Protein expression profiling identifies cyclophilin A as a molecular target in Fhit-mediated tumor suppression

Loss of fragile histidine triad (Fhit) expression is often associated with human malignancies, and Fhit functions as a tumor suppressor in controlling cell growth and apoptosis, although specific signal pathways are still undefined. We have used a proteomic approach to define proteins in the Fhit-mediated tumor suppression pathway. Because substitution of Tyr(114) (Y114) with phenylalanine (Y114F) diminishes Fhit functions, we did protein expression profiling to identify proteins differentially expressed in Fhit-negative H1299 lung cancer cells infected with wild-type (Ad-FHIT-wt) and Y114 mutant FHIT-expressing (Ad-FHIT-Y114F) adenoviruses. Among 12 distinct proteins that exhibited 4-fold differences in expression on comparison of the two infected cell lysates, cyclophilin A, the intracellular reporter of the immunosuppressive drug cyclosporine A, showed a remarkably decreased protein level in cells infected with Ad-FHIT-wt versus Ad-FHIT-Y114F. Conversely, loss of Fhit expression resulted in increased cyclophilin A expression in mouse tissues and cell lines. Restoration of Fhit expression led to down-regulated cyclophilin A protein expression and subsequently prevented cyclophilin A-induced up-regulation of cyclin D1, Cdk4, and resultant cell cycle progression (G(1)-S transition), which was independent of Ca(2+)/calmodulin-dependent kinase inhibitor, KN-93. Interestingly, Fhit down-modulation of phosphatase activity of calcineurin, which controls cyclin D1/Cdk4 activation, was reversed by cyclophilin A treatment in a concentration-dependent manner, a reversal that was inhibited by additional cyclosporine A treatment. Thus, cyclophilin A is a downstream target in Fhit-mediated cessation of cell cycle progression at late G(1) phase. Elucidation of the protein effectors of Fhit signaling may lead to identification of targets for lung cancer therapy.     

Characterization of astrocyte-specific conditional knockouts

Conditional gene knockouts are a very powerful tool for elucidating gene function in animal physiology and behavior. To obtain cell-specific knockouts, a promoter is utilized that drives expression of Cre recombinase specifically to the cell population of interest. We describe several transgenic lines of mice that were created in an attempt to obtain astrocyte-specific gene recombination. A 2 kb fragment from the human glial fibrillary acidic protein promoter is utilized to drive expression of inducible Cre recombinase, with both the Tet-Off and tamoxifen responsive systems. We show data obtained from crosses with two Cre reporter lines, ROSA26R and an astrocyte Cre reporter created in our laboratory, to assess the cell specificity of gene recombination. Additionally, our system is shown to successfully recombine a floxed Connexin43 locus, although recombination is not as extensive as seen in crosses with reporter lines.     

FK-506, a potent novel immunosuppressive agent, binds to a cytosolic protein which is distinct from the cyclosporin A-binding protein, cyclophilin

A novel macrolide antibiotic, FK-506, isolated from Streptomyces tsukubaensis, has been shown to be a potent immunosuppressive agent in vivo and in vitro. FK-506 shares a number of immunosuppressive properties with the cyclic peptide, cyclosporin A (CsA), although 10 to 100 times more potent in this regard. These similarities suggest that both agents may share a similar mechanism(s) of action at the biochemical level. We have identified a cytoplasmic binding protein for FK-506 in the human T cell line, JURKAT, using [3H]FK-506. The FK-506 binding protein has a mr of 10 to 12 kDa (as determined by gel filtration), is heat stable and does not bind CsA. This contrasts with the CsA binding protein, cyclophilin, in that cyclophilin is heat labile and has a mr of 15 to 17 kDa. Our data suggest that FK-506 binds to a low m.w. protein(s) in JURKAT cells, which is distinct from cyclophilin. This protein may mediate the immunosuppressive effects of FK-506 in T cells. In addition, our results suggest that the immunosuppressive activity of FK-506, as with CsA, is mediated by an intracellular mechanism.     

Molecular cloning and characterization of two isoforms of cyclophilin A gene from Venerupis philippinarum

Cyclophilin A (CypA) is a ubiquitously distributed intracellular protein belonging to the immunophilin family, which is recognized as the cell receptor for the potent immunosuppressive drug cyclosporine A. In the present study, two isoforms of cyclophilin A gene (named as VpCypA1 and VpCypA2) were isolated and characterized from Venerupis philippinarum by RACE approaches. Both VpCypA1 and VpCypA2 possessed all conserved features critical for the fundamental structure and function of CypA, indicating that the two isoforms of cyclophilin A should be new members of CypA family. The expression of VpCypA2 mRNA in haemocytes was significantly up-regulated and the highest expression level was detected at 96 h post-infection with 7.7-fold increase compared with that in the blank group. On the contrary, the relative expression level of VpCypA1 mRNA was down-regulated rapidly at 6 h post-infection and reached 0.4-fold of the control group. They exhibited different expression profile and identical effect of immune modulation, which might suggest the two VpCypA isoforms exert their function in a manner of synergy. These results provide valuable information for further exploring the roles of cyclophilin A in the immune responses of V. philippinarum.     

Sequences within and flanking hypersensitive sites 3 and 2 of the beta-globin locus control region required for synergistic versus additive interaction with the epsilon-globin gene promoter.

