[CANCER RESEARCH 48, 2101-2106, April 15. 1988)
Effects of Hyperthermia on Cell Survival and Patterns of Protein Synthesis in
Endothelial Cells from Different Origins1
Nika V. Ketis,2 Richard L. Hoover, and Morris J. Karnovsky
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
ABSTRACT
Thermotolerance, transient resistance to heat induced by heat itself, is
generally thought to be linked to the accumulation of heat-shock proteins
in eukaryotic cells. The induction of thermotolerance and the synthesis
of heat-shock proteins in primary and passage cultures of bovine aortic
endothelium, passage cultures of bovine brain capillaries, and passage
cultures of rat epididymal capillaries were examined. Primary and pas
sage cultures of bovine aortic endothelial cells readily acquired thermo
tolerance; however, passage cultures of rat epididymal capillary cells and
bovine brain capillary cells were very heat sensitive. In all endothelial
cell types examined except rat epididymal capillary cells, the levels of
HSP71, the most inducible of the HSP70 family, correlated well with
thermotolerance. With prolonged passage, rat epididymal capillary cells
and bovine braincapillary cells lost their ability to acquire heat resistance.
Endothelial cells from different origins (aortic endothelium versuscapil
lary endothelium) but from the same species and about the same passage
number had a notably different response in terms of thermotolerance and
synthesis of proteins after exposure to hyperthermia. The results of this
study suggest that, while the expression of HSP71 may be a good
indicator of heat resistance, the reverse is not necessarily true. Further
more, the data show that endothelial cells from different origins are
dissimilar in their response to hyperthermia.
INTRODUCTION
The cells of most eukaryotes respond to mild heat treatment
by the induction of a set of proteins termed the HSPs.3 In
addition, these proteins are induced as a result of cellular
responses to other stresses (1, 2), such as glucose deprivation
and anoxia. In Drosophila, where the response was first re
ported, the induction process has been studied extensively (3,
for review). In these cells, the HSPs appear to have different
induction characteristics, and the controls exerted on both
transcription and translation are regulated to ensure that HSPs
are produced as rapidly as possible.
The commitment to heat-shock protein synthesis is fully
reversible when Drosophila cells (4) and other eukaryotic cells
(5) are returned to their normal temperatures. Translation and
transcription gradually return to their prestressed state (6). A
search for regulatory mechanisms controlling the expression of
heat-shock proteins at both the transcriptional and posttranscriptional level is providing present areas of active research.
In view of the physiological and pathological "stress" to
which endothelial cells are subjected, such as shear forces,
hypoxia, hypertension, acute inflammation, angiogenesis, and
neoplasia, the study of the expression and regulation of HSPs
Received 6/26/86; revised 10/22/86, 8/10/87, 12/18/87; accepted 1/20/88.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Grants HL-17747 and HL-26191 from the NIH.
Part of this work was presented at the Heat Shock Meeting at Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY, on August 29, 1985, and at the
American Society for Cell Biology Annual Meetings, St. Louis, MO, on Novem
ber 20, 1987, and appeared in abstract forms (20, 21).
1 Recipient of a fellowship from the Medical Research Council of Canada.
3The abbreviations used are: HSP, heat shock protein; BAEC, calf aortic
endothelial cells; REEC, rat epididymal endothelium; BBCE, calf brain capillary
endothelium; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid; SDS,
sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; IEF, isoelectric focusing.
in these cells is of great interest. To further understand the
biology of a mammalian cell that may undergo physiological
and pathological "stress," the synthesis of HSPs in endothelial
cells from different origins (primary and passage cultures of
bovine aortic endothelium, passage cultures of bovine brain
capillaries, and the passage cultures of rat epididymal capillar
ies) was examined by polyacrylamide gel electrophoresis after
cells had been exposed to various temperatures and durations
of heat stress. The results in this study suggest that endothelial
cells from different origins are dissimilar in their response to
hyperthermia.
