101
Murution Research, 230 (1990) 101-109 Elsevier
MUT 04855
A quantitative
assay for measuring the induction of mutations peripheral blood T-lymphocytes
in human
Toshiyuki Norimura *, Veronica M. Maher and J. Justin McCormick Carcinogenesis
Loboratory
-
Fee Hall, Department of Microbiology and Department East Lansing, MI 48824-1316 (U.S.A.)
of Biochemistry,
Michigan State University,
(Received 28 August 1989) (Revision received 24 November 1989) (Accepted 4 December 1989)
Keywords:
T lymphocytes; High-cloning efficiencies; Benzo[ alpyrene diol epoxide; Ethyl nitrosourea; HPRT gene
summiuy We optimized conditions for propagating freshly-isolated human peripheral blood T-lymphocytes and cells that had been stored in liquid nitrogen on Day 5 post-isolation, exposing them to mutagens in exponential growth, and measuring the cytotoxicity of the agent from the loss of colony-forming ability, and its mutagenicity from the increase in frequency of 6-thioguanine-resistant cells. Supernatant containing T-cell growth factor, from 60Co-irradiated peripheral mononuclear cells cultured in the presence of 60Co-irradiated B-lymphoblastoid human cells as allogeneic stimulators, supplied at a concentration of 10% along with 10% serum and lo5 allogeneic stimulator cells/ml, supported exponential growth (population doubling times of 22 h) for extended periods (> 30 d). It gave cloning efficiencies of 2 40%. T-lymphocytes stored in liquid nitrogen and returned to culture shortly before mutagen exposure exhibited the same sensitivity as freshly-isolated T-cells to killing by the agents tested, i.e., UV radiation, ethylnitrosourea, and (+)-7~,8~-dihydroxy-9(Y,19a,epoxy-7,8,9,lO-tetrahydrobenzo[ alpyrene. We showed that if mutagenized populations frozen during the expression period were thawed and assayed, they exhibited the same cloning efficiencies and frequencies of 6-thioguanine-resistant cells as did the corresponding populations that had been assayed directly without freezing. Use of these procedures should facilitate investigation of the frequency and kinds of mutations induced in the HPRT gene of peripheral blood T-lymphocytes in vivo and in vitro.
Correspondence: Dr. Veronica M. Maher, Carcinogenesis Laboratory - Fee Hall, Michigan State University, East Lansing, MI 488241316 (U.S.A.), telephone 517 353-7785; fax 517 353-9862.
* Present address: Toshiyuki Norimura, Department of Radiation Biology and Health, University of Occupational and Environmental Health Japan, l-l lseigaoka Yahatanishi-ku, Ritakyushu 807 (Japan). 0027-5107/90/%03.50
Diploid human fibroblasts derived from skin have been used extensively for studies of the cytotoxic and mutagenic effects of environmental agents on human cells in culture (see, for example, Ruijter and Simons, 1980; Arlett and Harcourt, 1982; Aust et al., 1984; Patton et al., 1984; Watanabe et al., 1985; McCormick and ‘Maher, 1985). Comparison of cells from normal persons
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
102
with those from patients with an inherited predisposition to cancer, e.g., xeroderma pigmentosum patients, whose cells are deficient in nucleotide excision repair, has been useful for examining the role of DNA repair on such processes. For many of these studies, resistance to 6-thioguanine (TG) has served as the genetic marker. Such resistance results from the loss of a functioning hypoxanthine(guanine)phosphoribosyltransferase (HPRT) gene, and molecular analysis of the kinds of changes induced in the HPRT gene has been carried out in a number of laboratories (e.g., Albertini et al., 1985; Turner et al., 1985; Skulimowski et al., 1986; Nicklas et al., 1987). More recently, these molecular studies have been facilitated by the development of methods for isolating and cloning TG-resistant diploid human peripheral blood T-lymphocytes (Albertini et al., 1982; Morley et al., 1983a,b; Sanderson et al., 1984; Vijayalaxmi and Evans, 1984; Messing and Bradley, 1985; Henderson et al., 1986; O’Neill et al., 1987). The use of human peripheral blood T-lymphocytes for investigation of spontaneous and mutagen (carcinogen)-induced mutations acquired in vivo or in vitro offers several advantages. One advantage concerns the availability of cell samples. It is much easier to obtain 20-ml samples of blood for isolating T-lymphocytes from control populations of various ages or from persons with an inherited predisposition to cancer or carcinogen-exposed populations than to secure skin biopsy material for fibroblasts. A second advantage of working with T-lymphocytes as the target cells is their ability to be propagated indefinitely using T-cell growth factor. A third advantage of T-cells over fibroblasts concerns ease of handling since T-cells can be manipulated and diluted to the appropriate cell densities without the need for enzymatic digestion. Our laboratory is interested in comparing diploid human T-lymphocytes and fibroblasts for their sensitivity to the cytotoxic and mutagenic effects of a variety of mutagens. In preparation for such studies, we have examined published methods for culturing T-lymphocytes and assaying them for colony-forming ability at limiting dilution. For our purposes it was often convenient to be able to store the cells in liquid nitrogen at various stages
in the experimental protocol. We here describe methods we currently use to produce T-cell growth factor capable of routinely supporting cloning efficiencies of 40% or greater with T-lymphocytes taken from liquid nitrogen storage, as well as with freshly-isolated T-cells. We outline the methods we use to maintain T-lymphocytes in exponential growth indefinitely, expose them to carcinogens (mutagens), assay them for cell survival, select the population for TG-resistant cells, and expand clonally-derived TG-resistant cells into populations of lo9 or more cells. Materials
and methods
Isolation of mononuclear
cells from peripheral
blood
The protocols used to isolate mononuclear cells from leukocyte residues “ buffy coats” obtained from normal donors through the American Red Cross were essentially as described by Albertini et al. (1982) with the following modifications. Approx. 20 ml of leukocyte residue at room temperature was diluted with an equal volume of phosphate-buffered saline (PBS). Each 20 ml of this suspension was layered over 20 ml of room temperature histopaque 1077 (Sigma Chemical Co., St. Louis, MO) in a 50-ml centrifuge tube without allowing mixing and the two tubes were centrifuged at 400 x g for 30 min. Each cell interface (3-5 ml) was transferred to a second 50-ml centrifuge tube, diluted with PBS to a 20-ml volume, again layered over histopaque and the mononuclear cells separated by centrifugation. The interface cells were diluted with PBS to 40 ml and centrifuged at 250 X g for 10 min at room temperature. (When recovery of large numbers of cells was not essential, 100 X g for 10 min was employed to obtain a pellet consisting purely of mononuclear cells.) The cells in the pellet were resuspended in 10 ml PBS and mixed by aspiration using a lo-ml pipette. The volume was increased to a total of 40 ml with PBS and centrifuged for 10 min at 250 X g (or 100 X g). The cells in this pellet were resuspended in 50 ml of counting medium (see below) and the number of viable mononuclear cells per ml was determined using a hemocytometer and a phase-contrast microscope at 100 X to 200 X magnification. The yield averaged 15520 X lo6 viable mononuclear cells/ml.
