Association between BRAF expression in circulating tumor cells and the clinical feature of patients with multiple myeloma.

Multiple myeloma (MM) is the most common hematological malignancy with uncontrolled proliferation of monoclonal plasma cells. Despite treatment improvements, MM remains an incurable disease for most patients. Therefore, promising molecular markers are required for MM treatment decisions. In the present study, we explored the relationship between the BRAF expression in circulating tumor cells (CTCs) and the clinical features of patients with MM. The results showed that CTCs were associated with MM staging, and the expression of BRAF was associated with different CTCs. Moreover, the BRAF gene was correlated with patients' white blood cells, blood albumin levels, and Eastern Cooperative Oncology Group (ECOG) score. BRAF expression positively correlated with total CTCs, hybrid CTCs, and mesenchymal CTCs. Taken together, CTCs tightly correlated with the clinical stages and characteristics of MM. Our findings may provide a promising prognosis biomarker for MM treatment decisions.

Isolation and functional characterization of three MiFTs genes from mango.

FLOWERING LOCUS T (FT) is a key integrator of environmental signals and internal cues and plays a central role in the photoperiod response mechanism in Arabidopsis. However, the function of FTs in Mangifera indica L. is unknown. In this study, we identified three MiFTs genes from mango and characterized their role in flowering regulation. The open reading frames of MiFT1, MiFT2, and MiFT3 are 540, 516, and 588 bp in length and encode 180, 172, and 196 amino acids, respectively; the genes belong to the PEBP family. MiFTs share the conserved exon/intron structure of FTs. The nucleotide sequence of MiFT1 is 90% identical to that of MiFT2 and 82% identical to that of MiFT3; MiFT2 and MiFT3 share 81% homology with each other. According to expression analysis, MiFTs were detected at different expression levels in all tested tissues. The expression levels of the three MiFTs were significantly different in leaves during flower development, and MiFT1 expression increased sharply in leaves and was significantly higher than that of the other two MiFTs during flower bud development. All three MiFTs showed daily cycles. Ectopic expression of the three MiFTs in transgenic Arabidopsis resulted in an earlier flowering genotype under long-day conditions, and MiFT1 had the strongest effect in promoting flowering. Additionally, overexpression of three MiFTs in Arabidopsis upregulated the expression levels of several flowering-related genes. Our results suggest that the three MiFTs have positive roles in promoting flowering and suggest that MiFT1 may acts as a key regulator in the flowering pathway.

Third-Order Nonlinear Optical Properties of InN Thin Films Under 532 nm Nanosecond Laser Pulses.

In order to investigate the third-order nonlinear optical properties of InN thin film, the sample was deposited on sapphire substrates by reactive RF magnetron sputtering. The prepared samples with a hexagonal wurtzite structure were confirmed by both X-ray diffraction (XRD) and scanning electron microscope (SEM). The optical absorption spectrum of the prepared samples was measured by a double beam UV/Visible spectrophotometer. The results show that the optical bandgap of deposited thin film is 2.06 eV. The third-order nonlinear optical coefficients of the film were measured by using the open and closed aperture transmission Z-scan (TZ-scan) technique under nanosecond laser pulses with a wavelength of 532 nm. The test results show that the prepared InN thin film performs strong saturation absorption, and the InN thin film with positive nonlinear refractive index coefficient is the self-focusing material under the conditions of the nanosecond laser pulses with the photon energy larger than the bandgap of prepared samples.

Involvement of IRS-1 interaction with ADAM10 in the regulation of neurite extension.

