Two Novel Variants of the v-srcOncogene Isolated from Low and High Metastatic RSV-Transformed Hamster Cells

AbstractFour different transformed cell lines were isolated as a result of independent infection of primary hamster fibroblasts by Rous sarcoma virus (RSV SR-D stocks). These lines differ by the level of their spontaneous metastatic activity: HET-SR-1, HET-SR-8, and HET-SR-10 cell lines induced 70–200 metastatic nodules in the lung and/or lymph nodes of inoculated animals (high metastatic lines, HM). Metastatic activity was not identified after injection of HET-SR cells (low metastatic line, LM). All cell lines contained one copy of integrated and expressed intact RSV provirus. The difference in the amount of v-srcprotein in cell lines was not correlated with their metastatic potentialin vivo.Complete v-srcHM and v-srcLM genes were cloned from corresponding gene libraries and sequenced. In the unique region of both v-srcisoforms a GC-rich insert of 60 nucleotides (20 a.a.) was found. The presence of this insert explains the unusual apparent molecular weight of protein encoded by v-srcHM and v-srcLM: 62 kDa. Both genes had 10 identical amino acid changes when compared to the known RSV SR-D v-srcsequence. v-srcHM and v-srcLM differ by several amino acid changes. Most of them are localized in the unique domain and the extreme carboxy-terminal region of the oncoprotein. Both v-srcvariants and chimeric v-srcwith mutually substituted parts were subcloned in a retroviral vector and introduced into avian neuroretina cells. Significant differences in the morphology of transformed neuroretina cells were associated with the mutations in the carboxy-terminal region of the v-srconcogene. Low metastatic HET-SR cells transfected with v-srcHM and the chimeric gene v-src-LH remarkably increased their metastatic potential. In contrast, this effect was not observed when the same cells were transfected with v-srcLM and the chimeric v-srcHL gene. Specific changes in the distribution of fibronectin matrix typical for high metastatic cells were found in the lines transfected with v-srcHM

Low dose IR-induced IGF-1-sCLU expression: a p53-repressed expression cascade that interferes with TGFß1 signaling to confer survival

To better understand tissue responses after low IR doses, we generated a reporter system using human clusterin promoter fused to firefly luciferase (hCLUp-Luc). Secretory clusterin (sCLU), an extra-cellular molecular chaperone, induced by low doses of cytotoxic agents, clears cell debris promoting survival. Low dose IR (>2 cGy) exposure induced hCLUp-Luc activity with peak levels at 96 h, consistent with endogenous sCLU levels. As doses increased (>1 Gy), sCLU induction amplitudes increased and time to peak response decreased. sCLU expression was stimulated by IGF-1, but suppressed by p53. Responses in transgenic hCLUp-Luc reporter mice after low IR doses showed that specific tissues (i.e., colon, spleen, mammary, thymus, bone marrow) of female mice induced hCLUp-Luc activity more than male mice after whole body >10 cGy. Tissue-specific, non-linear dose- and time-responses of hCLUp-Luc and endogenous sCLU levels were noted. Colon maintained homeostatic balance after 10 cGy. Bone marrow responded with delayed, but prolonged and elevated expression. Intraperitoneal administration of the α-TGFß1 (1D11) antibody, but not a control antibody (13C4), immediately following IR exposure abrogated CLU induction responses. Induction in vivo also correlated with Smad signaling via activated TGFß1 after IR. Mechanistically, media with elevated sCLU levels suppressed signaling, blocked apoptosis and increased survival of TGFß1-exposed tumor or normal cells. Thus, sCLU is a TGF-ß1-induced pro-survival, potential bystander factor expressed in certain exposed tissues that, in turn, abrogates TGFß1 signaling and may promote wound healing and likely contributes to a pro-tumor growth microenvironment

Recruitment of the CoREST transcription repressor complexes by Nerve Growth factor IB-like receptor (Nurr1/NR4A2) mediates silencing of HIV in microglial cells.

