Cytometric methods to analyze thermal effects

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Cytometric Methods to Analyze Thermal Effects Robert P. VanderWaal, Ryuji Higashikubo, Mai Xu, Douglas R. Spitz, William D. Wright, and Joseph L. Roti Roti Mallinckrodt Institute of Radiology Radiation Oncology Center Section of Cancer Biology Washington University School of Medicine St. Louis, Missouri 63108

I. Introduction II. Application III. Methods to Measure Nuclear and Nuclear Matrix Protein Content A. Nuclear Protein Content by Flow Cytometry B. Nuclear Matrix Protein Content by Flow Cytometry C. Nuclear Matrix Stability Assay Following Heat Shock Using Flow Cytometry IV. Identification of Altered DNA Replication Patterns Following Heat Shock V. Nuclear Localization of hsp70 A. Flow Cytometric Detection of Nuclear hsp/hscT0 B. Nuclear Localization of hsp70 by in Situ Immunofluorescence VI. Prooxidant Measurement VII. Results A. Nuclear Protein Content B. Pattern of DNA Replication C. hsp/hsc70 D. Prooxidant Production References

I. I n t r o d u c t i o n T h e s t u d y o f t h e r m a l effects on cells is o f critical i n t e r e s t to at least t h r e e d i v e r s e fields: (1) studies of the r e g u l a t i o n o f g e n e e x p r e s s i o n in t h e r e s p o n s e ( s ) METHODS IN CELL BIOLOGY, VOL. 64 Copyright © 2001 by Academic Press. All rights of reproduction in any forln reserved. 0091-679X/(}1 $3500

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of cells to environmental stress; (2) the application of hyperthermia as a single or combined modality in the treatment of cancer (Urano and Douple, 1988, 1989); and (3) the identification of thermal artifacts (or effects) in studies of the biological effects of physical agents such as radiofrequency radiation and ultrasound (Miller and Ziskin, 1989). In these studies thermal effects have been detected at temperatures as low as 39°C and up to 47°C (Henle and Roti Roti, 1988; A. Laszlo, unpublished, 1996). For most cell lines the study of temperatures above 48°-50°C are not biologically relevant owing to rapid cytotoxicity and/or massive protein denaturation (Wright et al., 1998). For the most part, the effects described in this chapter are generally studied following (or during) heat shocks at temperatures between 41 ° and 46°C. Hyperthermia in the 41°-45°C range causes a plethora of effects on most cellular organelles and functions. These effects range from the interaction of the plasma membrane with the extracellular matrix, to protein aggregation with the nuclear matrix (Roti Roti and Laszlo, 1988; Laszlo, 1992; Kampinga, 1993). Functional changes can be seen in cytosol and the nucleoplasm. In this sense most, if not all, of the methods described in this book can be used to detect differences between heat-shocked and normal cells. The challenge for the workers in this field is to find out those changes that contribute mechanistically to the effects of interest. Thus, we have selected parameters that reflect thermal cytotoxicity uniquely, as far as we know, distinguish between heat-sensitive and heatresistant cells, are markers for transient heat resistance, thermotolerance, and those that show interrelationships between thermal stress and oxidative stress. The last parameter is included since there are a number of studies that suggest that both the oxidative and thermal stress responses, while not identical, share some overlapping features. The methods described in this chapter, therefore, should have a wide variety of applications in the study of cells and their responses to environmental stress, particularly heat shock.

II. Application As stated earlier, several cytometric methods have been used to assay the effects of hyperthermia on cells. The first assay, the amount of protein that coisolates with nuclei or nuclear matrices, measures a parameter that correlates with numerous heat effects on nuclear function such as inhibition of DNA repair, DNA synthesis, transcription, and processing of hnRNA. This parameter correlates very well with cell death in many cell lines, particularly those that do not die by apoptosis. Because the mode of cell death induced by nuclear protein aggregation appears to involve progression through S phase, we will describe methods to determine the pattern of DNA replication factories which changes in a time-dependent manner. The next parameter will be the expression of heatshock proteins whose nuclear localization and delocalization correlates with the development of thermotolerance and heat resistance. Finally, we will describe

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a method to measure intracellular prooxidant capacity. One of the effects that can be induced by the exposure of cells to hyperthermia is an imbalance between prooxidant and antioxidant status either by a reduced antioxidant function or an increased prooxidant function, or both. An increased prooxidant level may, in turn, lead to a myriad of biochemical alterations that disrupt normal cellular processes and even lead to cell death.

