The objective of the proposed studies is to elucidate the mechanism by which butyltins (BTs) (widespread environmental contaminants) decrease the functional capacity of human natural killer (NK) cells. Humans have significant exposure to BTs, and they are found in human blood and other tissues. BTs reduce the function of human natural killer (NK) cells, a primary immune defense against cancer. There is significant BT-induced activation of mitogen activated protein kinases (MAPKs). MAPKs are required for the tumordestroying function of NK cells. Thus, spurious BT-induced activation of MAPKs could leave the NK cell unresponsive to subsequent tumor cells. The hypothesis that alterations of MAPK activation pathways are central to the BT-induced loss of NK function will be addressed. The following specific aims are designed to investigate this hypothesis: 1. Determine the effect(s) of BT (tributyltin and dibutyltin) exposures on key upstream activating signaling proteins for each class of MAPK (p44/42, p38, and JNK) and the physiological inhibitors of MAPKs (MAPK phosphatases). The upstream activating signals to be examined are monomeric GTP-binding proteins (ras and rac), MAPK kinase kinases (MAP3K) (raf-1 and ASK-1), and MAPK kinases (MAP2K) (MEK1/2, MKK3/6, MKK4, and MKK7). Activation states of small G proteins will be monitored using an affinity binding assay followed by western blot; activating phosphorylations of MAP3Ks and MAP2Ks will be measured using western blot and kinase assays. A phosphatase assay will be used to monitor the activation state of the MAPK and MAP2K phosphatases (PPA-2/PP1 and MKP-1) 2. Determine if direct activation of MAPK pathways by selective p44/42 an p38/JNK pathway activators is able to affect NK cytotoxic function, cytolytic protein expression, and cell surface protein expression in a manner similar to that seen with BT exposures. Cytotoxic function is measured using a chromium release assay, cytolytic protein expression is determined using western blot (mRNA levels are measured using RT-PCR), and cell surface protein expression is measured using flow cytometry. 3. Determine if direct inhibition of MAPK pathways by selective inhibitors is able to block the effects of BT exposures on MAPKs, NK cytotoxic funciton, cytolytic protein expression, and cell surface protein expression, using western blot and the methods described in aim 2. 4. Determine the effects of BT exposures on transcription regulators regulated by MAPKs. These include, ELK-1 phosphorylation, Jun phosphorylation, and overall levels of Jun and Fos. Phosphorylation state and levels of transcription regulators will be assessed using western blot and RT-PCR; ability to bind DNA elements will be assessed using an ELISA and EMSA. The proposed studies will elucidate the role that environmental contaminants, such as BTs, may play in increasing susceptiblity to cancer by their ability to diminish the critical immune defense against cancer that is provided by NK cells.
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