LETTER TO THE EDITOR
Simpkins et al. (l, 2) and Pearlman et al. (3) reflect to which
degree (and to some degree where) the drug is bound to the
polynucleotide. To get any information on the distribution of
the drug in question, however, simulated fluorescence decay
curves for different distributions must be compared with the
measured ones. Quantitation of drug binding to polynucleotides
can be studied by much simpler and more accurate methods.
Great caution must therefore be paid when using Tb3+ as a
probe for guanine/drug interactions. We refer interested readers
to our previous work (4) for a summation of the criteria that
must be fulfilled when using Tb3+ for such investigations.
Fig. 2. Time-resolved fluorescence of Tb3* bound to DNA at two Tb'VDNA
ratios in the absence or presence of doxorubicin (D/P = 1/500). Fluorescence at
S44 nm (slit width, 20 uni) was measured with a gating time window of 0.02 ms
in an Hitachi Perkin-Elmer LS-5 fluorescence spectrometer in the phosphoresc
ence mode. Excitation was performed at 290 nm (slit width, 10 nm). SO »AI
(phosphate) calf thymus DNA (Sigma) in 20 HIMTris (pH = 7.4) were incubated
without (D. O) or with (•.•¿)
0.1 «Mdoxorubicin (Farmitalia Carlo Erba). Ti><ï,
(Riedel-de Haen) was thereafter added to a final concentration of 25 JIM(O, •¿)
or 100 MM(D, •¿).
Tb3* fluorescence at zero time of solutions with 100 MMTb3*
was arbitrarily set to 1.0. The excitation pulse profile was narrow and is not
shown (see Ref. 4).
RNA. The concentration of free actinomycin D is far too low
to quench the fluorescence of RNA bound Tb3+.
Also in sentence 4 of their "Note" the authors say "the results
reported here showing no effect of AD-32 on terbium:poly(rG),
rRNA, and poly(dG dC) •¿
poly(dG •¿
dC) fluorescence
" As
far as we can see from Figs. 2 and 3 of this article (3), there
was an effect of AD-32 on Tb3* fluorescence.
We have shown quite explicitly, in this communication and
a previous one (4), that resonance energy transfer from Tb3+ to
drugs absorbing in the visible region is the main reason for
quenching of Tb3+ fluorescence irrespective of D/P ratio. Since
energy may be transferred from Tb3+ to doxorubicin molecules
situated as far as 13 basepairs away, Tb3+ fluorescence does not
tell anything about the "guanine-specificity" of anthracyclines.
Others have also shown that doxorubicin binds as well to A-T
as to G-C basepairs (10). Hence, we suggest that the results of
Trond Stokke
Department of Biophysics
The Norwegian Radium Hospital
Mon tebello
Oslo 3, Norway
References
1. Simpkins, H., Pearlman, L. F., and Thompson, L. M. Effects of Adriamycin
on supercoiled DNA and calf thymus nucleosomes studied with fluorescent
probes. Cancer Res.. ¿¥.-613-618,1984.
2. Simpkins, H., and Pearlman, L. F. The binding of actinomycin D and
Adriamycin to supercoiled DNA, single-stranded DNA and polynucleotides.
Biochim. Biophys. Acta, 783: 293-300, 1984.
3. Pearlman, L. F., Chuang, R. Y., Israel, M., and Simpkins, H. Interaction of
three second-generation anthracyclines with polynucleotides, RNA, DNA
and nucleosomes. Cancer Res., 46: 341-346, 1986.
4. Stokke, T., and Steen, H. B. Neither Adriamycin nor actinomycin D displaces
Tb3* from DNA. Biochim. Biophys. Acta, «25:416-418, 1985.
5. Gersanovski, D., Colson, P., Houssier, C., and Fredericq, E. Terbium(3+) as
a probe of nucleic acids structure. Does it alter the DNA conformation in
solution? Biochim. Biophys. Acta, 824: 313-323, 1985.
6. Widom, J. and Baldwin, R. L. Cation-induced toroidal condensation of DNA.
J. Mol. Biol., 144:431-453, 1980.
7. Stokke, T., and Steen, H. B. Multiple binding modes for Hoechst 33258 to
DNA. J. Histochem. Cytochem., 33: 333-338, 1985.
8. Horrocks, W. D., and Sudnick, D. R. Lanthanide ion probes in biology.
Laser-induced luminescence decay constants provide a direct measure of the
number of metal-coordinated water molecules. J. Am. Chem. Soc., 101:334340, 1979.