III. M e t h o d s t o M e a s u r e N u c l e a r and N u c l e a r M a t r i x Protein Content A. Nuclear Protein Content by Flow Cytometry Two methods for isolating nuclei have been described in the previous volume of this series (Higashikubo et al., 1990). We describe here another method that works very well for cultured cells that grow on monolayer. All reagents are from Sigma, St. Louis, MO. 1. Reagents 1. Spinner salt solution, pH 7.0; 270-300 mOs/kg; per lml double-distilled water (ddH20): 0.4 mg KC1, 2.2 mg NaHCO3, 0.1 mg MgSO4, 1.4 mg NaH2PO4, 6.8 mg NaC1, and 1.0 mg D-glucose. 2. Nuclear isolation buffer: 0.5% Nonidet P-40, 50 mM Tris base/Tris-HC1 (pH 7.4), 10 mM EDTA, 50 mM NaC1. 3. Bicarbonate buffer (pH 8.1): 0.03 M NaC1, 0.03 M NaHCO3. 4. Staining solution: (a) 1 mg/ml RNase A in ddH20 (boil for 5 rain before each use), (b) 3 /~g/ml fluorescein isothiocyanate (F1TC) in bicarbonate buffer, and (c) 70 ~g/ml propidium iodide (PI) in bicarbonate buffer.

2. Nuclear Isolation and Staining Procedure 1. Detach cells by trypsinization or other enzymatic methods and neutralize by the addition of complete medium. Make sure cells are well monodispersed. Centrifuge at 150 g and decant medium. Adjust the cell number to 11 . 5 X ] 0 6 per sample. . Wash twice with Spinner salt solution, each followed by centrifugation at 150 g. 3. Resuspend in 5 ml of nuclear isolation buffer. Keep on ice. The duration for this treatment depends on the cell type. For example, Chinese hamster ovary K1 cells need 5 min, while some cell lines of epithelial origin may need longer treatment.

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4. 5. 6. 7.

Collect nuclei by centrifugation at 900 g. Wash twice with bicarbonate buffer. Resuspend in 400/xl of the same buffer. Add 50/xl of RNase A solution and incubate for 30 rain at room temperature. Add 50/zl of FITC stock solution and 500/zl of PI solution. Stain for at least 1 hr before analysis.

3. Special Considerations 1. The FITC fluorescence intensity of nuclei is sensitive to the concentration of nuclei and pH of the final suspension. The concentration of nuclei in the final staining solution should not exceed 1.5 x 106 per ml. The isolated nuclei should be washed clean of cellular debris and organelles that may cause pH change of the final staining solution. 2. The stained nuclei should be examined under a fluorescence microscope to ensure that they are free of cytoplasmic contamination. However, the loss of cellular particles during the nuclear isolation procedure and the subsequent washes should be kept at a minimum, for example, no more than 20%. A low nuclear yield indicates that the isolation procedure is too harsh and may have extracted some nuclear proteins. 4. Instruments 1. Both dyes can be excited with the 488 nm line of an argon laser. Green FITC fluorescence is detected through a 535 nm band-pass filter, whereas red PI fluorescence can be detected through a 640 nm long-pass filter. 2. Very strong FITC signals, unlike those from immunofluorescence staining, may necessitate fluorescence compensation on the red fluorescence channel. B. Nuclear Matrix Protein Content by Flow Cytornetry The nuclear matrix is perhaps the most heat labile structure in eukaryotic cells. As the result of denaturation, soluble nuclear proteins bind to the nuclear matrix altering its structure (vanderWaal et al., 1996). One result of this protein binding is that nuclei isolated from heat-shocked cells (see earlier) have a higher protein content, compared to controls (Warters and Roti Roti, 1982). As another result of this protein binding, many nuclear functions are compromised. These include DNA replication, and RNA transcription and processing (Higashikubo and Roti Roti, 1993). 1. Reagents 1. HeLa cells in tissue culture flasks with watertight caps. Alternatively, the flasks can be sealed with Parafilm during the heat-shock steps. Another

