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Studies on genomic interactions in hybrids between enzymic variants of a human cell line
Cell Differentiation 2,325
335 (1974). © North-Holland Publishing Company
STUDIES ON GENOMIC INTERACTIONS IN HYBRIDS BETWEEN ENZYMIC VARIANTS OF A HUMAN CELL LINE S. BARLATI, A.S. SANTACHIARA-BENERECETTI, S. BALACCO*, C. ULLU, R. DI LERNIA***, C. ROSSI** and L. DE CARLI*** Laboratorio di Genetica Biochimica ed Evoluzionistica del Consiglio Nazionale delle Ricerche, Istituto di Genetica, Universitb di Pavia, 27100 Pavia, Italy Accepted 11 December 1973
The chromosomal complement and a number of enzymic activities have been studied and found to be stable in hybrids obtained from variants of the human line EUE. The parental lines utilized were characterized by high or low levels of alkaline phosphatase (AP), an enzyme which is a mixture of a thermostable (Ts) and a thermolabile (T1) component, the former being increased and the latter reduced by the addition of prednisolone to the cell culture. In the hybrids, AP activity exhibited a small amount of Ts component as in the low level parental line and a large amount of the TI component as in the high level parental line. The response of the two fractions to prednisolone indicates that, in tbe hybrids, they maintain their induction or repression properties unaltered.
The formation of hybrids between clonal derivatives of a human heteroploid line, the EUE, varying in their alkaline phosphatase (AP) levels, has been previously reported (Simoni et al., 1970). The purpose of this work is to determine the dominance relationships between the regulatory mechanisms of the two parental genomes when combined in the same cytoplasm. A similar approach has been used by other authors for studying the regulation of a number of specialized cellular functions (Davidson et al., 1968, 1970; Weiss et al., 1971). The experiments reported here are performed with clonal variants of the same cell line selected for differences in a few enzymic activities. One of these activities, namely AP, is susceptible to control by exogenous agents.
* 24.7.70. ** Present address: Istituto di Genetica, Universith di Parma, 42100 Parma, Italy. *** Present address: Istituto di Biologia Generale, Facult~ di Medicina, Universit~ di Milano, 20100 Milano, Italy.
326
Genomic interactions in human hybrM lines
Under these conditions the product of interclonal hybridization should be simpler to examine and more similar, in a way, to the system provided by the merozygotes in bacteria. The results should then have a simpler interpretation because most of the interactions due to gross differences in cell types can be eliminated. We have studied in particular AP, an enzyme which is subjected to regulation (Maio et al., 1963; Santachiara-Benerecetti et al., 1967a) and contains at least two components: one thermostable (Ts) and one thermolabile (T1) (Santachiara-Benerecetti et al., 1967b), which can be separated by starch gel electrophoresis. In addition to AP and to the enzymes involved in the system for the selection of the hybrids, like thymidine kinase (TK) and hypoxanthine-guanine-phosphoribosyltransferase (HGPRT), we tested the triosophosphate isomerase (TPI) and glucose6-phosphate dehydrogenase (G6PD) activities. The TPI and G6PD activities were chosen because of the available information on their cytological location. Since G6PD and HGPRT are known to be linked the levels of these two enzymic activities could be compared.
MATERIALS AND METHODS Cell lines
The two parental lines were characterized as follows: E/HGPRT-AP+: and EUE (Terni et al., 1958) subline resistant to 100 ~tg/ml of 8-azaguanine (AG), obtained by successive cultures with increasing concentrations of the analogue and isolated as a clone; the level of HGPRT activity was below detection. The specific activity of alkaline phosphatase was 18-26 units/mg protein and the modal number of chromosomes was 57. E 6 D / T K - A P - : and EUE subline resistant to 20/~g/ml of bromodeoxyuridine (BUdR) showing a small residual thymidine kinase activity; the specific activity of AP was about 5 units/rag protein and the modal number of chromosomes was 53. The experiments of AP induction have been carried out after a period of storage at -80°C. Culture techniques
The stock cultures were maintained as monolayers in Eagle medium with 10% calf serum. The resistant lines were cultured in the presence of the appropriate concentration of AG or BUdR and the medium containing the analogues was substituted with normal medium 24 hr before harvesting the cells for enzymic assays. The hybrid lines were derived from a single fusion experiment by using Sendal virus as previously described (Simoni et al., 1970). The hybrids, selected in HAT medium, were all primary clones presumably originated from different fusion events. The hybrid lines E18/15 and E11/1 and the two parental lines were grown in parallel cultures in Eagle medium; the E11/1 line was also grown in HAT medium
S. BARLATI et al.
327
(Eagle supplemented with 5 ~g/ml thymidine 5/ag/ml hypoxanthine and 1/~g/ml aminopterin). When the cultures were nearly confluent the medium was changed and the cells harvested the following day. An aliquot of l0 s cells was inoculated into a bottle (44 cm 2) which was used for the karyotype analysis. The remaining cells were divided in 15 aliquots of 3 X 10 6 to 4 X 10 6 cells in centrifuge tubes and washed with Hanks buffer. The pellets were stored frozen at -80°C until needed for the enzyme assays.
