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Ann Thorac Surg 2000;69:1675-1680
© 2000 The Society of Thoracic Surgeons

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Original articles: General thoracic

Oncogenic ras results in increased cell kill due to defective thermoprotection in lung cancer cells

^a Department of Surgery, The University of Texas Medical Branch, Galveston, Texas, USA
^b Department of Pathology, The University of Texas Medical Branch, Galveston, Texas, USA

Address reprint requests to Dr Vertrees, Department of Surgery, The University of Texas Medical Branch, 301 University Blvd, Galveston, TX 77555-0528
e-mail: rvertree{at}utmb.edu

Presented at the Forty-sixth Annual Meeting of the Southern Thoracic Surgical Association, San Juan, Puerto Rico, Nov 4–6, 1999.

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. The survival response of normal cells to heat stress is an upregulation of heat shock proteins and ras protein activation. We hypothesized that in lung cancer cells the presence of oncogenic ras interferes with thermoprotective mechanisms resulting in cell death.

Methods. An equal number of lung tissue culture cells (normal and cancerous) were subjected to either heat stress and then recovery (43°C for 180 minutes, 37°C for 180 minutes) or recovery alone (37°C for 360 minutes). End points were surviving number of cells, cell-death time course, heat shock protein (HSP70, HSC70, HSP27) expression before and after heat stress, and time course for HSP70 expression during heat stress and recovery. Heated cells were compared with unheated control cells, then this difference was compared between cell types.

Results. Heat stress in normal cells caused an 8% decrease in cell number versus a 78% ± 5% decrease in cancer cells (p < 0.05). In normal cells, heat stress caused a 4.4-fold increase in HSP70, no change in HSC70, and a 1.7-fold increase in HSP27. In contrast, cancer cells initially contained significantly less HSP70 (p < 0.05), and there was a 27-fold increase in HSP70 and a 2-fold increase in HSC70 with no HSP27 detected (comparison significant, p < 0.05). HSP70 time course in normal cells showed that HSP70 increased 100-fold, reaching a vertex at 2 hours and remaining elevated for 24 hours; in cancer cells, HSP70 maximum expression (100-fold) peaked at 5 hours, then decreased to slightly elevated at 24 hours.

Conclusions. Cancer cells with oncogenic ras have defective thermoprotective mechanism(s) causing increased in vitro cell death, which provides an opportunity for thermal treatment of lung cancer.

	Introduction

Top Abstract Introduction Material and methods Results Comment References

 
The survival response to heat stress in normal cells which confers thermoprotection consists of both ras^* activation [1, 2] and heat shock protein upregulation [3]. In contrast, the survival response is less well orchestrated in cancer cells and the outcome less predictable [4]. If cancer cells are more susceptible or can be made more susceptible to heat-induced cell death than normal cells, or if hyperthermia could potentiate the cell-killing ability of radiation or chemotherapy, then hyperthermia could become an adjuvant in the treatment of cancer [5, 6].

The Ras proteins (ras, p21) are 21-kDa GTPases which function as molecular switches relaying signals from the plasma membrane through protein pathways to the nucleus. The Ras superfamily of proteins is separated into three subfamilies (Ras, Rho, and Rab), each further divided into groups according to amino acid sequence homology. The Ras subfamily contains various specific isoforms (eg, H-Ras, K-Ras, N-Ras).

The result of the ras-initiated cell survival response is dependent upon the specificity of downstream signaling pathways. That is, ras can stimulate either the mitogen-activated protein kinase (MAPK) family of kinases resulting in cell growth and differentiation, or the stress-activated protein kinase (SAPK) family of kinases resulting in cell death [7]. Ras, the predominant oncogene in lung cancer, was the first human oncogene discovered [8]. Mutant forms of ras have an increased transforming (cancer inducing) capacity and a stimulating effect on downstream protein pathways [9]. Evidence suggests that the effect of heat shock on cells with abnormal ras expression is an increased cell death (thermosensitivity) due to a reduction in heat shock gene expression [10].

