Analysis of transforming activity of human synovial sarcoma-associated chimeric protein SYT-SSX1 bound to chromatin remodeling factor hBRM/hSNF2α

Human synovial sarcoma has been shown to exclusively harbor the chromosomal translocation t(X;18) that produces the chimeric gene SYT-SSX. However, the role of SYT-SSX in cellular transformation remains unclear. In this study, we have established 3Y1 rat fibroblast cell lines that constitutively express SYT, SSX1, and SYT-SSX1 and found that SYT-SSX1 promoted growth rate in culture, anchorage-independent growth in soft agar, and tumor formation in nude mice. Deletion of the N-terminal 181 amino acids of SYT-SSX1 caused loss of its transforming activity. Furthermore, association of SYT-SSX1 with the chromatin remodeling factor hBRM/hSNF2α, which regulates transcription, was demonstrated in both SYT-SSX1-expressing 3Y1 cells and in the human synovial sarcoma cell line HS-SY-II. The binding region between the two molecules was shown to reside within the N-terminal 181 amino acids stretch (aa 1–181) of SYT-SSX1 and 50 amino acids (aa 156–205) of hBRM/hSNF2α and we found that the overexpression of this binding region of hBRM/hSNF2α significantly suppressed the anchorage-independent growth of SYT-SSX1-expressing 3Y1 cells. To analyze the transcriptional regulation by SYT-SSX1, we established conditional expression system of SYT-SSX1 and examined the gene expression profiles. The down-regulation of potential tumor suppressor DCC was observed among 1,176 genes analyzed by microarray analysis, and semi-quantitative reverse transcription–PCR confirmed this finding. These data clearly demonstrate transforming activity of human oncogene SYT-SSX1 and also involvement of chromatin remodeling factor hBRM/hSNF2α in human cancer.

Neurotrophic factors [activity-dependent neurotrophic factor (ADNF) and basic fibroblast growth factor (bFGF)] interrupt excitotoxic neurodegenerative cascades promoted by a PS1 mutation

Although an excitotoxic mechanism of neuronal injury has been proposed to play a role in chronic neurodegenerative disorders such as Alzheimer’s disease, and neurotrophic factors have been put forward as potential therapeutic agents, direct evidence is lacking. Taking advantage of the fact that mutations in the presenilin-1 (PS1) gene are causally linked to many cases of early-onset inherited Alzheimer’s disease, we generated PS1 mutant knock-in mice and directly tested the excitotoxic and neurotrophic hypotheses of Alzheimer’s disease. Primary hippocampal neurons from PS1 mutant knock-in mice exhibited increased production of amyloid β-peptide 42/43 and increased vulnerability to excitotoxicity, which occurred in a gene dosage-dependent manner. Neurons expressing mutant PS1 exhibited enhanced calcium responses to glutamate and increased oxyradical production and mitochondrial dysfunction. Pretreatment with either basic fibroblast growth factor or activity-dependent neurotrophic factor protected neurons expressing mutant PS1 against excitotoxicity. Both basic fibroblast growth factor and activity-dependent neurotrophic factor stabilized intracellular calcium levels and abrogated the increased oxyradical production and mitochondrial dysfunction otherwise caused by the PS1 mutation. Our data indicate that neurotrophic factors can interrupt excitotoxic neurodegenerative cascades promoted by PS1 mutations.

Angiogenesis promoted by vascular endothelial growth factor: Regulation through α1β1 and α2β1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors—the α1β1 and α2β1 integrins—through induction of mRNAs encoding the α1 and α2 subunits. In contrast, VEGF did not induce increased expression of the α3β1 integrin, which also has been implicated in collagen binding. Integrin α1-blocking and α2-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and α1-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of α1β1 and α2β1 expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of α1-blocking and α2-blocking Abs. In vivo, a combination of α1-blocking and α2-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of α1β1 and α2β1 expression by EC is an important mechanism by which VEGF promotes angiogenesis and that α1β1 and α2β1 antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.

