Carl Y. Sasaki

Comparison of the Effect of Mutant and Wild-Type p53 on Global Gene Expression

The mechanisms for “gain-of-function” phenotypes produced by mutant p53s such as enhanced proliferation, resistance to transforming growth factor-β–mediated growth suppression, and increased tumorigenesis are not known. One theory is that these phenotypes are caused by novel transcriptional regulatory events acquired by mutant p53s. Another explanation is that these effects are a result of an imbalance of functions caused by the retention of some of the wild-type transcriptional regulatory events in the context of a loss of other counterbalancing activities. An analysis of the ability of DNA-binding domain mutants A138P and R175H, and wild-type p53 to regulate the expression levels of 6.9 × 103 genes revealed that the mutants retained only <5% of the regulatory activities of the wild-type protein. A138P p53 exhibited mostly retained wild-type regulatory activities and few acquired novel events. However, R175H p53 possessed an approximately equal number of wild-type regulatory events and novel activities. This is the first report that, after examination of the regulation of a large unfocused set of genes, provides data indicating that remaining wild-type transcriptional regulatory functions existing in the absence of counterbalancing activities as well as acquired novel events both contribute to the gain-of-function phenotypes produced by mutant p53s. However, mutant p53s are likely to be distinct in terms of the extent to which each mechanism contributes to their gain-of-function phenotypes.

Interleukin-12-induced Interferon-γ Production by Human Peripheral Blood T Cells Is Regulated by Mammalian Target of Rapamycin (mTOR)

Depending on the type of external signals, T cells can initiate multiple intracellular signaling pathways that can be broadly classified into two groups based on their sensitivity to the immunosuppressive drug cyclosporin A (CsA). Interleukin (IL)-12-mediated interferon (IFN)-γ production by activated T cells has been shown to be CsA-insensitive. In this report, we demonstrate that the IL-12-induced CsA-resistant pathway of IFN-γ production is sensitive to rapamycin. Rapamycin treatment resulted in the aberrant recruitment of Stat3, Stat4, and phospho-c-Jun to the genomic promoter region resulting in decreased IFN-γ transcription. IL-12-induced phosphorylation of Stat3 on Ser-727 was affected by rapamycin, which may be due to the effect of rapamycin on the IL-12-induced interaction between mammalian target of rapamycin (mTOR) and Stat3. In accordance with this, reduction in the mTOR protein level by small interfering RNA resulted in suppression of Stat3 phosphorylation and decreased production of IFN-γ after IL-12 stimulation. These results suggest that mTOR may play a major role in IL-12-induced IFN-γ production by activated T cells.

Expression of E-cadherin reduces Bcl-2 expression and increases sensitivity to etoposide-induced apoptosis†

Expression of Bcl‐2 is important in determining cancer cell resistance to chemotherapy. However, it is not clear whether cell–cell interactions regulate Bcl‐2 expression. Using rat breast carcinoma cells selected for loss of hormone responsiveness, we found that parental E‐cadherin–expressing cells (E cells) were more sensitive to etoposide‐induced apoptosis than hormone–non‐responsive cells (F cells), which failed to express E‐cadherin. Expression of β‐catenin and pp120 src substrate proteins, which associate with E‐cadherin, was unaffected. To determine whether re‐expression of E‐cadherin in F cells would restore etoposide sensitivity, F cells were transfected with an expression vector coding for the mouse E‐cadherin gene. Stable clonal isolates expressing E‐cadherin (F.Cad) showed increased sensitivity to etoposide treatment compared with control clones (F.Neo). Expression of E‐cadherin resulted in a redistribution of β‐catenin from the cytoskeletal/nuclear fraction to the cytoplasmic/membrane fraction of the cells. E‐cadherin–expressing clones also showed reduced invasion through basement membrane. Etoposide‐induced apoptosis was characterized by morphological changes (nuclear blebbing) and DNA fragmentation. Induction of CPP32‐like caspase activity was also observed in F.Cad transfectants but not F.Neo cells. Unlike F cells, F.Cad transfectants were not able to express Bcl‐2, but transient transfection of bcl‐2 resulted in re‐expression and resistance to etoposide treatment. Therefore, E‐cadherin may negatively regulate Bcl‐2 expression by altering the availability of nuclear β‐catenin. Loss of E‐cadherin in invasive tumor cells may lead to increased Bcl‐2 expression and resistance to chemotherapeutic drugs. Int. J. Cancer 86:660–666, 2000. Published 2000 Wiley‐Liss, Inc.

p(⁷⁰S⁶K¹) in the TORC1 pathway is essential for the differentiation of Th17 Cells, but not Th1, Th2, or Treg cells in mice.

