Robert P. Vanderwaal

Nuclear matrix as a target for hyperthermic killing of cancer cells

The nuclear matrix organizes nuclear DNA into operational domains in which DNA is undergoing replication, transcription or is inactive. The proteins of the nuclear matrix are among the most thermal labile proteins in the cell, undergoing denaturation at temperatures as low as 43-45 degrees C, i.e. relevant temperatures for the clinical treatment of cancer. Heat shock-induced protein denaturation results in the aggregation of proteins to the nuclear matrix. Protein aggregation with the nuclear matrix is associated with the disruption of many nuclear matrix-dependent functions (e.g. DNA replication, DNA transcription, hnRNA processing, DNA repair, etc.) and cell death. Heat shock proteins are believed to bind denatured proteins and either prevents aggregation or render aggregates more readily dissociable. While many studies suggest a role for Hsp70 in heat resistance, we have recently found that nuclear localization/delocalization of Hsp70 and its rate of synthesis, but not its amount, correlate with a tumor cells ability to proliferate at 41.1 degrees C. These results imply that not only is the nuclear matrix a target for the lethal effects of heat, but it also is a target for the protective, chaperoning and/or enhanced recovery effects of heat shock proteins.

The mTOR inhibitor rapamycin suppresses DNA double-strand break repair.

Abstract mTOR (mammalian target of rapamycin) signaling plays a key role in the development of many tumor types. Therefore, mTOR is an attractive target for cancer therapeutics. Although mTOR inhibitors are thought to have radiosensitization activity, the molecular bases remain largely unknown. Here we show that treating MCF7 breast cancer cells with rapamycin (an mTOR inhibitor) results in significant suppression of homologous recombination (HR) and nonhomologous end joining (NHEJ), two major mechanisms required for repairing ionizing radiation-induced DNA DSBs. We observed that rapamycin impaired recruitment of BRCA1 and Rad51 to DNA repair foci, both essential for HR. Moreover, consistent with the suppressive role of rapamycin on both HR and NHEJ, persistent radiation-induced DSBs were detected in cells pretreated with rapamycin. Furthermore, the frequency of chromosome and chromatid breaks was increased in cells treated with rapamycin before and after irradiation. Thus our results show that radiosensitization by mTOR inhibitors occurs via disruption of the major two DNA DSB repair pathways.

A dual role for A-type lamins in DNA double-strand break repair

A-type lamins are emerging as regulators of nuclear organization and function. Changes in their expression are associated with cancer and mutations are linked to degenerative diseases -laminopathies-. Although a correlation exists between alterations in lamins and genomic instability, the molecular mechanisms remain largely unknown. We previously found that loss of A-type lamins leads to degradation of 53BP1 protein and defective long-range non-homologous end-joining (NHEJ) of dysfunctional telomeres. Here, we determined how loss of A-type lamins affects the repair of short-range DNA double-strand breaks (DSBs) induced by ionizing radiation (IR). We find that lamins deficiency allows activation of the DNA damage response, but compromises the accumulation of 53BP1 at IR-induced foci (IRIF), hindering the fast phase of repair corresponding to classical-NHEJ. Importantly, reconstitution of 53BP1 is sufficient to rescue long-range and short-range NHEJ. Moreover, we demonstrate an unprecedented role for A-type lamins in the maintenance of homologous recombination (HR). Depletion of lamins compromises HR by a mechanism involving transcriptional downregulation of BRCA1 and RAD51 by the repressor complex formed by the Rb family member p130 and E2F4. In line with the DNA repair defects, lamins-deficient cells exhibit increased radiosensitivity. This study demonstrates that A-type lamins promote genomic stability by maintaining the levels of proteins with key roles in DNA DSBs repair by NHEJ and HR. Our results suggest that silencing of A-type lamins by DNA methylation in some cancers could contribute to the genomic instability that drives malignancy. In addition, lamins-deficient tumor cells could represent a good target for radiation therapy.

The role of RPA2 phosphorylation in homologous recombination in response to replication arrest.

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.

Heat-induced modifications in the association of specific proteins with the nuclear matrix.

