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Tissue-specificity of reactivity to concanavalin-A of human cells in culture
ExperimentalGerontology, Vol. 18, pp. 293-301, 1983 Printed in the USA. All rights reserved.
0531-5565/83 $3.00 + .00 Copyright©1983 Pergamon Press Ltd
TISSUE-SPECIFICITY OF REACTIVITY TO CONCANAVALIN-A OF HUMAN CELLS IN CULTURE
SADAYUKI BAN*'"~ a n d S H o z o IIDA'~ Department of Pathology* and Clinical Laboratoriest, Radiation Effects Research Foundation, Hiroshima 730, Japan
(Received 12 May 1983) A b s t r a c t - Concanavalin-A (Con-A) reactivity was studied to identify the tissue-specificity of cells established from various normal human tissues. Cells were treated with Con-A-labelled human red blood cells (C-RBC). C-RBC was not adsorbed on the cells derived from the bone marrow, skin and liver. Lung-derived fibroblast cells showed weak C-RBC adsorption. Kidney-derived cells showed epithelial morphology and easily adsorbed C-RBC. These suggest that a large number of Con-A receptors exists on the membrane surface of kidney cells.
INTRODUCTION ESTAaLISn~ENT OF a simple method for identifying the neoplastic nature of cultured cells is important for the advancement of cancer research. The indices of neoplastic transformations most frequently used at present include: a) loss of post-confluent inhibition of cell proliferation (Holley and Kierman, 1968; Pollack et al., 1968); b) proliferative ability in soft agar (MacPherson, 1963; Freedman and Shin, 1974; Tucker et al., 1977; Sanford et al., 1967; Persky, 1982); c) cellular immortality (Igel et al., 1975); d) loss of diploidy (Sumner, 1981; Trent and Salmon, 1981; Yunis, 1981); and e) changes in Con-A reactivity (Oishi et al., 1981). Of these, examining changes in the reactivity of cells to concanavalinA (Con-A) by adsorbing red blood cells treated with Con-A (abbreviated as C-RBC) (Con-A hemadsorption test) has been employed because of the simplicity of the procedure and the specificity for neoplastic transformation is high. The method is especially useful to detect tumor-associated characteristics of fibroblasts easily obtained in culture. Recently, Aizawa et al. (1980 a,b) succeeded in employing C-RBC adsorption as an index for the aging of cells in vitro. On examining C-RBC adsorption of cultured fibroblasts derived from normal human fetal lungs, they found that absorption was higher in older cells than in young cells. The number of C-RBC adsorbed by the cells was proportional to the age-related increase of the cell membrane surface area. There was no change in the number of Con-A receptors per unit area of the membrane surface of the cells used in their study. We examined whether C-RBC adsorption of the membrane surface of cultured human 'Present address: Biology Department, Brookhaven National Laboratory, NY 11973. Address all correspondence to Dr. S. Ban. 293
294
BAN AND n D A
cells had tissue-specificity. Cells established from tissue samples of lung, liver, skin, and bone-marrow had a fibroblast morphology (or fibroblast-like cells) and hardly adsorbed any C-RBC. However, most of the cells established from the kidney showed an epithelial morphology with very strong C-RBC adsorption. Materials used in this study were obtained from organs which had been confirmed at autopsy to have neither tumors nor degeneration. METHODS AND PROCEDURE Tissue and cells All cells used in this study were established in our laboratory and were obtained from resected tissue during autopsies. Tissue sections were washed thoroughly in a buffer solution containing 500 # g / m l of streptomycin and 500 U / m l of penicillin. These sections were minced into 1 m m 3 or smaller pieces in 5 cm plastic dishes (Falcon Co. USA) containing a small a m o u n t of culture solution. The small pieces were put into Eagle's M E M culture solution (Nissui Pharmaceuticals, Tokyo) containing fetal bovine serum (Hy-Clone Co. USA), and cultured at 37°C in 5% Co~-95% air. After 3-10 days, the culture medium was replaced with Eagle's M E M medium containing 10% fetal bovine serum. W h e n numerous cells began to proliferate from the pieces of tissue, subcultures were initiated according to methods previously reported (Ban et al., 1980 a, 1982). Eagle's M E M supplemented with 10% fetal bovine serum was used for the maintenance and subculture of cells. A solution containing 0.125% trypsin (Difco, USA) and 0.01% E D T A (Wako Chemicals, Tokyo) was used for cell harvesting. Figure 1 shows the tissues used and the ages of the donors.
Preparation o f Con-A-labelled red blood cells (C-RBC) The m e t h o d of Oishi et al. (1981) was employed in the preparation of C o n - A labelled RBC. Two ml of h u m a n male blood (type A or type O) was collected by venepuncture. After suspending in 5 ml of PBS ( - ) , the sample was washed twice by centrifugation for five minutes at 1,000 rpm. Then 0.03 ml of the red blood precipitate was added to 3 ml of PBS, in which concanavalin-A (Con-A; Sigma, USA) had been dissolved at various concentrations. Cells and Con-A were allowed to react at 37°C for 30 minutes. After reaction, the red blood cells were washed and centrifuged twice, and 3 ml of the PBS ( - ) was dispensed into each test tube. The C-RBC were freshly prepared for each experiment.