9. Muller, W., and Gautier, F. Interactions of heteroaromatic compounds with
nucleic acids. Eur. J. Biochem., 54: 385-394, 1975.
10. Graves, D. E.. and Krugh, T. R. Adriamycin and daunorubicin bind in a
cooperative manner to deoxyribonucleic acid. Biochemistry, 22: 3941-3947,
1983.
Reply
The initial report of Stokke and Steen (1) and the above letter
are interesting additional experimental data but suffer from
overinterpretation. The authors show that the addition of dox
orubicin depresses the phosphorescence decay curve of the
DNA-Tb3+ complex significantly. The resultant curve is not
parallel to that of the control as would be expected if displace
ment of Tb3* was the sole event occurring, indicating that
energy transfer must also occur. The observed effects on the
phosphorescence decay curve can be explained by: (a) energy
transfer (quenching) in solution from Tb3+ directly to the drug
[this has recently been shown to occur by Candida et al. (T)}
and/or (b) energy transfer from Tb3+ to the drug/polynucleotide
complex. The latter alternative assumes that the drug is bound
to the DNA within a short distance from the Tb3+ ion to enable
energy transfer to take place. In addition, displacement of Tb3+
Added in Proof (3) referred to process (a) which does not
involve drug binding to the polynucleotide.
It must be pointed out that displacement of Tb3* can occur
in the presence of energy transfer (quenching); however it is
impossible to determine unambiguously if this is the case by
phosphorescence decay measurements since the decay curves
will be parallel only if terbium displacement is the sole process
that occurs. If displacement and energy transfer are occurring
together, then the curves will not be parallel and it becomes
difficult to establish whether energy transfer is the sole expla
nation of the process or not. To resolve these questions, nonspectroscopic techniques must be employed. Equilibrium di
alysis has been employed to determine Tb3+ binding to poly
nucleotides (4) but has not been used to investigate the effects
of the anthracyclines on the terbium binding process.
Extrapolation of phosphorescence decay curves to zero time,
may also occur in addition to (a) and/or (b). Unfortunately it
was not clear in Stokke and Steen's original publication whether
as described by Stokke and Steen (1) and in the above letter, to
process (a) or (b) was supposed to occur and thus our "Note
determine if the curves extrapolate back to the same point are
also liable to error. The curves presented below commence at
Received 6/7/88, revised 8/15/88; accepted 8/23/88.
the first experimental point and are not arbitrarily extrapolated
6963
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LETTER TO THE EDITOR
back to the same point at zero time. Our results show that
doxorubicin does affect the Tb3+:DNA decay curve, as does
100.0J
(A)
actinomycin D to a lesser extent (Fig. 1). When poly(rG) was
employed, similar results were obtained with doxorubicin (Fig.
2A), but only a minor effect was observed with actinomycin.
This is predictable in light of actinomycin D nonreactivity with
ribonucleotides. Oligo(dG)4, when allowed to react with doxo
rubicin, also exhibited marked changes on the decay curve, but
not with actinomycin D (Fig. 2B) probably reflecting the pro
pensity of this drug to bind to —¿GpC—
sequences
The results with both poly(rG) and oligo(dG)4 are interesting
since, at the concentrations employed (25 fi\t), neither can form
a double helix. Additionally, it is difficult to imagine that
oligoUK. ).,will form any structure at all at this concentration.
In fact, Tb3+ fluorescence enhancement is maximal with this
length oligomer (4), an observation ascribed to the formation
of secondary structure with longer chain polynucleotides.
Poly(rG) at this concentration forms a stacked structure but
not a helix. Thus doxorubicin can alter the Tb3+ fluorescence
intensity and phosphorescence decay curve without intercalat
ing within a helical structure. This result is consistent with
results reported in our first publication (5) which noted a
decrease in Tb3+ fluorescence with the monomer, 5'-GMP.
Thus the statement made in the letter above "lack of quenching
... is of course due to the fact that actinomycin D does not
bind to RNA" does not hold true since displacement of terbium
or energy transfer from the nonbound drug could occur (or a
100.0
.6
8
t<j(msec)
Fig. 2. A, phosphorescence decay curves of poly(rG) (25 ^M) (O) treated with
Adriamycin for 30 min at 37'C at D/P of 1/500 (•),1/50 (»)and actinomycin
D at D/P of 1/500 (•)and 1/50 (A). The samples were then reacted with Tb'*
(50 JIM)for 15 min at 25"C. The spectroscopy was performed as described in Fig.