The insulin-like growth factor-1 (IGF-1) signaling pathway plays an important role in neuronal cell differentiation. Recent studies have shown that IGF-1 has the capacity to counteract the retraction of neuronal processes in response to inflammatory cytokines such as TNF-α, which is a known factor for neuronal injury in the central nervous system. This event is thought to be mediated via interference of TNF-α-induced interaction of ß1-integrin with insulin receptor substrate-1 (IRS-1). Here, we demonstrate the interaction of IRS-1 with disintegrin and metalloproteinase ADAM10 through the N-terminal domain of IRS-1 and that this is involved in the regulation of neurite extension and retraction by IGF-1 and TNF-α, respectively. PC12 cells expressing the N-terminal domain show enhanced neurite extension after IGF-1 treatment and reduced neurite depletion relative to control cells after TNF-α treatment. The level of ADAM10 was found to be increased in immunohistochemical studies of HIV encephalitis clinical samples and is present with TNF-α and TNFR1 in both astrocytes and neurons. Altogether, these observations suggest a role for ADAM10 in the mechanism for IGF1/IRS-1 signaling pathway in sustaining the stability of neuronal processes.

Insulin-like growth factor-I-forkhead box O transcription factor 3a counteracts high glucose/tumor necrosis factor-α-mediated neuronal damage: implications for human immunodeficiency virus encephalitis.

In HIV patients, antiretroviral medications trigger metabolic abnormalities, including insulin resistance. In addition, the inflammatory cytokine tumor necrosis factor-α (TNFα), which is elevated in human immunodeficiency virus encephalitis (HIVE), also induces insulin resistance and inflicts neuronal damage in vitro. In differentiated PC12 cells and rat cortical neurons, high glucose (HG; 25 mM) triggers reactive oxygen species (ROS) accumulation, contributing to the retraction of neuronal processes, with only a minimal involvement of neuronal apoptosis. In the presence of TNFα, HG-treated neurons undergo massive apoptosis. Because mammalian homolog of the Forkhead family of transcription factors, Forkhead box O transcription factor 3a (FOXO3a), controls ROS metabolism, we asked whether FOXO3a could affect the fate of differentiated neurons in the paradigm of HIVE. We observed FOXO3a nuclear translocation in HG-treated neuronal cultures, accompanied by partial loss of mitochondrial potential and gradual retraction of neuronal processes. Addition of TNFα to HG-treated neurons increased expression of the FOXO-dependent proapoptotic gene Bim, which resulted in extensive apoptotic death. Insulin-like growth factor-I (IGF-I) significantly lowered intracellular ROS, which was accompanied by IGF-I-mediated FOXO3a nuclear export and decrease in its transcriptional activity. The clinical relevance of these findings is supported by detection of nuclear FOXO3a in TUNEL-positive cortical neurons from HIVE, especially in brain areas characterized by elevated TNFα.

Inhibition of p66ShcA longevity gene rescues podocytes from HIV-1-induced oxidative stress and apoptosis.

Glomerular visceral epithelial cells (podocytes) play a critical role in the pathogenesis of human immunodeficiency virus (HIV)-associated nephropathy. A key question concerns the mechanism(s) by which the HIV-1 genome alters the phenotype of the highly specialized, terminally differentiated podocytes. Here, using an in vitro system of conditionally immortalized differentiated human podocytes (CIDHPs), we document a pivotal role for the p66ShcA protein in HIV-1-induced reactive oxygen species generation and CIDHP apoptosis. CIDHP transfected with truncated HIV-1 construct (NL4-3) exhibit increased reactive oxygen species metabolism, DNA strand breaks, and a 5-fold increase in apoptosis, whereas the opposite was true for NL4-3/CIDHP co-transfected with mu-36p66ShcA (micro-36) dominant negative expression vector or isoform-specific p66-small interfering RNA. Phosphorylation at Ser-36 of the wild type p66ShcA protein, required for p66ShcA redox function and inhibition of the potent stress response regulator Foxo3a, was unchanged in micro-36/NL4-3/CIDHP but increased in NL4-3/CIDHP. Acute knockdown of Foxo3a by small interfering RNA induced a 50% increase in micro-36/NL4-3/CIDHP apoptosis, indicating that Foxo3a-dependent responses promote the survival phenotype in micro-36 cells. We conclude that inhibition of p66ShcA redox activity prevents generation of HIV-1 stress signals and activation of the CIDHP apoptosis program.