All mammalian cells examined to date respond to heat stress (5C° to 6C° above the ambient optimal growth temperature) by upregulation of a large family of proteins—heat shock proteins; this has been positively correlated with increased cell survival [11]. Heat shock proteins are present in the cell in two forms, constitutive and inducible. When not stimulated, constitutive heat shock proteins are in a complex with the heat shock transcription factor (HSF); when stimulated by the presence of denatured proteins, the heat shock protein is released from the HSF. The HSF migrates to the nucleus, where it stimulates the synthesis of the inducible form of the heat shock protein in a negative-feedback cycle [12]. Heat shock proteins perform a dual role in cell survival; the immediate upregulation of heat shock proteins results in protection against the initial heat insult (thermoresistance), whereas an accumulation causes a transiently prolonged increased resistance to subsequent heat insults (thermotolerance) [13]. Heat shock protein expression in cancerous pancreatic and gastrointestinal tissue varies from that of normal pancreatic and gastrointestinal tissue [4, 14]. Therefore, we speculated that a difference in heat shock protein expression exists between normal and cancerous lung cells, which if present, may facilitate the selective destruction of lung cancer cells by hyperthermia.

The purpose of this study was to determine if ras-transformed lung cells are more thermosensitive than their normal counterparts and to evaluate the role of heat shock proteins in this increased thermosensitivity. Both oncogenic ras-transformed (cancer cells) and normal lung cells were exposed to a thermal dose of 43°C for 180 minutes; studies included cell-death time course, appropriate thermal dose for heat stress, and correlation of cell death to HSP70 time course. We demonstrate that lung cells with the ras oncogene present showed a decreased cell survival due to a defective thermoprotective mechanism.

	Material and methods

Top Abstract Introduction Material and methods Results Comment References

 
Experimental design
In all studies, results were compared between the noncancerous (BEAS2-B) and cancerous (BZR-T33) human lung cell lines. Initially Western blotting was used to determine and monitor levels of H-ras, p21 in the cell lines. The first in the viability series of studies counted and compared the number of cells surviving heat shock between cancerous and noncancerous groups. Follow-up studies were used to clarify these results and included (1) the use of molecular stains for both live and dead cells, (2) morphologic studies with a light microscope, and (3) viability assessment of detached cells. Once heat-induced cell death had been quantified, the intracellular expression of various heat shock proteins was evaluated. This study was carried out in two types of experiments: (1) expression of heat shock proteins compared before and after heat stress and (2) time course of HSP70 expression. Once an increased thermosensitivity was demonstrated in the transformed cell line, we determined its frequency in 12 different cancerous and normal lung tissue cell lines.

Cell culture
All studies were conducted with the following two cell lines. BEAS2-B (a family of cell lines generously donated by Curtis C. Harris, MD, National Cancer Institute, Bethesda, MD) is an SV40 T-antigen-positive immortalized normal human bronchial epithelial cell line. This cell line was neoplastically transformed by the insertion of the coding region of the v-Ha-ras oncogene into the Zip-NEOSV(x) retroviral vector resulting in a new line, BZR-T33. BZR-T33 displays a malignant phenotype in cell culture and animal studies [15–17]. The following cell lines were studied for thermosensitivity. Human lung cancer cells used were CaLU-3 (ATCC HTB 55), adenocarcinoma of the lung; ChagoK-1 (ATCC HTB 168), undifferentiated bronchogenic carcinoma; and NCI-H596 (ATCC HTB 178), adenosquamous carcinoma of the lung. Normal human lung cells used were CCD-19Lu (ATCC CCL 210), a fibroblast-like cell line derived from normal human lung tissue, and WI-38 (ATCC CCL 75), normal human embryonic lung tissue. Transformed cell lines of human origin were NeoD₄, a transfectant of NCl H484 (ATCC HTB 175), a small cell lung cancer, and three ras (c-Ha-ras, G12V) mutants of LB9, L27, and S₃D₃.

The cells were cultured in 95% humidified air, 5% CO₂ incubators at 37°C in 90% recommended media, 10% fetal bovine serum. Heat shock treatments were performed by placing prepared culture vessels into a preheated incubator maintained at 43.3°C, 5% CO₂, and 95% relative humidity. Cell count was accomplished using the Coulter counter (Model Z_F, Coulter Electronics, Inc, Hialeah, FL).

