The goal of this review is to provide a brief introduction to the effects of hyperthermia on cellular structures and physiology. The review focuses on the effects of hyperthermia thought to contribute to the enhancement of cancer therapy namely the mechanisms of cell killing and the sensitization of cells to ionizing radiation or chemotherapeutic agents. Specifically the review addresses four topics: hyperthermia induced cell killing, mathematical models of cell killing, mechanisms of thermal effects in the hyperthermia temperature range and effects on proteins that contribute to resistance to other stresses, i.e., DNA damage. Hyperthermia has significant effects on proteins including unfolding, exposing hydrophobic groups, and aggregation with proteins not directly altered by hyperthermia. Protein aggregation has effects throughout the cell but has a significant impact within the nucleus. Changes in the associations of nuclear proteins particularly those involved in DNA replication cause the stalling of DNA replication forks and lead to the induction of DNA damage such as double strand breaks. It has long been recognized that heat has effects on plasma membrane protein distribution alters the permeability of plasma membranes resulting in a calcium spike and disrupts the mitochondrial membrane potential resulting in the change in the redox status of cells. These effects contribute to the protein unfolding effects of hyperthermia and contribute to effects observed in the nucleus. Thus heat effects on multiple cellular targets can be integrated through global effects on protein folding to affect specific end points such as cell killing and sensitization to additional stresses.
All cells have intricately coupled sensing and signaling mechanisms that regulate the cellular outcome following exposure to genotoxic agents such as ionizing radiation (IR). In the IR-induced signaling pathway, specific protein events, such as ataxia-telangiectasia mutated protein (ATM) activation and histone H2AX phosphorylation (;-H2AX), are mechanistically well characterized. How these mechanisms can be altered, especially by clinically relevant agents, is not clear. Here we show that hyperthermia, an effective radiosensitizer, can induce several steps associated with IR signaling in cells. Hyperthermia induces ;-H2AX foci formation similar to foci formed in response to IR exposure, and heat-induced ;-H2AX foci formation is dependent on ATM but independent of heat shock protein 70 expression. Hyperthermia also enhanced ATM kinase activity and increased cellular ATM autophosphorylation. The hyperthermia-induced increase in ATM phosphorylation was independent of Mre11 function. Similar to IR, hyperthermia also induced MDC1 foci formation; however, it did not induce all of the characteristic signals associated with irradiation because formation of 53BP1 and SMC1 foci was not observed in heated cells but occurred in irradiated cells. Additionally, induction of chromosomal DNA strand breaks was observed in IR-exposed but not in heated cells. These results indicate that hyperthermia activates signaling pathways that overlap with those activated by IR-induced DNA damage. Moreover, prior activation of ATM or other components of the IR-induced signaling pathway by heat may interfere with the normal IRinduced signaling required for chromosomal DNA doublestrand break repair, thus resulting in increased cellular radiosensitivity. [Cancer Res 2007;67(7):3010-7]
The protein products of several rad checkpoint genes of Schizosaccharomyces pombe (rad1؉ , rad26 ؉ , and hus1 ؉ ) play crucial roles in sensing changes in DNA structure, and several function in the maintenance of telomeres. When the mammalian homologue of S. pombe Rad9 was inactivated, increases in chromosome end-to-end associations and frequency of telomere loss were observed. This telomere instability correlated with enhanced S-and G 2 -phase-specific cell killing, delayed kinetics of ␥-H2AX focus appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase-specific DNA damage repair. Furthermore, mammalian Rad9 interacted with Rad51, and inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S and G 2 phases of the cell cycle. Together, these findings provide evidence of roles for mammalian Rad9 in telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.
Failure to reactivate stalled or collapsed DNA replication forks is a potential source of genomic instability. Homologous recombination (HR) is a major mechanism for repairing the DNA damage resulting from replication arrest. The single-strand DNA (ssDNA)-binding protein, replication protein A (RPA), plays a major role in multiple processes of DNA metabolism. However, the role of RPA2 hyperphosphorylation, which occurs in response to DNA damage, had been unclear. Here, we show that hyperphosphorylated RPA2 associates with ssDNA and recombinase protein Rad51 in response to replication arrest by hydroxyurea (HU) treatment. In addition, RPA2 hyperphosphorylation is critical for Rad51 recruitment and HR-mediated repair following HU. However, RPA2 hyperphosphorylation is not essential for both ionizing radiation (IR)-induced Rad51 foci formation and I-Sce-I endonuclease-stimulated HR. Moreover, we show that expression of a phosphorylation-deficient mutant of RPA2 leads to increased chromosomal aberrations following HU treatment but not after exposure to IR. Finally, we demonstrate that loss of RPA2 hyperphosphorylation results in a loss of viability when cells are confronted with replication stress whereas cells expressing hyperphosphorylation-defective RPA2 or wild-type RPA2 have a similar sensitivity to IR. Thus, our data suggest that RPA2 hyperphosphorylation plays a critical role in maintenance of genomic stability and cell survival after a DNA replication block via promotion of HR.
Topoisomerase II is a multifunctional protein required during DNA replication, chromosome disjunction at mitosis, and other DNA-related activities by virtue of its ability to alter DNA supercoiling. The enzyme is encoded by two similar but nonidentical genes: the topoisomerase II␣ and II genes. In HeLa cells synchronized by mitotic shake-off, topoisomerase II␣ mRNA levels were found to vary as a function of cell cycle position, being 15-fold higher in late S phase (14 to 18 h postmitosis) than during G 1 phase. Also detected was a corresponding increase in topoisomerase II␣ protein synthesis at 14 to 18 h postmitosis which resulted in significantly higher accumulation of the protein during S and G 2 phases. Topoisomerase II␣ expression was not dependent on DNA synthesis during S phase, which could be inhibited without effect on the timing or level of mRNA expression. Mechanistically, topoisomerase II␣ expression appears to be coupled to cell cycle position mainly through associated changes in mRNA stability. When cells are in S phase and mRNA levels are maximal, a half-life of greater than 4 h was observed. However, during G 1 phase, when cellular levels are lowest, the half-life of topoisomerase II␣ mRNA was determined to be approximately 30 min. A similar decrease in mRNA stability was also induced by two external factors known to delay cell cycle progression. Treatment of S-phase cells, at the time of maximum topoisomerase II␣ mRNA stability, with either ionizing radiation (5 Gy) or heat shock (45؇C for 15 min) caused the accumulated topoisomerase II␣ mRNA to decay. This finding suggests a potential relationship between stress-induced decreases in topoisomerase II␣ expression and cell cycle progression delays in late S/G 2 .The double-stranded nature of the DNA molecule presents special problems during chromosomal replication and segregation. Movement of the replication fork along the DNA molecule generates positive supercoiling in advance while the replicated parental strands in its wake are negatively supercoiled. Topological problems also arise near the end of replication when adjacent replication forks converge. Depending on the relative unwinding rate of the parental or progeny strands, either an intertwined or a gapped structure is formed (47). Unless untangled, the former structure leads to chromosome breakage, nondisjunction, and cell death (20). DNA topology in the cell is controlled through the action of two types of topoisomerase enzymes (see references 20 and 46 for reviews). Type I topoisomerases introduce into the duplex DNA molecule transient single-strand nicks which act as swivels during DNA replication or relieve the torsional stress associated with RNA transcription. Topoisomerase II enzymes relax, catenate, or decatenate DNA by introducing transient double-strand breaks which allow another DNA segment to pass through. While topoisomerase II is also capable, in theory, of relaxing transcription and replication induced supercoiling, it appears that topoisomerase I is mainly responsible for ...
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