MiR-7-5p targeted Rb regulating cell cycle is involved in hydroquinone-induced malignant progression in human lymphoblastoid TK6 cells.

MiR-7-5p has been demonstrated to inhibit tumorigenesis by limiting tumor cell proliferation, migration and invasion. However, its role in countering hydroquinone (HQ)-induced malignant phenotype of TK6 cells has remained unclear. The present study aimed to investigate whether miR-7-5p overexpression could restrain the malignant phenotype in TK6 cells exposed to HQ. The results displayed that HQ suppressed the expression of miR-7-5p and promoted cell cycle progression. Further investigations confirmed that miR-7-5p could decelerate the cell cycle progression by targeting Rb after acute HQ exposure. Through the regulation of the Rb/E2F1 signaling pathway, the overexpression of miR-7-5p mitigated HQ-induced malignant phenotype in TK6 cells by impeding cell cycle progression. In conclusion, miR-7-5p overexpression appears to be involved in HQ-induced malignant transformation by suppressing Rb/E2F1 signaling pathway, resulting in a deceleration of the cell cycle progression.

JNK1 activated pRb/E2F1 and inhibited p53/p21 signaling pathway is involved in hydroquinone-induced pathway malignant transformation of TK6 cells by accelerating the cell cycle progression.

Hydroquinone (HQ) is an important metabolites of benzene in the body, and it has been found to result in cellular DNA damage, mutation, cell cycle imbalance, and malignant transformation. The JNK1 signaling pathway plays an important role in DNA damage repair. In this study, we focused on whether the JNK1 signaling pathway is involved in the HQ-induced cell cycle abnormalities and the underlying mechanism. The results showed that HQ induced abnormal progression of the cell cycle and initiated the JNK1 signaling pathway. We further confirmed that JNK1 suppression decelerated the cell cycle progression through inhibiting pRb/E2F1 signaling pathway and triggering p53/p21 pathway. Therefore, we concluded that JNK1 might be involved in HQ-induced malignant transformation associated with activating pRb/E2F1 and inhibiting p53/p21 signaling pathway which resulting in accelerating the cell cycle progression.

Nrf2 affects hydroquinone-induces cell cycle arrest through the p16/pRb signaling pathway and antioxidant enzymes.

Hydroquinone (HQ), a well-known carcinogenic agent, induces oxidative stress, cell cycle arrest, apoptosis, and malignant transformation. As an antioxidant actor, the nuclear factor erythroid 2-related factor 2 (Nrf2) drives adaptive cellular protection in response to oxidative stress. The human lymphoblastoid cell line (TK6 cells) is widely used as a model for leukemia researches. In the present study, we focused on exploring whether Nrf2 regulatory cell cycle in TK6 cells upon HQ treatment and the underlying mechanisms. The results showed that the cell cycle arrest in TK6 cells induced by hydroquinone was accompanied by activation of the Nrf2 signaling pathway. We further clarified that Nrf2 loss accelerated cell cycle progression from G0/G1 to S and G2/M phases and promoted ROS production by downregulating the expression of SOD and GSH. Western blotting analysis indicated that Nrf2 regulated cell cycle progression via p16/pRb signaling pathways. Therefore, we conclude that Nrf2 is engaged in HQ-induced cell cycle arrest as well through p16/pRb and antioxidant enzymes.

Poly(ADP-ribose)polymerase-1 affects hydroquinone-induced aberrant cell cycle and apoptosis through activation of p16/pRb signaling pathway in TK6 cells.

Hydroquinone (HQ), a key metabolite of benzene, affects cell cycle and apoptosis. Poly (ADP-ribose) polymerase-1 (PARP-1) plays an important role in DNA damage repair. To explore whether PARP-1 is involved in HQ-induced cell cycle and apoptosis, we assessed the effect of PARP-1 suppression and overexpression on induction of cell cycle and apoptosis analyzed by flow cytometry analysis. We observed that HQ induced aberrant cell cycle progression and apoptosis. We further confirmed that PARP-1 suppression accelerated the cell cycle progression and inhibited cell apoptosis via inhibiting p16/pRb signal pathway after acute HQ exposure, while overexpression of PARP-1 displayed the opposite results. Therefore, we concluded that HQ-induced cell cycle and apoptosis were regulated by PARP-1 through activation of p16/pRb signaling pathway.