Cell viability
Experiments used a Live/Dead Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR). Live cells stain positively when the nonfluorescent cell-permeant calcein AM is enzymatically converted by intracellular esterase activity to the green fluorescent calcein (ex/em 495 nm/515 nm). Ethidium homodimer that is excluded by intact cell membranes enters cells with damaged membranes where it undergoes a 40-fold increase in fluorescence when binding to nucleic acids and produces a bright red fluorescence (ex/em 495 nm/~635 nm).

Western blot analysis
Briefly, cells were harvested by scraping and protein was extracted and stored at -80°C. The Bradford assay was used for protein quantitation. Each lane was loaded with 25 µL of cell lysate. Gels used were 12% Tris-HCl Bio-Rad Ready Gels (Bio-Rad Laboratories, Hercules, CA) with standards. The primary antibodies (1:100) used were anti-H-ras specific for H-Ras 21, anti-HSP70, p72 specific for HSP70 p72, anti-HSC70 specific for HSC70, p73, and anti-HSP27 specific for HSP27, p27, (Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were incubated at room temperature for 2 hours with a compatible secondary antibody (1:1,000 to 1:5,000). Bands were visualized with chemiluminescence luminal reagent (sc-2048; Santa Cruz Biotechnology). Each individual blot result was analyzed by UNSCAN-IT software (version 5.1; Silk Scientific, Orem, UT). This software provides a densimetric analysis and comparison between lanes of the relative intensity of the bands formed. Results are displayed both as bands and as graphed data.

Statistical analyses
Values are expressed as the mean ± standard error of the mean. When comparing cell numbers before and after heat shock, the paired t test was used. When comparing heated cells with unheated control cells, or when comparing results between cell lines, either the unpaired Student’s t test or one-way analysis of variance (ANOVA) was used. Significant differences were detected at p < 0.05.

	Results

Top Abstract Introduction Material and methods Results Comment References

 
Confirmation of increased H-ras, p21 in BZR-T33 cell lines
The ratio of H-ras, p21 expression between BEAS2-B and BZR-T33 cell lines was consistent as monitored approximately every five passages throughout these experiments. The immunoblot revealed that there was four times more H-ras, p21 in the unheated BZR-T33 (75,009 pixels) than in the unheated BEAS2-B (19,022 pixels) cells, and that heat stress did not affect this difference (p > 0.05).

Viability of BEAS2-B is greater than BZR-T33 cells after heat stress
More BEAS2-B cells survived heat stress time course
Cell count was procured hourly during the treatment interval (6 hours) and 18 hours later and compared between BEAS2-B and BZR-T33 cell lines. The heat treatment appeared to have had no deleterious effect on the BEAS2-B cell line, whereas there was an immediate reduction in the number of BZR-T33 cells after 1 hour of heat exposure (Fig 1). At 24 hours the BEAS2-B cells essentially doubled in number, whereas the BZR-T33 cells showed a 50% heat-related reduction in cell number.

View larger version (17K): [in this window] [in a new window]   Fig 1. The time course relating cell survival (percentage of starting number of cells) to heat dose revealed that there was no deleterious effect of heat stress on the BEAS2-B cell line. In contrast, the BZR-T33 cell line displayed a reduction in cell number after 1 hour of heat stress which eventually reduced this population by 50%.

Detached cells are nonviable
After the standard heat stress treatment and the 3-hour reincubation period, detached (floating) cells were removed and placed into new T-175 flasks. Newly plated floating cells were allowed to grow for 7 days and then a final count was determined. For both cell lines, survival of detached cells after 7 days was less than 1% of the original number of plated cells, indicating that detached cells were not viable.

Heat shock protein levels are different in BZR-T33 compared with BEAS2-B cells
More HSP70, p72 is present in BEAS2-B cells before heat shock
HSP70 was quantitatively assessed in both cell lines before and after heat shock and recovery and then this difference was compared between cell lines. Figure 2A shows the result of the Western blot for HSP70 and reveals that there is significantly more HSP70 present in BEAS2-B cells under nonheated conditions (BEAS2-B [2^C], 6,021 pixels versus BZR-T33 [T33^C], 1,567 pixels, p < 0.05). After the recovery phase of heat stress, BEAS2-B (2^H) cells displayed a 4.4-fold increase (from 6,021 to 26,487 pixels, p < 0.05), whereas BZR-T33 (T33^H) cells displayed a 27.9-fold increase (from 1,567 to 42,175 pixels, p < 0.05) (significant differences, ANOVA, p < 0.05).

