Mark Baguma-Nibasheka

Basic Fibroblast Growth Factor (FGF-2) Overexpression Is a Risk Factor for Esophageal Cancer Recurrence and Reduced Survival, which Is Ameliorated by Coexpression of the FGF-2 Antisense Gene

Purpose: The basic fibroblast growth factor (FGF-2) gene is bidirectionally transcribed to generate overlapping sense and antisense (FGF-AS) mRNAs. FGF-AS has been implicated in the post-transcriptional regulation of FGF-2 expression. The aim of this study was to characterize FGF-2 and FGF-AS in esophageal cancer and to correlate their expression with clinicopathologic findings and outcome. Experimental Design: Reverse transcription-PCR was used to study FGF-2 and FGF-AS mRNA expression (normalized to glyceraldehyde-3-phosphate dehydrogenase) in 48 esophageal cancers relative to matched histologically normal esophageal epithelia (internal control). We used Cox proportional hazards analysis to calculate hazard ratios for recurrence and survival of patients with underexpression relative to the overexpression of FGF-2 and/or FGF-AS. Results: Overexpression of FGF-2 mRNA, by comparison with tumors underexpressing FGF-2, was associated with significantly increased risk for tumor recurrence (hazard ratio, 3.80; 95% confidence interval, 1.64-8.76) and reduced overall survival (hazard ratio, 2.11; 95% confidence interval, 1.0-4.58). When the effects of FGF-2 and FGF-AS were considered simultaneously, the association of FGF-2 mRNA overexpression with recurrence and mortality was even more pronounced, whereas FGF-AS mRNA overexpression was associated with reduced risk for recurrence and improved survival. Conclusions: Overexpression of FGF-2 mRNA is associated with tumor recurrence and reduced survival after surgical resection of esophageal cancer and that these risks are reduced in tumors coexpressing the FGF-AS mRNA. These data support the hypothesis that FGF-AS is a novel tumor suppressor that modulates the effect of FGF-2 expression and may have potential clinical application to the development of novel therapeutic strategies.

Pulmonary Hypoplasia in the Connective Tissue Growth Factor (Ctgf) Null Mouse

Connective tissue growth factor (CTGF) is a mediator of growth factor activity, and Ctgf knockouts die at birth from respiratory failure due to skeletal dysplasia. Previous microarray analysis revealed Ctgf down‐regulation in the hypoplastic lungs of amyogenic mouse embryos. This study, therefore, examined pulmonary development in Ctgf−/− mouse fetuses to investigate if respiration could also have been impaired by lung abnormalities. The Ctgf−/− lungs were hypoplastic, with reduced cell proliferation and increased apoptosis. PDGF‐B, its receptor and IGF‐I, were markedly attenuated and the TTF‐1 gradient lost. Type II pneumocyte differentiation was perturbed, the cells depicting excessive glycogen retention and diminished lamellar body and nuclear size, though able to synthesize surfactant‐associated protein. However, type I pneumocyte differentiation was not affected by Ctgf deletion. Our findings indicate that the absence of Ctgf and/or its protein product, CTGF, may induce pulmonary hypoplasia by both disrupting basic lung developmental processes and restricting thoracic expansion. Developmental Dynamics 237:485–493, 2008.

The fibroblast growth factor-2 antisense gene inhibits nuclear accumulation of FGF-2 and delays cell cycle progression in C6 glioma cells

