effects of hyperthermia on cell survival and patterns of protein … · [cancer research 48,...

[CANCER RESEARCH 48, 2101-2106, April 15. 1988)

Effects of Hyperthermia on Cell Survival and Patterns of Protein Synthesis inEndothelial 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, isgenerally thought to be linked to the accumulationof heat-shock proteinsin eukaryotic cells. The induction of thermotolerance and the synthesisof heat-shock proteins in primary and passage cultures of bovine aorticendothelium, passage cultures of bovine brain capillaries, and passagecultures of rat epididymal capillaries were examined. Primary and passage cultures of bovine aortic endothelial cells readily acquired thermotolerance; however, passage cultures of rat epididymal capillary cells andbovine brain capillary cells were very heat sensitive. In all endothelialcell types examined except rat epididymal capillary cells, the levels ofHSP71, the most inducible of the HSP70 family, correlated well withthermotolerance. With prolonged passage, rat epididymal capillary cellsand bovine braincapillary cells lost their ability to acquire heat resistance.Endothelial cells from different origins (aortic endothelium versuscapillary endothelium) but from the same species and about the same passagenumberhad a notably different response in terms of thermotoleranceandsynthesis of proteins after exposure to hyperthermia. The results of thisstudy suggest that, while the expression of HSP71 may be a goodindicator of heat resistance, the reverse is not necessarily true. Furthermore, the data show that endothelial cells from different origins aredissimilar in their response to hyperthermia.

INTRODUCTION

The cells of most eukaryotes respond to mild heat treatmentby the induction of a set of proteins termed the HSPs.3 In

addition, these proteins are induced as a result of cellularresponses to other stresses (1, 2), such as glucose deprivationand anoxia. In Drosophila, where the response was first reported, the induction process has been studied extensively (3,for review). In these cells, the HSPs appear to have differentinduction characteristics, and the controls exerted on bothtranscription and translation are regulated to ensure that HSPsare produced as rapidly as possible.

The commitment to heat-shock protein synthesis is fullyreversible when Drosophila cells (4) and other eukaryotic cells(5) are returned to their normal temperatures. Translation andtranscription gradually return to their prestressed state (6). Asearch for regulatory mechanisms controlling the expression ofheat-shock proteins at both the transcriptional and posttran-scriptional 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, andneoplasia, 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 inaccordance 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 SpringHarbor Laboratory, Cold Spring Harbor, NY, on August 29, 1985, and at theAmerican Society for Cell Biology Annual Meetings, St. Louis, MO, on November 20, 1987, and appeared in abstract forms (20, 21).

1Recipient 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 capillaryendothelium; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; SDS,sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; IEF, isoelec-tric focusing.

in these cells is of great interest. To further understand thebiology of a mammalian cell that may undergo physiologicaland pathological "stress," the synthesis of HSPs in endothelial

cells from different origins (primary and passage cultures ofbovine aortic endothelium, passage cultures of bovine braincapillaries, and the passage cultures of rat epididymal capillaries) was examined by polyacrylamide gel electrophoresis aftercells had been exposed to various temperatures and durationsof heat stress. The results in this study suggest that endothelialcells from different origins are dissimilar in their response tohyperthermia.

MATERIALS AND METHODS

Endothelial Cells and Cultures. BAEC were isolated according tomethods described by Booyse et al. (7). When passage cells were desired,primary endothelial cells were cloned, passaged, and grown in Dulbec-co's modified Eagle's medium with 5% fetal bovine serum and 5% Nu-

Serum (Collaborative Research, Waltham, MA) plus antibioticsâ€”penicillin, 100 Mgper ml; streptomycin, 100 ng per ml; and amphotericin,0.25 fig per ml. The parent primary cells were cultured in the samegrowth medium. BBCE was isolated according to the method of Spatzet al. (8), and REEC was isolated according to Wagner and Matthews(9). Cells were identified as endothelium morphologically by theircobblestone appearance (10), Â¡mmunologicallyby staining with fluores-cently labeled anti-Factor VIII (10), and enzymatically by assaying forangiotensin Il-converting enzyme. All late passage clones used in thisstudy retained a cobblestone appearance and contained Factor VIII-related antigen when examined by immunofluorescence microscopy.Early passages of REEC expressed low levels of angiotensin II converting enzyme (e.g., passage 9; 9,371 units per IO6cells) compared totheir 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 ofBAEC and BBCE also showed a decay of angiotensin H-convertingenzyme activity with prolonged passage. However, the decay appearedto be less dramatic than the patterns generated by REEC (data notshown). Angiotensin Il-converting enzyme activity was assessed asdescribed in the technical bulletin provided by Ventrex Laboratories(Portland, ME), the supplier of the radioactive substrate. In heat-shockexperiments, 24-well cluster dishes (2 cm2 per well) with confluentmonolayers of endothelium were floated in a constant-temperaturewater bath for the times indicated. The temperature in the wells of thegrowth medium was 37Â°Cwhen the cells were placed into the waterbath whose temperature was either 4 PC, 42Â°C,43'C, or 45Â°C.Thetemperature of the growth medium (500 ui) progressed from 37'C-4PC, 42Â°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 morebasic with the increase in length of incubation (starting at 7.5; final,8.1 for cells transferred from 37Â°C-45Â°Cfor 2 h).

