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Background Evidence suggests that tumor cells exposed to some DNA damaging agents are more likely to die if they retain microscopically visible γH2AX foci that are known to mark sites of double-strand breaks. This appears to be true even after exposure to the alkylating agent MNNG that does not cause direct double-strand breaks but does produce γH2AX foci when damaged DNA undergoes replication. Methods To examine this predictive ability further, SiHa human cervical carcinoma cells were exposed to 8 DNA damaging drugs (camptothecin, cisplatin, doxorubicin, etoposide, hydrogen peroxide, MNNG, temozolomide, and tirapazamine) and the fraction of cells that retained γH2AX foci 24 hours after a 30 or 60 min treatment was compared with the fraction of cells that lost clonogenicity. To determine if cells with residual repair foci are the cells that die, SiHa cervical cancer cells were stably transfected with a RAD51-GFP construct and live cell analysis was used to follow the fate of irradiated cells with RAD51-GFP foci. Results For all drugs regardless of their mechanism of interaction with DNA, close to a 1:1 correlation was observed between clonogenic surviving fraction and the fraction of cells that retained γH2AX foci 24 hours after treatment. Initial studies established that the fraction of cells that retained RAD51 foci after irradiation was similar to the fraction of cells that retained γH2AX foci and subsequently lost clonogenicity. Tracking individual irradiated live cells confirmed that SiHa cells with RAD51-GFP foci 24 hours after irradiation were more likely to die. Conclusion Retention of DNA damage-induced γH2AX foci appears to be indicative of lethal DNA damage so that it may be possible to predict tumor cell killing by a wide variety of DNA damaging agents simply by scoring the fraction of cells that retain γH2AX foci.

MacPhail, S. H., Banáth, J. P., Yu, Y. T., Chu, E. and Olive, P. L. Cell Cycle-Dependent Expression of Phosphorylated Histone H2AX: Reduced Expression in Unirradiated but not X-Irradiated G1-Phase Cells. Radiat. Res. 159, 759–767 (2003). Exposure of cells to ionizing radiation causes phosphorylation of histone H2AX at sites flanking DNA double-strand breaks. Detection of phosphorylated H2AX (γH2AX) by antibody binding has been used as a method to identify double-strand breaks. Although generally performed by observing microscopic foci within cells, flow cytometry offers the advantage of measuring changes in γH2AX intensity in relation to cell cycle position. The importance of cell cycle position on the levels of endogenous and radiation-induced γH2AX was examined in cell lines that varied in DNA content, cell cycle distribution, and kinase activity. Bivariate analysis of γH2AX expression relative to DNA content and synchronization by centrifugal elutriation were used to measure cell cycle-specific expression of γH2AX. With the exception of xrs5 cells, γH2AX level was approximately 3 times lower in unirradiated G1-phase cells than S- and G2-phase cells, and the slope of the G1-phase dose–response curve was 2.8 times larger than the slope for S-phase cells. Cell cycle differences were confirmed using immunoblotting, indicating that reduced antibody accessibility in intact cells was not responsible for the reduced antibody binding in G1-phase cells. Early apoptotic cells could be easily identified on flow histograms as a population with 5–10-fold higher levels of γH2AX, although high expression was not maintained in apoptotic cells by 24 h. We conclude that expression of γH2AX is associated with DNA replication in unirradiated cells and that this reduces the sensitivity for detecting radiation-induced double-strand breaks in S- and G2-phase cells.

Evidence suggests that tumor cells exposed to some DNA damaging agents are more likely to die if they retain microscopically visible [gamma]H2AX foci that are known to mark sites of double-strand breaks. This appears to be true even after exposure to the alkylating agent MNNG that does not cause direct double-strand breaks but does produce [gamma]H2AX foci when damaged DNA undergoes replication. To examine this predictive ability further, SiHa human cervical carcinoma cells were exposed to 8 DNA damaging drugs (camptothecin, cisplatin, doxorubicin, etoposide, hydrogen peroxide, MNNG, temozolomide, and tirapazamine) and the fraction of cells that retained [gamma]H2AX foci 24 hours after a 30 or 60 min treatment was compared with the fraction of cells that lost clonogenicity. To determine if cells with residual repair foci are the cells that die, SiHa cervical cancer cells were stably transfected with a RAD51-GFP construct and live cell analysis was used to follow the fate of irradiated cells with RAD51-GFP foci. For all drugs regardless of their mechanism of interaction with DNA, close to a 1:1 correlation was observed between clonogenic surviving fraction and the fraction of cells that retained [gamma]H2AX foci 24 hours after treatment. Initial studies established that the fraction of cells that retained RAD51 foci after irradiation was similar to the fraction of cells that retained [gamma]H2AX foci and subsequently lost clonogenicity. Tracking individual irradiated live cells confirmed that SiHa cells with RAD51-GFP foci 24 hours after irradiation were more likely to die. Retention of DNA damage-induced [gamma]H2AX foci appears to be indicative of lethal DNA damage so that it may be possible to predict tumor cell killing by a wide variety of DNA damaging agents simply by scoring the fraction of cells that retain [gamma]H2AX foci.