Cytological preparations The chromosomal counts were performed on a suspension of cells from trypsinized cultures according to Hsu et al. (1960). Biochemical assays The assay of HGPRT activity was done according to Littlefield (1964); the frozen cell pellet was suspended in 1 ml of 0.05 M phosphate buffer and lysed by freezing and thawing three times in liquid nitrogen; the resulting extract was centrifuged for 10 min at 20,000 g. The supernatant was collected and immediately used for enzymic assays or stored at -20°C. The activity was expressed as ~tmoles of hypoxanthine converted in 10 rain per mg protein. TK was assayed according to Bollum et al. (1959) with the modifications introduced by Migeon et al. (1969); the extracts were prepared as for the HGPRT assay but in this case the buffer was 10-a M Tris-HC1 pH 8, 10-1 M KC1 and 0.0029 M /3-mercaptoethanol. The activity was expressed as/amoles of thymidine converted in 10 min per mg protein. The H 3 thymidine used in the TK assay was purified, before utilization, by elution for 8 hr on descending chromatography on Whatman D.E. 81 paper. The assays for TPI and G6PD were performed by using the standard kits of Boehringer und Sohne GMBH Co., Mannheim (Germany). The activity was expressed as A O.D. at 366 nm/min per mg protein. The preparation of the extracts was done as described above but with 0.3 M triethanolamine at pH 7.6 as resuspending buffer. All the assays were done in duplicate and the mean values are reported in the tables. The extraction procedure and the enzymic assay for alkaline phosphatase were performed as previously reported (Santachiara-Benerecetti et al., 1967b). The AP thermostable fraction was determined by heating, for 30 min at 70°C, 0.1 ml aliquots of cell extracts diluted with 0.4 ml of 0.05 M Tris-HC1 pH 7.1 preheated at 70°C. The heated samples, together with the controls, were extracted after cooling with butanol according to the Morton technique as modified by Herz et al. (1962). AP activity was assayed on the aqueous phase and the thermostable component was determined as percent of the control and expressed as Ts specific activity. The T1 specific activity was determined by subtracting the Ts activity from the total AP specific activity. Both the treatments and the assays were done in triplicate.
328
Genomic interactions in human hybrid lines
Induction experiments One day after trypsinisation of the cells the medium was substituted with Eagle's containing 10/ag/ml of prednisolone dissolved in absolute ethyl alcohol. Maximum induction of AP was obtained after 4 to 6 days of incubation at 37°C in the presence of the hormone; this treatment did not significantly affect the growth rate of the cells. The medium was checked for pH daily and changed one day before harvesting the cells. Also in this case the cultures were nearly confluent at the time of harvesting. Chemicals 8-azaguanine, 5-bromo-2'-deoxyuridine, aminopterin and prednisolone were obtained from Sigma Chemical Co., while thymidine and hypoxanthine were from Calbiochem. H 3 thymidine (1 mc/ml; 5.6 X 10-2 M) was obtained from New England Nuclear.
RESULTS Cytological controls The chromosome distributions for the various lines observed at three successive times on samples of fifty mitoses are reported in fig. 1. No gross alteration in chromosome number was detected for the two parental and for the hybrid lines during the time interval of two and a half months. A wider spread in the chromosome number was observed in the hybrids; for the hybrid line EI1/1 the relative stability of the total number of chromosomes is evident both in the presence and in the absence of the selective medium containing aminopterin. A detailed chromosomal analysis of all the lines examined was virtually impossible and in any case it would not have given an unambiguous answer. Therefore, this stability refers only to the total chromosome number and not to their karyotypes. The difference in the number of small acrocentric chromosomes persisted in the parental line as previously described (De Carli et al., 1968); all the marker chromosomes of the parental lines could be recognized in the hybrids. Stability o f enzymic activities in the hybrM and in the parental lines The quantitative determinations of HGPRT, TK, TPI and G6PD were performed on the cell pellets harvested at different times of culture and kept at -80°C. The pellets were obtained from triplicate bottles of a single cell line, frozen independently. The specific activity of these enzymes did not change when the assay was done on extracts of fresh or of frozen pellets. The specific activities of the various enzymes of the parental and of hybrid lines, assayed at different time intervals, are reported in table 1. The four enzymic activities did not show any systematic change
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330
Genomic interactions in human hybrid lines
Table 1 HGPRT, TK, TPI, G6PD specific activities in the parental and hybrid lines at three different times. Cell lines Enzyme
Parentals Time
E6D/ TK AP
Hybrids E/HGPRTAP+
El
El
EI1/1
8/15
1/1
HAT
HGPRT*
0 1 month 2½ months
92.4 92.3 97.0
0 0 0
32.3 53.3 40.8
72.8 60.3
54.1 46.6 67.7
TK *
0 1 month 2½ months
0.4 0.8 1.1
20.7 11.5 21.1
15.7 13.7 8.0
16.5 22.4 16.3
23.2 28.0 -
TPI**
0 1 month 21½months
9.7 19.2 17.03
17.1 14.4 19.8
25.4 23.2 22.0
28.2 28.1 25.6
25.4 20.7 -
G6PD
0 1 month 21/2months
48.7 74.3 57.8
20.9 21.2 23.4
30.3 30.0 37.4
52.6 46.5 51.0
30.0 29.3 .
o
* The specific activity is expressed as: ~moles of substrate converted in 10 ram, at 37 C, per ** mg protein. The specific activity is expressed as: ~ O.D. at 366 nm/min per mg protein.