MATERIALS AND METHODS
Endothelial Cells and Cultures. BAEC were isolated according to
methods described by Booyse et al. (7). When passage cells were desired,
primary endothelial cells were cloned, passaged, and grown in Dulbecco's modified Eagle's medium with 5% fetal bovine serum and 5% NuSerum (Collaborative Research, Waltham, MA) plus antibiotics—pen
icillin, 100 Mgper ml; streptomycin, 100 ng per ml; and amphotericin,
0.25 fig per ml. The parent primary cells were cultured in the same
growth medium. BBCE was isolated according to the method of Spatz
et al. (8), and REEC was isolated according to Wagner and Matthews
(9). Cells were identified as endothelium morphologically by their
cobblestone appearance (10), ¡mmunologicallyby staining with fluorescently labeled anti-Factor VIII (10), and enzymatically by assaying for
angiotensin Il-converting enzyme. All late passage clones used in this
study retained a cobblestone appearance and contained Factor VIIIrelated antigen when examined by immunofluorescence microscopy.
Early passages of REEC expressed low levels of angiotensin II con
verting enzyme (e.g., passage 9; 9,371 units per IO6cells) compared to
their parental cells (e.g., passage 8; 23,138 units per 10' cells). REEC
lost as much as 60% of activity in a single passage. Passage cultures of
BAEC and BBCE also showed a decay of angiotensin H-converting
enzyme activity with prolonged passage. However, the decay appeared
to be less dramatic than the patterns generated by REEC (data not
shown). Angiotensin Il-converting enzyme activity was assessed as
described in the technical bulletin provided by Ventrex Laboratories
(Portland, ME), the supplier of the radioactive substrate. In heat-shock
experiments, 24-well cluster dishes (2 cm2 per well) with confluent
monolayers of endothelium were floated in a constant-temperature
water bath for the times indicated. The temperature in the wells of the
growth medium was 37°Cwhen the cells were placed into the water
bath whose temperature was either 4 PC, 42°C,43'C, or 45°C.The
temperature of the growth medium (500 ui) progressed from 37'C4PC, 42°C,or 43'C in about 3 min and to 45'C in about 5 min and
appeared to remain there. The pH of the growth medium became more
basic with the increase in length of incubation (starting at 7.5; final,
8.1 for cells transferred from 37°C-45°C
for 2 h).
Labeling and Accumulation of Newly Synthesized Proteins. Endothe
lial cells were grown in 24-well culture dishes to a density of 5 x IO4
cells per cm2. The cell monolayers were washed with growth medium
and replaced with 200 ¡i\of fresh medium containing 80 pCi of [35S]methionine (specific activity, 1,066 ¿iCi/mmo!;New England Nuclear,
Boston, MA). Labeling times were as indicated in "Results." The cells
were subsequently washed with Hanks' balanced salt solution buffered
with HEPES (15 min), pH 7.4, and lysed in 200 n\ of 5% sodium
dodecyl sulfate, 50% sucrose, 40 mM dithiothreitol, and 213 mM TrisHC1 (pH 6.8).
Gel Electrophoresis and Autoradiography. Both uniform concentra2101
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HEAT SHOCK PROTEIN SYNTHESIS IN ENDOTHEL1AL CELLS
tion SDS-PAGE as described by Laemmli (11) and two-dimensional
IEF gels described by O'Farrell (12) were used to examine the labeling
BAEC-3
P-7
and accumulation of newly synthesized protein by endothelial cells.
Three % polyacrylamide isofocusing gels were prepared with LKB
ampholines (LKB Instruments, Inc., Gaithersburg, \I I)) to form linear
gradients in the range of pH 4 to 8. Focusing was done at 400 V
overnight (15 to 18 h) followed by l h at 800 V. The IEF gels were
equilibrated for l h in SDS-sample buffer, and the proteins were further
separated on SDS-polyacrylamide gels (11). The gels were stained with
Coomassie Brilliant Blue R-250 (0.1%) in 10% isopropanol and 10%
acetic acid at room temperature overnight and destained in 10% isopropanol/70% acetic acid for 8 h. Polyacrylamide gels containing
radiolabeled proteins were processed for fluorography by treatment in
Enlightning solution (New England Nuclear, Boston, MA) for IS to 30
min. The gels were dried on filter paper using a slab gel drier and
exposed to X-ray film (Kodak; XAR-2).
