orn the concentration
of t e amino acid tested. Exptl Cd Res 59
62 J. Laissue et al. A significant decline in cell multiplication concomitant with an increase in the relative number of dead cells was observed on the 2nd to 3rd day of incubation in media deficient in leucine or histidine. Upon omission of tryptophan from the culture medium, cell multiplication continued at a rate comparable with that in control medium during the incubation period of 4 days. In experiments of longer duration, however, omission of tryptophan resulted in a decrease in growth rate beginning approx. 6 days after incubation in the deficient medium. In addition to the effects on cell multiplication, a decrease in mean cell size was observed in cultures containing reduced concentrations of leucine or histidine, or lacking tryptophan. This phenomenon may be attributed to the effects of amino acid depletion on cellular protein metabolism and/or to changes in cell cycle characteristics induced by deficient media [5]. The possibility might be considered that the changes in average cellular amine content and sulfate incorporation, as observed after 4 days’ incubation in amino acid deficient media, have occurred as a result of selection of subpopulations which, under the relatively unfavorable culture conditions, would outgrow the majority of the original cell population. This would imply that the expression of mast cell function, as determined in our studies, is transmitted from parent cell to progeny in the process of cell proliferation. In view of the short duration of the experiments, however, it appears unlikely that the observed modifications in mast cell characteristics are attributable primarily to a selection process. Furthermore, omission of tryptophan or reduction of the histidine concentration to 25 pmoles/l resulted in pronounced changes in average 5-HT or histamine content without a significant decrease in cell multiplication. These findings are in favour of an Exptl Cell Res 69
adaptive change of specific mast cell functions under modified culture conditions. On the other hand, despite the recent clonal origin of the HCb, line, a considerable heterogeneity of the cell population with respect to the amount of metachromatic material per individual cell was observed. This is in agreement with the reported variability in 5-HT contents of individual mastocytoma cells [12, 141. Furthermore, variations in amine content during the cell division cycle, as well as differentiation of “precursor” cells into more mature cell types, cannot be excluded. As long as the relationships between proliferation kinetics and specific functions of mastocytoma cells are not known, it is difficult to draw any conclusions as to the mechanisms underlying the induced changes in average amine content and sulfate incorporation. Cellular amine levels are a function of the synthesis rate and, in addition, depend on the rate of cell multiplication and possibly rates of degradation and/or release of amines into the medium. Mastocytoma cells form 5-HT and histamine from the precursor amino acids, tryptophan and histidine, respectively [7, 91. Free L-tryptophan appears to be the major, if not the sole, precursor of 5-HT [2, 161.It is, therefore, not surprising that reduction of the concentration of tryptophan or histidine in the medium resulted in a decrease of cellular 5-HT or histamine contents, respectively, as seen in figs 4 and 5. On the other hand, n.o increase in cellular amine levels was observed in mastocytoma cells cultured in media containing tryptophan or histidine in concentrations exceeding those providing for optimal growth conditions [2]. In cultures of mastocytoma cells, no catabolites of 5-HT or histamine were detected intracellularly or in the medium [7]. Loss of amines and, to a lesser extent, of heparin from cells into the culture medium has been
