[CANCER RESEARCH 35, 2921 2927, November 1975]
Enhanced Cell Killing by Bieomycin and 43 ~ Hyperthermia and the
Inhibition of Recovery from Potentially Lethal Damage
Jonathan Braun and George M. Hahn
Department of Radiology, Stanford University School o[ Medicine, Stan/ord, Cali[ornia 94305
SUMMARY
The effect of hyperthermia on bleomycin (BLEO) toxicity
and repair was studied in -unfed" monolayer cultures of
Chinese hamster cells. Synergy of toxicities was observed
with simultaneous exposure to BLEO and 43 ~ For exampie, when cells were exposed for I hr to BLEO (40 ug/ml) at
43", survival was reduced to 4 • 10-s; separately, hyperthermia and 37 ~ BLEO exposurc each resulted in a survival
of 20%. Heating at 43" prior to drug exposure at 37 ~ also
produced substantial sensitization, indicating that the primary sensitizing effect involved cell damage rathcr than an
increased rate of drug action: 41 ~ produced only modest cell
sensitization to BLEO and the effect was not retained in
cells heated prior-to drug exposure.
No increasc in [x4C]BLEO uptake was observcd at 43 ~
over that at 37 ~ and thus the increased cytotoxicity was not
correlated with a gross change in cell permeability to
BLEO, although increased drug availability to particular
sensitive targets could not be ruled out. Studies of the
repair kinetics alter different 43 ~ BLEO protocols demonstrated that most of the cells sustaining potentially lethal
damage rapidly rccovered. However, 43 ~ hyperthermia inhibited this recovery and, with increasing durations of 43 ~
exposure, the fixation of potentially lethal damage was
enhanced. Because of the substantial repair of BLEO
damage observed in vivo, the possible usefulness of hypcrthermia as an adjunct to BLEO therapy is discussed.
INTRODUCTION
Recently, a great deal of activity has centered on the
potential of hyperthermia (temperatures above 37 ~ as an
adjunct modality in cancer therapy. Increased heat sensitivity of malignant over normal tissue has been observed in
several instances (6, 36). In vitro, selective heat sensitivity
has also been observed (11, 23, 24, 34), and was recently
quantified for normal and SV40-transformed human fibroblasts (20). However, this response may not be universal
(19, 31).
Cell sensitivity to hyperthermia has been related to the
effects of growth phase, nutritive state, and ambient medium in Chinese hamster cells in vitro (12). Plateau phase
cultures were found to be more heat sensitive than exponen1This work was supported by Grants CA-04542, CA-10372, and
CA- 15769.
Received April 21, 1975; accepted July 17, 1975.
tial cultures, with the sensitivity increasing in the order:
exponential < unfed plateau < fed plateau (in fed plateau
cultures, the medium is exchanged daily as cells approach
confluence). Recovery from heat damage proceeded rapidly
(Tv, --~30 rain in full medium) in plateau cultures, but its
magnitude in unfed cultures was greatly reduced (G. Hahn,
E. Shiu, G. Li, and J. Braun, unpublished data).
Exactly how mild hyperthermia kills cells has not been
established with certainty. Protein damage has received the
greatest amount of interest, and a significant amount of
evidence has mounted in its favor. In Chinese hamster ovary
cells, an Arrhenius plot of rate of cell killing versus
temperature-x yielded a cell inactivation energy of 140,800
cal/mole, a value which corresponds to denaturation energy
for several proteins (42). Other workers observed 185,000
cal/mole inactivation energy for Chinese hamster V-79
cells, which was also explained in terms of protein denaturation (18).
Combined toxicity of heat and drugs has been reported
for several agents and may involve both an increase of initial
damage and an inhibition of repair. In exponential cultures of Chinese hamster V-79 cells, rate constants for
tris(aziridinyl)phosphine sulfide toxicity increased regularly for both the drug concentration and temperature of
the drug exposure (18), presumably reflecting an increase
in the reaction rate for alkylation. Increased drug toxicity
at elevated temperatures, due either to additive or synergistic interactions, was also observed for cordccypin, 2mercapto- l-(13-4-pyridethyl)benzimidazole, I,-erythro-c~,C~dihydroxybutyraldehyde (10, 32), actinomycin D, t,-phenylalanine mustard (10, 35), DL-glyceraldehyde, and
sodium oxamate (10).
