William D. Wright

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.

DNA Loop Structure and Radiation Response

Publisher Summary This chapter discusses new approaches to investigate the role of nuclear and chromatin structure in the effects of ionizing radiation on mammalian cells. The nucleus of eukaryotic cells is the critical organelle in which interactions with ionizing radiation result in reproductive cell death. The first level of higher-order organization of DNA in chromatin is the nucleosome. Each nucleosome contains two molecules each of the four core histones that form a disk around which is wrapped ∼ 146 base pairs (bp) of DNA. Each nucleosome is separated from its neighbor by approximately 54 bp of “linker” DNA making the nucleosome repeat unit equal to ∼ 200 bp. Packaging of DNA into nucleosomes results in a fiber of approximately 11 nm in diameter. In the next level of organization, polynucleosomes are packaged into a solenoidal array of highly condensed DNA, partly because of the interaction of histone HI with the internucleosomal or “linker” DNA and nucleosome core particles. The chapter presents a study wherein effects of radiation on nucleoid sedimentation behavior were studied with two approaches: first, alterations in the ethidium bromide response were studied as a function of dose; second, alterations in the sedimentation distance were investigated. The sedimentation technique can detect changes in nucleoids induced by heat shock or by radiation with great sensitivity. However, the interpretation of what these changes mean in terms of DNA supercoiling is compromised by the absence of independent nucleoid sedimentation and varying amounts of protein co-sedimenting with the nucleoid.

THE INTERACTION OF HEAT AND RADIATION AFFECTING THE ABILITY OF NUCLEAR-DNA TO UNDERGO SUPERCOILING CHANGES

DNA damage (putatively strand breaks) from ionizing radiation inhibits the ability of intercalating dyes to induce right-handed supercoils in the DNA loops of HeLa nucleoids [Cook and Brazelle, J. Cell Sci. 22, 287-302 (1976); Roti Roti and Wright, Cytometry 8, 461-467 (1987)] while heat-induced changes in the nuclear matrix enhance this ability [Roti Roti and Painter, Radiat. Res. 89, 166-175 (1982)]. Since heat and radiation interact synergistically or additively on most cellular functions which they affect, the rewinding of DNA supercoils is unusual in that these agents alone affect it in an antagonistic manner. When HeLa cells were exposed to 45 degrees C for 30 min and immediately irradiated with 10 Gy of 137Cs gamma rays, the rewinding response was intermediate between that for cells which had been exposed to 10 Gy only and control. When repair of this damage was assayed in control cells, 97% of the initial damage had been repaired at 30 min postirradiation; at the same time only 10% of the initial damage had been repaired in the heat-shocked cells. This apparent dose reduction effect and the inhibition of repair were interpreted to indicate that heat-induced changes in nuclear structure were masking DNA damage from the assay and the repair system. These effects correlated with the amount of heat-induced excess protein associated with the nucleus and the nucleoid.

Radiation sensitivity correlates with changes in DNA supercoiling and nucleoid protein content in cells of three Chinese hamster cell lines.

We have investigated the composition of nuclear matrix proteins and DNA supercoiling characteristics of cell lines expressing altered radiation sensitivity. Chinese hamster ovary cell lines 4364 (wild-type), XR-1 (DSB repair-deficient, radiosensitive) and XR-122 (a radioresistant variant of XR-1 bearing human chromosome 5) were used as a model to study the relationship between intrinsic radiation sensitivity and the level of DNA supercoiling ability within chromatin loops and the composition of nuclear matrix proteins. Analysis of the ability of DNA loop domains to undergo changes in DNA supercoiling in the presence of DNA damage revealed that the degree of inhibition of loop rewinding was greater in the radiation-sensitive cells (XR-1) compared to the radiation-resistant cells (4364 and XR-122). Furthermore, the loop-rewinding characteristics correlated inversely with the clonogenic survival of these cells after exposure to ionizing radiation. Since DNA loops are anchored to the nuclear matrix by protein-DNA anchor points, a study of the nuclear matrix proteins by high-resolution 2D-PAGE was conducted for these cells to determine whether differential inhibition of loop rewinding could be due to differences in the DNA loop-protein anchor points in these cells. The XR-1 cells showed an overall absence of 13 proteins compared to the 4364 cells. Of these 13, 5 were restored in XR-122 cells. These results are consistent with the hypothesis that stability of the DNA loop domains in the presence of DNA damage contributes to the expression of potentially lethal damage by ionizing radiation.

DNA supercoiling changes and nucleoid protein composition in a group of L5178Y cells of varying radiosensitivity

Cell of the radioresistant L5178Y-R, -S35, -SR and M10(neo 5)-1 and radiosensitive L5178Y-S, M10 and LX830 cell lines were used to investigate the relationship between radiosensitivity and DNA supercoiling ability mediated by the nuclear matrix within chromatin loops containing DNA damage. The ability of DNA loops to undergo changes in supercoiling in the presence of radiation-induced damage revealed that in all cases the degree of inhibition of supercoil rewinding was greater in the radiosensitive cells. Since the amount of DNA damage induced per unit dose is known to be equal in all these cell lines, the same number of DNA lesions produced a greater loss of topological constraint in the radiosensitive cells. The differential loss of DNA supercoiling ability could be due to differences in DNA-nuclear matrix anchor points. High-resolution two-dimensional gel electrophoresis of nucleoid proteins showed numerous reproducible differences in nuclear matrix protein between the cell lines studied. A total of nine proteins were associated with nucleoids from L5178Y-R cells and absent from L5178Y-S nucleoids. None of them, however, correlated absolutely with radioresistance. Thus, unlike previous studies in CHO cells, no candidates for the conveyance of cellular radiosensitivity that were single proteins were detected. However, these results are consistent with the hypothesis that stability of DNA loop domains in the presence of DNA damage is a determinant of the outcome of radiation-induced DNA damage.

