Angeline Tan, Tan Wee Keong
School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, Singapore
Student Internship Programme (SIP), Final Year Project (8 months)

Presented as a requirement for the successful completion of the Diploma in Biotechnology in the School of Life Sciences at Ngee Ann Polytechnic, 6 April 2004

Charles Gullo, Research Scientist, Department of Clinical Research, SGH

Multiple Myeloma Research Laboratory, SingHealth

In the bone marrow microenvironment, multiple myeloma (MM) cells interact with the underlying bone marrow stromal cells (BMSCs) and secrete cytokines such as Interleukin-6 (IL-6) and Vascular Endothelial Growth Factor (VEGF).¹ When soluble human CD40L is added to MM cells, it induces growth and proliferation of MM cells in vitro and in turn up-regulate the expression of IL-6 and VEGF expression in an autocrine fashion.² In addition, IL-6 which is secreted by BMSCs will in turn up-regulate VEGF expression in a paracrine fashion.³ In order to test this, the in vivo bone marrow microenvironment was mimicked by co-culturing MM cells with the stromal cell line, AA101. The in vitro co-culture system was established by using RPMI-8226 and SGH-MM5 cell lines and then activating the human MM cells with different concentrations of sCD40L and/or IL-6. These cytokines were also used to stimulate human MM cells in the absence of bone marrow stromal cells as well. After a defined period of time, the non-adherent MM cells were then harvested and RNA was extracted using Qiagen RNeasy Mini Kit (Qiagen GmBh, Hilden Germany). VEGF expression from the adherent bone marrow stromal cells was not addressed in this study. The extracted RNA was then used to perform Quantitative-Reverse Transcriptase Polymerase Chain Reaction (Q-RT-PCR) using the RNA Master SYBR Green Kit (Roche LighCycler®, Roche Diagnostics GmbH, Germany). Concomitantly, supernatants harvested from the co-culture experiments were used to perform VEGF ELISA using Human VEGF DuoSet (R&D systems, Wisconsin USA) for detection of VEGF at protein level. Our hope here at the Multiple Myeloma Research Laboratory (MMRL) is to one day use these assays as a screening tool for advanced stages of MM.

RPMI MM cells were plated on a confluent layer of AA101 cells for several hours before soluble CD40L was added at the indicated concentrations. Total RNA was then harvested and Q-RT-PCR was performed. Figure 1a indicates the number of cycles needed to detect of VEGF or Actin mRNA (during amplification) while Figure 1b indicates the specificity of those products as measured by melting point analysis (the loss of signal as the products are melted and the amplicon is denatured). Higher dose of soluble CD40L resulted in the detection of more products during amplification for VEGF and a higher melting peak as well. The housekeeping gene Actin was unchanged following stimulation with either high or low does of soluble CD40L in RPMI, suggesting CD40L upregulation of VEGF was specific.

Fig. 1.

We would like to thank our supervisor Dr Charles Gullo for all his assistance throughout this project. This project was supported by a Department of Clinical Research grant #DCR/PO7/2004.

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2. Urashima, M, Chauhan D, Uchiyama, H, Freeman, GJ, Anderson KC. CD40 ligand triggered interleukin-6 secretion in multiple myeloma cells. Blood 1995; 85:1903-12.
3. Dankbar B, Padro T, Leo R, Feldmann B, Kropff M, Mesters RM, Serve H, et al. Vascular endothelial growth factor and intereukin-6 in paracrine tumor-stromal cell interactions in multiple myeloma. Blood 2000; 95:2630-6.