1. B, phosphorescence decay curves of oligo(dG)* (25 nM) (O) treated with
Adriamycin for 20 min at 37"C at D/P of 1/500 (•),1/50 (A), and actinomycin
D at D/P of 1/500 (•)and 1/50 (A). The samples were then reacted with Tb3*
(50 >iM).The spectroscopy was performed as described in Fig. 1.
mixture of the two). If Stokke and Steen's criterion is employed
(that the phosphorescence decay curve of the drug-treated polynucleotide must be extrapolated to zero time) and if this
calculated zero time phosphorescence is decreased compared
with that of the (non-drug-treated) control and they assume
Tb3'1"displacement has occurred, then it is hard to be certain
that the curves of the drug-treated samples presented in their
letter above and Figs. 1 and 2 must all extrapolate back to the
same point at zero time. In addition, it should be pointed out
Fig. I. Phosphorescence decay cune of DNA (50 <IM)(O) treated with Adrithat DNA-Tb3+ fluorescence and phosphorescence is approxi
amycin for 30 min at 37'C at D/P ratios of 1/500 (•).1/50 (»)and actinomycin
mately 2-4% ofthat observed with poly(rG) and oligo(dG)4 (4),
D at D/P ratios of 1/500 <•)
and 1/50 (A). The samples were then reacted with
Tb3* (100 fiM) for 15 min at 25'C. The gate time was 0.05 ms. the delay time
and thus liable to far greater error. Also since the Y axis of a
was varied between 0.01 to 2 ms. The excitation wavelength was 290 nm, the
decay curve is logarithmic, not linear in scale, then extrapola
emission wavelength was 544 nm. and the slit widths were 20 nm (emission) and
10 nm (excitation), respectively.
tion to zero time is subject to more error. The curve presented
6964
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LETTER TO THE EDITOR
in Fig. 2B for oligo(dG)4 treated with actinomycin D at a D/P
of 1/500 lies above the curve of the nontreated oligomer,
subsequent experiments resulted in curves slightly above and
slightly below the control. This datum is presented to show that
both fluorescence and phosphorescence measurements are sub
ject to some degree of experimental error.
Thus it is difficult to determine precisely what processes are
occurring. Certainly energy transfer takes place, but in view of
the experiments above it is not clear whether this is the sole
explanation, and displacement of Tb3+ may also occur to a
greater or lesser extent. As previously suggested, other, nonspectroscopic techniques will have to be employed to unravel
this problem. It is evident that more than one process is involved
and the statement made in our initial publications, that doxorubicin displaces terbium, was an oversimplification of the
fluorescence intensity data. Sophisticated techniques such as
phosphorescence decay show the process to be more complex.
However, the same criticism can be leveled at the interpretation
of phosphorescence decay data. This holds true in the above
letter where a calculation was made as to the distance between
Tb3+ and the drug binding site on the polynucleotide (32 A).
its second generation derivatives [including 3'-deamino-3'-(3cyano-4-morpholinyl)doxorubicin],
an anthrapyrazole, and ac
tinomycin D were compared to the relative decrease in Tb3+
This calculation must make an assumption as to the orientation
factor K, which is dependent on whether the energy acceptor
and donor have complete rotational freedom (one value can be
calculated) or if one is fixed (the value now ranges from 0.33
to 1.33 depending on the angle between the transition moments
of the acceptor and donor) (6). Thus, whether this calculation
is meaningful for an elongated and highly charged polynucleo
tide can be disputed especially since actinomycin D has base
specificity. Therefore the drugs may not bind randomly along
the DNA chain and when they do bind, are they fixed or not
with respect to Tb3+? In addition, energy transfer from Tb3+ to
described by Gross and Simpkins (4).
H. Simpkins
M. Figliomeni
L. Pearlman
Department of Pathology
Staten Island Hospital and
Department of Pathology
State University of New York (Downsîate)
Brooklyn, New York, 11203-2098
the nonbound drug cannot be ignored and will affect the cal
culation in an ill-defined manner.
