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Human HPRT Mutation Analysis 155

15
Methods for Detecting Somatic Mutations In Vitro

The Human T-Cell Cloning Assay Selecting for HPRT Mutants

Sai-Mei Hou

Summary
The T-cell cloning assay, which detects mutations in the gene for hypoxanthine-gua-
nine phosphoribosyltransferase (HPRT), is the most well-developed reporter system for
studying specific locus mutation in human somatic cells. The assay is based on a mito-
gen- and growth factor-dependent clonal expansion of peripheral T lymphocytes in which
the 6-thioguanine-resistant HPRT mutants can be selected, enumerated, and collected
for molecular analysis of their mutational nature. The assay provides a unique tool for
studying in vivo and in vitro mutagenesis and for investigating the functional impact of
common polymorphisms in metabolism and repair genes. The present chapter presents a
simple and reliable method for the enumeration of HPRT mutant frequency induced in
vitro without using any source of recombinant interleukin-2. The other main feature is
that only truly induced and unique mutants are collected for further analysis.
Key Words: Cloning assay; T lymphocytes; HPRT gene; mutations.

1. Introduction
The T-cell cloning assay, which enables the enumeration and molecular analysis of
peripheral T lymphocytes with mutations in the X-linked hypoxanthine-guanine
phosphoribosyl transferase (HPRT) gene, has been extensively used for studying
human somatic gene mutation in vivo. The assay combines mitogen- and growth fac-
tor-dependent expansion of lymphocyte clones with 6-thioguanine (TG) selection of
mutant cells. Resistance to TG identifies cells lacking the HPRT enzyme owing to
inactivation or loss of the HPRT gene (1). Knowledge of the entire human HPRT gene
sequence has further enabled analysis of the molecular nature of HPRT mutations and
the establishment of background and induced mutational spectra in various cell types
(2). Inherited mutations in the HPRT gene can also be studied in patients with the
Lesch-Nyhan syndrome, which makes it possible to compare the mechanisms for
mutagenesis in somatic and germline cells. Methods for molecular analysis of HPRT
mutations have been described in detail (3) (see also Chap. 16).

From: Methods in Molecular Biology, vol. 291, Molecular Toxicology Protocols


Edited by: P. Keohavong and S. G. Grant Humana Press Inc., Totowa, NJ

155
156 Hou

A wide range of mean background HPRT mutant frequencies (MFs) have been
reported for normal nonexposed adult donors (1.116.5 106) (4). Much of the con-
siderable variation can be explained by interlaboratory variation in experimental meth-
odologies and donor attributes such as age and smoking. The age effect may be
associated with a decrease in DNA repair capacity, an increase in the mutation rate, or
an accumulation of mutations over time. Differences in individual susceptibility to
environmental mutagens owing to common inherited polymorphisms in metabolism
and repair enzymes may also contribute to such variation (5). The assay may thus
provide a unique tool for studying the functional impact of common polymorphisms in
metabolism and repair genes, especially under controlled treatment conditions in vitro.
For example, human N-acetyltransferase (NAT2) and glutathione S-transferase
(GST-) are known to exhibit marked genetic polymorphisms. At least 50% of most
Caucasian populations are slow acetylators or completely lack GST- activity
(GSTM1 null genotype). NAT2 is involved in the metabolic activation of 2-
nitrofluorene to the known carcinogen N-acetyl-2-aminofluorene. Further metabolism
results in deactivation through glutathione conjugation. We obtained a clear dose-
related increase in the HPRT mutant frequency after treating mitogen-stimulated lym-
phocytes isolated from a normal blood donor with 2-nitrofluorene (up to fivefold at
400 g/mL, 24-h exposure; Fig. 1). No such mutant induction was seen when using
cells from another donor. The susceptible cells turned out to have the NAT2 rapid and
GSTM1 null genotype combination (capable of activation, with insufficient deactiva-
tion), whereas the resistant cells had NAT2 slow and GSTM1-positive genotypes
(incapable of activation). This finding suggests that functional polymorphisms in
metabolism and repair genes may indeed affect mutant induction in T cells, both in
vitro and in vivo.
The possibility of using primary T lymphocytes for in vitro mutational analysis has
only been utilized in a few studies (69). Many attempts have been made to improve
the T-cell cloning assay (reviewed in refs. 5 and 10). Most laboratories use different
concentrations of recombinant interleukin-2 (IL-2) with or without addition of condi-
tioned medium or lymphokine-activated killer cell (LAK) supernatant (1020% in
growth medium). The LAK supernatant is basically used in culture medium with a
large amount of recombinant IL-2 added to stimulate the proliferation of killer cells
from cancer patients. However, this medium is not available in most laboratories. The
present chapter describes a T-cell cloning protocol (5) using only a conditioned me-
dium that is easily prepared from X-irradiated lymphocytes with lethally irradiated
TK6 cells as allogenic stimulators (modified from ref. 11). The procedure for the enu-
meration and collection of HPRT mutants induced in vitro has been described recently
(9). In brief, pre-existing in vivo HPRT mutants are removed before in vitro treatment,
and independent mutants are collected from different subcultures for molecular analy-
sis. The mutational spectra obtained should thus be considered as the true in vitro
spontaneous or induced spectra in the T cells of the blood donor without any in vivo
background mutants or in vitro sibling mutants.
Human HPRT Mutation Analysis 157