Protein concentrations were determined by the method of Bradford
(13).
Cell Survival Determination. After appropriate treatment, confluent
monolayers of endothelial cells were trypsinized with 0.25% trypsin in
Hanks' balanced salt solution buffered with HEPES, pH 7.O. After
BAECI0
2 4 8,24
24
\
* I
trypsinization, the cells were suspended in 3 ml of growth medium
containing 2 mg per ml of egg white albumin, a trypsin inhibitor. Cell
counts were determined with a particle cell counter (Coulter Counter
Electronics). An appropriate amount of cells was diluted and plated for
cell survival determination. Each determination was performed in trip
licate. Plating efficiency was between 60 and 90%.
8,24
*/
BBCE-3 REEC-IOIIM
P-13
P-36
2
4 8,24
*/
2
4 8,24
*/
B
— IOOK
— 90K
71K
RESULTS
Patterns of Protein Synthesis at Various Temperatures. To
test whether endothelial cells from different origins varied in
their response to heat stress, we measured both the induction
of the HSPs as well as the expression of normal cellular protein
synthesis as a function of temperature of incubation and dura
tion of heat treatment.
The fluorographs shown in Fig. 1 display the effect of heat
treatment on overall protein accumulation in endothelial cells
from different origins. Radiolabel was added to cultures prior
to incubating cells at either 37°C(Fig. \A), 41'C (Fig. IB), or
43°C(Fig. 1C). Cultures were harvested for analysis of radiolabeled protein after either 2, 4, or 8 h of incubation at these
temperatures, or after cultures labeled for 8 h at 37"C, 41°C,
or 43"C were transferred to 37°Cin the presence of label for
an additional 24 h. This latter group was included to ensure
recovery of normal protein synthesis. Not unlike other cell
types, endothelial cells incubated under normal growth condi
tions (37°C)incorporated [35S]methionine into numerous polypeptides spanning a broad spectrum of molecular weights. As
the temperature of incubation was increased either to 41°Cor
4VC, the production of a group of heat-shock proteins of
71,000, 73,000, 80,000, 90,000, and 100,000 became evident.
However, accumulation of HSPs as well as the decrease of
normal cellular proteins appeared to vary depending on the
origin of the endothelial cells.
Endothelial cells from different origins were clearly different
in their response to a given range of temperatures and duration
of treatment for the expression of a given stress protein (Fig.
1). First, the pattern of both heat-shock and normal cellular
proteins synthesized by passage cultures of bovine aortic endo
thelial cells (BAEC-3, P-7) and primary cultures (BAEC T)
after a 41*C heat stress (Fig. IB) appeared qualitatively similar.
However, the polypeptide patterns of primary versus passaged
BAEC subjected to a 43°Cheat challenge (Fig. 1C) were differ
ent. Passage cultures (BAEC-3, P-7) showed a decrease in total
cellular protein synthesis at 43°Cin comparison to primary
Fig. 1. Effect of heat treatment on overall protein accumulation in endothelial
cells from different origins. Endothelial cells from primary (BAEC 1') and passage
(BAEC) cultures of bovine aortic endothelium, passage cultures of bovine capil
laries (BBCE), and passage cultures of rat epididymal capillaries (REEC) at 37'C
(A), 41'C (B), and 43'C (Q were labeled continuously with |"S]methionine
(-numeral equals clone number, P-numeral equals passage number). Radiolabel
was added to cells prior to incubating cells at 37'C, 41'C, or 43'C, and the label
was present throughout the experiment. Cultures were harvested for protein
analysis after either 2, 4, or 8 h at the various temperatures, or after return to
37'C (noted by the downward-pointing arrow) for an additional 24 h. Proteins
were analyzed by SDS-gel electrophoresis and fluorography. Each lane contained
equal numbers of cell equivalents. Position of the heat-shock proteins is indicated,
on the right. In this and subsequent figures, 71K, 73K, 80K, 90K, and IOOKrefer
to molecular weight (e.g., 7IK means M, 71,000). The bold arrowhead, on the
right, in all three panels indicates the M, 71,000 position.