We must also answer the postulate in the above letter that
doxorubicin has AT base specificity. The author's rationale is
that methyl green, an apparently AT-specific drug (7), results
in a similar decrease in fluorescence intensity to that produced
by doxorubicin. However, the decrease in fluorescence reported
with methyl green is 9% at a D/P of 1/500, whereas a decrease
of approximately 30% was reported with Adriamycin. Also at
a high D/P ratio (1/50), quenching by the nonbound drug
becomes important, and any decreases in fluorescence intensity
at this drug concentration may not solely reflect drug binding
to the polynucleotide. Secondly, the authors ignore the wide
spread literature which suggests that doxorubicin may exhibit
slight guanine sensitivity (8) and more precisely that actino
mycin D has —¿GpC—
specificity (9, 10). In both our publica
tions (3, 5) we included the possibility that the drug could react
with AT regions proximal to guanine residues.
Another point made in the original publication of Stokke and
Steen (1) was that drugs with absorption spectra in the 500600-nm region will quench terbium fluorescence. This means
that the degree of spectral overlap with Tb3+ should be related
to the decrease in fluorescence intensity observed with each
drug. When the relative absorbance at 544 nm of doxorubicin,
fluorescence that each produced, no correlation was observed.
In addition, our initial results with ethidium bromide (5), which
are misquoted by Stokke and Steen (1), showed that this inter
calating dye did not affect Tb3+-DNA fluorescence but does
absorb in this region.
In conclusion, our initial reports which attributed the de
crease in Tb3+ fluorescence intensity to displacement of terbium
were reasonable at the time they were published, since phos
phorescence decay instrumentation was not then commonly
available. It is now clear employing this more sophisticated
technique that the process is more complex. However, phos
phorescence decay alone cannot differentiate between the many
different processes which may bring about a decrease in terbium
fluorescence. It is our feeling that the only means by which the
interaction of doxorubicin and its derivatives with Tb3+ and
polynucleotides can be studied and the results interpreted un
ambiguously is by employment of additional, nonspectroscopic
techniques such as equilibrium dialysis employing 160Tb3+as
M. Rosen
Department of Biology
City University of New York
Staten Island, New York
References
1. Stokke, T., and Steen, H. B. Neither Adriamycin nor actinomycin-D displaces
Tb3* from DNA. Biochem. Biophys. Acta, 825: 416-418, 1985.
2. Canada, R. G., Saway, W., and Thompson. E. Interaction of Adriamycin
with a calcium binding site. Biochem. Biophys. Res. Commun., ¡51:679685, 1988.
3. Pearlman, L. F., Chung, R. Y., Israel, M., and Simpkins. H. Interaction of
three second-generation anthracyclines with polynucleotides. RNA, DNA,
and nucleosomes. Cancer Res., 46: 341-346, 1986.
4. Gross, D. S., and Simpkins, H. Evidence for two-site binding in the Terbium
(III)—Nucleic Acid interaction. J. Biol. Chem., 256: 9593-9598, 1981.
5. Simpkins, H., Pearlman, L. F., and Thompson, L. M. Effects of Adriamycin
on supercoiled DNA and calf thymus nucleosomes studied with fluorescent
probes. Cancer Res., 44: 613-618, 1984.
6. Wu, C. W., and Stryer. L. Proximity relationships in Rhodopsin. Proc. Nati.
Acad. Sci. USA, 60: 1104-1108, 1972.
7. Muller, W., and Gautier, F. Interactions of heteroaromatic compounds with
nucleic acids. Eur. J. Biochem., 54: 385-394, 1975.
8. Duvernay. V. H., Pachter. J., and Crooke, T. S. Deoxyribonucleic acid
binding studies on several new anthracycline antitumor antibiotics. Sequence
preference and structure activity relationship of marcellomycin and its ana
logues as compared to Adriamycin. Biochemistry', 18:4024-4030. 1979.
9. Wilkins, R. J. Selective binding of actinomycin-D and distamycin A to DNA.
Nucleic Acid Res., 10: 7273-7282, 1982.
10. Lane, M. J., Dabrowiak, J. C., Vournakis, J. N. Sequence specificity of
actinomycin-D and Netropsin binding to pBR322 DNA analyzed by protec
tion from DNase 1. Proc. Nati. Acad. Sci. USA, 80: 3260-3264, 1983.
6965
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.
Correspondence re: L. F. Pearlman et al., Interaction of Three
Second-Generation Anthracyclines with Polynucleotides, RNA,
DNA, and Nucleosomes. Cancer Res., 46: 341−346, 1986−−Reply
H. Simpkins, M. Figliomeni, L. Pearlman, et al.
Cancer Res 1988;48:6963-6965.
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