Kidney
Lung
~
Bone-
o
marrow
--
0
0
VV
- -
Skin
--
Liver
--
0
•
] Fetus
~
•
//
i
0
~ V
dis
!
I
!
0
i
I
!
!
50
Age
(~)0
of
Tissue
Donors
!
I
[ i00
(years)
FIG. 1. Origin of established cells and ages of cell donors. Each organ was confirmed to have neither tumor nor cellular degeneration at the time of the autopsy.
TISSUE-SPECIFICITY
OF
295
REACTIVITY TO CONCANAVAL|N-A
Con-A hemadsorption test For all cell types, the fifth to the tenth generation were used in the tests. The target cells were cultured in advance for two days in aMEM culture solution supplemented with 10% fetal bovine serum. 2.5 × 10~ cells were inoculated in wells with a growing surface area of 1.6 cm 2 (Lab-Tek, USA), and cultured overnight at 37°C in 5% CO2. After the cells were washed twice in PBS ( + ) , 1 ml of C-RBC was added to each well. After the mixture was allowed to react at 37°C for 10 minutes, the cells were washed three times in PBS ( + ) . If C-RBC remained on the surface of the culture chamber, the washing procedure was repeated. Before the ceils dried, they were fixed for three minutes in aceton-formalin fixative (pH 6.6). After washed in water, they were stained iu Giemsa's staining solution, and enclosed with a cover glass. Vermilion-colored human red cells were adsorbed on cells stained with Giemsa. The number of C-RBC adsorbed on the surface of the 200 or more cells in each slide was counted under a microscope. RESULTS F i g u r e 2 s h o w s t h e n u m b e r a n d f r e q u e n c y o f C - R B C a d s o r b e d per cell u s i n g v a r i o u s o r g a n d e r i v e d cells o b t a i n e d f r o m t h e s a m e i n d i v i d u a l ( b o y f o u r y e a r s o l d ) . 50 40
5 ug/ml
Con-A
30 20 10
40 30 20 z uJ i0 ac u-
u~ LU
O--
0 40 30 20 i0
40 30 20
10
i
i
I
i
i
i
i
i
NUMBER OF RBC PER CELL
i
i
(a)
FIG. 2. Distribution of C-RBC adsorption (human red blood cells labelled with Con-A) by culture cells established from the various organs of a four-year-old boy. Concentrations of Con-A used to label human red blood cells are shown, a) Kidney derived cells, b) Lung derived cells, c) Femoral bone marrow derived cells, d) Skin derived cells, e) Liver derived cells.
g_
0
10
2O
30
40
0
lO
20
30
lO
~ 4x.
w
20
~
30
40
0
i0
20
30
40
50
60
70
80
i
Jg/ml Con-A
20 ~g/~l Con-A
i0 ug/ml Con-A
NUMBER OF RBC PER CELL
.L
_I
5 ~g/ml Con-A
(b)
~_
g
a:
~
0
1Q
20
30
4O
50
6O
7O
80
90
0
2o
30
40
50
60
70
80
90
i00
o
i
i
~
i
NUMBER OF RBC PER
I
20 pg/ml C o n - A
i
5 pg/ml C o n - A
CELL
I
I
I
40 ug/ml C o n - A
I
io pg/ml C o n - A
(c)
bJ
-
0
LP*
0
u'1
(d)
4o
~o
0-
0
1
I I
NUMBER OF RBC PER CELL
I
0
lo
2o
3o
10
4o
5o
30
--
0 6o
lo
20
40
50-
6O
2o
~_
~-
~
Con-A
60
0 70
i0
20
30
40
50
60
70
7O
~g/ml
90 80
3o
40
Con-A
~: ul
--
Con-A
ug/ml
80
~g/ml
i0
90
20
Con-A
~
0-
U-
5 ~g/ml
~
2o
~
30
50-
60
.o!