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possibility is to use HeLa cells grown in suspension and transfer the culture to an appropriately sized centrifuge tube prior to immersion. Medium prewarmed to the temperature selected for the heat shock experiment. Trypsin-EDTA. 0.01 M phosphate-buffered saline (PBS) (pH 7.4): For 1 liter of 10× stock solution, 80 g NaC1, 2 g KC1, 11.5 g Na2HPO4" 7H20, 2 g KH2PO4. Digestion Buffer: 10 mM PIPES, pH 6.8, 50 m M NaC1, 300 mM sucrose, 3 mM MgC12, 1 mM EGTA, 0.5% (v/v) Triton X-100, 4 mM vanadyl riboside complex (add just before use), 1.2 mM phenylmethylsulfonyl fluoride (PMSF) (add just before use). 5 mg/ml deoxyribonuclease I (DNase I) stock: Add l ml 50% glycerol to 5 mg DNase I (Worthington Biochemical, Freehold, NJ, DPRF grade), store at -20°C. 1 M N H 4 S O 4 in water. Cytoskeleton buffer: 0.292 g NaC1 in 100 ml digestion buffer. FITC stain: 3/zg FITC (Calbiochem, La Jolla, CA) per ml in digestion buffer.

2. Nuclear Matrix Isolation and Staining Procedure 1. Heat shock cells by submerging flasks in a water bath set at the desired temperature and incubate. 2. Immediately after removal from the water bath, aspirate medium, and replace with trypsin/EDTA. Keep at room temperature for 3 rain. 3. Centrifuge cells at 235 g for 5 min. 4. Wash cells with PBS at 4°C and centrifuge. 5. Extract cells with cytoskeleton buffer for 3 rain at 4°C at a density of 4 × 106 cells per ml. Centrifuge for 5 min at 940 g. 6. Resuspend pellet in digestion buffer, using same volume as in Step 5. Add DNase I to a final concentration of 150/xg DNase I per ml of digestion buffer. Incubate 30 rain at room temperature. 7. Add 1 M N H 4 S O 4 stock to cell suspension to 250 mM final concentration, incubate 5 min at room temperature, and centrifuge for 5 min at 940 g. 8. Resuspend pellet with 0.9 ml digestion buffer, add 0.1 ml stock FITC stain, and incubate at 4°C for 1 hr to overnight. 9. Analyze nuclear matrices by flow cytometry (FCM). 3. Instruments For flow cytometry using a 488 nm excitation line from an argon laser, FITC fluorescence is detected through a 535 nm band-pass filter.

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C. Nuclear Matrix Stability Assay Following Heat Shock Using Flow Cytometry Evidence is accumulating which suggests that the nuclear matrix and its associated functions are a primary target for the effects of heat shock (for review, see Roti Roti et al., 1998). This subnuclear organelle is composed mostly of R N A and protein. Evidence for the structural role of R N A in the matrix resides in the fact that the matrix fragments as a result of RNase treatment (for review, see Nickerson et al., 1990). In contrast, the nuclear matrices from heated cells are stable following exhaustive digestion with RNase (Wright et al., 1989).

1. Reagents 1. Exponentially growing HeLa cells in suspension with Joklik modified minimal essential medium (MEM), supplemented with 3.5% calf serum and 3.5% fetal bovine serum. 2. 0.01 M PBS (pH 7.4): For 1 liter of 10× stock solution, 80 g NaCI, 2 g KC1, 11.5 g Na2HPO4" 7H20, 2 g KH2PO4. 3. Lysis buffer: 1% Triton X-100, 80 mM NaCI, 10 mM EDTA, pH 7.2. 4. TMNP buffer: 10 mM Tris, pH 7.4, 5 mM Mg2C1, 0.1 mM PMSF. 5. 5 mg/ml DNase I stock: Add 1 ml 50% glycerol to 5 mg DNase I (Worthington Biochemical, DPRF grade), store at -20°C. 6. High salt buffer: 3 M NaCI, 10 mM EDTA, 10 mM Tris, pH 9.0, 20 mM /3-mercaptoethanol. 7. RNase A: 1 mg/ml in TMNP, boil for 5 min before use. 8. Staining solution: TMNP buffer with 3 mg/ml FITC and 35/xl/ml PI (Sigma, St. Louis, MO).

2. Procedures 1. Collect HeLa cells growing in suspension by centrifugation, then resuspend in prewarmed 45°C medium at 10 times their original concentration, then submerse in a 45°C water bath. 2. Place cells in an ice bath for 2 min. 3. Centrifuge at 240 g for 5 min. 4. Wash three times with PBS by resuspending the pellet, and centrifuging at 240 g. 5. Wash pellet three times in lysis buffer by resuspending the pellet, and centrifuging at 1000 g. 6. Wash pellet twice with TMNP buffer, by resuspending the pellet, and centrifuging at 1000 g.