Reitsema, T. J., Banáth, J. P., MacPhail, S. H. and Olive,P. L. Hypertonic Saline Enhances Expression of Phosphorylated Histone H2AX after Irradiation. Radiat. Res. 161, 402– 408 (2004). Phosphorylation of histone H2AX at serine 139 occurs at sites surrounding DNA double-strand breaks, producing discrete spots called “foci” that are visible with a microscope after antibody staining. This modification is believed to create changes in chromatin structure and assemble various repair proteins at sites of DNA damage. To examine the role of chromatin structure, human SiHa cells were exposed to hypertonic salt solutions that are known to condense chromatin and sensitize cells to chromosome damage and killing by ionizing radiation. Postirradiation incubation in 0.5 M Na+ increased γH2AX expression about fourfold as measured by flow cytometry and immunoblotting, and loss of γH2AX was inhibited in the presence of high salt. Focus size rather than the number of radiation-induced γH2AX foci was also increased about fourfold. When high-salt treatment was delayed for 1 h after irradiation, effects on focus size and retention were reduced. The increase in focus size was associated with a decrease in the rate of rejoining of double-strand breaks as measured using the neutral comet assay. We conclude that γH2AX expression after irradiation is sensitive to salt-induced changes in chromatin structure during focus formation, and that a large focus size may be an indication of a reduced ability to repair DNA damage.

MacPhail, S. H. and Olive, P. L. RPA Foci are Associated with Cell Death after Irradiation. Radiat. Res. 155, 672–679 (2001). Complexes containing replication protein A (RPA) were observed in human TK6 and WIL-2NS lymphoblast cells and SiHa cervical carcinoma cells exposed to 250 kV X rays. Image analysis of individual cells with fluorescence-tagged anti-RPA antibodies was used to measure numbers of discrete foci per cell. RPA foci formed in S-phase cells in response to radiation doses as low as 0.5 Gy, and the number of foci/nucleus was linearly related to dose up to 50 Gy. The maximum number of cells with foci occurred 4–8 h after exposure to 4 Gy, and subsequently declined. However, the number of RPA foci per nucleus (in those cells with foci) reached a maximum after 2–4 h. Apoptotic nuclei from irradiated TK6 and WIL-2NS cells initially contained foci, but these were lost as degradation continued. Radiation-induced micronuclei in SiHa cells were greatly enriched for RPA foci, and cells with nuclei without foci often contained micronuclei with multiple RPA foci. In SiHa cells examined up to 7 days after 4 Gy, RPA foci reappeared in one or more cells in up to 90% of the surviving colonies, and some cells contained 150 or more distinct foci. Reappearance of these complexes could be indicative of radiation-induced genomic instability. These results are consistent with the idea that RPA foci observed several hours after irradiation represent irreparable lesions and as such might be useful in identifying radiosensitive cells.

Sex work, and ideas about women in the trade, have long been represented as tragic and/or threatening. However, such portrayals tell us very little about how women think about themselves and the kinds of work they do. The data for this paper come from an ethnographic, community-based study in London, Ontario, that involves women in street-based, indoor and transactional sex work. This discussion focuses on how women develop different individual identities, including the management of multiple selves, their sexual identities and what we have termed the 'good junkie' identity. We also examine how these women employ aspects of dominant representation of sex workers, namely the low status accorded to those in street-based work and the defamatory term 'whore' or 'ho', when negotiating the moral hierarchies that exist within various kinds of sex work (i.e., stripping, massage parlours) and making sense of their professional and personal lives. The work that goes into the creation and maintenance of the women's divergent identities sheds important light on this complicated and tremendously demanding, yet inadequately understood, aspect of life as women in the sex trade.

This paper presents findings from an ethnographic, community-based research project with women in the sex trade in London, Ontario, the first of its kind in this Canadian city. Drawing upon 19 semi-structured life-history interviews with women between 24 and 60 years of age who have taken part in various aspects of the sex trade (i.e. street-based, indoor, transactional), this discussion focuses on how they entered the trade, the types of sex trade work done and the relationships with their male clients. Among the most important findings is the diversity that informs the women’s experiences across these fundamental aspects of the trade, which is best understood against the trajectory of these women’s lives and their oscillating, complicated involvement in sex work over time. Unlike most studies of sex work entry, which contend that this is a singular occurrence, our participants experienced entry and re-entry many times as they transition between different types of sex work. Their involvement in the trade follows a basic pattern of beginning in privately arranged situations (i.e. exotic dancing, sugar daddies) and moving to primarily street-based work, a significant change that is mediated by structural and inter-personal factors like the loss of their children, inability to access social and health services and deepening addictions to drugs. The women’s experiences with their male clients are also characterized by diversity and change according to the kind of sex work done and a constellation of mutable forces like socioeconomic need and drug dependency. Our findings, especially those regarding the factors affecting the women’s transition to the most dangerous and criminalized form of sex work, street-based, represent important and much-needed information with which to develop tailored, effective social policies and service provision to make London’s sex trade more supported and safer for the women who work here.