80
90
i00
5
~g/ml
J
i
l
l
l
;
I0 pg/ml Con-A
NUMBEROF RBC PER CELL
40 ~g/ml C o n - A
_L b
U..--
|
20 pg/ml Con-A
Con-A
(e)
E Z #
© Z > Z >
0
-i
;m n~ > c~
o
.<
C m
298
BAN AND
liDA
Cells derived from the femoral bone-marrow, skin and liver showed almost no C-RBC adsorption ability, while lung cells showed weak C-RBC adsorption ability. A slight concentration dependency was observed up to a Con-A concentration of 20/~g/ml. The above four cell types showed a morphology quite similar with that of fibroblasts. Kidney-derived cells showed epithelial morphology and remarkably high C-RBC adsorption ability. Con-A unlabelled RBC were not adsorbed in the cells (data not shown). RBC labelled with Con-A at low concentrations (5 ~g/ml) were easily adsorbed in kidney cells. However, even when the concentration of Con-A was increased to more than 10 #g/ml, adsorption distribution of C-RBC underwent little change. It is evident that cell populations having no C-RBC adsorption exist at a fixed ratio. As a result of the data shown in Figure 2 (c-e), cells adsorbing six or more C-RBC were regarded as Con-A reactivity positive and cells adsorbing no RBC as Con-A reactivity negative. The patterns of C-RBC adsorption of cells obtained from various organs of the individuals (Figure 1) mostly showed, with the exception of kidney ceils, the trends observed in Figure 2. Figure 3 shows in summary the ability to adsorb RBC labelled with Con-A at a concentration of 40/zg/ml. A comparison was made of the percent frequency levels of Con-A positive cells (cells adsorbing six or more C-RBC per cell) and Con-A negative cells (cells adsorbing no C-RBC). On comparison by age of cell donor (those less than four indicated as y in the figure and those more than 44 indicated as a in the figure), there was
~6 I00
RBC/cell
0 RBC/cell
--
af ~
oaf 0 af 0 am Oam
af
<7 af ~af
oym
s g~
V~ tsym
@ym
•ym
50
oaf
oaf OY m
Oym vym
AYm @ym ~af af
V ym
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l
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I
af af ~/~ am V yra af
l
y~ ~f%0
!
I
I
I
I
am
!
mN
Cell
Origins
FIO. 3. C-RBC adsorption ability of culture cells established from a variety of normal human tissues. The left panel demonstrates the percent frequency of cells adsorbing six or more C-RBC per cell, and the right panel shows the percent "frequency of cells adsorbing no C-RBC. a: aged donor (donor age 44 years and over), y: young donor (donor age four years and under), m: male, f: female
TISSUE-SPECIFICITY OF REACTIVITY TO CONCANAVALIN-A
299
no association between Con-A reactivity and donor age for skin and kidney fibroblasts. Lung fibroblasts showed a tendency for Con-A reactivity to be higher in younger cells than in aged cells. However, a large number of cases are needed in order to detect any cell type association between Con-A reactivity and donor age. Con-A reactivity of skin fibroblasts was very low. Con-A reactivity of liver and bone marrow cells was also low. In Figure 4, the percent frequency of kidney cells adsorbing six or more C-RBC per cell is shown in relation to the concentration of Con-A, The cells were established from a portion of kidney tissues collected from a premature fetus (chromosome karyotype, 44 + XX), four-year-old boy, 62-year-old male, 70-year-old female, 79-year-old female, 80-year-old male, and 85-year-old male. C-RBC adsorption abilities for two cases of subjects four years and under, and those for five cases of 62 years and over were distributed rather widely. That is, there seems to be no correlation between the age of cell donors and Con-A reactivity.
100
50
i0
Concentration
20
30
of C o n - A
40 (~g/ml)
FIG. 4. C o n - A concentration-dependency in C-RBC adsorption of kidney derived culture cells. The percent fre-
quency of cells adsorbing six or more C-RBC per cell was obtained. There was no difference in C-RBC adsorption ability between tissue donors of four years and under ( © ) and those of 62 years and over ( O ) .
300
BAN AND [IDA
DISCUSSION Normal human diploid cells, which have a finite doubling potential in culture, are widely used as an in vitro aging model. Especially, fibroblasts established from lung (Ban et al., 1980 a,b, 1981) and skin of human fetuses (Nikaido et al., 1980) and skin tissue of human adults (Nikaido et al., 1980) are frequently employed because they are easily available, can be established easily in culture, and have high proliferative ability. Cells derived from other organs are also excellent material for studies on aging (Ban et al., 1982). Identification of species-specificity or tissue-specificity of the proliferating fibroblasts and other cells is very meaningful for the advancement of studies on cellular aging. Furthermore, identifying the presence or absence of tumor-specificity in fibroblasts established from tumor tissues is of importance for promoting cancer studies. Hayflick et al. (1961, 1965) suggested the presence of tissue-specificity in the in vitro life-span of fibroblasts. Oishi et al. (1981) reported that