Cytometric methods to analyze ionizing-radiation effects

Four cytometric assays for the assessment of radiation-induced DNA damage in individual cells are presented. Two of these, the alkaline and neutral comet assays, are useful for the detection of DNA damage due to very low radiation doses and promise to be useful for the quantitation of genomic damage after clinically or environmentally relevant exposures. The other two, the halo and halo-comet assays, reveal aspects of chromatin structure in the presence of DNA damage that reflect differences in intrinsic cellular radiosensitivity. Further development of these assays used alone, or in combination, should eventually lead to the definition of readily measurable cytometric parameters that will be useful as predictive markers for cellular responses to DNA damaging agents.

Resolution of DNA Topoisomerase II by two-dimensional polyacrylamide gel electrophoresis and western blotting

Eukaryotic DNA Topoisomerase II (Topo II) has been studied using high-resolution two-dimensional polyacrylamide electrophoresis (2D-PAGE) and immunodetection of resolved proteins using specific antisera (Western blotting). Traditional methods of 2D-PAGE failed to resolve Topo II and neither nonequilibrium nor equilibrium pH gradients allowed Topo II to enter the first dimension gel. Exhaustive nuclease digestion and alternate protein solubilization strategies also produced negative results. We have developed altered first dimension pH gradient profiles and employed a more aggressive protein solubilization procedure which resulted in the resolution of Topo II. The 170-kDa polypeptide focuses with an apparent isoelectric point of approximately 6.5.

Effects of irradiation on nuclear protein synthesis in G2 phase of the cell cycle.

A method was developed to determine the synthesis of nuclear proteins throughout the cell cycle which was resolved into six compartments on the basis of DNA and nuclear protein content (i.e., early and late G1, early and late S, etc). Using this technique cell-cycle-specific synthesis of certain nuclear proteins was observed. Of particular interest was a 170-kDa protein(s) whose synthesis was initiated in early S phase and reached a maximum rate in late G2. Following irradiation with 6.8 Gy of 137Cs gamma rays the synthesis of the 170-kDa protein(s) declined in the G2 population with near total inhibition seen by 24 h. Synthesis of the 170-kDa protein(s) appeared to be slightly enhanced, and the postirradiation inhibition of its synthesis was reversed, in the presence of 3 mM caffeine. Also, the synthesis of 55-kDa nuclear protein(s) was stimulated throughout the cell cycle in the presence of 3 mM caffeine. These observations suggest new possibilities regarding the mechanism of the X-ray-induced G2 block and its reversal by caffeine. However, the exact role of these nuclear proteins in cellular events remains to be ascertained.

Flow cytometric methods for studying isolated nuclei: DNA accessibility to DNase I and protein-DNA content.

Two FCM methods utilizing isolated nuclei were described. A DNase I sensitivity assay, employing changes in binding and digestion kinetics of the enzyme as well as the binding of intercalating fluorochrome was used to observe structural changes of chromatin rendered by physical and chemical agents. A nuclear DNA-protein staining method was used to study changes in nuclear protein content, redistribution of populations in the cell cycle, and unbalanced growth, manifested as an extraordinary accumulation of nuclear protein brought about by physical and chemical perturbation.

Effects of radiation on TNFα-mediated cytolysis of cell lines derived from cervical carcinomas

Abstract The effect of radiation, a primary mode of treatment for cervical malignancies, on the tumor necrosis alpha (TNFα)-mediated cytolysis of five cell lines derived from human cervical carcinoma cell lines (C-33 A, ME-180, HT-3, MS751, and SiHa) was analyzed. Results of this analysis showed that all of the cell lines were resistant to the cytolytic effects of TNFα. Although resistant when protein synthesis proceeds normally, ME-180, HT-3, MS751, and SiHa cells were sensitive to TNFα-mediated cytolysis in the presence of protein synthesis inhibitors. The cytolytic response of these cells to radiation was heterogeneous, with C-33 A cells being the most radiosensitive and SiHa cells being the least radiosensitive. The cell lines ME-180, MS751, and HT-3 were intermediate in their sensitivities to radiation. Because radiation is known to inhibit protein synthesis, the ability of radiation to enhance TNFα cytolytic activity was examined. The cell lines with intermediate sensitivities to radiation (ME-180, HT-3, and MS751) demonstrated statistically significant synergistic increases in cytolysis when exposed to TNFα in combination with radiation. Neither the radioresistant SiHa cell line nor the radiosensitive C-33 A cell line displayed increased cytolysis with increasing concentrations of TNFα at any dose of radiation. Possible mechanisms which may explain the synergy in ME-180, HT-3, and MS751 cells and lack of synergy in C-33 A and SiHa cells by TNFα and radiation are discussed.