Human immune deficiency virus (HIV) infection in the brain leads to chronic neuroinflammation due to the production of pro-inflammatory cytokines, which in turn promotes HIV transcription in infected microglial cells. However, powerful counteracting silencing mechanisms in microglial cells result in the rapid shutdown of HIV expression after viral reactivation to limit neuronal damage. Here we investigated whether the Nerve Growth Factor IB-like nuclear receptor Nurr1 (NR4A2), which is a repressor of inflammation in the brain, acts directly to restrict HIV expression. HIV silencing following activation by TNF-α, or a variety of toll-like receptor (TLR) agonists, in both immortalized human microglial cells (hμglia) and induced pluripotent stem cells (iPSC)-derived human microglial cells (iMG) was enhanced by Nurr1 agonists. Similarly, overexpression of Nurr1 led to viral suppression, while conversely, knock down (KD) of endogenous Nurr1 blocked HIV silencing. The effect of Nurr1 on HIV silencing is direct: Nurr1 binds directly to the specific consensus binding sites in the U3 region of the HIV LTR and mutation of the Nurr1 DNA binding domain blocked its ability to suppress HIV-1 transcription. Chromatin immunoprecipitation (ChIP) assays also showed that after Nurr1 binding to the LTR, the CoREST/HDAC1/G9a/EZH2 transcription repressor complex is recruited to the HIV provirus. Finally, transcriptomic studies demonstrated that in addition to repressing HIV transcription, Nurr1 also downregulated numerous cellular genes involved in inflammation, cell cycle, and metabolism, further promoting HIV latency and microglial homoeostasis. Nurr1 therefore plays a pivotal role in modulating the cycles of proviral reactivation by potentiating the subsequent proviral transcriptional shutdown. These data highlight the therapeutic potential of Nurr1 agonists for inducing HIV silencing and microglial homeostasis and ultimately for the amelioration of the neuroinflammation associated with HIV-associated neurocognitive disorders (HAND)

Roscovitine suppresses CD4+ T cells and T cell-mediated experimental uveitis.

T cells are essential for the development of uveitis and other autoimmune diseases. After initial activation, CD4+ lymphocytes express the co-stimulatory molecule OX40 that plays an important role in T cell proliferation. Cyclin dependent kinase 2 (CdK2) plays a pivotal role in the cell cycle transition from G1 to S phase. In addition, recent research has implicated CdK2 in T cell activation. Thus, we sought to test the immunosuppressive effect of roscovitine, a potent CdK2 inhibitor, on CD4+ T cell activation, proliferation, and function.Mouse CD4+ T cells were activated by anti-CD3 and anti-CD28 antibodies. The expression of OX40, CD44, and CdK2 were analyzed by flow cytometry. In addition, cell cycle progression and apoptosis of control and roscovitine-treated T lymphocytes were measured by BrdU incorporation and annexin V assay, respectively. Furthermore, the immunoregulatory effect of roscovitine was evaluated in both ovalbumin-induced uveitis and experimental autoimmune uveitis (EAU) models.In this study, we found that T cell activation induced OX40 expression. Cell cycle analysis showed that more CD4+OX40+ cells entered S phase than OX40- T cells. Concurrently, CD4+OX40+ cells had a higher level of CdK2 expression. Roscovitine treatment blocked activated CD4+ cells from entering S phase. In addition, roscovitine not only reduced the viability of CD4+ lymphocytes but also suppressed T cell activation and cytokine production. Finally, roscovitine significantly attenuated the severity of T cell-dependent, OX40-enhanced uveitis.These results implicate CdK2 in OX40-augmented T cell response and expansion. Furthermore, this study suggests that roscovitine is a novel, promising, therapeutic agent for treating T cell-mediated diseases such as uveitis

Induction of CD4+ T lymphocyte apoptosis and cell death by roscovitine.

<p>Splenocytes from C57BL/6 mice were activated with anti-CD3 and anti-CD28 antibodies <i>in </i><i>vitro</i> in the presence of 0, 10, and 50 μM roscovitine. Then, the cells were labeled with annexin V and PI at 24, 48, and 72 hours. Shown are representative plots of a cell viability assay on gated CD4+ lymphocytes from 3 independent studies. </p

Efficient suppression of secretory clusterin levels by polymer-siRNA nanocomplexes enhances ionizing radiation lethality in human MCF-7 breast cancer cells in vitro-1

<p><b>Copyright information:</b></p><p>Taken from "Efficient suppression of secretory clusterin levels by polymer-siRNA nanocomplexes enhances ionizing radiation lethality in human MCF-7 breast cancer cells in vitro"</p><p></p><p>International Journal of Nanomedicine 2006;1(2):155-162.</p><p>Published online Jan 2006</p><p>PMCID:PMC2426783.</p><p>© 2006 Dove Medical Press Limited. All rights reserved</p

Expression of CdK2 in activated CD4+CD44+ cells (A).

<div><p>C57BL/6 splenocytes were activated with anti-CD3 and anti-CD28 antibodies <i>in </i><i>vitro</i>. Seventy-two hours later, surface CD4, CD44 and intracellular CdK2 were stained and analyzed by flow cytometry. Shown are representative plots of CdK2 and CD44 expression in gated CD4+ lymphocytes from 3 independent studies. </p> <p>Higher CdK2 expression in CD4+OX40+ T cells (B). Splenocytes from C57BL/6 mice were activated with anti-CD3 and anti-CD28 antibodies <i>in </i><i>vitro</i> for 72 hours. Cell surface CD4 and OX40 expression were analyzed by flow cytometry. Gated CD4+OX40- and CD4+OX40+ cells were further analyzed for the intracellular expression of CdK2. Shown are representative plots from 3 independent studies. </p></div