IGF-IR in neuroprotection and brain tumors.

The IGF-IR is a multifunctional tyrosine kinase receptor involved in several biological processes including cell proliferation, differentiation, DNA repair, and cell survival. In the brain IGF-I plays a critical role during embryonic and early postnatal development. In the mature brain, IGF-I binding sites have been found in different regions of the brain, and multiple reports confirmed a strong neuroprotective action of the IGF-IR against different pro-apoptotic insults. When the IGF-IR signaling system is insufficiently deployed, either by low level of expression in elderly individuals, or by the inhibition associated with inflammatory cytokines, neuronal function and survival could be compromised. The examples of such CNS pathologies include HIV associated dementia, diabetic neuropathies, and Alzheimer's disease. On the other hand, elevated expression activity of the IGF-IR may support uncontrolled cell proliferation and protection from apoptosis. Probably the best example of the IGF-IR involvement in brain tumors is medulloblastomas in which functional cooperation between viral oncoprotein, JC virus large T-antigen, and IGF-IR has been recently established. Therefore, better understanding of the beneficial and potentially harmful aspects of the IGF-IR can be critical for the development of new clinical regimens against neurodegenerative disorders and brain tumors.

Interaction between serine phosphorylated IRS-1 and beta1-integrin affects the stability of neuronal processes.

Tumor necrosis factor-alpha (TNFalpha) released in the brain by HIV-activated macrophages/microglia is suspected to compromise neuronal survival. Previously, we have demonstrated that activated receptor for insulin-like growth factor I (IGF-IR) protects neurons from TNFalpha-induced neuronal damage (Wang et al. [ 2006] J. Neurosci. Res. 83:7-18). Because TNFalpha triggers phosphorylation of insulin receptor substrate 1 (IRS-1) on serine residues (pS-IRS-1; Rui et al. [ 2001] J. Clin. Invest. 107:181-189), and pS-IRS-1 binds integrins (Reiss et al. [ 2001] Oncogene 20:490-500), we asked how these events affect neuronal processes. We show that beta1-integrin and pS-IRS-1 colocalize in PC12 cells and in primary cortical neurons. TNFalpha treatment elevated membrane-associated pS-IRS-1, enhanced pS-IRS-1 interaction with beta1-integrin, and attenuated cell attachment to collagen IV. In contrast, IGF-I inhibited pS-IRS-1-beta1-integrin complexes and improved cell attachment. The domain of IRS-1 involved in beta1-integrin binding mapped between amino acids 426 and 740, and the expression of 426-740/IRS-1 mutant attenuated neuronal outgrowth. Our results indicate that TNFalpha facilitates the interaction of pS-IRS-1 and beta1-integrin and destabilizes neuronal processes. IGF-I counteracts TNFalpha-mediated accumulation of pS-IRS-1-beta1-integrin complexes supporting the stability of neuronal processes.

IRS-1-Rad51 nuclear interaction sensitizes JCV T-antigen positive medulloblastoma cells to genotoxic treatment.

The large T-antigen from human polyomavirus JC (JCV T-antigen) is suspected to play a role in malignant transformation. Previously, we reported that JCV T-antigen requires the presence of a functional insulin-like growth factor I receptor (IGF-IR) for transformation of fibroblasts and for survival of medulloblastoma cell lines; that IGF-IR is phosphorylated in medulloblastoma biopsies and that JCV T-antigen inhibits homologous recombination-directed DNA repair, causing accumulation of mutations. Here we are evaluating whether JCV T-antigen positive and negative mouse medulloblastoma cell lines, which significantly differ in their tumorigenic properties, are also different in their abilities to repair double strand breaks of DNA (DSBs). Our results show that despite much stronger tumorigenic potential, JCV T-antigen positive medulloblastoma cells are more sensitive to genotoxic agents (cisplatin and gamma-irradiation). Subsequent analysis of DNA repair of DSBs indicated that homologous recombination-directed DNA repair (HRR) was selectively attenuated in JCV T-antigen positive medulloblastoma cells. JCV T-antigen did not affect HRR directly. In the presence of JCV T-antigen, insulin receptor substrate 1 (IRS-1) translocated to the nucleus where it co-localized with Rad51, possibly attenuating HRR.

