Mutation Research, 240 (1990) 135-142 Elsevier
135
MUTGEN 01516
In vitro induction, expression and selection of thioguanine-resistant mutants with human T-lymphocytes J.P. O'Neill, L.M. S u l l i v a n a n d R.J. A l b e r t i n i Department of Medicine and Vermont Regional Cancer Center, University of Vermont, Genetics Laboratory, 32 N. Prospect St., Burlington, VT 05401 (U.S.A.) (Received 12 May 1989) (Revision received 25 September 1989) (Accepted 30 September 1989)
Keywords: Human T-lymphocytes; Lymphocytes, T, human; Gamma-irradiation; Hprt locus; Hypoxanthine-guanine phosphoribosyl transferase locus; TCGF; Crude T-cell growth factor
Summary Conditions have been defined to measure the in vitro induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human T-lymphocytes by a cell cloning assay. The in vitro growth of mass cultures as well as cell cloning is accomplished by the use of crude T-cell growth factor (TCGF) and irradiated human lymphoblastoid feeder cells. These initial studies employed irradiation of G O phase peripheral blood mononuclear cells from a single individual. After exposure to T-irradiation from a 137Cs source, the cells were stimulated with the mitogen phytohemagglutinin (PHA) and maintained in exponential growth with exogenous TCGF to allow phenotypic expression of the 6-thioguanine-resistant (TG r) mutants. The mutant frequency was determined by measuring cell cloning efficiency in microtiter dishes in the absence and presence of TG, with an optimal selection density of 1 x 10 4 cells/well. The development of this in vitro assay should allow direct study of susceptibility to T-irradiation in the human population in terms of both cytotoxicity and mutation induction.
The development of methods for the in vitro cloning of human T-lymphocytes from peripheral blood samples has allowed the measurement of the in vivo frequency of 6-thioguanine-resistant (TG r) mutants in human somatic ceils (Albertini et al., 1982; Morley et al., 1983; Henderson et al.,
Correspondence: Dr. J.P. O'Neill, Department of Medicine and Vermont Regional Cancer Center, University of Vermont, Genetics Laboratory, 32 N. Prospect St., Burlington, VT 05401
(U.S.A.).
1986; O'Neill et al., 1987a). Such an assay should prove useful in human genotoxicity monitoring (Hakoda et al., 1988). The use of T-lymphocytes also allows one to define the relationship between the measured mutant frequency and the actual in vivo mutation frequency (Nicklas et al., 1987; Hakoda et al., 1989). Knowledge of the latter is the ultimate goal of human genetic monitoring studies. However, interpretation of these mutation frequency values will be complicated by the probable heterogeneity of mutagen susceptibility in the human population. One possible means to assess this heterogeneity would be the use of human
0165-1218/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
136 T-lymphocyte cultures in in vitro mutation induction assays (O'Neill et al., 1987b). The development of a quantitative in vitro measurement of mutation induction would allow determination of individual susceptibility to a particular mutagenic agent. In addition, molecular analysis of mutations induced by a specific agent in vitro might aid in the interpretation of in vivo events occurring in humans exposed to that same agent. An example of this would be the comparison of the types of mutations induced in vitro by v-irradiation with those found occurring in vivo in individuals exposed to irradiation, either accidentally or for therapeutic reasons (Messing et al., 1986). Preliminary studies of in vitro induction of mutations in T-lymphocytes have demonstrated the feasibility of this approach (Sanderson et al., 1984; Vijayalaxmi and Evans, 1984; Shulimowsky et al., 1986; O'Neill and Albertini, 1986). This report presents progress in the development of a quantitative assay of in vitro mutagenicity in human T-lymphocytes. Materials and methods
Cells and culture conditions These studies employed blood samples from a single individual. The mononuclear cell fraction containing the T-lymphocytes was isolated as described previously (O'Neill et al., 1987a). All cell culture employed medium RPMI 1640 (Mediatech) containing 20% nutrient medium HL-1 (Ventrex) and 5% prescreened, heat inactivated (56 ° C, 30 min) fetal bovine serum (FBS; Sterile Systems) in humidified incubators at 37-38°C in an atmosphere of 5-6% CO 2 in air. T-cell growth factor (TCGF) was prepared as described previously (O'Neill et al., 1987a) or purchased from Collaborative Research (human T-cell polyclone). All cell culture employed y-irradiated human lymphoblastoid feeder cells described in detail previously (O'Neill et al., 1987). The optimal amount of T C G F and feeder cells was defined as described in the results section. The feeder cells were exposed to 9 krad of "/-radiation from a 137Cs source at approx. !100 r a d / m i n . Mutation induction, expression and selection The M N C fraction was suspended at 2 x 106 cells/ml in RPMI 1640 and exposed to irradiation
from the 137Cs source. Then an equal volume of medium RPMI 1640 containing 40% HL-1 and 10% FBS was added, the cells aliquoted at 20 ml per 25-cm2 flask and PHA added at 1 /~g/ml. After 36-40 h incubation (designated day 2) the cell number was determined and the cells plated for cloning efficiency determinations at 1, 2 and 4 cells/well in 96 well (round bottom) microtiter plates (Nunc). Cells were plated at 2 x 104 cells/well in 10 /xM T G for mutant selection (O'Neill et al., 1987a). Cells were also plated in mass culture at 1 x 105 cells/ml in growth medium containing 2.5 x 105 irradiated feeder cells/cm 2 and optimal amounts of T C G F and incubated for 3 days. On day 5, and at 3-day intervals thereafter, cells were subcultured, and plated for cloning efficiency (1 and 2 cells/well) and mutant selection (1 x 104 cells/well). Colony growth was determined by use of an inverted phase-contrast microscope after 10-15 days incubation. Cloning efficiency was calculated by the P0 distribution and the ratio of these values in the presence and absence of T G yields the mutant frequency. Results and discussion
In order to quantify in vitro induction of mutations at the hprt locus in human T-lymphocytes, conditions must be defined to allow optimal proliferation of mass cultures for expression of the T G r phenotype, as well as optimal cell cloning for mutant selection. These parameters were not addressed in two previous preliminary studies (Vijayalaxmi and Evans, 1984; Sanderson et al., 1984). Lack of this information renders meaningful study of heterogeneity in the response in the human population difficult, if not impossible, because such comparisons require quantitative and reproducible determinations. Our investigations of in vitro growth and cloning of T-cell cultures have employed samples from 10 individuals to define the optimal conditions. Based on previous studies, the medium used was RPMI 1640 containing 20% medium HL-1 and 5% prescreened FBS (O'Neill et al., 1987a). The mononuclear cell (MNC) fraction from peripheral blood samples was incubated in this medium at 1 x 10 6 cells/ml in the presence of 1 # g / m l puff-
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Fig. 1. Effect of T C G F amount on T-lymphocyte colony growth. Primed MNC were plated at 1 cell/well in the indicated amount of crude TCGF, either prepared as described (left panel) or purchased from commercial sources (right panel). Cloning efficiencies (circles) and fraction of large colonies (squares) were determined after 14 days incubation.