Labeling and Accumulation of Newly Synthesized Proteins. Endothelial cells were grown in 24-well culture dishes to a density of 5 x IO4cells per cm2. The cell monolayers were washed with growth mediumand replaced with 200 Â¡i\of fresh medium containing 80 pCi of [35S]-

methionine (specific activity, 1,066 Â¿iCi/mmo!;New England Nuclear,Boston, MA). Labeling times were as indicated in "Results." The cellswere subsequently washed with Hanks' balanced salt solution buffered

with HEPES (15 min), pH 7.4, and lysed in 200 n\ of 5% sodiumdodecyl sulfate, 50% sucrose, 40 mM dithiothreitol, and 213 mM Tris-HC1 (pH 6.8).

Gel Electrophoresis and Autoradiography. Both uniform concentra-

2101

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HEAT SHOCK PROTEIN SYNTHESIS IN ENDOTHEL1AL CELLS

tion SDS-PAGE as described by Laemmli (11) and two-dimensionalIEF gels described by O'Farrell (12) were used to examine the labeling

and accumulation of newly synthesized protein by endothelial cells.Three % polyacrylamide isofocusing gels were prepared with LKBampholines (LKB Instruments, Inc., Gaithersburg, \I I)) to form lineargradients in the range of pH 4 to 8. Focusing was done at 400 Vovernight (15 to 18 h) followed by l h at 800 V. The IEF gels wereequilibrated for l h in SDS-sample buffer, and the proteins were furtherseparated on SDS-polyacrylamide gels (11). The gels were stained withCoomassie Brilliant Blue R-250 (0.1%) in 10% isopropanol and 10%acetic acid at room temperature overnight and destained in 10% iso-propanol/70% acetic acid for 8 h. Polyacrylamide gels containingradiolabeled proteins were processed for fluorography by treatment inEnlightning solution (New England Nuclear, Boston, MA) for IS to 30min. The gels were dried on filter paper using a slab gel drier andexposed to X-ray film (Kodak; XAR-2).

Protein concentrations were determined by the method of Bradford(13).

Cell Survival Determination. After appropriate treatment, confluentmonolayers of endothelial cells were trypsinized with 0.25% trypsin inHanks' balanced salt solution buffered with HEPES, pH 7.O. After

trypsinization, the cells were suspended in 3 ml of growth mediumcontaining 2 mg per ml of egg white albumin, a trypsin inhibitor. Cellcounts were determined with a particle cell counter (Coulter CounterElectronics). An appropriate amount of cells was diluted and plated forcell survival determination. Each determination was performed in triplicate. Plating efficiency was between 60 and 90%.

RESULTS

Patterns of Protein Synthesis at Various Temperatures. Totest whether endothelial cells from different origins varied intheir response to heat stress, we measured both the inductionof the HSPs as well as the expression of normal cellular proteinsynthesis as a function of temperature of incubation and duration of heat treatment.

The fluorographs shown in Fig. 1 display the effect of heattreatment on overall protein accumulation in endothelial cellsfrom different origins. Radiolabel was added to cultures priorto incubating cells at either 37Â°C(Fig. \A), 41'C (Fig. IB), or43Â°C(Fig. 1C). Cultures were harvested for analysis of radio-

labeled protein after either 2, 4, or 8 h of incubation at thesetemperatures, or after cultures labeled for 8 h at 37"C, 41Â°C,or 43"C were transferred to 37Â°Cin the presence of label for

an additional 24 h. This latter group was included to ensurerecovery of normal protein synthesis. Not unlike other celltypes, endothelial cells incubated under normal growth conditions (37Â°C)incorporated [35S]methionine into numerous poly-

peptides spanning a broad spectrum of molecular weights. Asthe temperature of incubation was increased either to 41Â°Cor

4VC, the production of a group of heat-shock proteins of71,000, 73,000, 80,000, 90,000, and 100,000 became evident.However, accumulation of HSPs as well as the decrease ofnormal cellular proteins appeared to vary depending on theorigin of the endothelial cells.

