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[CANCER
RESEARCH
38, 1031-1035,
Drug-induced
April 1978]
Changes in DMA Fluorescence
Microfluorometry
and Their Implications
Intensity Detected by Flow
for Analysis of DNA Content
Distributions
Oliver Alabaster,1 Eric Tannenbaum, Mary Cassidy Habbersett, Ian Magrath, and Chester Herman
Laboratory ol Pathology ¡O.A., E. T., M. C. H., C. H.) and Pediatrie Oncology Branch ¡I.M.], National Cancer Institute, NIH, Bethesda, Maryland 20014
ABSTRACT
Chicken erythrocytes, which contain less DNA than
mammalian diploid cells, were used as an internal stan
dard to control instrumental and staining variables during
flow microfluorometric analysis. With the DNA stain, mithramycin, and with an EPICS II flow microfluorometer,
ratios between the modal G, fluorescence of experimental
cells and that of chicken erythrocytes were determined.
The results indicate that unperturbed cell populations of
L1210 and HeLa cells in vitro and L1210 ascites cells in
vivo have relatively stable fluorescence ratios, although
there is a significant difference between the ratios of one
L1210 cell line in vitro and another in vivo.
In contrast, L1210 ascites treated in vivo with different
schedules of cyclophosphamide and Adriamycin showed
wide fluctuations in the fluorescence intensity ratios for
96 hr after treatment. Also, differences in the fluorescence
ratios were observed between less advanced and more
advanced L1210 ascites after treatment with the same
schedule.
These effects indicate an alteration in DNA staining with
mithramycin, brought about by drug treatment that could
seriously affect the interpretation of DNA histogram data.
Nevertheless, changes in mithramycin staining may prove
to be a very important probe to detect persistent drug
effects.
INTRODUCTION
Flow microfluorometry is used currently for rapid deter
mination of relative DNA content distribution by a variety of
stains that are presumed to be stoichiometric for DNA (4, 5,
11, 12, 17). These stains bind to DNA and fluoresce under
appropriate light excitation in direct proportion to the DNA
mass. This technique has provided relative DNA content
distributions in a variety of tumor systems in perturbed and
unperturbed growth conditions (21, 23, 27-30). These DNA
content distributions have then been used to determine the
relative position of cells in the cell cycle, and a variety of
mathematical techniques have been used to derive cell
cycle parameters (1-3, 14, 15, 19, 20, 22, 39).
Hitherto, DNA content distributions have been obtained
without the use of an internal standard. At best, serial
histograms have been obtained when instrumental settings
were left constant. Subsequent variations in fluorescence
therefore could be partly due to instrumental or staining
variables.
1 To whom requests for reprints should be addressed, at Room 1A21,
Building 10, National Cancer Institute, NIH, Bethesda, Md. 20014.
Received September 16, 1977; accepted January 16, 1978.
APRIL
Chicken erythrocytes, which have a lower DNA content
than mammalian cells, fluoresce much less intensely than
do normal mammalian diploid cells and therefore are well
suited for use as an internal standard. The difference in
fluorescence between the G, peak of mammalian cells and
the G, peak of chicken erythrocytes can be used to deter
mine a ratio of absolute fluorescence intensity and, hence,
the DNA dry mass of the cell population (35). Furthermore,
this ratio of fluorescence can be used to determine the
influence of chemotherapeutic agents on the relative fluo
rescence of treated cell populations.
Until now it has been assumed that the DNA stoichiometry
of mithramycin (and other fluorochromes) is constant even
in drug-treated cell populations. However, the demonstra
tion of drug-induced changes in fluorescence properties of
cells must call this assumption into question. For example,
if cells are not equally affected because a drug is cell cycle
phase dependent, cells in protected phases of the cycle
may not change their fluorescence properties as much as
cells in sensitive phases of the cell cycle. Thus, under
certain conditions the assumptions on the fluorescence
stoichiometry of all cells in each cell cycle phase may be
invalid.
