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Length of human prematurely condensed chromosomes during G0 and G1 phase
Preliminary
notes
461
18. Bisulputra, T & Burton. H, J ultrastruct res 29 (1969) 224. Received December 29, 1980 Revised version received March 30, 1981 Accepted April 2, 1981
Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827/81/080461-05%02.00/0
Length of human prematurely condensed chromosomes during GO and Gl phase HARD1 SCHMIADY’
a
and KARL SPERLING, In-
stitut far Humangenetik. Freie Universit6t Herrhnml~eg 6, D-1000 Berlin 19, Gerrntrn>
Berlin.
Summary. Length measurements on C-banded prematurely condensed no. 1 human chromosomes of GO and Gl lymphocytes, as well as of synchronized Gl HEp cells revealed that (i) no length difference exists between mitotic chromosomes and GO chromosomes; (ii) 1 h after PHA stimulation a clear increase in length is detectable; (iii) in isolated cases an increase by the factor 5 can be observed during Gl; (iv) the increase is significantly less for constitutive heterochromatin than for euchromatin. The possibility is discussed that these conformational changes of chromatin reflect physiological differences, i.e. the rate of RNA synthesis during interphase.
Ceil cycle-related changes/inrhe confor a-;’ tional patterns of chromatin have been CT ‘emonstrated by biochemical and histochemical techniques, but more directly by application of the phenomenon of premature chromosome condensation (PCC) [l-7]. After fusion between mitotic and interphase cells the chromatin of the latter enters into a mitosis-like condensation *r process. The morphology of prematurely condensed chromosomes clearly reflects their position in the cell cycle at the time of fusion, Gl, S or G2 phase [S, 91. However, a wide range of variability in chromosome length is observed within individual Fig. 1. (a) Human lymphocyte metaphase chromoGl and G2 cells. These length differences somes: (b) prematurely condensed chromosomes of lymphocytes; and (c) G1 lymphocytes after 20 h are influenced to only a minor degree by GO PHA stimulation. Arrows indicate the no. 1 C-banded I
1 Present address: Fritz-Haber-Institut f. Elektronenmikroskopie, Faradayweg 3-4; D-1000 Berlin 33, Germany.
chromosomes.
Exp Cd Res 134 (1981)
462
Preliminary notes 35 -
TI I I
-i I I I, I I I
‘1 I.35
nn -”
2.5 h
C
I
-
-
-I
7h
-
M I G, Fig. 3. Average length-including S.D. of HEp mitotic chromosomes (M) and prematurely condensed chromosomes 24, 5, and 7 h after release of the arrest at metaphase. n, no. of chromosomes I analysed.
5
4
6
8
10
12
14
16
18
as cells move through Gl phase, whereas an increasing condensation from early to Fig. 2. Length distribution of (a) human lymphocyte late G2 was found after length measurechromosomes no. 1 at metaphase; (b) in prematurely condensed chromosomes of GO lymphocytes; (c) Cl ments of prematurely condensed Microtus lymphocytes after 1 h PHA stimulation; and (d) after agrestis chromosomes [ 111. Moreover, it 20 h PHA stimulation. was shown that within individual chromosomes euchromatic and heterochromatic repreparational conditions, e.g. the length of gions may independently undergo distinct the induction process at 37°C or the mitotic : conformational changes [7, 121.As no quaninterphase ratio of the fused cells [lo]. titative data on the degree of chromatin There is, on the other hand, accumulating condensation during GO and Gl phase are evidence that chromosome length reflects hitherto available, we have measured the the position of the cell within each phase total length of prematurely condensed huof the cell cycle. Using a semi-quantita- man chromosome 1 as well as its heterotive classification [6], a progressive decon- chromatic region in GO and Gl lymphodensation of chromatin has been observed cytes and in synchronized Gl-HEp cells. Abs. length
of chromosomes
(urn)
Preliminary notes
463
Table 1. Lqigth of euchromatin and of centromeric heterochromatin of human no. 1 chromosome, including S.D., in mitotis and in prematurely condensed GI chromosomes ,fiom HEp cells and lymphocytes Average length, in pm Heterochromatin
Euchromatin
HEp cells Mitosis 2.5 h Gl-PCC 5 h GI-PCC 7 h GI-PCC Lymphocytes Mitosis GO-PCC I h Gl-PCC 20 h GI-PCC
N
Abs.
