Cellular stress triggers the human topoisomerase I damage response independently of DNA damage in a p53 controlled manner

The 'human topoisomerase I (htopoI) damage response' was reported to be triggered by various kinds of DNA lesions. Also, a high and persistent level of htopoI cleavage complexes correlated with apoptosis. In the present study, we demonstrate that DNA damage-independent induction of cell death using colcemid and tumor necrosis factor is also accompanied by a strong htopoI response that correlates with the onset of apoptotic hallmarks. Consequently, these results suggest that htopoI cleavage complex formation may be caused by signaling pathways independent of the kind of cellular stress. Thus, protein interactions or signaling cascades induced by DNA damage or cellular stress might lead to the formation of stabilized cleavage complexes rather than the DNA lesion itself. Finally, we show that p53 not only plays a key role in the regulation of the htopoI response to UV-C irradiation but also to treatment with colcemid.

The human topoisomerase I damage response plays a role in apoptosis

Previous studies have shown that human topoisomerase I cleavage complexes form as a response to various DNA damages in vivo, the so called human topoisomerase I “damage response”. It was suggested that this damage response may play a role in DNA repair as well as in apoptosis, but only very few investigations have been done and the significance of the damage response still remains unclear. Here we demonstrate that human topoisomerase I cleavage complexes induced by high doses of UV irradiation are highly stable for up to 48 h. Furthermore, we show that human topoisomerase I cleavage complexes correlate with apoptosis. However, at low UV doses the cleavage complex level was very low and the complexes were repaired. Surprisingly, we found that high levels of stable cleavage complexes were not only found in UV-irradiated cells but also in untreated cells that underwent apoptosis. A possible role of human topoisomerase I in apoptosis is discussed.

CD151 is associated with prostate cancer cell invasion and lymphangiogenesis in vivo

CD151, a member of the tetraspanin family, is associated with regulation of migration of normal and tumour cells via cell surface microdomain formation. CD151 was found in our laboratory to have a prognostic value in prostate cancer and is a promoter of prostate cancer migration and invasion. These roles involve association with integrins on both cell-cell and cell-stroma levels. Furthermore, CD151 plays a role in endothelial cell motility. CD151 expression was examined in three commonly used prostate cancer cell lines. We investigated CD151 expression, angiogenesis (microvessel density; MVD) and lymphangiogenesis (lymphatic vessel density; LVD) in an orthotopic xenograft model of prostate cancer in matched tumours from primary and secondary sites. CD151 was found to be heterogeneously expressed across different prostate cancer cell lines and the levels of CD151 expression were significantly higher in the highly tumorigenic, androgen-insensitive cells PC-3 and DU-145 compared to the androgen-sensitive cell line LNCaP (P<0.05). The majority of in vivo xenografts developed pelvic lymph node metastases. Importantly, primary tumours that developed metastasis had significantly higher CD151 expression and MVD compared to those which did not develop metastasis (P<0.05). We identified, for the first time, that CD151 expression is associated with LVD in prostate cancer. These findings underscore the potential role of CD151 and angiogenesis in the metastatic potential of prostate cancer. CD151 has a prognostic value in this mouse model of prostate cancer and may play a role in lymphangiogenesis. CD151 is likely an important regulator of cancer cell communication with the surrounding microenvironment.

Isolation of human lymphatic malformation endothelial cells, their in vitro characterization and in vivo survival in a mouse xenograft model