fibroblasts established from human prostate cancer show high reactivity to Con-A. We studied Con-A reactivity of various cells for the purpose of identifying tissuespecificity of cells proliferating from normal human tissues. Materials were collected after they were confirmed to be free of either tumor or degradation. Cells proliferating from the kidney, lung, and skin are easily established, and proliferation of the established cells is good. Con-A reactivity of skin fibroblasts (Figures 2d and 3) is very low, and that of the liver cells (Figures 2e and 3) and femoral bone marrow cells (Figures 2c and 3) also seems to be low. Con-A reactivity of lung fibroblasts is slightly higher in the younger cells (Figures 2b and 3). However, kidney cells showed remarkably high C-RBC adsorption ability compared with the four types of cells mentioned above. Kidney cells are characterized by their Con-A reactivity at a low concentration (5 ~g/ml) and the ratio of cells adsorbing 50 or more C-RBC per cell is high. However, many kidney cells do not adsorb C-RBC even when the concentration of Con-A used to label human red blood cells is increased (Figure 2). Some of the kidney cells that do not adsorb C-RBC demonstrate fibroblast-like morphology, but most are difficult to classify morphologically. From the above observations, it is evident that Con-A reactivity is tissue-specific, and especially, that a large number of Con-A receptors exist on the membrane surface of kidney cells. However, Con-A reactivity of the individual kidney cells differed greatly, whiCh suggests that kidney cells represent a mixed population of proliferating cells. However, an overwhelmingly large number of these cells have many Con-A receptors on their membrane surface. The number of Con-A receptors may possibily change during the cell cycle, but Aizawa et al. (1980, b) do not believe this to be true. Most of the kidney cells had an epithelial morphology, which remained stable throughout the in vitro life-span of the cells. With some exceptions, it is very difficult to morphologically differentiate culture into epithelial cells and fibroblasts. Particularly, morphological kinetic changes, frequently observed as subcultures, are repeated after the first generation of culture cells is established. The Con-A hemadsorption test is useful as an indicator of changes in the growth kinetics of culture cells. SUMMARY An examination was made of the ability of culture cells that were established from various normal human tissues to adsorb concanavalin-A treated red blood cells (Con-A hemadsorption test). As most tumor cells have a high reactivity to Con-A, the Con-A
TISSUE-SPECIFICITY OF REACTIVITY TO CONCANAVALIN-A
301
h e m a d s o r p t i o n test is o f t e n used as a c o n v e n i e n t i n d i c a t o r for i d e n t i f y i n g the n e o p l a s t i c c h a r a c t e r i s t i c o f f i b r o b l a s t s . This s t u d y r e v e a l e d , h o w e v e r , t h a t c u l t u r e d cells f r o m norm a l k i d n e y tissue have a r e m a r k a b l y high reactivity to C o n - A . T h e m o r p h o l o g y o f the k i d n e y cells was similar to t h a t o f e p i t h e l o i d cells. H o w e v e r , C o n - A reactivity o f f i b r o b l a s t s (or f i b r o b l a s t - l i k e cells) d e r i v e d f r o m lung, skin, b o n e - m a r r o w , a n d liver was low. T h e C o n - A h e m a d s o r p t i o n test is n o t o n l y useful for i d e n t i f i c a t i o n o f n e o p l a s t i c cells, b u t also p r o v i d e s i n f o r m a t i o n o f cellular specificity. REFERENCES AIZAWA,S., MITSUI,Y. and KURIMOTO,F. (1980) (a) Exp. Cell Res. 125, 287. AIZAWA,S., MITSUI, Y., KURIMOTO,F. and MATSUOKA,K. (1980) (b) Exp. CellRes. 125, 297. BAN, S., NIKAIDO,O. and SUGAnARA,T. (1980) (a) Radiat. Res. 81, 120. BAN, S., NIKAIDO,O. and SUGA~L~RA,T, (1980) (b) Exp. Gerontol. 15, 539. BAN, S., IKUSmMA,T. and SUGAnARA,T. (1981) Radiat. Res. 87, 1. BAN, S., hDA, S., CHIN, N. and AwA, A.A. (1982) J. Radiat. Res. 23, 88. FREEDMAN,V.H. and SHIN, S.I. (1974) Cell, 3, 355. HAVFLICK,L. and MOORHEAD,P.S. (1961) Exp. Cell Res. 25, 585. HAVFLICK,L. (1965) Exp. Cell Res. 37, 614. HOLLEr, R.W. and KIERNAN,J.A. (1968) Proc. Nat. Acad. Sci. USA, 60, 300. IGEL, H.J., FREEMAN,A.E., SPmWAK,J.E. and KLEINFELD,K.L. (1975) In vitro, 2, 117. MAcPHERSON, I. (1963) J. Nat. Cancer Inst. 30, 795. NXKAIDO,O., BAN, S. and SUGAHARA,T. (1980) Adv. Exp. Med. Biol. 129, 303. OIsnI, K., ROMmN, J.C. and SCHRSDER, F.H. (1981) The Prostate, 2, 11. PERSKV, B., THOMPSON,S.P., MEVSKENS,JR., F.L. and HENDRIX, M.J.C. (1981) In vitro, 18, 929. POtLACK, R.E., GREEN H. and TODAROG.J. (1968) Proc. Nat. Acad. Sci. USA, 60, 126. SANFORD,K.K., BARKER,B., WOODS,M.W., PARSHAD,R. and LAW, L.W. (1967) J. Nat. Cancerlnst. 39, 705. SUMNER, A.T. (1981) Anticancer Res. 1,205. TRENT, J.M. and SAtMON, S.E. (1981) Cancer Genet. Cytogenet. 3, 279. TUCKER, R.W., SANrORD,K.K., HANDLEMAN,S.L. and JONES, G.M. (1977) Cancer Res. 37, 1571. YUNIS, J.J. (1981) Hum. PathoL 12, 494.