In the present study we have measured the fluorescent
intensity of unperturbed and drug-treated cell populations
and compared it to the fluorescent intensity of the internal
standard. The results indicate that unperturbed cell popu
lations have a reasonably stable ratio, although there is a
difference between the ratio obtained from different L1210
lines in vivo and in vitro. However, in CTX2- and ADRtreated populations, there are major changes in mithramy
cin staining characteristics.
The possibility of drug-induced changes in stoichiometry
must therefore be considered before attempting to interpret
data on DNA content distributions, since assumptions re
garding the relative position of all G,, S, and G2 + M cells
within the DNA histogram cannot be made with any degree
of certainty. However, changes in the fluorescence proper
ties of mithramycin-stained, drug-treated cells could be
used to estimate the duration of drug effects on both host
and target tissues.
MATERIALS
AND METHODS
Chicken erythrocytes were obtained from heparinized
whole chicken blood; WBC were not removed. After centrif* The abbreviations used are: CTX, cyclophosphamide; ADR, Adriamycin;
CTX/ADR, concurrent administration of both drugs, with CTX given first
followed immediately by ADR.
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O. Alabaster et al.
ugation at 360 x g for 10 min, the plasma was removed,
and the cells were washed with phosphate-buffered 0.85%
NaCI solution, fixed in 50% ethanol, and stored at 4°.The
same pool of chicken erythrocytes was used for all studies.
HeLa cells grown in monolayers in Eagle's essential
medium supplemented with 10% fetal calf serum were
harvested daily by trypsinization [0.5% trypsin in EDTA
(0.2% solution); incubated 7 to 10 min]. Growth media were
changed daily.
L1210 cells grown in RPMI Culture Medium 1630 (Grand
Island Biological Co., Grand Island, N. Y.) were harvested
during the exponential growth phase, fixed in 70% ethanol,
and stored at 4°.
For the in vivo studies, 5 x 105 L1210 cells i.p. of a
different line, which was routinely carried in BALB/c x
DBA/2 F, (hereafter called CD2F,) mice, were implanted
into groups of male CD2F, mice (average weight, 21 g).
On Days 5, 6, and 7 of tumor growth, 5 control mice were
sacrificed by cervical dislocation, and the L1210 cells were
harvested by peritoneal lavage with cold 1% sodium citrate
and placed in centrifuge tubes on ice. The cells then were
centrifuged at 360 x g for 5 min, the supernatant was
removed, and the cell pellet was resuspended in cold 70%
ethanol. All samples were stored at 4°.
Cyclophosphamide (Meade Johnson Laboratory, Evansville, Ind.) was dissolved in sterile water and was Injected at
a dose of 100 mg/kg i.p. Adriamycin (Adria Laboratories,
Inc., Del.) was dissolved in sterile 0.85% NaCI solution and
injected at a dose of 3 mg/kg i.p. Groups of CD2F! mice
carrying L1210 ascites were then treated in the following 3
ways. In the first way, ADR alone was administered, and
groups of 5 mice were sacrificed at 8, 16, 24, 48, 72 hr,
respectively. In the second way, CTX was given first, fol
lowed by ADR at 24 hr. In the third way, both drugs were
given together; CTX was given first, followed immediately
by ADR (CTX/ADR). Subgroups of 5 mice were then sacri
ficed at 8, 16, 24, 48, 72, 96 hr, respectively.
Two other groups of CD2F, mice were also treated with
CTX/ADR, but the treatment differed only in that the drugs
were injected 3 days after tumor inoculation in one group
and 5 days after tumor inoculation in the other. Subgroups
of 5 tumor-carrying mice treated on the third day postinoc
ulation were then sacrificed at 24-hr intervals from Days 5
to 11; subgroups of 5 tumor-carrying mice treated 5 days
postinoculation were similarly sacrificed from Days 5 to 9.
Cells were harvested from the treated mice in the same way
as were cells from the controls.
