SAHA-induced TRAIL-sensitisation of Multiple Myeloma cells is enhanced in 3D cell culture.

BACKGROUND: Multiple Myeloma (MM) is currently incurable despite many novel therapies. Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is a potential anti-tumour agent although effects as a single agent are limited. In this study, we investigated whether the Histone Deacetylase (HDAC) inhibitor SAHA can enhance TRAIL-induced apoptosis and target TRAIL resistance in both suspension culture, and 3D cell culture as a model of disseminated MM lesions that form in bone. METHODS: The effects of SAHA and/or TRAIL in 6 Multiple Myeloma cell lines were assessed in both suspension cultures and in an Alginate-based 3D cell culture model. The effect of SAHA and/or TRAIL was assessed on apoptosis by assessment of nuclear morphology using Hoechst 33342/Propidium Iodide staining. Viable cell number was assessed by CellTiter-Glo luminescence assay, Caspase-8 and -9 activities were measured by Caspase-Glo™ assay kit. TRAIL-resistant cells were generated by culture of RPMI 8226 and NCI-H929 by acute exposure to TRAIL followed by selection of TRAIL-resistant cells. RESULTS: TRAIL significantly induced apoptosis in a dose-dependent manner in OPM-2, RPMI 8226, NCI-H929, U266, JJN-3 MM cell lines and ADC-1 plasma cell leukaemia cells. SAHA amplified TRAIL responses in all lines except OPM-2, and enhanced TRAIL responses were both via Caspase-8 and -9. SAHA treatment induced growth inhibition that further increased in the combination treatment with TRAIL in MM cells. The co-treatment of TRAIL and SAHA reduced viable cell numbers all cell lines. TRAIL responses were further potentiated by SAHA in 3D cell culture in NCI-H929, RPMI 8226 and U266 at lower TRAIL + SAHA doses than in suspension culture. However TRAIL responses in cells that had been selected for TRAIL resistance were not further enhanced by SAHA treatment. CONCLUSIONS: SAHA is a potent sensitizer of TRAIL responses in both TRAIL sensitive and resistant cell lines, in both suspension and 3D culture, however SAHA did not sensitise TRAIL-sensitive cell populations that had been selected for TRAIL-resistance from initially TRAIL-sensitive populations. SAHA may increase TRAIL sensitivity in insensitive cells, but not in cells that have specifically been selected for acquired TRAIL-resistance.

Blood donors' attitudes towards voluntary non-remunerated donation in Trinidad and Tobago.

OBJECTIVES: To assess the attitude towards voluntary non-remunerated blood donation among blood donors in Trinidad and Tobago (TRT). BACKGROUND: Blood donors in TRT are either family replacement (F/R, 87%) or remunerated (13%). There is chronic blood shortage and high seroreactivity for transfusion-transmissible infections (TTI) in donors. Converting existing to voluntary non-remunerated donors (VNRD) reduces the need to recruit news donors in achieving 100% VNRD. METHODS: A questionnaire-based, cross-sectional survey was conducted at two blood collection centres at an interval of 8 years. Donors were surveyed for sociodemographic characteristics, awareness of the blood shortage, previous donation behaviour, donor-beneficiary linkage if F/R, willingness to become VNRD and choice of motivators for converting to VNRD. RESULTS: A total of 400 and 595 donors respectively participated in Surveys 1 and 2, of whom 92·8 and 86·3% were F/R (P < 0·001), respectively. In both surveys, 52% of participants were unaware of an existing blood shortage (P = 0·983). Only 9·8 and 9·1% of participants expressed unwillingness to become VNRD (P = 0·720). The main motivators to convert to VNRD were reminders from the centre (84%) and extended opening hours (78%) in Survey 1 as compared to confidence that donated blood was used properly (73%) and shortened waiting times to donate (73%) in Survey 2. CONCLUSION: Despite low awareness of blood shortage, willingness to become VNRD was high among existing donors. Accountability and donor convenience underpinned the main motivators for converting to VNRD.

Osteolytica: An automated image analysis software package that rapidly measures cancer-induced osteolytic lesions in in vivo models with greater reproducibility compared to other commonly used methods.