0531-5565/83 $3.00 + .00 Copyright©1983 Pergamon Press Ltd
TISSUE-SPECIFICITY OF REACTIVITY TO CONCANAVALIN-A OF HUMAN CELLS IN CULTURE
SADAYUKI BAN*'"~ a n d S H o z o IIDA'~ Department of Pathology* and Clinical Laboratoriest, Radiation Effects Research Foundation, Hiroshima 730, Japan
(Received 12 May 1983) A b s t r a c t - Concanavalin-A (Con-A) reactivity was studied to identify the tissue-specificity of cells established from various normal human tissues. Cells were treated with Con-A-labelled human red blood cells (C-RBC). C-RBC was not adsorbed on the cells derived from the bone marrow, skin and liver. Lung-derived fibroblast cells showed weak C-RBC adsorption. Kidney-derived cells showed epithelial morphology and easily adsorbed C-RBC. These suggest that a large number of Con-A receptors exists on the membrane surface of kidney cells.
INTRODUCTION ESTAaLISn~ENT OF a simple method for identifying the neoplastic nature of cultured cells is important for the advancement of cancer research. The indices of neoplastic transformations most frequently used at present include: a) loss of post-confluent inhibition of cell proliferation (Holley and Kierman, 1968; Pollack et al., 1968); b) proliferative ability in soft agar (MacPherson, 1963; Freedman and Shin, 1974; Tucker et al., 1977; Sanford et al., 1967; Persky, 1982); c) cellular immortality (Igel et al., 1975); d) loss of diploidy (Sumner, 1981; Trent and Salmon, 1981; Yunis, 1981); and e) changes in Con-A reactivity (Oishi et al., 1981). Of these, examining changes in the reactivity of cells to concanavalinA (Con-A) by adsorbing red blood cells treated with Con-A (abbreviated as C-RBC) (Con-A hemadsorption test) has been employed because of the simplicity of the procedure and the specificity for neoplastic transformation is high. The method is especially useful to detect tumor-associated characteristics of fibroblasts easily obtained in culture. Recently, Aizawa et al. (1980 a,b) succeeded in employing C-RBC adsorption as an index for the aging of cells in vitro. On examining C-RBC adsorption of cultured fibroblasts derived from normal human fetal lungs, they found that absorption was higher in older cells than in young cells. The number of C-RBC adsorbed by the cells was proportional to the age-related increase of the cell membrane surface area. There was no change in the number of Con-A receptors per unit area of the membrane surface of the cells used in their study. We examined whether C-RBC adsorption of the membrane surface of cultured human 'Present address: Biology Department, Brookhaven National Laboratory, NY 11973. Address all correspondence to Dr. S. Ban. 293
294
BAN AND n D A
cells had tissue-specificity. Cells established from tissue samples of lung, liver, skin, and bone-marrow had a fibroblast morphology (or fibroblast-like cells) and hardly adsorbed any C-RBC. However, most of the cells established from the kidney showed an epithelial morphology with very strong C-RBC adsorption. Materials used in this study were obtained from organs which had been confirmed at autopsy to have neither tumors nor degeneration. METHODS AND PROCEDURE Tissue and cells All cells used in this study were established in our laboratory and were obtained from resected tissue during autopsies. Tissue sections were washed thoroughly in a buffer solution containing 500 # g / m l of streptomycin and 500 U / m l of penicillin. These sections were minced into 1 m m 3 or smaller pieces in 5 cm plastic dishes (Falcon Co. USA) containing a small a m o u n t of culture solution. The small pieces were put into Eagle's M E M culture solution (Nissui Pharmaceuticals, Tokyo) containing fetal bovine serum (Hy-Clone Co. USA), and cultured at 37°C in 5% Co~-95% air. After 3-10 days, the culture medium was replaced with Eagle's M E M medium containing 10% fetal bovine serum. W h e n numerous cells began to proliferate from the pieces of tissue, subcultures were initiated according to methods previously reported (Ban et al., 1980 a, 1982). Eagle's M E M supplemented with 10% fetal bovine serum was used for the maintenance and subculture of cells. A solution containing 0.125% trypsin (Difco, USA) and 0.01% E D T A (Wako Chemicals, Tokyo) was used for cell harvesting. Figure 1 shows the tissues used and the ages of the donors.
Preparation o f Con-A-labelled red blood cells (C-RBC) The m e t h o d of Oishi et al. (1981) was employed in the preparation of C o n - A labelled RBC. Two ml of h u m a n male blood (type A or type O) was collected by venepuncture. After suspending in 5 ml of PBS ( - ) , the sample was washed twice by centrifugation for five minutes at 1,000 rpm. Then 0.03 ml of the red blood precipitate was added to 3 ml of PBS, in which concanavalin-A (Con-A; Sigma, USA) had been dissolved at various concentrations. Cells and Con-A were allowed to react at 37°C for 30 minutes. After reaction, the red blood cells were washed and centrifuged twice, and 3 ml of the PBS ( - ) was dispensed into each test tube. The C-RBC were freshly prepared for each experiment.