We have studied the combined effect of BLEO 2 and
hyperthermia on cell survival. BLEO, a complex glycopeptide, has proven valuable in the treatment of squamous cell
carcinoma, lymphomas, and testicular carcinoma (4), perhaps as a result of its relative specificity for noncycling cells
(2, 14, 39). However, in vitro cell survival studies have
shown that it elicits a biphasic response for both dose (1, 2)
and duration of exposure (1, 2, 39), so that its range of effective drug concentration is limited. In addition, recovery
of cells of the EMT-6 tumor in vivo has been observed
following treatment with B I E O (16), and this involves
primarily noncycling cells (14).
For these reasons we became interested in different
methods which might enhance BLEO toxicity and inhibit
2The abbreviation used is: BLEO, bleomycin.
NOVEMBER 1975
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2921
J. Braun and G. M . Hahn
the recovery of cells from BLEO damage. Studying hyperthermia in this regard, we found that in Chinese hamster
ceils exposed to BLEO killing proceeded much more rapidly
at 43 ~ than at 41 ~. This finding was difficult to explain on
the basis of changes in activation energies, and it therefore
suggested a qualitative difference in cell response at these
temperatures (13).
We describe in this paper the response of cells to different
sequences of 43 ~ and BLEO exposure, and detail the
kinetics of cell recovery from the damage produced by the
combined action of these agents. Our conclusion is that
inhibition of cell recovery by hyperthermia probably represents a major cause of the enhanced toxicity that we
observed after the combined action of BLEO and 43 ~
hyperthermia.
solution and allowed to dry. The bottoms of the plastic
dishes were then punched out, and the radioactivity of the
retained [~4C]BLEO was determined by a planchet-type
gas-flow counter.
RESULTS
Dose and Time Response. 3 The survival for unfed cells
was determined for various drug concentrations at 37 ~, 41 ~,
and 43 ~ (Chart 1). The results, normalized to controls that
had been exposed to the appropriate temperature in the
absence of drug, show that the rate of cell killing increased
appreciably between 41 ~ and 43 ~ One hr of exposure at 43 ~
in the absence of BLEO prior to drug exposure at 37 ~ also
produced a substantial degree of sensitization. In contrast,
unfed cells preheated for 1 hr at 41 ~ did not retain
sensitization (data not shown).
M A T E R I A L S AND M E T H O D S
To determine whether cytotoxicity at 43 ~ was due to
Cell Culture. HA 1 cells, a pseudodiploid Chinese hamster damage similar to that responsible for heat toxicity itself,
cell line not known to be neoplastic, were regularly screened cells were preheated for 1 hr at 43 ~ and then incubated for 1
for Mycoplasma and grown in monolayers as previously hr at 37 ~ as a "heat repair" period (in this paper, we dedescribed (15). " U n f e d " cultures were obtained by seeding fine repair as the increase of survival observed when cells
105 cells into 60-mm Petri dishes and were used on Day 8. are allowed to incubate at 37 ~ for a period of time between
The cell density was approximately 3 • l0 b cells/sq cm; no treatment and the stimulation of cell proliferation by
significant change in cell number was observed after Day 6. trypsin). After the heat repair period, the cells were exposed
The culture consisted primarily of G~-like cells (15), al- at 37 ~ to various drug concentrations (Chart 1, A). Repair
though up to 20% of the cells may have been in G2; the of heat damage under these conditions is essentially complete within 1 hr after hyperthermia. However, the degree of
plating efficiency on the 8th day was approximately 60%.