Rel.
Abs.
Rel
35 123 20 15
5.8+1.2 9.2k2.1 17.5k4.2 19.8+2.3
I.0 I.58 3.0 3.41
l.lliO.3 0.9t0.2 1.?fOA I .4+0.5
1.0 0.82 1.09 I .27
57 27 20 I6
6.6k1.3 6.1+1.1 7.6f 1.6 I l.Ok2.6
I.0 0.92 1.15 1.66
1.2io.2 0.9+0.2 I .0&0.2 1.1*0.3
I .o 0.75 0.83 0.92
The measurements on the lvmohocvte chromosomes were restricted to the homologue with the larger C-band N, no. of chromosomes aniysed. .
Material and Methods HEp cells and human lymphocytes were employed in this study. Synchronized Gl populations of HEp cells were obtained after application of a single thymidine block to logarithmically growing cells and subsequent arrest of the mitotic cells by an N,O block (4.2 kp/cmz for 8 h) [13]. After release of the NZO block the mitotic cells were harvested by gentle shaking (mitotic index >85%) and plated into 6 cm Petri dishes. Two hours later about 70% had entered into Gl phase. At 24, 5, and 7 h after the reversal of the N,O block the cells were fused with colcemidarrested mitotic HEp cells for the induction of PCC. Human lymphocytes of a normal male donor were isolated from whole blood by Ficoll-Metrizoate (Seromed) density centrifugation. The lymphocytes were then washed twice with Hanks solution and either fused without PHA stimulation to mitotic HEp cells or cultured in Ham’s F-10 medium supplemented with 15% fetal calf serum (FCS) and phytohemagglutinin (PHA) (Difco) at 37°C for 1 h and 20 h respectively and then induced into PCC. The detailed procedure for PCC induction has been described earlier [9]. In brief: mitotic HEp cells and HEp or lymphocyte interphase cells were mixed at a ratio of 2 : I (IX IO” cells total) and incubated with 1000 HAU of UV-inactivated Sendai virus at 4°C for 15 min. The virus-cell mixture was then transferred into a water bath of 3PC for 40 min and thereafter treated with hypotonic KC1 (0.075 M) for I5 min. The cells were then fixed in three changes of methanol : acetic acid (3 : I), dropped on wet slides, and airdried. Metaphase chromosomes of HEp cells and lymphocytes were obtained after 4 h treatment with 0.2 pg/ml colcemid. C-banding of the preparations were performed according to Sumner [l4]. Photographs were taken of randomly selected cells with clearly
identifiable no. I chromosomes. The film negatives were enlarged by a film reader and length measurements were then performed from drawings of a no. 1 chromosome and its heterochromatic region.
Results and Discussion As can be seen from figs 1, 2 the length of prematurely condensed GO chromosomes is no different from that of mitotic chromosomes. Moreover, the variability in length may be even less than in the mitotic population. However, when lymphocytes are stimulated with PHA and PCC is induced 1 h later, a bimodal distribution with respect to chromosome length is observed. Whereas one peak nearly coincides with the original peak of GO lymphocytes, the other represents a population with a 40% increase in chromosome length. These data point to the existence of at least two lymphocyte populations which differ in their PHA response. Obviously they correspond to the fast-reacting subpopulation of Tlymphocytes and to T-lymphocytes with delayed PHA response and B-lymphocytes [1.5-171. Thus, our data demonstrate that Exp
Cell
Res
134 (1981)
464
Preliminary
notes
conformational changes as indicated by the length of prematurely condensed chromosomes can serve as a sensitive indicator of physiological differences between cells, i.e. genetically inactive and active cells. Twenty hours after PHA stimulation all PCCs are still of the Gl type, their average length has increased by 60% and the most elongated chromosomes are about 2-3 times the length of the GO elements. The existence of different lymphocyte subpopulations and hence the complex patterns of cell cycle kinetics render this system impractical for the study of the condensation process of mid and late Gl phase quantitatively. We have therefore utilized synchronized HEp cells to analyse the length of human chromosome 1 at early, mid, and late Gl phase. As shown in fig. 3 a progressive and significant increase in length is observed as HEp cells traverse through Gl. The early Gl PCCs, 2h h after release of the NzO block, are about 50% longer than the corresponding metaphases (PC1 %); at midGl phase they are about twice as long. After another 2 h the chromosome is further lengthened, though the difference from midGl is statistically not significant. One hour later, i.e. 8 h after release of the N,O block, most of the cells have already moved into S phase. Evidently our data do not cover the whole range of chromosome length at late Gl stage, as the PCCs become more and more diffused. It was thus impossible to identify chromosome no. 1 regularly. It may be stressed, however, that in individual cases, late Gl phase chromosomes are five times longer than metaphase chromosomes. This observation corresponds completely with the length variability found by Rohme [18] for muntjac Gl chromosomes. In addition, our data clearly illustrate Exp Cd Res 134 (19.811
