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Department of Biochemistry and Molecular Biology, The George Washington University, School of Medicine and Health Sciences, Washington, D.C. 20037, USA
1Correspondence: Department of Biochemistry and Molecular Biology, The George Washington University, School of Medicine and Health Sciences, Ross Hall, Room 526, 2300 Eye St., N.W., Washington, DC 20037, USA. E-mail: bcmvwh{at}gwumc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONAPPENDIX 1APPENDIX 2REFERENCES |
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Key Words: metabolic radiolabeling • beta emitters • cell cycle arrest • apoptosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONAPPENDIX 1APPENDIX 2REFERENCES |
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and
elevation of p53 (2
, 3)
, a tumor suppressor protein that
is a key cell cycle checkpoint regulator that monitors DNA damage. One
group associated the radioisotope-induced elevation of p53 with
sustained growth arrest or apoptosis, depending on the cell type
(3)
. Such harmful effects of radiolabels may not be
apparent in the absence of deliberate attempts to induce cells to
divide in a synchronous manner, which may explain why there is
relatively little documentation of this phenomenon. On the other hand,
the radiotoxicity of low-energy beta emitters has been long appreciated
by radiobiologists (4
5
6)
, although not investigated at
the molecular level or within the context of cell cycle progression.
Although the implications of radiation damage induced by radioisotopes
during metabolic labeling have not been widely considered, many studies
have focused on the molecular and cellular effects of 
- and UV
radiation (7
8
9
10
11)
. For example, UV irradiation of cells
has been shown to cause G1 as well as G2 checkpoint arrest involving
p53-dependent induction of p21Cip1/Waf1 or
phosphorylation of inhibitory residues of cdc2, respectively (7
, 8)
. -Irradiation has also been shown to induce p53-dependent
cell cycle arrest that can be manifested over multiple cell cycles
(11)
. In addition, -irradiation has been associated
with reduced long-term survival or clonogenicity of cells in culture
(11)
. In both cases, the primary target of radiation
damage is DNA, thus activating the DNA damage molecular sensors at both
the G1 (p53) and G2 (cdc2) checkpoints. With respect to low-energy
ß-emitting radioisotopes, the targets as well as mechanism(s) of
radiation damage are neither well understood nor well investigated. We
report here that [35S]methionine at normal
doses used in metabolic labeling studies substantially interferes with
cell cycle progression and survival.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONAPPENDIX 1APPENDIX 2REFERENCES |
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Cells and culture media
Two different lines of rabbit smooth muscle cells (A6 and B1)
were kindly provided by Dr. Gene Liau (Novartis Pharmaceuticals). The
A6 line is a cell line transfected with the gene for human acidic
fibroblast growth factor (FGF-121–154) in
plasmid p267–3. The B1 line was transfected with the plasmid alone.
Both of these cell lines were cultured in M199 medium supplemented with
10% fetal bovine serum (FBS, Atlanta Biologicals, Atlanta, Ga.), 2 mM
glutamine, antibiotic/antimycotic solution to final concentrations of
100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml
amphotericin B, and geneticin at 250 µg/ml. The NIH 3T3 murine
fibroblasts were obtained from Dr. Patricia Berg (GWUMC) and were
cultured in Dulbecco’s minimum essential medium (DMEM) supplemented
with 10% FBS. The human prostate carcinoma cell line, DU145, was
obtained from Dr. Mahnaz Badamchian (GWUMC) and cultured in DMEM:F12
(1:1) medium supplemented with 10% FBS. Other than the experiments
described in Fig. 2
, the A6 rabbit smooth muscle line was used for all
experiments described in this report.