Unfed cultures are sensitive to stimulation by fresh sensitization was unaffected by this period. Thus, it was
medium, and heat sensitivity varies markedly with the type unlikely that the combined effect was related only to the
of medium in which the cells are exposed (12). All type of damage responsible for heat toxicity.
The survival of cells to a fixed dose (20 #g/ml) but
experiments reported were performed with depleted medium collected from replicate unfed cultures and passed variable time of BLEO exposure was measured at 37 ~ and
43 ~ (Chart 2). Again we observed that the biphasic part of
through a 0.22-#m filter prior to use.
BLEO and Hyperthermia. Concentrated solutions of the BLEO response was less noticeable at the higher
BLEO (Blenoxane; Bristol Laboratories, Syracuse, N. Y.) temperature.
Preheating Response. To determine the dependence of
were prepared in Dulbecco's phosphate-buffered saline
(Grand Island Biological Co., Santa Clara, Calif.), and sensitization on the duration of 43 ~ exposure, we heated
dilutions were prepared in depleted medium within 6 hr of cells for various periods of time immediately prior to a fixed
use. Unless otherwise indicated, BLEO exposures were for 1 drug dose (50 #g/ml) at 37 ~ (Chart 3) and then assayed for
hr, following which the cells were rinsed twice with phos- clonogenicity. Results were normalized to unheated control
groups; thus, if no sensitization occurred, the BLEO
phate-buffered saline.
Heat exposures were carried out in specially designed response would coincide with the heat toxicity. However,
water baths which retained the temperatures +0.1 ~ in a 95% preheating and BLEO reduced cell survival geometrically
air-5% CO2 atmosphere (12). Three different sequences of with the duration of heat exposure. At 41 ~ we also observed
heat and drug exposure were used: (a) exposure at 43 ~ slight sensitization, which developed only over a period of
without drug, followed by exposure to BLEO at 37 ~ several hr (data not shown).
[~4C]BLEO Uptake. The sensitization described in the
("preheating"), (b) exposure to BLEO at 37 ~ followed by
exposure at 43 ~ without drug ("postheating"), and (c) previous sections could have involved hyperthermic ensimultaneous exposure to both agents.
Duplicate dishes were used at each experimental point.
s Surviving fraction is defined as:
Cell survival was measured using Puck's cloning assay.
[14C]BLEO. [~4C]BLEO A-2 (Nippon Kayaku Co., Ltd.,
(Plating efficiency of experimental group)
Tokyo, Japan) was obtained at a specific activity of 38.59
(Plating efficiency of untreated control).
#Ci/mg. The identical procedure for unlabeled BLEO
exposure was used in the [~4C]BLEO experiments, except Surviving fraction ratio is defined as:
that 2.5 rather than 5.0 ml of the [14C]BLEO-medium
(Plating efficiency of experimental group)
solution were added to each dish. After [I~C]BLEO expo(Plating efficiency of specially designated experimental group).
sure, cultures were rinsed 3 times with Hanks' balanced salt
2922
CANCER RESEARCH VOL. 35
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Bleomycin and Hyperthermia
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were incubated at 37 ~ for 16 hr following the heat and drug
exposure (Chart 3). The survival shoulder was extended by
repair; however, the same final rate of cell killing was
observed both with or without the repair interval, suggesting
that durations of exposure at 43 ~ in excess of about 45 min
inhibited repair.
In an attempt to distinguish between sensitization and
repair inhibition by 43 ~ h y p e r t h e r m i a , we exposed cultures
at 37 ~ to B L E O (50 # g / m l ) , followed i m m e d i a t e l y by
various durations of 43 ~ exposure. Replicate dishes were
subcultured to d e t e r m i n e survival i m m e d i a t e l y after the
drug-heat exposure, or after an additional 16 hr of incubation at 37 ~ (Chart 5).
