Neil Cross

Osteoprotegerin (OPG) produced by bone marrow stromal cells protects breast cancer cells from TRAIL-induced apoptosis

Advanced breast cancer is often associated with metastatic bone disease, causing a number of serious complications for the patients such as hypercalceamia, pain, nerve compression and fractures. The formation of bone metastases depends on complex interactions between tumour cells and the cells of the bone microenvironment, but the precise molecular mechanisms involved in the development of tumour-induced bone disease have not been identified. We have investigated the ability of bone marrow stromal cells (BMSC) isolated from breast cancer patients to generate osteoprotegerin (OPG), a molecule involved both in bone turnover and cell survival. The potential survival effects of OPG are mediated through binding to a member of the TNF super family, TNF-related Apoptosis Inducing Ligand (TRAIL), preventing association between TRAIL and its death-inducing receptors present on a number of tumour cell types. In the present report we show that bone marrow stromal cells isolated from breast cancer patients produce OPG when grown in culture. The levels of OPG present in BMSC conditioned medium is sufficient to protect breast cancer cells from undergoing TRAIL induced apoptosis. Our data suggest that bone-derived OPG may increase survival of breast cancer cells that reach the bone microenvironment as part of the metastatic process.

Osteoprotegerin (OPG) Expression by Breast Cancer Cells in vitro and Breast Tumours in vivo – A Role in Tumour Cell Survival?

SummaryIn addition to its role in bone turnover, osteoprotegerin (OPG) has been reported to bind to and inhibit Tumour necrosis factor-related apoptosis inducing ligand (TRAIL). TRAIL is produced in tumours by invading monocytes, inducing apoptosis in neoplastic cells sensitive to this cytokine. OPG production by tumour cells would therefore be a novel mechanism whereby cancer cells evade host defences and gain a growth advantage. In this study we show that OPG produced by breast cancer cells enhances tumour cell survival by inhibiting TRAIL-induced apoptosis. OPG expression by breast cancer cells (MDA-MB 436/231) grown in vitro was examined using PCR and ELISA, and the sensitivity of these cells to TRAIL was determined. The effects of OPG on TRAIL induced apoptosis was investigated by exposing MDA-MB 436 cells to TRAIL, in the presence or absence of OPG, followed by assessment of nuclear morphology. We found that the levels of OPG produced were sufficient to inhibit TRAIL-induced apoptosis, suggesting that OPG may play a role in tumour cell survival. We also examined the expression pattern of OPG in a selection of breast tumours (n=400) by immunohistochemistry, and related OPG expression to the clinico-pathological data for each tumour. OPG expression was found to be negatively correlated with increasing tumour grade. To our knowledge these results are the first to demonstrate that OPG can act as an endocrine survival factor for breast cancer cells, as well as reporting the expression patterns of OPG in a large cohort of human breast tumours.

Blood vessel maturation and response to vascular-disrupting therapy in single vascular endothelial growth factor-A isoform-producing tumors

Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer therapy but the factors that influence tumor susceptibility to these agents are poorly understood. We evaluated the consequences of modifying tumor vascular morphology and function on vascular and therapeutic response to combretastatin-A4 3-O-phosphate (CA-4-P), which was chosen as a model VDA. Mouse fibrosarcoma cell lines that are capable of expressing all vascular endothelial growth factor (VEGF) isoforms (control) or only single isoforms of VEGF (VEGF120, VEGF164, or VEGF188) were developed under endogenous VEGF promoter control. Once tumors were established, VEGF isoform expression did not affect growth or blood flow rate. However, VEGF188 was uniquely associated with tumor vascular maturity, resistance to hemorrhage, and resistance to CA-4-P. Pericyte staining was much greater in VEGF188 and control tumors than in VEGF120 and VEGF164 tumors. Vascular volume was highest in VEGF120 and control tumors (CD31 staining) but total vascular length was highest in VEGF188 tumors, reflecting very narrow vessels forming complex vascular networks. I.v. administered 40 kDa FITC-dextran leaked slowly from the vasculature of VEGF188 tumors compared with VEGF120 tumors. Intravital microscopy measurements of vascular length and RBC velocity showed that CA-4-P produced significantly more vascular damage in VEGF120 and VEGF164 tumors than in VEGF188 and control tumors. Importantly, this translated into a similar differential in therapeutic response, as determined by tumor growth delay. Results imply differences in signaling pathways between VEGF isoforms and suggest that VEGF isoforms might be useful in vascular-disrupting cancer therapy to predict tumor susceptibility to VDAs.

In vitro propagation and characterization of neoplastic stem/progenitor-like cells from human prostate cancer tissue

According to the cancer stem cell hypothesis, tumor growth is sustained by a subpopulation of cancer stem/progenitor‐like cells. Self‐renewal and high clonogenic potential are characteristics shared by normal stem and neoplastic stem/progenitor‐like cells. We investigated whether human prostate cancer specimens contain cells with these properties.

Human Bone Marrow Stromal Cells Protect Prostate Cancer Cells From TRAIL‐Induced Apoptosis

Tumor‐derived OPG has recently been shown to protect prostate cancer cells from apoptosis. This study has confirmed that bone marrow stromal cell‐derived OPG also suppresses cytokine‐induced apoptosis in this tumor type, suggesting that it may be the presence of bone‐derived OPG that is responsible for the observed preference of these cells in colonizing the skeleton.