Identification of diverse nerve growth factor-regulated genes by serial analysis of gene expression (SAGE) profiling

Neurotrophic factors such as nerve growth factor (NGF) promote a wide variety of responses in neurons, including differentiation, survival, plasticity, and repair. Such actions often require changes in gene expression. To identify the regulated genes and thereby to more fully understand the NGF mechanism, we carried out serial analysis of gene expression (SAGE) profiling of transcripts derived from rat PC12 cells before and after NGF-promoted neuronal differentiation. Multiple criteria supported the reliability of the profile. Approximately 157,000 SAGE tags were analyzed, representing at least 21,000 unique transcripts. Of these, nearly 800 were regulated by 6-fold or more in response to NGF. Approximately 150 of the regulated transcripts have been matched to named genes, the majority of which were not previously known to be NGF-responsive. Functional categorization of the regulated genes provides insight into the complex, integrated mechanism by which NGF promotes its multiple actions. It is anticipated that as genomic sequence information accrues the data derived here will continue to provide information about neurotrophic factor mechanisms.

Plant growth in elevated CO2 alters mitochondrial number and chloroplast fine structure

With increasing interest in the effects of elevated atmospheric CO2 on plant growth and the global carbon balance, there is a need for greater understanding of how plants respond to variations in atmospheric partial pressure of CO2. Our research shows that elevated CO2 produces significant fine structural changes in major cellular organelles that appear to be an important component of the metabolic responses of plants to this global change. Nine species (representing seven plant families) in several experimental facilities with different CO2-dosing technologies were examined. Growth in elevated CO2 increased numbers of mitochondria per unit cell area by 1.3–2.4 times the number in control plants grown in lower CO2 and produced a statistically significant increase in the amount of chloroplast stroma (nonappressed) thylakoid membranes compared with those in lower CO2 treatments. There was no observable change in size of the mitochondria. However, in contrast to the CO2 effect on mitochondrial number, elevated CO2 promoted a decrease in the rate of mass-based dark respiration. These changes may reflect a major shift in plant metabolism and energy balance that may help to explain enhanced plant productivity in response to elevated atmospheric CO2 concentrations.

From transforming growth factor-β signaling to androgen action: Identification of Smad3 as an androgen receptor coregulator in prostate cancer cells

Although transforming growth factor-β (TGF-β) has been identified to mainly inhibit cell growth, the correlation of elevated TGF-β with increasing serum prostate-specific antigen (PSA) levels in metastatic stages of prostate cancer has also been well documented. The molecular mechanism for these two contrasting effects of TGF-β, however, remains unclear. Here we report that Smad3, a downstream mediator of the TGF-β signaling pathway, functions as a coregulator to enhance androgen receptor (AR)-mediated transactivation. Compared with the wild-type AR, Smad3 acts as a strong coregulator in the presence of 1 nM 5α-dihydrotestosterone, 10 nM 17β-estradiol, or 1 μM hydroxyflutamide for the LNCaP mutant AR (mtAR T877A), found in many prostate tumor patients. We further showed that endogenous PSA expression in LNCaP cells can be induced by 5α-dihydrotestosterone, and the addition of the Smad3 further induces PSA expression. Together, our findings establish Smad3 as an important coregulator for the androgen-signaling pathway and provide a possible explanation for the positive role of TGF-β in androgen-promoted prostate cancer growth.

Insulin-like growth factor II mediates epidermal growth factor-induced mitogenesis in cervical cancer cells.