Methods currently used to analyse osteolytic lesions caused by malignancies such as multiple myeloma and metastatic breast cancer vary from basic 2-D X-ray analysis to 2-D images of micro-CT datasets analysed with non-specialised image software such as ImageJ. However, these methods have significant limitations. They do not capture 3-D data, they are time-consuming and they often suffer from inter-user variability. We therefore sought to develop a rapid and reproducible method to analyse 3-D osteolytic lesions in mice with cancer-induced bone disease. To this end, we have developed Osteolytica, an image analysis software method featuring an easy to use, step-by-step interface to measure lytic bone lesions. Osteolytica utilises novel graphics card acceleration (parallel computing) and 3-D rendering to provide rapid reconstruction and analysis of osteolytic lesions. To evaluate the use of Osteolytica we analysed tibial micro-CT datasets from murine models of cancer-induced bone disease and compared the results to those obtained using a standard ImageJ analysis method. Firstly, to assess inter-user variability we deployed four independent researchers to analyse tibial datasets from the U266-NSG murine model of myeloma. Using ImageJ, inter-user variability between the bones was substantial (±19.6%), in contrast to using Osteolytica, which demonstrated minimal variability (±0.5%). Secondly, tibial datasets from U266-bearing NSG mice or BALB/c mice injected with the metastatic breast cancer cell line 4T1 were compared to tibial datasets from aged and sex-matched non-tumour control mice. Analyses by both Osteolytica and ImageJ showed significant increases in bone lesion area in tumour-bearing mice compared to control mice. These results confirm that Osteolytica performs as well as the current 2-D ImageJ osteolytic lesion analysis method. However, Osteolytica is advantageous in that it analyses over the entirety of the bone volume (as opposed to selected 2-D images), it is a more rapid method and it has less user variability.

A review of current murine models of multiple myeloma used to assess the efficacy of therapeutic agents on tumour growth and bone disease.

Pre-clinical in vivo models of multiple myeloma are essential tools for investigating the pathophysiology of multiple myeloma and for testing new therapeutic agents and strategies prior to their potential use in clinical trials. Over the last five decades, several different types of murine models of multiple myeloma have been developed ranging from immunocompetent syngeneic models, e.g. the 5 T series of myeloma cells, to immunocompromised models including the SCID xenograft models, which use human myeloma cell lines or patient-derived cells. Other models include hybrid models featuring the implantation of SCID mice with bone chips (SCID-hu or SCID-rab) or 3-D bone scaffolds (SCID-synth-hu), and mice that have been genetically engineered to develop myeloma. Bearing in mind the differences in these models, it is not surprising that they reflect to varying degrees different aspects of myeloma. Here we review the past and present murine models of myeloma, with particular emphasis on their advantages and limitations, characteristics, and their use in testing therapeutic agents to treat myeloma tumour burden and bone disease.

A systems biology approach to Down syndrome: identification of Notch/Wnt dysregulation in a model of stem cells aging.

Stem cells are central to the development and maintenance of many tissues. This is due to their capacity for extensive proliferation and differentiation into effector cells. More recently it has been shown that the proliferative and differentiative ability of stem cells decreases with age, suggesting that this may play a role in tissue aging. Down syndrome (DS), is associated with many of the signs of premature tissue aging including T-cell deficiency, increased incidence of early Alzheimer-type, Myelodysplastic-type disease and leukaemia. Previously we have shown that both hematopoietic (HSC) and neural stem cells (NSC) in patients affected by DS showed signs of accelerated aging. In this study we tested the hypothesis that changes in gene expression in HSC and NSC of patients affected by DS reflect changes occurring in stem cells with age. The profiles of genes expressed in HSC and NSC from DS patients highlight pathways associated with cellular aging including a downregulation of DNA repair genes and increases in proapoptotic genes, s-phase cell cycle genes, inflammation and angiogenesis genes. Interestingly, Notch signaling was identified as a potential hub, which when deregulated may drive stem cell aging. These data suggests that DS is a valuable model to study early events in stem cell aging.