fied P H A for 36-40 h to achieve mitogen stimulation. These primed cells were then used to measure cell cloning and mass culture proliferation. Optimal amounts of T C G F for cell cloning were defined as described previously (O'Neill et al., 1987a) employing limiting dilution in 96-well mierotiter dishes. Fig. 1 shows the results obtained with the two batches of crude T C G F used in the studies described. These results with one individual are similar to those obtained with the other 9 individuals' samples in that maximal cloning efficiencies and colony size were obtained with 50% (left panel) and 10% (right panel) T C G F for these two batches, respectively. The primed cells were also used for mass culture proliferation in 2-cm 2 wells in the presence of different amounts of T C G F . The cells were plated at log dilutions in order to define any possible cell density effect, primarily because "/-irradiation will result in cytotoxicity. Therefore, irradiated cultures will have decreased numbers of viable cells and might respond differently to in vitro growth conditions in comparison with non-irradiated cultures. It is essential that culture conditions allow equivalent growth, regardless of viable cell inoculum size. In addition, P H A was also added to the medium since the use of different amounts of T C G F also results in the dilution of the P H A employed in the production of the crude T C G F . Fig. 2 (panel A) shows the increase in cell number as a function of time in culture for cells in 50% T C G F , the amount found optimal by cell cloning
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Fig. 2. Effect of PHA and T C G F on the in vitro growth of T-lymphocyte mass cultures. Cultures were incubated for 36 h with 1 # g / m l PHA and then plated at 1×105 (triangles), 1 × 104 (squares), 1 ×103 (circles) or 1 × 102 (diamonds) cells per 2-cm 2 wells in 2 ml in the absence (closed symbols) or presence (open symbols) of 1 /~g/ml PHA and the indicated amount of TCGF. All wells also contained 2 x 1 0 5 irradiated lymphoblastoid feeder cells. Panel A shows the increase in cell number as a function of time in culture for cells in 50% TCGF. Panels B and C show the maximum cell number obtained with 0-50% T C G F in the absence and presence of PHA, respectively. The maximal cell densities were usually reached on days 4, 7, 10 and 13, for cells plated at 105 , 104 , 103 and 102 cells/well, respectively. A cell count of 0.05 x 106 was obtained with feeder cells alone and has not been subtracted.
5 A
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% TCGF Fig. 3. Effect of amount of T C G F on the in vitro growth of T-lymphocytes. T-lymphocytes were cultured as described in vitro for 2 days (panel A) or 4 days (panel B) and then plated at 1 ×10 5 (open triangles), 1 ×10 4 (closed triangles), 1),¢J z laJ m i, i, ~J
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Fig. 5. In vitro growth of T-lymphocytes. T-lymphocytes were incubated for 24 h with 1 #g/ml PHA and then subcultured at the indicated times in medium containing optimal amounts of TCGF and 2.5 × l0 s irradiated feeder cells/cm2. Cell number was determined daily and the cells subcultured at 1 × 105 cells per ml every four days.
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Fig. 8. Cytotoxicity of y-irradiation of T-lymphocytes. Cells were exposed to 0-400 rad of y-irradiation, incubated with PHA for 36-40 h and plated in 96-well microtiter dishes at 1, 2 and 4 cells/well. Cloning efficiencies are expressed relative to the unirradiated control. The symbols represent the mean ( + SD) values for 3-5 independent determinations.
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Fig. 6. Effect of amount of TCGF on T-cell cloning. Tlymphocytes were subcultured in vitro for 2 (panel A), 5 (panel B) or 8 (panel C) days and then plated at 1 ceil/well in 2130ttl of medium containing the indicated amount of TCGF and 1 × 104 irradiated feeder cells. The two symbols represent two different individuals.
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Fig. 7. Limiting dilution analysis of T-lymphocyte cloning. Cells were subcultured for 11 (closed circles) or 13 days (open circles) and plated for cloning at 0.5-8 cells/well, based on limiting dilution. The mean cloning efficiencies+S.D, were 0.316+0.071 (open circles) and 0.796-1-0.081 (dosed circles) and the lines are drawn based on these mean values.