that, in contrast to euchromatin, C-bandpositive constitutive heterochromatin exhibits only a minor length increase during Gl (table 1). This obviously corresponds to the more compact state of these regions in interphase nuclei. Altogether our findings are in accordance with the existence of a ‘chromosome cycle’ during interphase based on a condensationdecondensation mechanism [ 191. With respect to the biological significance of these morphological alterations, two observations are of interest: (i) the significant length increase found soon after PHA stimulation in prematurely condensed lymphocyte chromosomes; (ii) the more distinct reaction of euchromatin as compared with human constitutive heterochromatin during Gl. In both cases the length increase is correlated with increased RNA synthesis [17, 201. Moreover, the length increase found during Gl is obviously correlated with the synthesis rate of all RNA in HeJ.a cells through Gl [21, 221. We may thus conclude that the cyclic conformational changes of the chromatin during interphase are at least to some extent the expression of varying functional states of the interphase nucleus. This work was supported by the Deutsche Forschungsgemeinschaft.
References 1. Pederson, T & Robbins, E, J cell biol 55 (1972) 322. 2. Pederson, T, Proc natl acad sci US 69 (1972)2224. 3. Schor. S L, Johnson, R T & Waldren, C A, J cell sci 17 (1975) 539. 4. Hittelman, W N & Rao, P N, Exp cell res 100 (1976) 219. 5. Rao, P N, Wilson, B & Puck, T T, J cell physio191 (1977) 131. 6. Hittelman, W N & Rao, P N, J cell physiol 95 (1978) 333. 7. Marcus, M & Sperling, K, Exp cell res 123 (1979) 406. 8. Johnson, R T & Rao, P N, Nature 226 (1970) 717. 9. Sperling, K & Rao, P N, Humangenetik 23 (1974) 235.
Preliminary
notes
465
ing cells have been characterized by their ultrastructure and membrane profile as being epithelial and of mammary origin [3, 41. An important characteristic of these cells is that they show selectivity in junctional communication, forming communicating junctions with homologous cells and lens epithelium, but not with breast-derived [5] and other fibroblasts. This property has served to distinguish the normal breast epithelium from breast cancer cell lines and strains derived from primary and metastatic tumours, which were found to be either unable to form communicating junctions with any cell type (non-communicators) or Received January 12, 1981 Revised version received March 23, 1981 did so non-selectively [6, 71. Accepted April 14, 1981 We have found that inclusion of growth factors [8] and human serum [l, 3, 91 increases the division potential of the milk cells in vitro, and it is now possible to carry Copyright @ 1981 by Academic Press. Inc. All rights of reproduction in any form reserved them through several subcultures. To deter0014-4827/81/080465-061Eo2.00/0 mine whether the cells in subculture show the same phenotype as those in primary Junctional communication pattern of cells culture, we have examined their pattern of cultured from human milk junctional communication. The results I. A. MCKAY and J. TAYLOR-PAPADIMITRIOU, show that with subculture, the population Imperial Cancer Research Fund, Lincoln’s Inn Fields, of cells which emerges exhibits a pattern London, WCZA 3PX, UK of non-selective communication which Summary. Junctional communication between cultured human milk cells and fibroblasts derived from differs from that seen with either HumE human foreskin or mammary gland has been investior fibroblasts. gated. The dividing cells in primary cultures from milk, IO. Schmiady, H, Dissertation, Freie Universitat, Ber-
lin (1979). 11. Sperling, K & Rao, P N, Chromosoma 45 (1974) 121. 12. Schmiady, H & Sperling, K, Human genet 35 (1976) 107. 13. Rao, P N, Science 160 (1968) 774. 14. Sumner, A T, Exp cell res 75 (1972) 304. 15. Levis, W R & Robbins, J H, Exp cell res 61 (1970) 153. 16. Dudin, G, Beek, B & Obe, G, Mutat res 23 (1974) 279. 17. Ling, N R & Kay, 3 E, Lymphocyte stimulation. North-Holland, Amsterdam (1975). 18. Rohme, D, Hereditas 76 (1974) 251. 19. Mazia, D, J cell camp physio162 (1963) 123. 20. Comings, D E, Adv human genet 3 (1972) 237. 21. Pfeiffer, S E & Tolmach, L J, J cell physiol 71 (1968) 77. 22. Scharff, M D & Robbins, E, Nature 208 (1976)464.
