Boquan Jin

The PI3K/Akt pathway and its downstream transcriptional factors as targets for chemoprevention.

The PI3K/Akt signalling pathway and its downstream transcription factors have been intensively studied for their role in cell proliferation, survival, cycle control, as well as other cellular functions. There is growing evidence showing that dysregulation of this pathway also plays an essential role in cancer development. The overexpression or permanent activation of RTKs and GPCRs, as well as the exposure to environmental carcinogens cause constant activation of PI3K/Akt. On the other hand, PI3K/Akt themselves can also become hyperactivated due to gene amplification or PTEN inactivation. Consequently, the targets downstream of PI3K/Akt can be abnormally activated, which promote proliferation and survival of cancer cells in carcinogenesis. Among these targets we find that the NFkappaB and AP-1 are the most interesting. Therefore, methods and compounds aiming to inhibit the altered components of this pathway can simultaneously prevent the proliferation of tumor cells and sensitize them toward apoptosis. To this regard, the natural compounds from vegetables and fruits with high affinity and non toxicity to target the PI3K/Akt pathway and prevent cancer are attractive.

Cancer/testis antigen CT45: Analysis of mRNA and protein expression in human cancer

CT45 is a cancer/testis gene that we previously identified by massively parallel signature sequencing. Encoded by a multigene family on chromosome X, CT45 showed restricted mRNA expression to normal testis and various cancers. In this study, monoclonal antibodies were generated against recombinant CT45 protein, and CT45 protein expression in normal and tumor tissues was evaluated by immunohistochemical analysis. In adult normal tissue, CT45 expression was restricted to testicular germ cells, detected as a nuclear protein mainly at the stage of primary spermatocytes. In tumors, CT45 protein expression correlated with the mRNA levels detected by quantitative RT‐PCR, and most lung cancer and ovarian cancers with CT45 mRNA at levels >1% of testicular expression were CT45 protein‐positive. In positive cases, CT45 showed expression patterns that ranged from diffuse strong staining to heterogeneous and patchy expression. In lung cancer, CT45 expression was least frequent in adenocarcinoma, more frequent in squamous cell carcinoma and neuroendocrine tumors. Using tissue microarrays, 376 lung cancer, 219 ovarian cancer and 155 breast cancer were evaluated for CT45 protein expression. The expression frequency was highest in ovarian cancer (37%), followed by lung cancer (13%) and lowest in breast cancer (<5%). Given the focal nature of CT45 expression in many cases, these numbers represented the minimal frequency of expression in these tumor types. In summary, the expression frequency and characteristics of CT45 expression are similar to other CT cancer vaccine targets currently in clinical trials, e.g., NY‐ESO‐1 and MAGE‐A, suggesting CT45 as a potentially useful cancer target.

TLiSA1 (PTA1) activation antigen implicated in T cell differentiation and platelet activation is a member of the immunoglobulin superfamily exhibiting distinctive regulation of expression.

T lineage-specific activation antigen 1 (TLiSA1) antigen was initially described as a T lineage-specific activation antigen involved in the differentiation of human cytotoxic T cells. Subsequently, the antigen was identified on platelets and was shown to be involved in platelet activation, hence it was renamed platelet and T cell antigen 1 (PTA1), although identity between the two antigens was not established. In the present study we have cloned the cDNA encoding TLiSA1 from Jurkat cells and show it to be a novel member of the immunoglobulin superfamily with the unusual structure of two V domains only. Identity between TLiSA1 and platelet PTA1 is established by immunological criteria, by internal peptide sequences obtained from the purified platelet glycoprotein and by sequencing the platelet transcript after reverse transcriptase-polymerase chain reaction. In Jurkat cells, TLiSA1/PTA1 mRNA and surface protein expression is greatly stimulated by treatment of the cells with phorbol ester, but the T cell proliferative signal of phorbol ester and ionophore combined greatly reduces or abrogates this response, and this suppressive effect of the ionophore is not reversed by incorporating FK506 to inhibit calcineurin. Together with the known signaling role of PTA1, these data substantiate the notion that this molecule is implicated in T cell differentiation, perhaps by engagement of an adhesive ligand.