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Mitotic arrest and cell synchronization protocols
Unsynchronized cells were incubated with the radiolabel at 100
µCi/ml for 2 h in prewarmed Met-deficient DMEM supplemented with
10% dialyzed FBS, 2 mM L-Gln, antibiotic/antimycotic, and 1 mg/l cold
Met before addition of nocodazole (Sigma) at 0.04 µg/ml and overnight
incubation to accumulate mitotic cells (~15 h). The control cells
were treated with equivalent volumes of the respective carrier buffers
instead of the label. The number of mitotic cells (those that were
visibly rounded on the culture dish) were counted within several 20x
fields on an Olympus CK inverted phase contrast microscope.
Cells were synchronized to the G1/S interface by a thymidine/mimosine
double block procedure (12)
. This involved an intial
12 h treatment with 2 mM thymidine, followed by a wash and further
culture in complete M199 medium (10–12 h). A second block was induced
with mimosine (400 µM) for 12–14 h, at which time the cells were
arrested in G1 as confirmed by PI staining and scanning laser cytometry
(see Fig. 3A
).
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Cell cycle analysis of anchored cells
Cell cycle analyses were performed on propidium iodide (PI)
-stained anchored cells as described previously (13)
. To
quantitate cell-associated PI fluorescence, cells were fixed at 2 h intervals after release from the mimosine block with 50% cold
methanol/phosphate-buffered saline (PBS) for 0.5 h at 4°C and
then stained with PI (0.05 mg/ml in 0.1% sodium citrate, pH 7) for 4
min at 4°C. The PI staining was done either with or without prior
RNase treatment (1 mg/ml for 30 min at 37°C) of the fixed cells with
the same results. Fluorescence analyses were performed using a Meridian
ACAS 570 Interactive Laser Cytometer and the Image Analysis program,
with excitation of the fluor at 488 nm. The program automatically
collects and processes fluorescence values over a programmable region
of the culture dish. Basically, 5–10 areas (720 µ x 720 µ)
were analyzed per sample for cell-associated fluorescence. Histograms
depicting the fluorescence profile of a minimum of 1956 cells were
prepared using the Meridian Instrument’s ‘Histogram’ program, which
combines data from the different fields examined. Figure 3
depicts the
relative number of cells as a function of integrated fluorescence.
Proliferation assays
In a short-term assay, cells were labeled for 2 h with
35S-Trans label at 100 µCi/ml, washed, and then
left in culture for the indicated number of days before counting viable
cells with a hemacytometer. Trypan blue staining immediately after
labeling did not reveal any significant decrease in cell viability.
For the colony-forming efficiency assays, cells were labeled for 2 h at the indicated doses. The labeled cells were then trypsinized,
washed to remove label, and replated in duplicate culture dishes at
1000 and 2000 cells/dish. Cells were fed with complete M199 medium
every other day for 7 days, after which they were fixed and stained
with 0.25% crystal violet in 2% formaldehyde/80% methanol. The
colonies (which were defined as a minimum of 100 cells in a cluster)
were counted manually for both labeled and unlabeled (control) samples
and percent colony formation was corrected according to the plating
efficiency (PE) of the control cells. Percent colony formation =
100 x number of colonies established/number of cells seeded x PE, where PE = number of colonies established by unlabeled
cells/total number of cells seeded. PE for the experiment shown in Fig. 5
was ~36%.
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Morphological analyses
Cell morphology after radiolabeling was examined by both
bright-field and fluorescence microscopy using an Olympus IX70-inverted
system microscope equipped with an IX-FLA inverted reflected light
fluorescence observation attachment. Pictures were recorded with an
Olympus PM-20 photomicrographic system using both 20x and 40x
objectives on the microscope. Some samples were fixed and stained with
PI as described above to better identify morphological changes in cell
nuclei.
Apoptosis assay
Cells were washed with PBS and stained with annexin V-Oregon
Green (Trevigen, Inc.) [250 µg/ml] for 15 min in the dark. The live
cells were then examined with an inverted fluorescence microscope, and
both phase contrast and fluorescence images were recorded
photographically. To quantitate apoptosis, the number of Oregon
Green-stained cells were counted and divided by the total number of
cells in the area examined. To facilitate cell count and to preserve
the cell-associated fluroescence for a longer period of time, the
radiolabeled cells were fixed with cold methanol after staining live
cells with annexin V-Oregon Green and also stained with PI to highlight
the nuclei. The number of Oregon Green-stained cells in both
radiolabeled and control samples were counted within a 10 x 10
grid on the fluorescence microscope and divided by the number of nuclei
(or total cell number) in the same area.