Flow Microfluorometry. Chicken erythrocyteswere added
to each cell sample so that they represented approximately
10 to 20% of the total cell number. The samples were then
filtered through a 62-/¿mnylon mesh filter (Small Parts, Inc.,
Miami, Fla.). Mithramycin staining solution (12) at 1 x 106
cells/ml, after staining for a minimum of 20 min, was then
examined under the fluorescence microscope for doublets,
which never exceed 6%. Each sample was then analyzed
with an EPICS II flow microfluorometer and cell sorter
(Particle Technology, Inc., Los Alamos, N. M.). The princi
ples of this instrument have been well described elsewhere
(34). An argon ion laser at 457 nm 200 (power output, milli
watts) was used to excite fluorescence. The fluorescent sig
nals (at 495 nm) were then displayed on a 128-channel pulse
1032
height analyzer as a frequency distribution histogram. The
photomultiplier voltage was adjusted so that the chicken
erythrocytes appeared in Channel 15 (Chart 1); this ensured
that the ratio was calculated well within the linear range of
the amplifier. The G, peak of the chicken erythrocytes was
made steady and symmetrical prior to recording the chan
nel number of the G, peak of the sample. The optical
alignment of the EPICS II was maintained with fluorescent
microspheres (Particle Technology), which gives a coeffi
cient of variation of 3.73%.
When all samples had been analyzed, the ratios were
calculated by dividing the cell sample G, peak channel
number by the chicken erythrocyte peak channel number.
The mean ratios ±S.D. were then plotted graphically; the
plots are shown in Charts 2, 3, and 4.
EPICS II
*
4096
128
Chart 1. A typical fluorescence intensity histogram demonstrates a small
chicken erythrocyte G, peak in Channel 15 (the internal standard) and the
large G, peak of L1210 cells, which in this example is in Channel 72.
Ordinate, cells per channel ; abscissa, increasing fluorescence intensity. The
iate-S-G. + M region is off scale because the chicken erythrocytes were
deliberately placed far enough upscale to be well within the linear range of
the amplifier. All tumor samples were analyzed with the internal standard
symmetrically placed in Channel 15 to calculate the ratio of the fluorescence
of the 2 G, peaks.
1
2
3
4
5
6
?
8
!
DAYS (Turn« Growth)
Chart 2. A, fluorescence of L1210 ascites cells in vivo on Days 5, 6, and 7
of tumor growth is compared to that of chicken erythrocytes used as an
internal standard. The fluorescence ratios are plotted •
S.D. Cells from 2
separate cultures of L1210 cells in exponential growth, at the same cell
density, have an identical fluorescence ratio, which is lower that that
obtained in vivo. B, HeLa cells grown in monolayer for 9 days. The G,
fluorescence ratio is compared to that of the internal standard on each day
of growth. The mean ratio is 4.99 ±0.25.
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VOL. 38
Drug-Induced Changes in DNA Fluorescence
W
72
96
HOURS(PostTreatment)
Chart 3. A, G, fluorescence
ratio of L1210 ascites cells ¡nexponential
growth treated with CTX (100 mg/kg) and then with ADR (3 mg/kg) 24 hr
later is compared to the ratios obtained with CTX/ADR. B, G, fluorescence
ratio of L1210 ascites at the same stage of tumor growth as in A. treated with
ADR (3 mg/kg) alone. The mice did not survive beyond 72 hr. Bars, ±S.D.
6
7
B
9
10
11
DAYS(PostTreatment)
Chart 4. Longer term effect of CTX (100 mg/kg)/ADR
(3 mg/kg) given
concurrently to L1210 ascites tumor on Day 3 and Day 5 of tumor growth. No
regrowth of S-phase cells was observed on Days 10 and 11 after treatment of
the Day 3 tumor. S-Phase cells reappeared on Day 8 after treatment of the
Day 5 tumor, and the mice did not survive beyond Day 9. Bars, ±S.D.
