Cell of origin determines tumor phenotype in an oncogenic Ras/p53 knockout transgenic model of high-grade glioma.

Human high-grade gliomas (HGGs) are known for their histologic diversity. To address the role of cell of origin in glioma phenotype, transgenic mice were generated in which oncogenic Ras and p53 deletion were targeted to neural stem/progenitor cells (NSPCs) and mature astrocytes. The hGFAP-Cre/Kras/p53 mice develop multifocal HGGs that vary histopathologically and with respect to the expression of markers associated with NSPCs. One HGG pattern strongly expressed markers of NSPCs and arose near the subventricular zone. Additional nonoverlapping patterns that recapitulate human HGG variants were present simultaneously in the same brain. These neoplastic foci were more often cortical or leptomeningeal based, and the neoplastic cells lacked expression of NSPC markers. To determine whether cell of origin determines tumor phenotype, astrocytes and NSPCs were harvested from neonatal mutant pups. Onorthotopic transplantation, early-passage astrocytes and NSPCs formed tumors that differed in engraftment rates, latency to clinical signs, histopathology, and protein expression. Astrocyte-derivedtumors were more aggressive, had giant-cell histology, and glial fibrillary acidic protein expression. The NSPC-derived tumors retained NSPC markers and showed evidence of differentiation along astrocytic, oligodendroglial, and neuronal lineages. These results indicate that identical tumorigenic stimuli produce markedly different glioma phenotypes, depending on the differentiation status of the transformed cell.

Dibutyltin activates MAP kinases in human natural killer cells, in vitro.

Previous studies have shown that dibutyltin (DBT) interferes with the function of human natural killer (NK) cells, diminishing their capacity to destroy tumor cells, in vitro. DBT is a widespread environmental contaminant and has been found in human blood. As NK cells are our primary immune defense against tumor cells, it is important to understand the mechanism by which DBT interferes with their function. The current study examines the effects of DBT exposures on key enzymes in the signaling pathway that regulates NK responsiveness to tumor cells. These include several protein tyrosine kinases (PTKs), mitogen-activated protein kinases (MAPKs), and mitogen-activated protein kinase kinases (MAP2Ks). The results showed that in vitro exposures of NK cells to DBT had no effect on PTKs. However, exposures to DBT for as little as 10 min were able to increase the phosphorylation (activation) of the MAPKs. The DBT-induced activations of these MAPKs appear to be due to DBT-induced activations of the immediate upstream activators of the MAPKs, MAP2Ks. The results suggest that DBT-interference with the MAPK signaling pathway is a consequence of DBT exposures, which could account for DBT-induced decreases in NK function.

Increases in cytosolic calcium ion levels in human natural killer cells in response to butyltin exposure.

This study investigated whether exposures to butyltins (BTs), tributylin (TBT), and dibutyltin (DBT) were able to alter cytosolic calcium levels in human natural killer (NK) cells. Additionally, the effects of cytosolic calcium ion increases on the activation state of mitogen-activated protein kinases (MAPKs) in NK cells were also investigated. NK cells are an intital immune defense against the development of tumors or viral infections. TBT and DBT are widespread environmental contaminants, due to their various industrial applications. Both TBT and DBT have been shown to decrease the ability of NK cells to lyse tumor cells (lytic function). TBT has also been shown to activate MAPKs in NK cells. The results of this study indicated that TBT increased cytosolic calcium levels by as much as 100% after a 60-min exposure to 500 nM TBT, whereas DBT increased cytosolic calcium levels to a much smaller extent (and required higher concentrations). The results also indicated that increases in cytosolic calcium could activate MAPKs but only for a short period of time (5 min), whereas previous studies showed that activation of MAPKs by TBT last for at least 6 h. Thus, it appears that TBT-stimulated increases in cytosolic calcium might contribute to, but are not fully responsible for, TBT-induced activation of MAPKs.

