**4. Continuous low-dose exposure to asbestos fibers in MT-2 cells: Resistance to cytotoxicity**

The initial aim of asbestos exposure on the MT-2 cell line was to establish a model of continuous, recurrent, and relatively low-dose exposure on human T cells and observe which kind of alterations occur under conditions of continuous and low-dose exposure as found with human populations exposed to asbestos, such as the occurrence of cancers.

Exposed doses for continuous exposure were then determined as doses which caused apoptosis in less than half of MT-2 cells. Doses included 5 or 10 μg/ml in culture flasks, as shown on the left side of **Figure 1**. A dose of 10 μg/ml of chrysotile caused various apoptotic cellular events, although the degrees of these events were less than those resulting from exposure to 25 μg/ml of chrysotile [31, 34]. Since MT-2 cells were derived from T cells, they were grown by floating in the culture. Thus, the concentration of asbestos fibers was determined using μg/ml, although various adherent cells such as epithelial and mesothelial cells were measured using μg/cm<sup>2</sup> in the culture. The MT-2 cell culture was continued with a subculture twice a week and substitute asbestos fibers according to the determined concentrations. To monitor cellular alteration, the asbestos fibers were removed from continuous culture by density gradient centrifugation using LymphoPrep (gradient = 1.077) and continuously exposed cells were analyzed for the occurrence of apoptosis after transient exposure to highdose asbestos fibers (which caused apoptosis in most of the MT-2 cells such as 25–50 μg/ml) [31, 32, 34]. After 8 months of continuous exposure, the appearance of apoptosis was reduced in the continuously exposed subline. This acquisition of resistance to asbestos-induced apoptosis was sustained in long-term cultures (until now, the sublines were cultured with asbestos fibers) [31, 34].

After 1 year of continuous exposure, the cellular features of the continuously exposed subline (MT-2 CE) were compared to those of the original MT-2 cells (MT-2Org, which were never exposed to asbestos fibers) as shown on the right side of **Figure 1**. A consideration of cytokine production showed that there was an excess production of interleukin (IL)-10 in MT-2 CE relative to MT-2Org. The regulation of IL-10 production was mediated by Src kinase, since PPT, the Src inhibitor, reduced IL-10 production in both MT-2Org and MT-2 CE cells. The excessively produced IL-10 was then utilized by the autocrine mechanism, since MT-2 possesses the IL-10 receptor (R) at its surface. As a result of IL-10 utilization, the signaling molecule, signal transducers, and the activator of transcription (STAT)-3, located downstream of IL-10R, were phosphorylated to a higher level in MT-2 CE compared to MT-2Org. Since the antiapoptotic molecule Bcl-2 is located downstream of STAT3, the expression of Bcl2 was upregulated in MT-2 CE compared to that in MT-2Org. The Bax/Bcl2 ratio was lower in MT-2 CE than in MT-2Org, as shown on the right side of **Figure 1**. In order to investigate the importance of Bcl-2 in MT-2 CE concerning acquisition of resistance to asbestos-induced apoptosis, siRNA for Bcl-2 was introduced into MT-2 CE cells and the occurrence of apoptosis and growth inhibition following transient and high-dose exposure to asbestos fibers were examined. As

**Figure 1.** Schematic representation of the effects of the asbestos fibers chrysotile (CH) and crocidolite (CR) on MT-2 cells, a human T cell leukemia virus (HTLV)-1 immortalized human polyclonal T cell line [44–46]. Left side: Cellular and molecular alterations in MT-2 cells following transient and relatively high-dose exposure to CH or CR are summarized

c was released from mitochondria to the cytoplasm, and caspases 9 and 3 were truncated into active forms. Cells then proceeded to apoptosis. A comparison of the effects caused by CH or CR showed that reactive oxygen species (ROS) production was greater with CR exposure, whereas growth inhibition and the level of apoptosis were greater following CH exposure. Right side: The effects of continuous and relatively low-dose exposure are summarized. MT-2 CE (a continuously exposed subline) revealed excess IL-10 production via Src kinase and phosphorylation of STAT3 resulting in upregulation of Bcl-2 [34]. In addition, the transcription factor FoxO1 was reduced in MT-2 CE, causing a reduction of proapoptotic molecules such as puma, bim, and FasL [36]. Both exposures contributed to the development of resistance

, proapoptotic signaling molecules such as JNK and p38 were phosphorylated, cytochrome

[32, 33]. Cells produced O<sup>2</sup>

12 Cytotoxicity

−

to asbestos-induced apoptosis in MT-2 CE cells [44–46].

suspected, Bcl-2 silenced cells exhibited much higher apoptosis and growth inhibition. Thus, the Src ➔ IL-10 ➔ STAT3 ➔ Bcl-2 axis was considered important for the acquisition of resistance to asbestos-induced apoptosis in MT-2 CE [31, 34]. Additionally, CD4-positive peripheral blood cells from asbestos-exposed patients exhibiting pleural plaque (PP) or malignant mesothelioma (MM) showed enhanced expression of Bcl-2 compared with that of healthy volunteers (HV). Thus, the MT-2 CE model may express events occurring within T cells of asbestos-exposed patients [31, 34].