There is increasing evidence that activation of the insulin-like growth factor I (IGF-I) receptor plays a major role in the control of cellular proliferation of many cell types. We studied the mitogenic effects of IGF-I, IGF-II, and epidermal growth factor (EGF) on growth-arrested HT-3 cells, a human cervical cancer cell line. All three growth factors promoted dose-dependent increases in cell proliferation. In untransformed cells, EGF usually requires stimulation by a "progression" factor such as IGF-I, IGF-II, or insulin (in supraphysiologic concentrations) in order to exert a mitogenic effect. Accordingly, we investigated whether an autocrine pathway involving IGF-I or IGF-II participated in the EGF-induced mitogenesis of HT-3 cells. With the RNase protection assay, IGF-I mRNA was not detected. However, IGF-II mRNA increased in a time-dependent manner following EGF stimulation. The EGF-induced mitogenesis was abrogated in a dose-dependent manner by IGF-binding protein 5 (IGFBP-5), which binds to IGF-II and neutralizes it. An antisense oligonucleotide to IGF-II also inhibited the proliferative response to EGF. In addition, prolonged, but not short-term, stimulation with EGF resulted in autophosphorylation of the IGF-I receptor, and coincubations with both EGF and IGFBP-5 attenuated this effect. These data demonstrate that autocrine secretion of IGF-II in HT-3 cervical cancer cells can participate in EGF-induced mitogenesis and suggest that autocrine signals involving the IGF-I receptor occur "downstream" of competence growth factor receptors such as the EGF receptor.

Myelin-associated neurite growth-inhibitory proteins and suppression of regeneration of immature mammalian spinal cord in culture.

Neurite outgrowth across spinal cord lesions in vitro is rapid in preparations isolated from the neonatal opossum Monodelphis domestica up to the age of 12 days. At this age oligodendrocytes, myelin, and astrocytes develop and regeneration ceases to occur. The role of myelin-associated neurite growth-inhibitory proteins, which increase in concentration at 10-13 days, was investigated in culture by applying the antibody IN-1, which blocks their effects. In the presence of IN-1, 22 out of 39 preparations from animals aged 13-17 days showed clear outgrowth of processes into crushes. When 34 preparations from 13-day-old animals were crushed and cultured without antibody, no axons grew into the lesion. The success rate with IN-1 was comparable to that seen in younger animals but the outgrowth was less profuse. IN-1 was shown by immunocytochemistry to penetrate the spinal cord. Other antibodies which penetrated the 13-day cord failed to promote fiber outgrowth. To distinguish between regeneration by cut neurites and outgrowth by developing uncut neurites, fibers in the ventral fasciculus were prelabeled with carbocyanine dyes and subsequently injured. The presence of labeled fibers in the lesion indicated that IN-1 promoted regeneration. These results show that the development of myelin-associated growth-inhibitory proteins contributes to the loss of regeneration as the mammalian central nervous system matures. The definition of a critical period for regeneration, coupled with the ability to apply trophic as well as inhibitory molecules to the culture, can permit quantitative assessment of molecular interactions that promote spinal cord regeneration.

Insulin-like growth factor I treatment reduces demyelination and up-regulates gene expression of myelin-related proteins in experimental autoimmune encephalomyelitis.

To compare effects of insulin-like growth factor I (IGF-I) and placebo treatment on lesions that resemble those seen during active demyelination in multiple sclerosis, we induced experimental autoimmune encephalomyelitis in Lewis rats with an emulsion containing guinea pig spinal cord and Freund's adjuvant. On day 12-13, pairs of rats with the same degree of weakness were given either IGF-I or placebo intravenously twice daily for 8 days. After 8 days of placebo or IGF-I (200 micrograms/day or 1 mg/day) treatment, the spinal cord lesions were studied by in situ hybridization and with immunocytochemical and morphological methods. IGF-I produced significant reductions in numbers and areas of demyelinating lesions. These lesions contained axons surrounded by regenerating myelin segments instead of demyelinated axons seen in the placebo-treated rats. Relative mRNA levels for myelin basic protein, proteolipid protein (PLP), and 2',3'-cyclic nucleotide 3'-phosphodiesterase in lesions of IGF-I-treated rats were significantly higher than they were in placebo-treated rats. PLP mRNA-containing oligodendroglia also were more numerous and relative PLP mRNA levels per oligodendrocyte were higher in lesions of IGF-I-treated rats. Finally, a significantly higher proportion of proliferating cells were oligodendroglia-like cells in lesions of IGF-I-treated rats. We think that IGF-I effects on oligodendrocytes, myelin protein synthesis, and myelin regeneration reduced lesion severity and promoted clinical recovery in this experimental autoimmune encephalomyelitis model. These IGF-I actions may also benefit patients with multiple sclerosis.