Effects of tributyltin on protein tyrosine kinases and phospholipase C gamma in human natural killer cells.

ABSTRACT Tributyltin (TBT) has been used in wood preservation, marine antifouling paints, disinfection of circulating industrial cooling waters, and slime control in paper mills. Detectable levels have been found in human blood. Exposure to TBT decreases the tumor cell lysing (lytic) function of human natural killer (NK) lymphocytes. In this study we assessed the effects of concentrations of TBT that have been shown to decrease NK lytic function on protein tyrosine kinases (PTKs) (Syk, Zap-70, Src, and Pyk) and phospholipase C gamma (PLC-gamma) in NK cells. Exposure to 500 nM TBT caused no change in phosphorylation of any of the PTKs. A 60-min exposure of NK cells to 500 nM TBT did not significantly affect the phosphorylation state of PLC-gamma at any of the lengths of exposure. However, total levels of PLC-gamma were increased by almost 50% after this exposure. Exposure of NK cells to 300 nM TBT for 5 to 60 min caused no significant changes in the phosphorylation state PTKs or PLC-gamma. Exposure of NK cells to 200 nM TBT for 24 h caused no significant changes in the PTK phosphorylation state or total levels. Cells that were exposed to 300 nM TBT for 1 h followed by 24 h or 48 h in TBT-free media showed a significant increase in the phosphorylated forms of Syk and Zap-70 after 24 h in TBT-free media but not after 48 h. These data indicate that in vitro exposure to TBT caused no changes in PTK or PLC-gamma phosphorylation under most conditions.

Pattern of MAP kinases p44/42 and JNK activation by non-lethal doses of tributyltin in human natural killer cells.

Tributyltin (TBT) has been shown to disrupt the ability of natural killer (NK) cells to destroy tumor targets in vitro even at exposures of 25 nM for 24 h, but cell viability was not significantly impacted. Thus, evaluation of intracellular molecular events that regulate cell viability in TBT exposed NK cells are of interest. It has been suggested that activation of the mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), may promote apoptosis while activation of the MAPK p44/42 may be crucial in mediating anti-apoptotic stimuli. However, it is well established that increases in pro-apoptotic BCL-2 family members, such as Bax, results in cell death. We have set out to study the effects of a range of TBT concentrations on the MAPKs, JNK and p44/42. Additionally, we examined the effects of TBT on the levels of pro-apoptotic proteins Bax and p53 as well as anti-apoptotic protein Bcl-2. The results show that 300-25 nM TBT activated JNK within 10 min. MAPK p44/42 was also activated by 300-50 nM TBT within 10 min. These data show that while 300-200 nM TBT activates p44/42 significantly more than JNK, the pattern of 100-25 nM TBT activation of these MAPKs may be similar. TBT exposure alters neither pro-apoptotic proteins Bax and p53 nor anti-apoptotic protein Bcl-2 levels at any exposure studied. The results suggest that exposure to TBT activated the anti-apoptotic regulatory p44/42 pathway to a greater extent than the pro-apoptotic JNK pathway, which may explain to some extent how NK cell viability is maintained.

Effect of tributyltin (TBT) on ATP levels in human natural killer (NK) cells: relationship to TBT-induced decreases in NK function.