which have been shown to be epithelial by immunological and morphological criteria, showed selectivity in communication and did not transfer 3H-labelled nucleotides to either type of fibroblast. With passage the communication phenotype changed, selectivity was lost and a morpholoaically homogeneous cell type emerged which communicated not only with both types of fibroblast, but also with the milk epithelial cells found in primary culture.
In working with cell cultures derived from animal tissues or secretions the identification of specific cell types is a primary concern. In recent years methods have been developed for culturing cells from human milk [l]. In primary cultures fibroblasts are not present [2, 31 and the divid-
Materials
and Methods
Culture and passage of Human Mammary Epithelial (HumE) cells. Epithelial cells from pooled samples
of early lactation milks were grown on 93 mm tissue culture- Petri dishes (NUNC) ;n RPM1 1640 medium supplemented with 15% fetal calf serum (FCS), 10% pooled human serum, 10 &ml insulin, 5 pg/ml hydrocortisone and 50 rig/ml cholera toxin. The medium was changed every 3-4 days until cultures reached confluence. Unfractionated milks contain many tissue macrophages which, however, do not divide, and are displaced after IO-14 days [2, 4,9]. While attached to the plates the macrophages act as feeders for the epithelial cells [9]. The primary cultures used in the work reported here were taken 14 days after seeding, when 90% of the macrophages had been displaced. The few remaining do not re-attach on subculture and in any event have been shown to be non-communicators . Exp Cell Res 134 (1981)
notes
461
18. Bisulputra, T & Burton. H, J ultrastruct res 29 (1969) 224. Received December 29, 1980 Revised version received March 30, 1981 Accepted April 2, 1981
Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827/81/080461-05%02.00/0
Length of human prematurely condensed chromosomes during GO and Gl phase HARD1 SCHMIADY’
a
and KARL SPERLING, In-
stitut far Humangenetik. Freie Universit6t Herrhnml~eg 6, D-1000 Berlin 19, Gerrntrn>
Berlin.
Summary. Length measurements on C-banded prematurely condensed no. 1 human chromosomes of GO and Gl lymphocytes, as well as of synchronized Gl HEp cells revealed that (i) no length difference exists between mitotic chromosomes and GO chromosomes; (ii) 1 h after PHA stimulation a clear increase in length is detectable; (iii) in isolated cases an increase by the factor 5 can be observed during Gl; (iv) the increase is significantly less for constitutive heterochromatin than for euchromatin. The possibility is discussed that these conformational changes of chromatin reflect physiological differences, i.e. the rate of RNA synthesis during interphase.
Ceil cycle-related changes/inrhe confor a-;’ tional patterns of chromatin have been CT ‘emonstrated by biochemical and histochemical techniques, but more directly by application of the phenomenon of premature chromosome condensation (PCC) [l-7]. After fusion between mitotic and interphase cells the chromatin of the latter enters into a mitosis-like condensation *r process. The morphology of prematurely condensed chromosomes clearly reflects their position in the cell cycle at the time of fusion, Gl, S or G2 phase [S, 91. However, a wide range of variability in chromosome length is observed within individual Fig. 1. (a) Human lymphocyte metaphase chromoGl and G2 cells. These length differences somes: (b) prematurely condensed chromosomes of lymphocytes; and (c) G1 lymphocytes after 20 h are influenced to only a minor degree by GO PHA stimulation. Arrows indicate the no. 1 C-banded I
1 Present address: Fritz-Haber-Institut f. Elektronenmikroskopie, Faradayweg 3-4; D-1000 Berlin 33, Germany.
chromosomes.