allow the i n vitro growth a n d single cell c l o n i n g of T - l y m p h o c y t e s from h u m a n peripheral b l o o d sampies. T o m e a s u r e the i n d u c t i o n of m u t a t i o n s at the hprt locus b y y-irradiation, the G O phase p e r i p h eral b l o o d M N C were irradiated, p r i m e d with 1 / ~ g / m l P H A for 3 6 - 4 0 h a n d then s u b c u l t u r e d at 3-day intervals at 1 × 10 5 c e l l s / m l in growth m e d i u m c o n t a i n i n g o p t i m a l a m o u n t s of T C G F a n d 2.5 × 10 5 irradiated feeder c e l l s / c m 2. Cytotoxicity of the i r r a d i a t i o n was d e t e r m i n e d b y measuring the c l o n i n g efficiency of the p r i m e d cells. M u t a n t frequency was d e t e r m i n e d b y m e a s u r i n g the c l o n i n g efficiency i n the a b s e n c e a n d presence of 1 0 / ~ M T G , usually with 1, 2 a n d 4 c e l l s / w e l l a n d 1 x 10 4 cells/well, respectively. I n these initial experiments, cells from a single i n d i v i d u a l were employed, utilizing i n d e p e n d e n t b l o o d sampies. Fig. 8 shows the cytotoxicity of 7 - i r r a d i a t i o n of G O phase M N C as d e t e r m i n e d b y the cell c l o n i n g assay. The p l a t i n g of cells after i r r a d i a t i o n a n d p r i m i n g allows m e a s u r e m e n t of cell c l o n i n g before a n y cell division occurs a n d reflects i n d i v i d u a l G O cell response to the treatment. Based o n the cytotoxicity, a dose of 300 rad was chosen for s t u d y of the p h e n o t y p i c expression of i n d u c e d m u t a n t s . At each time interval (after p r i m i n g which is design a t e d day 2 a n d at 3-day intervals of s u b c u l t u r e
140 150
TABLE 1
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EFFECT OF SELECTION CELL DENSITY ON MEASURED TG r M U T A N T F R E Q U E N C Y
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Mutant frequency at
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EXPRESSION TIME (doy,)
Fig. 9. Expression time of the TG r phenotype. Cells were exposed to 300 rad of "c-irradiation, incubated with PHA for 40 h and plated in the absence of TG at 1, 2 and 4 cells/well and in the presence of TG at 1 x 104 cells/well. (Time designated day 2 after irradiation.) Cells were also subcultured at 1 x l0 s cells/ml in 20-ml cultures and plated as above for mutant selection at 3-day intervals. The measured mutant frequency is graphed as a function of expression time (days).
thereafter) cells were placed in the absence and presence of T G in 96-well microtiter dishes and the cloning efficiencies determined by the P0 distribution as described previously for in vivo derived mutants (O'Neill et al., 1987a). The ratio of cloning efficiencies defines the T G r mutant frequency. Fig. 9 shows the results of one determination of the expression of the T G r phenotype during postirradiation subculture. The unirradiated culture shows little change in the T G r mutant frequency during the 14 days of subculture (range = 1.5-7.5 x 10-6). However, cells exposed to 300 rad show an increase in mutant frequency at 5 days, reaching the highest value at 8 days with a subsequent decline thereafter. This time course of phenotypic expression is similar to that described previously with cells from another individual (O'Neill et al., 1987b). Based on these and other determinations, an interval of 8 days of culture in vitro appears to allow maximal phenotypic expression. However, the observed decline with longer subculture suggests that there may be some type of selection against T G ~ mutants which occurs during the growth of mass cultures. Whether this is a consequence of the loss of H P R T enzyme activity or is a manifestation of an immunological response to the mutant cells is not known. However, this
Day Day Day Day
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a A minimum of 1.92 x 106 cells or 4 plates were employed at each selection cell density.
clearly warrants further study. At present, the use of an 8-day expression time probably allows a reasonable measure of mutation induction with T-cells. Table 1 shows that the use of a selection cell density of 1 x 10 4 cells/well does allow quantitative cloning of T G r mutants since similar mutant frequencies are obtained at lower cell densities. This selection density of 1 x 10 4 cells/well does not result in the loss of mutants due to metabolic cooperation or media depletion. In addition, these results with cell densities of 1 x 10 3 tO 2 X 10 4 cells/well suggest that T G r mutant cell cloning follows the expected Poisson distribution as was shown for cell cloning in the absence of T G (Fig. 7).
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Fig. 10. Dose-response of 7-irradiation induced T G r mutant frequency. The measured mutant frequency after 8 days phenotypic expression is graphed as a function of radiation dose. The symbols represent the mean ( + SD) values for 3-5 independent determinations.
141 75
Acknowledgements
%
Research supported by NCI ROI CA30688 and DOE DE-ACO283ER60147A003. The latter does not constitute an endorsement by DOE of the views expressed in this manuscript.
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.
.
.
.
.
.
.
.
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SURVIVAL
Fig. 11. Induced mutant frequency versus cell survival. Induced mutant frequency (Fig. 10) is graphed as a function of initial cell survival (Fig. 8).
Employing a phenotypic expression time of 8 days and a selection cell density of 1 x 10 4 cells/well, the dose-response for y-irradiation was determined and is shown in Fig. 10. The T G r mutant frequency increases with irradiation dose in a non-linear fashion as has been reported with other cell systems (cf. Thacker and Cox, 1975). These types of dose-response relationships are thought to involve the repair of potentially mutagenic damage in a manner analogous to the repair of potentially lethal damage (reviewed in Sankaranarayanan, 1982). The relationship between induced mutant frequency and cytotoxicity appears to be linear (Fig. 11) as described originally by Munson and Goodhead (1977). These results demonstrate that conditions can be defined which allow measurement of mutation induction by y-irradiation in human T-cells in vitro. The type of dose-response study shown in Figs. 8 and 10 requires a blood sample of 60 ml. Recent improvements in T-lymphocyte culture will reduce the sample size needed for such an analysis. This assay could then be employed to measure heterogeneity of sensitivity to irradiation in the human population for both cytotoxicity (cf. Miyakoshi et al., 1987) and T G r mutation induction. In addition, the ability to induce mutants allows molecular analysis of the nature of these mutations, as described in the accompanying article (O'Neill et al., 1990).