The purpose of this study was to investigate the role that tributyltin (TBT)-induced decreases in ATP levels may play in TBT-induced decreases in the tumor lysing (lytic) function of natural killer (NK) cells. NK cells are a subset of lymphocytes that act as an initial immune defense against tumor cells and virally infected cells. TBT is an environmental contaminant that has been detected in human blood, which has been shown to interfere with ATP synthesis. Previous studies have shown that TBT is able to decrease very significantly the lytic function of NK cells. In this study NK cells were exposed to various concentrations of TBT and to two other compounds that interfere with ATP synthesis (rotenone a complex I inhibitor and oligomycin an ATP synthase inhibitor) for various lengths of time before determining the levels of ATP and lytic function. Exposures of NK cells to 10, 25, 50 and 100 nm TBT did not significantly reduce ATP levels after 24 h. However, these same exposures caused significant decreases in cytotoxic function. Studies of brief 1 h exposures to a range of TBT, rotenone and oligomycin concentrations followed by 24 h, 48 h and 6 day periods in compound-free media prior to assaying for ATP levels or cytotoxic function showed that each of the compounds caused persistent decreases in ATP levels and lytic function of NK cells. Exposures to 0.05-5 microm rotenone or oligomycin for 1 h reduced ATP levels by 20-25% but did not have any measurable effect on the ability of NK cells to lyse tumor cells. ATP levels were also decreased by about 20-25% after 24 h or 48 h exposures to rotenone or oligomycin (0.5 microm ), and the lytic function was decreased by about 50%. The results suggest that TBT-induced decreases in ATP levels were not responsible for the loss of cytotoxic function seen at 1 h and 24 h. However, TBT-induced decreases of NK-ATP levels may be at least in part responsible for losses of NK-cytotoxic function seen after 48 h and 6 day exposures.

Alteration of an essential NK cell signaling pathway by low doses of tributyltin in human natural killer cells.

Tributyltin (TBT), a toxic and widespread environmental contaminant, has been shown to inhibit natural killer (NK) cell cytotoxic function significantly. Inhibition of NK cell cytotoxic function has the potential to increase viral infections and tumor growth. Upon NK cell binding to lysis-sensitive tumor cells, an intracellular pathway is activated, which generally begins with activation of non-receptor protein tyrosine kinases (PTKs) and ends with mitogen-activated protein kinase (MAPK)-mediated release of lytic granules toward the contacted target cell. In the current studies, we used a cytotoxicity assay to examine how low doses (200nM or lower) of TBT affect cytotoxic function. Additionally, we investigated how low doses of TBT modulate the signaling pathway that dictates lytic granule exocytosis. A 1h exposure to 200, 100, 50 and 25nM TBT significantly decreased cytotoxic function 6d later. We also saw significant activation of p38 and p44/42 by as low as 50nM TBT within ten minutes of exposure. The observed activation of MAPKs, p38 and p44/42, implicated their upstream activators MAPK kinases (MAP2Ks). On examining MAP2Ks, MKK3/6 and MEK1/2, activation was seen within ten minutes. However, when the most upstream signaling molecules in this pathway, non-receptor protein tyrosine kinases (PTKs) such as Syk, ZAP-70, Pyk2 and Src were examined, no significant activation was seen. These data imply that upstream activators of MAP2Ks, MAP2K kinases (MAP3Ks), are activated by TBT exposures and/or that MAP2K phosphatases are being inhibited by TBT. Taken together, these data suggest that TBT-induced activation of MAPKs, p38 and p44/42, is caused by their upstream activators MAP2Ks, MKK3/6 and MEK1/2, respectively.

Persistent immunotoxic effects of tributyltin in human natural killer cells can be reversed by interleukin 2.

Tributyltin (TBT) is a widespread environmental contaminant due to its use in marine antifouling paints and as an anti-microbial agent in other applications. There are measurable levels of TBT in some samples of human blood. Natural killer (NK) cells are lymphocytes capable of killing tumor and virally infected cells. Previously, we have shown that a 1h exposure to 300nM TBT caused a permanent decrease in the ability of human NK cells to bind to and destroy tumor target cells and in their expression of certain functionally relevant cell surface markers. The present study investigates the effect of the NK-stimulatory cytokine, interleukin (IL) 2 on TBT-induced decreases in NK cytotoxicity, binding function, and expression of CD16 and CD56. A 1h exposure to 300nM TBT followed by 24h in TBT-free media decreased cytotoxic function by 80%, and expression of CD16 by 16%. When 10ng/mL IL-2 was present during the 24h incubation there was no statistically significant decrease in cytotoxicity or expression of CD16. A 96h incubation in TBT-free media produced decreases in cytotoxicity (99%), binding function (65%), and expression of CD16 (48%) and CD56 (51%). IL-2 was able to reverse the TBT-induced decreases in each of these parameters seen after 96h. These results suggest that IL-2 restoration of NK ability to bind target cells correlates with its ability to restore CD16 and CD56 expression. Additionally, neither the loss of cytotoxic function (seen in the 24 or 48h periods following TBT exposure) nor its restoration by IL-2 can be substantially explained by the effects of TBT exposure on CD16 and CD56 expression or target binding.

