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Specific Aim 1:

1.1. The effects of a neutralizing Flt-1 antibody (Genentech) on (1) VEGF triggered ERK-activation and (2) VEGF-induced migration were investigated. VEGF-triggered ERK activation was inhibited in a concentration- and time-dependent fashion in the presence of the neutralizing Flt-1 antibody. Furthermore, VEGF-triggered migrational activity was also inhibited in a concentration-dependent manner. These results further support the specific activation of the VEGFR Flt-1 I have suggested previously (Podar et al., 2001; Podar et al., 2002).

1.2. After having shown the efficacy of this drug in MM cell migration, I further investigated whether PI3-kinase activity is blocked. I observed a partial block of PIP2 phosphorylation.

1.3. As proposed, I am now investigating the role of the small Rho/ GTPases (Rho, Rac, Cdc42) in MM cell adhesion, migration, and invasion, also focusing on participation of cytoskeletal proteins

1.4. Since the inhibitory effect of this neutralizing antibody is only partial, I will also test another VEGFR-inhibitor, newly developed by GlaxoBeecham Kline.

Specific Aim 2:

Raf-1 expression was shown in several MM cell line cells and patient MM cells. Furthermore, I confirmed the 3-fold increase of Raf-1 activity upon stimulation with VEGF, and an ~ 8-fold increase of Raf-1 activity upon stimulation with IL-6. Interestingly, the use of the PI3-kinase inhibitor LY, although blocking Raf-1 activity, did not block MEK and ERK-1,2 phosphorylation. These preliminary data suggest that Raf-1 might not be the major MEKK in MM cells. I am now attempting to identify a possible alternative MEKK. I have available the Raf-1 antisense oligonucleotides (ISIS 5132/ CGP 69846A) to specifically block Raf-1 activation. In ongoing studies I will investigate the role of Raf-1 in MM cell proliferation, survival, and migration in more detail.

Specific Aim 3:

As proposed, I investigated whether VEGF, like IL-6, can protect against Dex- and IR-induced apoptosis. Preliminary results show no/ weak antiapoptotic effects of VEGF on Dex- and IR- induced apoptosis in MM cells. In ongoing studies including also other apoptotic triggers, I will confirm these results. Furthermore, I will investigate the effects of antisense-Raf-1, RAFTK and SHP2 on IL-6- , IGF-I- , and VEGF- treated MM cells after exposure to IR, Dex, and and different drugs that are currently investigated in this lab.

In order to investigate VEGF-induced expression pattern in MM cells, I additionally isolated mRNA at different times after VEGF stimulation to perform oligonucleotide microarray analysis. By clustering the data, I aim to define further genes that are involved in VEGF-induced MM cell growth, survival, and migration.

New Specific Aim 4:

In extension to Specific Aim 3, I investigated in more detail the initial events of IL-6- and IGF-I- mediated signal transduction pathways leading to MM cell survival. Caveolae, specialized flask-shaped lipid rafts on the cell surface, are mainly composed of cholesterol, sphingolipids, and structural proteins, termed caveolins; functionally, these plasma membrane microdomains have been implicated in signal transduction and transmembrane transport. My preliminary data show for the first time the existence of caveolae, usually absent in blood cells, in MM cells by RT-PCR, western blot and transmission electron microscopy. My results also seem to indicate a possible role of caveolin-1 as a diagnostic and prognostic factor in MM, as previously suggested for some solid tumors (prostate cancer, oesophageal cancer, …). Fractions of plasma membrane rafts obtained by a discontinuous sucrose gradient contain the IL-6-receptor signal transducing chain gp130 and IGF-IR, as well as Caveolin-1. The constitutive complex-formation of gp130 and IGF-IR with Caveolin-1 was further supported by co-immunoprecipitation. The functional relevance of these lipid raft microdomains in IL-6- and IGF-I- signal transduction was next investigated. Importantly, IL-6- and IGF-I- treatment triggered the recruitment of PI3-kinase to caveolae. To disrupt caveolae-biogenesis I first targeted cholesterol and second, caveolin-1. Cholesterol depletion by b-cyclodextrin inhibits PI3-kinase/ receptor/ Caveolin-1 complex-formation and eventually the activation of PI3-kinase by IL-6 and IGF-I. Consequently, b-cyclodextrin and inhibition of cholesterol biosynthesis by lovastatin also cause blockade of IL-6- and IGF-I- triggered Akt-1/ PKB activation, leading to a decrease in overall MM cell survival. With the use of an inducible antisense-caveolin-1 containing vector, I am now trying to support these data demonstrating a key role of caveolae in the regulation of IL-6- and IGF-I-triggered survival mechanisms. Optimal plasmid-concentrations and transfection-timepoints were determined by usage of an eGFP-vector. Co-transfections with eGFP and Caveolin-1 will be performed next. After sorting positive cells, I will either develop stable cell lines or use transiently transfected cells directly.