Albertini, R.J. (1985) Somatic gene mutations in vivo as indicated by the 6-thioguanine resistant T-lymphocytes in human blood, Mutation Res., 150, 411-422. Albertini, R.J., K.S. Castle and W.R. Borcherding (1982) T-Cell cloning to detect the mutant 6-thioguanine resistant lymphocytes present in human peripheral blood, Proc. Natl. Acad. Sci. (U.S.A.), 79, 6617-6621. Albertini, R.J., J.P. O'Neill, J.A. Nicklas, N.H. Heintz and P.C. Kelleher (1985) Alterations of the hprt gene in human 6-thioguanine resistant T-lymphocytes arising in vivo, Nature (London), 316, 369-371. Hakoda, M., M. Ahiyama, S. Kyoizumi, A.A. Awa, M. Yamahido and M. Otake (1988) Increased cell mutant frequency in atomic bomb survivors, Mutation Res., 201, 39-48. Hakoda, M., Y. Hirai, S. Kyoizumi and M. Ahiyama (1989) Molecular analysis of in vivo hprt mutant T-cells from atomic bomb survivors, Environ. Mol. Mutagen., 13, 25-33. Henderson, L., H. Cole, S.E. James and M. Green (1988) Detection of somatic mutations in man: evaluation of the microtitre cloning assay for T-lymphocytes, Mutagenesis, 1, 195-200. Messing, K., A.M. Seifert and W.E.C. Bradley (1986) In vivo mutant frequency of technicians professionally exposed to ionizing radiation, in: M. Sorsa and H. Norppa (Eds.), Monitoring of Occupational Genetoxicants, Liss, New York, pp. 87-97. Miyakoshi, J., K. Tatsumi and H. Takebe (1987) Radiation sensitivity of T-lymphocytes grown with recombinant human interleukin-2, Mutation Res., 192, 163-167. Morley, A.A., K.J. Trainor, R. Seshadri and R.G. Ryall (1983) Measurement of in vivo mutations in human lymphocytes, Nature (London), 302, 155-156. Munson, R.J., and D.T. Goodhead (1977) The relation between mutation frequency and cell survival, a theoretical induced approach and an examination of experimental data for eukaryotes, Mutation Res., 42, 145-160. Munzer, J.S., S.K. Jones, J.P. O'Neill, J.N. Hartshorn and S.H. Robison (1988) Detection of DNA damage and repair by alkaline elution using human lymphocytes, Mutation Res., 194, 101-108. Nicklas, J.A., J.P. O'Neill and R.J. Albertini (1986) Use of T-cell receptor gene probes to quantify the in vivo hprt mutations in human T-lymphocytes, Mutation Res., 173, 65-72. Nicklas, J.A., T.C. Hunter, L.M. Sullivan, J.K. Berman, J.P. O'Neill and R.J. Albertini (1987a) Molecular analysis of in vivo hprt mutations in human T-lymphocytes, I. Studies of
142 low frequency spontaneous mutants by Southern blots, Mutagenesis, 2, 341-347. Nicklas, J.A., J.P. O'Neill, L.M. Sullivan, T.C. Hunter, M. Allegretta, B.F. Chastenay, B.L. Libbus and R.J. Albertini (1987b) Molecular analyses of in vivo hypoxanthine-guanine phosphoribosyltransferase mutations in human T-lymphocytes, II. Demonstration of a clonal amplification of hprt mutant T-lymphocytes in vivo, Environ. Mol. Mutagen., 12, 271-284. O'Neill, J.P., and R.J. Albertini (1986) Human somatic cell mutation in vitro: development of a clonal assay for T-cell mutants, Environ. Mol. Mutagen., 8 (Suppl. 6), 62. O'Neill, J.P., M.J. McGinniss, J.K. Berman, L.M. Sullivan, J.A. Nicklas and R.J. Albertini (1987a) Refinement of a T-lymphocytes cloning assay to quantify the in vivo thioguanine mutant frequency in humans, Mutagenesis, 2, 87-94. O'Neill, J.P., J.A. Nicklas, T.C. Hunter, L.M. Sullivan and R.J. Albertini (1987b) Molecular analysis of mutations at the hprt locus in human T-lymphocytes, Banbury Rept., 28, 249-261. O'Neill, J.P., L.M. Sullivan, J.A. Nicklas, T.C. Hunter and R.J. Albertini (1987c) In vitro mutation induction by gamma irradiation in human T-lymphocytes, Environ. Mutagen., 9 (Suppl. 8), 8.
O'Neill, J.P., T.C. Hunter, L.M. Sullivan, J.A. Nicklas and R.J. Albertini (1990) Southern-blot analysis of human T-lymphocyte mutants induced in vitro by y-irradiation, Mutation Res., 240, 143-149. Sanderson, B.J.S., J.L. Dempsey and A.A. Morley (1984) Mutations in human lymphocytes: Effect of X- and UV-radiation, Mutation Res., 140, 223-227. Sankaranarayanan, K. (1982) Genetic Effects of Ionizing Radiation on Multicellular Eukaryotes and the Assessment of Genetic Radiation in Man, Elsevier, Amsterdam. Skulimowski, A.W., D.R. Turner, A.A. Morley, F.J.S. Sanderson and M. Haliandros (1986) Molecular basis of X-ray induced mutations at the HPRT locus in human lymphocytes, Mutation Res., 162, 223-227. Subak-Sharpe, J.H., R.R. Burk and J.P. Pitts (1969) Metabolic cooperation between biochemically marked mammalian cells in tissue culture, J. Cell. Sci., 353-367. Thacker, J., and R. Cox (1975) Mutation induction and inactivation in mammalian cells exposed to ionizing radiation, Nature (London), 258, 429-431. Vijayalaxmi and H.J. Evans (1984) Measurement of spontaneous and X-irradiation induced 6-thioguanine resistant human blood lymphocytes using a T-cell cloning technique, Mutation Res., 129, 283-289.