Our investigations showed that the CXC chemokine receptor 3 (CXCR3) exhibited reduced expression in MT-2 CEs compared to MT-2Org [38, 39]. CXCR3 is known to play an important role in antitumor immunity because it summons antitumor T cells with IFN-γ. As shown in the cell line model, investigation of freshly isolated CD4-positive T cells revealed decreased expression of CXCR3 when activated *in vitro* with CH fibers. Furthermore, peripheral blood CD4-positive cells from patients with PP or MM showed a reduction of CXCR3 and an inhibited potential for IFN-γ production when stimulated *in vitro* [31, 38, 39]*.* These investigations indicated that the cell line model for continuous and low-dose exposure to asbestos using the MT-2 cell line was suitable for analysis of immune alteration in asbestos-exposed human

Cytotoxicity Caused by Asbestos Fibers and Acquisition of Resistance by Continuous Exposure…

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As the MT-2 cell line was known to possess a Treg function [40, 41], the Treg function was estimated in MT-2Org and MT-2 CE. In regard to cell-cell contact, MT-2 CE enhanced its suppressive function onto Tresp cells [42]. In addition, MT-2 CE produced higher TGF-β and IL-10 in comparison to MT-2Org as described earlier. These two cytokines are the typical soluble factors for Treg in order to manifest its function. Following the knockdown of each cytokine in MT-2 CE, the suppressive function was reduced relative to that in MT-2 CE [42]. These results indicated that asbestos exposure enhanced Treg function by cell-cell contact and an increase of soluble factors [42]. In addition, FoxO1 reduced its expression in MT-2 CE as described earlier, and is known to regulate the cell cycle to suppress the accelerating genes, such as cyclins, as well as to enhance the breaking genes, such as cyclin-dependent kinase inhibitors (CDK-Is) [43]. As a consequence, cyclins were enhanced and the expression of CDK-I2 was reduced in MT-2 CE because of the reduced expression of FoxO1. Cell cycle progression was, therefore, enhanced in MT-2 CE [43]. These overall results suggest that Treg volume may also be enhanced in asbestos-exposed human populations and patients exhibiting PP or MM [42, 43]. These findings indicated that asbestos exposure causes reduction of antitumor immunity.

Investigation of cytotoxicity in human T cells caused by asbestos exposure indicated that the production of ROS and activation of the mitochondrial apoptotic pathway were the main causes for apoptosis of T cells following a transient and relatively high-dose exposure [32], similar to known mechanisms investigated previously using alveolar epithelial and pleural mesothelial cells [5–9, 44–46]. However, the continuous and relatively low-dose exposure of T cells to asbestos altered cellular and molecular events that caused acquisition of resistance against asbestosinduced cytotoxicity. Investigations revealed the importance of the Src ➔ IL-10 ➔ STAT3 ➔ Bcl-2 axis as well as the reduced expression of FoxO1 [31, 33–35]. These changes induce the reduction of antitumor immunity in an asbestos-exposed population and create an increased risk of carcinogenicity due to the transforming activity associated with asbestos fibers [44–46]. Considering the most important issue in asbestos-exposed population, the occurrence of malignancies such as mesothelioma and lung cancer after long-term latent period should be explored the mechanisms as well as be prevented [1–4]. Thus, regarding the cytotoxic effects

populations and patients with PP or MM [31, 38, 39].

**6. Conclusion**

Sublines continuously exposed to asbestos were established independently and comprised six sublines exposed to CH and three sublines exposed to CR. The profiles of cytokine production in these MT-2 CEs (exposed to CH or CR) were similar [31, 33, 35]. Continuous exposure caused excess production of IL-10 (as mentioned earlier) and transforming growth factor (TGF)-β, whereas interferon (IFN)-γ production was reduced in MT-2 CEs compared to that in MT-2Org. In addition, Bcl-2 upregulation was found in all MT-2 CEs and there were no differences between exposure to CH and CR [31, 33, 35]. In fact, a cDNA microarray assay using MT-2 CEs and MT-2org indicated that most of the upregulated and downregulated genes were similar in MT-2 CEs. Therefore, these MT-2 CEs could be used as a continuously asbestos-exposed immune T cell model.

The cDNA microarray assay revealed that the transcription factor forkhead box O1 (FoxO1) was expressed to a lesser degree in MT-2 CE compared to MT-2Org [36]. FoxO1 is known to regulate various genes in apoptosis, metabolism, cell growth and differentiation, and so on. In particular, FoxO1 controls various proapoptotic genes such as the p53 upregulated modulator of apoptosis (Puma), bcl-2 interacting mediator of cell death (Bim), and the Fas ligand (FasL) [36, 37]. The message expression of these proapoptotic molecules was reduced in MT-2 CE compared with that in MT-2Org (shown on the right side of **Figure 1**). In addition, following knockdown of the FoxO1 gene in MT-2Org, the level of apoptotic cells caused by transient and high-dose exposure to asbestos was reduced. Furthermore, when the expression of FoxO1 was forced in MT-2 CE, the ratio of apoptosis increased following transient and high-dose exposure to asbestos and the expression of Puma was recovered [36].

These results indicated that acquisition of resistance to asbestos-induced apoptosis by continuous and low-dose exposure to asbestos was regulated by the FoxO1 transcription factor in addition to the Src ➔ IL-10 ➔ STAT3 ➔ Bcl-2 axis [31–36].