Fibroblast growth factor-2 (FGF-2) is a potent heparin-binding protein with growth-promoting and anti-apoptotic activity. Transcription of the GFG/NUDT6 gene on the opposite DNA strand generates an overlapping antisense RNA (FGF-AS) implicated in the post-transcriptional regulation of FGF-2. C6 glioma cells coordinately express FGF-2 and FGF-AS mRNA in a cell cycle-dependent manner. Cellular FGF-2 immunoreactivity was also cell cycle-dependent, with marked nuclear accumulation during S-phase. Stable transfection and overexpression of the FGF-AS RNA resulted in suppression of total cellular FGF-2, and a reduction in nuclear accumulation of FGF-2 isoforms. Serum stimulation of growth-arrested wild-type cells evoked a rapid nuclear translocation of FGF-2, and cell cycle re-entry. FGF-AS transfectants, in contrast, showed a significant delay in recovery of both nuclear FGF-2 staining and S-phase re-entry. Similar results were observed when cells were released from aphidicolin-induced G1 arrest or subjected to heat shock. These findings indicate that FGF-AS RNA inhibits expression and cell cycle-dependent nuclear accumulation of FGF-2, and this is associated with a marked delay in S-phase progression. The results suggest that the endogenous FGF antisense RNA may play a significant functional role in the regulation of FGF-2 dependent cell proliferation in FGF-2 expressing cells.

Alternative splicing and differential subcellular localization of the rat FGF antisense gene product

BackgroundGFG/NUDT is a nudix hydrolase originally identified as the product of the fibroblast growth factor-2 antisense (FGF-AS) gene. While the FGF-AS RNA has been implicated as an antisense regulator of FGF-2 expression, the expression and function of the encoded GFG protein is largely unknown. Alternative splicing of the primary FGF-AS mRNA transcript predicts multiple GFG isoforms in many species including rat. In the present study we focused on elucidating the expression and subcellular distribution of alternatively spliced rat GFG isoforms.ResultsRT-PCR and immunohistochemistry revealed tissue-specific GFG mRNA isoform expression and subcellular distribution of GFG immunoreactivity in cytoplasm and nuclei of a wide range of normal rat tissues. FGF-2 and GFG immunoreactivity were co-localized in some, but not all, tissues examined. Computational analysis identified a mitochondrial targeting sequence (MTS) in the N-terminus of three previously described rGFG isoforms. Confocal laser scanning microscopy and subcellular fractionation analysis revealed that all rGFG isoforms bearing the MTS were specifically targeted to mitochondria whereas isoforms and deletion mutants lacking the MTS were localized in the cytoplasm and nucleus. Mutation and deletion analysis confirmed that the predicted MTS was necessary and sufficient for mitochondrial compartmentalization.ConclusionPrevious findings strongly support a role for the FGF antisense RNA as a regulator of FGF2 expression. The present study demonstrates that the antisense RNA itself is translated, and that protein isoforms resulting form alternative RNA splicing are sorted to different subcellular compartments. FGF-2 and its antisense protein are co-expressed in many tissues and in some cases in the same cells. The strong conservation of sequence and genomic organization across animal species suggests important functional significance to the physical association of these transcript pairs.

Selective cyclooxygenase-2 inhibition suppresses basic fibroblast growth factor expression in human esophageal adenocarcinoma

Inhibition of cyclooxygenase (COX)‐2 is reported to suppress growth and induce apoptosis in human esophageal adenocarcinoma (EADC) cells, although the precise biologic mechanism is unclear. In this study we tested the hypothesis that the antitumor activity of COX‐2 inhibitors may involve modulation of basic fibroblast growth factor (FGF‐2), which is overexpressed in EADC. We evaluated the effects of NS‐398, a selective COX‐2 inhibitor, on FGF‐2 expression and proliferation of EADC cell lines that express COX‐2 and those that do not. We also correlated COX‐2 and FGF‐2 expression with clinico‐pathologic findings and outcome in a well‐characterized series of surgically resected EADC tissues. Seg‐1 cells robustly expressed COX‐2 and FGF‐2, whereas Bic‐1 cells expressed neither transcript. FGF‐2 was reduced to undetectable levels in Seg‐1 cells following NS‐398 treatment, but increased within 4 h of drug removal. NS‐398 significantly inhibited the growth of Seg‐1 cells, and this effect was ameliorated by addition of exogenous FGF‐2. In contrast, NS‐398 had no effect on Bic‐1 cell proliferation and FGF‐2 alone had no effect on proliferation of either cell line. NS‐398, or a neutralizing anti‐FGF‐2 antibody, induced apoptosis in Seg‐1 cells, and these effects were inhibited by addition of exogenous FGF‐2. COX‐2 protein was strongly expressed in 46% (10/22) of EADCs, and was associated with a trend towards reduced disease‐free survival. These findings indicate that the antitumor effects of COX‐2 inhibition in EADC cells may be mediated via suppression of FGF‐2, and that COX‐2 may be a clinically relevant molecular marker in the management of human EADC.