Human lymphatic vascular malformations (LMs), also known as cystic hygromas or lymphangioma, consist of multiple lymphatic endothelial cell-lined lymph-containing cysts. No animal model of this disease exists. To develop a mouse xenograft model of human LM, CD34NegCD31Pos LM lymphatic endothelial cells (LM-LEC) were isolated from surgical specimens and compared to foreskin CD34NegCD31Pos lymphatic endothelial cells (LECs). Cells were implanted into a mouse tissue engineering model for 1, 2 and 4 weeks. In vitro LM-LECs showed increased proliferation and survival under starvation conditions (P < 0.0005 at 48 h, two-way ANOVA), increased migration (P < 0.001, two-way ANOVA) and formed fewer (P = 0.029, independent samples t test), shorter tubes (P = 0.029, independent samples t test) than foreskin LECs. In vivo LM-LECs implanted into a Matrigel™-containing mouse chamber model assembled to develop vessels with dilated cystic lumens lined with flat endothelium, morphology similar to that of clinical LMs. Human foreskin LECs failed to survive implantation. In LM-LEC implanted chambers the percent volume of podoplaninPos vessels was 1.18 ± 2.24 % at 1 week, 6.34 ± 2.68 % at 2 weeks and increasing to 7.67 ± 3.60 % at 4 weeks. In conclusion, the significantly increased proliferation, migration, resistance to apoptosis and decreased tubulogenesis of LM-LECs observed in vitro is likely to account for their survival and assembly into stable LM-like structures when implanted into a mouse vascularised chamber model. This in vivo xenograft model will provide the basis of future studies of LM biology and testing of potential pharmacological interventions for patients with lymphatic malformations.

Strategies and challenges for systematically mapping biologically significant molecular pathways regulating carcinoma epithelial-mesenchymal transition

Enormous progress has been made towards understanding the role of specific factors in the process of epithelial-mesenchymal transition (EMT); however, the complex underlying pathways and the transient nature of the transition continues to present significant challenges. Targeting tumour cell plasticity underpinning EMT is an attractive strategy to combat metastasis. Global gene expression profiling and high-content analyses are among the strategies employed to identify novel EMT regulators. In this review, we highlight several approaches to systematically interrogate key pathways involved in EMT, with particular emphasis on the features of multiparametric, high-content imaging screening strategies that lend themselves to the systematic discovery of highly significant modulators of tumour cell plasticity.

Differential expression of VEGF ligands and receptors in prostate cancer

BACKGROUND Prostate cancer disseminates to regional lymph nodes, however the molecular mechanisms responsible for lymph node metastasis are poorly understood. The vascular endothelial growth factor (VEGF) ligand and receptor family have been implicated in the growth and spread of prostate cancer via activation of the blood vasculature and lymphatic systems. The purpose of this study was to comprehensively examine the expression pattern of VEGF ligands and receptors in the glandular epithelium, stroma, lymphatic vasculature and blood vessels in prostate cancer. METHODS The localization of VEGF-A, VEGF-C, VEGF-D, VEGF receptor (VEGFR)-1, VEGFR-2, and VEGFR-3 was examined in cancerous and adjacent benign prostate tissue from 52 subjects representing various grades of prostate cancer. RESULTS Except for VEGFR-2, extensive staining was observed for all ligands and receptors in the prostate specimens. In epithelial cells, VEGF-A and VEGFR-1 expression was higher in tumor tissue compared to benign tissue. VEGF-D and VEGFR-3 expression was significantly higher in benign tissue compared to tumor in the stroma and the endothelium of lymphatic and blood vessels. In addition, the frequency of lymphatic vessels, but not blood vessels, was lower in tumor tissue compared with benign tissue. CONCLUSIONS These results suggest that activation of VEGFR-1 by VEGF-A within the carcinoma, and activation of lymphatic endothelial cell VEGFR-3 by VEGF-D within the adjacent benign stroma may be important signaling mechanisms involved in the progression and subsequent metastatic spread of prostate cancer. Thus inhibition of these pathways may contribute to therapeutic strategies for the management of prostate cancer.

