Heat shock proteins, thermotolerance, and their relevance to clinical hyperthermia.

Mammalian cells, when exposed to a non-lethal heat shock, have the ability to acquire a transient resistance to subsequent exposures at elevated temperatures, a phenomenon termed thermotolerance. The mechanism(s) for the development of thermotolerance is not well understood, but earlier experimental evidence suggests that protein synthesis may play a role in its manifestation. On the molecular level, heat shock activates a specific set of genes, so-called heat shock genes, and results in the preferential synthesis of heat shock proteins. The heat shock response, specifically the regulation, expression and functions of heat shock proteins, has been extensively studied in the past decades and has attracted the attention of a wide spectrum of investigators ranging from molecular and cell biologists to radiation and hyperthermia oncologists. There is much data supporting the hypothesis that heat shock proteins play important roles in modulating cellular responses to heat shock, and are involved in the development of thermotolerance. This review summarizes some current knowledge on thermotolerance and the functions of heat shock proteins, especially hsp70. The relationship between thermotolerance development and hsp70 synthesis in tumours and in normal tissues is examined. The possibility of using hsp70 as an indicator for thermotolerance is discussed.

Correlation of heat resistance and HSP-70A mRNA levels in human tumor cells measured by competitive quantitative polymerase chain reaction.

PURPOSE: Several HSP-70 genes have been cloned and sequenced in human cells. Among these genes, the HSP-70A mRNA and protein levels correlate best with the development and decay of thermotolerance and intrinsic thermal sensitivity. Leukemic and nonleukemic human tumor cells express low levels of the normally heat inducible HSP-70A mRNA in control nonheated cells. Using a competitive quantitative polymerase chain reaction, we have measured the levels of mRNA for this gene and have correlated it with both transient and intrinsic thermal sensitivity of tumor cells. Such studies were also extended to tumor samples obtained from patients. METHODS AND MATERIALS: In these studies, the plasmid phHSP-70 which contains the entire human HSP-70A gene was modified by the insertion of the T7 promoter at the 5'-end untranslated region as well as the insertion of a 23 bp synthetic linker at the BamH1 site in the promoter region of the HSP-70A gene. The PCR primers were located such that the amplified fragment contained the linker. Using the T7 polymerase, the HSP-70A mRNA was transcribed from this plasmid (phHSP-70L) in vitro. A known amount of HSP-70A mRNA was then added to the total RNA prepared from the cell samples or from the tumor tissues obtained from patients. Using the components of the PCR reaction plus known amounts of HSP-70A mRNA synthesized from phHSP-70L and unknown amounts of total cellular RNA, the samples were amplified and analysed on a denaturing acrylamide gel. The PCR products obtained from phHSP-70L were 23 bp larger than the PCR products obtained from the cell samples due to the addition of the synthetic linker to the HSP-70A gene in phHSP-70L and therefore, the two products could be easily distinguished from each other and quantitated. The alpha-32P-dCTP incorporated in each sample was quantitated by AMBIS Scanner. When the 32P-counts were equal in the known and the unknown samples, the amount of the HSP-70A mRNA was taken to be equal in the known and the unknown sample. RESULTS: The results show that HSP-70A mRNA levels can be used to predict the survival levels during the development and decay of thermotolerance. In nonleukemic human tumor cell lines, there are as much as 40-50-fold induction of HSP-70A mRNA levels during the peak of thermotolerance. In leukemic cell lines, however, HSP-70A mRNA levels are induced only by three-fold during the same time period. These differences between the levels of HSP-70A mRNA positively correlate with the amount of tolerance development in leukemic and nonleukemic tumor cells. HSP-70A mRNA levels also vary in different tumor cells under nonheated conditions and there is a positive correlation between HSP-70A mRNA levels in nonleukemic human tumor cells and the level of their intrinsic thermal resistance. CONCLUSION: HSP-70A mRNA levels can be used to predict the intrinsic thermal sensitivity of nonleukemic human tumor cells.

Expression of HSP-28 and three HSP-70 genes during the development and decay of thermotolerance in leukemic and nonleukemic human tumors.

Leukemic cells appear to develop less thermotolerance and then to lose their thermotolerance more rapidly than do other tumor cell lines. The reason for this phenomenon is not known. After heat shock (or other environmental stresses), mammalian cells preferentially synthesize a set of proteins known as heat shock proteins (HSPs). HSP-28 and the various isoforms of HSP-70 have been suggested as being responsible for the development of thermotolerance. In these studies, we have attempted to determine by their expression with HSPs positively correlate with the development and decay of thermotolerance and whether the expression of these genes could explain the differing thermotolerance response observed between leukemic and nonleukemic tumor cells. Polymerase chain reaction was used to detect the expression of HSP-28 and several HSP-70 genes. Our data indicate that the expression of all three heat-inducible HSP-70 genes, 70A (Hunt and Morimoto, Proc. Natl. Acad. Sci. USA, 82: 6455-6459, 1985), 70B (Voellmy et al., Proc. Natl. Acad. Sci. USA, 82: 4949-4953, 1985), and 70B' (Leung et al., Biochem J., 267: 125-132, 1990) correlate with the development and decay of thermotolerance in nonleukemic tumor cell lines after heat or arsenite treatment. HSP-28 (Hickey et al., Nucleic Acids Res., 4: 4127-4145, 1986) failed to correlate with thermotolerance development; it was not induced after 45 degrees C primary heat shock. In leukemic cells, however, none of the HSPs were induced for extended periods of time. The lack of coordinate expression of HSP genes in cells of myeloid origin may explain the poor induction and maintenance of thermotolerance that is observed in these cells.