View larger version (82K): [in this window] [in a new window]   Fig 2. Comparison between cell lines for the effect of heat stress on expression of three specific heat shock proteins. (A) More HSP70, p72 is present in the normal cell line (2C) before heat shock. Heat shock resulted in an increase in both cell lines; the increase in the BZR-T33 (T33H) was significantly greater. (B) More HSC70, p73 is present in the normal cell line (2C). Heat stress resulted in a significant increase in the expression of the transformed cell line (T33H). (C) Heat shock resulted in a significant increase in only the BEAS2-B (2H) cells.

No HSP27, p27 was detected either before or after treatment in BZR-T33 cells
Figure 2C shows no detectable amounts of HSP27, p27 in either cell line before heat shock. As a result of heat shock and recovery there was minimally a 173% increase (from 470 [background] to 8,120 pixels, p < 0.05) in HSP27, p27 in BEAS2-B (2^H) cells and still no detectable presence in the BZR-T33 (T33^H) cells.

HSP70, p72 time course is different between cell lines
The time course for expression of HSP70, p72 during heat shock and recovery in BEAS2-B and BZR-T33 cell lines is shown in Figure 3. Plates with equal numbers of cells were removed hourly (1, 2, and 3 hours of heat stress and 1, 2, and 3 hours of recovery, and 18 hours later), and cell count, protein extraction, and Western blot analysis were performed. By correlating band densities to cell number per time point it was possible to determine that the BEAS2-B cell line contained significantly (p < 0.05) more HSP70 initially, expressed significantly higher levels of HSP70 sooner, and sustained elevated levels longer compared with the BZR-T33 cell line. The amount of HSP70 in BZR-T33 cells appeared to reach maximum expression slowly.

View larger version (15K): [in this window] [in a new window]   Fig 3. Comparison of the time course for production of HSP70, p72. Cells were harvested and protein collected for each hour of heat stress (1, 2, and 3 hours) and recovery (1, 2, and 3 hours) and 18 hours later. Densimetric analysis of the Western blot showed two different responses to the heat stress treatment. Optical density (OD)/cell number was significantly (p < 0.05) higher in BEAS2-B cells. The results showed a delayed and more feeble heat stress response mustered by the BZR-T33 cell line at 1 to 5 hours which declined over the next 19 hours. In BEAS2-B cells, the response was immediate, prolonged, and maximal 24 hours later.

View larger version (48K): [in this window] [in a new window]   Fig 4. Results of survey technique revealed that in human lung cancer cell lines a mutant (mt.) ras was associated with a significant reduction (*p < 0.05) in the number of cells surviving heat stress (43°C for 180 minutes). The ras status of ChagoK-1 and NCl-H596 cell lines is currently undetermined, however, their response to the heat stress was very similar to those cell lines with mutant ras. (wt = wild type.)

	Comment

Top Abstract Introduction Material and methods Results Comment References

We quantified the number of surviving cells and compared this number between various cancerous and noncancerous cell lines and in all cases determined that noncancerous cells had a greater number of surviving cells (Fig 4). Our data are in agreement with Li and colleagues [19], who reported that rat embryo cells transfected with c-myc gene and resulting in a malignant phenotype were more susceptible to heat-induced cell death than their parental cell line. In addition, Haveman and associates [20] reported a reduced cloning ability of human lung cancer cells after 1 hour at 43°C. Mitsudomi and coworkers [21] found variable thermosensitivity when they transfected rat fibroblast cell lines with seven known oncogenic agents (including Ha-ras); in all cases the parental cell line (noncancerous) was least thermosensitive. They also determined that the increased thermosensitivity in the transformed cell lines was not due to characteristics usually associated with this phenotype such as higher saturation density, faster growth rate, or anchorage independence.