Abnormal Retinal Development in the Btrc Null Mouse

Previous microarray analysis revealed beta‐transducin repeat containing (Btrc) down‐regulation in the retina of mouse embryos specifically lacking cholinergic amacrine cells (CACs) as a result of the absence of skeletal musculature and fetal ocular movements. To investigate the role of Btrc in the determination of retinal cell fate, the present study examined retinal morphology in Btrc−/− mouse fetuses. The Btrc−/− retina showed a normal number of cell layers and number of cells per layer with normal cell proliferation and apoptosis. However, there was a complete absence of CACs and a decrease in tyrosine hydroxylase‐expressing amacrine cells. The population of other amacrine cell subtypes was normal, whereas that of the precursor cells was decreased. There was also a reduction in the number of retinal ganglion cells, whereas their progenitors were increased. These findings suggest a role for Btrc in regulating the eventual ratio of resulting differentiated retinal cell types. Developmental Dynamics 238:2680–2687, 2009.

Atrial natriuretic peptide inhibits cell cycle activity of embryonic cardiac progenitor cells via its NPRA receptor signaling axis

The biological effects of atrial natriuretic peptide (ANP) are mediated by natriuretic peptide receptors (NPRs), which can either activate guanylyl cyclase (NPRA and NPRB) or inhibit adenylyl cyclase (NPRC) to modulate intracellular cGMP or cAMP, respectively. During cardiac development, ANP serves as an early maker of differentiating atrial and ventricular chamber myocardium. As development proceeds, expression of ANP persists in the atria but declines in the ventricles. Currently, it is not known whether ANP is secreted or the ANP-NPR signaling system plays any active role in the developing ventricles. Thus the primary aims of this study were to 1) examine biological activity of ANP signaling systems in embryonic ventricular myocardium, and 2) determine whether ANP signaling modulates proliferation/differentiation of undifferentiated cardiac progenitor cells (CPCs) and/or cardiomyocytes. Here, we provide evidence that ANP synthesized in embryonic day (E)11.5 ventricular myocytes is actively secreted and processed to its biologically active form. Notably, NPRA and NPRC were detected in E11.5 ventricles and exogenous ANP stimulated production of cGMP in ventricular cell cultures. Furthermore, we showed that exogenous ANP significantly decreased cell number and DNA synthesis of CPCs but not cardiomyocytes and this effect could be reversed by pretreatment with the NPRA receptor-specific inhibitor A71915. ANP treatment also led to a robust increase in nuclear p27 levels in CPCs compared with cardiomyocytes. Collectively, these data provide evidence that in the developing mammalian ventricles ANP plays a local paracrine role in regulating the balance between CPC proliferation and differentiation via NPRA/cGMP-mediated signaling pathways.

Effects of β-adrenergic receptor drugs on embryonic ventricular cell proliferation and differentiation and their impact on donor cell transplantation