in the time interval examined, both in the parental and in the hybrid lines. However, considerable fluctuations can be observed in the values obtained in successive assays of the same enzyme. The variations do not follow a regular pattern; however, the degree of variability appears to be unequally distributed among the hybrids and the parental lines. Also, if we consider the size of the error of the enzymic determinations we can hardly attribute any significance to the above change. Differences in enzymic activities other than those involved in the selection can also be observed in the parental cell lines. The G6PD activity in the E6D/TK-AP-line is almost three times as high as in the E/HGPRT-AP ÷ line. This might be explained by the linkage between G6PD and HGPRT, which are both located on the X chromosome. Differences can also be recognized between hybrids and parental lines: TPI and TK activities are higher in the hybrids than one would expect from the mean values of the two parentals. These results will be discussed. A P activity The results of the enzymic assays are reported in table 2. It can be seen that, during the culture period considered the total AP activity does not change significantly in the parental and in the hybrid lines grown in normal medium; a decrease has been observed at later times for the hybrid EI1/1 grown in HAT medium. The
S. BARLATI et al.
331
Table 2 Total and thermostable AP specific activities expressed as ~rnoles of substrate converted in 30 min per mg protein in the parental and hybrid lines at three different times. Cell lines
Parentals
Hybrids
AP
Time
E6D/TKAP-
E/HGPRTAP+
EI8/15
EI1/I
El1/1 HAT
Total activity (units/mg)
0 1 month 21/2months
5.4 5.4 3.5
19.0 18.0 26.8
13.7 8.9 16.3
21.1 27.6 16.1
32.1 12.5 9.4
Thermostable fraction (units/mg)
0 1 month 21/2months
2.6 2.4 1.7
9.6 7.5 12.1
3.8 3.3 5.4
2.8 2.2 0.8
1.7 1.0 1.1
Thermolabile fraction (units/mg)
0 1 month 21/2months
2.8 3.0 1.8
9.4 10.5 14.7
9.9 5.6 10.9
18.3 25.1 15.3
30.4 11.5 8.3
loss of activity seems to be due to a reduction of the T1 fraction which is the major component of the AP activity. A loss of the Ts component was observed when the hybrid was grown in normal medium; this loss is not evident when the total AP activity is considered since the Ts fraction represents only a small percentage of the total activity. However, no relationship can be envisaged between changes in the AP components and the presence or absence of the selective medium. At every time the total AP activity is equal to or higher than the average of the activities of the parental lines. The T1 component, however, shows values which are close to or higher than the ones in the AP ÷ parental line. On the other hand the Ts component appears to be consistently lower than that expected from an additive contribution of the two parental lines and is very close to the activity present in the AP- parental line. In order to establish whether this pattern was a general feature of all hybrid lines derived from crosses between our AP ÷ and AP- clonal variants, the study was extended to a larger number of hybrid clones. Hybrids with the most stable and characteristic karyotype were chosen. The results of the assays of the AP fraction on these lines are reported in table 3. It can be seen that, out of ten hybrids analyzed, nine showed a level of Ts AP activity very close to or lower than that of the low activity parental line. Only one hybrid, the EI8/15 exhibited a level o f activity, for the same fraction, higher than that o f the parental line E 6 D / T K - A P , but lower than that expected from the additive contribution of the two parental lines. The presence of endogenous activators or inhibitors o f this fraction of AP activity was tested by mixing in various proportions the crude cell extracts from the two
Genomic interactions in human hybrid lines
332
Table 3 Total and thermostable AP activity in hybrid lines expressed as ~moles of substrate in 30 rain per mg protein. Clones
Subclones number
E/HGPRT- AP÷ E6D/TK- APTheoretical hybrid EI8/15 El1/1 EI8/9 E18/16 EI8/17 E18/23 EI8/5
{ 14 16 4 4 6 15 2
AP total units
AP thermostable units
AP thermolabile units
Modal chromosome number
21.3 4.8 13.1 13.0 21.6 15.8 16.1 19.1 11.2 28.3 10.1 35.9 25.8
10.5 2.3 6.4 4.1 1.9 0.4 0.4 0.9 1.0 1.0 1.3 2.8 1.0
10.8 2.5 6.7 8.9 19.7 15.4 15.7 18.2 10.2 27.3 8.8 33.1 24.8
57 53 110 99-100 104 105 105 102 106 102 98-99 103 102-103
parental lines and from the hybrids. The activity obtained in all cases was the sum of the activities as determined in the same volume of unmixed extracts. In conclusion, the Ts and T1 components behave in all the hybrids tested like the Ts component of the AP-parental line and the T1 component o f the AP÷parental line respectively.