Unstained drug-treated cells did not fluoresce when they
were examined under the fluorescence microscope and in
the EPICS II with the laser line at 457 nm.
ratios in unperturbed cells, CTX- and ADR-treated cells
showed major changes in the absolute fluorescence inten
sity of the G, peak.
Chart 3A demonstrates the effect of CTX alone at 8, 16,
and 24 hr (open circles). At 24 hr, ADR was added so that
the open circles from 48 to 96 hr represent the effect of the
2 drugs in a sequential schedule. The closed circles repre
sent the effects of the 2 drugs given simultaneously. The 2
schedules produced parallel changes in the fluorescence
ratio from 24 to 96 hr. When CTX was given first, however,
its effect was greater and led to a persistently higher G,
fluorescent intensity despite the subsequent addition of
ADR. Chart 3B shows the effect of ADR alone; for the first
48 hr, the curve resembles that seen after both drugs had
been given simultaneously.
The effect of CTX/ADR over a longer period of time is
seen in Chart 4. In this experiment the drugs were given to
mice that bore L1210 ascites 3 days (closed circles) and 5
days (open circles) after inoculation of tumor, and the
effects were monitored for 11 and 9 days, respectively. The
Day 3 tumor-bearing mice survived more than 11 days after
treatment and, at this time, have had no regrowth of S
phase cells. The Day 5 tumor-bearing mice, however, had
regrowth of S phase cells by Day 8 after treatment, and
none survived to Day 10. Despite these differences in regrowth characteristics, the ratios were similarly depressed
in both groups for the last 2 days of each study.
In Chart 5, examples of histograms of different cell cycle
distribution although of increased and decreased ratios are
shown. In all these histograms the chicken erythrocytes are
in Channel 8 so that the complete DNA content distribution
of the tumor cells can be demonstrated. The Chart 5A
histogram shows the cytokinetic response of the ascites
tumor 16 hr after CTX was given, with a ratio of 6.25 and a
large proportion of cells in the late S-G2 + M region. The
Chart 56 histogram, 48 hr after CTX and ADR were given in
sequence, also shows an increased ratio of 5.75, although
it shows fewer S and G2 + M cells. However, the Chart 5C
histogram, 96 hr after CTX/ADR treatment had been given
simultaneously, also shows fewer S and Gz + M cells, but
the ratio is below the control at 3.75.
RESULTS
The ratio of the modal G, fluorescence of the tumor cells
to the modal G! fluorescence of the internal standard was
calculated for each sample by histogram displays similar to
that seen for untreated control cells in Chart 1. The coeffi
cient of variation of the G, peak of the tumor samples
ranged from 2.9 to 5.6%, whereas that of the chicken
erythrocytes was 8.2 to 11.3%.
In Chart 2A the mean ratio ±S.D. is plotted for 5 samples
of L1210 ascites on Days 5, 6, and 7 of tumor growth. The
in vitro ratio was derived from cell samples taken from 2
independent cultures of a different line of L1210 cells in
exponential growth. The 2 ratios obtained from the in vitro
cells were identical. Both in vivo and in vitro ratios therefore
were reproducible and differed significantly. However, ¡n
Chart 20 the ratio obtained from HeLa cells grown 9 days in
culture showed minor fluctuations with a mean ±S.D. of
4.99 ±0.25.
In contrast to the relative stability of the G, fluorescence
DISCUSSION
The data presented in this paper indicate that the use of
chicken erythrocytes as an internal standard is a very effec
tive wasy to determine relative changes in the fluorescence
intensity of a cell population, with mithramycin as the DNA
stain.
The untreated cell populations possess relatively stable
ratios (Chart 2). In contrast, the drug-treated cell popula
tions show significant changes in mithramycin staining
intensity, which persist for at least 11 days (Charts 3 and 4).
These changes are important because of assumptions
usually made about the persistence of DNA staining stoichiometry after interference with cell growth. Although the
presence of a biomodal distribution of cells with a fluores
cence ratio of 1:2 and an intermediate cell population
suggests normal stoichiometry, the detection of a change
in the fluorescence intensity of cells after drug treatment
must raise the question as to whether all cells in the
APRIL 1978
1033
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O. Alabaster et al.