Endothelial cells from different origins were clearly differentin their response to a given range of temperatures and durationof treatment for the expression of a given stress protein (Fig.1). First, the pattern of both heat-shock and normal cellularproteins synthesized by passage cultures of bovine aortic endothelial 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 passagedBAEC subjected to a 43Â°Cheat challenge (Fig. 1C) were differ

ent. Passage cultures (BAEC-3, P-7) showed a decrease in totalcellular protein synthesis at 43Â°Cin comparison to primary

2102

BAEC-3 BBCE-3 REEC-IOIIMP-7 BAECI0 P-13 P-36

B

2 4 8,24 24 8,24 2 4 8,24 2 4 8,24\ * I */ */ */

â€” IOOKâ€” 90K

71K

Fig. 1. Effect of heat treatment on overall protein accumulation in endothelialcells from different origins. Endothelial cells from primary (BAEC 1') and passage

(BAEC) cultures of bovine aortic endothelium, passage cultures of bovine capillaries (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). Radiolabelwas added to cells prior to incubating cells at 37'C, 41'C, or 43'C, and the labelwas present throughout the experiment. Cultures were harvested for proteinanalysis after either 2, 4, or 8 h at the various temperatures, or after return to37'C (noted by the downward-pointing arrow) for an additional 24 h. Proteinswere analyzed by SDS-gel electrophoresis and fluorography. Each lane containedequal 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 IOOKreferto molecular weight (e.g., 7IK means M, 71,000). The bold arrowhead, on theright, in all three panels indicates the M, 71,000 position.



HEAT SHOCK PROTEIN SYNTHESIS IN ENDOTHELIAL CELLS

BAEC cultures. The level of heat-shock polypeptides synthesized at this temperature was also reduced relative to primarycultures. 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 passageBAEC. After a 4TC heat stress, the amount of heat-shock andnormal cellular proteins was reduced in BBCE and REECrelative to both primary and passage BAEC (Fig. IB). Whencultures of endothelial cells from different origins were returnedto 37Â°Cfor 24 h, after an initial exposure of 8 h at 41Â°C,total

cellular protein synthesis of all four cell types recovered to100%. Protein synthesis was significantly inhibited in BBCE-3cells at 43Â°Cand was undetectable in REEC cells at thistemperature. Protein synthesis did recover in both these 43Â°C-treated cell lines when cultures were returned to 37Â°C.The

reason for the apparent nonlinear accumulation of radiolabelin protein in 43Â°C-treated BBCE-3 cells is unknown. Quantifi

cation of HSPs and total cellular protein synthesis on autora-diograms was analyzed by densitometric tracing (not shown).All data were normalized to the level of protein synthesis inunstressed cells.

Fig. 2 shows the effect of heat treatment on the rate of proteinsynthesis in endothelial cells from different origins. Cells werepulse labeled for l h ending at the time (in hours) indicated inthe figure. Thus, cells were labeled for l h either at 37Â°Corfrom 1-2, 3-4, or 7-8 h after transfer to 43Â°C,or for l h aftercultures which had been treated for 8 h at 43Â°Cwere incubatedat 37Â°Cfor an additional 23 h. Cells cultured at 37Â°C(Fig. 2A)incorporated [3SS]methionine into a wide range of polypeptidesspanning a broad spectrum of molecular weights. At 43Â°C,rates

of protein synthesis from endothelial cells from different originswere strikingly different. Likewise, the effect of this temperatureon the rates of protein synthesis of primary versus passageBAEC were notably different. Passage cells showed a greaterrepression of total cellular protein synthesis in comparison toBAEC T, and the level of HSPs synthesis was reduced relativeto primary cultures. BBCE, as well, demonstrated a dramatic

repression of total cellular protein synthesis relative to controltemperature (Fig. 2/4). But unlike passage cultures of BAEC,BBCE synthesize lower levels of HSPs. REEC were mostsensitive to heat stress in that polypeptide synthesis is severelyrepressed when cells are exposed to 43Â°Cfor up to 4 h, whereas

BBCE still synthesize polypeptides. When primary and passageBAEC and passage BBCE are returned to 37Â°Cfor 24 h, afteran initial exposure of 8 h at 43Â°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 proteinsynthesis rate after 24 h at 37Â°C.