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The cells were then diluted into the appropriate medium depending upon the purpose for which they were to be used and at the designated density (see below). To isolate T-lymphocytes from small volumes of whole blood (20 ml), the freshly-drawn blood from donors was centrifuged in two 15-ml tubes at 400 x g for 30 min at room temperature. The top layer was removed by aspiration to within 50 mm of the interface. The interface “buffy coat” was removed, placed into a 50-ml tube, diluted to 20 ml with PBS and purified as above. The yield averaged 12 X lo6 mononuclear cells per 10 ml fresh whole blood. Media and culture conditions The medium used for counting the cells (counting medium) and to stimulate the cells to begin blast formation (priming medium) or to propagate the T-lymphocytes (growth medium) was RPM1 1640 supplemented with Hepes pH 7.2 (25 mM, Sigma Chemical Co.), penicillin (100 U/ml), streptomycin (100 pg/ml), L-glutamine (2 mM), sodium pyruvate (2 mM). Counting medium lacks serum and T-cell growth factor (TCGF). The priming medium and growth medium were supplemented with 10% heat-inactivated fetal calf serum (HI-FCS) (Irvine Scientific, Santa Ana, CA), and TCGF used at 10% unless otherwise indicated and prepared as described below. Bulk cultures were propagated at 37OC in flasks slanted at an 8” angle on trays in humidified incubators containing 5% CO,/95% air. The volume of medium was 20 ml for the 75-ml flasks (T25’s) and 60-100 ml for the 250-ml flasks (T75’s). Slanting the flasks was found to facilitate cell growth. HPRT-deficient cells clonally derived from an established human B-lymphoblastoid cell line (Call et al., 1986) were included in the priming medium and growth medium at the designated cell densities to serve as allogeneic stimulators. These TK6derived cells were irradiated with the designated doses of 6oCo and used immediately or stored until use in liquid nitrogen at 5 X lo7 cells/vial in growth medium containing 15% fetal calf serum and 10% dimethyl sulfoxide. The abbreviation, X-TK6, is used to refer to these irradiated allogeneic stimulators. They were present at a density of 2 X 10’ cells/ml during the process of
priming for blast formation; at 5 X lo5 cells/ml during generation of growth factor; at 1 X lo5 cells/ml during growth (expansion) of T-cells; and at 2.5 x lo4 cells/ml when used with T-cells plated into 96 well rounded-bottom microtiter plates (Nunc, Denmark) for cloning assays. Production of T-cell growth factor To produce TCGF, freshly-isolated mononuclear cells were suspended at lo6 cells/ml of counting medium and irradiated with 10 Gy of 6oCo. The cells were mixed with an equal volume of TK6 cells previously irradiated with 55 Gy and suspended at lo6 cells/ml of culture medium containing 2% HI-FCS and 2% phytohemagglutinin (PHA-M) (Difco, Detroit, MI). The two kinds of cells, suspended in T75 flasks at a final density of 5 X lo5 cells/ml with 1% HI-FCS and 1% PHA-M, were incubated for 72 h with agitation at least once/day and checked periodically by phase-contrast microscopy for blast formation. The conditioned medium from these cells, which contained TCGF, was harvested by centrifugation of the cells at 1500 X g for 30 min and filtration of the supernatant through two Whatman 9F/D filters and a GF/F glass fiber filter in a Buchner filter funnel. A lo-ml sample was sterilized by filtration through a 22+m syringe filter and tested for ability to support growth of T-lymphocytes at cloning density (see below). The rest was stored at - 20°C until the quality had been determined. Once this information was known, the supernatants containing good quality TCGF were pooled, sterilized with a 0.22-pm Corning batch filtration system supplemented with a Millipore AP prefilter, and stored at -20°C until used. The material was stable for months. Priming cells for blast formation Freshly-isolated mononuclear cells were suspended at 2 X lo5 cells/ml of priming medium in T25 or T75 flasks with X-TK6 cells also present at 2 x lo5 cells/ml. They were incubated at 37 “C, agitated at least once a day for 3 days and checked for blast formation. Testing rate of growth of cells to high density Replicating T-cells were diluted to 0.5 X lO’/ml in RPM1 1640 medium containing the designated
104
concentrations of TCGF or HI-FCS and containing X-TK6 cells at the designated densities, and were allowed to replicate for several days. The number of T-lymphocytes per ml was determined daily using a phase contrast microscope, which allows recognition of “phase positive” viable mononuclear cells having a halo of refracted light. When the density exceeded 4 or 5 X 10’ cells/ml, the cells were centrifuged at 100 x g for 10 min and resuspended in fresh growth medium at 0.5 X lo5 cells/ml and daily counts were made. Unless specified, the concentration of HI-FCS and TCGF in this medium was 10% and the density of X-TK6 cells was 1 x lo5 cells/ml. In our hands, the XTK6 cells did not disintegrate during the few days before replicating T-lymphocytes had to be diluted again. Assaying cloning efficiency Proliferating T-lymphocytes were diluted to the desired density and combined with an equal volume of growth medium containing X-TK6 cells at a density of 5 X lo4 cells/ml and 200 ~1 of the combined medium was delivered by micropipette to each of 96 wells of a microtiter plate. The plates were tilted at an 8” angle and incubated as described above for flasks. To maintain replication of the T-cells, 100 ~1 of the growth medium was replaced after 7 d with 100 ~1 of fresh growth medium adjusted to have the concentration of fresh HI-FCS and TCGF in the well be 10% and the density of fresh X-TK6 cells be 2.5 X lo4 cells/ml. The number of wells containing clones was determined microscopically after 2 wk. The mean number of clonable cells per well was calculated from the fraction of wells containing no clones, using the Poisson distribution function, and that number, divided by the mean number of cells plated per well, yielded the cloning efficiency (Albertini et al., 1982, 1985). Treatment with UV The protocols for irradiation of cells with Mineralight UV SL-54 germicidal lamp (254 nm radiation) have been described (Patton et al., 1984). Exponentially growing T-lymphocytes in bulk culture (Day 3 post-isolation) were centrifuged (100 x g, 10 min) and suspended at 5 X lo6 cells/ml in Hanks’ balanced salt solution lacking Ca2+, MgZf
and phenol red. 1 ml of the cell suspension was spread out in the center of a 100 mm-diameter petri dish and irradiated at room temperature for specified times. Immediately after irradiation, the cells were diluted 1 : 5 with counting medium. Cells to be assayed for survival were further diluted appropriately in counting medium and combined with an equal volume of growth medium containing 20% HI-FCS and X-TK6 cells at 5 x lo4 cells/ml and plated into 96 microtiter wells to determine decrease in cloning efficiency as described above. Cells to be assayed for UV-induced mutations were diluted into growth medium at lo5 cells/ml in flasks and allowed an expression period. Treatment with ethylnitrosourea (ENV) or ( _t )7P,8a-dihydroxy-9~,10ar,epoxy-7,8,9,lO-tetrahydrobenzo[a]pyrene (BPDE) These carcinogenic chemicals, obtained from the Chemical Carcinogenesis Programs of the National Cancer Institute, were stored desiccated at -20°C. They were weighed out and dissolved in anhydrous dimethyl sulfoxide (ENU) or anhydrous acetone (BPDE) immediately before use. The maximum concentration of the solvent in the medium was 0.5%. An 0.5% concentration of the appropriate solvent also was included in the medium for the untreated controls. On the day of treatment, i.e., Day 3 post-isolation, exponentially-growing T-lymphocytes were centrifuged and resuspended in counting medium at a concentration of 1 x lo6 cells/ml. For the ENU treatment, the concentration of Hepes in the medium was lowered to 15 mM; for BPDE, it was omitted. The cells were exposed to mutagen for 1 h at 37°C in a humidified atmosphere of 95% air and 5% CO,. After treatment, they were diluted appropriately as described above for UV, and allowed to begin the expression period and/or assayed for survival. Mutagenicity assay During the expression period following treatment with mutagens, the cells were incubated at 37°C and subcultured in fresh growth medium every 2-3 d to maintain exponential growth between lo5 and lo6 cells/ml for 7-11 days. To assay for TG resistance, the cells were centrifuged
105
(2 cells per well). 100 ~1 of medium in each well was replaced weekly with selective or non-selective medium and the cultures were scored for growing colonies after 14 days. The cloning efficiencies of the cells was determined as described above, and the frequency of TG’ cells was calculated from the ratio of the cloning efficiencies of the cells in the presence and absence of TG. Results and discussion Optimization
TIME (days)
Fig. 1. Relative rate of growth of T-lymphocytes as a function of the concentration of growth factor. The growth medium was supplemented with 10% heat inactivated fetal calf serum and 1 x 10’ X-TK6 cells/ml. Pruned T-cells were initially diluted to 0.5 ~10’ cells/ml in growth medium containing various concentrations of TCGF. After several days of growth they were again diluted to that density (designated Day 0 in the figure) in their respective growth media. Cell counts were taken daily using a phase contrast microscope. Concentration of TCGF in the media: ,I, 0%; o, 2.5%; 0, 5%; A, 7.5%; 0, 10%; n, 15%.