EPICS II
B
4*M
Chart 5. Typical histograms that illustrate different ratios and cell cycle
distributions. In each example the chicken erythrocytes are in Channel 8. A
small peak is seen in Channel 16, which is produced by doublets. A, a
histogram obtained 16 hr after a single dose of CTX. The majority of cells are
in late S-G2 + M, and the ratio is increased to 6.25 (control ratio, 4.46 ±
0.21). B, a histogram obtained 48 hr after CTX and ADR were given in
sequence. The majority of cells are in Q,, and the ratio is also increased at
5.25. C, a histogram obtained 96 hr after treatment with CTX/ADR. The
majority of cells are also in G,, but the ratio is below the control value at
3.75. There was no evidence in any histogram that the G, fluorescence
intensity ratio was influenced by the cell cycle distribution. Ordinate, cells
per channel; abscissa, increasing fluorescence intensity.
population have been equally affected. The G.:G, channel
ratio of the control histograms had a range of 1.95 to 1.98,
whereas the range in the drug-treated cell populations was
1.83 to 2.13. The wider variability of the G2:Gi ratio in the
drug-treated group therefore supports the possibility of an
unequal cell response.
As can be seen from Chart 3A, administration of CTX
alone raises the fluorescence ratio substantially at 8, 16,
and 24 hr. CTX, which is an alkylating agent thought to
affect cells in all phases of the cell cycle (10, 26, 37), is
known to induce cross linkages in DNA either by linking
adjacent areas of 1 strand or by joining together opposite
strands of the helix (7). Furthermore, alkylating agents can
1034
induce not only development of giant cells with increased
amounts of DNA and protein (8, 9) but also chromosomal
aberrations (32, 33, 36).
However, ADR binds to DNA, and this could influence the
availability of mithramycin-binding sites. From Chart 3A it is
evident that simultaneous treatment with ADR limits the
increased fluorescence induced by CTX, so that the effects
more closely resemble those seen with ADR alone (Chart
3B).
The changes seen in both groups from 48 to 96 hr (Chart
3A) are very difficult to explain simply in terms of drug
binding because fluorescence both increases and de
creases in relation to the controls after treatment. There
fore, it seems more likely that these changes in fluores
cence are due to structural alteration of the chromatin.
Furthermore, nothing is known of the precise metabolic
interaction of CTX and ADR, which might explain these
observations.
An increase in the fluorescence ratio in unperturbed cell
populations is consistent with an increase in absolute DNA
dry mass (35). However, in drug-treated populations, the
greatest increase in the fluorescence ratio is within 8 hr,
and this is therefore probably not due to a true increase in
DNA mass. Also, it is probably not due to quantitative
changes in mitochondrial DNA since this represents such a
small proportion of the total cellular DNA.
The subsequent fall in the fluorescence ratio (Charts 3
and 4) below the control value is also probably not due to
changes in DNA mass because subsequent increases in
DNA mass would have to have occurred so rapidly. This is
evident in Chart 3/4 from 72 to 96 hr.
Interpretation of all these observations is further compli
cated by the data seen in Chart 4. These data indicate a
different pattern of response in L1210 tumor cell popula
tions treated at different intervals after implantation. Not
only is there a difference in the fluorescence ratios, but also
there is a difference in the regrowth characteristics. The
more advanced tumor resumed cell growth (reappearance
of S-phase cells) on Day 8, whereas the less advanced
tumor had no evidence of regrowth by Day 11. However,
the fluorescence ratios in the advanced tumor on Days 8
and 9 were very similar to those seen on Days 10 and 11 in
the less advanced tumor. Therefore, the fluorescence
changes appear to be independent of cell proliferation and
dependent on other factors.