Statistical analyses
All data are expressed as means ± SE. The
Student’s t test was used to determine statistical
significance of the difference between labeled samples vs. matched
controls.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONAPPENDIX 1APPENDIX 2REFERENCES |
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. Figure 1
shows that metabolic labeling of rabbit smooth muscle cells with
[35S]methionine at 100 µCi/ml prevents the
normal accumulation of mitotic cells in the presence of nocodazole.
Neither the commercial source of the label nor the composition of the
carrier buffer was a factor in this inhibition, as shown in Fig. 1
. In
separate studies, removal of the label after an overnight or a 2 h
pulse labeling period also failed to reverse the inhibition (data not
shown). The effect of a 30 min prelabeling amino acid depletion
protocol was considered as a possible cause of mitotic inhibition
despite ample mitotic cell accumulation in the similarly starved
unlabeled control. This possibility was ruled out by the observations
that decreasing the preincubation time with depleted medium to 5 min
and labeling of cells in complete medium (without any starvation
period) had no influence on the inhibitory effects of the radiolabel on
mitotic induction. Furthermore, the effect of
[35S]methionine was not cell type specific, as
several different cell lines were similarly inhibited in terms of entry
into mitosis (Fig. 2
). Thus, we postulated that exposure to standard amounts of
[35S]methionine induced a form of cell cycle
arrest.
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35S-methionine induces cell cycle arrest
This postulate was confirmed by cell cycle analyses using PI
staining for DNA content and adapted for analysis of anchored cells
using scanning laser cytometry (13)
. As shown in
Fig. 3A
, unlabeled cells synchronized at the G1/S transition by a
thymidine-mimosine double block procedure (12)
are
characterized by a single peak in the fluorescence profile of
PI-stained cells, representing cells with 2n DNA content immediately
after washout of mimosine. Figure 3B
shows the appearance of
the G2 peak 8 h after release from the mimosine block, with the G2
peak having a mean fluorescence value approximately twice that of the
G1 peak, corresponding to cells with 4n DNA content. Figure 3C
shows that 24 h after the mimosine washout, a large
fraction of unlabeled control cells had returned to G1 and were
proceeding once again into S phase. At the same time point, a sample
that had been labeled for 2 h with
[35S]methionine immediately after washout of
mimosine and chased with cold complete medium for 22 h was still
predominantly in the initial G2 phase (Fig. 3D
), having
never rounded up. Since all cells in this experiment had been
previously synchronized to the G1/S interface, these studies suggest
that [35S]methionine incorporation into cells
can induce cell cycle arrest, even though DNA replication is not
inhibited by this protocol. This G2 arrest was independent of the
presence of nocodazole in the culture medium since the radiolabeled
sample had not been treated with nocodazole. Two earlier experiments
(2
, 3)
had associated radiolabeling with
[35S]methionine with an increase in p53 levels,
normally associated with G1 arrest. In light of our observations of a
[35S]methionine-induced G2 arrest, several
possibilities can be considered: 1) elevated p53 levels can
also induce G2 arrest, as reported elsewhere (15
16
17
18)
;
2) a p53-independent mechanism is involved in
radioisotope-induced cell cycle arrest, as has been suggested for other
forms of irradiation (3
, 5)
; 3) the stage
and/or mechanism of cell cycle arrest may be dependent on the cell
cycle compartment in which the label is introduced or on the cell type
involved. The resolution of these possibilities awaits further
experimentation.