To further substantiate that HSPs expression is different invarious endothelial cells, [35S]methionine-labeled total cellular

protein from endothelial cells of different origins treated for 2h either at 37Â°Cor 42Â°Cwas evaluated by two-dimensional gel

electrophoresis. The data (Fig. 3) demonstrate that inductionof heat-shock proteins in endothelial cells after a 42Â°Chyper-

thermic treatment is expressed in a cell-specific manner. Primary BAEC cultures were less responsive to 42Â°Cheat-shock

treatment than BAEC-3 passage cells. Under conditions ofcontinuous heat treatment at 42Â°Cfor 2 h, HSP71, 73, 80, 90,

and 100 were identified in passage BAEC (Fig. 3d), while inprimary BAEC, only HSP71, 73, and 80 were induced (Fig.3Ã„).

Examination of protein synthesis of heat-stressed endothelialcells from different origins suggests that these cells are dissimilar in their response to hyperthermia. BBCE responded toconditions of continuous heat treatment (42Â°C,2 h) by the

induction of three stress proteinsâ€”HSP71, 73, and 90 (Fig.3/). Stressed BBCE synthesized higher amounts of these proteins than stressed primary BAEC cultures and did not appearto induce HSP90 or 100, which were clearly evident in stressedpassage BAEC. Late passage REEC (P-36) synthesized theleast amount of HSP71 under stressed conditions (Fig. 3h) anddid not express HSP71 at normal temperatures (37Â°C).Thesynthesis of many of the HSPs was detectable at 37Â°C,but in

much reduced amounts (Fig. 3, a, c, c. and g). Cell-specific

BAEC-1 BBCE-0227C1 REEC-10I2JBAECI0 P-18 P-15 P-15

Fig. 2. Effect of temperature on the rate ofprotein synthesis in endothelial cells from different origins. Endothelial cells from primary(BAEC D and passage (BAEC) cultures ofbovine aortic endothelium, passage cultures ofbovine capillaries (BBCE), and passage cultures of rat epididymal capillaries (REEC)(-numeral equals clone number; P-numeralequals passage number) were labeled for 1 hwith ("S]methionine of either 37'C (A) or43'C (/<). Radiolabel was added to the cells 1

h prior to the end of the treatment time at43'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 24h in these cultures). Proteins were analyzed asin Fig. 1 by single-dimensional SDS-gel electrophoresis and fluorography.

71K-Â»

2 4 8,24 2 4 8^24 248

2103

24 8 .24



HEAT SHOCK PROTEIN SYNTHESIS IN ENDOTHELIAL CELLS

37Â°C

r-IF-

HS\

I -IF-

* â€¢'-73K7tK

BAECI0

2hr

BAECI0

42Â°C

2hr

Fig. 3. Two-dimensional gel analysis ofnormal and stressed endothelial cells from different origins. To label cellular proteins, endothelial cells from different origins were incubated with ("Sjmethionine for 2 h at either37'C or 42'C. Cells were solubilized. and the

proteins were analyzed by isoelectric focusing(//1 in a pH 4-8 gradient (acid on the right)followed by electrophoresis into an SDS-poly-acrylamide gel and fluorography. The stressproteins are indicated by arrows. To controlfor differences in migration between gel states,equal numbers of cells grown at 37'C were runwith each heat-shock condition, a and b, BAECT labeled for 2 h at 37'C or 42'C for 2 h,respectively; c and d, passage BAEC at 37"Cor 42'C for 2 h, respectively; e and /, passageBBCE at 37'C or 42"C for 2 h, respectively; gand A, passage REEC at 37'C or 42'C for 2 h,respectively. Note: -numeral equals clone number; P-numeral equals passage number. IIS.heat shock.

r:

Â»a- - â€¢^

BAEC-3P-7

2hr- d

.IOOK,90K

fcr-eOK*^73K

7IK

BAEC-3P-7

42Â°C2hr

BBCE-3P-13

2hr

BBCE-3P-13

REECtoil MP-36

2hr

71 K

REECIOIIMP-36

h 42Â°C

2hr

differences were observed and are highlighted by the arrows. survival (Figs. 4 and 5) was examined to ascertain whetherHeat-shock Survival Responses and the Development of Ther- endothelial cells from different origins displayed similar sur

motolerance in Endothelial Cells from Different Origins. Cell vivai characteristics in response to elevated temperatures and2104