Multiple locations on chromosome 3 are the targets of specific deletions in uveal melanoma

PurposeLoss of chromosome 3 is a frequent event in uveal melanomas, which is associated with hepatic metastases and a poor prognosis. The entire copy of chromosome 3 is usually lost (monosomy 3); however, a small subset of tumours demonstrate partial deletions of chromosome 3. Analysis of these tumours may allow the identification of tumour suppressor genes (TSGs) that are the molecular target of monosomy 3. Therefore, the purpose of this investigation was to determine the location of these partial deletions of chromosome 3 in uveal melanomas.MethodsMicrosatellite analysis and restriction fragment-length polymorphism analysis were performed on 52 primary uveal melanomas using 19 markers located on both arms of chromosome 3. Cytogenetic analysis and fluorescence in situhybridisation were performed, where possible, to confirm molecular findings.ResultsOf 52 tumours studied, five tumours (10%) demonstrated LOH at one or more informative markers, but retention of heterozygosity was observed at other loci on chromosome 3, consistent with the presence of structural abnormalities to chromosome 3. Consistent with previous findings, the pattern of LOH in these tumours indicates the presence of deletions around 3p25–26 and on 3q, and that a new target region at 3p11–14 is preferentially deleted.ConclusionsThese results indicate the presence of several tumour suppressor loci on chromosome 3 and support the notion that the high rate of monosomy 3 in uveal melanoma is driven by disruption of several TSGs located on both arms of chromosome 3.

Real-time polymerase chain reaction shows that density centrifugation does not always remove Chlamydia trachomatis from human semen

OBJECTIVE To evaluate the efficiency of sperm washing procedures to remove Chlamydia trachomatis from semen both in clinical samples and experimental inoculations. DESIGN Laboratory-based study. SETTING Research laboratory in a university hospital. PATIENT(S) One hundred men attending for diagnostic semen analysis as part of infertility investigations and three sperm donors providing ejaculates for research purposes. MAIN OUTCOME MEASURE(S) Number of DNA copies of C. trachomatis, infectivity in an HeLa cell monolayer, and immunofluorescence. RESULT(S) Of the 100 semen samples examined, 13 contained detectable levels of C. trachomatis DNA (675-15,920 copies/mL) and in only 7 was this completely removed after sperm washing. In the remaining six DNA-positive samples, the number of copies in the postwash preparation ranged from 36-455 per mL. Experimental inoculations found that postwash preparations containing C. trachomatis DNA as low as 61 copies/mL were able to establish an infection in vitro. CONCLUSION(S) Undiagnosed C. trachomatis infections in men attending for assisted conception could potentially lead to infection or contamination of the IVF culture system as sperm washing methods are not 100% effective.

Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH(Lo) cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH(Hi) population, or whether all ALDH(Hi) cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH(Hi) cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH(Hi) cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH(Lo) population can develop ALDH(Hi) populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH(Hi) cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in defined medium favouring stem cell characteristics. Although ALDH(Hi) status enriches for holoclone formation, this activity may be mediated by a minority of ALDH(Hi) cells.

Differential Effects of Polyphenols on Proliferation and Apoptosis in Human Myeloid and Lymphoid Leukemia Cell Lines

Background: Mortality rates for leukemia are high despite considerable improvements in treatment. Since polyphenols exert pro-apoptotic effects in solid tumors, our study investigated the effects of polyphenols in haematological malignancies. The effect of eight polyphenols (quercetin, chrysin, apigenin, emodin, aloe-emodin, rhein, cis-stilbene and trans-stilbene) were studied on cell proliferation, cell cycle and apoptosis in four lymphoid and four myeloid leukemic cells lines, together with normal haematopoietic control cells. Methods: Cellular proliferation was measured by CellTiter-Glo® luminescent assay; and cell cycle arrest was assessed using flow cytometry of propidium iodide stained cells. Apoptosis was investigated by caspase-3 activity assay using flow cytometry and apoptotic morphology was confirmed by Hoescht 33342 staining. Results: Emodin, quercetin, and cis-stilbene were the most effective polyphenols at decreasing cell viability (IC50 values of 5-22 µM, 8-33 µM, and 25-85 µM respectively) and inducing apoptosis (AP50 values (the concentration which 50% of cells undergo apoptosis) of 2-27 µM, 19-50 µM, and 8-50 µM respectively). Generally, lymphoid cell lines were more sensitive to polyphenol treatment compared to myeloid cell lines, however the most resistant myeloid (KG-1a and K562) cell lines were still found to respond to emodin and quercetin treatment at low micromolar levels. Non-tumor cells were less sensitive to all polyphenols compared to the leukemia cells. Conclusions: These findings suggest that polyphenols have anti-tumor activity against leukemia cells with differential effects. Importantly, the differential sensitivity of emodin, quercetin, and cis-stilbene between leukemia and normal cells suggests that polyphenols are potential therapeutic agents for leukemia.

Anti-angiogenic properties of ADAMTS-4 in vitro

Angiogenesis is an indispensable mechanism in development and in many pathologies, including cancer, synovitis and aberrant wound healing. Many angiogenic stimulators and inhibitors have been investigated, and some have progressed to the clinic. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) is a group of multifunctional proteinases. ADAMTS‐1 and ADAMTS‐8 have been reported to be anti‐angiogenic. Here, we provide evidence that ADAMTS‐4, like ADAMTS‐1, is expressed by endothelial cells and binds to vascular endothelial groth factor (VEGF). Moreover, ADAMTS‐4 inhibited human dermal microvascular endothelial cells (HuDMEC) VEGF‐stimulated VEGF receptor (R) R2 phosphorylation, differentiation and migration, suggesting that ADAMTS‐4 may be a novel anti‐angiogenic molecule.