35S-metabolic labeling inhibits cell proliferation
To investigate the effect of metabolic labeling on cell
proliferation, rabbit smooth muscle cells labeled for 2 h with
[35S]methionine were washed thoroughly to
remove label and cultured for additional periods of time. Figure 4
shows that whereas the unlabeled control cells increased in number over
the course of 3 days, the labeled cells showed no increase and, in some
experiments, decreased in number (data not shown). Colony-forming
efficiency assays were conducted at different concentrations of
35S-Trans label to assess the effects of varying
doses of radiolabel on long-term survival and proliferation of cells.
The results (Fig. 5A
) not only show that cells labeled for 2 h at 100
µCi/ml failed to form colonies over a period of 7 days, but also that
a 1000-fold reduction in the radioisotope concentration to 0.1 µCi/ml
yields a 40% reduction in the number of colonies formed relative to
unlabeled control cells. When expressed as a function of pCi
uptake/cell (Fig. 5B
), it is clear that the inhibition of
colony formation correlates directly with the amount of radioactivity
incorporated. Furthermore, relatively few atoms of
35S [0.07 pCi/cell corresponds to ~36,000
molecules of radiolabel] can cause substantial inhibition of cell
proliferation (~ 40%), suggesting that critical targets may be
damaged by this low-energy beta emitter. It will be interesting to
determine the exact nature of the target(s) as well as its relation to
cell cycle progression and apoptosis. It is noteworthy that there were
still some live cells in the dishes labeled at 100 µCi/ml, some of
which appeared to have undergone a few rounds of replication. However,
these cells were altered in morphology (see below), and the number of
such cells in a cluster did not meet the criterion set for a
‘colony’. It remains to be seen whether the surviving cells in these
small clusters are transiently growth-arrested, terminally
differentiated, or altered with respect to the length of the cell
cycle. The colony-forming efficiency assays thus indicate that even
very low levels of radiolabeling (0.1–1 µCi/ml) can induce
inhibition of cell proliferation and/or cell death.
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Radiolabeling dramatically alters cell morphology
Along with the inhibition of cell proliferation, we observed
dramatic changes in cell morphology for the radiolabeled smooth muscle
cells from predominantly spindle-shaped cells (Fig. 6A
) to enlarged, multinucleated, irregularly shaped cells
(Figs. 6B, C
). Yeargin and Haas (3)
also
reported that radiolabeling of human foreskin fibroblasts induced the
appearance of large, senescent-like cells that remained quiescent for
17 days. PI-staining confirmed the presence of multinucleated cells as
well as cells with fragmented nuclei (Figs. 6E, F
). These
morphological changes prompted us to examine the effects of
[35S]methionine on apoptosis.
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Radiolabeling induces delayed apoptosis of cells
Labeled and unlabeled cells were examined for signs of apoptosis
using a fluorescent conjugate of annexin V, a protein that strongly and
specifically binds to phophatidylserine (PS), which flips from the
inner to the outer monolayer of the plasma membrane as the cell
undergoes apoptosis. The resulting exposure of PS on the cell surface
as detected by annexin V binding is often taken as a marker for early
stages of apoptosis and occurs well before DNA fragmentation or trypan
blue staining is detectable (19)
. Figure 7
shows that radiolabeled cells showed an enhancement of apoptosis in
comparison to unlabeled control cells. The amount of apoptosis in
radiolabeled cell cultures was comparable to or exceeded the amount of
apoptosis induced by exposure of these cells to chromium, an
environmental toxin previously shown to induce apoptosis
(20)
. The degree of apoptosis increased as a function of
time after a 2 h exposure to radiolabel, suggesting that metabolic
radiolabeling and incorporated [35S]methionine
can induce delayed apoptosis. By contrast, trypan blue staining
immediately after the 2 h labeling period did not show any
significant decrease in cell viability (data not shown).