(100 X g, 10 min) and diluted into growth medium containing 10 PM TG (selective medium). Cultures were distributed into four microtiter 96-well plates at a mean concentration of 5000 cells/well in 200 pi/well. X-TK6 cells were present at 2.5 X lo4 cells/ml. At the same time the cloning efficiency of the cells in growth medium lacking TG was determined by plating cells into 96 microwells
ing human
of the culture conditions for propagat-
T-lymphocytes
T-lymphocytes were isolated from fresh leukocyte residues packs of normal adult blood donors or from small volumes of freshly-drawn blood, as described in the Methods section. The volume of the original blood sample did not affect the ability of the cells to form blasts. The rate of increase in the number of T-cells was used to determine the optimal concentration of growth factor and serum to include in the growth medium, as well as the optimum density of allogeneic stimulator X-TK6 cells. A representative growth curve from assaying the optimal concentration of TCGF is shown in Fig. 1. After being diluted to 0.5 x lo5 cells/ml, the T-cells given TCGF exhibited exponential growth until they attained densities of between 5 X 10’ and 10 X 105, depending on the concentration of TCGF. There was no significant advantage of using a concentration above 10%.
i~~~~~~:
k!!
0
5 IO 15 GROWTH FACTOR (%)
0
5 SERUM
IO (%)
15 0.1
0.2 X-TK6
0.5
I.0
2.0
(ccl Is x m?x IC?) (20ml/T25)
Fig. 2. Relative T-cell propagation as a function of: (A) the concentration of T-cell growth factor; (B) the percent of fetal calf serum in the growth medium; (C) the density of allogeneic stimulator cells. The data in Fig. 2A are taken from Day 4 (0) and Day 5 (0) of the growth curve shown in Fig. 1 with the data expressed as a fraction of the highest cell densities obtained for a particular day. For the assay shown in 2B, the medium contained TCGF at 10% and X-TK6 cells at 1 X 10’ cells/ml. The data are taken from Day 3 (0) and Day 4 (0) of a growth curve similar to that shown in Fig. 1, expressed as a fraction of the highest value. For the assay shown in 2C, the concentration of TCGF and HI-FCS was 10%. The data are taken from Day 4 of a growth curve like that in Fig. 1.
106
0.3 I
0
I
I
I
I
8 TIME
I
I
II
16 (days)
I
I
I
I
24 TIME
(days)
Fig. 3. Rate of growth of T-cells during expansion under optimal conditions, i.e., TCGF and HI-FCS at 10% and X-TK6 cells at 1 x 10’ cells/ml, renewed with each refeeding. (A) Actively-growing primed T-cells were diluted from high density to 0.5 x lo5 cells/ml (open circles) when the density reached - 4X lo5 cells/ml, and the number of cells per ml was determined daily (closed circles) over a period of a month. (B) Comparison of rate of growth (population doubling time) of T-cells derived from three individual donors over a 20-28 day period post-isolation. Determination of the number of population doublings was begun on Day 3 when the primed cells were diluted to 0.5 X lo5 cells/ml. The symbols distinguish individual cell lines.
The results obtained on Days 4 and 5 of this particular assay are shown in Fig. 2A as relative increase in cell density. The results of similar assays for optimal serum concentration and density of X-TK6 cells are shown in Figs. 2B and 2C. The data indicated that concentrations of 10% TCGF and 10% HI-FCS, and a density of X-TK6 cells of 1 X 10’ cells/ml were optimal for propagating T-lymphocytes. Using those conditions, we determined the ability of freshly-isolated T-lymphocyte cultures to be expanded over a period of 28 days. An example is shown in Fig. 3A and 3B. The results showed that it was routinely possible to maintain the cultures in exponential growth for at least that length of time. This indicated that it would be possible to isolate cells, prime them for blast formation for 3 d, expose them to mutagens on Day 3, allow an 8-10 day period of growth for expression of TG resistance and still have cells able to replicate vigorously in the presence or absence of TG for an additional 14 days to form large-sized colonies in 96-well plates. Therefore, this protocol was routinely used. Experience, however, indicated the critical importance of maintaining the level of fresh TCGF, HI-FCS, and X-TK6 cells in the
growth medium throughout the entire period of growth. Testing batches of T-cell growth factor The most stringent assay for TCGF is to determine its ability to support clonal growth of the cells at limiting dilutions, i.e., diluted to l-10 cells per microwell. Therefore, when new batches of TCGF were prepared and filtered as described in the Methods section, a lo-ml sample from each batch was assayed for this property and compared with the batch of TCGF currently in use at the time. Results over several years have shown that the majority of the batches of TCGF generated as described yield T-cell cloning efficiencies greater than 30%, with many giving values of 50% or more (data not shown). This was true for primed T-cells assayed for clonal growth on Day 3 post-isolation and for cells assayed at later times post-isolation. Optimal conditions for treating cells with various mutagens The protocols used to obtain reproducible survival curves with T-lymphocytes treated in culture with UV-radiation, ENU, and BPDE were adapted from those used with diploid human
107
cells to the level of the background frequency TG-resistant cells, i.e., 5 X 10e6 cells.
I
0
0.04
I
0.08
I
0.12
BPDE (p.M)
Fig. 4. Comparison of freshly-isolated T-cells (0) and cells thawed from liquid nitrogen storage (A) for their sensitivity to the cytotoxic effect of BPDE. Survival was determined from the colony forming ability of cells plated into 96-well plates at densities ranging from 2 to 20 cells per well. For T-cells plated at cloning densities, the density of X-TK6 cells in the growth medium was 2.5 x lo4 cells/ml. 100 gl of medium was replaced on day 7, and the clones were counted on day 14. Cell survival was determined from the cloning efficiency of the treated cells relative to that of the untreated cells.
fibroblasts (Aust et al., 1984, Patton et al., 1984). Because the T-cells had to be treated in suspension, rather than attached to the surface of the dishes, the conditions required to obtain reproducible killing curves were determined empirically over a period of months. The optimal conditions now used routinely are described in the Methods section. It is sometimes more convenient to prime populations of T-cells and then store them in liquid nitrogen for use later. We tested if there were any significant differences in the cytotoxicity of the mutagens if cells were treated as freshly-isolated primed cells, or were frozen on Day 5 post-isolation and treated 2 days after being thawed and returned to culture. No differences were found. Representative data for BPDE are shown in Fig. 4. Similar results were obtained with ENU and uv. Optimization of the conditions for selection of TGresistant cells The concentration of TG to be used for selection under our experimental conditions was determined from the decrease in the cells’ colonyforming ability. The results (Fig. 5) showed that a concentration of 10 PM was sufficient to reduce the frequency of colony-formation of TG-resistant
of
Ability to freeze mutagen-treated populations prior to TG selection If one can store mutagen-treated cells during the expression period before assaying them for resistance to TG, one can determine the cytotoxic effect of the original exposure and only assay suitable populations for frequency of TG-resistant populations” refers to cells. The term “suitable those with survival levels between 80% and 10% of the untreated control populations. To see if it were possible to freeze T-lymphocytes during the g-day expression period post-treatment without affecting the results obtained in later assays, T-cells were treated with ENU or UV. Aliquots of cells were assayed for survival and the rest were propagated for several days. On Day 4 or Day 6 post-treatment, a portion of each population was stored frozen. The rest was propagated to Day 8 posttreatment and assayed for cloning efficiency and frequency of TG-resistant cells. After several weeks, the frozen populations were thawed, and the cells were allowed to replicate through the same number of population doublings as their counterpart before being assayed for cloning and for TG resistance. The results (Table 1) showed that freezing did not have a significant effect on the cloning efficiency (columns 4 and 6) or
lo+’
’ ’ ’ ’ ’ 20 30 40 TG (CM) Fig. 5. Cytotoxic effect of 6-thioguanine as a function of applied concentration. T-lymphocytes were plated into 96-well plates at densities ranging from 2 cells/well to 2 X lo4 cells/well in a total volume of 200 pi/well using the growth medium and culture conditions described in the legend to Fig. 4, except for the density of X-TK6 cells which was 1 x 10’ cells/ml. 0
’
’
IO
’
108 TABLE EFFECT
1 OF FREEZING
Agent used
Dose
Control ENU uv
_ 0.7 mM 4 J/m2
MUTAGENIZED Cytotoxicity
100 59k6 63+5
POPULATIONS a
OF T-LYMPHOCYTES
DURING
THE EXPRESSION
PERIOD
Not frozen before selection
Stored frozen before selection ’
Cloning efficiency b (%) * S.D.