Induction o f AP in the parental and hybrid lines Upon treatment with prednisolone the Ts fraction of the E 6 D / T K - A P - c a n be increased 4 - 5 fold while it is increased only 1.7 fold in the E/HGPRT-AP * line. The treatment with the hormone, on the other hand, decreases the T1 component by a factor o f 2 in the E 6 D / T K - A P - l i n e and by a factor between 4 to 20 in the
Table 4 AP activity in the parental lines before and after PRD treatment. Total Control
Ts fraction
T1 fraction
Treated
Control
Treated
Control
E6D/TK- AP-
Exp. I Exp. II
0.69 0.78
1.6 1.7
0.27 0.29
1.4 1.4
AZA, HGPRT- AP÷
Exp. 1 Exp. I1
19.8 17.9
9.1 8.0
4.2 2.3
8.3 5.3
0.42 0.49 15.6 15.6
Treated 0.22 0.34 0.70 2.7
333
S. BARLATI et al. Table 5 AP activity in three hybrid lines before and after PRD treatment. Total
EI8/9-14 E18/16 -4 El8/23 -2
Ts fraction
T1 fraction
Control
Treated
Control
Treated
Control
Treated
8.9 27.2 13.5
2.5 7.3 7.3
0.37 1.3 0.72
1.3 6.4 3.6
8.5 25.8 12.8
1.2 1.9 3.7
E/HGPRT-AP + line. The results of such induction-repression experiments are reported in table 4. Since the hybrid lines seem to retain a low Ts fraction as in the E 6 D / T K - A P parental line and a high T1 fraction as in the E/HGPRT-AP ÷parental line, one would expect that the treatment with prednisolone would cause an increase of the Ts fraction similar to that found in the AP-parental line and a decrease of the T1 fraction comparable to that of the AP ÷ parental line. The hormone treatment was performed on three different hybrid lines: E I 8 / 9 - 1 4 , E I 8 / 1 6 - 4 and E I 8 / 2 3 - 2 ; the results obtained are shown in table 5. As expected the Ts and T1 fractions of AP behave, in the hybrids, like independent characters maintaining unaltered their induction or repression properties. DISCUSSION The hybrids under study show a chromosomal stability sufficient to allow an investigation of the interactions between the genomes of the two clonal variants of the EUE line. The modal number of chromosomes is, in the hybrids, always lower than the sum of the modal values of the two parental lines; this could be a consequence of chromosomal loss, which occurs immediately after the fusion and which continues although at a much lower rate in the successive subcultures. Alternatively, this could be due to the fact that cells with a submodal chromosomal number have a higher probability of fusion or of recovery of hybrids. In either case the possibility exists that the chromosomal loss involved genes concerned with the enzymes under investigation; however, of the five enzymes studied, only AP, when analyzed in its Ts and T1 component, showed remarkable and constant deviations from the values expected from a simple additive effect. The possibility of a preferential loss of chromosomes carrying structural and/or regulatory genes for the various AP components should be taken into consideration. However, it appears more probable that the observed variations have a regulatory origin, in view of the observed induction-repression phenomenon for AP in cultured cells.
334
Genomic interactions in human hybrid lines
Nine out of the ten independent hybrids tested showed a low AP Ts fraction as in the E6D/TK-AP- and a high T1 fraction as in the E/HGPRT-AP ÷ parental line. As far as the T1 component of AP is concerned this is often higher in the hybrids than in the AP + parental line; however, as we have seen for the other enzymic activity tested (TPI, TK) the levels in the hybrids are generally higher than expected. Similar observations have been made by other authors (Littlefield, 1964). What seems to be a unique feature here is the defect in the AP Ts component which can be interpreted in terms of negative control. It should be noted that the Ts component maintains, in the hybrids, its inducibility by prednisolone which is a prevalent regulatory characteristic of the parent with low AP activity. These results could be explained by assuming that the A P - a n d AP + parental lines contain respectively a high and a low level of a hypothetical repressor substance; in the hybrids, therefore, the amount of repressor, contributed by one of the two combined genomes, would be enough to keep the overall AP thermostable activity low. The T1 fraction o f AP also seems to be subjected to regulation, since it decreases in the hybrid after prednisolone treatment, in the same way as the T1 fraction of the AP ÷ parental line. However, a more extensive analysis is needed before any interpretation of the relationships which seems to exist between variation of AP Ts and T1 fractions could be attempted. A disadvantage of this kind of study carried out on heteroploid cell lines is that the parental lines used for hybridization are not genotypically and phenotypically homogenous. The hybrids could therefore derive from the occasional fusion of E/HGPRT-AP ÷ cell with a low Ts and a high T1 AP activity with E6D/TK-AP- cells. This should not be the case since the determination of AP activity by histochemical staining in the E/HGPRT-AP + line showed a frequency of AP-cells lower than 1%. Therefore, the production of ten independent hybrids with the characteristic described here should be an extremely rare event.
ACKNOWLEDGEMENTS C. Ullu and C. Rossi were supported during this work by a postdoctoral fellowship of the 'Ciclo biennale di ricerche per la specializzazione in Genetica e Biochimica applicate alia microbiologia industriale' sponsored by C. Erba S.p.A., Farmitalia S.p.A.; Italseber S.p.A., Lepetit S.p.A. and Pierrel S.p.A.
REFERENCES Bollum, F.J. and V.R. Potter: Cancer Res. 19, 561-565 (1959). Davidson, R., B. Ephrussi and K. Yamamoto: J. Cellular Physiol. 72, 115-128 (1968). Davidson, R. and P. Benda: Proc. Natl. Acad. Sci., U.S. 67, 1870-1877 (1970). De Carli, L., F. Nuzzo, A.S. Santachiara-Benerecetti and J. Morrow: Ann. N.Y. Acad. Sci., 155, 1003-1019 (1968).