135
MUTGEN 01516
In vitro induction, expression and selection of thioguanine-resistant mutants with human T-lymphocytes J.P. O'Neill, L.M. S u l l i v a n a n d R.J. A l b e r t i n i Department of Medicine and Vermont Regional Cancer Center, University of Vermont, Genetics Laboratory, 32 N. Prospect St., Burlington, VT 05401 (U.S.A.) (Received 12 May 1989) (Revision received 25 September 1989) (Accepted 30 September 1989)
Keywords: Human T-lymphocytes; Lymphocytes, T, human; Gamma-irradiation; Hprt locus; Hypoxanthine-guanine phosphoribosyl transferase locus; TCGF; Crude T-cell growth factor
Summary Conditions have been defined to measure the in vitro induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human T-lymphocytes by a cell cloning assay. The in vitro growth of mass cultures as well as cell cloning is accomplished by the use of crude T-cell growth factor (TCGF) and irradiated human lymphoblastoid feeder cells. These initial studies employed irradiation of G O phase peripheral blood mononuclear cells from a single individual. After exposure to T-irradiation from a 137Cs source, the cells were stimulated with the mitogen phytohemagglutinin (PHA) and maintained in exponential growth with exogenous TCGF to allow phenotypic expression of the 6-thioguanine-resistant (TG r) mutants. The mutant frequency was determined by measuring cell cloning efficiency in microtiter dishes in the absence and presence of TG, with an optimal selection density of 1 x 10 4 cells/well. The development of this in vitro assay should allow direct study of susceptibility to T-irradiation in the human population in terms of both cytotoxicity and mutation induction.
The development of methods for the in vitro cloning of human T-lymphocytes from peripheral blood samples has allowed the measurement of the in vivo frequency of 6-thioguanine-resistant (TG r) mutants in human somatic ceils (Albertini et al., 1982; Morley et al., 1983; Henderson et al.,
Correspondence: Dr. J.P. O'Neill, Department of Medicine and Vermont Regional Cancer Center, University of Vermont, Genetics Laboratory, 32 N. Prospect St., Burlington, VT 05401
(U.S.A.).
1986; O'Neill et al., 1987a). Such an assay should prove useful in human genotoxicity monitoring (Hakoda et al., 1988). The use of T-lymphocytes also allows one to define the relationship between the measured mutant frequency and the actual in vivo mutation frequency (Nicklas et al., 1987; Hakoda et al., 1989). Knowledge of the latter is the ultimate goal of human genetic monitoring studies. However, interpretation of these mutation frequency values will be complicated by the probable heterogeneity of mutagen susceptibility in the human population. One possible means to assess this heterogeneity would be the use of human
0165-1218/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
136 T-lymphocyte cultures in in vitro mutation induction assays (O'Neill et al., 1987b). The development of a quantitative in vitro measurement of mutation induction would allow determination of individual susceptibility to a particular mutagenic agent. In addition, molecular analysis of mutations induced by a specific agent in vitro might aid in the interpretation of in vivo events occurring in humans exposed to that same agent. An example of this would be the comparison of the types of mutations induced in vitro by v-irradiation with those found occurring in vivo in individuals exposed to irradiation, either accidentally or for therapeutic reasons (Messing et al., 1986). Preliminary studies of in vitro induction of mutations in T-lymphocytes have demonstrated the feasibility of this approach (Sanderson et al., 1984; Vijayalaxmi and Evans, 1984; Shulimowsky et al., 1986; O'Neill and Albertini, 1986). This report presents progress in the development of a quantitative assay of in vitro mutagenicity in human T-lymphocytes. Materials and methods
Cells and culture conditions These studies employed blood samples from a single individual. The mononuclear cell fraction containing the T-lymphocytes was isolated as described previously (O'Neill et al., 1987a). All cell culture employed medium RPMI 1640 (Mediatech) containing 20% nutrient medium HL-1 (Ventrex) and 5% prescreened, heat inactivated (56 ° C, 30 min) fetal bovine serum (FBS; Sterile Systems) in humidified incubators at 37-38°C in an atmosphere of 5-6% CO 2 in air. T-cell growth factor (TCGF) was prepared as described previously (O'Neill et al., 1987a) or purchased from Collaborative Research (human T-cell polyclone). All cell culture employed y-irradiated human lymphoblastoid feeder cells described in detail previously (O'Neill et al., 1987). The optimal amount of T C G F and feeder cells was defined as described in the results section. The feeder cells were exposed to 9 krad of "/-radiation from a 137Cs source at approx. !100 r a d / m i n . Mutation induction, expression and selection The M N C fraction was suspended at 2 x 106 cells/ml in RPMI 1640 and exposed to irradiation
from the 137Cs source. Then an equal volume of medium RPMI 1640 containing 40% HL-1 and 10% FBS was added, the cells aliquoted at 20 ml per 25-cm2 flask and PHA added at 1 /~g/ml. After 36-40 h incubation (designated day 2) the cell number was determined and the cells plated for cloning efficiency determinations at 1, 2 and 4 cells/well in 96 well (round bottom) microtiter plates (Nunc). Cells were plated at 2 x 104 cells/well in 10 /xM T G for mutant selection (O'Neill et al., 1987a). Cells were also plated in mass culture at 1 x 105 cells/ml in growth medium containing 2.5 x 105 irradiated feeder cells/cm 2 and optimal amounts of T C G F and incubated for 3 days. On day 5, and at 3-day intervals thereafter, cells were subcultured, and plated for cloning efficiency (1 and 2 cells/well) and mutant selection (1 x 104 cells/well). Colony growth was determined by use of an inverted phase-contrast microscope after 10-15 days incubation. Cloning efficiency was calculated by the P0 distribution and the ratio of these values in the presence and absence of T G yields the mutant frequency. Results and discussion
In order to quantify in vitro induction of mutations at the hprt locus in human T-lymphocytes, conditions must be defined to allow optimal proliferation of mass cultures for expression of the T G r phenotype, as well as optimal cell cloning for mutant selection. These parameters were not addressed in two previous preliminary studies (Vijayalaxmi and Evans, 1984; Sanderson et al., 1984). Lack of this information renders meaningful study of heterogeneity in the response in the human population difficult, if not impossible, because such comparisons require quantitative and reproducible determinations. Our investigations of in vitro growth and cloning of T-cell cultures have employed samples from 10 individuals to define the optimal conditions. Based on previous studies, the medium used was RPMI 1640 containing 20% medium HL-1 and 5% prescreened FBS (O'Neill et al., 1987a). The mononuclear cell (MNC) fraction from peripheral blood samples was incubated in this medium at 1 x 10 6 cells/ml in the presence of 1 # g / m l puff-
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PERCENT TCGF"
Fig. 1. Effect of T C G F amount on T-lymphocyte colony growth. Primed MNC were plated at 1 cell/well in the indicated amount of crude TCGF, either prepared as described (left panel) or purchased from commercial sources (right panel). Cloning efficiencies (circles) and fraction of large colonies (squares) were determined after 14 days incubation.