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HEAT SHOCK PROTEIN SYNTHESIS IN ENDOTHELIAL CELLS
BAEC cultures. The level of heat-shock polypeptides synthe
sized at this temperature was also reduced relative to primary
cultures. Second, bovine brain capillary endothelial cells
(BBCE-3) and rat epididymal endothelial cells (REEC-101 IM)
were more sensitive to heat stress than either primary or passage
BAEC. After a 4TC heat stress, the amount of heat-shock and
normal cellular proteins was reduced in BBCE and REEC
relative to both primary and passage BAEC (Fig. IB). When
cultures of endothelial cells from different origins were returned
to 37°Cfor 24 h, after an initial exposure of 8 h at 41°C,total
repression of total cellular protein synthesis relative to control
temperature (Fig. 2/4). But unlike passage cultures of BAEC,
BBCE synthesize lower levels of HSPs. REEC were most
sensitive to heat stress in that polypeptide synthesis is severely
repressed when cells are exposed to 43°Cfor up to 4 h, whereas
BBCE still synthesize polypeptides. When primary and passage
BAEC and passage BBCE are returned to 37°Cfor 24 h, after
an initial exposure of 8 h at 43°C,the cells can recover the rate
of protein synthesis to essentially control levels. However,
REEC were able to recover only 50% of the control protein
synthesis rate after 24 h at 37°C.
cellular protein synthesis of all four cell types recovered to
100%. Protein synthesis was significantly inhibited in BBCE-3
To further substantiate that HSPs expression is different in
cells at 43°Cand was undetectable in REEC cells at this various endothelial cells, [35S]methionine-labeled total cellular
temperature. Protein synthesis did recover in both these 43°C- protein from endothelial cells of different origins treated for 2
treated cell lines when cultures were returned to 37°C.The h either at 37°Cor 42°Cwas evaluated by two-dimensional gel
reason for the apparent nonlinear accumulation of radiolabel
in protein in 43°C-treated BBCE-3 cells is unknown. Quantifi
cation of HSPs and total cellular protein synthesis on autoradiograms was analyzed by densitometric tracing (not shown).
All data were normalized to the level of protein synthesis in
unstressed cells.
Fig. 2 shows the effect of heat treatment on the rate of protein
synthesis in endothelial cells from different origins. Cells were
pulse labeled for l h ending at the time (in hours) indicated in
the figure. Thus, cells were labeled for l h either at 37°Cor
from 1-2, 3-4, or 7-8 h after transfer to 43°C,or for l h after
cultures which had been treated for 8 h at 43°Cwere incubated
at 37°Cfor an additional 23 h. Cells cultured at 37°C(Fig. 2A)
incorporated [3SS]methionine into a wide range of polypeptides
spanning a broad spectrum of molecular weights. At 43°C,rates
of protein synthesis from endothelial cells from different origins
were strikingly different. Likewise, the effect of this temperature
on the rates of protein synthesis of primary versus passage
BAEC were notably different. Passage cells showed a greater
repression of total cellular protein synthesis in comparison to
BAEC T, and the level of HSPs synthesis was reduced relative
to primary cultures. BBCE, as well, demonstrated a dramatic
electrophoresis. The data (Fig. 3) demonstrate that induction
of heat-shock proteins in endothelial cells after a 42°Chyperthermic treatment is expressed in a cell-specific manner. Pri
mary BAEC cultures were less responsive to 42°Cheat-shock
treatment than BAEC-3 passage cells. Under conditions of
continuous heat treatment at 42°Cfor 2 h, HSP71, 73, 80, 90,
and 100 were identified in passage BAEC (Fig. 3d), while in
primary BAEC, only HSP71, 73, and 80 were induced (Fig.
3Ä).
Examination of protein synthesis of heat-stressed endothelial
cells from different origins suggests that these cells are dissim
ilar in their response to hyperthermia. BBCE responded to
conditions of continuous heat treatment (42°C,2 h) by the
induction of three stress proteins—HSP71, 73, and 90 (Fig.