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Herz, F. and H.M. Nitowsky: Arch. Biochem. Biophys. 9 6 , 5 0 6 - 5 1 5 (1962). Hsu, T.C. and D.S. Kellog: J. Natl. Cancer Inst. 2 5 , 2 2 1 - 2 3 4 (1960). Littlefield, J.W.: Science 145, 7 0 9 - 7 1 0 (1964). Maio, J. and L. de Carli: Biochem. Biophys. Res. Commun., 1 1 , 3 3 5 - 3 4 2 (1963). Migeon, B.R., S.W. Smith and C.L. Leddy: Biochem. Genet. 3 , 5 8 3 - 5 9 0 (1969). Santachiara-Benerecetti, A.S. and I. Cesari: Atti Ass. Genet. Ital. 12, 188-190 (1967a). Santachiara-Benerecetti, A.S., I. Cesari and L. de Carli: J. Cellular Physiol. 69, 169-176 (1967b). Simoni, G. and L. De Carli: Europ. J. Cancer, 6, 1 5 (1970). Terni, M. and G.B. Lo Monaco: Sperimentale 108, 177-185 (1958). Weiss, M. and M. Chaplain: Proc. Natl. Acad. Sci., U.S. 68, 3 0 2 6 - 3 0 3 0 (1971).
335 (1974). © North-Holland Publishing Company
STUDIES ON GENOMIC INTERACTIONS IN HYBRIDS BETWEEN ENZYMIC VARIANTS OF A HUMAN CELL LINE S. BARLATI, A.S. SANTACHIARA-BENERECETTI, S. BALACCO*, C. ULLU, R. DI LERNIA***, C. ROSSI** and L. DE CARLI*** Laboratorio di Genetica Biochimica ed Evoluzionistica del Consiglio Nazionale delle Ricerche, Istituto di Genetica, Universitb di Pavia, 27100 Pavia, Italy Accepted 11 December 1973
The chromosomal complement and a number of enzymic activities have been studied and found to be stable in hybrids obtained from variants of the human line EUE. The parental lines utilized were characterized by high or low levels of alkaline phosphatase (AP), an enzyme which is a mixture of a thermostable (Ts) and a thermolabile (T1) component, the former being increased and the latter reduced by the addition of prednisolone to the cell culture. In the hybrids, AP activity exhibited a small amount of Ts component as in the low level parental line and a large amount of the TI component as in the high level parental line. The response of the two fractions to prednisolone indicates that, in tbe hybrids, they maintain their induction or repression properties unaltered.
The formation of hybrids between clonal derivatives of a human heteroploid line, the EUE, varying in their alkaline phosphatase (AP) levels, has been previously reported (Simoni et al., 1970). The purpose of this work is to determine the dominance relationships between the regulatory mechanisms of the two parental genomes when combined in the same cytoplasm. A similar approach has been used by other authors for studying the regulation of a number of specialized cellular functions (Davidson et al., 1968, 1970; Weiss et al., 1971). The experiments reported here are performed with clonal variants of the same cell line selected for differences in a few enzymic activities. One of these activities, namely AP, is susceptible to control by exogenous agents.
* 24.7.70. ** Present address: Istituto di Genetica, Universith di Parma, 42100 Parma, Italy. *** Present address: Istituto di Biologia Generale, Facult~ di Medicina, Universit~ di Milano, 20100 Milano, Italy.
326
Genomic interactions in human hybrM lines
Under these conditions the product of interclonal hybridization should be simpler to examine and more similar, in a way, to the system provided by the merozygotes in bacteria. The results should then have a simpler interpretation because most of the interactions due to gross differences in cell types can be eliminated. We have studied in particular AP, an enzyme which is subjected to regulation (Maio et al., 1963; Santachiara-Benerecetti et al., 1967a) and contains at least two components: one thermostable (Ts) and one thermolabile (T1) (Santachiara-Benerecetti et al., 1967b), which can be separated by starch gel electrophoresis. In addition to AP and to the enzymes involved in the system for the selection of the hybrids, like thymidine kinase (TK) and hypoxanthine-guanine-phosphoribosyltransferase (HGPRT), we tested the triosophosphate isomerase (TPI) and glucose6-phosphate dehydrogenase (G6PD) activities. The TPI and G6PD activities were chosen because of the available information on their cytological location. Since G6PD and HGPRT are known to be linked the levels of these two enzymic activities could be compared.
MATERIALS AND METHODS Cell lines
The two parental lines were characterized as follows: E/HGPRT-AP+: and EUE (Terni et al., 1958) subline resistant to 100 ~tg/ml of 8-azaguanine (AG), obtained by successive cultures with increasing concentrations of the analogue and isolated as a clone; the level of HGPRT activity was below detection. The specific activity of alkaline phosphatase was 18-26 units/mg protein and the modal number of chromosomes was 57. E 6 D / T K - A P - : and EUE subline resistant to 20/~g/ml of bromodeoxyuridine (BUdR) showing a small residual thymidine kinase activity; the specific activity of AP was about 5 units/rag protein and the modal number of chromosomes was 53. The experiments of AP induction have been carried out after a period of storage at -80°C. Culture techniques
The stock cultures were maintained as monolayers in Eagle medium with 10% calf serum. The resistant lines were cultured in the presence of the appropriate concentration of AG or BUdR and the medium containing the analogues was substituted with normal medium 24 hr before harvesting the cells for enzymic assays. The hybrid lines were derived from a single fusion experiment by using Sendal virus as previously described (Simoni et al., 1970). The hybrids, selected in HAT medium, were all primary clones presumably originated from different fusion events. The hybrid lines E18/15 and E11/1 and the two parental lines were grown in parallel cultures in Eagle medium; the E11/1 line was also grown in HAT medium
S. BARLATI et al.
327
(Eagle supplemented with 5 ~g/ml thymidine 5/ag/ml hypoxanthine and 1/~g/ml aminopterin). When the cultures were nearly confluent the medium was changed and the cells harvested the following day. An aliquot of l0 s cells was inoculated into a bottle (44 cm 2) which was used for the karyotype analysis. The remaining cells were divided in 15 aliquots of 3 X 10 6 to 4 X 10 6 cells in centrifuge tubes and washed with Hanks buffer. The pellets were stored frozen at -80°C until needed for the enzyme assays.