Fig. 1. Hprt mutant frequency in human peripheral lymphocytes exposed in vitro to 2-


nitrofluorene. Different dose responses were obtained using cells from blood donors with
different genotypes. Donor 1, N-acetyltransferase 2 (NAT2) slow and glutathione S-transferase
M1 (GSTM1)-positive; Donor 2, NAT2 rapid and GSTM1-null.

2. Materials
2.1. Cells
1. Buffy coats (leukocyte preparations, each from 0.5 L whole blood centrifuged at 2700g
for 10 min; obtained from hospital blood center).
2. TK6 cells (kindly provided by Dr. William Thilly at the Massachusetts Institute of Tech-
nology, Center for Environmental Health Sciences, Cambridge, MA).
3. Feeder cells: lymphoblastoid RJK853 cells (kindly provided by Dr. Richard Gibbs at
Baylor College of Medicine, Houston, TX), lethally X-irradiated (40 Gy) prior to use.

2.2. Culture Media


1. Basic medium (BM): RPMI-1640 (Dutch modification) supplemented with 0.3 mg/mL L-
glutamine, 150 IU/mL benzylpenicillin, and 150 g/mL streptomycin (all from Gibco-
BRL, Life Technologies, Gaithersburg, MD).
2. Nutrient medium (NM): BM with 5% heat inactivated (56C, 30 min) fetal calf serum
(FCS; Gibco BRL, Life Technologies) and 5% heat-inactivated human AB serum (HS;
supplied by hospital blood center, pooled).
3. Growth medium (GM): NM with 0.3% (3 g/mL) phytohemagglutinin (PHA; BD Bio-
sciences, Franklin Lakes, NJ) and 20% T cell growth factor-enriched conditioned me-
dium (CM; prepared according to Subheading 3.2.).
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2.3. Hprt Assay


1. Phosphate-buffed saline (PBS).
2. 50X Concentrated hypoxanthine, aminopterin, and thymidine (HAT) supplement (Gibco
BRL, Life Technologies).
3. Stock solution of 6-TG (Sigma): 1 mg/mL in 0.01 M NaOH made immediately before use.
4. Microplates (96 wells, 7-mm round bottom; Nunc, Weisbaden, Germany).
5. 24-Well plates (Nunc).
6. UNI-SEP tubes with Ficoll-Paque (Wak-Chemie Medical, Steinbach, Germany).

3. Methods
3.1. Preparation of Mononuclear Cells
1. Obtain a buffy coat from a hospital blood center.
2. Dilute the content threefold with PBS.
3. Isolate the mononuclear cell fraction by Ficoll-Paque density separation in UNI-SEP
tubes, according to the manufacturers instructions.
4. Wash the cells twice in PBS.
5. Resuspend the cells in nutrient medium.
3.2. Preparation of Conditioned Medium
1. Isolate mononuclear cells from three buffy coats according to Subheading 3.1.
2. Resuspend the cells in BM to 3 106 cells/mL.
3. Irradiate with 10 Gy of X-rays.
4. Mix these cells with an equal volume of lymphoblastoid TK6 cells that have been X-
irradiated at the same density in BM, but with 40 Gy.
5. Dilute the cell mixture three times with BM to make the final density of each type of cells
5 105/mL.
6. Supplement with 2% FCS.
7. Stimulate with 1% PHA.
8. Incubate the cells for 72 h at 37C with 5% CO2 in the air.
9. Collect the supernatant by centrifugation at 1500 rpm for 30 min.
10. Store at 80C.
3.3. Testing for Quality of Conditioned Medium and Human Serum
1. Use a T-cell culture that has been grown in GM for 10 d or more to test for the ability of
CM or HS to promote the long-term proliferation of activated T cells on a microplate.
2. Seed each well with 1 104 cells in 200 L GM containing 0.3% PHA and various con-
centrations of CM or HS. Use an internal laboratory standard batch that has been prepared
previously as control.
3. Incubate the plate for at least 1 wk.
4. Compare the cell growth between different wells, both visually by using an inverted
microscope and quantitatively by Trypan blue staining and cell counting.
3.4. Mutant Induction
1. Purify lymphocytes from the buffy coat of a healthy blood donor according to Subheading 3.1.
2. Wash and resuspend the cells in NM supplemented with 0.3% PHA to a density of 1.5
106 cells/mL.
Human HPRT Mutation Analysis 159