3/). Stressed BBCE synthesized higher amounts of these pro
teins than stressed primary BAEC cultures and did not appear
to induce HSP90 or 100, which were clearly evident in stressed
passage BAEC. Late passage REEC (P-36) synthesized the
least amount of HSP71 under stressed conditions (Fig. 3h) and
did not express HSP71 at normal temperatures (37°C).The
synthesis of many of the HSPs was detectable at 37°C,but in
much reduced amounts (Fig. 3, a, c, c. and g). Cell-specific
BAECI0
Fig. 2. Effect of temperature on the rate of
protein synthesis in endothelial cells from dif
ferent origins. Endothelial cells from primary
(BAEC D and passage (BAEC) cultures of
bovine aortic endothelium, passage cultures of
bovine capillaries (BBCE), and passage cul
tures of rat epididymal capillaries (REEC)
(-numeral equals clone number; P-numeral
equals passage number) were labeled for 1 h
with ("S]methionine of either 37'C (A) or
43'C (/<). Radiolabel was added to the cells 1
BAEC-1
P-18
BBCE-0227C1
P-15
REEC-10I2J
P-15
71K-»
h prior to the end of the treatment time at
43'C, indicated (hours). After 8 h at 43'C,
some cells (marked byJ) were returned to 37'C
for 24 h (the label was present from 23 to 24
h in these cultures). Proteins were analyzed as
in Fig. 1 by single-dimensional SDS-gel elec
trophoresis and fluorography.
2 4 8,24
2 4
8^24
248
24
8 .24
2103
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HEAT SHOCK PROTEIN SYNTHESIS IN ENDOTHELIAL CELLS
37°C
r
HS
I
-IF-
\
-IF-
*
•'73K
7tK
BAECI0
BAECI0
42°C
2hr
2hr
r:
Fig. 3. Two-dimensional gel analysis of
normal and stressed endothelial cells from dif
ferent origins. To label cellular proteins, endo
thelial cells from different origins were incu
bated with ("Sjmethionine for 2 h at either
37'C or 42'C. Cells were solubilized. and the
»a^
.IOOK
,90K
fcr-eOK
*^73K
7IK
- •
proteins were analyzed by isoelectric focusing
(//1 in a pH 4-8 gradient (acid on the right)
followed by electrophoresis into an SDS-polyacrylamide gel and fluorography. The stress
proteins are indicated by arrows. To control
for differences in migration between gel states,
equal numbers of cells grown at 37'C were run
with each heat-shock condition, a and b, BAEC
T labeled for 2 h at 37'C or 42'C for 2 h,
respectively; c and d, passage BAEC at 37"C
or 42'C for 2 h, respectively; e and /, passage
BBCE at 37'C or 42"C for 2 h, respectively; g
and A, passage REEC at 37'C or 42'C for 2 h,
respectively. Note: -numeral equals clone num
ber; P-numeral equals passage number. IIS.
heat shock.
BAEC-3
P-7
2hr
BAEC-3
P-7
- d
42°C
2hr
BBCE-3
P-13
BBCE-3
P-13
2hr
71 K
REEC
toil M
P-36
2hr
differences were observed and are highlighted by the arrows.
Heat-shock Survival Responses and the Development of Thermotolerance in Endothelial Cells from Different Origins. Cell
REEC
IOIIM
P-36
h
42°C
2hr
survival (Figs. 4 and 5) was examined to ascertain whether
endothelial cells from different origins displayed similar sur
vivai characteristics in response to elevated temperatures and
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HEAT SHOCK PROTEIN SYNTHESIS IN ENDOTHEL1AL CELLS
45°C
stress as compared to other endothelial cell lines described
herein. These cells acquired heat resistance after prolonged
heating at 45°C(2 h) if they were first exposed to a primary
OBAECI°
•
BAEC-IP-12
•
BBCE-0227CI P-9
»REEC-IOI2J P-8
Fig. 5. Cell survival of endothelial cells from different origins and late passage
numbers. Endothelial cells were exposed to 45"C or preincubated at 42'C for l h
followed by a second treatment at 45'C for the times indicated as in Fig. 4. Note
nonlethal heat dose. It is important to note that late passages
(16 passages) of REEC and BBCE appeared not to express
thermotolerance under the test conditions described herein.