The radiation therapy patient as a student assessor

Background Radiation Therapy students at Queensland University of Technology undertake clinical placement across a wide range of sites Interpersonal skills with clinical staff and patients are an essential component: – Lectures – Role playing – Expert patient input

Selective regulation of p38β protein and signaling by integrin-linked kinase mediates bladder cancer cell migration

Integrin-linked kinase (ILK) and p38MAPK are protein kinases that transduce extracellular signals regulating cell migration and actin cytoskeletal organization. ILK-dependent regulation of p38MAPK is critical for mammalian kidney development and in smooth muscle cell migration, however, specific p38 isoforms has not been previously examined in ILK-regulated responses. Signaling by ILK and p38MAPK is often dysregulated in bladder cancer, and here we report a strong positive correlation between protein levels of ILK and p38β, which is the predominant isoform found in bladder cancer cells, as well as in patient-matched normal bladder and tumor samples. Knockdown by RNA interference of either p38β or ILK disrupts serum-induced, Rac1-dependent migration and actin cytoskeletal organization in bladder cancer cells. Surprisingly, ILK knockdown causes the selective reduction in p38β cellular protein level, without inhibiting p38β messenger RNA (mRNA) expression. The loss of p38β protein in ILK-depleted cells is partially rescued by the 26S proteasomal inhibitor MG132. Using co-precipitation and bimolecular fluorescent complementation assays, we find that ILK selectively forms cytoplasmic complexes with p38β. In situ proximity ligation assays further demonstrate that serum-stimulated assembly of endogenous ILK–p38β complexes is sensitive to QLT-0267, a small molecule ILK kinase inhibitor. Finally, inhibition of ILK reduces the amplitude and period of serum-induced activation of heat shock protein 27 (Hsp27), a target of p38β implicated in actin cytoskeletal reorganization. Our work identifies Hsp27 as a novel target of ILK–p38β signaling complexes, playing a key role in bladder cancer cell migration.

ATF3 suppresses metastasis of bladder cancer by regulating gelsolin-mediated remodeling of the actin cytoskeleton

Bladder cancer is associated with high recurrence and mortality rates due to metastasis. The elucidation of metastasis suppressors may offer therapeutic opportunities if their mechanisms of action can be elucidated and tractably exploited. In this study, we investigated the clinical and functional significance of the transcription factor activating transcription factor 3 (ATF3) in bladder cancer metastasis. Gene expression analysis revealed that decreased ATF3 was associated with bladder cancer progression and reduced survival of patients with bladder cancer. Correspondingly, ATF3 overexpression in highly metastatic bladder cancer cells decreased migration in vitro and experimental metastasis in vivo. Conversely, ATF3 silencing increased the migration of bladder cancer cells with limited metastatic capability in the absence of any effect on proliferation. In keeping with their increased motility, metastatic bladder cancer cells had increased numbers of actin filaments. Moreover, ATF3 expression correlated with expression of the actin filament severing protein gelsolin (GSN). Mechanistic studies revealed that ATF3 upregulated GSN, whereas ATF3 silencing reduced GSN levels, concomitant with alterations in the actin cytoskeleton. We identified six ATF3 regulatory elements in the first intron of the GSN gene confirmed by chromatin immunoprecipitation analysis. Critically, GSN expression reversed the metastatic capacity of bladder cancer cells with diminished levels of ATF3. Taken together, our results indicate that ATF3 suppresses metastasis of bladder cancer cells, at least in part through the upregulation of GSN-mediated actin remodeling. These findings suggest ATF3 coupled with GSN as prognostic markers for bladder cancer metastasis.

Pancreatic cancer – a patient and his doctor balance hope and truth [Book Review]

This book comprises 11 chapters, alternating between two authors (a patient with metastatic pancreatic cancer and an oncologist)...