Dual Processing of FAT1 Cadherin Protein by Human Melanoma Cells Generates Distinct Protein Products

The giant cadherin FAT1 is one of four vertebrate orthologues of the Drosophila tumor suppressor fat. It engages in several functions, including cell polarity and migration, and in Hippo signaling during development. Homozygous deletions in oral cancer suggest that FAT1 may play a tumor suppressor role, although overexpression of FAT1 has been reported in some other cancers. Here we show using Northern blotting that human melanoma cell lines variably but universally express FAT1 and less commonly FAT2, FAT3, and FAT4. Both normal melanocytes and keratinocytes also express comparable FAT1 mRNA relative to melanoma cells. Analysis of the protein processing of FAT1 in keratinocytes revealed that, like Drosophila FAT, human FAT1 is cleaved into a non-covalent heterodimer before achieving cell surface expression. The use of inhibitors also established that such cleavage requires the proprotein convertase furin. However, in melanoma cells, the non-cleaved proform of FAT1 is also expressed at the cell surface together with the furin-cleaved heterodimer. Moreover, furin-independent processing generates a potentially functional proteolytic product in melanoma cells, a persistent 65-kDa membrane-bound cytoplasmic fragment no longer in association with the extracellular fragment. In vitro localization studies of FAT1 showed that melanoma cells display high levels of cytosolic FAT1 protein, whereas keratinocytes, despite comparable FAT1 expression levels, exhibited mainly cell-cell junctional staining. Such differences in protein distribution appear to reconcile with the different protein products generated by dual FAT1 processing. We suggest that the uncleaved FAT1 could promote altered signaling, and the novel products of alternate processing provide a dominant negative function in melanoma.

Chromosome X-encoded cancer/testis antigens show distinctive expression patterns in developing gonads and in testicular seminoma.

BACKGROUND Cancer/testis (CT) antigens are cancer antigens normally expressed in adult testicular germ cells. The expression of chromosome X-encoded CT antigens (CT-X antigens) in human fetal gonads and in testicular seminomas was examined. METHODS The expression of 10 CT-X antigens (MAGEA, NY-ESO-1, GAGE, CT7/MAGEC1, CT10/MAGEC2, CT45, SAGE1, SSX2, NXF2 and SPANX) was studied immunohistochemically. RESULTS In adult human testis, SPANX is expressed in late spermatids and spermatozoa, whereas all other CT-X antigens are predominantly expressed in spermatogonia or primary spermatocytes. All CT-X antigens except SPANX are expressed in human fetal germ cells. CT-X-positive germ cells appear as early as 13 weeks after gestation, increase with age and reach a plateau at around 22 weeks. In the fetal ovary, CT-X-positive oogonia are most abundant at around 24 weeks and sharply decrease subsequently. CT-X antigens are almost exclusively expressed in OCT3/4-negative gonocytes and their expression appears to coincide with the loss of pluripotency. Spermatocytic seminoma, a neoplasm derived from adult pre-meiotic germ cells, showed uniform expression of all CT-X antigens except SPANX. In contrast, most seminomas (>80%) express CT7, CT45, GAGE and CT10 but express MAGEA, NXF2 and NY-ESO-1 at lower frequency, and very rarely express SSX2 and SAGE1. CONCLUSIONS Most CT-X antigens are expressed in human fetal germ cells after they have lost stem cell characteristics, with predominant expression in pre-meiotic germ cells. Spermatocytic seminomas showed expression of all CT-X antigens except SPANX, whereas classical seminomas only express some CT-X antigens, reflecting their different origins from adult versus fetal germ cells.

Pb exposure attenuates hypersensitivity in vivo by increasing regulatory T cells

Pb is a common environmental pollutant affecting various organs. Exposure of the immune system to Pb leads to immunosuppression or immunodysregulation. Although previous studies showed that Pb exposure can modulate the function of helper T cells, Pb immunotoxicity remains incompletely understood. In this study, we investigated the effect of Pb exposure on T cell development, and the underlying mechanism of Pb-induced suppression of the delayed-type hypersensitivity (DTH) response in vivo. Sprague-Dawley rats were exposed to 300 ppm Pb-acetate solution via the drinking water for six weeks, and we found that Pb exposure significantly increased Pb concentrations in the blood by 4.2-fold (p<0.05) as compared to those in the control rats. In Pb-exposed rats, the amount of thymic CD4(+)CD8(-) and peripheral CD4(+) T cells was significantly reduced, whereas, CD8(+) population was not affected. In contrast to conventional CD4(+) T cells, Foxp3(+) regulatory T cells (Tregs) were increased in both the thymus and peripheral lymphoid organs of Pb-exposed rats. In line with the increase of Tregs, the DTH response of Pb-exposed rats was markedly suppressed. Depletion of Tregs reversed the suppression of DTH response by Pb-exposed CD4(+) T cells in an adoptive transfer model, suggesting a critical role of the increased Tregs in suppressing the DTH response. Collectively, this study revealed that Pb-exposure may upregulate Tregs, thereby leading to immunosuppression.

Enzyme-linked immunosorbent assay of changes in serum levels of growth hormone (cGH) in common carps (Cyprinus carpio).

The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1–14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1–6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1–7, 9 and 10) were used in fluorescent staining and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions.