TG-resistant cells per lo6 clonable cells
Cloning efficiency (%)
TG-resistant cells per lo6 clonable cells
33+17 25klO 34* 6
5* 3 134*33 26513
58 34 35
4 120 34
a Cell survival determined by a colony-forming assay immediately after the treatment with the mutagenic agent. b Determined by limiting dilution colony-forming assay at the same time as selection in TG was begun., ’ Instead of being propagated directly to Day 8 post-treatment and assayed for TG-resistance, the T-cells were stored frozen in liquid nitrogen on Day 4 or Day 6 post-treatment and subsequently assayed after the same total number of population doublings as cells propagated directly to Day 8.
frequency of TG-resistant cells (columns 5 and 7). These results with T-cells agree with what this laboratory has found to be the case for diploid human fibroblasts treated with mutagens/ carcinogens, allowed to replicate for 3 or 4 d, stored frozen, and assayed at a later time. Expansion of TG-resistant clones to large populations Molecular analysis of the specific kinds of mutations present in the HPRT gene of TG-resistant T-cells or determination of the restriction fragment length polymorphisms of T-cell receptor genes of a series of clonally isolated TG-resistant cells requires extensive expansion of such clones. Fig. 6 shows a representative curve depicting the exponential growth of an individual clone of TGresistant T-cells (- 2 X lo5 cells) isolated after 3 wk of TG selection and propagated from that time (designated Day 0 in Fig. 6) through 25 additional days of growth, with dilution to the optimal cell density at the appropriate times and with daily cell counts. The results indicated that in contrast to diploid human fibroblasts, clones of T-cells could be expanded 2*‘-fold in 20 days to yield 2 X 10” progeny cells. Approximately 5 X 107cells is sufficient for molecular analysis. To test for the stability of the TG-resistant phenotype, 18 clonally-derived populations of TG-resistant T-cells were expanded for 64 days in the presence or absence of TG, and then assayed for colony formation in the presence of concentrations of TG from 10 PM to 33 PM. The results
(data not shown) indicated that growth in the absence of TG made no significant difference. The ratio of the cloning efficiencies of the two cultures assayed in TG ranged from 0.85 to 1.15 with a mean of 1.10. In summary, the results obtained using the protocols and procedures described here emphasize the feasibility and ease of using human peripheral blood T-lymphocytes as the target cells
DAYS AFTER ISOLATION Fig. 6. Rate of replication and extent of clonal expansion of a TG-resistant cell. A TG-resistant clone was isolated from an individual well on Day 22 after TG selection was begun. The cells were diluted to 0.5 x 10’ cells/ml in growth medium as described in the legend to Fig. 3. The cell density was monitored daily and the cells were diluted to 0.5 X lOs/ml as required whenever they reached - 5 x 10s cells/ml. The number of population doublings was determined from the daily increase in cell number as shown in Fig. 3A.
109
for investigating the frequency and kinds of mutations induced by environmental mutagens. We are currently applying these procedures in studies designed to compare the sensitivity of human Tlymphocytes and skin fibroblasts to the cytotoxic and mutagenic effect of a series of carcinogenic mutagens. Acknowledgements The authors wish to thank Shu-Chen Liu, Linda Stafford Kimball and Sonya Michaud for their excellent technical assistance during the course of these investigations. The research was supported by U.S. Public Health Service grants CA21253 and CA37838 from the National Cancer Institute. References Albertini, R.J., K.L. 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 in v&o-derived 6-thioguanine-resistant T lymphocytes, Nature (London), 316, 369-371. Arlett, C.F., and S.A. Harcourt (1981) Variation in response. to mutagens amongst normal and repair-defective human cells, C.W. Lawrence (Ed.), Induced Mutagenesis, Plenum, New York, pp. 249-290. Aust, A.E., N.R. Drinkwater, K.C. Debien, V.M. Maher and J.J. McCormick (1984) Comparison of the frequency of diphtheria toxin and thioguanine resistance induced by a series of carcinogens to analyze their mutational specificities in diploid human fibroblasts, Mutation Res., 125, 955 104. Call, K.M., J.C. Jensen, H.L. Liber and W.G. Thilly (1986) Studies of mutagenicity and clastogenicity of 5-azacytidine in human lymphoblasts and SalmoneNa typhimurium, Mutation Res., 160, 249-257. Henderson, L., H. Cole, J. Cole, S.E. James and M. Green (1986) Detection of somatic mutations in man: evaluation of the microtitre cloning assay for T-lymphocytes, Mutagenesis, 1, 195-200. McCormick, J.J., and V.M. Maher (1985) Use of human cells in human mutagenicity and carcinogenicity determination, in: A.P. Li (Ed.), New Approaches in Toxicity Testing and
their Application to Human Risk Assessment, Raven, New York, pp. 17-35. Messing, K., and W.E.C. Bradley (1985) In viva mutant frequency rises among breast cancer patients after exposure to high doses of y-radiation, Mutation Res., 152, 107-112. Morley, A.A., K.J. Trainor and R.S. Scshadri (1983a) Cloning of human lymphocytes using limiting dilution, Exp. Hematol., 11, 418-424. Morley, A.A., K.J. Trainor, R. Seshadri and R.G. Ryall(1983b) Measurement of in vitro mutations in human lymphocytes, Nature (London), 302, 155-156. Nicklas, J.A., T.C. Hunter, L.M. Sullivan, J.K. Berman, J.P. G’Neill and R.J. Albertini (1987) Molecular analyses of in viva hprt mutations in human T-lymphocytes, I. Studies of low frequency “spontaneous” mutants by Southern blots, Mutagenesis, 2, 341-347. G’Neill, J.P., M.J. McGinmss, J.K. Berman, L.M. Sullivan, J.A. Nicklas and R.J. Albertini (1987) Refinement of a Tlymphocyte cloning assay to quantify the in vivo thioguanine-resistant mutant frequency in humans, Mutagenesis, 2, 87-94. Patton, J.D., L.A. Rowan, A.L. Mendrala, J.N. Howell, V.M. Maher and J.J. McCormick (1984) Xeroderma pigmentosum (XP) fibroblasts including cells from XP variants are abnormally sensitive to the mutagenic and cytotoxic action of broad spectrum simulated sunlight, Photochem. Photobiol., 39, 37-42. Ruijter, Y.C.E.M. de, and J.W.I.M. Simons (1980) Determination of the expression time and the dose-response relationship for mutations at the HGPRT (hypoxanthine-guaninephosphotibosyl transferase) locus induced by X-irradiation in human diploid skin fibroblasts, Mutation Res., 69, 325332. Sanderson, B.J.S., J.L. Dempsey and A.A. Morley (1984) Mutations in human lymphocytes: effect of X- and UV-irradiation, Mutation Res., 140, 223-227. Skulimowski, A.W., D.R. Turner, A.A. Morley, B.J.S. Sanderson and M. Haliandros (1986) Molecular basis of X-ray-induced mutation at the HPRT locus in human lymphocytes, Mutation Res., 162, 105-112. Turner, D.R., A.A. Morley, M. Haliandros, R. Kutlaca and B.J.S. Sanderson (1985) In viva somatic mutations in human lymphocytes frequently result from major gene alterations, Nature (London), 315, 343-345. Vijayalaxmi and H.J. Evans (1984) Induction of 6thioguanine-resistant mutants and SCEs by 3 chemical mutagens (EMS, ENU and MMC) in cultured human blood lymphocytes, Mutation Res., 129, 283-289. Watanabe, M., V.M. Maher and J.J. McCormick (1985) Excision repair of UV- or benzo[ alpyrene diol epoxide-induced lesions in xeroderma pigmentosum variant cells is “errorfree”, Mutation Res., 146, 285-294.
