Purpose: Osteosarcoma (OS) is the most common type of cancer in children. OS demonstrates aggressive growth with a high risk of early, pulmonary metastasis. Here we investigated expression of malignancy-related factors in six osteosarcoma cell lines (SaOS, MNNG/ HOS, MG63, SW1353, SKES, SJSA), ten biopsies of primary OS and OS derived cells.

Methods: For this project, we are using RT-PCR and immunohistochemistry to detect PARP, ET-1, ETA and ETB receptor. We also, examined the expression of osteocrin by in situ hybridization. Zymography and Northern Blot were use to observe the presence of gela-tinases (MMP-2 and MMP-9) and finally, we showed the presence of ET-1(1–31) by Elisa-immunoassay.

Results: In OS tissues and cells, we observed ET-1, MMP-2, and ETA receptor overexpression, in contrast to under-expression of MMP-9 and ETB receptor. Additionally, in high malignant OS cells, MMP-2, MMP-3, MMP-13 and PARP were overexpressed and TIMP-1, TIMP-2 and TIMP-4 expressed at low levels. Using a new specific immunoassay for ET-1 (1–31) we showed presence of this alternative form of ET-1 in OS-derived cells. We also showed that, in cells exposed to ET-1, the inhibition of NF-kB pathway (NF-kB is an ubiquitous transcription factor playing a central role in differentiation, proliferation and malign transformation) drastically blocked MMP-2 production and activity, and that ET-1 induces NF-kB p65 unit translocation to the nucleus.

Conclusions: Thus, we concluded that expression of malignancy-related factors in parallel to the histological evaluation of these tumors, could be beneficial for malignancy understanding. Tumor cells invasion and aggressiveness of OS cells are the results, in part, of the tumorigenic potential of alternative forms of ET-1 generated by MMPs and enhanced in malignant microenvironment. These factors could be predictive for tumoral progression. Funding: Educational Grant from the Canadian Orthopaedic Foundation. Funding Parties: Canadian Orthopaedic Foundation (Carrol A. Laurin Award) and MENTOR program of CIHR

Purpose: Recently, we highlighted a dysfunction in the melatonin signalling pathway in the osteoblasts from adolescent idiopathic scoliosis patients (AIS). The objective of this project is to verify if in the cells coming from the SIA patients, estrogens interfere with melatonin signalling pathways and to identify mechanisms through which these effects are carried out.

Methods: The effects of estrogens on the melatonin signalling pathway, in osteoblasts from AIS patients (n=7), were determined by measuring the capacity of the Gi proteins to inhibit the accumulation of cAMP. The osteoblasts were incubated in the presence of increasing amounts of melatonin (10–11 to 10–5 M) with or without 17-& #946;-estradiol in physiological concentrations (10–10 M) (n=7). Moreover, coimmunoprecipitations using anti-phosphoserine antibodies were carried out and then followed with a Western blot in order to detect melatonin receptors (MT1 and MT2).

Results: The intracellular level of cAMP is higher in the presence of a physiological concentration of 17-& #946;-estradiol among scoliotic patients compared to the level observed in the presence of melatonin alone. Moreover, the preliminary results of the coimmunoprecipitations seem to show an increase in the phosphorylation of proteins interacting with MT1 and MT2 receptors. The precise nature of these proteins remains to be identified.

Conclusions: These results seem to show the antagonistic effects of the 17-& #946;-estradiol on the melatonin signalling pathway in the osteoblasts from AIS patients. However, more cAMP dosages in the presence and absence of 17-& #946;-estradiol are underway so as to increase the number of patients. The results of this study could contribute to the development of the first molecular screening tests as well as the development of new therapeutic approaches.