PARP-1 participates in regulation of cell cycle signaling in the hydroquinone-induced TK6 malignant transformation / 中国职业医学

@#To investigate the expression of polyadenosine diphospho-ribose polymerase 1 (PARP-1) and p16/ retinoblastoma (Rb) protein in hydroquinone (HQ)-induced TK6 cells and their regulatory mechanisms. Methods According to the 2×2 factorial design model, TK6 cells were divided into PBS-TK6 group and HQ-TK6 group based on HQ exposure, and then sub-divided into non-DOX intervention subgroup and DOX intervention subgroup based on DOX intervention, a total of four groups. The PBS-TK6 group was treated with phosphate buffer saline, and the HQ-TK6 group was treated with HQ at a final concentration of 20.0 μmol/L. The non-DOX intervention subgroup was added with 0.05% dimethyl sulfoxide; and the DOX intervention subgroup was added with PARP-1 agonist DOX at a final concentration of 0.5 μmol/L. The distribution of cell cycle was detected by flow cytometry. The protein expression of p16/Rb, cyclin D1 (cyclinD1), multifunctional protein E2 transcription factor 1 (E2F1), Rb, and p-Rb were detected by Western blot, and the level of p16 ribosylation was detected by immunofluorescence and immunoprecipitation. Results Compared with the PBS-TK6 group, the cell cycle distribution percentage in G0/G1 phase and the relative expression levels of p16 proteins were decreased in the cells of the HQ-TK6 group (all P<0.05). The cell cycle distribution percentage in S phase and the relative expression levels of cyclinD1 and p-Rb proteins were up-regulated (all P<0.05). Compared with the non-DOX intervention group, the cell cycle distribution percentage in G0/G1 and G2/M phases and the relative expression level of p16 protein increased in the DOX intervention group (all P<0.05). The percentage of cells in S phase and the relative expression levels of cyclinD1 and p-Rb proteins were down-regulated (all P< 0.05). The results of interaction effect analysis showed that compared with the non-DOX PBS-TK6 cells, the relative expression levels of Rb and E2F1 protein in the DOX PBS-TK6 cells intervention group were down-regulated (all P<0.05). The relative expression level of Rb protein in non-DOX HQ-TK6 cell group was down-regulated (P<0.05), and the relative expression of E2F1 protein was up-regulated (P<0.05). Compared with DOX PBS-TK6 cell group, the relative expression level of Rb protein in DOX HQ-TK6 cell group was down-regulated and that of E2F1 protein was up-regulated (all P<0.05). Compared with the non-DOX HQ-TK6 cell group, the relative expression level of Rb protein in the DOX HQ-TK6 cell group was up-regulated and that of E2F1 protein was down-regulated (all P<0.05). Conclusion PARP-1 participates in cell cycle regulation by regulating the p16/Rb signaling pathway in TK6 cells.

CD4(+)CD25 (+) regulatory T cells are not required for mesenchymal stem cell function in fully MHC-mismatched mouse cardiac transplantation.

Although the immunomodulative properties of mesenchymal stem cells (MSCs) open up attractive possibilities in solid-organ transplantation, information concerning the optimal dose, route, timing of administration, major histocompatibility complex (MHC)-restriction and relevant mechanisms is currently lacking. Therefore, better characterization of MSC immunoregulatory activity and elucidation of its mechanisms are crucial. In this study, we confirmed that MSCs did not elicit proliferation by allogeneic CD4(+) T cells, suggesting that MSCs were not immunogenic. By using C57BL/6 mouse MSCs as donor-derived or recipient-derived or as third-party MSCs, we discovered that MSCs suppressed CD4(+) T cell proliferation and prolonged mouse cardiac allograft survival in a dose-dependent and non-MHC-restricted manner. We also found that intraperitoneal administration favored survival prolongation, although this prolongation was weaker than that via the intravenous route. Only infusion at earlier time points favored survival prolongation. Depletion of CD4(+)CD25(+) T cells did not affect the immunosuppression of MSCs on CD4(+) T cells. Moreover, MSCs did not induce regulatory T cells. The in vivo data revealed that MSCs did not increase the percentage of CD4(+)CD25(+) T cells and FoxP3 expression. More importantly, we demonstrated for the first time that depletion of CD4(+)CD25(+) T cells did not hinder MSC-induced survival prolongation, indicating that CD4(+)CD25(+) regulatory T cells were not essential for the prolongation of MSC-mediated allograft survival.

Transplant long-surviving induced by CD40-CD40 ligand costimulation blockade is dependent on IFN-γ through its effect on CD4(+)CD25(+) regulatory T cells.