The effects of hyperthermia on tumor carcinoembryonic antigen expression.

The effects of hyperthermia on carcinoembryonic antigen (CEA) expression were investigated. The human colon adenocarcinoma cell line, LS174T, was heated in vitro for 42 degrees C/1 hr, 43 degrees C/1 hr, or 45 degrees C/30 min. Carcinoembryonic antigen membrane expression was assayed by live cell radioimmunoassay 0-6 days after heating. A heat exposure of 45 degrees C/30 min resulted in an initial decrease in carcinoembryonic antigen membrane expression 1 day post-heating followed by a 2-3 fold increase which peaked 3 days post-heating. Carcinoembryonic antigen expression began returning to normal by the sixth day. Heat exposures of 42 degrees C/1 hr and 43 degrees C/1 hr also resulted in increased carcinoembryonic antigen expression but this increase was of lesser magnitude and of shorter duration. Carcinoembryonic antigen shed into the medium, as measured by enzyme immunoassay, also increased after heating in a temperature-dependent fashion. Flow cytometry analysis demonstrated that cells in all phases of the cell cycle expressed this increase. We conclude that hyperthermia results in significant changes in carcinoembryonic antigen membrane expression and shedding. The implications that these findings have with regards to clinical hyperthermia and radioimmunotherapy are discussed.

Enhancement of murine bone marrow ablation by combining whole body hyperthermia with total body irradiation and cyclophosphamide.

Bone marrow ablation using combined whole body hyperthermia (WBH), total body irradiation (TBI), and cyclophosphamide (Cy) was investigated in C3H f/Sed mice to demonstrate cytotoxic synergism between the three modalities. TBI was given on day 0. WBH treatment was for 1 hr at 41.8 degrees C, given in daily sessions for 1, 2 or 3 modalities. TBI was given on day 0. WBH treatment was for 1 hr at 41.8 degrees C, given in daily sessions for 1, 2 or 3 consecutive days following TBI. Total cyclophosphamide doses were 160 and 240 mg/kg given in 2 daily injections on days 1 and 2 following TBI. Polymorphonuclear leukocyte and lymphocyte numbers were determined by differential cell counts. The total peripheral blood cell counts were also determined. WBH alone, given in daily sessions for 3 days, did not reduce the total peripheral blood cell counts. However, when WBH was added to TBI (6.3 Gy) peripheral blood cellularity was reduced on day 2, but no significant heat/radiosensitization was evident after day 2. WBH (3 daily sessions) significantly reduced the peripheral blood cellularity and resulted in bone marrow ablation when it was combined with TBI and Cy. CY (160-240 mg/kg) combined with TBI (5.4 Gy) resulted in bone marrow ablation and subsequent death in 14-22% of mice treated; 60-100% of mice died from bone marrow ablation when WBH was added to TBI (5.4 Gy) and Cy (160-240 mg/kg). Femoral and vertebral tissue sections showed total loss of progenitor cells when WBH, TBI (5.4 Gy), and Cy (240 mg/kg) were combined whereas lessor treatment was associated with histologically verified reconstitution of progenitor cells inside the marrow cavities. These studies indicate that bone marrow ablation can be achieved when using WBH in combination with lower doses of TBI and Cy.

Effect of irradiation on thermal sensitivity of bone marrow progenitors.

The heat sensitivity of murine CFU-GM and CFU-E following 2.5 Gy of total body irradiation (TBI) was studied. C3H f/Sed female mice were treated with 2.5 Gy TBI and femoral bone marrow was heated in vitro at 43 degrees C. CFU-GM show heat radiosensitization when bone marrow was heated immediately following irradiation. There was a brief decline in heat and radiation interaction when cells were heated 3 hours following 2.5 Gy of TBI, but heat radiosensitization returned to its maximum from 1 to 2 days following irradiation and remained significantly different from the control on days 5 and 7 following irradiation. The heat and radiation interaction disappeared by 30 days. CFU-E shows significant heat radiosensitization only on day 2 following 2.5 Gy of TBI. Total nucleated cells per femur showed a decrease by 70 per cent in days 1 to 2 following TBI, recovered to control values by day 5, and did not correlate with the changes in heat radiosensitization. Cell cycle analysis of CFU-GM using hydroxyurea showed no significant changes in cell cycle parameters on days 1 and 2 following 2.5 Gy, when maximum heat sensitization was observed. It is concluded that bone marrow progenitors may respond in a different way from other normal tissues to heat and irradiation sequencing, and that these differences must be considered when designing clinical trials.