Murution Research, 230 (1990) 101-109 Elsevier
MUT 04855
A quantitative
assay for measuring the induction of mutations peripheral blood T-lymphocytes
in human
Toshiyuki Norimura *, Veronica M. Maher and J. Justin McCormick Carcinogenesis
Loboratory
-
Fee Hall, Department of Microbiology and Department East Lansing, MI 48824-1316 (U.S.A.)
of Biochemistry,
Michigan State University,
(Received 28 August 1989) (Revision received 24 November 1989) (Accepted 4 December 1989)
Keywords:
T lymphocytes; High-cloning efficiencies; Benzo[ alpyrene diol epoxide; Ethyl nitrosourea; HPRT gene
summiuy We optimized conditions for propagating freshly-isolated human peripheral blood T-lymphocytes and cells that had been stored in liquid nitrogen on Day 5 post-isolation, exposing them to mutagens in exponential growth, and measuring the cytotoxicity of the agent from the loss of colony-forming ability, and its mutagenicity from the increase in frequency of 6-thioguanine-resistant cells. Supernatant containing T-cell growth factor, from 60Co-irradiated peripheral mononuclear cells cultured in the presence of 60Co-irradiated B-lymphoblastoid human cells as allogeneic stimulators, supplied at a concentration of 10% along with 10% serum and lo5 allogeneic stimulator cells/ml, supported exponential growth (population doubling times of 22 h) for extended periods (> 30 d). It gave cloning efficiencies of 2 40%. T-lymphocytes stored in liquid nitrogen and returned to culture shortly before mutagen exposure exhibited the same sensitivity as freshly-isolated T-cells to killing by the agents tested, i.e., UV radiation, ethylnitrosourea, and (+)-7~,8~-dihydroxy-9(Y,19a,epoxy-7,8,9,lO-tetrahydrobenzo[ alpyrene. We showed that if mutagenized populations frozen during the expression period were thawed and assayed, they exhibited the same cloning efficiencies and frequencies of 6-thioguanine-resistant cells as did the corresponding populations that had been assayed directly without freezing. Use of these procedures should facilitate investigation of the frequency and kinds of mutations induced in the HPRT gene of peripheral blood T-lymphocytes in vivo and in vitro.
Correspondence: Dr. Veronica M. Maher, Carcinogenesis Laboratory - Fee Hall, Michigan State University, East Lansing, MI 488241316 (U.S.A.), telephone 517 353-7785; fax 517 353-9862.
* Present address: Toshiyuki Norimura, Department of Radiation Biology and Health, University of Occupational and Environmental Health Japan, l-l lseigaoka Yahatanishi-ku, Ritakyushu 807 (Japan). 0027-5107/90/%03.50
Diploid human fibroblasts derived from skin have been used extensively for studies of the cytotoxic and mutagenic effects of environmental agents on human cells in culture (see, for example, Ruijter and Simons, 1980; Arlett and Harcourt, 1982; Aust et al., 1984; Patton et al., 1984; Watanabe et al., 1985; McCormick and ‘Maher, 1985). Comparison of cells from normal persons
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
102
with those from patients with an inherited predisposition to cancer, e.g., xeroderma pigmentosum patients, whose cells are deficient in nucleotide excision repair, has been useful for examining the role of DNA repair on such processes. For many of these studies, resistance to 6-thioguanine (TG) has served as the genetic marker. Such resistance results from the loss of a functioning hypoxanthine(guanine)phosphoribosyltransferase (HPRT) gene, and molecular analysis of the kinds of changes induced in the HPRT gene has been carried out in a number of laboratories (e.g., Albertini et al., 1985; Turner et al., 1985; Skulimowski et al., 1986; Nicklas et al., 1987). More recently, these molecular studies have been facilitated by the development of methods for isolating and cloning TG-resistant diploid human peripheral blood T-lymphocytes (Albertini et al., 1982; Morley et al., 1983a,b; Sanderson et al., 1984; Vijayalaxmi and Evans, 1984; Messing and Bradley, 1985; Henderson et al., 1986; O’Neill et al., 1987). The use of human peripheral blood T-lymphocytes for investigation of spontaneous and mutagen (carcinogen)-induced mutations acquired in vivo or in vitro offers several advantages. One advantage concerns the availability of cell samples. It is much easier to obtain 20-ml samples of blood for isolating T-lymphocytes from control populations of various ages or from persons with an inherited predisposition to cancer or carcinogen-exposed populations than to secure skin biopsy material for fibroblasts. A second advantage of working with T-lymphocytes as the target cells is their ability to be propagated indefinitely using T-cell growth factor. A third advantage of T-cells over fibroblasts concerns ease of handling since T-cells can be manipulated and diluted to the appropriate cell densities without the need for enzymatic digestion. Our laboratory is interested in comparing diploid human T-lymphocytes and fibroblasts for their sensitivity to the cytotoxic and mutagenic effects of a variety of mutagens. In preparation for such studies, we have examined published methods for culturing T-lymphocytes and assaying them for colony-forming ability at limiting dilution. For our purposes it was often convenient to be able to store the cells in liquid nitrogen at various stages
in the experimental protocol. We here describe methods we currently use to produce T-cell growth factor capable of routinely supporting cloning efficiencies of 40% or greater with T-lymphocytes taken from liquid nitrogen storage, as well as with freshly-isolated T-cells. We outline the methods we use to maintain T-lymphocytes in exponential growth indefinitely, expose them to carcinogens (mutagens), assay them for cell survival, select the population for TG-resistant cells, and expand clonally-derived TG-resistant cells into populations of lo9 or more cells. Materials
and methods
Isolation of mononuclear
cells from peripheral
blood
The protocols used to isolate mononuclear cells from leukocyte residues “ buffy coats” obtained from normal donors through the American Red Cross were essentially as described by Albertini et al. (1982) with the following modifications. Approx. 20 ml of leukocyte residue at room temperature was diluted with an equal volume of phosphate-buffered saline (PBS). Each 20 ml of this suspension was layered over 20 ml of room temperature histopaque 1077 (Sigma Chemical Co., St. Louis, MO) in a 50-ml centrifuge tube without allowing mixing and the two tubes were centrifuged at 400 x g for 30 min. Each cell interface (3-5 ml) was transferred to a second 50-ml centrifuge tube, diluted with PBS to a 20-ml volume, again layered over histopaque and the mononuclear cells separated by centrifugation. The interface cells were diluted with PBS to 40 ml and centrifuged at 250 X g for 10 min at room temperature. (When recovery of large numbers of cells was not essential, 100 X g for 10 min was employed to obtain a pellet consisting purely of mononuclear cells.) The cells in the pellet were resuspended in 10 ml PBS and mixed by aspiration using a lo-ml pipette. The volume was increased to a total of 40 ml with PBS and centrifuged for 10 min at 250 X g (or 100 X g). The cells in this pellet were resuspended in 50 ml of counting medium (see below) and the number of viable mononuclear cells per ml was determined using a hemocytometer and a phase-contrast microscope at 100 X to 200 X magnification. The yield averaged 15520 X lo6 viable mononuclear cells/ml.