The data presented in Chart 4 indicate that drug effects
may persist for some time. It is known that both CTX and
ADR may cause long-term chromosomal damage and are
capable of inducing malignancy (6, 16, 18, 25, 33, 35).
Thus, it is possible that persistent changes in fluorescence
ratios might be a way to detect permanent drug effects
on host tissues as well as the persistence of drug effects on
tumor cell populations.
Finally, what is of most immmediate significance is that
changes in cell fluorescence may effect the interpretation
of DNA histograms. Not only may some drugs have selective
effects on cells in different phases of the cell cycle in
lethality or arrest of cell cycle progression, but also these
effects may be absent or reduced in resistant subpopulations of cells. More sensitive cells might then change their
fluorescence properties to a greater extent than would less
CANCER
RESEARCH
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VOL. 38
Drug-Induced Changes in DNA Fluorescence
15. Dethlefsen, L. A., Gray, J. M., George, Y. S., and Johnson, S. Flow
Cytometric Analysis of the Perturbed Cellular Kinetics of Solid Tumors:
Problems and Promises. In: W. Go'hde, J. Schumann, and T. H. Büchner
(eds.), Second International Symposium on Pulse-Cytophotometry, pp.
188-200. Ghent, Belgium: European Press Medikon, 1976.
16. DiPaolo, J. A. Teratogenic Agents: Mammalian Test Systems and Chem
icals. Ann. N. Y. Acad. Sci., Õ63:801-812, 1969.
17. Dittrich, W., and Göhde,W. Impulse Cytophotometry of Single Cells in
Suspension. Z. Naturforsch., 24b: 360-361, 1969.
18. Fahmy, 0. G., and Fahmy, M. The Genetic Effects of the Biological
Alkylating Agents with Reference to the Pesticides. Ann. N. Y. Acad.
Sci., 160: 228-243, 1969.
19. Fried, J. Method for Quantitative Evaluation of Data from Flow Micro
fluorometry. Computers Biomed. Res., 9: 263-276, 1976.
20. Fried, J., Yataganas, X., Kitahara, T., Perez, A., Ferguson, R., Sullivan,
S., and Clarkson, B. Quantitative Analysis of Flow Microfluorometric
Data from a Synchronous and Drug Treated Cell Populations. Computer
Biomed. Res., 9: 277-290, 1976.
ACKNOWLEDGMENTS
21. Göhde,W., J. Schumann, and T. H. Büchner(eds.). Second Interna
tional Symposium on Pulse-Cytophotometry. Ghent, Belgium: European
The authors express their appreciation to Dr. Kwang Woo for the cultured
Press Medikon, 1976.
Hela cells, Donna Roberts for her secretarial assistance, and Ralph Isenberg
22. Gray, J. W. Cell Cycle Analysis from Computer Synthesis of Deoxyribo
for his photographic services.
nucleic Acid Histograms. J. Histochem. Cytochem., 22: 642-650, 1974.
23. C. A. M. Haanen, H. F. P. Hillen, and J. M. C. Wessels (eds.). First
International Symposium on Pulse-Cytophotometry. Ghent, Belgium:
European Press Medikon, 1975.
REFERENCES
24. Liedeman, R., and Bolund, L. Acridine Orange Binding to Chromatin of
Individual Cells and Nuclei under Different Staining Conditions. I and II.
1. Alabaster, 0., and Bunnag, B. Flow Microfluorimetric Analysis of Sensi
Exptl. Cell Res., 101: 164-183, 1976.
tive and Resistant Leukemia L1210 following 1-/3-Arabinofuranosylcytosine in Vivo. Cancer Res., 36. 2744-2749, 1976.
25. Loveless, A. Genetic and Allied Effects of Alkylating Agents. London:
2. Baisch, H., Göhde,W., and Linden, W. A. Mathematical Analysis of ICPButterworth & Co., 1966.
26. Lundlum, D. B. Molecular Biology of Alkylation: An Overview. In: A. C.
Data to Determine the Fraction of Cells in the Various Phases of Cell
Sartorelli and D. G. Johns (eds.), Antineoplastic and Immunosuppressive
Cycle. In: C. A. M. Haanen, H. F. P. Hillen, and J. M. C. Wessels, (eds.),
Agents, Part 2, Chap. 31, pp. 6-17. Berlin: Springer-Verlag, 1975.