fied P H A for 36-40 h to achieve mitogen stimulation. These primed cells were then used to measure cell cloning and mass culture proliferation. Optimal amounts of T C G F for cell cloning were defined as described previously (O'Neill et al., 1987a) employing limiting dilution in 96-well mierotiter dishes. Fig. 1 shows the results obtained with the two batches of crude T C G F used in the studies described. These results with one individual are similar to those obtained with the other 9 individuals' samples in that maximal cloning efficiencies and colony size were obtained with 50% (left panel) and 10% (right panel) T C G F for these two batches, respectively. The primed cells were also used for mass culture proliferation in 2-cm 2 wells in the presence of different amounts of T C G F . The cells were plated at log dilutions in order to define any possible cell density effect, primarily because "/-irradiation will result in cytotoxicity. Therefore, irradiated cultures will have decreased numbers of viable cells and might respond differently to in vitro growth conditions in comparison with non-irradiated cultures. It is essential that culture conditions allow equivalent growth, regardless of viable cell inoculum size. In addition, P H A was also added to the medium since the use of different amounts of T C G F also results in the dilution of the P H A employed in the production of the crude T C G F . Fig. 2 (panel A) shows the increase in cell number as a function of time in culture for cells in 50% T C G F , the amount found optimal by cell cloning
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Fig. 2. Effect of PHA and T C G F on the in vitro growth of T-lymphocyte mass cultures. Cultures were incubated for 36 h with 1 # g / m l PHA and then plated at 1×105 (triangles), 1 × 104 (squares), 1 ×103 (circles) or 1 × 102 (diamonds) cells per 2-cm 2 wells in 2 ml in the absence (closed symbols) or presence (open symbols) of 1 /~g/ml PHA and the indicated amount of TCGF. All wells also contained 2 x 1 0 5 irradiated lymphoblastoid feeder cells. Panel A shows the increase in cell number as a function of time in culture for cells in 50% TCGF. Panels B and C show the maximum cell number obtained with 0-50% T C G F in the absence and presence of PHA, respectively. The maximal cell densities were usually reached on days 4, 7, 10 and 13, for cells plated at 105 , 104 , 103 and 102 cells/well, respectively. A cell count of 0.05 x 106 was obtained with feeder cells alone and has not been subtracted.
5 A
o
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% TCGF Fig. 3. Effect of amount of T C G F on the in vitro growth of T-lymphocytes. T-lymphocytes were cultured as described in vitro for 2 days (panel A) or 4 days (panel B) and then plated at 1 ×10 5 (open triangles), 1 ×10 4 (closed triangles), 1),¢J z laJ m i, i, ~J
::'° l J:/ uJ 0
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Fig. 5. In vitro growth of T-lymphocytes. T-lymphocytes were incubated for 24 h with 1 #g/ml PHA and then subcultured at the indicated times in medium containing optimal amounts of TCGF and 2.5 × l0 s irradiated feeder cells/cm2. Cell number was determined daily and the cells subcultured at 1 × 105 cells per ml every four days.
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Fig. 8. Cytotoxicity of y-irradiation of T-lymphocytes. Cells were exposed to 0-400 rad of y-irradiation, incubated with PHA for 36-40 h and plated in 96-well microtiter dishes at 1, 2 and 4 cells/well. Cloning efficiencies are expressed relative to the unirradiated control. The symbols represent the mean ( + SD) values for 3-5 independent determinations.
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Fig. 6. Effect of amount of TCGF on T-cell cloning. Tlymphocytes were subcultured in vitro for 2 (panel A), 5 (panel B) or 8 (panel C) days and then plated at 1 ceil/well in 2130ttl of medium containing the indicated amount of TCGF and 1 × 104 irradiated feeder cells. The two symbols represent two different individuals.
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Fig. 7. Limiting dilution analysis of T-lymphocyte cloning. Cells were subcultured for 11 (closed circles) or 13 days (open circles) and plated for cloning at 0.5-8 cells/well, based on limiting dilution. The mean cloning efficiencies+S.D, were 0.316+0.071 (open circles) and 0.796-1-0.081 (dosed circles) and the lines are drawn based on these mean values.