T-antigen of the human polyomavirus JC attenuates faithful DNA repair by forcing nuclear interaction between IRS-1 and Rad51.

JC polyomavirus (JCV), which infects 90% of the human population, is detectable in human tumors. Its early protein, JCV T-antigen, transforms cells in vitro and is tumorigenic in experimental animals. Although T-antigen-mediated transformation involves genetic alterations of the affected cells, the mechanism underlying this genomic instability is not known. We show that JCV T-antigen inhibits homologous recombination DNA repair (HRR), which results in an accumulation of mutations. T-antigen does not operate directly but utilizes a cytosolic molecule, insulin receptor substrate 1 (IRS-1). Following T-antigen-mediated nuclear translocation, IRS-1 binds Rad51 at the site of damaged DNA. This T-antigen-mediated inhibition of HRR does not function in cells lacking IRS-1, and can be reproduced in the absence of T-antigen by IRS-1 with artificial nuclear localization signal. Our observations define a new mechanism by which viral protein utilizes cytosolic molecule to inhibit faithful DNA repair, and suggest how polyomaviruses could compromise stability of the genome. (c) 2005 Wiley-Liss, Inc.

Involvement of alpha1beta1 integrin in insulin-like growth factor-1-mediated protection of PC12 neuronal processes from tumor necrosis factor-alpha-induced injury.

Insulin-like growth factor 1 receptor (IGF-1R) supports neuronal survival against a wide variety of insults. This includes tumor necrosis factor-alpha (TNFalpha)-mediated neuronal damage, which represents one of the factors suspected to play a role in HIV-associated dementia (HAD). PC12 neurons engineered to express human IGF-1R (PC12/IGF-1R) maintain neuronal processes on collagen IV for several weeks. However, prolonged treatment with TNFalpha caused degeneration of neuronal processes, with no apparent signs of apoptosis. In this process, TNFalpha did not affect IGF-1-mediated phosphorylation of IRS-1, IRS-2, Akt, or Erks. In addition, PC12/IGF-1R cells were found to express predominantly alpha1beta1 integrin, which has high affinity to collagen IV. The treatment of PC12/IGF-1R neurons with a specific alpha1beta1 integrin inhibitor, obtustatin, also caused loss of neuronal processes, accompanied by a quick cell detachment and extensive apoptosis. In the presence of IGF-1, both TNFalpha-induced and obtustatin-induced degeneration of neuronal processes were effectively inhibited. Furthermore, TNFalpha-mediated neuronal degeneration correlated with decreased attachment of PC12/IGF-1R cells to collagen IV and with a reduced level of alpha1beta1 integrin, consistent with a role for this surface protein in the maintenance of neuronal processes. Thus the neuroprotective effects of IGF-1 are not restricted to its antiapoptotic properties but also involve an additional neuroprotective mechanism, by which IGF-1 counteracts the negative effect of TNFalpha on alpha1beta1 integrin-mediated attachment to collagen IV.

Impaired homologous recombination DNA repair and enhanced sensitivity to DNA damage in prostate cancer cells exposed to anchorage-independence.