Exp Cd Res 134 (1981)
462
Preliminary notes 35 -
TI I I
-i I I I, I I I
‘1 I.35
nn -”
2.5 h
C
I
-
-
-I
7h
-
M I G, Fig. 3. Average length-including S.D. of HEp mitotic chromosomes (M) and prematurely condensed chromosomes 24, 5, and 7 h after release of the arrest at metaphase. n, no. of chromosomes I analysed.
5
4
6
8
10
12
14
16
18
as cells move through Gl phase, whereas an increasing condensation from early to Fig. 2. Length distribution of (a) human lymphocyte late G2 was found after length measurechromosomes no. 1 at metaphase; (b) in prematurely condensed chromosomes of GO lymphocytes; (c) Cl ments of prematurely condensed Microtus lymphocytes after 1 h PHA stimulation; and (d) after agrestis chromosomes [ 111. Moreover, it 20 h PHA stimulation. was shown that within individual chromosomes euchromatic and heterochromatic repreparational conditions, e.g. the length of gions may independently undergo distinct the induction process at 37°C or the mitotic : conformational changes [7, 121.As no quaninterphase ratio of the fused cells [lo]. titative data on the degree of chromatin There is, on the other hand, accumulating condensation during GO and Gl phase are evidence that chromosome length reflects hitherto available, we have measured the the position of the cell within each phase total length of prematurely condensed huof the cell cycle. Using a semi-quantita- man chromosome 1 as well as its heterotive classification [6], a progressive decon- chromatic region in GO and Gl lymphodensation of chromatin has been observed cytes and in synchronized Gl-HEp cells. Abs. length
of chromosomes
(urn)
Preliminary notes
463
Table 1. Lqigth of euchromatin and of centromeric heterochromatin of human no. 1 chromosome, including S.D., in mitotis and in prematurely condensed GI chromosomes ,fiom HEp cells and lymphocytes Average length, in pm Heterochromatin
Euchromatin
HEp cells Mitosis 2.5 h Gl-PCC 5 h GI-PCC 7 h GI-PCC Lymphocytes Mitosis GO-PCC I h Gl-PCC 20 h GI-PCC
N
Abs.
Rel.
Abs.
Rel
35 123 20 15
5.8+1.2 9.2k2.1 17.5k4.2 19.8+2.3
I.0 I.58 3.0 3.41
l.lliO.3 0.9t0.2 1.?fOA I .4+0.5
1.0 0.82 1.09 I .27
57 27 20 I6
6.6k1.3 6.1+1.1 7.6f 1.6 I l.Ok2.6
I.0 0.92 1.15 1.66
1.2io.2 0.9+0.2 I .0&0.2 1.1*0.3
I .o 0.75 0.83 0.92
The measurements on the lvmohocvte chromosomes were restricted to the homologue with the larger C-band N, no. of chromosomes aniysed. .
Material and Methods HEp cells and human lymphocytes were employed in this study. Synchronized Gl populations of HEp cells were obtained after application of a single thymidine block to logarithmically growing cells and subsequent arrest of the mitotic cells by an N,O block (4.2 kp/cmz for 8 h) [13]. After release of the NZO block the mitotic cells were harvested by gentle shaking (mitotic index >85%) and plated into 6 cm Petri dishes. Two hours later about 70% had entered into Gl phase. At 24, 5, and 7 h after the reversal of the N,O block the cells were fused with colcemidarrested mitotic HEp cells for the induction of PCC. Human lymphocytes of a normal male donor were isolated from whole blood by Ficoll-Metrizoate (Seromed) density centrifugation. The lymphocytes were then washed twice with Hanks solution and either fused without PHA stimulation to mitotic HEp cells or cultured in Ham’s F-10 medium supplemented with 15% fetal calf serum (FCS) and phytohemagglutinin (PHA) (Difco) at 37°C for 1 h and 20 h respectively and then induced into PCC. The detailed procedure for PCC induction has been described earlier [9]. In brief: mitotic HEp cells and HEp or lymphocyte interphase cells were mixed at a ratio of 2 : I (IX IO” cells total) and incubated with 1000 HAU of UV-inactivated Sendai virus at 4°C for 15 min. The virus-cell mixture was then transferred into a water bath of 3PC for 40 min and thereafter treated with hypotonic KC1 (0.075 M) for I5 min. The cells were then fixed in three changes of methanol : acetic acid (3 : I), dropped on wet slides, and airdried. Metaphase chromosomes of HEp cells and lymphocytes were obtained after 4 h treatment with 0.2 pg/ml colcemid. C-banding of the preparations were performed according to Sumner [l4]. Photographs were taken of randomly selected cells with clearly
identifiable no. I chromosomes. The film negatives were enlarged by a film reader and length measurements were then performed from drawings of a no. 1 chromosome and its heterochromatic region.