Prognostic impact of vascular and lymphovascular invasion in early lung cancer

Background The prognostic significance of vascular and lymphatic invasion in non-small-cell lung cancer is under continuous debate. We analyzed the effect of tumor aggressiveness (lymphatic and/or vessel invasion) on survival and relapse in stage I and II non-small-cell lung cancer. Methods We retrospectively analyzed prospectively collected data of 457 patients with stage I and II non-small-cell lung cancer from 1998 to 2008. Specimens were analyzed for intratumoral vascular invasion and lymphovascular space invasion. Overall survival and disease-free survival were estimated using the Kaplan-Meier method, and differences were determined by the logrank test. Cox regression analysis was performed to identify independent risk factors. Results: The incidence of intratumoral vascular invasion was 23.4%, and this correlated significantly with grade of differentiation, visceral pleural involvement, lymphovascular space invasion, and N status. The incidence of lymphovascular space invasion was 5.5%, and this correlated significantly with grade of differentiation, lymph nodes involved, and intratumoral vascular invasion. On multivariate analyses, intratumoral vascular invasion proved to be an significant independent risk factor for overall survival but not for disease-free survival. Lymphovascular space invasion was associated significantly with early tumor recurrence but not with overall survival. Conclusions: Vascular and lymphatic invasion can serve as independent prognostic factors in completely resected nonsmall- cell lung cancer. Intratumoral vascular invasion and lymphovascular space invasion in early stage non-small-cell lung cancer are important factors in overall survival and early tumor recurrence. Further large scale studies with more recent patient cohorts and refined histological techniques are warranted.

IL-23R is epigenetically regulated and modulated by chemotherapy in non-small cell lung cancer

The Interleukin-23 (IL-23)/IL-23R signaling axis is an important inflammatory pathway, involved in the stimulation and regulation of the T helper (Th) 17 lymphocytes, resulting in the production of IL-17. Aside from auto-immunity, this cytokine has also been linked to carcinogenesis and polymorphisms in the IL-23R gene are associated with an increased risk for the development of a number of different cancers. Activation of the IL-23 pathway results in the up-regulation of STAT3 and it is thought that the pathological consequences associated with this are in part due to the production of IL-17. We have previously identified IL-23A as pro-proliferative and epigenetically regulated in non-small cell lung cancer (NSCLC). The current study aims to evaluate IL-23R in greater detail in NSCLC. We demonstrate that IL-23R is expressed and epigenetically regulated in NSCLC through histone post-translation modifications and CpG island methylation. In addition, Gemcitabine treatment, a chemotherapy drug used in the treatment of NSCLC, resulted in the up-regulation of the IL-23R. Furthermore, Apilimod (STA 5326), a small molecule which blocks the expression of IL-23 and IL-12, reduced the proliferative capacity of NSCLC cells, particularly in the adenocarcinoma (A549) sub-type. Apilimod is currently undergoing investigation in a number of clinical trials for the treatment of auto-immune conditions such as Crohn's disease and Rheumatoid Arthritis. Our results may have implications for treating NSCLC patients with Gemcitabine or epigenetic targeted therapies. However, Apilimod may possibly provide a new treatment avenue for NSCLC patients. Work is currently ongoing to further delineate the IL-23/IL-23R axis in this disease.

Epigenetic regulation of chemokine/chemokine receptor expression

Epigenetic regulation of gene expression is an important event for normal cellular homeostasis. Gene expression may be "switched" on or "turned" off via epigenetic means through adjustments in DNA architecture. These structural alterations result from changes to the DNA methylation status in addition to histone posttranslational modifications such as acetylation and methylation. Drugs which can alter the status of these epigenetic markers are currently undergoing clinical trials in a wide variety of diseases, including cancer.We illustrate the treatment of cell lines with histone deacetylase (HDi) and DNA methyltransferase inhibitors and the subsequent RNA isolation and reverse transcriptase polymerase chain reaction for several members of the CXC (ELR(+)) chemokine family. In addition we describe a chromatin immunoprecipitation assay to determine the association between chromatin transcription markers and DNA following pretreatment of cell cultures with an HDi, Trichostatin A (TSA). This assay allows us to determine whether treatment with TSA dynamically remodels the promoter region of our selected genes, as judged by the differences in the PCR product between our treated and untreated samples.