103
The cells were then diluted into the appropriate medium depending upon the purpose for which they were to be used and at the designated density (see below). To isolate T-lymphocytes from small volumes of whole blood (20 ml), the freshly-drawn blood from donors was centrifuged in two 15-ml tubes at 400 x g for 30 min at room temperature. The top layer was removed by aspiration to within 50 mm of the interface. The interface “buffy coat” was removed, placed into a 50-ml tube, diluted to 20 ml with PBS and purified as above. The yield averaged 12 X lo6 mononuclear cells per 10 ml fresh whole blood. Media and culture conditions The medium used for counting the cells (counting medium) and to stimulate the cells to begin blast formation (priming medium) or to propagate the T-lymphocytes (growth medium) was RPM1 1640 supplemented with Hepes pH 7.2 (25 mM, Sigma Chemical Co.), penicillin (100 U/ml), streptomycin (100 pg/ml), L-glutamine (2 mM), sodium pyruvate (2 mM). Counting medium lacks serum and T-cell growth factor (TCGF). The priming medium and growth medium were supplemented with 10% heat-inactivated fetal calf serum (HI-FCS) (Irvine Scientific, Santa Ana, CA), and TCGF used at 10% unless otherwise indicated and prepared as described below. Bulk cultures were propagated at 37OC in flasks slanted at an 8” angle on trays in humidified incubators containing 5% CO,/95% air. The volume of medium was 20 ml for the 75-ml flasks (T25’s) and 60-100 ml for the 250-ml flasks (T75’s). Slanting the flasks was found to facilitate cell growth. HPRT-deficient cells clonally derived from an established human B-lymphoblastoid cell line (Call et al., 1986) were included in the priming medium and growth medium at the designated cell densities to serve as allogeneic stimulators. These TK6derived cells were irradiated with the designated doses of 6oCo and used immediately or stored until use in liquid nitrogen at 5 X lo7 cells/vial in growth medium containing 15% fetal calf serum and 10% dimethyl sulfoxide. The abbreviation, X-TK6, is used to refer to these irradiated allogeneic stimulators. They were present at a density of 2 X 10’ cells/ml during the process of
priming for blast formation; at 5 X lo5 cells/ml during generation of growth factor; at 1 X lo5 cells/ml during growth (expansion) of T-cells; and at 2.5 x lo4 cells/ml when used with T-cells plated into 96 well rounded-bottom microtiter plates (Nunc, Denmark) for cloning assays. Production of T-cell growth factor To produce TCGF, freshly-isolated mononuclear cells were suspended at lo6 cells/ml of counting medium and irradiated with 10 Gy of 6oCo. The cells were mixed with an equal volume of TK6 cells previously irradiated with 55 Gy and suspended at lo6 cells/ml of culture medium containing 2% HI-FCS and 2% phytohemagglutinin (PHA-M) (Difco, Detroit, MI). The two kinds of cells, suspended in T75 flasks at a final density of 5 X lo5 cells/ml with 1% HI-FCS and 1% PHA-M, were incubated for 72 h with agitation at least once/day and checked periodically by phase-contrast microscopy for blast formation. The conditioned medium from these cells, which contained TCGF, was harvested by centrifugation of the cells at 1500 X g for 30 min and filtration of the supernatant through two Whatman 9F/D filters and a GF/F glass fiber filter in a Buchner filter funnel. A lo-ml sample was sterilized by filtration through a 22+m syringe filter and tested for ability to support growth of T-lymphocytes at cloning density (see below). The rest was stored at - 20°C until the quality had been determined. Once this information was known, the supernatants containing good quality TCGF were pooled, sterilized with a 0.22-pm Corning batch filtration system supplemented with a Millipore AP prefilter, and stored at -20°C until used. The material was stable for months. Priming cells for blast formation Freshly-isolated mononuclear cells were suspended at 2 X lo5 cells/ml of priming medium in T25 or T75 flasks with X-TK6 cells also present at 2 x lo5 cells/ml. They were incubated at 37 “C, agitated at least once a day for 3 days and checked for blast formation. Testing rate of growth of cells to high density Replicating T-cells were diluted to 0.5 X lO’/ml in RPM1 1640 medium containing the designated
104
concentrations of TCGF or HI-FCS and containing X-TK6 cells at the designated densities, and were allowed to replicate for several days. The number of T-lymphocytes per ml was determined daily using a phase contrast microscope, which allows recognition of “phase positive” viable mononuclear cells having a halo of refracted light. When the density exceeded 4 or 5 X 10’ cells/ml, the cells were centrifuged at 100 x g for 10 min and resuspended in fresh growth medium at 0.5 X lo5 cells/ml and daily counts were made. Unless specified, the concentration of HI-FCS and TCGF in this medium was 10% and the density of X-TK6 cells was 1 x lo5 cells/ml. In our hands, the XTK6 cells did not disintegrate during the few days before replicating T-lymphocytes had to be diluted again. Assaying cloning efficiency Proliferating T-lymphocytes were diluted to the desired density and combined with an equal volume of growth medium containing X-TK6 cells at a density of 5 X lo4 cells/ml and 200 ~1 of the combined medium was delivered by micropipette to each of 96 wells of a microtiter plate. The plates were tilted at an 8” angle and incubated as described above for flasks. To maintain replication of the T-cells, 100 ~1 of the growth medium was replaced after 7 d with 100 ~1 of fresh growth medium adjusted to have the concentration of fresh HI-FCS and TCGF in the well be 10% and the density of fresh X-TK6 cells be 2.5 X lo4 cells/ml. The number of wells containing clones was determined microscopically after 2 wk. The mean number of clonable cells per well was calculated from the fraction of wells containing no clones, using the Poisson distribution function, and that number, divided by the mean number of cells plated per well, yielded the cloning efficiency (Albertini et al., 1982, 1985). Treatment with UV The protocols for irradiation of cells with Mineralight UV SL-54 germicidal lamp (254 nm radiation) have been described (Patton et al., 1984). Exponentially growing T-lymphocytes in bulk culture (Day 3 post-isolation) were centrifuged (100 x g, 10 min) and suspended at 5 X lo6 cells/ml in Hanks’ balanced salt solution lacking Ca2+, MgZf
and phenol red. 1 ml of the cell suspension was spread out in the center of a 100 mm-diameter petri dish and irradiated at room temperature for specified times. Immediately after irradiation, the cells were diluted 1 : 5 with counting medium. Cells to be assayed for survival were further diluted appropriately in counting medium and combined with an equal volume of growth medium containing 20% HI-FCS and X-TK6 cells at 5 x lo4 cells/ml and plated into 96 microtiter wells to determine decrease in cloning efficiency as described above. Cells to be assayed for UV-induced mutations were diluted into growth medium at lo5 cells/ml in flasks and allowed an expression period. Treatment with ethylnitrosourea (ENV) or ( _t )7P,8a-dihydroxy-9~,10ar,epoxy-7,8,9,lO-tetrahydrobenzo[a]pyrene (BPDE) These carcinogenic chemicals, obtained from the Chemical Carcinogenesis Programs of the National Cancer Institute, were stored desiccated at -20°C. They were weighed out and dissolved in anhydrous dimethyl sulfoxide (ENU) or anhydrous acetone (BPDE) immediately before use. The maximum concentration of the solvent in the medium was 0.5%. An 0.5% concentration of the appropriate solvent also was included in the medium for the untreated controls. On the day of treatment, i.e., Day 3 post-isolation, exponentially-growing T-lymphocytes were centrifuged and resuspended in counting medium at a concentration of 1 x lo6 cells/ml. For the ENU treatment, the concentration of Hepes in the medium was lowered to 15 mM; for BPDE, it was omitted. The cells were exposed to mutagen for 1 h at 37°C in a humidified atmosphere of 95% air and 5% CO,. After treatment, they were diluted appropriately as described above for UV, and allowed to begin the expression period and/or assayed for survival. Mutagenicity assay During the expression period following treatment with mutagens, the cells were incubated at 37°C and subcultured in fresh growth medium every 2-3 d to maintain exponential growth between lo5 and lo6 cells/ml for 7-11 days. To assay for TG resistance, the cells were centrifuged
105
(2 cells per well). 100 ~1 of medium in each well was replaced weekly with selective or non-selective medium and the cultures were scored for growing colonies after 14 days. The cloning efficiencies of the cells was determined as described above, and the frequency of TG’ cells was calculated from the ratio of the cloning efficiencies of the cells in the presence and absence of TG. Results and discussion Optimization
TIME (days)
Fig. 1. Relative rate of growth of T-lymphocytes as a function of the concentration of growth factor. The growth medium was supplemented with 10% heat inactivated fetal calf serum and 1 x 10’ X-TK6 cells/ml. Pruned T-cells were initially diluted to 0.5 ~10’ cells/ml in growth medium containing various concentrations of TCGF. After several days of growth they were again diluted to that density (designated Day 0 in the figure) in their respective growth media. Cell counts were taken daily using a phase contrast microscope. Concentration of TCGF in the media: ,I, 0%; o, 2.5%; 0, 5%; A, 7.5%; 0, 10%; n, 15%.