First International Symposium on Pulse-Cytophotometry, pp. 68-76.
27. D. Lutz (ed.), Third International Symposium on Pulse-Cytophotometry.
Ghent, Belgium: European Press Medikon, 1975.
3. Baisch, H., Linden, W. A. Different Mathematical Models for PulseGhent, Belgium: European Press Medikon, in press.
28. Mayall,, B. H. (ed.). Papers from the Third Engineering Foundation
Cytophotometric Evaluations Applied to Asynchronous and Partially
Conference on Automated Cytology. J. Histochem. Cytochem., 22: 451Synchronised Cell Populations. In: C. A. M. Haanen, H. F. P. Hillen, and
J. M. C. Wessels (eds.), First International Symposium on Pulse-Cyto
765.
photometry, pp. 61-67. Ghent, Belgium: European Press Medikon, 1975.
29. Mayall, B. H. (ed.). Papers from the Fourth Engineering Foundation
Conference on Automated Cytology. J. Histochem. Cytochem., 24: 14. Barlogie, B., Zante, J., Drewinko, B., Schumann, J., Búchner, T.,
Göhde,W., Hart, J. S., and Johnston, D. A. The Use of Mithramycin in
414,1976.
Pulse-Cytophotometry. In: W. Göhde,J. Schumann, and T. H. Büchner 30. Mayall, B. H., and Gledhill, b. L. (eds.). Papers from the Fifth Engineer
(eds.), Second International Symposium on Pulse-Cytophotometry, pp.
ing Foundation Conference on Automated Cytology. J. Histochem.
125-136. Ghent, Belgium: European Press Medikon, 1976.
Cytochem., 25: 479-952, 1977.
5. Berhan, E. Pulse-Cytophotometry as a Method for Rapid Photometer
31. Nicolini, C., and Baserga, R. Circular Dichroism Spectra and Ethidium
Bromide binding of 5-Deoxybromouridine-substituted Chromatin. Bio
Analysis of Cells. In: C. A. M. Haanen, H. F. P. Hillen, and J. M. C.
Wessels (eds.), First International Symposium on Pulse-Cytophotometry,
chem., Biophys. Res. Commun., 64: 189-195, 1975.
pp. 15-21. Ghent, Belgium: European Press Medikon, 1975.
32. Ochoa, M., Jr., and Hirschberg, E. Alkylating Agents. In: R. J. Schnitzer
and F. Hawking (eds.), Experimental Chemotherapy, Vol. 5, pp. 1-132.
6. Brookes, P., and Lawley, P. D. The Reaction of Mustard Gas with
Nucleic Acids in Vivo and in Vitro. Biochem. J., 77: 478-484, 1960.
New York: Academic Press, Inc., 1967.
7. Brookes, P., and Lawley, P. D. The Reaction of Mono and Di-Functional
33. Sieber, S. M., and Adamson, R. M. The Clastogenic, Mutagenic, Tera
Alkylating Agents with Nucleic Acids. Biochem. J., 80: 496-503, 1961.
togenic, and Carcinogenic Effects of Various Antineoplastic Agents. In:
8. Caspersson, T., Farber, S., Foley, G. E., and Killander, D. Cytochemical
Pharmacological Basis of Cancer Chemotherapy, The University of
Observations on the Nucleolus-Ribosome System. Effects of ActinomyTexas System Cancer Center M. D. Anderson Hospital and Tumor
cin D and Nitrogen Mustard. Exptl. Cell Res.. 32: 528-552, 1963.
Institute, pp. 401-468. Baltimore: Williams & Wilkins, 1975.