Recovery by cells exposed to heat alone was observed in
this experiment, suggesting an inconsistency with the apparent lack of such repair observed in the corresponding
preheating experiment ( C h a r t 3). However, initial survival
in the postheating e x p e r i m e n t was lower than in the
preheating experiment, reflecting the fact that, in the
former, survival was assayed i m m e d i a t e l y after heat exposure while, in the latter, the period of drug exposure
following the heat exposure provided a l-hr repair interval
for the heat controls (which were incubated in drug-free
medium). The heat control results were therefore consistent
with the rapid rate of repair of heat-induced d a m a g e noted
in the introduction.
k
BLEO (~g/ml)
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C h a r t 1. B L E O dose response for 1 hr of exposure at 37 ~ (O), 41 ~ (11),
and 43 ~ (O). One hr of preheating at 43 ~ followed by a period of
incubation at 37~, and then by 1 hr of exposure to BLEO (37~ i , no 37~
incubation; A, 1 hr of incubation at 37~ All values normalized to heat
toxicitics. Verticallines, • 1 S.E. for 5 replicate dishes.
h a n c e m e n t of cell permeability to BLEO. We therefore
r e e x a m i n e d the dose response experiments described in the
previous section but used [I4C]BLEO. The cultures used in
these labeling experiments were treated in a fashion similar
to those of the corresponding survival experiments except
that, after rinsing, the labeled groups were not trypsinized
for the survival assay but instead were allowed to dry so
that the intracellular [14C]BLEO could be d e t e r m i n e d
(Chart 4). In all groups, uptake was a linear function of
drug concentration. Surprisingly, the group exposed at 37 ~
consistently retained more drug than the groups exposed
to 43 ~ . In addition, the group preheated at 43 ~ prior to
B L E O exposure retained less drug than the group exposed simultaneously to 43 ~ and BLEO. Therefore, we
find no correlation of B L E O uptake with survival under
these various conditions.
Repair. As previously reported (33, 38), repair of potentially lethal B L E O d a m a g e is observed with plateau phase
cells (Chart 6). We first attempted to inhibit B L E O repair
by heating cells to 41 ~ i m m e d i a t e l y following a 37 ~ B L E O
exposure. However, B L E O repair occurred even during the
heat " b l o c k , " proceeding at 41~ as rapidly as at 37 ~ (data
not shown).
In order to determine the extent of repair of the d a m a g e
produced by 43 ~ preheating and BLEO, replicate groups
NOVEMBER
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EXPOSURE IN BLEO (MIN)
Chart 2. Time response for BLEO (20 ~g/ml): 37~ (Q) and 43 ~ (O).
All values normalized to corresponding heat toxicities.
1975
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2923
J. Braun and G. M . H a h n
consider the i m p o r t a n c e of each factor in determining cell
survival.
A simple explanation for 43 ~ sensitization involves the
possible increase of cell permeability to B L E O . Effects of
h y p e r t h e r m i a on m e m b r a n e permeability have been shown
in several e x p e r i m e n t a l systems (5); the suggestion that cell
permeability m a y be a limiting factor in B L E O toxicity was
m a d e previously (7, 39) and has received support f r o m
several lines of evidence. C h a r a c t e r i z a t i o n of a BLEO-sensitive m u t a n t strain of Escherichia coli correlated the phenotype with an increased rate of drug u p t a k e (30, 43); the
g r e a t e r toxicity of B L E O against mouse s q u a m o u s cell
c a r c i n o m a versus mouse skin s a r c o m a (both induced by
m e t h y l c h o l a n t h r e n e ) was correlated with a higher rate of
drug u p t a k e by the c a r c i n o m a (although this sensitivity also
paralleled reduced activity for a B L E O - i n a c t i v a t i n g enzyme) (41).
The m e a s u r e m e n t s reported in this paper indicate that no
gross change in cell permeability can be correlated with the
toxicity produced by exposure to B L E O and 43 ~ However,
this observation does not exclude the possibility that the
change in permeability is more localized. An illustration of
this point was shown in the differential effects of various
polyene antibiotics on B L E O - i n d u c e d inhibition of m a c r o -
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75
90
E X P O S U R E A T 43 ~ (MIN)
140
Chart 3. Response to preheating at 43 ~. Cells were heated at 43 ~ for
the periods of time indicated on the abscissa and then were exposed to
BLEO (50 #g/ml) for 1 hr at 37~ Survival immediately after BLEO: O,
control (no drug); A, BLEO. Survival after an additional 17-hr incubation
period at 37~ O, control; A, BLEO. Results normalized to corresponding
unheated groups.