allow the i n vitro growth a n d single cell c l o n i n g of T - l y m p h o c y t e s from h u m a n peripheral b l o o d sampies. T o m e a s u r e the i n d u c t i o n of m u t a t i o n s at the hprt locus b y y-irradiation, the G O phase p e r i p h eral b l o o d M N C were irradiated, p r i m e d with 1 / ~ g / m l P H A for 3 6 - 4 0 h a n d then s u b c u l t u r e d at 3-day intervals at 1 × 10 5 c e l l s / m l in growth m e d i u m c o n t a i n i n g o p t i m a l a m o u n t s of T C G F a n d 2.5 × 10 5 irradiated feeder c e l l s / c m 2. Cytotoxicity of the i r r a d i a t i o n was d e t e r m i n e d b y measuring the c l o n i n g efficiency of the p r i m e d cells. M u t a n t frequency was d e t e r m i n e d b y m e a s u r i n g the c l o n i n g efficiency i n the a b s e n c e a n d presence of 1 0 / ~ M T G , usually with 1, 2 a n d 4 c e l l s / w e l l a n d 1 x 10 4 cells/well, respectively. I n these initial experiments, cells from a single i n d i v i d u a l were employed, utilizing i n d e p e n d e n t b l o o d sampies. Fig. 8 shows the cytotoxicity of 7 - i r r a d i a t i o n of G O phase M N C as d e t e r m i n e d b y the cell c l o n i n g assay. The p l a t i n g of cells after i r r a d i a t i o n a n d p r i m i n g allows m e a s u r e m e n t of cell c l o n i n g before a n y cell division occurs a n d reflects i n d i v i d u a l G O cell response to the treatment. Based o n the cytotoxicity, a dose of 300 rad was chosen for s t u d y of the p h e n o t y p i c expression of i n d u c e d m u t a n t s . At each time interval (after p r i m i n g which is design a t e d day 2 a n d at 3-day intervals of s u b c u l t u r e
140 150
TABLE 1
%
EFFECT OF SELECTION CELL DENSITY ON MEASURED TG r M U T A N T F R E Q U E N C Y
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100
Mutant frequency at
b.
0.
0
5
10
115
EXPRESSION TIME (doy,)
Fig. 9. Expression time of the TG r phenotype. Cells were exposed to 300 rad of "c-irradiation, incubated with PHA for 40 h and plated in the absence of TG at 1, 2 and 4 cells/well and in the presence of TG at 1 x 104 cells/well. (Time designated day 2 after irradiation.) Cells were also subcultured at 1 x l0 s cells/ml in 20-ml cultures and plated as above for mutant selection at 3-day intervals. The measured mutant frequency is graphed as a function of expression time (days).
thereafter) cells were placed in the absence and presence of T G in 96-well microtiter dishes and the cloning efficiencies determined by the P0 distribution as described previously for in vivo derived mutants (O'Neill et al., 1987a). The ratio of cloning efficiencies defines the T G r mutant frequency. Fig. 9 shows the results of one determination of the expression of the T G r phenotype during postirradiation subculture. The unirradiated culture shows little change in the T G r mutant frequency during the 14 days of subculture (range = 1.5-7.5 x 10-6). However, cells exposed to 300 rad show an increase in mutant frequency at 5 days, reaching the highest value at 8 days with a subsequent decline thereafter. This time course of phenotypic expression is similar to that described previously with cells from another individual (O'Neill et al., 1987b). Based on these and other determinations, an interval of 8 days of culture in vitro appears to allow maximal phenotypic expression. However, the observed decline with longer subculture suggests that there may be some type of selection against T G ~ mutants which occurs during the growth of mass cultures. Whether this is a consequence of the loss of H P R T enzyme activity or is a manifestation of an immunological response to the mutant cells is not known. However, this
Day Day Day Day
5 8 11 14
Selection cell density (cells/weU × 10 4)
a
0.1
0.25
0.5
1.0
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Mean + SD
35 105 70 63
35 207 140 42
46 91 70 85
36 126 108 53
30 124 111 26
36.4+ 5.2 130.6+40.3 99.8+26.7 53.9 + 19.8
a A minimum of 1.92 x 106 cells or 4 plates were employed at each selection cell density.
clearly warrants further study. At present, the use of an 8-day expression time probably allows a reasonable measure of mutation induction with T-cells. Table 1 shows that the use of a selection cell density of 1 x 10 4 cells/well does allow quantitative cloning of T G r mutants since similar mutant frequencies are obtained at lower cell densities. This selection density of 1 x 10 4 cells/well does not result in the loss of mutants due to metabolic cooperation or media depletion. In addition, these results with cell densities of 1 x 10 3 tO 2 X 10 4 cells/well suggest that T G r mutant cell cloning follows the expected Poisson distribution as was shown for cell cloning in the absence of T G (Fig. 7).
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Fig. 10. Dose-response of 7-irradiation induced T G r mutant frequency. The measured mutant frequency after 8 days phenotypic expression is graphed as a function of radiation dose. The symbols represent the mean ( + SD) values for 3-5 independent determinations.
141 75
Acknowledgements
%
Research supported by NCI ROI CA30688 and DOE DE-ACO283ER60147A003. The latter does not constitute an endorsement by DOE of the views expressed in this manuscript.
x
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25
References 0
.
.
.
.
.
.
.
.
1.00
, 0.10
SURVIVAL
Fig. 11. Induced mutant frequency versus cell survival. Induced mutant frequency (Fig. 10) is graphed as a function of initial cell survival (Fig. 8).
Employing a phenotypic expression time of 8 days and a selection cell density of 1 x 10 4 cells/well, the dose-response for y-irradiation was determined and is shown in Fig. 10. The T G r mutant frequency increases with irradiation dose in a non-linear fashion as has been reported with other cell systems (cf. Thacker and Cox, 1975). These types of dose-response relationships are thought to involve the repair of potentially mutagenic damage in a manner analogous to the repair of potentially lethal damage (reviewed in Sankaranarayanan, 1982). The relationship between induced mutant frequency and cytotoxicity appears to be linear (Fig. 11) as described originally by Munson and Goodhead (1977). These results demonstrate that conditions can be defined which allow measurement of mutation induction by y-irradiation in human T-cells in vitro. The type of dose-response study shown in Figs. 8 and 10 requires a blood sample of 60 ml. Recent improvements in T-lymphocyte culture will reduce the sample size needed for such an analysis. This assay could then be employed to measure heterogeneity of sensitivity to irradiation in the human population for both cytotoxicity (cf. Miyakoshi et al., 1987) and T G r mutation induction. In addition, the ability to induce mutants allows molecular analysis of the nature of these mutations, as described in the accompanying article (O'Neill et al., 1990).