The TORC1 pathway is necessary for ribosomal biogenesis and initiation of protein translation. Furthermore, the differentiation of Th1 and Th17 cells requires TORC1 activity. To investigate the role of the TORC1 pathway in the differentiation of Th1 and/or Th17 cells in more detail, we compared the differentiation capacity of naïve T cells from wild type and p70S6K1 knockout mice. Expression of many of the genes associated with Th17‐cell differentiation, such as IL17a, IL17f, and IL‐23R, were reduced in p70S6K1 knockout mice. In contrast, the development of Th1, Th2, and Treg cells was unaffected in the absence of p70S6K1. Furthermore, expression of the major transcription factor in Th17‐cell differentiation, retinoic acid receptor‐related orphan receptor gamma T, remained unchanged. However, the acetylation of histone 3 at the promoters of IL17a and IL17f was reduced in the absence of p70S6K1. In accordance with the in vitro data, the kinetics, but not the development, of EAE was affected with the loss of p70S6K1 expression. Collectively, our findings suggested that both in vitro and in vivo differentiation of Th17 cells were positively regulated by p70S6K1.

Traf1 Induction and Protection from Tumor Necrosis Factor by Nuclear Factor-κB p65 Is Independent of Serine 536 Phosphorylation

Abnormal nuclear factor-kappaB (NF-kappaB) signaling has been attributed to the initiation and progression of cancer. Posttranslational modification of p65 facilitates optimal NF-kappaB signaling after activation. Here, we show that the phosphorylation of serine 536 was required for p65-mediated transcription and I kappa B alpha expression in fibroblasts. Furthermore, tumor necrosis factor (TNF) treatment slightly induced p65 phosphorylation, and both unphosphorylated and phosphorylated p65 translocated into the nucleus. The phosphorylation of serine 536 was not required for p65-mediated protection from TNF cytotoxicity and Traf1 induction in fibroblasts. Also, the corecruitment of p65 and RNA polymerase II to the Traf1 enhancer region did not require p65 phosphorylation. However, the corecruitment of p65 and RNA polymerase II to the Csf2 promoter required the phosphorylation of serine 536. These findings suggested that the requirement of serine phosphorylation at residue 536 and the distance between the NF-kappaB response element and the start of transcription may influence which genes will be transcribed.

Regulation of urokinase plasminogen activator (uPA) activity by E-cadherin and hormones in mammary epithelial cells.

Urokinase plasminogen activator (uPA) is involved in proteolysis of extracellular matrix during development and tumor cell invasion. In the present study, we examined the regulation of uPA in hormone‐responsive, noninvasive mammary epithelial cells by using fibrinolytic and caseinolytic enzyme activity assays. Urokinase PA expression was activated after contact with fibrin and initiation of cell–cell interactions that were mediated by E‐cadherin. Fibrinolysis occurred in zones surrounding cellular aggregates. Stromal matrix proteins that disrupted aggregation or anti‐E‐cadherin antibodies that inhibited cellular compaction inhibited fibrinolysis perhaps by increasing cell–matrix adhesion or preventing E‐cadherin signaling, respectively. Aggregation required the presence of divalent cations and was inhibited by serum and ethylene diaminetetraacetic acid, whereas serine protease inhibitors reduced uPA activity without affecting aggregation. Inhibitors of PA (type 2; PAI‐2) and a specific antisense uPA oligonucleotide also reduced enzymatic activity, suggesting that fibrinolysis depends on translational regulation of uPA. In addition, the activation of plasmin from plasminogen was inhibited by anti‐E‐cadherin antibodies and PAI‐2, consistent with a role for uPA. The data also support a role for transcriptional regulation of uPA activity because treatment of cells with progesterone, hydrocortisone, or dexamethasone inhibited uPA activation on fibrin without affecting cellular aggregation. Estradiol and insulin did not alter, whereas human chorionic gonadotropin and prolactin increased uPA activity. The expression of the 55‐kDa uPA activity was consistent with specific hormone action and correlated with protein expression by immunoblotting. Therefore, the alteration of downstream signaling events by hormones may affect uPA production. These results indicate that uPA is an enzyme that may be important in the degradation of extracellular matrix during development and that specific E‐cadherin interactions and hormones can regulate its activity. Investigation of the regulation of uPA in these cells may be useful in understanding and manipulating mammary gland remodeling. J. Cell. Physiol. 181:1–13, 1999. Published 1999 Wiley‐Liss, Inc.

Combination therapy with lenalidomide and nanoceria ameliorates CNS autoimmunity

OBJECTIVE Multiple sclerosis (MS) is a debilitating neurological disorder involving an autoimmune reaction to oligodendrocytes and degeneration of the axons they ensheath in the CNS. Because the damage to oligodendrocytes and axons involves local inflammation and associated oxidative stress, we tested the therapeutic efficacy of combined treatment with a potent anti-inflammatory thalidomide analog (lenalidomide) and novel synthetic anti-oxidant cerium oxide nanoparticles (nanoceria) in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. METHODS C57BL/6 mice were randomly assigned to a control (no EAE) group, or one of the four myelin oligodendrocyte glycoprotein-induced EAE groups: vehicle, lenalidomide, nanoceria, or lenalidomide plus nanoceria. During a 23 day period, clinical EAE symptoms were evaluated daily, and MRI brain scans were performed at 11-13 days and 20-22 days. Histological and biochemical analyses of brain tissue samples were performed to quantify myelin loss and local inflammation. RESULTS Lenalidomide treatment alone delayed symptom onset, while nanoceria treatment had no effect on symptom onset or severity, but did promote recovery; lenalidomide and nanoceria each significantly attenuated white matter pathology and associated inflammation. Combined treatment with lenalidomide and nanoceria resulted in a near elimination of EAE symptoms, and reduced white matter pathology and inflammatory cell responses to a much greater extent than either treatment alone. INTERPRETATION By suppressing inflammation and oxidative stress, combined treatment with lenalidomide and nanoceria can reduce demyelination and associated neurological symptoms in EAE mice. Our preclinical data suggest a potential application of this combination therapy in MS.