Nuclei isolated from heat-shocked mammalian cells have an increased protein content which reflects an enhanced protein binding to nuclear structures. These nuclear changes are correlated with cell survival and inhibition of DNA replication, transcription and repair of DNA damage. It appears that most of the altered protein binding occurs in association with the nuclear matrix. The present study was conducted to determine if measurements of specific proteins in isolated nuclei reflect changes that occur at the nuclear matrix. The amounts of various proteins associated with HeLa cell nuclei and nuclear matrices after heat shock were measured by (1) densitometric scans of Coomassie blue-stained gels, (2) immunoblotting with antibodies to nuclear proteins and (3) antisera raised against nuclear matrix proteins from heated cells. These measurements revealed heat-induced increases in the levels of many nuclear matrix proteins. While a number of proteins show similar changes in both nuclei and nuclear matrices, for many the extent of increased association with the nuclear matrix is not reflected in the measured changes in the nuclei. These results are essential for understanding and studying further the relationships between the cellular response to hyperthermia and heat-altered associations of specific proteins with either nuclei or nuclear matrices.

Delaying S-phase progression rescues cells from heat-induced S-phase hypertoxicity.

The mechanism by which a cell protects itself from the lethal effects of heat shock and other stress‐inducing agents is the subject of much research. We have investigated the relationship between heat‐induced damage to DNA replication machinery and the lethal effects of heat shock, in S‐phase cells, which are more sensitive to heat shock than either G1 or G2. We found that maintaining cells in aphidicolin, which prevents the passage of cells through S‐phase, can rescue S‐phase HeLa cells from the lethal effects of heat shock. When S‐phase, HeLa cells were held for 5–6 h in 3 μM aphidicolin the measured clonogenic survival was similar to that for exponentially growing cells. It is known, that heat shock induces denaturation or unfolding of proteins, rendering them less soluble and more likely to co‐isolate with the nuclear matrix. Here, we show that enhanced binding of proteins involved in DNA replication (PCNA, RPA, and cyclin A), with the nuclear matrix, correlates with lethality of S‐phase cells following heat shock under four different experimental conditions. Specifically, the amounts of RPA, PCNA, and cyclin A associated with the nuclear matrix when cells resumed progression through S‐phase correlated with cell killing. Heat‐induced enhanced binding of nuclear proteins involved with other aspects of DNA metabolism, (Mrell, PDI), do not show this correlation. These results support the hypothesis that heat‐induced changes in the binding of proteins associated with DNA replication factories are the potentially lethal lesions, which become fixed to lethal lesions by S‐phase progression but are repairable if S‐phase progression is delayed.

A Comparison of the Modes and Kinetics of Heat-Induced Cell Killing in HeLa and L5178Y Cells

The mode and kinetics of cell killing in HeLa and L5178Y cells were investigated to elucidate possible relationships between the mechanisms and modes of heat-induced cell death. L5178Y cells were heat-shocked for either 24 min at 43 degrees C or 30 min at 45 degrees C, while HeLa cells were given only the latter treatment. The degree of heat-induced excess nuclear protein correlated with cell death in HeLa cells but not in L5178Y cells. This difference suggests that the mechanism of cell death differs between these cell lines. With L5178Y cells the main mode of death after treatment at 43 degrees C was apoptosis with little progression of cells through the cell cycle. However, after treatment at 45 degrees C these cells died by necrosis without progressing through the cell cycle. HeLa cells were found to die by a mechanism other than apoptosis after significant progression of cells through the cell cycle and perturbation of the normal distribution of cells in the phases of the cell cycle (specifically, the fraction of cells in S and G2 phase increased 50% and 30%, respectively, prior to the occurrence of measurable cell death). These results are consistent with the hypothesis that the response to injury which has the potential to be lethal varies between different cell types, and results in the induction of different pathways leading to cell death.

Evidence that protein disulfide isomerase (PDI) is involved in DNA-nuclear matrix anchoring.