3. Incubate for 20 h.
4. Remove pre-existing in vivo HPRT mutants by treating the cells with 2% HAT for 24 h.
5. Wash the cells with PBS and resuspend them in GM to a cell density of 1.5 106/mL.
6. Expose the cells to the chemical agent over a day or night.
7. Wash the cells with PBS and resuspend them in GM to a cell density of 1.5 106/mL.
8. Seed two 96-well plates with two test cells and 2 104 feeder cells per well in GM for
determination of relative cloning efficiency (CE: relative survival, treated vs control).
9. Subculture the remaining cells on 24-well plates, in 2 mL GM/well.
10. Incubate for 8 d for mutant expression. Keep approx 2 106 cells in each well by cell
counting every second day.
3.5. Estimation of the Average Cloning Efficiency and Mutant Frequency
1 Mix 2 105 cells from each subculture.
2. Make a limited (stepwise) dilution of cells.
3. Seed on two microplates two mixed test cells and 2 104 irradiated feeder cells per well
without TG.
4. Inoculate 10 selection plates with 2 104 mixed test cells and 1 104 feeder cells per well.
5. Wrap the plates in plastic foil to avoid evaporation and incubate at 37C in 5% CO2 and
95% humidity for 2 wk without medium change.
6. Score all plates visually using an inverted microscope.
7. Calculate the CE in plates with and without TG from the proportion of negative wells (P0)
assuming a Poisson distribution: CE = lnP0/number of cells seeded per well.
8. Obtain the MF by dividing the cloning efficiency in the presence of TG by that in the
absence of TG.
3.6. Mutant Selection for Molecular Analysis
1. Prepare for mutant selection of each subculture on a half 96-well plate.
2. Seed in each microwell 2 104 test cells and 1 104 feeder cells in GM supplemented
with TG (2 g/mL).
3. Incubate for 2 wk.
4. To avoid sibling mutants in the mutational spectrum, only one TG-resistant clone is to be
collected from each microplate (subculture) for molecular analysis.
4. Notes
1. The growth-supporting potency of the CM is usually highest when it is used at 1520% in
GM. The CM should produce consistently high CE, but only when combined with human
serum (5). The addition of 5% HS together with 5% FCS in GM has been shown to give a
remarkable increase in CE (5). Total replacement of FCS, i.e., use of 10% HS, did not
give any further increase, nor did addition of IL-2 (Boehringer Mannheim Biochemical,
Mannheim, Germany; 1020 U/mL) to GM (5).
2. In the present protocol, cells are primed with PHA for 20 h before treatment for up to 24
h. This 44 h of incubation before plating of T cells should not allow any cell division,
which may give rise to sibling clones.
3. Cell counting may affect the plating efficiency, since differently experienced technicians
may count cells in different ways. In particular, counting only the large stimulated cells
may introduce an overestimation of CE. However, theoretically, this should not affect the
calculated MF, since the CE in the selection plates is affected to the same extent.
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4. Inclusion of feeder cells in selection plates promotes the growth of TG-resistant cells.
Use of lethally irradiated RJK853 lymphoblastoid cells originated from a Lesch-Nyhan
patient with a total deletion of the HPRT gene as feeder cells excludes any cross-contami-
nation of mutant HPRT DNA by the remaining HPRT sequence from feeder cells in the
molecular analysis of HPRT mutation. Lethally irradiated lymphoblastoid TK6-derived
36X4 cells with a total deletion of the HPRT gene can also be used as feeder cells in both
nonselection and selection plates.
5. Donor genotypes for enzymes involved in activation and detoxification of mutagenic
agents may affect both background and induced MF and thereby the overall mutagenic
potency as judged from the doseresponse relationship. Knowledge of the metabolic path-
ways of the chemical agent and relevant genotypes of the blood donors should thus be
taken into consideration. Use of cell mixtures made from buffy coats of several different
donors or repeated experiments using cells from different donors may be necessary.
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