Activities forgone because of chronic breathlessness: a cross-sectional, population prevalence study

BackgroundChronic breathlessness is a prevalent, disabling syndrome affecting many people for years. Identifying the impact of chronic breathlessness on people’s activities in the general population is pivotal for designing symptom management strategies. ObjectiveThis study aimed to evaluate the association between chronic breathlessness and activities respondents identify can no longer be undertaken (‘activities foregone’). DesignThis population-based, cross sectional, online survey used a market research company’s database of 30,000 registrants for each sex, generating the planned sample size - 3,000 adults reflecting Australia’s 2016 Census by sex, age group, state of residence and rurality. Setting/Subjects The population of focus (n=583) reported a modified Medical Research Council (mMRC) breathlessness scale >1 and experienced this breathlessness for >3 months. MeasurementsActivities forgone were categorised by mMRC using coding derived from the Dyspnea Management Questionnaire domains. Activities were classified as ‘higher/lower intensity’ using Human Energy Expenditure scale.ResultsRespondents were: male 50.3%; median age 50.0 (IQR 29.0); with 66% living in metropolitan areas; reporting 1,749 activities forgone. For people with mMRC 1 (n=533), 35% had not given up any activity, decreasing to 9% for mMRC 2 (n=38) and 3% for mMRC 3-4 (n=12). Intense sport (e.g. jogging, bike riding) was the top activity forgone: 42% (mMRC 1); 32% (mMRC 2); and 36% (mMRC 3-4). For respondents with mMRC 3-4, the next most prevalent activities foregone were ‘sexual activities’ (14%); ‘lower intensity sports’ (11%) and ‘other activities’ (11%).ConclusionsPeople progressively reduce a wide range of activities because of their chronic breathlessness

Investigating the effects of novel iron-binding anti-cancer agents on p21Cip1/Waf1 regulation in cancer

The cyclin dependent kinase (CDK) inhibitor, p21, is well known for its role in cell cycle arrest and tumour suppression [1]. However, recent studies suggest that, under certain conditions, p21 can promote proliferation, oncogenicity and display anti-apoptotic functions [2, 3]. Given the pivotal role that p21 plays in cancer [1] it is emerging as a promising therapeutic target. In recent years, novel Fe-binding anti-cancer agents have demonstrated significant regulation of p21 expression in a variety of cancer cells [4-8]. Studies demonstrated that these agents significantly induced the expression of p21 mRNA independently of p53 [4, 6]. However, the expression of p21 protein in response to Fe chelators varied depending on the cell line being examined [5, 7, 8]. The exact molecular mechanisms by which chelators regulate p21 expression remain unknown and are important to establish. CHAPTER 3: Initial studies examined p21 mRNA and protein expression levels in five cancer cell lines with varying p53 status, including MCF-7 (WT p53), LNCaP (WT p53), SK-MEL-28 (mutant p53), CFPAC-1 (mutant p53) and SK-N-MC (p53 null). Incubation with Fe chelators significantly induced p21 mRNA expression in all cell-types examined. However the expression of p21 protein varied between the cell lines. Consistent with previous studies from our group [5, 8], p21 was significantly down-regulated in MCF-7 cells in response to chelators. Unlike MCF-7 cells, incubation with chelators significantly induced p21 expression in SK-MEL-28 and CFPAC-1 cells, and demonstrated no significant change in p21 expression in LNCaP or SK-N-MC cells. Therefore these findings clearly indicated that chelators are regulating p21 mRNA and protein independently of p53 status. Considering the latter finding, the expression of proteins known to modulate p21 independently of p53 were examined. Numerous isoforms of the murine double minute 2 (MDM2) protein were detected in four of the five cell lines examined. Importantly, a close correlation between the expression of p21 and the dominant negative p75MDM2 isoform was observed in MCF-7, LNCaP and SK-MEL-28 cells treated with chelators. This data suggested that MDM2 may be one mechanism by which chelators regulate p21 in these cell-types. Further studies examining the sub-cellular localisation of p21 demonstrated that Fe chelators induced its nuclear localisation in SK-MEL-28 and CFPAC-1 cell lines. Considering the anti-proliferative function of p21 when localised to the nucleus [9], its nuclear accumulation induced by chelators may be one mechanism by which these agents achieve their anti-proliferative activity. In contrast, incubating MCF-7 cells with these agents led to a reduction in the nuclear expression of p21. Taking into account that p21 is also known to aid in cell cycle progression by stabilising interactions between cyclins and CDKs [9-11], reducing the endogenously high levels of p21 in MCF-7 cells treated with chelators may partially account for the anti-proliferative activity of these agents in these cells. CHAPTER 4: Interestingly, the transcriptional regulation of the p21 promoter by Fe chelators was demonstrated to be dependent on the chelator and cell line being examined. The novel chelator, Dp44mT, was shown to induce p21 promoter activity in SK-MEL-28 cells but not MCF-7 cells. Further analysis of the p21 promoter identified a 50 bp region between -104 and -56 that was required for its Dp44mT-induced activation in SK-MEL-28 cells. Importantly, this region contained several binding sites for the Sp1 transcription factor. Mutational analysis of the region revealed that the Sp1-3 binding site played a significant role in Dp44mT-induced activation of p21 in SK-MEL-28 cells. Furthermore, a marked increase in the interactions between Sp1 and other transcription factors, namely oestrogen receptor-alpha (ER-α) and c-Jun, were observed in SK-MEL-28 cells treated with Dp44mT. Considering previous studies identifying that these interactions may enhance p21 promoter activation via the Sp1-3 binding site [12-14], the latter finding suggested their involvement in the Dp44mT-induced activation of p21 in SK-MEL-28 cells. CHAPTER 5: In addition to the transcriptional regulation of p21, Fe chelators have previously been shown to regulate p21 mRNA post-transcriptionally in MCF-7 cells [5]. Interestingly, in a recent study from our group, 311 and DFO were found to induce stress granule (SG) formation in MCF-7 cells [15]. Considering the previously reported involvement of SGs in the post-transcriptional regulation of p21 [16, 17], their involvement in chelator-induced regulation of p21 was examined. Similarly to MCF-7 cells [15], chelators induced the formation of SGs in SK-MEL-28 cells. Furthermore, the expression of the CUG triplet repeat RNA-binding protein 1 (CUGBP1) was significantly induced in SK-MEL-28 cells incubated with these agents. The co-localisation of CUGBP1 and SGs was also observed in SK-MEL-28 cells following chelator treatment. Interestingly, CUGBP1 has previously been shown to be involved in recruiting p21 mRNA to SGs [16]. However, upon CUGBP1 depletion, no significant change in p21 expression was observed in response to chelators. Collectively these data suggested that CUGBP1 does not play a major role in the post-transcriptional regulation of p21 in response to chelators. In summary, the data presented within this thesis demonstrated that chelators regulate p21 at multiple levels, including transcriptionally and post-transcriptionally depending on the cell line being examined. The findings presented within have enhanced our understanding of the anti-cancer function of chelators via the regulation of p21