Kidney
Lung
~
Bone-
o
marrow
--
0
0
VV
- -
Skin
--
Liver
--
0
•
] Fetus
~
•
//
i
0
~ V
dis
!
I
!
0
i
I
!
!
50
Age
(~)0
of
Tissue
Donors
!
I
[ i00
(years)
FIG. 1. Origin of established cells and ages of cell donors. Each organ was confirmed to have neither tumor nor cellular degeneration at the time of the autopsy.
TISSUE-SPECIFICITY
OF
295
REACTIVITY TO CONCANAVAL|N-A
Con-A hemadsorption test For all cell types, the fifth to the tenth generation were used in the tests. The target cells were cultured in advance for two days in aMEM culture solution supplemented with 10% fetal bovine serum. 2.5 × 10~ cells were inoculated in wells with a growing surface area of 1.6 cm 2 (Lab-Tek, USA), and cultured overnight at 37°C in 5% CO2. After the cells were washed twice in PBS ( + ) , 1 ml of C-RBC was added to each well. After the mixture was allowed to react at 37°C for 10 minutes, the cells were washed three times in PBS ( + ) . If C-RBC remained on the surface of the culture chamber, the washing procedure was repeated. Before the ceils dried, they were fixed for three minutes in aceton-formalin fixative (pH 6.6). After washed in water, they were stained iu Giemsa's staining solution, and enclosed with a cover glass. Vermilion-colored human red cells were adsorbed on cells stained with Giemsa. The number of C-RBC adsorbed on the surface of the 200 or more cells in each slide was counted under a microscope. RESULTS F i g u r e 2 s h o w s t h e n u m b e r a n d f r e q u e n c y o f C - R B C a d s o r b e d per cell u s i n g v a r i o u s o r g a n d e r i v e d cells o b t a i n e d f r o m t h e s a m e i n d i v i d u a l ( b o y f o u r y e a r s o l d ) . 50 40
5 ug/ml
Con-A
30 20 10
40 30 20 z uJ i0 ac u-
u~ LU
O--
0 40 30 20 i0
40 30 20
10
i
i
I
i
i
i
i
i
NUMBER OF RBC PER CELL
i
i
(a)
FIG. 2. Distribution of C-RBC adsorption (human red blood cells labelled with Con-A) by culture cells established from the various organs of a four-year-old boy. Concentrations of Con-A used to label human red blood cells are shown, a) Kidney derived cells, b) Lung derived cells, c) Femoral bone marrow derived cells, d) Skin derived cells, e) Liver derived cells.
g_
0
10
2O
30
40
0
lO
20
30
lO
~ 4x.
w
20
~
30
40
0
i0
20
30
40
50
60
70
80
i
Jg/ml Con-A
20 ~g/~l Con-A
i0 ug/ml Con-A
NUMBER OF RBC PER CELL
.L
_I
5 ~g/ml Con-A
(b)
~_
g
a:
~
0
1Q
20
30
4O
50
6O
7O
80
90
0
2o
30
40
50
60
70
80
90
i00
o
i
i
~
i
NUMBER OF RBC PER
I
20 pg/ml C o n - A
i
5 pg/ml C o n - A
CELL
I
I
I
40 ug/ml C o n - A
I
io pg/ml C o n - A
(c)
bJ
-
0
LP*
0
u'1
(d)
4o
~o
0-
0
1
I I
NUMBER OF RBC PER CELL
I
0
lo
2o
3o
10
4o
5o
30
--
0 6o
lo
20
40
50-
6O
2o
~_
~-
~
Con-A
60
0 70
i0
20
30
40
50
60
70
7O
~g/ml
90 80
3o
40
Con-A
~: ul
--
Con-A
ug/ml
80
~g/ml
i0
90
20
Con-A
~
0-
U-
5 ~g/ml
~
2o
~
30
50-
60
.o!