120
r-
S h o r t d u r a t i o n s of exposure at 43 ~ produced an additive
effect on both initial B L E O survival and on survival after
the repair interval, i.e., the normalized heat-drug response
coincided with the response to heat alone. However, postheating for periods longer than ~ 6 0 min reduced the
m a g n i t u d e of repair, and after 100 min of postheating,
repair was essentially abolished.
To c o m p a r e pre- and postheating sensitization, we determined the kinetics of repair following 40- or 80-rain
exposures at 43 ~ ( C h a r t 6), In both cases, the preheated
groups sustained a substantially larger initial toxicity;
however, the preheated groups rapidly recovered from the
lower survival level, so that their final survival closely
corresponded to the survival of the corresponding postheated cells.
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DISCUSSION
This report has considered 3 aspects involved in the
combined action of heat and B L E O : (a) the effect of 43 ~ on
cell permeability, (b) the qualitative difference between 41 ~
and 43 ~ on cell sensitization to B L E O , and (c) the kinetics
of repair observed after the heat and drug exposure. We now
2924
1
I
I
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I
2
4
6
8
10
BLEO (,ug/ml)
Chart 4. Dose response for [~4C]BLEO. Cells were exposed for l hr to
2.5-ml aliquots of various ['4C]BLEO concentrations, then were rinsed 3
times with Hanks' balanced salt solution: cpm/dish were determined with
planchet-type gas flow counter. Uptake at: 37 ~ (Q), 43 ~ (O); l hr
preheating at 43 ~ followed by 37~ [14C]BLEO (&). Each point represents
average of duplicate dishes.
C A N C E R R E S E A R C H VOL. 35
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Bleomycin and Hyperthermia
:
1
10--1
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10-3
action of this drug. Cell killing by B L E O perhaps involves
strand scission (9, 17, 26, 28), and this action is unique in
that it appears to be dependent on a specific c o n f o r m a t i o n
of the B L E O molecule about the D N A substrate (27, 37).
A l t h o u g h h y p e r t h e r m i a m a y increase the reaction rate,
different factors related to drug action m a y p r e d o m i n a t e
at different t e m p e r a t u r e s . The 43 ~ p r e h e a t i n g e x p e r i m e n t
d e m o n s t r a t e d that sensitization involved a lesion that was
not rapidly repaired (cf. C h a r t 1), while at 41 ~ t h e r e was no
evidence of the persistence of sensitizing d a m a g e . Thus, the
response of cells to B L E O at 41 ~ m a y p r i m a r i l y involve
e n h a n c e m e n t of the reaction rate for drug d a m a g e , while the
response at 43 ~ seems to depend m a i n l y on a different sort
of heat effect.
R e c o v e r y after BLEO exposure described here and
elsewhere (16, 33, 38) involves a repair m e c h a n i s m that is
p r o b a b l y e n z y m a t i c (25, 38, 40), and we have seen that
repair m a y be t e m p o r a r i l y or p e r m a n e n t l y blocked by
increasing d u r a t i o n s at 43 ~ . Suppose that cell survival
depends on the c o n t i n u o u s function of this repair mecha-
10-4
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EXPOSURE AT 43 ~ (MIN)
1
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Chart 5. Response to 43 ~ postheating. Cells were exposed to BLEO (50
ug/ml) for I hr at 37~ followed by 43 ~ postheating for the durations
indicated on the abscissa. Survival immediately after 43~ O, control (no
drug); I , BLEO. Survival after additional 16-hr incubation period at 37~
O, control; F1, BLEO. Results normalized to corresponding unheated
group.
molecular synthesis. These antibiotics all d e m o n s t r a t e a
selective affinity for sterols in the m e m b r a n e (3, 21) and, as
a result, they reduce m e m b r a n e integrity. O f the drugs
tested, however, synergy of p e n t a m y c i n and B L E O was
observed (29) while no such interaction was observed with
a m p h o t e r i c i n B (22). In addition, a u t o r a d i o g r a p h y was
recently used to suggest that the integrity of the nuclear
m e m b r a n e as a drug barrier might d e t e r m i n e cell sensitivity
to B L E O (8).
