The p14 alternate reading frame (ARF) tumor suppressor plays a central role in cancer by binding to mdm2 (Hdm2 in humans) and enhancing p53-mediated apoptosis following DNA damage and oncogene activation. It is unclear, however, how ARF initiates its involvement in the p53/mdm2 pathway, as p53 and mdm2 are located in the nucleoplasm, whereas ARF is largely nucleolar in tumor cells. We have used immunofluorescence and coimmunoprecipitation to examine how the subnuclear distribution and protein-protein interactions of ARF change immediately after DNA damage and over the time course of the DNA damage response in human tumor cells. We find that DNA damage disrupts the interaction of ARF with the nucleolar protein B23(nucleophosmin) and promotes a transient p53-independent translocation of ARF to the nucleoplasm, resulting in a masking of the ARF NH 2 terminus that correlates with the appearance of ARF-Hdm2 complexes. The translocation also results in an unmasking of the ARF COOH terminus, suggesting that redistribution disrupts a nucleolar interaction of ARF involving this region. By 24 hours after irradiation, DNA repair has ceased and the pretreatment immunofluorescence patterns and complexes of ARF have been restored. Although the redistribution of ARF is independent of p53 and likely to be regulated by interactions other than Hdm2, ARF does not promote UV sensitization unless p53 is expressed. The results implicate the nucleolus and nucleolar interactions of the ARF, including potentially novel interactions involving its COOH terminus as sites for early DNA damage and stress-mediated cellular events. (Cancer Res 2005; 65(21): 9834-42)

p14ARF (ARF) and topoisomerase I play central roles in cancer and have recently been shown to interact. The interaction activates topoisomerase I, an important target for camptothecin-like chemotherapeutic drugs, but the regulation of the interaction is poorly understood. We have used the H358 and H23 lung cancer cell lines and purified recombinant human topoisomerase I to demonstrate that the ARF/topoisomerase I interaction is regulated by topoisomerase I serine phosphorylation, a modification that regulates topoisomerase I activity. Both cell lines express wild-type ARF and topoisomerase I proteins at equivalent levels, but H23 topoisomerase I, unlike that of H358 cells, is largely devoid of serine phosphorylation, has low activity, and complexes poorly with ARF. The ability of H23 topoisomerase I to complex with ARF can be restored by treatment with the serine kinase, casein kinase II. Consistent with these observations, we show that the response of H23 cells to camptothecin treatment is unaffected by changes in intracellular levels of ARF. However, in H358 and PC-3 cells, which express a serine phosphorylated topoisomerase I that complexes with ARF, ectopic overexpression of ARF causes sensitization to camptothecin, and siRNA-mediated down-regulation of endogenous ARF causes desensitization to camptothecin. These biological responses correlate with increased and decreased levels, respectively, of ARF/topoisomerase I complex and DNA-bound topoisomerase I. Thus, ARF is a serine phosphorylation-dependent coregulator of topoisomerase I in vivo, and it regulates cellular sensitivity to camptothecin by interacting with topoisomerase I. Certain cancer associated defects affecting ARF/topoisomerase I complex formation could contribute to cellular resistance to camptothecin.

Restoration of apoptosis is an important therapeutic strategy for cancer, but bystander effects may be crucial to treatment success. We examined the involvement of bystander effects in the outcome of pro-apoptotic treatments and investigated the role of macrophages. Using a murine N202 breast cancer chamber model and intravital microscopy, we observed bystander apoptosis in vivo in mixed spheroids consisting of bystander N202 cells plus modified N202 cells overexpressing the p14ARF N-terminal region, which promotes p53-mediated apoptosis. The effect was not observed in cocultures in vitro, and could not be transferred through conditioned medium from modified N202 cells. However, if macrophages were also included in the N202 co-cultures, bystander apoptosis was restored, and correlated with elevated surface expression of phosphatidyl serine, a macrophage recognition molecule, on the modified N202 cells. Bystander killing was not observed in cocultures of N202 cells plus macrophages plus cisplatin-treated or 5-fluorouracil-treated N202 cells, where apoptosis induction in the target cell population was inefficient, suggesting that specific activation of macrophages by apoptotic tumor cells was required. The results suggest that pro-apoptotic therapies benefit both from the intrinsic vulnerability of cancer cells to apoptosis and from an innate immune response that amplifies the therapeutic effect.

Condom use among female sex workers (FSWs) is dynamic. We conducted a survey, by anonymous questionnaire, of condom use among FSWs routinely attending our clinical service in Northern Sydney. Logistic regression models determined associations with inconsistent condom use. All 201 women approached completed a questionnaire. Chinese-born women comprised 67% of participants. The median age was 35 years. Clients requesting unprotected fellatio was reported by 95% of participants, and approximately two-thirds felt pressure to have unprotected fellatio and vaginal sex (VSI). Inconsistent condom use at work was reported by 74% for fellatio and by 24% for VSI. The strongest adjusted association with inconsistent condom use for fellatio was monetary inducement by clients (aOR = 40.7, 95% CI 4.87–340, p = 0.001). The only other significant adjusted association was age ≥ 30 years ( p = 0.02). The strongest adjusted association with inconsistent condom use for VSI was also monetary inducement by clients (aOR = 56.1, 95% CI 9.26–340, p < 0.001). Other significant adjusted associations were Chinese-speaking participants ( p = 0.03) and clients requesting unprotected VSI ( p = 0.02). We report high levels of inconsistent condom use. Health promotion should assist FSWs, particularly those of Chinese ethnicity, develop skills in dealing with pressure for unprotected sex, particularly fellatio.