Our data indicate that 60% more transformed and cancerous than noncancerous cells were killed outright by the heat. In experiments designed to determine the viability of remaining adherent cells, results in cancerous cells showed a significant outright cell kill with a delayed growth of the surviving cells. In noncancerous cells, there was a slight growth delay, no increased cell death, and a normal growth curve when growth resumed. Detached cells were not functional, which is in agreement with Iwagami [22].

The data from our cell-death time course experiments (Fig 1) showed that heat shock had effects on the transformed/cancer cells that when added together could explain the decreased survival reported for these cells. First, more transformed/cancerous cells were killed outright by the heat. Second, more transformed/cancerous cells than noncancerous cells were damaged by the heat treatment. Finally, we observed an imposed growth delay in the transformed/cancerous cells that survived.

Harmful conditions such as heat shock, oxidative stress, and ultraviolet light stimulate the activation of a family of homologous stress-activated protein kinases resulting in programmed cell death [23]. In addition, these same conditions also resulted in the increased expression of heat shock proteins, which has been shown to confer thermoprotection to the cell [3]. The presence of denatured proteins causes the dissociation of HSC70 from the heat shock factor, the increased availability of HSC70 thus conferring thermoresistance. Our Western blot analysis before heat shock showed significantly less HSC70 and HSP70 present in the ras-transformed cells (compared with the normal cells), thus potentially not providing a sufficient amount of thermoprotection to these cells. This could explain our observation that during the initial heating interval a large number of the transformed cells died, whereas in the noncancerous cell line the numbers continued to increase. This observation is supported by the HSP70 time course graph (Fig 3) compared between cell lines and correlated to the cell-death time course curve (Fig 1). A comparison of these curves reveals a feeble heat shock response present in the BZR-T33 cells compared with the BEAS2-B cells. This feeble response is characterized as heat shock protein: levels starting significantly lower, requiring longer for increased expression, reaching vertex substantially later, significantly reduced 24 hours later. BZR-T33 cells produced substantial amounts of HSP70, however, this occurred 2 hours after removal of the heat stimulus. Additionally, the HSP27 response was not detected in the transformed cells, whereas in the noncancerous cells HSP27 expression was increased after heat shock.

Thermotolerance is a transient resistance to subsequent heat insults and is the result of accumulation of heat shock proteins within the cytoplasm of the cell. Again referring to both the cell-death time course graph (Fig 1) and the HSP70 time course graph (Fig 3) reveals that the amount of HSP70 remains elevated within the noncancerous cell 24 hours later. However, this is not the case in the transformed cell; levels are almost back to baseline values within this time. Therefore, thermotolerance in the ras-transformed cell would potentially be significantly decreased.

A shortcoming of this research is that the majority of the experiments were conducted with only two cell lines and that using different cell lines might possibly give different answers. It is equally inappropriate to suggest that these "normal" cells are fully representative of normal lung as it is to suggest that the ras-transformed cells are fully representative of lung cancer. Conversely, a strength of this study is that the two cell lines used differed only in the presence of a known oncogene—ras. Therefore, confidence that the observed phenomena are related to this oncogene is strong. This is an early report on the effects of hyperthermia, however, the results are encouraging. Future studies will be directed at further definition of this apparent ras-associated thermosensitivity and will seek to determine the underlying cause; the eventual goal is to determine the therapeutic utility of heat in lung cancer.

In summary, these experiments revealed that in these lung cells the presence of a ras oncogene was associated with an increased susceptibility to heat-induced cell death. The ras-transformed and malignant cell lines showed an increased amount of the H-ras protein present which was unaffected by heat stress. In addition, we showed that in this ras-transformed cell line thermoprotection was defective. Thermoresistance (immediate increase in heat shock proteins) and thermotolerance (sustained increased level of heat shock proteins) were both quantitatively less and delayed in the ras-transformed cell lines as opposed to the noncancerous cell lines.

	Acknowledgments

 
We thank Steve Schuenke, Karen Martin, and Eileen Figueroa for preparing the manuscript, and Mary Treinen Moslen, PhD, B. Mark Evers, MD, and Mark R. Hellmich, PhD, for critically reviewing the manuscript.

	Footnotes

 
^* When the word ras is italicized it refers to the ras gene; when ras is not italicized as in ras, p21 this refers to the product of the ras gene, which is the protein ras, p21.

	References

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