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The results described collectively suggest that low level beta
emitters, particularly [35S]methionine at the
concentration typically used in metabolic labeling experiments, can
cause cell cycle arrest, long-term inhibition of proliferation, and
apoptosis of cells. These results may not be so surprising if one
considers the amount of radioactive decay energy the cell is exposed to
during labeling as well as after metabolic incorporation of the
radiolabel. An estimate of the initial dose rate or energy emitted by
100 µCi 35S in a 1 ml volume (typical
concentration of [35S]methionine in metabolic
labeling experiments) is 1.8 x 105 MeV/s-g
or ~10 rad/h (See Appendix 1 for calculations). For comparison, 10 R
(~10 rad) of whole body radiation can cause an elevated number of
chromosome aberrations (though no detectable injury) in peripheral
blood cells, and an exposure of 20 R to the reproductive system can
cause a doubling of spontaneous mutations (21)
. After a
cellular uptake of ~40% of the label (present initially at 100
µCi/ml), the amount of energy released per cell/h is 230 MeV per
cell/h or 2.3 x 10-4 erg per cell/h
(Appendix 2). Our colony-forming assays also show that even very low
levels of radioisotope uptake (e.g.,. 0.07 pCi/cell) can cause
significant inhibition of cell proliferation. Thus, these results and
calculations suggest a substantial potential for DNA and/or cellular
damage by ionizing beta radiation from incorporated
35S atoms, as evidenced by earlier radiotoxicity
assays (5)
. In view of this and earlier studies (2
, 3
, 5)
, we suggest that caution should be exercised in using
metabolic radiolabeling techniques (especially for experiments
involving cycling cells) as well as in evaluating and interpreting the
literature involving the application of these methods. Furthermore,
efforts should be directed toward finding alternative methods for
following the biosynthesis, modification, and degradation of proteins
in vivo. One possible alternative might involve the use of
stable isotopes to follow protein expression and modification
(22)
. Aside from the obvious impact on experimental
design, these results would also suggest the reevaluation of acceptable
exposure levels to low-energy beta-emitting radioisotopes in the
workplace, clinic, and environment.
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APPENDIX 1 |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONAPPENDIX 1APPENDIX 2REFERENCES |
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Estimated mass of region irradiated during first hour: ~1 g [1 ml medium + cells]
Concentration of label: 100 µCi of 35S-methionine/ml ~ 100 µCi/g
Average beta energy of 35S: 0.049 MeV/disintegration
1 millirad = 62,500 MeV/g
Energy emitted/s
= Concentration of label/gm x (disintegrations/s) x (energy/disintegration)
= 100 µCi/g x 3.7 x 104 dps x 0.049 MeV/disintegration
= 1.8 x 105 MeV/s-g
Initial dose rate
= [1.8 x 105 MeV/s-g]/62,500 MeV/g-mrad
= 2.92 mrad/s ~ 10,500 mrad/h = 10 rad/h (in culture dish)
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APPENDIX 2 |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONAPPENDIX 1APPENDIX 2REFERENCES |
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Total added: 100 µCi [specific activity = 1175 Ci/mmol]
Number of cells: 106
Amount of 35S-Met taken up by cells:
40 µCi/1.175 x 109 µCi/mmol
= 34 x 10-9 mmol/106 cells
= 34 x 10-18 mol/cell x 6.02 x 1023 molecules/mol/cell
= 2 x 107 molecules/cell
Fractional disintegration in first hour = 1/2112, given 88 day half-life of 35S
Average beta energy = 0.049 MeV
Amount of energy released per cell/h:
0.5 x 2 x 107 35S atoms/cell x 1/2112 x 0.049 MeV/ disintegration = 230 MeV per cell/h or 2.3 x 10-4 erg per cell/h
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONAPPENDIX 1APPENDIX 2REFERENCES |
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-irradiation. Cancer Res 57,1171-1179This article has been cited by other articles:
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V. W. HU, D. S. HEIKKA, P. B. DIEFFENBACH, and L. HA Metabolic radiolabeling: experimental tool or Trojan horse? 35S-Methionine induces DNA fragmentation and p53-dependent ROS production FASEB J, July 1, 2001; 15(9): 1562 - 1568. [Abstract] [Full Text] [PDF]
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