(100 X g, 10 min) and diluted into growth medium containing 10 PM TG (selective medium). Cultures were distributed into four microtiter 96-well plates at a mean concentration of 5000 cells/well in 200 pi/well. X-TK6 cells were present at 2.5 X lo4 cells/ml. At the same time the cloning efficiency of the cells in growth medium lacking TG was determined by plating cells into 96 microwells
ing human
of the culture conditions for propagat-
T-lymphocytes
T-lymphocytes were isolated from fresh leukocyte residues packs of normal adult blood donors or from small volumes of freshly-drawn blood, as described in the Methods section. The volume of the original blood sample did not affect the ability of the cells to form blasts. The rate of increase in the number of T-cells was used to determine the optimal concentration of growth factor and serum to include in the growth medium, as well as the optimum density of allogeneic stimulator X-TK6 cells. A representative growth curve from assaying the optimal concentration of TCGF is shown in Fig. 1. After being diluted to 0.5 x lo5 cells/ml, the T-cells given TCGF exhibited exponential growth until they attained densities of between 5 X 10’ and 10 X 105, depending on the concentration of TCGF. There was no significant advantage of using a concentration above 10%.
i~~~~~~:
k!!
0
5 IO 15 GROWTH FACTOR (%)
0
5 SERUM
IO (%)
15 0.1
0.2 X-TK6
0.5
I.0
2.0
(ccl Is x m?x IC?) (20ml/T25)
Fig. 2. Relative T-cell propagation as a function of: (A) the concentration of T-cell growth factor; (B) the percent of fetal calf serum in the growth medium; (C) the density of allogeneic stimulator cells. The data in Fig. 2A are taken from Day 4 (0) and Day 5 (0) of the growth curve shown in Fig. 1 with the data expressed as a fraction of the highest cell densities obtained for a particular day. For the assay shown in 2B, the medium contained TCGF at 10% and X-TK6 cells at 1 X 10’ cells/ml. The data are taken from Day 3 (0) and Day 4 (0) of a growth curve similar to that shown in Fig. 1, expressed as a fraction of the highest value. For the assay shown in 2C, the concentration of TCGF and HI-FCS was 10%. The data are taken from Day 4 of a growth curve like that in Fig. 1.
106
0.3 I
0
I
I
I
I
8 TIME
I
I
II
16 (days)
I
I
I
I
24 TIME
(days)
Fig. 3. Rate of growth of T-cells during expansion under optimal conditions, i.e., TCGF and HI-FCS at 10% and X-TK6 cells at 1 x 10’ cells/ml, renewed with each refeeding. (A) Actively-growing primed T-cells were diluted from high density to 0.5 x lo5 cells/ml (open circles) when the density reached - 4X lo5 cells/ml, and the number of cells per ml was determined daily (closed circles) over a period of a month. (B) Comparison of rate of growth (population doubling time) of T-cells derived from three individual donors over a 20-28 day period post-isolation. Determination of the number of population doublings was begun on Day 3 when the primed cells were diluted to 0.5 X lo5 cells/ml. The symbols distinguish individual cell lines.
The results obtained on Days 4 and 5 of this particular assay are shown in Fig. 2A as relative increase in cell density. The results of similar assays for optimal serum concentration and density of X-TK6 cells are shown in Figs. 2B and 2C. The data indicated that concentrations of 10% TCGF and 10% HI-FCS, and a density of X-TK6 cells of 1 X 10’ cells/ml were optimal for propagating T-lymphocytes. Using those conditions, we determined the ability of freshly-isolated T-lymphocyte cultures to be expanded over a period of 28 days. An example is shown in Fig. 3A and 3B. The results showed that it was routinely possible to maintain the cultures in exponential growth for at least that length of time. This indicated that it would be possible to isolate cells, prime them for blast formation for 3 d, expose them to mutagens on Day 3, allow an 8-10 day period of growth for expression of TG resistance and still have cells able to replicate vigorously in the presence or absence of TG for an additional 14 days to form large-sized colonies in 96-well plates. Therefore, this protocol was routinely used. Experience, however, indicated the critical importance of maintaining the level of fresh TCGF, HI-FCS, and X-TK6 cells in the
growth medium throughout the entire period of growth. Testing batches of T-cell growth factor The most stringent assay for TCGF is to determine its ability to support clonal growth of the cells at limiting dilutions, i.e., diluted to l-10 cells per microwell. Therefore, when new batches of TCGF were prepared and filtered as described in the Methods section, a lo-ml sample from each batch was assayed for this property and compared with the batch of TCGF currently in use at the time. Results over several years have shown that the majority of the batches of TCGF generated as described yield T-cell cloning efficiencies greater than 30%, with many giving values of 50% or more (data not shown). This was true for primed T-cells assayed for clonal growth on Day 3 post-isolation and for cells assayed at later times post-isolation. Optimal conditions for treating cells with various mutagens The protocols used to obtain reproducible survival curves with T-lymphocytes treated in culture with UV-radiation, ENU, and BPDE were adapted from those used with diploid human
107
cells to the level of the background frequency TG-resistant cells, i.e., 5 X 10e6 cells.
I
0
0.04
I
0.08
I
0.12
BPDE (p.M)
Fig. 4. Comparison of freshly-isolated T-cells (0) and cells thawed from liquid nitrogen storage (A) for their sensitivity to the cytotoxic effect of BPDE. Survival was determined from the colony forming ability of cells plated into 96-well plates at densities ranging from 2 to 20 cells per well. For T-cells plated at cloning densities, the density of X-TK6 cells in the growth medium was 2.5 x lo4 cells/ml. 100 gl of medium was replaced on day 7, and the clones were counted on day 14. Cell survival was determined from the cloning efficiency of the treated cells relative to that of the untreated cells.
fibroblasts (Aust et al., 1984, Patton et al., 1984). Because the T-cells had to be treated in suspension, rather than attached to the surface of the dishes, the conditions required to obtain reproducible killing curves were determined empirically over a period of months. The optimal conditions now used routinely are described in the Methods section. It is sometimes more convenient to prime populations of T-cells and then store them in liquid nitrogen for use later. We tested if there were any significant differences in the cytotoxicity of the mutagens if cells were treated as freshly-isolated primed cells, or were frozen on Day 5 post-isolation and treated 2 days after being thawed and returned to culture. No differences were found. Representative data for BPDE are shown in Fig. 4. Similar results were obtained with ENU and uv. Optimization of the conditions for selection of TGresistant cells The concentration of TG to be used for selection under our experimental conditions was determined from the decrease in the cells’ colonyforming ability. The results (Fig. 5) showed that a concentration of 10 PM was sufficient to reduce the frequency of colony-formation of TG-resistant
of
Ability to freeze mutagen-treated populations prior to TG selection If one can store mutagen-treated cells during the expression period before assaying them for resistance to TG, one can determine the cytotoxic effect of the original exposure and only assay suitable populations for frequency of TG-resistant populations” refers to cells. The term “suitable those with survival levels between 80% and 10% of the untreated control populations. To see if it were possible to freeze T-lymphocytes during the g-day expression period post-treatment without affecting the results obtained in later assays, T-cells were treated with ENU or UV. Aliquots of cells were assayed for survival and the rest were propagated for several days. On Day 4 or Day 6 post-treatment, a portion of each population was stored frozen. The rest was propagated to Day 8 posttreatment and assayed for cloning efficiency and frequency of TG-resistant cells. After several weeks, the frozen populations were thawed, and the cells were allowed to replicate through the same number of population doublings as their counterpart before being assayed for cloning and for TG resistance. The results (Table 1) showed that freezing did not have a significant effect on the cloning efficiency (columns 4 and 6) or
lo+’
’ ’ ’ ’ ’ 20 30 40 TG (CM) Fig. 5. Cytotoxic effect of 6-thioguanine as a function of applied concentration. T-lymphocytes were plated into 96-well plates at densities ranging from 2 cells/well to 2 X lo4 cells/well in a total volume of 200 pi/well using the growth medium and culture conditions described in the legend to Fig. 4, except for the density of X-TK6 cells which was 1 x 10’ cells/ml. 0
’
’
IO
’
108 TABLE EFFECT
1 OF FREEZING
Agent used
Dose
Control ENU uv
_ 0.7 mM 4 J/m2
MUTAGENIZED Cytotoxicity
100 59k6 63+5
POPULATIONS a
OF T-LYMPHOCYTES
DURING
THE EXPRESSION
PERIOD
Not frozen before selection
Stored frozen before selection ’
Cloning efficiency b (%) * S.D.