Cytological preparations The chromosomal counts were performed on a suspension of cells from trypsinized cultures according to Hsu et al. (1960). Biochemical assays The assay of HGPRT activity was done according to Littlefield (1964); the frozen cell pellet was suspended in 1 ml of 0.05 M phosphate buffer and lysed by freezing and thawing three times in liquid nitrogen; the resulting extract was centrifuged for 10 min at 20,000 g. The supernatant was collected and immediately used for enzymic assays or stored at -20°C. The activity was expressed as ~tmoles of hypoxanthine converted in 10 rain per mg protein. TK was assayed according to Bollum et al. (1959) with the modifications introduced by Migeon et al. (1969); the extracts were prepared as for the HGPRT assay but in this case the buffer was 10-a M Tris-HC1 pH 8, 10-1 M KC1 and 0.0029 M /3-mercaptoethanol. The activity was expressed as/amoles of thymidine converted in 10 min per mg protein. The H 3 thymidine used in the TK assay was purified, before utilization, by elution for 8 hr on descending chromatography on Whatman D.E. 81 paper. The assays for TPI and G6PD were performed by using the standard kits of Boehringer und Sohne GMBH Co., Mannheim (Germany). The activity was expressed as A O.D. at 366 nm/min per mg protein. The preparation of the extracts was done as described above but with 0.3 M triethanolamine at pH 7.6 as resuspending buffer. All the assays were done in duplicate and the mean values are reported in the tables. The extraction procedure and the enzymic assay for alkaline phosphatase were performed as previously reported (Santachiara-Benerecetti et al., 1967b). The AP thermostable fraction was determined by heating, for 30 min at 70°C, 0.1 ml aliquots of cell extracts diluted with 0.4 ml of 0.05 M Tris-HC1 pH 7.1 preheated at 70°C. The heated samples, together with the controls, were extracted after cooling with butanol according to the Morton technique as modified by Herz et al. (1962). AP activity was assayed on the aqueous phase and the thermostable component was determined as percent of the control and expressed as Ts specific activity. The T1 specific activity was determined by subtracting the Ts activity from the total AP specific activity. Both the treatments and the assays were done in triplicate.
328
Genomic interactions in human hybrid lines
Induction experiments One day after trypsinisation of the cells the medium was substituted with Eagle's containing 10/ag/ml of prednisolone dissolved in absolute ethyl alcohol. Maximum induction of AP was obtained after 4 to 6 days of incubation at 37°C in the presence of the hormone; this treatment did not significantly affect the growth rate of the cells. The medium was checked for pH daily and changed one day before harvesting the cells. Also in this case the cultures were nearly confluent at the time of harvesting. Chemicals 8-azaguanine, 5-bromo-2'-deoxyuridine, aminopterin and prednisolone were obtained from Sigma Chemical Co., while thymidine and hypoxanthine were from Calbiochem. H 3 thymidine (1 mc/ml; 5.6 X 10-2 M) was obtained from New England Nuclear.
RESULTS Cytological controls The chromosome distributions for the various lines observed at three successive times on samples of fifty mitoses are reported in fig. 1. No gross alteration in chromosome number was detected for the two parental and for the hybrid lines during the time interval of two and a half months. A wider spread in the chromosome number was observed in the hybrids; for the hybrid line EI1/1 the relative stability of the total number of chromosomes is evident both in the presence and in the absence of the selective medium containing aminopterin. A detailed chromosomal analysis of all the lines examined was virtually impossible and in any case it would not have given an unambiguous answer. Therefore, this stability refers only to the total chromosome number and not to their karyotypes. The difference in the number of small acrocentric chromosomes persisted in the parental line as previously described (De Carli et al., 1968); all the marker chromosomes of the parental lines could be recognized in the hybrids. Stability o f enzymic activities in the hybrM and in the parental lines The quantitative determinations of HGPRT, TK, TPI and G6PD were performed on the cell pellets harvested at different times of culture and kept at -80°C. The pellets were obtained from triplicate bottles of a single cell line, frozen independently. The specific activity of these enzymes did not change when the assay was done on extracts of fresh or of frozen pellets. The specific activities of the various enzymes of the parental and of hybrid lines, assayed at different time intervals, are reported in table 1. The four enzymic activities did not show any systematic change
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Genomic interactions in human hybrid lines
Table 1 HGPRT, TK, TPI, G6PD specific activities in the parental and hybrid lines at three different times. Cell lines Enzyme
Parentals Time
E6D/ TK AP
Hybrids E/HGPRTAP+
El
El
EI1/1
8/15
1/1
HAT
HGPRT*
0 1 month 2½ months
92.4 92.3 97.0
0 0 0
32.3 53.3 40.8
72.8 60.3
54.1 46.6 67.7
TK *
0 1 month 2½ months
0.4 0.8 1.1
20.7 11.5 21.1
15.7 13.7 8.0
16.5 22.4 16.3
23.2 28.0 -
TPI**
0 1 month 21½months
9.7 19.2 17.03
17.1 14.4 19.8
25.4 23.2 22.0
28.2 28.1 25.6
25.4 20.7 -
G6PD
0 1 month 21/2months
48.7 74.3 57.8
20.9 21.2 23.4
30.3 30.0 37.4
52.6 46.5 51.0
30.0 29.3 .