Results and Discussion As can be seen from figs 1, 2 the length of prematurely condensed GO chromosomes is no different from that of mitotic chromosomes. Moreover, the variability in length may be even less than in the mitotic population. However, when lymphocytes are stimulated with PHA and PCC is induced 1 h later, a bimodal distribution with respect to chromosome length is observed. Whereas one peak nearly coincides with the original peak of GO lymphocytes, the other represents a population with a 40% increase in chromosome length. These data point to the existence of at least two lymphocyte populations which differ in their PHA response. Obviously they correspond to the fast-reacting subpopulation of Tlymphocytes and to T-lymphocytes with delayed PHA response and B-lymphocytes [1.5-171. Thus, our data demonstrate that Exp
Cell
Res
134 (1981)
464
Preliminary
notes
conformational changes as indicated by the length of prematurely condensed chromosomes can serve as a sensitive indicator of physiological differences between cells, i.e. genetically inactive and active cells. Twenty hours after PHA stimulation all PCCs are still of the Gl type, their average length has increased by 60% and the most elongated chromosomes are about 2-3 times the length of the GO elements. The existence of different lymphocyte subpopulations and hence the complex patterns of cell cycle kinetics render this system impractical for the study of the condensation process of mid and late Gl phase quantitatively. We have therefore utilized synchronized HEp cells to analyse the length of human chromosome 1 at early, mid, and late Gl phase. As shown in fig. 3 a progressive and significant increase in length is observed as HEp cells traverse through Gl. The early Gl PCCs, 2h h after release of the NzO block, are about 50% longer than the corresponding metaphases (PC1 %); at midGl phase they are about twice as long. After another 2 h the chromosome is further lengthened, though the difference from midGl is statistically not significant. One hour later, i.e. 8 h after release of the N,O block, most of the cells have already moved into S phase. Evidently our data do not cover the whole range of chromosome length at late Gl stage, as the PCCs become more and more diffused. It was thus impossible to identify chromosome no. 1 regularly. It may be stressed, however, that in individual cases, late Gl phase chromosomes are five times longer than metaphase chromosomes. This observation corresponds completely with the length variability found by Rohme [18] for muntjac Gl chromosomes. In addition, our data clearly illustrate Exp Cd Res 134 (19.811
that, in contrast to euchromatin, C-bandpositive constitutive heterochromatin exhibits only a minor length increase during Gl (table 1). This obviously corresponds to the more compact state of these regions in interphase nuclei. Altogether our findings are in accordance with the existence of a ‘chromosome cycle’ during interphase based on a condensationdecondensation mechanism [ 191. With respect to the biological significance of these morphological alterations, two observations are of interest: (i) the significant length increase found soon after PHA stimulation in prematurely condensed lymphocyte chromosomes; (ii) the more distinct reaction of euchromatin as compared with human constitutive heterochromatin during Gl. In both cases the length increase is correlated with increased RNA synthesis [17, 201. Moreover, the length increase found during Gl is obviously correlated with the synthesis rate of all RNA in HeJ.a cells through Gl [21, 221. We may thus conclude that the cyclic conformational changes of the chromatin during interphase are at least to some extent the expression of varying functional states of the interphase nucleus. This work was supported by the Deutsche Forschungsgemeinschaft.
References 1. Pederson, T & Robbins, E, J cell biol 55 (1972) 322. 2. Pederson, T, Proc natl acad sci US 69 (1972)2224. 3. Schor. S L, Johnson, R T & Waldren, C A, J cell sci 17 (1975) 539. 4. Hittelman, W N & Rao, P N, Exp cell res 100 (1976) 219. 5. Rao, P N, Wilson, B & Puck, T T, J cell physio191 (1977) 131. 6. Hittelman, W N & Rao, P N, J cell physiol 95 (1978) 333. 7. Marcus, M & Sperling, K, Exp cell res 123 (1979) 406. 8. Johnson, R T & Rao, P N, Nature 226 (1970) 717. 9. Sperling, K & Rao, P N, Humangenetik 23 (1974) 235.