45Â°C

OBAECIÂ°â€¢BAEC-IP-12â€¢BBCE-0227CI P-9Â»REEC-IOI2J P-8

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 hfollowed by a second treatment at 45*C for the times indicated. Cells were platedand survival measured as described in "Materials and Methods." Clone numberis expressed as -numeral and passage number as P-numeral. Bars, SD.

oBAECIÂ°â€¢BAEC-I P-19â€¢BBCE-0227CI P-16Â»REEC-IOI2J P-16

012 0123Incubation Time(hrs)

Fig. 5. Cell survival of endothelial cells from different origins and late passagenumbers. Endothelial cells were exposed to 45"C or preincubated at 42'C for l hfollowed by a second treatment at 45'C for the times indicated as in Fig. 4. Note

that later passage number cultures were used in these studies, compared to thosepresented in Fig. 4.

whether resistance due to a prior nonlethal heat dose was cell-line or passage-number dependent. Cell survival was measuredby colony formation of cells after hyperthermic treatments forvarious times. Passage BBCE and REEC appeared more sensitive to heat treatment than primary and passage BAEC (Figs.4 and 5, left). Minor passage number-dependent differenceswere observed after acute exposure to 45Â°C,which were similar

in magnitude to the variation between experiments for theprimary BAEC cells (BAEC 1").

Figs. 4 and 5 (right) show that cell survival was higher, forequivalent 45Â°Cexposure times, when cultures were first treatedwith 42Â°Cfor 1 h, except in late passage BBCE and REEC

cells.

DISCUSSION

Several salient features emerge from this study. It is clearthat HSPs were differentially expressed in endothelial cellsfrom different origins. In all endothelial cell types examined,except perhaps in REEC, the accumulation of HSP71 wasrelated to the extent of stress. Endothelial cells from differentorigins differed in response to a given range of temperaturesand duration of treatment. REEC were very sensitive to heat

stress as compared to other endothelial cell lines describedherein. These cells acquired heat resistance after prolongedheating at 45Â°C(2 h) if they were first exposed to a primary

nonlethal heat dose. It is important to note that late passages(16 passages) of REEC and BBCE appeared not to expressthermotolerance under the test conditions described herein.REEC, the rat epididymal endothelial cells, might be expectedto be more thermosensitive due to the site of their origin.Finally, the patterns of protein synthesis after heat shock variedconsiderably between cell lines, and no clear relationship between heat-shock protein synthesis and thermotolerance wasapparent, although such a relationship cannot be ruled out bythe data presented here. Several groups have correlated HSPexpression with thermotolerance (survival) (14, 15), but this isnot a general finding (16, 17).

The basic functions performed by endothelial cells are similarin blood vessels of all sizes (18, 19). Despite functional similarities, there is reason to believe that there may be significantphysiological differences between endothelial cells from largearteries and veins and those from small capillaries. In neoplasia,arthritis, and psoriasis, there is a rapid proliferation of newcapillaries with relatively little change in the large vessels. It isinteresting, then, to note that endothelial cells from aorticendothelium versus capillary endothelium but from the samespecies and about the same passage number appear to havenotably different response in terms of thermotolerance andsynthesis of proteins after exposure to hyperthermia.

In summary, we present several novel observations pertainingto the effects of hyperthermia on cell survival and patterns ofprotein synthesis in endothelial cells from different origins;namely, (a) endothelial cells from different origins differ in theexpression 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 passagecultures of BAEC readily acquire thermotolerance, but REECand BBCE lose this function with increased passage number.This information may be useful in determining the thermalresponse of neoplastic tissue involving capillary endothelium.

ACKNOWLEDGMENTS

We would like to thank Ruth G. Schillig and Brenda Rogers for theirassistance with cell cultures and Barbara K. Gricus for typing thismanuscript.

REFERENCES

1. Ashburner, M , and Bonner. J. J. The induction of gene activity in Drosophilaby heat shock. Cell, 17: 241-245, 1979.

2. Schlesinger, M. J., Ashburner, M., and Tissieres, A. (eds.). Heat Shock: fromBacteria to Man, p. 440. Cold Spring Harbor, NY: Cold Spring HarborLaboratories, 1982.