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7. Resuspend the final pellet in TMNP to a concentration of 2 × 106 cell nuclei per ml. 8. Add DNase I to a final concentration of 130/zg/ml. Incubate for 2 hr at 37°C. 9. Centrifuge 5 min at 1000 g. 10. Resuspend pellet to original volume with TMNP, and centrifuge again. 11. Resuspend pellet in 1/8 starting volume of TMNP and add 5 volumes of high salt buffer. Incubate 10 min at room temperature. 12. Centrifuge as in Step 9. 13. Wash twice using original volume of TMNP. 14. Digest the nuclear matrices with RNase by resuspending 2-3 × 106 particles in 200 ffl TMNP. Separate samples are digested with a final RNase concentration of 0.001, 0.01, 0.1, 1, 10, and 100/,g/ml RNase. Digestion is carried out at 0°C for 30 min. 15. Collect nuclear matrices by centrifugation at 1000 g for 5 min. 16. Resuspend pellet in the staining solution, and incubate on ice for at least 1 hr. 17. Analyze matrices by FCM, collecting FITC fluorescence, light scatter, and PI fluorescence.

3. Special Considerations 1. The differential stability between nuclear matrices from control and heated cells can cause yield differences, which, if large enough, can alter dye binding characteristics. Therefore, it is important to count the matrices recovered with a Coulter counter or a hemocytometer as a means to compute yield differences to adjust the number of particles in each sample. Alternatively, cell sorting can be used to ensure that the same number of matrices are compared in samples from control and heated cells. 2. Nuclear matrices from rodent cell lines tend to be less stable than those from human cell lines. 3. This protocol can be adapted for cells growing in monolayer.

4. Instruments 1. Nuclear matrices from HeLa cells retain enough double-stranded RNA to be detected with PI. Thus, the excitation and detection configuration described earlier can be used. However, fluorescence compensation is a must because the PI signal will be weak.

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2. Even when both the PI and FITC signals become weak, the nuclear matrices can be detected by forward light scatter (unless they have become dissociated).

IV. Identification o f Altered D N A Replication Patterns Following Heat Shock DNA replication is organized temporally and spatially within the nuclei during S phase (Ma et al., 1998). To study this process following heat shock, aphidicolin (APH) is used to synchronize HeLa cells at the G1/S phase border. Cells are given heat shock (i.e., 15-30 min at 45°C) and then allowed to move through S phase. Periodically during the recovery from heat shock, cells are pulse-labeled with bromodeoxyuridine (BrdU), harvested, and the morphology of DNA replication determined using immunohistochemical methods. Our choice of primary antibody permits the immunostaining of incorporated BrdU without the harsh DNA denaturation steps with HC1 or NaOH required by other procedures that may alter the morphology of DNA replication factories (Taragi et al., 1993).

1. Reagents 1. HeLa cells cultured glass coverslips in 25-mm tissue culture dishes, in F10 medium supplemented with 10% calf serum. One dish with several coverslips are required for each time point. 2. 30 /,M APH (10× stock): 1 mg APH is dissolved in a small volume of DMSO, then brought to 98 ml with F10 medium. The stock solution can be divided into small volumes and stored at -20°C for several months. The stock solution is diluted 10-fold with complete F-10 medium (i.e., supplemented with 10% calf serum) immediately before use. 3. Fixing solution: Histochoice (Amresco, Solon, OH) fixative supplemented with 5% acetic acid and 0.5% Triton X-100. 4. 0.01 M PBS (pH 7.4): For 1 liter of 10× stock solution, 80 g NaC1, 2 g KC1, 11.5 g Na:HPO4-7HeO, 2 g KH2PO4. 5. 1 m M BrdU (100×). 6. Blocking reagent: 1% bovine serum albumin, 10% normal goat serum in PBS. 7. Primary antibody: Anti-bromodeoxyuridine monoclonal antibody mouse plus nuclease (Amersham, Arlington Heights, IL, RPN 202). 8. Secondary antibody: Sheep anti-mouse immunoglobulin G (IgG), Cy-3 conjugate (Sigma), diluted 1 : 1000 with blocking reagent. 9. FITC staining solution: 10 txg/ml FITC in PBS.