Identification and Characterization of the CD226 Gene Promoter

CD226 is one of the main activating receptors on natural killer cells, and it can induce cytotoxicity to target cells through interaction with its ligands CD155 or CD112. CD226 is also involved in T cell differentiation, activation, and cytotoxicity. The expression of CD226 on natural killer cells and T cells can be regulated by cytokines and chemical stimuli; however, the mechanism of the regulation of the CD226 gene is still unknown. In this study, we have identified two promoters in the human CD226 gene named P1 and P2, which are located at –810 to –287 bp and +33 to +213 bp, respectively, and a negative regulation element between P1 and P2. Both P1 and P2 can be regulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) and calcium ionophore (A23187). Bioinformatics analysis shows that, within this CD226 gene region, there are putative binding sites for transcription factors AP-1, Sp1, PEA3, and Ets-1. We have found that transcription factor activating protein-1 (AP-1) can up-regulate CD226 promoters P1 and P2 in human hepatocarcinoma cells, a hepatocarcinoma cell line with low expression of endogenous AP-1 and Ets-1. Interestingly, the transcription factor Ets-1 promotes AP-1-induced P2 activity but inhibits AP-1-induced P1 activity for which a 10-bp AP-1/Ets-1 composite site (CCTTCCTTCC) in P1 may be responsible.

CD226 is expressed on the megakaryocytic lineage from hematopoietic stem cells/progenitor cells and involved in its polyploidization

Abstract:  Objectives:  In this study, we examined the expression of CD226 on megakaryocytic, granulocytic and erythroid lineage from hematopoietic stem cells/progenitor cells in adult and fetus and its potential role in megakaryocytic maturation. Methods: CD34+ cells from adult and fetus were induced to differentiate toward the megakaryocytic lineage by thrombopoietin (TPO) and the granulocytic lineage by granulocyte colony‐stimulating factor (G‐CSF), respectively. Mononuclear cells from fetal liver and CD34+ cells from adult were induced to differentiate toward erythroid‐lineage by erythropoiesis (EPO). We investigated the expression of CD226 and lymphocyte function associated antigen‐1 (LFA‐1) (CD11a) during hemopoiesis. We also studied the effect of CD226 monoclonal antibody (MoAb) and LFA‐1 MoAb on megakaryocyte with antibody cross‐liking technique. Results: CD34+ cells from adult and fetus and TPO‐induced CD41+ cells all expressed CD226 molecule. CD226 was not expressed on erythroid progenitor cells and erythroblasts and most cells of granulocytic lineage although G‐CSF induced a significant increase of the expression of CD226 on CD34+ cells in early period of time. CD226 MoAb acts on megakaryocytes by inducing intracellular calcium mobilization. The expression of LFA‐1 decreased significantly at late stage of differentiation and maturation of fetal megakaryocytes whereas the expression of LFA‐1 on adult megakaryocytes retained at a high level. CD226 MoAb in combination with LFA‐1 MoAb shifted the ploidy of generated megakaryocytes from adult‐derived CD34+ cells to higher classes significantly although CD226 and LFA‐1 MoAb slightly increased the ploidy of the generated megakaryocytes individually. CD226 MoAb or LFA‐1 MoAb or CD226 MoAb plus LFA‐1 MoAbs did not increase the ploidy of the generated megakaryocytes from fetus‐derived CD34+ cells. Conclusion: CD226 molecules play an important role in maturation of the megakaryocytes in combination with LFA‐1.

Identification and characterization of CD226 gene promoter

CD226 is one of the main activating receptors on natural killer cells, and it can induce cytotoxicity to target cells through interaction with its ligands CD155 or CD112. CD226 is also involved in T cell differentiation, activation, and cytotoxicity. The expression of CD226 on natural killer cells and T cells can be regulated by cytokines and chemical stimuli; however, the mechanism of the regulation of the CD226 gene is still unknown. In this study, we have identified two promoters in the human CD226 gene named P1 and P2, which are located at –810 to –287 bp and +33 to +213 bp, respectively, and a negative regulation element between P1 and P2. Both P1 and P2 can be regulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) and calcium ionophore (A23187). Bioinformatics analysis shows that, within this CD226 gene region, there are putative binding sites for transcription factors AP-1, Sp1, PEA3, and Ets-1. We have found that transcription factor activating protein-1 (AP-1) can up-regulate CD226 promoters P1 and P2 in human hepatocarcinoma cells, a hepatocarcinoma cell line with low expression of endogenous AP-1 and Ets-1. Interestingly, the transcription factor Ets-1 promotes AP-1-induced P2 activity but inhibits AP-1-induced P1 activity for which a 10-bp AP-1/Ets-1 composite site (CCTTCCTTCC) in P1 may be responsible.