This article presents findings from a qualitative research project that sought to explore the organization of sex work and women's experiences in the trade in a medium-size Canadian city. Drawing upon 14 semi-structured life-history interviews with women between 24 and 60 years of age who have taken part in sex work, primarily street-based, this discussion examines the women's accounts of childhood and early family experiences. Findings related to childhood highlight the role of the women's mothers, fathers, and a range of non-normative conditions in shaping their social experience of childhood. Our participants' accounts of family dynamics feature discussions of loving familial relations, inter-generational involvement in the sex trade, and feelings of exclusion. These findings support existing studies on childhood and family experiences among street-based sex workers and contribute new data to this relatively under-developed area of study within the sex work literature. One of the most unique insights is that although the women's accounts reveal difficult experiences across the spheres of socialization related to childhood and family, they did not identify them as the root cause of their sex trade participation. We discuss the practical significance of this finding for health care and social service professionals (i.e., case worker, support staff, community clinic workers), who work with women in the sex trade but may be uncertain how to broach and/or navigate the sensitive issues of childhood and family in their work with these marginalized groups of women.

Evidence suggests that tumor cells exposed to some DNA damaging agents are more likely to die if they retain microscopically visible gammaH2AX foci that are known to mark sites of double-strand breaks. This appears to be true even after exposure to the alkylating agent MNNG that does not cause direct double-strand breaks but does produce gammaH2AX foci when damaged DNA undergoes replication. To examine this predictive ability further, SiHa human cervical carcinoma cells were exposed to 8 DNA damaging drugs (camptothecin, cisplatin, doxorubicin, etoposide, hydrogen peroxide, MNNG, temozolomide, and tirapazamine) and the fraction of cells that retained gammaH2AX foci 24 hours after a 30 or 60 min treatment was compared with the fraction of cells that lost clonogenicity. To determine if cells with residual repair foci are the cells that die, SiHa cervical cancer cells were stably transfected with a RAD51-GFP construct and live cell analysis was used to follow the fate of irradiated cells with RAD51-GFP foci. For all drugs regardless of their mechanism of interaction with DNA, close to a 1:1 correlation was observed between clonogenic surviving fraction and the fraction of cells that retained gammaH2AX foci 24 hours after treatment. Initial studies established that the fraction of cells that retained RAD51 foci after irradiation was similar to the fraction of cells that retained gammaH2AX foci and subsequently lost clonogenicity. Tracking individual irradiated live cells confirmed that SiHa cells with RAD51-GFP foci 24 hours after irradiation were more likely to die. Retention of DNA damage-induced gammaH2AX foci appears to be indicative of lethal DNA damage so that it may be possible to predict tumor cell killing by a wide variety of DNA damaging agents simply by scoring the fraction of cells that retain gammaH2AX foci.

Exposure of cells to ionizing radiation causes phosphorylation of histone H2AX at sites flanking DNA double-strand breaks. Detection of phosphorylated H2AX (γH2AX) by antibody binding has been used as a method to identify double-strand breaks. Although generally performed by observing microscopic foci within cells, flow cytometry offers the advantage of measuring changes in γH2AX intensity in relation to cell cycle position. The importance of cell cycle position on the levels of endogenous and radiation-induced γH2AX was examined in cell lines that varied in DNA content, cell cycle distribution, and kinase activity. Bivariate analysis of γH2AX expression relative to DNA content and synchronization by centrifugal elutriation were used to measure cell cycle-specific expression of γH2AX. With the exception of xrs5 cells, γH2AX level was approximately 3 times lower in unirradiated G1-phase cells than S- and G2-phase cells, and the slope of the G1-phase dose-response curve was 2.8 times larger than the slope for S-phase cells. Cell cycle differences were confirmed using immunoblotting, indicating that reduced antibody accessibility in intact cells was not responsible for the reduced antibody binding in G1-phase cells. Early apoptotic cells could be easily identified on flow histograms as a population with 5-10-fold higher levels of γH2AX, although high expression was not maintained in apoptotic cells by 24 h. We conclude that expression of γH2AX is associated with DNA replication in unirradiated cells and that this reduces the sensitivity for detecting radiation-induced double-strand breaks in S- and G2-phase cells.