During metastases, cancer cells are temporarily exposed to the condition in which interactions with extracellular environment can be restricted (anchorage-independence). We demonstrate that the sensitivity of prostate cancer cell lines, DU145 and PC-3, to genotoxic treatment (cisplatin and gamma-irradiation) increased several folds when cells were forced to grow in anchorage-independence. This enhanced drug sensitivity was associated with a severe impairment of homologous recombination-directed DNA repair (HRR). The mechanism involves Rad51, which is the major enzymatic component of HRR. The protein level of Rad51 and its recruitment to DNA double-strand breaks (DSBs) were both attenuated. Rad51 deficiency in anchorage-independence was not associated with Rad51 promoter activity, and was not compensated by a constitutive overexpression of Rad51 cDNA. Instead, Rad51 protein level and its ability to colocalize with DSBs were restored in the presence of proteosome inhibitors, or when cells from the suspension cultures were allowed reattachment. Presented results indicate that anchorage-independence sensitizes prostate cancer cells to genotoxic agents; however, it also attenuates faithful component of DNA repair by targeting stability of Rad51. This temporal attenuation of HRR may contribute to the accumulation mutations after DNA damage, and possibly the selection of new adaptations in cells, which survived genotoxic treatment.

HIV-Tat promotes cellular proliferation and inhibits NGF-induced differentiation through mechanisms involving Id1 regulation.

Id1 is a helix-loop-helix transcriptional factor that controls growth and survival of neuronal cells. Downregulation of Id1 expression is required to initiate differentiation and cell-cycle withdrawal in primary neuronal culture as well as in PC12 cells. The HIV-1 transactivating factor, Tat, has been suspected of causing neuronal dysfunction that often leads to the development of HIV-associated dementia in AIDS patients. We found that the expression of Tat in PC12 cells promotes serum-independent growth, formation of large colonies in soft agar, and the acceleration of tumor growth in nude mice. In addition, Tat showed the ability to inhibit the nerve growth factor (NGF)-induced neuronal differentiation of PC12 cells. Our results show that the Tat-mediated signaling events, which lead to serum-independent growth and the inhibition of NGF-induced differentiation, have a common cellular target: the upregulation of Id1 expression. In the absence of NGF, expression of Id1 is required to promote serum-independent proliferation of PC12/Tat cells, as the inhibition of Id1 by antisense DNA restored the serum-dependent growth of PC12/Tat cells. In the presence of NGF, Tat utilizes an additional pathway that involves phosphorylation of Stat5a, to upregulate Id1 expression and block neuronal cell differentiation. Suppression of Stat5a by use of its dominant-negative mutant reversed the transient expression of Id1 and the blockage of NGF-mediated differentiation in PC12/Tat cells. Finally, the treatment of PC12 cells with recombinant Tat also enhanced the NGF-induced Id1 expression, further pointing to Id1 as a target for Tat. Taken together, these studies suggest additional targets for Tat action in neuronal cells and provide new insights into the mechanisms involved in the dysregulation of neuronal functions.

Role of the insulin-like growth factor I/insulin receptor substrate 1 axis in Rad51 trafficking and DNA repair by homologous recombination.

The receptor for insulin-like growth factor I (IGF-IR) controls normal and pathological growth of cells. DNA repair pathways represent an unexplored target through which the IGF-IR signaling system might support pathological growth leading to cellular transformation. However, this study demonstrates that IGF-I stimulation supports homologous recombination-directed DNA repair (HRR). This effect involves an interaction between Rad51 and the major IGF-IR signaling molecule, insulin receptor substrate 1 (IRS-1). The binding occurs within the cytoplasm, engages the N-terminal domain of IRS-1, and is attenuated by IGF-I-mediated IRS-1 tyrosine phosphorylation. In the absence of IGF-I stimulation, or if mutated IGF-IR fails to phosphorylate IRS-1, localization of Rad51 to the sites of damaged DNA is diminished. These results point to a direct role of IRS-1 in HRR and suggest a novel role for the IGF-IR/IRS-1 axis in supporting the stability of the genome.

T-antigen of human polyomavirus JC cooperates withIGF-IR signaling system in cerebellar tumors of the childhood-medulloblastomas.