p52-independent nuclear translocation of RelB promotes LPS-induced attachment

The NF-kappaB signaling pathways have a critical role in the development and progression of various cancers. In this study, we demonstrated that the small cell lung cancer cell line (SCLC) H69 expressed a unique NF-kappaB profile as compared to other cancer cell lines. The p105/p50, p100/p52, c-Rel, and RelB protein and mRNA transcripts were absent in H69 cells but these cells expressed RelA/p65. The activation of H69 cells by lipopolysaccharide (LPS) resulted in the induction of RelB and p100 expression. The treatment also induced the nuclear translocation of RelB without the processing of p100 to p52. Furthermore, LPS-induced beta1 integrin expression and cellular attachment through an NF-kappaB-dependent mechanism. Blocking RelB expression prevented the increase in the expression of beta1 integrin and the attachment of H69. Taken together, the results suggest that RelB was responsible for the LPS-mediated attachment and may play an important role in the progression of some cancers.

Recruitment of RelB to the Csf2 Promoter Enhances RelA-mediated Transcription of Granulocyte-Macrophage Colony-stimulating Factor

Tumor necrosis factor (TNF) induces expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) but lymphotoxin β (LTβ) does not. Here we report that priming of cells with agonistic LTβ receptor antibody synergistically enhanced TNF-induced GM-CSF expression. The LTβ priming process was not due to an increase in TNF-mediated nuclear translocation of p65, p65 DNA binding, or NF-κB transactivational activity. The synergistic effect of LTβ priming was not observed with other TNF-responsive genes such as Ccl2 or RelB, which suggested that this effect was not a general increase in TNF signaling. Furthermore, RelB and p65 were both independently recruited to the GM-CSF promoter when cells were primed with LTβ followed by TNF treatment. As a consequence, an increase in both chromatin accessibility and the recruitment of RNA polymerase II were observed to the GM-CSF promoter. Taken together, these findings suggested that LTβ signaling amplified TNF-mediated GM-CSF expression by facilitating chromatin access and the co-recruitment of RNA polymerase II to increase gene transcription. Moreover, the novel priming process described here underscores the complexity of the interactions between the classical and alternative NF-κB signaling pathways.

Transforming Growth Factor β1 (TGF-β1) Suppresses Growth of B-cell Lymphoma Cells by p14ARF-dependent Regulation of Mutant p53

Background: TGF-β1 suppresses growth of B-cell lymphoma cells. Results: TGF-β1-induced down-regulation of mutant p53 via p14ARF renders B-cell lymphoma cells sensitive to TGF-β1. Conclusion: Overexpression of p14ARF possibly causes TGF-β1 resistance. Significance: p14ARF is a potential therapeutic target for B-cell lymphoma. Previously we reported that TGF-β1-induced growth suppression was associated with a decrease in mutant p53 levels in B-cell lymphoma cells. The goal of the present study was to understand the mechanism involved in TGF-β1-mediated down-regulation of mutant p53. In RL and CA46, two B-cell lymphoma cell lines, TGF-β1 treatment caused down-regulation of E2F-1 transcription factor resulting in the down-regulation of both p14ARF and mutant p53, leading to growth arrest. Experimental overexpression of E2F-1 increased p14ARF level and blocked TGF-β1-induced down-regulation of p14ARF. Overexpression of p14ARF blocked the down-regulation of mutant p53 and prevented growth arrest. p14ARF also attenuated TGF-β1-induced p21Cip1/WAF1 induction, which was reversible by p53 siRNA, indicating the involvement of mutant p53 in controlling the TGF-β1-induced expression of p21Cip1/WAF1. The interaction observed between phospho-Smad2 and mutant p53 in the nucleus could be the mechanism responsible for blocking the growth-suppressive effects of TGF-β1. In RL cells, p14ARF is present in a trimer consisting of mutant p53-Mdm2-p14ARF and in a dimer consisting of Mdm2-p14ARF. Because it is known that Mdm2 can degrade p53, it is possible that, in its trimeric form, p14ARF is able to stabilize mutant p53 by inhibiting Mdm2. In its dimeric form, p14ARF may be sequestering Mdm2, limiting its ability to degrade p53. Collectively, these data demonstrate a unique mechanism in which the inhibition of TGF-β1-mediated growth suppression by mutant p53 can be reversed by the down-regulation of its stabilizing protein p14ARF. This work suggests that the high levels of p14ARF often found in tumor cells could be a potential therapeutic target.