Perhaps the most interesting feature of the results we
have described involves the qualitative difference between
drug toxicity at 41 ~ and 43 ~. U n l i k e previous reports of
additive heat-drug toxicities at 42 ~ (10, 32) or of an
interaction yielding a linear Qto relationship, and thus no
qualitative change in the nature of the lethal event(s) (18),
the 43 ~ sensitization which we observed with B L E O is of
such great m a g n i t u d e that when c o m p a r e d to 41 ~ sensitization it suggests a novel h y p e r t h e r m i c effect. The possible
synergistic toxicity of heat with L-erythro-c~,/3-dihydroxy b u t y r a l d e h y d e (10) and a d r i a m y c i n (13) has also been
observed; however, the c o n t r a s t i n g actions of these drugs
m a k e it difficult to infer that in each case the same
sensitizing lesion is involved.
The effects of h y p e r t h e r m i a on the response of cells to
B L E O is p r o b a b l y due in part to the unusual mode of
NOVEMBER
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TiME BETWEEN T R E A T M E N T A N D SUBCULTURE (H)
Chart 6. Effect of heat-drug sequence on the kinetics of repair.
Recovery from potentially lethal damage was determined during incubation at 37~ after the following treatments: O, control (no heat); A, 40 rain
of preheating at 43~ followed by BEEO exposure; ZX, 80 min of preheating
at 43 ~ followed by BLEO; I , BLEO exposure followed by 40 min of
postheating at 43~ D, BLEO exposure followed by 80 rain of postheating
at 43 ~ All groups were exposed to BLEO (50 #g/ml) for 1 hr at 37~
exposure. Zero hr indicates start of 37~ repair period. All results are
normalized to corresponding 37~ plating efficiencies.
1975
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Research.
2925
J . B r a u n a n d G. M . H a h n
n i s m d u r i n g a n d a f t e r d r u g e x p o s u r e ; then, i n h i b i t i o n of the
p r o t e c t i v e f u n c t i o n of the r e p a i r m e c h a n i s m at 43 ~ w o u l d
offer an e x p l a n a t i o n of the 43 ~ s e n s i t i z a t i o n . S u c h a
m e c h a n i s m c a n n o t be d e t e r m i n e d d i r e c t l y f r o m survival
e x p e r i m e n t s . By d e f i n i t i o n , we m e a s u r e r e p a i r as the
i n c r e a s e in survival w h i c h follows d r u g e x p o s u r e ; the
m a g n i t u d e of the p r o t e c t i v e role of the r e p a i r s y s t e m d u r i n g
e x p o s u r e is difficult to assess.
H o w e v e r , the m a r k e d d r o p in survival o b s e r v e d in
B L E O - t r e a t e d cells p o s t h e a t e d at 43 ~ for p e r i o d s of exposure l o n g e r t h a n a b o u t 1 hr ( C h a r t 5) m a y reflect the
c o n v e r s i o n of p o t e n t i a l l y l e t h a l lesions into lethal lesions
due to the h y p e r t h e r m i c i n h i b i t i o n of such a p r o t e c t i v e
f u n c t i o n . T h i s e x p l a n a t i o n is s u p p o r t e d by the o b s e r v a t i o n
t h a t the survival level a f t e r the c o m p l e t i o n of r e p a i r does not
seem to d e p e n d on the s e q u e n c e of h e a t a n d d r u g e x p o s u r e .