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2. Procedures 1. Culture cells in the APH containing medium for 15 to 20 hr at 37°C to align them at the G1/S border. 2. Remove APH by three quick washes with prewarmed complete F10 medium. Replace the medium and return the cells to a 37°C incubator. Allow cells to recover from the APH block for approximately 30 min prior to exposure to heat shock. Without this recovery time, the cells fail to resume DNA replication. 3. For heating, seal the culture dishes with Parafilm, and submerge in a 45°C water bath for 15 or 30 min. At the completion of heat exposure, return culture dishes to a 37°C incubator. 4. At each time point, label cells for 5 rain by adding 10/xl of BrdU solution per milliliter of medium and wash with PBS. BrdU labeling is conducted immediately after heat shock and every 1-2 hr thereafter. 5. Fix cells by submerging coverslips in the fixative solution for 30 min. 6. Following fixation replace the fixative with PBS. The cells can be stored in PBS for several days at 4°C. 7. Remove the coverslips from the PBS solution. 8. Place 100 ~1 blocking reagent on the cells, and incubate cells for 15 rain. 9. Aspirate the blocking reagent and replace with 50 txl primary antibody for 1 hr. 10. Aspirate the primary antibody off the cells and quickly wash with blocking reagent three times, followed by three additional washes of 5 min each. 11. Apply diluted secondary antibody to the cells and incubate for 30 min. 12. Remove secondary antibody and wash the cells six times with blocking reagent as in Step 9, above. 13. Counterstain by applying FITC staining solution for 5 min, then rinse well with PBS. 14. For mounting on glass slides, place 7/xl of PBS on the slide, and gently place the coverslip, cell side down on the PBS. Seal the coverslip with fingernail polish. The slides can be stored in this condition for several months at 4°C. Alternatively, the mounting medium, described in the next section, can be used. 15. Visualize nuclear sites of DNA replication under a fluorescence microscope using a rhodamine filter cube. 3. Special Considerations 1. All steps are carried out at room temperature, except as noted otherwise. 2. Careful attention should be paid to ensure that the cells do not dry out at any time.

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4. Instrumentation A fluorescent microscope equipped with a rhodamine and FITC filter cubes.

V . N u c l e a r L o c a l i z a t i o n o f hsp70 A. Flow Cytometric Detection of Nuclear hsp/hsc70 1. Level of hsp/hsc 70 Associated with Nuclei Nuclei from heated and control cells are isolated with the procedures described earlier. Nuclei can be used immediately, or they can be fixed in 70% cold ethanol or methanol. It should be noted, however, that fixing nuclei in alcohol results in excessive clumping of nuclear particles, especially if they are not clean nuclei and have numerous cytoplasmic tabs remaining. 2. Reagents 1. Primary antibody: Mouse anti-hsp70 antibody clone C92F3A-5 (diluted 1:100 in PBS). 2. Secondary antibody: Goat anti-mouse IgG, FITC conjugate (diluted 1 : 100 in PBS). 3. RNase A: 100/xg/ml in ddH20, boil for 5 min before use. 3. Procedures 1. 2. 3. 4. 5. 6. 7.

Wash isolated nuclei (fixed or unfixed) with PBS. Label with the primary antibody for 1 hr at room temperature. Wash twice with PBS. Label with the secondary antibody for 1 hr at room temperature. Wash twice with PBS. Treat with RNase A for 30 min at room temperature. Stain with 25/zg/ml PI for 1 hr.

4. Special Considerations The assay can be applied to isolated nuclear matrix. Because the nuclear matrix is more fragile than the nucleus, more care should be paid in its handling as described earlier. 5. Instruments Flow cytometry can be set up for routine dual parameter PI/FITC data acquisition as described for nuclei (earlier).

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B. Nuclear Localization o f hsp70 by in Situ Imrnunofluorescence Although the FCM methods provide a rapid measurement of nuclear hsc/ hsp70 amounts, this method provides a visualization of intranuclear hsp70 localization and will allow a comparison of relative amounts in the cytoplasm and nucleus. Both methods provide unique information and complement each other well. 1. Reagents 1. 0.01 M PBS (pH 7.4): For 1 liter of 10X stock solution, 80 g NaC1, 2 g KC1, 11.5 g NazHPO4 • 7H20, 2 g KH2PO4. 2. 3.7% formaldehyde fixative solution: for 100 ml 3.7% formaldehyde solution, 10 ml 37% formaldehyde, 0.2 ml Triton X-100, and 89.8 ml 0.01 M PBS. 3. Absolute acetone: stored at -20°C. 4. Blocking solution: 0.01 M PBS with 10% goat serum. 5. Mouse anti-hsp70 monoclonal antibody (StressGen, Victoria, Canada, product SPA-810). Working concentration is 1:80 and diluted in blocking buffer. 6. FITC-conjugated goat anti-mouse antibody (Becton Dickinson, San Jose, CA, product 347580). Working concentration is 1 : 100 and diluted in blocking buffer. 7. Mounting medium: 2.4 g Mowiol 4-88 (Calbiochem 475904), 6 g glycerol (analytical grade), 6 ml doubly distilled H20, 12 ml 0.2 M Tris buffer, pH 8.5, mixed for 4 hr on a shaker; then the solution is incubated in water bath at 50°C for 10 min with occasional stirring to dissolve the Mowiol 4-88. Centrifuge the mixture at 5000 g for 15 rain, divide the supernatant into aliquots, and store in -20°C (Heimer and Taylor, 1974).