DNA–nuclear matrix (NM) anchoring plays a critical role in the organization of DNA within the nucleus and in functional access to DNA for transcription, replication, and DNA repair. The cellular response to oxidative stress involves both gene expression and DNA repair. We, therefore, determined if changes in the oxidative–reductive environment can affect DNA–NM anchoring. The present study used two approaches to study the effect of the reducing agent DTT on DNA–NM anchoring. First, the relative stringency of the DNA–NM attachment was determined by measuring the ability of NM attached DNA loops to undergo supercoiling changes. Second, the effects of DTT on the association of nuclear proteins with DNA were determined by cisplatin crosslinking. When nucleoids (nuclear matrices with attached DNA loops) were prepared from HeLa cells with 1 mM dithiothreitol (DTT), supercoiled DNA loops unwound more efficiently compared with control in the presence of increasing propidium iodide (PI) concentrations. In addition, the rewinding of DNA supercoils in nucleoids treated with DTT was inhibited. Both effects on DNA supercoiling ability were reversed by diamide suggesting that they are dependent on the oxidation state of the protein thiols. When DTT treated nucleoids were isolated from γ‐irradiated cells, the inhibition of DNA supercoil rewinding was equal to the sum of the inhibition due to DTT and γ‐rays alone. Nucleoids isolated from heat‐shocked cells with DTT, showed no inhibition of DNA rewinding, except a small inhibition at high PI concentrations. Nuclear DNA in DTT‐treated nuclei was digested faster by DNase I than in untreated nuclei. These results suggest that DTT is altering DNA–NM anchoring by affecting the protein component(s) of the anchoring complex. Extracting NM with increasing concentrations of DTT did not solubilize any protein to a significant extent until measurable NM disintegration occurred. Therefore, we determined if 1 mM DTT affected the ability of 1 mM cisplatin to crosslink proteins to DNA. Isolated nuclei were treated with 1 mM DTT for 30 min or left untreated prior to crosslinking with 1 mM cisplatin for 2 h at 4°C. The ability of capsulation to crosslink DNA to proteins per se, did not appear to be affected by 1 mM DTT because relative amounts of at least four proteins, 69, 60, 40, and 35 kDa, were crosslinked to DNA to the same extent in DTT‐treated and untreated nuclei. However, protein disulfide isomerase (PDI) crosslinked to DNA in untreated nuclei, but did not crosslink DNA in nuclei that were treated with 1 mM DTT; 1 mM DTT did not affect the intranuclear localization of PDI. Thus, DTT appears to alter the conformation of PDI, as suggested by the DTT‐induced change in DNA association, but not its NM association. These results also imply that DNA–NM anchoring involves the redox state of protein sulfhydryl groups. J. Cell. Biochem. 85: 689–702, 2002.

Nucleophosmin Redistribution following Heat Shock: A Role in Heat-Induced Radiosensitization

Cellular survival from radiation-induced DNA damage requires access to sites of damage for the assembly of repair complexes and the subsequent repair, particularly the repair of DNA double strand breaks (DSB). Hyperthermia causes changes in protein-protein/DNA interactions in the nucleus that block access to sites of DNA damage. Studies presented here indicate that the nucleolar protein, nucleophosmin (NPM), redistributes from the nucleolus following hyperthermia, increases its association with DNA, and blocks access to DNA DSBs. Reduction of NPM significantly reduces heat-induced radiosensitization, but reduced NPM level does not alter radiation sensitivity per se. NPM knockdown reduces heat-induced inhibition of DNA DSB repair. Also, these results suggest that NPM associates with nuclear matrix attachment region DNA in heat-shocked cells.

HSP27 phosphorylation increases after 45°C or 41°C heat shocks but not after non-thermal TDMA or GSM exposures

Purpose: Experiments with cultured HeLa, S3 and E.A. Hy296 cells were performed to determine if exposure to acute (30 min at 45°C) or chronic (2 h at 41°C) heat shocks or to non-thermal exposures of radiofrequency radiation (RF) induce changes in HSP27 phosphorylation. Materials and methods: The radiofrequency (RF) exposures used in this study were 847 MHz time division multiple access modulated (TDMA) at a specific absorption rate (SAR) of 5 W kg−1 for 1, 2 or 24 h or 900 MHz GSM modulated (GSM) at a SAR of 3.7 W kg−1 for 1, 2 or 5 h. HSP27 phosphorylation was evaluated by resolving the various phosphorylation forms using two-dimensional gel electrophoresis measuring the relative amount of each by densitometry. Alternatively, an antibody specific for phosphorylated HSP27 was used to detect changes in HSP27 phosphorylation levels. All heat shock and RF exposure conditions were analysed simultaneously along with a matched incubator control sample. Each experiment was repeated three times. Results: Following heat shock, the degree of phosphorylation of HSP27 varied with the heat dose, with acute hyperthermia (45°C) having an increased proportion of higher phosphorylated forms. Exposure of HeLa S3 cells to 5 W kg−1 TDMA for 1, 2 or 24 h did not induce significant differences in the levels of HSP27 phosphorylation compared to incubator control or sham. Exposure of E.A. Hy926 cells to 3.7 W kg−1 900 MHz GSM for 1, 2 or 5 h did not induce significant differences in the levels of HSP27 phosphorylation compared to sham exposed. Conclusions: Acute and moderate hyperthermia significantly increase HSP27 phosphorylation, but there was no significant change in the levels of HSP27 following non-thermal exposure to TDMA and GSM modulated RF radiations.