80
90
i00
5
~g/ml
J
i
l
l
l
;
I0 pg/ml Con-A
NUMBEROF RBC PER CELL
40 ~g/ml C o n - A
_L b
U..--
|
20 pg/ml Con-A
Con-A
(e)
E Z #
© Z > Z >
0
-i
;m n~ > c~
o
.<
C m
298
BAN AND
liDA
Cells derived from the femoral bone-marrow, skin and liver showed almost no C-RBC adsorption ability, while lung cells showed weak C-RBC adsorption ability. A slight concentration dependency was observed up to a Con-A concentration of 20/~g/ml. The above four cell types showed a morphology quite similar with that of fibroblasts. Kidney-derived cells showed epithelial morphology and remarkably high C-RBC adsorption ability. Con-A unlabelled RBC were not adsorbed in the cells (data not shown). RBC labelled with Con-A at low concentrations (5 ~g/ml) were easily adsorbed in kidney cells. However, even when the concentration of Con-A was increased to more than 10 #g/ml, adsorption distribution of C-RBC underwent little change. It is evident that cell populations having no C-RBC adsorption exist at a fixed ratio. As a result of the data shown in Figure 2 (c-e), cells adsorbing six or more C-RBC were regarded as Con-A reactivity positive and cells adsorbing no RBC as Con-A reactivity negative. The patterns of C-RBC adsorption of cells obtained from various organs of the individuals (Figure 1) mostly showed, with the exception of kidney ceils, the trends observed in Figure 2. Figure 3 shows in summary the ability to adsorb RBC labelled with Con-A at a concentration of 40/zg/ml. A comparison was made of the percent frequency levels of Con-A positive cells (cells adsorbing six or more C-RBC per cell) and Con-A negative cells (cells adsorbing no C-RBC). On comparison by age of cell donor (those less than four indicated as y in the figure and those more than 44 indicated as a in the figure), there was
~6 I00
RBC/cell
0 RBC/cell
--
af ~
oaf 0 af 0 am Oam
af
<7 af ~af
oym
s g~
V~ tsym
@ym
•ym
50
oaf
oaf OY m
Oym vym
AYm @ym ~af af
V ym
af a f m. -. -~ -y af !
l
0 ym
• ym
Z~ ym •
I
af af ~/~ am V yra af
l
y~ ~f%0
!
I
I
I
I
am
!
mN
Cell
Origins
FIO. 3. C-RBC adsorption ability of culture cells established from a variety of normal human tissues. The left panel demonstrates the percent frequency of cells adsorbing six or more C-RBC per cell, and the right panel shows the percent "frequency of cells adsorbing no C-RBC. a: aged donor (donor age 44 years and over), y: young donor (donor age four years and under), m: male, f: female
TISSUE-SPECIFICITY OF REACTIVITY TO CONCANAVALIN-A
299
no association between Con-A reactivity and donor age for skin and kidney fibroblasts. Lung fibroblasts showed a tendency for Con-A reactivity to be higher in younger cells than in aged cells. However, a large number of cases are needed in order to detect any cell type association between Con-A reactivity and donor age. Con-A reactivity of skin fibroblasts was very low. Con-A reactivity of liver and bone marrow cells was also low. In Figure 4, the percent frequency of kidney cells adsorbing six or more C-RBC per cell is shown in relation to the concentration of Con-A, The cells were established from a portion of kidney tissues collected from a premature fetus (chromosome karyotype, 44 + XX), four-year-old boy, 62-year-old male, 70-year-old female, 79-year-old female, 80-year-old male, and 85-year-old male. C-RBC adsorption abilities for two cases of subjects four years and under, and those for five cases of 62 years and over were distributed rather widely. That is, there seems to be no correlation between the age of cell donors and Con-A reactivity.
100
50
i0
Concentration
20
30
of C o n - A
40 (~g/ml)
FIG. 4. C o n - A concentration-dependency in C-RBC adsorption of kidney derived culture cells. The percent fre-
quency of cells adsorbing six or more C-RBC per cell was obtained. There was no difference in C-RBC adsorption ability between tissue donors of four years and under ( © ) and those of 62 years and over ( O ) .
300
BAN AND [IDA
DISCUSSION Normal human diploid cells, which have a finite doubling potential in culture, are widely used as an in vitro aging model. Especially, fibroblasts established from lung (Ban et al., 1980 a,b, 1981) and skin of human fetuses (Nikaido et al., 1980) and skin tissue of human adults (Nikaido et al., 1980) are frequently employed because they are easily available, can be established easily in culture, and have high proliferative ability. Cells derived from other organs are also excellent material for studies on aging (Ban et al., 1982). Identification of species-specificity or tissue-specificity of the proliferating fibroblasts and other cells is very meaningful for the advancement of studies on cellular aging. Furthermore, identifying the presence or absence of tumor-specificity in fibroblasts established from tumor tissues is of importance for promoting cancer studies. Hayflick et al. (1961, 1965) suggested the presence of tissue-specificity in the in vitro life-span of fibroblasts. Oishi et al. (1981) reported that fibroblasts established from