o
* The specific activity is expressed as: ~moles of substrate converted in 10 ram, at 37 C, per ** mg protein. The specific activity is expressed as: ~ O.D. at 366 nm/min per mg protein.
in the time interval examined, both in the parental and in the hybrid lines. However, considerable fluctuations can be observed in the values obtained in successive assays of the same enzyme. The variations do not follow a regular pattern; however, the degree of variability appears to be unequally distributed among the hybrids and the parental lines. Also, if we consider the size of the error of the enzymic determinations we can hardly attribute any significance to the above change. Differences in enzymic activities other than those involved in the selection can also be observed in the parental cell lines. The G6PD activity in the E6D/TK-AP-line is almost three times as high as in the E/HGPRT-AP ÷ line. This might be explained by the linkage between G6PD and HGPRT, which are both located on the X chromosome. Differences can also be recognized between hybrids and parental lines: TPI and TK activities are higher in the hybrids than one would expect from the mean values of the two parentals. These results will be discussed. A P activity The results of the enzymic assays are reported in table 2. It can be seen that, during the culture period considered the total AP activity does not change significantly in the parental and in the hybrid lines grown in normal medium; a decrease has been observed at later times for the hybrid EI1/1 grown in HAT medium. The
S. BARLATI et al.
331
Table 2 Total and thermostable AP specific activities expressed as ~rnoles of substrate converted in 30 min per mg protein in the parental and hybrid lines at three different times. Cell lines
Parentals
Hybrids
AP
Time
E6D/TKAP-
E/HGPRTAP+
EI8/15
EI1/I
El1/1 HAT
Total activity (units/mg)
0 1 month 21/2months
5.4 5.4 3.5
19.0 18.0 26.8
13.7 8.9 16.3
21.1 27.6 16.1
32.1 12.5 9.4
Thermostable fraction (units/mg)
0 1 month 21/2months
2.6 2.4 1.7
9.6 7.5 12.1
3.8 3.3 5.4
2.8 2.2 0.8
1.7 1.0 1.1
Thermolabile fraction (units/mg)
0 1 month 21/2months
2.8 3.0 1.8
9.4 10.5 14.7
9.9 5.6 10.9
18.3 25.1 15.3
30.4 11.5 8.3
loss of activity seems to be due to a reduction of the T1 fraction which is the major component of the AP activity. A loss of the Ts component was observed when the hybrid was grown in normal medium; this loss is not evident when the total AP activity is considered since the Ts fraction represents only a small percentage of the total activity. However, no relationship can be envisaged between changes in the AP components and the presence or absence of the selective medium. At every time the total AP activity is equal to or higher than the average of the activities of the parental lines. The T1 component, however, shows values which are close to or higher than the ones in the AP ÷ parental line. On the other hand the Ts component appears to be consistently lower than that expected from an additive contribution of the two parental lines and is very close to the activity present in the AP- parental line. In order to establish whether this pattern was a general feature of all hybrid lines derived from crosses between our AP ÷ and AP- clonal variants, the study was extended to a larger number of hybrid clones. Hybrids with the most stable and characteristic karyotype were chosen. The results of the assays of the AP fraction on these lines are reported in table 3. It can be seen that, out of ten hybrids analyzed, nine showed a level of Ts AP activity very close to or lower than that of the low activity parental line. Only one hybrid, the EI8/15 exhibited a level o f activity, for the same fraction, higher than that o f the parental line E 6 D / T K - A P , but lower than that expected from the additive contribution of the two parental lines. The presence of endogenous activators or inhibitors o f this fraction of AP activity was tested by mixing in various proportions the crude cell extracts from the two
Genomic interactions in human hybrid lines
332
Table 3 Total and thermostable AP activity in hybrid lines expressed as ~moles of substrate in 30 rain per mg protein. Clones
Subclones number
E/HGPRT- AP÷ E6D/TK- APTheoretical hybrid EI8/15 El1/1 EI8/9 E18/16 EI8/17 E18/23 EI8/5
{ 14 16 4 4 6 15 2
AP total units
AP thermostable units
AP thermolabile units
Modal chromosome number
21.3 4.8 13.1 13.0 21.6 15.8 16.1 19.1 11.2 28.3 10.1 35.9 25.8
10.5 2.3 6.4 4.1 1.9 0.4 0.4 0.9 1.0 1.0 1.3 2.8 1.0
10.8 2.5 6.7 8.9 19.7 15.4 15.7 18.2 10.2 27.3 8.8 33.1 24.8
57 53 110 99-100 104 105 105 102 106 102 98-99 103 102-103
parental lines and from the hybrids. The activity obtained in all cases was the sum of the activities as determined in the same volume of unmixed extracts. In conclusion, the Ts and T1 components behave in all the hybrids tested like the Ts component of the AP-parental line and the T1 component o f the AP÷parental line respectively.