2. Staining Procedure 1. Culture cells on the coverslips in the tissue culture dish for 48 hr, then shift to 41.1° from 37°C for different periods of time. 2. Wash in three changes of PBS at room temperature for 5 min each. 3. Fix cells with fixative solution for 15 min. 4. Wash in three changes of PBS at room temperature for 5 min each. 5. Incubate the cells with cold absolute acetone (keep at -20°C) for 10 rain for permeabilization at room temperature. 6. Wash in three changes of PBS at room temperature for 5 min each. 7. Incubate cells with blocking solution for 30 min. 8. Remove the blocking solution, add mouse anti-hsp70 antibody, and incubate for 1 hr at 37°C.

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9. Wash in three changes of PBS at room temperature for 5 min each. 10. Wash in three changes of cold PBS (4°C) for 5 min each. 11. Incubate the cells with FITC-conjugated goat anti-mouse antibody for 40 rain at room temperature. 12. Wash in three changes of room temperature PBS for 5 rain each. 13. Place a small drop of mounting medium with a needle on the slide and mount the coverslip on it. 14. Keep the slides in dark and observe under a fluorescence microscope.

3. Special Considerations 1. The cell number seeded in the dish containing coverslips has to be optimized considering the growth rate of cells and area of the dish bottom. Too many or too few cells will cause problems in the observation of the fluorescent cells. To achieve an optimal condition, we used 35 x 10 mm tissue culture dishes and placed four coverslips in each dish along with 2 ml medium containing 5 × 104 cells. 2. The washing step must be very gentle. When using the 24-well plate with one coverslip per well, wash each well with 1 ml of PBS for each washing step. Cells, especially heat sensitive cells, are more easily detached from the coverslip after heat treatment.

4. Instruments A fluorescent microscope equipped with an FITC fluorescence cube.

VI. P r o o x i d a n t M e a s u r e m e n t Much indirect evidence has suggested that heat stress and oxidative stress have some commonalties. We report here a method to directly measure the oxidative potential of the intracellular milieu following heat shock. 1. Reagents Labeling solution: 5-(and-6)-carboxy-2',7'dichlorodihydrofluorescein diacetate (carboxy-HaDCFDA): dissolved in DMSO and diluted to 10 tzg/ml in PBS (pH 7.2) containing 1 mg/ml glucose. 2. Staining Procedure 1. Suspend monodispersed cells (1 to 3 × 106) in 15 ml of labeling solution and incubate for 30 min at 37°C.

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2. Centrifuge and remove labeling solution, and resuspend cells in 1 ml of fresh labeling solution and keep on ice. 3. Special Considerations Three steps are involved for the probe to be used as an indicator of cellular oxidative activity. They are (1) the transport through the cellular membrane into the cell, (2) the cleavage of ester groups by intracellular esterases to deter its leakage out of the cell, and (3) the oxidation of the probe to become a fluorescent molecule. Hyperthermia is known to affect membrane permeability. To ensure that changes in fluorescence intensity is due to changes in oxidative activity and not due to other cellular properties, it is necessary to use an appropriate control. To this end the use of a probe such as 5-(and 6-)carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA), which does not require oxidation to become fluorescent but retains other characteristics of 5-(and 6-)carboxy-2',7'-dichlorodihydroftuorescein diacetate, is recommended as a parallel control. In some cases the fluorescence intensity from the oxidation of the probe is very weak. Therefore, it is also recommended that the background autofluorescence should be monitored at each time point and subtracted to obtain net fluorescence intensity. 4. Instruments The fluorescent dyes are excited with the 488 nm line of an argon laser. Fluorescence is detected through a 525 nm band-pass filter. VII. Results A. Nuclear Protein Content The amount of protein that coisolates with isolated nuclei from H e L a cells exposed to varying temperature is shown in Fig. 1. The increase in the amount of nuclear protein is dependent on exposure temperature and time, especially at the temperatures of 43°C or above. In many cell lines, the amount of protein correlates with cell killing. At 42°C or lower the increase is seen to plateau. The increase ceased immediately on the removal of elevated temperature, and the excess proteins are removed during incubation at 37°C. As the excess nuclear proteins are removed, macromolecular synthesis resumes; for example, when the nuclear protein content is reduced to 1.25 times that of the control the rate of D N A synthesis recovers to 25% of control (Higashikubo and Roti Roti, 1993). B. Pattern o f D N A Replication Three distinct patterns of D N A replication factories are evident as mammalian cells progress through S phase (Fig. 2). Type I occurs early to mid S phase, as