Proliferation reported 13, 6]; no data on the extent of cell death were, however, presented, although media containmg suboptimal concentrations of tryptophan or istidine were used. The pronounced increase in cellular 5-HT content without concomitant increase in histamine content in leucine-deficient media is of special interest. On the basis of the available data, release of amines by dead cells and their uptake by surviving cells cannot be excluded. The amounts of 5-HT that would thus become available are, however, barely sufficient, under the most favorable assumptions, to fully account for the observed increases in cellular 5-HT content. It appears likely, therefore, that under these suboptimal culture conditions, 5-HT metabolism by the neoplastic mast cells is characterized by an enhanced rate of synthesis and/or a reduced rate of release. A similar increase in cellular 5-HT content has been observed in mastocytoma cell cultures following incubation during 24 h in the presence of actinomycin D at an appropriate growthinhibitory concentration [l I]. Treatment of mice bearing a transplantable mastocytoma with doses of cyclophosphamide causing n also resulted in a markedly content of the surviving tumor cells [13]. Labeled sulfate has been shown to be incorporated into heparin by cultured mastocytoma cells [S]. Since inorganic sulfate, small-molecular weight sulfate derivatives and sulfolipids were removed during the extraction procedure, the radioactivity in the TCA-insoluble fraction may be considered to represent label incorporated into the protein-bound heparin fraction, and, therefore, to give an indication of the rate of heparin synthesis during the incubation period of 4 h. A marked increase in 35S0, incorporation was observed in media containing Eeucine at 25 ,umoles/l, i.e. under
and specific functkw conditions
resulting
of m~stQcytom~ cells
53
also in an increase in
In conclusion, the results obtained indicate that appropriate modifications of cell cdture conditions may affect ~ro~~ferat~v~ activity as well as specific functions of neoplastic mast cells. CelEular S-MT or histamine contents were preferentially affected In media containing the corresponding precm-sor amino acid at reduced concentrations: a decrease in amine content was already detectable at a precursor amino acid level which still provided for unimpaired cell proliferation. Reduction of the concentration of an amino acid not directly involved in amine synthesis, such as leucine, resulted in an inhibition of cell multiplication and an increase in ce?lular 5-HT content and sulfate incorporation into the protein-bound heparln fraction. Thus, the variations in the expression of specialized cell functions, as observed under modified culture conditions, do not necessarily paraM the changes in cell proliferation. In addition, 5-HT content, histamine content, and sulfate incorporation into the protein-bound begarm fraction, which are representative of three different functions of the same cell, may be modified independently from each other, This work was supported by the Swiss National Science Foundation -a& by research grant 5 ROT HE 01595 from the National Heart and Lung Institute, USPHS. The authors express their gra&ude tcp Professor H. Cottier for his encouragement and continued interest, and to Dr R. Keller,bermatoloaische Un~versitatsklinik. Zurich. for his kind offer to have the histamine dioassays’ carried out jn h,Es laboratory. One of ns (J. L.) is indebted to Dr W. Burkard and Dr F. Gey, Hoffmann-&a Roche & Co., Base], for being introduced into the technique of’ 5-HT determination. The expert technical assistance of Miss C. Hiirni is gratefully acknowledged.
1. Bogdanski, F, Pletscher, A, Brodie, B Udenfriend, S, 3 pharmacol expel ther I3 7 (I 956) 82. 2. Day, M & Green, J P, J physiol 164 (1962) 220. 3. - Biochem pharmaco: II (1962) 1043.
64
J. Laissue et al.
4. Furth, J, Hagen, P & Hirsch, E I, Proc sot exptl biol med 95 (1957) 824. 5. Gautschi, J R & Schindler, R, Experientia 26 (1970) 693. 6. Green, J P, Biochem pharmacoll2 (1963) 577. 7. - Europ j pharmacol 3 (1968) 68. 8. Green, J P & Day, M, Biochem pharmacol 3 (1960) 190. 9. Hagen, P & Lee, F L, J physiol 143 (1958) 7P. 10. $eller, R & Beeger, I, Int arch allergy 22 (1963) 11. Mannaioni, P F & Giarman, N J, Pharmacol res commun 1 (1969) 218. 12. Mengel, C E, Ann NY acad sci 103 (1963) 225. 13. Mengel, C E & Kelly, M G, Cancer res 21 (1961) 1545.
Exptl
Cell Res 69
14. Orden, L S van, Vugman, I, Bensch, K G & Giarman, N J, J pharmacol exptl ther 158 (1967) 195. 15. Schaer, J C & Schindler, R, Biochim biophys acta 147 (1967) 154. 16. Schindler, R, Biochem pharmacol 1 (1958) 323. 17. Schindler, R, Day, M & Fischer, G A, Cancer res 19 (1959) 47. 18. Schindler, R, Ramseier, L, Schaer, J C & Grieder, A, Exptl cell res 59 (1970) 90. 19. Thompson, J H, Spezia, C A & Angulo, M, Anal biochem 31 (1969) 321. Received March 24, 1971 Revised version received June 14, 1971