Induction o f AP in the parental and hybrid lines Upon treatment with prednisolone the Ts fraction of the E 6 D / T K - A P - c a n be increased 4 - 5 fold while it is increased only 1.7 fold in the E/HGPRT-AP * line. The treatment with the hormone, on the other hand, decreases the T1 component by a factor o f 2 in the E 6 D / T K - A P - l i n e and by a factor between 4 to 20 in the
Table 4 AP activity in the parental lines before and after PRD treatment. Total Control
Ts fraction
T1 fraction
Treated
Control
Treated
Control
E6D/TK- AP-
Exp. I Exp. II
0.69 0.78
1.6 1.7
0.27 0.29
1.4 1.4
AZA, HGPRT- AP÷
Exp. 1 Exp. I1
19.8 17.9
9.1 8.0
4.2 2.3
8.3 5.3
0.42 0.49 15.6 15.6
Treated 0.22 0.34 0.70 2.7
333
S. BARLATI et al. Table 5 AP activity in three hybrid lines before and after PRD treatment. Total
EI8/9-14 E18/16 -4 El8/23 -2
Ts fraction
T1 fraction
Control
Treated
Control
Treated
Control
Treated
8.9 27.2 13.5
2.5 7.3 7.3
0.37 1.3 0.72
1.3 6.4 3.6
8.5 25.8 12.8
1.2 1.9 3.7
E/HGPRT-AP + line. The results of such induction-repression experiments are reported in table 4. Since the hybrid lines seem to retain a low Ts fraction as in the E 6 D / T K - A P parental line and a high T1 fraction as in the E/HGPRT-AP ÷parental line, one would expect that the treatment with prednisolone would cause an increase of the Ts fraction similar to that found in the AP-parental line and a decrease of the T1 fraction comparable to that of the AP ÷ parental line. The hormone treatment was performed on three different hybrid lines: E I 8 / 9 - 1 4 , E I 8 / 1 6 - 4 and E I 8 / 2 3 - 2 ; the results obtained are shown in table 5. As expected the Ts and T1 fractions of AP behave, in the hybrids, like independent characters maintaining unaltered their induction or repression properties. DISCUSSION The hybrids under study show a chromosomal stability sufficient to allow an investigation of the interactions between the genomes of the two clonal variants of the EUE line. The modal number of chromosomes is, in the hybrids, always lower than the sum of the modal values of the two parental lines; this could be a consequence of chromosomal loss, which occurs immediately after the fusion and which continues although at a much lower rate in the successive subcultures. Alternatively, this could be due to the fact that cells with a submodal chromosomal number have a higher probability of fusion or of recovery of hybrids. In either case the possibility exists that the chromosomal loss involved genes concerned with the enzymes under investigation; however, of the five enzymes studied, only AP, when analyzed in its Ts and T1 component, showed remarkable and constant deviations from the values expected from a simple additive effect. The possibility of a preferential loss of chromosomes carrying structural and/or regulatory genes for the various AP components should be taken into consideration. However, it appears more probable that the observed variations have a regulatory origin, in view of the observed induction-repression phenomenon for AP in cultured cells.
334
Genomic interactions in human hybrid lines
Nine out of the ten independent hybrids tested showed a low AP Ts fraction as in the E6D/TK-AP- and a high T1 fraction as in the E/HGPRT-AP ÷ parental line. As far as the T1 component of AP is concerned this is often higher in the hybrids than in the AP + parental line; however, as we have seen for the other enzymic activity tested (TPI, TK) the levels in the hybrids are generally higher than expected. Similar observations have been made by other authors (Littlefield, 1964). What seems to be a unique feature here is the defect in the AP Ts component which can be interpreted in terms of negative control. It should be noted that the Ts component maintains, in the hybrids, its inducibility by prednisolone which is a prevalent regulatory characteristic of the parent with low AP activity. These results could be explained by assuming that the A P - a n d AP + parental lines contain respectively a high and a low level of a hypothetical repressor substance; in the hybrids, therefore, the amount of repressor, contributed by one of the two combined genomes, would be enough to keep the overall AP thermostable activity low. The T1 fraction o f AP also seems to be subjected to regulation, since it decreases in the hybrid after prednisolone treatment, in the same way as the T1 fraction of the AP ÷ parental line. However, a more extensive analysis is needed before any interpretation of the relationships which seems to exist between variation of AP Ts and T1 fractions could be attempted. A disadvantage of this kind of study carried out on heteroploid cell lines is that the parental lines used for hybridization are not genotypically and phenotypically homogenous. The hybrids could therefore derive from the occasional fusion of E/HGPRT-AP ÷ cell with a low Ts and a high T1 AP activity with E6D/TK-AP- cells. This should not be the case since the determination of AP activity by histochemical staining in the E/HGPRT-AP + line showed a frequency of AP-cells lower than 1%. Therefore, the production of ten independent hybrids with the characteristic described here should be an extremely rare event.
ACKNOWLEDGEMENTS C. Ullu and C. Rossi were supported during this work by a postdoctoral fellowship of the 'Ciclo biennale di ricerche per la specializzazione in Genetica e Biochimica applicate alia microbiologia industriale' sponsored by C. Erba S.p.A., Farmitalia S.p.A.; Italseber S.p.A., Lepetit S.p.A. and Pierrel S.p.A.
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