Albertini, R.J. (1985) Somatic gene mutations in vivo as indicated by the 6-thioguanine resistant T-lymphocytes in human blood, Mutation Res., 150, 411-422. Albertini, R.J., K.S. Castle and W.R. Borcherding (1982) T-Cell cloning to detect the mutant 6-thioguanine resistant lymphocytes present in human peripheral blood, Proc. Natl. Acad. Sci. (U.S.A.), 79, 6617-6621. Albertini, R.J., J.P. O'Neill, J.A. Nicklas, N.H. Heintz and P.C. Kelleher (1985) Alterations of the hprt gene in human 6-thioguanine resistant T-lymphocytes arising in vivo, Nature (London), 316, 369-371. Hakoda, M., M. Ahiyama, S. Kyoizumi, A.A. Awa, M. Yamahido and M. Otake (1988) Increased cell mutant frequency in atomic bomb survivors, Mutation Res., 201, 39-48. Hakoda, M., Y. Hirai, S. Kyoizumi and M. Ahiyama (1989) Molecular analysis of in vivo hprt mutant T-cells from atomic bomb survivors, Environ. Mol. Mutagen., 13, 25-33. Henderson, L., H. Cole, S.E. James and M. Green (1988) Detection of somatic mutations in man: evaluation of the microtitre cloning assay for T-lymphocytes, Mutagenesis, 1, 195-200. Messing, K., A.M. Seifert and W.E.C. Bradley (1986) In vivo mutant frequency of technicians professionally exposed to ionizing radiation, in: M. Sorsa and H. Norppa (Eds.), Monitoring of Occupational Genetoxicants, Liss, New York, pp. 87-97. Miyakoshi, J., K. Tatsumi and H. Takebe (1987) Radiation sensitivity of T-lymphocytes grown with recombinant human interleukin-2, Mutation Res., 192, 163-167. Morley, A.A., K.J. Trainor, R. Seshadri and R.G. Ryall (1983) Measurement of in vivo mutations in human lymphocytes, Nature (London), 302, 155-156. Munson, R.J., and D.T. Goodhead (1977) The relation between mutation frequency and cell survival, a theoretical induced approach and an examination of experimental data for eukaryotes, Mutation Res., 42, 145-160. Munzer, J.S., S.K. Jones, J.P. O'Neill, J.N. Hartshorn and S.H. Robison (1988) Detection of DNA damage and repair by alkaline elution using human lymphocytes, Mutation Res., 194, 101-108. Nicklas, J.A., J.P. O'Neill and R.J. Albertini (1986) Use of T-cell receptor gene probes to quantify the in vivo hprt mutations in human T-lymphocytes, Mutation Res., 173, 65-72. Nicklas, J.A., T.C. Hunter, L.M. Sullivan, J.K. Berman, J.P. O'Neill and R.J. Albertini (1987a) Molecular analysis of in vivo hprt mutations in human T-lymphocytes, I. Studies of
142 low frequency spontaneous mutants by Southern blots, Mutagenesis, 2, 341-347. Nicklas, J.A., J.P. O'Neill, L.M. Sullivan, T.C. Hunter, M. Allegretta, B.F. Chastenay, B.L. Libbus and R.J. Albertini (1987b) Molecular analyses of in vivo hypoxanthine-guanine phosphoribosyltransferase mutations in human T-lymphocytes, II. Demonstration of a clonal amplification of hprt mutant T-lymphocytes in vivo, Environ. Mol. Mutagen., 12, 271-284. O'Neill, J.P., and R.J. Albertini (1986) Human somatic cell mutation in vitro: development of a clonal assay for T-cell mutants, Environ. Mol. Mutagen., 8 (Suppl. 6), 62. O'Neill, J.P., M.J. McGinniss, J.K. Berman, L.M. Sullivan, J.A. Nicklas and R.J. Albertini (1987a) Refinement of a T-lymphocytes cloning assay to quantify the in vivo thioguanine mutant frequency in humans, Mutagenesis, 2, 87-94. O'Neill, J.P., J.A. Nicklas, T.C. Hunter, L.M. Sullivan and R.J. Albertini (1987b) Molecular analysis of mutations at the hprt locus in human T-lymphocytes, Banbury Rept., 28, 249-261. O'Neill, J.P., L.M. Sullivan, J.A. Nicklas, T.C. Hunter and R.J. Albertini (1987c) In vitro mutation induction by gamma irradiation in human T-lymphocytes, Environ. Mutagen., 9 (Suppl. 8), 8.
O'Neill, J.P., T.C. Hunter, L.M. Sullivan, J.A. Nicklas and R.J. Albertini (1990) Southern-blot analysis of human T-lymphocyte mutants induced in vitro by y-irradiation, Mutation Res., 240, 143-149. Sanderson, B.J.S., J.L. Dempsey and A.A. Morley (1984) Mutations in human lymphocytes: Effect of X- and UV-radiation, Mutation Res., 140, 223-227. Sankaranarayanan, K. (1982) Genetic Effects of Ionizing Radiation on Multicellular Eukaryotes and the Assessment of Genetic Radiation in Man, Elsevier, Amsterdam. Skulimowski, A.W., D.R. Turner, A.A. Morley, F.J.S. Sanderson and M. Haliandros (1986) Molecular basis of X-ray induced mutations at the HPRT locus in human lymphocytes, Mutation Res., 162, 223-227. Subak-Sharpe, J.H., R.R. Burk and J.P. Pitts (1969) Metabolic cooperation between biochemically marked mammalian cells in tissue culture, J. Cell. Sci., 353-367. Thacker, J., and R. Cox (1975) Mutation induction and inactivation in mammalian cells exposed to ionizing radiation, Nature (London), 258, 429-431. Vijayalaxmi and H.J. Evans (1984) Measurement of spontaneous and X-irradiation induced 6-thioguanine resistant human blood lymphocytes using a T-cell cloning technique, Mutation Res., 129, 283-289.