Preliminary
notes
465
ing cells have been characterized by their ultrastructure and membrane profile as being epithelial and of mammary origin [3, 41. An important characteristic of these cells is that they show selectivity in junctional communication, forming communicating junctions with homologous cells and lens epithelium, but not with breast-derived [5] and other fibroblasts. This property has served to distinguish the normal breast epithelium from breast cancer cell lines and strains derived from primary and metastatic tumours, which were found to be either unable to form communicating junctions with any cell type (non-communicators) or Received January 12, 1981 Revised version received March 23, 1981 did so non-selectively [6, 71. Accepted April 14, 1981 We have found that inclusion of growth factors [8] and human serum [l, 3, 91 increases the division potential of the milk cells in vitro, and it is now possible to carry Copyright @ 1981 by Academic Press. Inc. All rights of reproduction in any form reserved them through several subcultures. To deter0014-4827/81/080465-061Eo2.00/0 mine whether the cells in subculture show the same phenotype as those in primary Junctional communication pattern of cells culture, we have examined their pattern of cultured from human milk junctional communication. The results I. A. MCKAY and J. TAYLOR-PAPADIMITRIOU, show that with subculture, the population Imperial Cancer Research Fund, Lincoln’s Inn Fields, of cells which emerges exhibits a pattern London, WCZA 3PX, UK of non-selective communication which Summary. Junctional communication between cultured human milk cells and fibroblasts derived from differs from that seen with either HumE human foreskin or mammary gland has been investior fibroblasts. gated. The dividing cells in primary cultures from milk, IO. Schmiady, H, Dissertation, Freie Universitat, Ber-
lin (1979). 11. Sperling, K & Rao, P N, Chromosoma 45 (1974) 121. 12. Schmiady, H & Sperling, K, Human genet 35 (1976) 107. 13. Rao, P N, Science 160 (1968) 774. 14. Sumner, A T, Exp cell res 75 (1972) 304. 15. Levis, W R & Robbins, J H, Exp cell res 61 (1970) 153. 16. Dudin, G, Beek, B & Obe, G, Mutat res 23 (1974) 279. 17. Ling, N R & Kay, 3 E, Lymphocyte stimulation. North-Holland, Amsterdam (1975). 18. Rohme, D, Hereditas 76 (1974) 251. 19. Mazia, D, J cell camp physio162 (1963) 123. 20. Comings, D E, Adv human genet 3 (1972) 237. 21. Pfeiffer, S E & Tolmach, L J, J cell physiol 71 (1968) 77. 22. Scharff, M D & Robbins, E, Nature 208 (1976)464.
which have been shown to be epithelial by immunological and morphological criteria, showed selectivity in communication and did not transfer 3H-labelled nucleotides to either type of fibroblast. With passage the communication phenotype changed, selectivity was lost and a morpholoaically homogeneous cell type emerged which communicated not only with both types of fibroblast, but also with the milk epithelial cells found in primary culture.
In working with cell cultures derived from animal tissues or secretions the identification of specific cell types is a primary concern. In recent years methods have been developed for culturing cells from human milk [l]. In primary cultures fibroblasts are not present [2, 31 and the divid-
Materials
and Methods
Culture and passage of Human Mammary Epithelial (HumE) cells. Epithelial cells from pooled samples
of early lactation milks were grown on 93 mm tissue culture- Petri dishes (NUNC) ;n RPM1 1640 medium supplemented with 15% fetal calf serum (FCS), 10% pooled human serum, 10 &ml insulin, 5 pg/ml hydrocortisone and 50 rig/ml cholera toxin. The medium was changed every 3-4 days until cultures reached confluence. Unfractionated milks contain many tissue macrophages which, however, do not divide, and are displaced after IO-14 days [2, 4,9]. While attached to the plates the macrophages act as feeders for the epithelial cells [9]. The primary cultures used in the work reported here were taken 14 days after seeding, when 90% of the macrophages had been displaced. The few remaining do not re-attach on subculture and in any event have been shown to be non-communicators . Exp Cell Res 134 (1981)