3. Ashburner, M. Effects of heat shock and other stress on gene activity: anintroduction. In: M. J. Schlesinger, M. Ashburner, and A. Tissieres (eds.),Heat Shock: from Bacteria to Man, pp. 1-9. Cold Spring Harbor, NY: ColdSpring Harbor Laboratories, 1982.

4. DiDomenico, B. J., Bugaisky, G. E., and Linguist, S. The heat-shock responseis self-regulated at both the transcriptional and posttranscriptional level. Cell,31: 593-603, 1982.

5. Linguist, S. Varying patterns of protein synthesis in Drosophila during heatshock. Implication for regulation. Dev. Biol., 77: 463-479, 1980.

6. DiDomenico, B. J., Bugaisky, G. E., and Linguist, S. Heat shock and recoveryare mediated by different translational mechanisms. Proc. Nati. Acad. Sci.USA, 79:6181-6185, 1982.

7. Booyse, F. M., Sedlak, B. J., and Rafelson. M. E. Culture of arterialendothelial cells: characterization and growth of bovine aortic cells. Throm-bos. Diathes. Haemorrh. (Stutlg), 35: 825-839, 1975.

8. Spatz, M., Bembry, J., Dodson, R. F., Hervonen, H., and Murray. M. R.Endothelial cell cultures derived from isolated cerebral microvessels. BrainRes., 191: 577-582, 1980.

9. Wagner, R. C., and Matthews, M. A. The isolation and culture of capillary

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endothelium from epididymal fat. Microvasc. Res., 10: 286-297, I97S. on thermotolerance expression, heat shock protein synthesis, and heat shock10. Gimbrone, M. A., Cotran, R. S., and Folkman.J. Human vascular endothelial protein mRNA accumulation in rat fibroblasts. Mol. Cell. Biol., 6: 1088-

cells in culture. J. Cell Biol., 60:673-679, 1974. 1094, 1986.11. Laemmli, U. K. Cleavage of structural proteins during the assembly of the '7- Fisher, G. A., Anderson, R. L., and Hahn, G. M. Glucocorticoid-induced

head of bacteriophage T4. Nature (I mid.). 227:680-685, 1970. heat resistance in mammalian cells. J. Cell. Physiol., 72Â«:127-132, 1986.12. O'Farrell, P. H. High resolution two-dimensional electrophoresis of proteins. '8- Jaffe- E- A- Culture and identification of large vessel endothelial cells. In: E.

J. Biol. Chem., 280: 4007-4021, 1975. A- Jaffe (ed-'- B'Â°lÂ°gyof Endothelial Cells, pp. 1-13. Boston: Martinus13. Bradford, M. A rapid and sensitive method for the quantitation of microgram , Nijhoff Publishers, 1984.

quantities of protein utilizing the principle of protein-dye binding. Anal. 19' ^f"er- B, R ^"Â¿"Ã®Ã®*,Â°fCapillary endothelial cells. In: E. J. Jaffe (ed.),Brochera 72-248-2541976 Biology of Endothelial Cells, pp. 14-26. Boston: Martinus Nijhoff Publish-

14. Li C. C and Werb Z.'Correlation between synthesis of heat shock protein -,â€žJUJV, Hoover, R. L.,and Karnovsky, M. J. Varying patterns of protein

and developmen of thermotolerance m Chmese hamster libroblasts. Proc. symhesis .â€žendo(helia| cel|s from di(Terent ori ins e d ,â€žhvpertnermia.Nati Acad. Sci. USA, 79: 3219-3222, 1982. ,â€ž.Abs,rac! of Papers Presented at the 1985 Meeting on Hea, shock 42

15. Landry, J., Bermer, D., Chretien, P., Nicole, L. M., Tanguay, R. M., and Cold Spring Harbor, NY: Cold Spring Laboratory, 1985.Marceau, N. Synthesis and degradation of heat shock proteins during devel- 21. Ketis, N. V., and Karnovsky, M. J. Induction of heat-shock protein synthesisopment and decay of thermotolerance. Cancer Res., 42: 2457-2461, 1982. in endothelial cells from different origins during the development of ther-

16. Widelitz, R. B., Magun, B. E., and Gerner, E. W. Effects of cycloheximide motolerance. J. Cell. Biol., 105: 329a, 1987.

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1988;48:2101-2106. Cancer Res Nika V. Ketis, Richard L. Hoover and Morris J. Karnovsky Synthesis in Endothelial Cells from Different OriginsEffects of Hyperthermia on Cell Survival and Patterns of Protein

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