β-Adrenergic receptors (β-ARs) and catecholamines are present in rodents as early as embryonic day (E)10.5. However, it is not known whether β-AR signaling plays any role in the proliferation and differentiation of ventricular cells in the embryonic heart. Here, we characterized expression profiles of β-AR subtypes and established dose-response curves for the nonselective β-AR agonist isoproterenol (ISO) in the developing mouse ventricular cells. Furthermore, we investigated the effects of ISO on cell cycle activity and differentiation of cultured E11.5 ventricular cells. ISO treatment significantly reduced tritiated thymidine incorporation and cell proliferation rates in both cardiac progenitor cell and cardiomyocyte populations. The ISO-mediated effects on DNA synthesis could be abolished by cotreatment of E11.5 cultures with either metoprolol (a β1-AR antagonist) or ICI-118,551 (a β2-AR antagonist). In contrast, ISO-mediated effects on cell proliferation could be abolished only by metoprolol. Furthermore, ISO treatment significantly increased the percentage of differentiated cardiomyocytes compared with that in control cultures. Additional experiments revealed that β-AR stimulation leads to downregulation of Erk and Akt phosphorylation followed by significant decreases in cyclin D1 and cyclin-dependent kinase 4 levels in E11.5 ventricular cells. Consistent with in vitro results, we found that chronic stimulation of recipient mice with ISO after intracardiac cell transplantation significantly decreased graft size, whereas metoprolol protected grafts from the inhibitory effects of systemic catecholamines. Collectively, these results underscore the effects of β-AR signaling in cardiac development as well as graft expansion after cell transplantation.NEW & NOTEWORTHY β-Adrenergic receptor (β-AR) stimulation can decrease the proliferation of embryonic ventricular cells in vitro and reduce the graft size after intracardiac cell transplantation. In contrast, β1-AR antagonists can abrogate the antiproliferative effects mediated by β-AR stimulation and increase graft size. These results highlight potential interactions between adrenergic drugs and cell transplantation.

Regulation of fibroblast growth factor-2 expression and cell cycle progression by an endogenous antisense RNA.

Basic fibroblast growth factor (FGF2) is a potent wide-spectrum mitogen whose overexpression is associated with immortalization and unregulated cell proliferation in many tumors. The FGF2 gene locus is bi-directionally transcribed to produce FGF2 mRNA from the “sense” strand and a cis-antisense RNA (NUDT6) from the NUDT6 gene on the “antisense” strand. The NUDT6 gene encodes a nudix motif protein of unknown function, while its mRNA has been implicated in the post-transcriptional regulation of FGF2 expression. FGF2 and NUDT6 are co-expressed in rat C6 glioma cells, and ectopic overexpression of NUDT6 suppresses cellular FGF2 accumulation and cell cycle progression. However, the role of the endogenous antisense RNA in regulation of FGF2 is unclear. In the present study, we employed siRNA-mediated gene knockdown to examine the role of the endogenous NUDT6 RNA in regulation of FGF2 expression and cell cycle progression. Knockdown of either FGF2 or NUDT6 mRNA was accompanied by a significant (>3 fold) increase in the complementary partner RNA. Similar reciprocal effects were observed at the protein level, indicating that these two transcripts are mutually regulatory. Remarkably, knockdown of either FGF2 or NUDT6 significantly reduced cell proliferation and inhibited S-phase re-entry following serum deprivation, implicating both FGF2 and NUDT6 in the regulation of cell transformation and cell cycle progression.

Altered retinal cell differentiation in the AP-3 delta mutant (Mocha) mouse

Adaptor‐related protein complex 3 delta 1 (Ap3d1) encodes the delta 1 subunit of an adaptor protein regulating intracellular vesicle‐mediated transport, and the Ap3d‐deletion mutant (Mocha) mouse undergoes rapid photoreceptor degeneration leading to blindness soon after birth. Previous microarray analysis revealed Ap3d down‐regulation in the retina of mouse embryos specifically lacking cholinergic amacrine cells as a result of the absence of skeletal musculature. To investigate the role of Ap3d in the determination of retinal cell fate, the present study examined retinal morphology in newborn Ap3d−/− mice. The Ap3d−/− retina showed a complete absence of cholinergic amacrine cells and a decrease in parvalbumin‐expressing amacrine cells and syntaxin‐ and VC1.1‐expressing amacrine precursor cells, but had a normal number of cell layers and number of cells in each layer with no detectable difference in cell proliferation or apoptosis. These findings indicate that, despite having no apparent effect on the basic spatial organization of the retina at this stage of development, Ap3d could be involved in the regulation of progenitor cell competence and the eventual ratio of resulting differentiated cells. Finding the mouse mutant which phenocopies the eye defect seen in fetuses with no striated muscle was accomplished by the Systematic Subtractive Microarray Analysis Approach (SSMAA), explained in the discussion section.