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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Minority Biomedical Research Support - MBRS (S06)
Project #
5S06GM008092-35
Application #
8069161
Study Section
Minority Programs Review Committee (MPRC)
Project Start
Project End
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
35
Fiscal Year
2010
Total Cost
$123,558
Indirect Cost
Name
Tennessee State University
Department
Type
DUNS #
108814179
City
Nashville
State
TN
Country
United States
Zip Code
37209
Rana, Krupa; Whalen, Margaret (2015) Activation of protein kinase C and protein kinase D in human natural killer cells: effects of tributyltin, dibutyltin, and tetrabromobisphenol A. Toxicol Mech Methods 25:680-8
Hurd-Brown, Tasia; Udoji, Felicia; Martin, Tamara et al. (2013) Effects of DDT and triclosan on tumor-cell binding capacity and cell-surface protein expression of human natural killer cells. J Appl Toxicol 33:495-502
Sharow, Kyle A; Temkin, Boris; Asson-Batres, Mary Ann (2012) Retinoic acid stability in stem cell cultures. Int J Dev Biol 56:273-8
Hurd, Tasia; Walker, Jasmine; Whalen, Margaret M (2012) Pentachlorophenol decreases tumor-cell-binding capacity and cell-surface protein expression of human natural killer cells. J Appl Toxicol 32:627-34
Taylor, Thyneice R; Whalen, Margaret M (2011) Ziram activates mitogen-activated protein kinases and decreases cytolytic protein levels in human natural killer cells. Toxicol Mech Methods 21:577-84
Buchanan, FaMitah Q; Rochette-Egly, Cecile; Asson-Batres, Mary Ann (2011) Detection of variable levels of RAR? and RAR? proteins in pluripotent and differentiating mouse embryonal carcinoma and mouse embryonic stem cells. Cell Tissue Res 346:43-51
Hurd, Tasia; Whalen, Margaret M (2011) Tetrabromobisphenol A decreases cell-surface proteins involved in human natural killer (NK) cell-dependent target cell lysis. J Immunotoxicol 8:219-27
Udoji, Felicia; Martin, Tamara; Etherton, Rachel et al. (2010) Immunosuppressive effects of triclosan, nonylphenol, and DDT on human natural killer cells in vitro. J Immunotoxicol 7:205-12
Abraha, Abraham B; Rana, Krupa; Whalen, Margaret M (2010) Role of protein kinase C in TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells. Arch Environ Contam Toxicol 59:661-9
Hinkson, Natasha C; Whalen, Margaret M (2010) Hexabromocyclododecane decreases tumor-cell-binding capacity and cell-surface protein expression of human natural killer cells. J Appl Toxicol 30:302-9

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