TG-resistant cells per lo6 clonable cells
Cloning efficiency (%)
TG-resistant cells per lo6 clonable cells
33+17 25klO 34* 6
5* 3 134*33 26513
58 34 35
4 120 34
a Cell survival determined by a colony-forming assay immediately after the treatment with the mutagenic agent. b Determined by limiting dilution colony-forming assay at the same time as selection in TG was begun., ’ Instead of being propagated directly to Day 8 post-treatment and assayed for TG-resistance, the T-cells were stored frozen in liquid nitrogen on Day 4 or Day 6 post-treatment and subsequently assayed after the same total number of population doublings as cells propagated directly to Day 8.
frequency of TG-resistant cells (columns 5 and 7). These results with T-cells agree with what this laboratory has found to be the case for diploid human fibroblasts treated with mutagens/ carcinogens, allowed to replicate for 3 or 4 d, stored frozen, and assayed at a later time. Expansion of TG-resistant clones to large populations Molecular analysis of the specific kinds of mutations present in the HPRT gene of TG-resistant T-cells or determination of the restriction fragment length polymorphisms of T-cell receptor genes of a series of clonally isolated TG-resistant cells requires extensive expansion of such clones. Fig. 6 shows a representative curve depicting the exponential growth of an individual clone of TGresistant T-cells (- 2 X lo5 cells) isolated after 3 wk of TG selection and propagated from that time (designated Day 0 in Fig. 6) through 25 additional days of growth, with dilution to the optimal cell density at the appropriate times and with daily cell counts. The results indicated that in contrast to diploid human fibroblasts, clones of T-cells could be expanded 2*‘-fold in 20 days to yield 2 X 10” progeny cells. Approximately 5 X 107cells is sufficient for molecular analysis. To test for the stability of the TG-resistant phenotype, 18 clonally-derived populations of TG-resistant T-cells were expanded for 64 days in the presence or absence of TG, and then assayed for colony formation in the presence of concentrations of TG from 10 PM to 33 PM. The results
(data not shown) indicated that growth in the absence of TG made no significant difference. The ratio of the cloning efficiencies of the two cultures assayed in TG ranged from 0.85 to 1.15 with a mean of 1.10. In summary, the results obtained using the protocols and procedures described here emphasize the feasibility and ease of using human peripheral blood T-lymphocytes as the target cells
DAYS AFTER ISOLATION Fig. 6. Rate of replication and extent of clonal expansion of a TG-resistant cell. A TG-resistant clone was isolated from an individual well on Day 22 after TG selection was begun. The cells were diluted to 0.5 x 10’ cells/ml in growth medium as described in the legend to Fig. 3. The cell density was monitored daily and the cells were diluted to 0.5 X lOs/ml as required whenever they reached - 5 x 10s cells/ml. The number of population doublings was determined from the daily increase in cell number as shown in Fig. 3A.
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for investigating the frequency and kinds of mutations induced by environmental mutagens. We are currently applying these procedures in studies designed to compare the sensitivity of human Tlymphocytes and skin fibroblasts to the cytotoxic and mutagenic effect of a series of carcinogenic mutagens. Acknowledgements The authors wish to thank Shu-Chen Liu, Linda Stafford Kimball and Sonya Michaud for their excellent technical assistance during the course of these investigations. The research was supported by U.S. Public Health Service grants CA21253 and CA37838 from the National Cancer Institute. References Albertini, R.J., K.L. 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 in v&o-derived 6-thioguanine-resistant T lymphocytes, Nature (London), 316, 369-371. Arlett, C.F., and S.A. Harcourt (1981) Variation in response. to mutagens amongst normal and repair-defective human cells, C.W. Lawrence (Ed.), Induced Mutagenesis, Plenum, New York, pp. 249-290. Aust, A.E., N.R. Drinkwater, K.C. Debien, V.M. Maher and J.J. McCormick (1984) Comparison of the frequency of diphtheria toxin and thioguanine resistance induced by a series of carcinogens to analyze their mutational specificities in diploid human fibroblasts, Mutation Res., 125, 955 104. Call, K.M., J.C. Jensen, H.L. Liber and W.G. Thilly (1986) Studies of mutagenicity and clastogenicity of 5-azacytidine in human lymphoblasts and SalmoneNa typhimurium, Mutation Res., 160, 249-257. Henderson, L., H. Cole, J. Cole, S.E. James and M. Green (1986) Detection of somatic mutations in man: evaluation of the microtitre cloning assay for T-lymphocytes, Mutagenesis, 1, 195-200. McCormick, J.J., and V.M. Maher (1985) Use of human cells in human mutagenicity and carcinogenicity determination, in: A.P. Li (Ed.), New Approaches in Toxicity Testing and
their Application to Human Risk Assessment, Raven, New York, pp. 17-35. Messing, K., and W.E.C. Bradley (1985) In viva mutant frequency rises among breast cancer patients after exposure to high doses of y-radiation, Mutation Res., 152, 107-112. Morley, A.A., K.J. Trainor and R.S. Scshadri (1983a) Cloning of human lymphocytes using limiting dilution, Exp. Hematol., 11, 418-424. Morley, A.A., K.J. Trainor, R. Seshadri and R.G. Ryall(1983b) Measurement of in vitro mutations in human lymphocytes, Nature (London), 302, 155-156. Nicklas, J.A., T.C. Hunter, L.M. Sullivan, J.K. Berman, J.P. G’Neill and R.J. Albertini (1987) Molecular analyses of in viva hprt mutations in human T-lymphocytes, I. Studies of low frequency “spontaneous” mutants by Southern blots, Mutagenesis, 2, 341-347. G’Neill, J.P., M.J. McGinmss, J.K. Berman, L.M. Sullivan, J.A. Nicklas and R.J. Albertini (1987) Refinement of a Tlymphocyte cloning assay to quantify the in vivo thioguanine-resistant mutant frequency in humans, Mutagenesis, 2, 87-94. Patton, J.D., L.A. Rowan, A.L. Mendrala, J.N. Howell, V.M. Maher and J.J. McCormick (1984) Xeroderma pigmentosum (XP) fibroblasts including cells from XP variants are abnormally sensitive to the mutagenic and cytotoxic action of broad spectrum simulated sunlight, Photochem. Photobiol., 39, 37-42. Ruijter, Y.C.E.M. de, and J.W.I.M. Simons (1980) Determination of the expression time and the dose-response relationship for mutations at the HGPRT (hypoxanthine-guaninephosphotibosyl transferase) locus induced by X-irradiation in human diploid skin fibroblasts, Mutation Res., 69, 325332. Sanderson, B.J.S., J.L. Dempsey and A.A. Morley (1984) Mutations in human lymphocytes: effect of X- and UV-irradiation, Mutation Res., 140, 223-227. Skulimowski, A.W., D.R. Turner, A.A. Morley, B.J.S. Sanderson and M. Haliandros (1986) Molecular basis of X-ray-induced mutation at the HPRT locus in human lymphocytes, Mutation Res., 162, 105-112. Turner, D.R., A.A. Morley, M. Haliandros, R. Kutlaca and B.J.S. Sanderson (1985) In viva somatic mutations in human lymphocytes frequently result from major gene alterations, Nature (London), 315, 343-345. Vijayalaxmi and H.J. Evans (1984) Induction of 6thioguanine-resistant mutants and SCEs by 3 chemical mutagens (EMS, ENU and MMC) in cultured human blood lymphocytes, Mutation Res., 129, 283-289. Watanabe, M., V.M. Maher and J.J. McCormick (1985) Excision repair of UV- or benzo[ alpyrene diol epoxide-induced lesions in xeroderma pigmentosum variant cells is “errorfree”, Mutation Res., 146, 285-294.