human prostate cancer show high reactivity to Con-A. We studied Con-A reactivity of various cells for the purpose of identifying tissuespecificity of cells proliferating from normal human tissues. Materials were collected after they were confirmed to be free of either tumor or degradation. Cells proliferating from the kidney, lung, and skin are easily established, and proliferation of the established cells is good. Con-A reactivity of skin fibroblasts (Figures 2d and 3) is very low, and that of the liver cells (Figures 2e and 3) and femoral bone marrow cells (Figures 2c and 3) also seems to be low. Con-A reactivity of lung fibroblasts is slightly higher in the younger cells (Figures 2b and 3). However, kidney cells showed remarkably high C-RBC adsorption ability compared with the four types of cells mentioned above. Kidney cells are characterized by their Con-A reactivity at a low concentration (5 ~g/ml) and the ratio of cells adsorbing 50 or more C-RBC per cell is high. However, many kidney cells do not adsorb C-RBC even when the concentration of Con-A used to label human red blood cells is increased (Figure 2). Some of the kidney cells that do not adsorb C-RBC demonstrate fibroblast-like morphology, but most are difficult to classify morphologically. From the above observations, it is evident that Con-A reactivity is tissue-specific, and especially, that a large number of Con-A receptors exist on the membrane surface of kidney cells. However, Con-A reactivity of the individual kidney cells differed greatly, whiCh suggests that kidney cells represent a mixed population of proliferating cells. However, an overwhelmingly large number of these cells have many Con-A receptors on their membrane surface. The number of Con-A receptors may possibily change during the cell cycle, but Aizawa et al. (1980, b) do not believe this to be true. Most of the kidney cells had an epithelial morphology, which remained stable throughout the in vitro life-span of the cells. With some exceptions, it is very difficult to morphologically differentiate culture into epithelial cells and fibroblasts. Particularly, morphological kinetic changes, frequently observed as subcultures, are repeated after the first generation of culture cells is established. The Con-A hemadsorption test is useful as an indicator of changes in the growth kinetics of culture cells. SUMMARY An examination was made of the ability of culture cells that were established from various normal human tissues to adsorb concanavalin-A treated red blood cells (Con-A hemadsorption test). As most tumor cells have a high reactivity to Con-A, the Con-A
TISSUE-SPECIFICITY OF REACTIVITY TO CONCANAVALIN-A
301
h e m a d s o r p t i o n test is o f t e n used as a c o n v e n i e n t i n d i c a t o r for i d e n t i f y i n g the n e o p l a s t i c c h a r a c t e r i s t i c o f f i b r o b l a s t s . This s t u d y r e v e a l e d , h o w e v e r , t h a t c u l t u r e d cells f r o m norm a l k i d n e y tissue have a r e m a r k a b l y high reactivity to C o n - A . T h e m o r p h o l o g y o f the k i d n e y cells was similar to t h a t o f e p i t h e l o i d cells. H o w e v e r , C o n - A reactivity o f f i b r o b l a s t s (or f i b r o b l a s t - l i k e cells) d e r i v e d f r o m lung, skin, b o n e - m a r r o w , a n d liver was low. T h e C o n - A h e m a d s o r p t i o n test is n o t o n l y useful for i d e n t i f i c a t i o n o f n e o p l a s t i c cells, b u t also p r o v i d e s i n f o r m a t i o n o f cellular specificity. REFERENCES AIZAWA,S., MITSUI,Y. and KURIMOTO,F. (1980) (a) Exp. Cell Res. 125, 287. AIZAWA,S., MITSUI, Y., KURIMOTO,F. and MATSUOKA,K. (1980) (b) Exp. CellRes. 125, 297. BAN, S., NIKAIDO,O. and SUGAnARA,T. (1980) (a) Radiat. Res. 81, 120. BAN, S., NIKAIDO,O. and SUGA~L~RA,T, (1980) (b) Exp. Gerontol. 15, 539. BAN, S., IKUSmMA,T. and SUGAnARA,T. (1981) Radiat. Res. 87, 1. BAN, S., hDA, S., CHIN, N. and AwA, A.A. (1982) J. Radiat. Res. 23, 88. FREEDMAN,V.H. and SHIN, S.I. (1974) Cell, 3, 355. HAVFLICK,L. and MOORHEAD,P.S. (1961) Exp. Cell Res. 25, 585. HAVFLICK,L. (1965) Exp. Cell Res. 37, 614. HOLLEr, R.W. and KIERNAN,J.A. (1968) Proc. Nat. Acad. Sci. USA, 60, 300. IGEL, H.J., FREEMAN,A.E., SPmWAK,J.E. and KLEINFELD,K.L. (1975) In vitro, 2, 117. MAcPHERSON, I. (1963) J. Nat. Cancer Inst. 30, 795. NXKAIDO,O., BAN, S. and SUGAHARA,T. (1980) Adv. Exp. Med. Biol. 129, 303. OIsnI, K., ROMmN, J.C. and SCHRSDER, F.H. (1981) The Prostate, 2, 11. PERSKV, B., THOMPSON,S.P., MEVSKENS,JR., F.L. and HENDRIX, M.J.C. (1981) In vitro, 18, 929. POtLACK, R.E., GREEN H. and TODAROG.J. (1968) Proc. Nat. Acad. Sci. USA, 60, 126. SANFORD,K.K., BARKER,B., WOODS,M.W., PARSHAD,R. and LAW, L.W. (1967) J. Nat. Cancerlnst. 39, 705. SUMNER, A.T. (1981) Anticancer Res. 1,205. TRENT, J.M. and SAtMON, S.E. (1981) Cancer Genet. Cytogenet. 3, 279. TUCKER, R.W., SANrORD,K.K., HANDLEMAN,S.L. and JONES, G.M. (1977) Cancer Res. 37, 1571. YUNIS, J.J. (1981) Hum. PathoL 12, 494.