T h e effects of 43 ~ on cell r e c o v e r y f r o m B L E O d a m a g e
m a y be s u m m a r i z e d by o p e r a t i o n a l l y d e f i n i n g 2 t y p e s of
d a m a g e ( C h a r t 7): (a) a h y p e r s e n s i t i v e r e s p o n s e p r o d u c e d by
p r e h e a t i n g at 43 ~ , w h e r e survival of s t i m u l a t e d cells is
g e o m e t r i c a l l y d e p e n d e n t on h e a t d u r a t i o n , but repair is
efficient a n d relatively r e s i s t a n t to h e a t e x p o s u r e , and (b) a
t h r e s h o l d r e s p o n s e p r o d u c e d by b o t h h e a t i n g sequences and
o b s e r v e d c l e a r l y with 43 ~ p o s t h e a t i n g . I n c r e a s e d c o m b i n e d
cell k i l l i n g occurs w h e n the d u r a t i o n of 43 ~ exceeds 1 hr. It
is p a r a l l e l e d by the i n h i b i t i o n of r e p a i r a c t i v i t y which is
n e a r l y a b o l i s h e d by a 9 0 - m i n e x p o s u r e to 43 ~ As a model,
this s u m m a r y implies t h a t the final survival level for b o t h
g r o u p s is d e t e r m i n e d o n l y by the t h r e s h o l d response.
T h e relevance of our f i n d i n g s to cells in v i v o is n e c e s s a r i l y
subject to the l i m i t a t i o n s of an a r t i f i c i a l m o d e l . H o w e v e r ,
t h e i m p o r t a n c e of the i n h i b i t i o n o f B L E O r e p a i r at 43 ~ was
r e c e n t l y e m p h a s i z e d by a p r e l i m i n a r y s t u d y of the c o m b i n e d
effect of 43 ~ and B L E O on the survival o f cells f r o m the
E M T - 6 t u m o r t r e a t e d in v i v o . Cells f r o m t u m o r s excised 2
[-~
~
.
.
.
.
.
.
-,,..
M
Chart 7. Summary of combined heat and drug action. Viable cells (V)
initially enter an intermediate state immediately after exposure (-,) to
either 43 ~ (H) or BLEO (B). Exposure to the other agent places cells of the
1st intermediate state into a 2rid intermediate state (HB or BH) which is
unique to the sequence of agents which is used: thus, state HB is not
equivalent to state BH. However, cells in state HB efficiently repair (--,)
hypersensitive damage and thus enter to state BH. In contrast, repair of
threshold damage is inefficient and decreases with increasing durations of
exposure at 43 ~.
2926
hr a f t e r d r u g injection s h o w e d only additive h e a t and drug
cell killing. H o w e v e r , cells f r o m t u m o r s excised 24 hr l a t e r
s h o w e d no increase in survival; the r e p a i r seen in the
u n h e a t e d c o n t r o l ( f r o m 10 a survival to 50% at a dose of 40
m g / k g ) was c o m p l e t e l y b l o c k e d by h e a t i n g (G. H a h n , L.
G o r d o n , a n d D. K o z a k , u n p u b l i s h e d data).
T h u s w h a t we have called t h e r m o c h e m o t h e r a p y m a y offer
the p o t e n t i a l for s u b s t a n t i a l l y i n c r e a s i n g the t u m o r - t o - n o r m a l tissue l e t h a l i t y of isolated t u m o r s . W h a t is required is
the d e v e l o p m e n t of p r a c t i c a l units for t u m o r h e a t i n g in s i t u
b e f o r e clinical use of these f i n d i n g s can be s e r i o u s l y pursued.
ACKNOWLEDGMENTS
We wish to thank Dr. T. Terasima for his assistance in obtaining
[14C]BLEO, and Terry S. Marshall for her patient and excellent help in
preparing this manuscript.
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2927
Enhanced Cell Killing by Bleomycin and 43° Hyperthermia and
the Inhibition of Recovery from Potentially Lethal Damage
Jonathan Braun and George M. Hahn
Cancer Res 1975;35:2921-2927.
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