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Fig. 1 Effects of heat on nuclear protein content. HeLa cells were exposed to the various timetemperature combinations indicated on the graph, and their nuclei were isolated and stained for DNA and protein content according to published methods (Roti Roti et al., 1982). The relative nuclear protein content (control = 1) is plotted versus heating time for each temperature. The plotted points represent the mean of at least three repeated experiments; error bars have been omitted for clarity. Reproduced with permission from Roti Roti and Laszlo, 1988.

the cells are replicating euchromatin. N u m e r o u s small foci are evident over regions of euchromatin. Initiation of heterochromatin synthesis starting in mid S phase results in a Type II pattern where the replication factories are concentrated around the nuclear periphery, nucleoli, and other heterochromatin domains. During late S phase a Type III D N A replication pattern is seen where factories b e c o m e less numerous, are larger, and are found within the heterochromatin regions. Following heat shock, the distinctive T y p e II pattern is not observed. Instead, an altered pattern (Type II/III) appears, characterized by substantial D N A replication in heterochromatin regions. D u a l labeling will be n e e d e d to resolve T y p e II and Type III D N A replication patterns in heated cells. This technique is currently under development.

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DNA Replication Morphology

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Fig. 2 DNA replication patterns observed in control and heat-shockedHeLa cells. Type I replication pattern replicates euchromatin,leavingheterochromaticregions (nucleoli)bright green. Types II and III (and unresolvedII and III followingheat shock)are involvedin heterochromatinreplication, resulting in the yellow nucleolar region (red plus green). Photographed at 1000 × magnification. (See color plates.)

C. hsp/hsc70 Expression of hsp70 associated with nuclei and nuclear matrices following exposure of Chinese hamster ovary cells to heat shock of 45°C for 30 min is detected by flow cytometry and shown in Fig. 3. Heat-induced increases in the association of the proteins are clearly indicated in both cases. Although cellcycle dependent D N A content was preserved in nuclei, D N A content distribution is amorphous in nuclear matrices following exhaustive DNase digestion. The localization of hsp70 is a very useful parameter. Following heat shock, hsp70 is found in the nucleus. The rate of delocalization from the nucleus after heat shock correlates with the thermal resistance of the cell. In heat-resistant cells the delocalization is faster, whereas in heat-sensitive cells, it is slower (Ohtsuka and Laszlo, 1992), although hspT0 exhibits nuclear localization, the lack of nucleolar staining appears to correlate with survival for continuous heating at 41.1°C (see Fig. 4) (Xu et al., 1998). D. P r o o x i d a n t P r o d u c t i o n

The fluorescence intensity should be corrected by subtracting background and normalizing to that of control unheated cells. Prooxidant production is expected

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Fig. 4 (Right) Intensive cytoplasmic immunocytochemical staining pattern of hsp70 from NSY42129 cells heated at 41.1°C for 16 hr. (Left) Nucleolar punctate staining pattern of hsp70 from transformed fibroblast (TF) cells heated at 41.1°C for 16 hr. Photographed at 1000 x magnification.

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to v a r y d e p e n d i n g o n h e a t i n g p r o t o c o l , t h a t is, t e m p e r a t u r e a n d h e a t i n g d u r a t i o n , as w e l l as cell types. I n m o s t cases, h o w e v e r , w e f o u n d t h a t p r o o x i d a n t p r o d u c t i o n d e c r e a s e s i n i t i a l l y o n h e a t t r e a t m e n t a n d r e c o v e r s to a n d s u r p a s s e s t h e c o n t r o l l e v e l d u r i n g t h e p o s t h e a t r e c o v e r y f o l l o w i n g a c u t e h e a t s h o c k (i.e., 4 5 ° C f o r 15 m i n ) o r t h e a d j u s t m e n t p e r i o d d u r i n g l o n g d u r a t i o n m o d e r a t e h y p e r t h e r m i a (i.e., 24 h r at 41.5°C).