9. Cohen, L. S., and Studzinski, G. P. Correlation between Cell Enlarge
34. Steinkamp, J. S., Fulwyler, M. J., Coulter, J. R., Hiebert, R. D., Horney,
ment and Nucleic Acid and Protein Content of HeLa Cells in Unbalanced
J. L., and Mullaney. P. F. A New Multiparameter Separator of Micro
scopic Particles and Biological Cells. Rev. Sci. Instr., 44: 1301-1310,
Growth Produced by Inhibition of DNA Synthesis. J. Cellular Physiol.,
69: 331-339, 1967.
1973.
10. Connors, T. A. Mechanism of Action of 2-Chloroethylamine Derivatives,
35. Tannenbaum, E., Cassidy Habbersett, M., Alabaster, O., and Herman, C.
Sulfur Mustards, Epoxides, and Aziridines. In: A. C. Sartorelli and D. G.
J. Measurement of Cellular DNA Mass by Flow Microfluorometry Using a
Johns (eds.), Immunosuppressive and Antineoplastic Agents, Vol. 2,
Biological Internal Standard. J. Histochem. Cytochem., in press, 1978.
Chap. 32, pp. 18-34. Berlin: Springer-Verlag, 1975.
36. Vogel, F., and Roehrborn, G. (eds.). Chemical Mutagenesis in Mammals
and Man Berlin:Springer-Verlag, 1970.
11. Crissrnan, H. A., Oka, M. S. and Steinkamp, J. A. Rapid Staining
Methods for Analysis of Deoxyribonucleic Acid and Protein in Mamma
37. Wheeler, G. P. Studies Related to the Mechanisms of Action of Cytotoxic
lian Cells. J. Histochem. Cytochem, 24: 64-71, 1976.
Alkylating Agents: A Review. Cancer Res., 22: 651-688, 1962.
12. Crissrnan, H. A., and Tobey, R. A. Cell Cycle Analysis in 20 Minutes.
38. Wheeler, G. P. Alkylating Agents. In: J. F. Holland and E. Frei, III, (eds.),
Science, 184: 1297-1298, 1974.
Cancer Medicine, Chap. XIII-4, pp. 791-806, Philadelphia: Lea & Febiger,
13. Darzynkiewicz, Z., Tráganos, F., Sharpless, T. K., and Melamed, M. R.
1973.
Cell Cycle-related Changes in Nuclear Chromatin of Stimulated Lympho
39. Woo, K. B. The Discrete-Time Kinetic Model Analysis of DNA Content
cytes as Measured by Flow Cytometry. Cancer Res., 37: 4635-4640,
Distributions in Experimental Tumor Cells. Cell Tissue Kinet , in press
1977.
1978.
14. Dean, P. N., and Jett, J. H. Mathematical Analysis of DNA Distributions
40. Yonuschot, G., and Muschrush, G. W.Terbium as a Fluorescent Probe
Derived from Flow Microfluorometry. J. Cell Biol., 60: 523-527, 1974.
for DNA and Chromatin. Biochemistry 14: 1667-1681, 1975.
sensitive cells. Hypothetically, some G, cells could appear
within theG2 peak or wee versa. Under these circumstances
the detection and interpretation of cell cycle parameters
would be very difficult.
Further studies are required with well-established tech
niques (13, 24, 31, 40) used to assess whether these are
really changes in chromatin structure. It will also be neces
sary to examine other DNA dyes to determine whether these
fluorescence changes are a common finding or whether
mithramycin is a unique probe that could be used to detect
persistent drug effects and cell damage.
APRIL
1978
Downloaded from cancerres.aacrjournals.org on August 10, 2017. © 1978 American Association for Cancer
Research.
1035
Drug-induced Changes in DNA Fluorescence Intensity Detected
by Flow Microfluorometry and Their Implications for Analysis of
DNA Content Distributions
Oliver Alabaster, Eric Tannenbaum, Mary Cassidy Habbersett, et al.
Cancer Res 1978;38:1031-1035.
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