Interleukin-12 release from macrophages by hyaluronan, chondroitin sulfate A and chondroitin sulfate C oligosaccharides.

Mixtures of hyaluronan (HA), chondroitin sulfate (CS)-A and CS-C oligosaccharides were generated through the enzymatic digestion of the polysaccharides with either mammalian hyaluronidase or bacterial HA lyase or chondroitinase. Compared to mammalian enzymes, bacterial enzymes hydrolyze the polysaccharides through a different mechanism yielding chemically distinct sets of oligosaccharides. Peripheral leukocytes and a human monocytic cell line were exposed to these oligosaccharides and the amount of interleukin-12 released by the cells was measured. For all types of oligosaccharide tested, we found that the amount of interleukin-12 induced by oligosaccharides generated with bacterial enzyme was significantly lower than the amount of interleukin-12 induced by oligosaccharides generated with mammalian enzyme. In addition, we observed that CS oligosaccharides generated with bacterial enzyme were capable of reducing the lipopolysaccharide-induced interleukin-12 production in macrophages. Our results indicate that HA or CS oligosaccharides generated with mammalian enzymes might possess pro-inflammatory potential, while HA or CS oligosaccharides generated with bacterial enzymes might possess non- or anti-inflammatory properties. The implications of our findings in view of the ongoing investigation of the potential therapeutic benefits of HA and CS in arthritis or other inflammatory pathologies are discussed.

Expression of functionally relevant cell surface markers in dibutyltin-exposed human natural killer cells.

Butyltin (BT) compounds are known for their worldwide contamination. Dibutyltin (DBT) is used as a stabilizer in plastic products, and as a deworming agent in poultry. Poultry products have been shown to contain measurable levels of DBT. Drinking water has also been reported to contain BTs due to leaching from PVC pipes. We, and others, have found measurable levels of DBT in human blood. BTs appear to increase the risk of cancer and other viral infections in exposed individuals. In previous studies we have shown that the tumor killing function of natural killer (NK) lymphocytes was greatly diminished after as little as a 1 h exposure to DBT and the inhibition continued even after removal of the compound. We also showed that there was a significant decrease in NK cell lysis of K562 target cells after an exposure to 1.5 microM DBT for 24 h. This 24 h exposure also decreased the ability of NK cells to bind to tumor cells. Loss of binding function was not seen when NK cells were exposed to 5-10 microM DBT for 1 h. However, NK cells exposed to 5 microM DBT for 1 h and then incubated in DBT-free media for 24, 48, or 96 h, showed a significant loss of tumor-binding function within 24 h. The effects of DBT exposure on seven cell surface molecules that are involved in NK-cell interactions with target cells were investigated. The results indicated that the exposure of NK cells to 1.5 microM DBT for 24 h decreased the expression of CD2, CD11a, CD16, CD11c. There was no decrease in expression of any of the markers studied when NK cells were exposed to 5 microM DBT for 1 h, consistent with the fact that a 1-h exposure had no effect on the ability of NK cells to bind tumor cells. However, when NK cells were exposed to 5 microM DBT for 1 h followed by 24, 48 or 96 h incubations in DBT-free media there was decreased expression of several of the cells surface molecules with the most dramatic decreases being in CD16 and CD56.