References Heimer, C. V., and Taylor, C. E. (1974). Improved mountant for immunofluorescence preparations. J. Clin. Pathol. 27, 254-256. Henle, K. J., and Roti Roti, J. L. (1988). Response of cultured mammalian cells to hyperthermia. In "Hyperthermia and Oncology, Thermal Effects on Cells and Tissues" (M. Urano and E. Douple, eds.), Vol. 1, pp. 57-82. VSP, Utrecht, The Netherlands. Higashikubo, R., and Roti Roti, J. L. (1993). Alterations in nuclear protein mass and macromolecular synthesis following heat shock. Radiat. Res. 134, 193-201. Higashikubo, R., Wright, W. D., and Roti Roti, J. L. (1990). Flow cytometric methods for studying isolated nuclei: DNA accessibility to DNase I and protein-DNA content. In "Methods in Cell Biology" (Z. Darzynkiewicz and H. A. Crissman, eds.), Vol. 33, pp. 325-336. Academic Press, New York. Kampinga, H. H. (1993). Thermotolerance in mammalian cells: Protein denaturation and aggregation, and stress proteins. J. Cell Sci. 104, 11-17. Laszlo, A. (1992). The effects of hyperthermia on mammalian cell structure and function. J. Cell. Prolif. 25, 59-87. Ma, H., Sanmarabandu, S., Devdhan, R. S., Acharya, R., Cheng, P.-C., Meng, C., and Berezney, R. (1998). Spatial and temporal dynamics of DNA replication sites in mammalian cells. 3. Cell Biol. 143, 1415-1425. Miller, M. W., and Ziskin, M. C. (1989). Biological consequences of hyperthermia. Ultrasound Med. Biol. 15, 707-722. Nickerson, J. A., He, D., Fey, E. G., and Penman, S. (1990). The nuclear matrix. In "The Eukaryotic Nucleus" (P. R. Strauss and S. H. Wilson, eds.), Vol. 2, pp. 763-782. The Telford Press, Caldwell, NJ. Ohtsuka, K., and Laszlo, A. (1992). The relationship between hsp70 localization and heat resistance. Exp. Cell Res. 202, 507-518. Roti Roti, J.L., and Laszlo, A. (1988). The effects of hyperthermia on cellular macromolecules. In "Hyperthermia and Oncology, Thermal Effects on Cells and Tissues" (M. Urano and E. Douple, eds.), Vol. 1, pp. 13-56. VSP, Utrecht, The Netherlands. Roti Roti, J. L., Higashikubo, R., Blair, O. C., and Uygur, N. (1982). Cell-cycle position and nuclear protein content. Cytometry 3, 91-96. Roti Roti, J. L., Wright, W. D., and VanderWaal, R. (1997). The nuclear matrix: A target for heat shock effects and a determinant for stress response. Critical Rev. Eukaryotic Gene Expression 7, 343-360. Taragi, S., McFadden, M. L., Humphreys, R. E., Woda, B. A., and Sairenji, T. (1993). Detection of 5-bromo-2-deoxyuridine (BrdUrd) incorporation with monoclonal anti-BrdUrd antibody after deoxyribonuclease treatment. Cytometry 14, 640-648. Urano, M., and Douple, E. (eds.). (1988). Thermal effects on cells and tissues. In "Hyperthermia and Oncology," Vol. 1. VSP, Utrecht, The Netherlands. Urano, M., and Douple, E. (eds.). (1989). Biology of the thermal potentiation of radiotherapy. In "Hyperthermia and Oncology," Vol. 2. VSP, Utrecht, The Netherlands. VanderWaal, R., Thampy, G., Wright, W. D., and Roti Roti, J. L. (1996). Heat-induced modifications in the association of specific proteins with the nuclear matrix. Radiat. Res. 145, 746-753. Warters, R. L., and Roti Roti, J. L. (1982). Hyperthermia and the cell nucleus. Radiat. Res. 92, 458-462.

286

Robert P. VanderWaal et al. Wright, N. T., Chen, S. S., and Humphrey, J. D, (1998). Time-temperature equivalence of heatinduced changes in cells and proteins. Trans. A S M E 120, 2-26. Wright, W. D., Higashikubo, R., and Roti Roti, J. L. (1989). Flow cytometric studies of the nuclear matrix. Cytometry 10, 303-311. Xu, M., Wright, W. D., Higashikubo, R., and Roti Roti, J. L. (1998). Intracellular distribution of hsp70 during long duration moderate hyperthermia. Int. J. Hyperthermia 14, 211-225.