BACKGROUND: IFN-γ was documented to be commonly associated with acute rejection. In the present study, we investigated the role of IFN-γ in the transplant long-surviving induced by blocking CD40-CD40 ligand (CD40-CD40L) costimulation and its mechanisms. METHODS: IFN-γ expression in cardiac allografts and spleens from syngeneic and allogeneic recipients with or without anti-CD40L monoclonal antibody (MR-1) treatment was examined by real-time RT-PCR. The grafts survival time in Wild type (IFN-γ(+/+)) and IFN-γ deficient (IFN-γ(-/-)) recipients was investigated. Mixed lymphocyte reaction (MLR) of CD4(+) T cells and cytotoxic T lymphocyte (CTL) assay of CD8(+) T cells were also studied. FoxP3 expression in allografts and spleens from IFN-γ(+/+) or IFN-γ(-/-) recipients with MR-1 treatment was examined. Furthermore, FoxP3, IL-10 and CTLA-4 expressions and the suppressive capability of CD4(+)CD25(+) regulatory T cells were examined. RESULTS: Rejected allografts showed significantly higher IFN-γ expression than long-surviving allografts. Allograft survival was not prolonged in nonimmunosuppressed IFN-γ(-/-) mice. Administration of MR-1 induced long-term survival in 90.1% of IFN-γ(+/+) recipients (98±6.6 days) but failed to do so in IFN-γ(-/-) group (16.2±4.0 days). IFN-γ(-/-) recipients facilitated the proliferation and CTL generation of T cells. The allografts and spleens from IFN-γ(+/+) recipients contained higher FoxP3 expression than IFN-γ(-/-) recipients. Moreover, CD4(+)CD25(+) T cells from IFN-γ(+/+) recipients displayed a higher FoxP3 and IL-10 expression and suppressive capability. CONCLUSION: IFN-γ plays an important role in the long-surviving induced by blocking CD40-CD40L through inhibiting the function of activated T cells and increasing suppressive capability of CD4(+)CD25(+) regulatory T cells.

Cardiac allograft acceptance induced by blockade of CD40-CD40L costimulation is dependent on CD4+CD25+ regulatory T cells.

BACKGROUND: We have demonstrated previously that CD4(+)CD25(+) regulatory T cells (Treg) are important for spontaneous hepatic allograft tolerance. In this study, we examine the role of Treg in cardiac allograft acceptance induced by blockade of the CD40-CD40L pathway. METHODS: A heterotopic heart transplant model of major histocompatibility complex-mismatched mice was performed. Expression of forkhead/winged helix transcription factor (FoxP3) and/or the number of CD4(+)CD25(+) T cells in allografts and spleens were examined. The effect of Treg from the recipient or the donor on the induction and maintenance of long-term allograft survival was determined. Histologic analyses were also performed. The effects of Treg on CD4(+) and CD8(+) T cells were assessed. RESULTS: The levels of FoxP3 and/or CD4(+)CD25(+) T cells increased in long-surviving allografts and spleens. Depletion of Treg in the recipients but not the donors before transplantation caused rejection. Histologic analyses of allografts with Treg depletion showed extensive leukocyte infiltration and tissue destruction. However, delayed depletion of Treg in long-surviving recipients did not shorten their survival. Treg depletion increased the function of CD4(+) and CD8(+) T cells. CONCLUSION: Treg in the recipient but not in the donor is essential for long-term survival induced by CD40-CD40L blockade by inhibiting the function of CD4(+) and CD8(+) T cells; however, Treg are not important for maintenance. Both allograft and spleen are critical for induction of successful long-term survival.

CD40-CD40L costimulation blockade induced the tolerogenic dendritic cells in mouse cardiac transplant.

We aimed to investigate whether tolerogenic dendritic cells (DCs) were induced in the tolerant recipients with the blockade of CD40-CD40L costimulation. Mouse heterotopic heart transplantation was performed. DCs were sorted from rejected and tolerant recipients using magnetic-activated cell sorting. Their expression of CD40, CD80, and CD86 was examined using fluorescence-activated cell sorting. DCs were stimulated with lipopolysaccharide in vitro, and interleukin 10 (IL-10) and IL-12 levels in the supernatants were evaluated using enzyme-linked immunosorbent assay. By using mixed leukocyte reaction, we investigated the stimulatory capacities and tolerogenic capability of DCs. DCs from tolerant recipients expressed lower level of costimulatory molecules, including CD40, CD80, and CD86 and released higher levels of IL-10 and lower levels of IL-12. In addition, DCs from tolerant recipients were weak stimulators of the mixed leukocyte reaction and inhibited the proliferation of splenocytes. IL-10(high)IL-12(low) DCs with immature phenotype were induced in the tolerant recipients with the blockade of CD40-CD40L costimulation, and they obtained the tolerogenic function.

Expansion of CD25+CD4+ regulatory T cells by natural mature dendritic cells.

Because of the anergy of CD25+CD4+ regulatory T cells, it is unclear how the number of these regulatory T cells is sustained and expanded in normal physiologic circumstances. In the present study, we examined the effect of natural allogeneic mature dendritic cells (DCs) on the proliferation and function of CD25+CD4+ T cells. Our data showed that natural allogeneic mature DCs stimulated CD25+CD4+ T-cell growth vigorously, whereas immature DCs had little effect on the proliferation of CD25+CD4+ T cells. After expansion by mature DCs, CD25+CD4+ T cells maintained their expression of Foxp3 and suppressed the proliferation of CD25- CD4+ T cells similar to freshly isolated CD25+CD4+ T cells. Our results introduce a potentially critical role played by natural allogeneic mature DCs, which exist in normal physiologic circumstances, in controlling CD25+CD4+ regulatory T-cell expansion and function.