Polyomaviruses are implicated in a number of cancers, and the transforming activity of their early protein, large T-antigen, has been documented in a variety of cell types and in experimental animals (1). Although the pathways by which T-antigen induces uncontrolled cell growth are not fully defined, T-antigen mediated inactivation of tumor suppressors, p53 and pRB, is well-documented in some malignancies (2). Here we postulate that functional interaction between the insulin-like growth factor (IGF-IR) and the T-antigen of human polyomavirus JC (JCV T-antigen) may contribute to the process of malignant transformation in medulloblastomas: (i) the IGF-IR signaling system is strongly activated in medulloblastoma cell lines and medulloblastoma biopsies; (ii) the cytoplasmic protein, insulin receptor substrate 1 (IRS-1), is translocated to the nucleus in the presence of JCV T-antigen; (iii) molecular characterization of the interaction between IRS-1 and JCV T-antigen indicates that the binding involves the N-terminal portion of IRS-1 (PH/PTB domain) and the C-terminal region of JCV T-antigen (aa 411-628); and finally (iv) competition for the IRS-1-JCV T-antigen binding attenuates anchorage-independent growth of T-antigen positive medulloblastoma cells in culture. Based on these findings, we propose a novel role for IRS-1 in JCV T-antigen-mediated deregulation of cellular equilibrium, which may involve uncoupling of IRS-1 from the surface receptor and translocation of its function to the nuclear compartment of the cell.

Insulin-like growth factor I receptor signaling system in JC virus T antigen-induced primitive neuroectodermal tumors--medulloblastomas.

Medulloblastomas represent about 25% of all pediatric intracranial neoplasms. These highly malignant tumors arise from the cerebellum, affecting mainly children between ages 5 and 15. Although the etiology of medulloblastomas has not yet been elucidated, several reports suggest that both the cellular protein insulin-like growth factor I (IGF-I) and the early protein of the human polyomavirus JC (JCV T antigen) may contribute to the development of these tumors. The results of this study show a potential functional cooperation between these two proteins in the process of malignant transformation. Both medulloblastoma cell lines and medulloblastoma biopsies are characterized by the abundant presence of the IGF-I receptor (IGF-IR) and its major signaling molecule, insulin receptor substrate 1 (IRS-1). Importantly, IRS-1 is translocated to the nucleus in the presence of the JCV T antigen. Nuclear IRS-1 was detected in T antigen-positive cell lines and in T antigen-positive biopsies from patients diagnosed with medulloblastoma. The IRS-1 domain responsible for a direct JCV T antigen binding was localized within the N-terminal portion of IRS-1 molecule and the competition for IRS-1 T antigen binding by a dominant-negative mutant of IRS-1 inhibited growth and survival of JCV T antigen-transformed cells in anchorage-independent culture condition.

Insulin receptor substrate 1 translocation to the nucleus by the human JC virus T-antigen.

Insulin receptor substrate 1 (IRS-1) is the major signaling molecule for the insulin and insulin-like growth factor I receptors, which transduces both metabolic and growth-promoting signals, and has transforming properties when overexpressed in the cells. Here we show that IRS-1 is translocated to the nucleus in the presence of the early viral protein-T-antigen of the human polyomavirus JC. Nuclear IRS-1 was detected in T-antigen-positive cell lines and in T-antigen-positive biopsies from patients diagnosed with medulloblastoma. The IRS-1 domain responsible for a direct JC virus T-antigen binding was localized within the N-terminal portion of IRS-1 molecule, and the binding was independent from IRS-1 tyrosine phosphorylation and was strongly inhibited by IRS-1 serine phosphorylation. In addition, competition for the IRS-1-T-antigen binding by a dominant negative mutant of IRS-1 inhibited growth and survival of JC virus T-antigen-transformed cells in anchorage-independent culture conditions. Based on these findings, we propose a novel role for the IRS-1-T-antigen complex in controlling cellular equilibrium during viral infection. It may involve uncoupling of IRS-1 from its surface receptor and translocation of its function to the nucleus.