REEC, the rat epididymal endothelial cells, might be expected
to be more thermosensitive due to the site of their origin.
Finally, the patterns of protein synthesis after heat shock varied
considerably between cell lines, and no clear relationship be
tween heat-shock protein synthesis and thermotolerance was
apparent, although such a relationship cannot be ruled out by
the data presented here. Several groups have correlated HSP
expression with thermotolerance (survival) (14, 15), but this is
not a general finding (16, 17).
The basic functions performed by endothelial cells are similar
in blood vessels of all sizes (18, 19). Despite functional similar
ities, there is reason to believe that there may be significant
physiological differences between endothelial cells from large
arteries and veins and those from small capillaries. In neoplasia,
arthritis, and psoriasis, there is a rapid proliferation of new
capillaries with relatively little change in the large vessels. It is
interesting, then, to note that endothelial cells from aortic
endothelium versus capillary endothelium but from the same
species and about the same passage number appear to have
notably different response in terms of thermotolerance and
synthesis of proteins after exposure to hyperthermia.
In summary, we present several novel observations pertaining
to the effects of hyperthermia on cell survival and patterns of
protein synthesis in endothelial cells from different origins;
namely, (a) endothelial cells from different origins differ in the
expression of a given stress protein in response to heat stresses;
(b) the accumulation of HSP71 is related to the extent of stress,
except in rat epididymal capillaries; and (c) primary and passage
cultures of BAEC readily acquire thermotolerance, but REEC
and BBCE lose this function with increased passage number.
This information may be useful in determining the thermal
response of neoplastic tissue involving capillary endothelium.
that later passage number cultures were used in these studies, compared to those
presented in Fig. 4.
ACKNOWLEDGMENTS
2
0
Incubation Time(hrs)
Fig. 4. Cell survival of endothelial cells from different origins and early passage
numbers. Endothelial cells were exposed to 45'C or preincubated at 42*C for l h
followed by a second treatment at 45*C for the times indicated. Cells were plated
and survival measured as described in "Materials and Methods." Clone number
is expressed as -numeral and passage number as P-numeral. Bars, SD.
oBAECI°
•
BAEC-I P-19
•
BBCE-0227CI P-16
»REEC-IOI2J P-16
012
0123
Incubation Time(hrs)
whether resistance due to a prior nonlethal heat dose was cellline or passage-number dependent. Cell survival was measured
by colony formation of cells after hyperthermic treatments for
various times. Passage BBCE and REEC appeared more sen
sitive to heat treatment than primary and passage BAEC (Figs.
4 and 5, left). Minor passage number-dependent differences
were observed after acute exposure to 45°C,which were similar
in magnitude to the variation between experiments
primary BAEC cells (BAEC 1").
for the
Figs. 4 and 5 (right) show that cell survival was higher, for
equivalent 45°Cexposure times, when cultures were first treated
with 42°Cfor 1 h, except in late passage BBCE and REEC
cells.
DISCUSSION
Several salient features emerge from this study. It is clear
that HSPs were differentially expressed in endothelial cells
from different origins. In all endothelial cell types examined,
except perhaps in REEC, the accumulation of HSP71 was
related to the extent of stress. Endothelial cells from different
origins differed in response to a given range of temperatures
and duration of treatment. REEC were very sensitive to heat
We would like to thank Ruth G. Schillig and Brenda Rogers for their
assistance with cell cultures and Barbara K. Gricus for typing this
manuscript.
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.
Effects of Hyperthermia on Cell Survival and Patterns of Protein
Synthesis in Endothelial Cells from Different Origins
Nika V. Ketis, Richard L. Hoover and Morris J. Karnovsky
Cancer Res 1988;48:2101-2106.
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.