The locus control region is required for high-level, position-independent expression of mammalian beta-globin genes. It is marked by five major DNase hypersensitive sites (HSs) in a 16 kb region of chromatin, and the protein-DNA complexes that form these HSs may interact in a holocomplex that carries out the full function of the locus control region. Previous studies showed that a large rabbit DNA fragment containing both HS2 and HS3 in their native sequence context and spacing produced a much larger increase in expression of a linked reporter gene than the sum of the largest effects observed with DNA fragments containing HS2 or HS3 individually. To test whether this reflected a synergistic interaction between the 200-400 bp cores of the HSs or if this effect required additional sequences outside the cores, combinations of different restriction fragments containing HS2 or HS3 were tested for their ability to increase the expression of a hybrid epsilon-globin-luciferase reporter gene in transfected K562 cells. The results show that the human HS2 and HS3 cores do not interact either additively or synergistically with the reporter gene when juxtaposed, and separation by spacer DNA has little effect on their function. Fragments of human DNA containing cores plus flanking sequences for HS3 or HS2 show an additive effect in combination, whereas homologous fragments of rabbit DNA containing HS3 and HS2 interact synergistically. At least part of this difference localizes to the rabbit DNA fragment containing HS3, which can interact synergistically with the human DNA fragment containing HS2. The region 5' to the HS3 core plays a role both in the cooperative interaction observed with the rabbit DNA fragment and the domain-opening observed with the human DNA. A minor DNase HS maps to this region, and the pattern of sequence conservation is consistent with some difference in function between species.     

DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: implications for the androgen receptor functions and regulation

The majority of human prostate cancer cell lines, including the two "classical" cell lines DU-145 and PC-3, are reported to be androgen receptor (AR)-negative. However, other studies have provided evidence that the DU-145 and PC-3 cell lines express AR mRNA. These contradictory observations prompted us to investigate whether DU-145 and PC-3 cell lines express the androgen receptor. Using antipeptide antibodies directed against three distinct regions of the human AR protein and an improved method to detect AR protein in immunoblotting, we report that DU-145 and PC-3 cell lines express AR protein. We found that the relative levels of the AR mRNA and protein that were detected in DU-145 and PC-3 cell lines were lower than the LNCaP, an AR-positive cell line. Moreover, the antibody directed against the non-variant region (amino acids 299-315), but not the variant N- or C-terminal region (amino acids 1-20 and 900-919, respectively) of the human AR protein, detected the expression of AR in all prostate cancer cell lines. Notably, treatment of these cell lines with dihydrotestosterone (DHT) resulted in measurable increases in the AR protein levels and considerable nuclear accumulation. Although, treatment of DU-145 and PC-3 cells with DHT did not result in stimulation of the activity of an AR-responsive reporter, knockdown of AR expression in PC-3 cells resulted in decreases in p21(CIP1) protein levels, and a measurable decrease in the activity of the p21-luc-reporter. Our observations demonstrate the expression of AR protein in DU-145 and PC-3 prostate cancer cell lines.     

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.     

Wortmannin potentiates integrase-mediated killing of lymphocytes and reduces the efficiency of stable transduction by retroviruses

Retroviral infection induces integrase-dependent apoptosis in DNA-PK-deficient murine scid lymphocytes. Furthermore, the efficiency of stable transduction of reporter genes is reduced in adherent cell lines that are deficient in cellular DNA-repair proteins known to mediate nonhomologous end joining (NHEJ), such as DNA-PK and XRCC4 (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644-647, 1999). Here we report that wortmannin, an irreversible inhibitor of phosphatidylinositol 3-kinase (PI-3K)-related PKs, including the catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) and ATM, sensitizes normal murine lymphocytes to retrovirus-mediated cell killing. We also show that the efficiency of stable transduction of reporter genes in human (HeLa) cells, mediated by either an avian sarcoma virus or a human immune deficiency virus type 1 vector, is reduced in the presence of wortmannin. The dose dependence of such reduction correlates with that for inhibition of PI-3K-related protein kinase activity in these cells. Results from wortmannin treatment of a panel of cell lines confirms that formation and/or survival of transductants is dependent on components of the NHEJ pathway. However, stable transduction is virtually abolished by wortmannin treatment of cells that lack ATM. These results suggest that ATM activity is required for the residual transduction observed in the NHEJ-deficient cells. Our studies support the hypothesis that DNA repair proteins of the NHEJ pathway and, in their absence, ATM are required to avoid integrase-mediated killing [corrected] and allow stable retroviral DNA transduction. The studies also suggest that cells can be sensitized to such killing and stable retroviral DNA integration blocked by drugs that inhibit cellular DNA repair pathways.     

人淋巴细胞CD2基因5’侧翼序列的克隆和鉴定

本文报告以ＣＤ２ｃＤＮＡ５’端的片段作探针,从人Ｔ淋巴细胞基因组文库筛选阳性重组克隆,经限制性内切酶降解和Ｓｏｕｔｈｅｒｎ杂交分析,证明其中一个阳性克隆的插入片段中含ＣＤ２基因５’侧翼顺序。经插入片段的亚克隆、限制性内切酶图谱及ＤＮＡ序列分析,鉴定出一含转录起始点及其上游序列的４．０ｋｂ片段。将此片段中含转录起始点和两个ＤＮ（ａｓｅ）Ⅰ高敏感位点的２．５ｋｂ片段定向克隆到以虫萤光素酶为报告基因的表达载体ｐＭＧ３中,并用限制性内切酶对此２．５ｋｂ片段作不同程度缺失,构成一系列突变子。这些重组的表达质粒转染人ＪｕｒｋａｔＴ细胞后,以瞬时表达实验分析各突变子驱动虫萤光素酶基因的表达,结果发现在ＣＤ２基因５’上游具有很弱的启动子活性,初步测定该启动子位于－１．２ｋｂ～－９８ｂｐ域。ＣＤ２基因具有弱启动子、强增强子的特点与Ｔ细胞表面其它抗原分子基因是相似的。