Retinoblastoma Susceptibility Research Articles

Interstitial deletion of 13q associated with polymicrogyria

Interstitial deletion of the long arm of chromosome 13 is a rare condition characterized by multiple clinical findings. We report a male dizygotic twin with an interstitial deletion of 13q and failure to thrive, hypotonia, polymicrogyria, bilateral foci of retinoblastoma, hearing loss, bilateral inguinal hernias, submucous cleft palate, and dysmorphic features including a triangular shaped face, broad forehead, small chin, prominent eyes, downslanting palpebral fissures, and a downturned mouth. Chromosome analysis showed an interstitial deletion of chromosome 13 which was confirmed by fluorescence in situ hybridization analysis to include the Rb locus, but spare the 13q subtelomeric region. The karyotype was 46,XY,del(13)(q14.1q31.2).ish del(13)(RB1-,D13S327+) de novo. Breakpoints were further characterized by SNP-based microarray. Retinoblastoma tumors are a well-known complication of deletion of the retinoblastoma susceptibility gene, located at chromosome 13q14.2. Growth retardation is another common feature that has been described in other patients with a deletion of 13q. Additionally, this patient had brain findings on MRI consistent with bilateral polymicrogyria with predominance of the frontal lobes, as well as prominent infratentorial and supratentorial vasculature. There are a variety of polymicrogyria syndromes that are distinguished by the cortical location of the abnormal folding. Several of the subtypes have known genetic loci associated with them. To our knowledge, this is the only report of polymicrogyria in association with a deletion of chromosome 13.

Molecular mechanisms of cellular senescence and immortalization of human cells

Cellular senescence was originally described as a phenomenon observed in cultured human cells. Accumulating lines of evidence now indicate that the same processes also take place in vivo, suggesting important implications for tumor development. Telomere shortening is the most well-established cause of cellular senescence that can be induced by many other intrinsic and extrinsic factors. The retinoblastoma susceptibility gene product is a convergent target that is downstream of these factors. p53, p38MAPK and cyclin-dependent kinase inhibitors p16INK4a (p16) and p21CIP1 (p21) are key mediators. As most stresses that induce cellular senescence are also known causes of cancer, a common strategy might be applied to the development of cancer chemopreventive agents and anti-ageing drugs.

Crystal Structure of the Retinoblastoma Protein N Domain Provides Insight into Tumor Suppression, Ligand Interaction, and Holoprotein Architecture

The retinoblastoma susceptibility protein, Rb, has a key role in regulating cell-cycle progression via interactions involving the central "pocket" and C-terminal regions. While the N-terminal domain of Rb is dispensable for this function, it is nonetheless strongly conserved and harbors missense mutations found in hereditary retinoblastoma, indicating that disruption of its function is oncogenic. The crystal structure of the Rb N-terminal domain (RbN), reveals a globular entity formed by two rigidly connected cyclin-like folds. The similarity of RbN to the A and B boxes of the Rb pocket domain suggests that Rb evolved through domain duplication. Structural and functional analysis provides insight into oncogenicity of mutations in RbN and identifies a unique phosphorylation-regulated site of protein interaction. Additionally, this analysis suggests a coherent conformation for the Rb holoprotein in which RbN and pocket domains directly interact, and which can be modulated through ligand binding and possibly Rb phosphorylation.

C. elegansorthologs of components of the RB tumor suppressor complex have distinct pro-apoptotic functions

To obtain insight into the role of the retinoblastoma susceptibility gene (Rb; also known as Rb1) in apoptosis, we analyzed Caenorhabditis elegans mutants lacking a functional lin-35 RB gene. We found that the loss of lin-35 function results in a decrease in constitutive germ cell apoptosis. We present evidence that lin-35 promotes germ cell apoptosis by repressing the expression of ced-9, an anti-apoptotic C. elegans gene that is orthologous to the human proto-oncogene BCL2. Furthermore, we show that the genes dpl-1 DP, efl-1 E2F and efl-2 E2F also promote constitutive germ cell apoptosis. However, in contrast to lin-35, dpl-1 (and probably also efl-1 and efl-2) promotes germ cell apoptosis by inducing the expression of the pro-apoptotic genes ced-4 and ced-3, which encode an APAF1-like adaptor protein and a pro-caspase, respectively. Based on these results, we propose that C. elegans orthologs of components of the RB tumor suppressor complex have distinct pro-apoptotic functions in the germ line and that the transcriptional regulation of components of the central apoptosis machinery is a critical determinant of constitutive germ cell apoptosis in C. elegans. Finally, we demonstrate that lin-35, dpl-1 and efl-2, but not efl-1, function either downstream of or in parallel to cep-1 p53 (also known as TP53) and egl-1 BH3-only to cause DNA damage-induced germ cell apoptosis. Our results have implications for the general mechanisms through which RB-like proteins control gene expression, the role of RB-, DP- and E2F-like proteins in apoptosis, and the regulation of apoptosis.

Proteomic profiling of forskolin-induced differentiated BeWo cells: an in-vitro model of cytotrophoblast differentiation

Placental trophoblastic differentiation is characterized by the fusion of monolayer cytotrophoblasts into syncytiotrophoblasts. During this process of differentiation, several morphological and biochemical changes are known to occur, and this model has been employed to investigate the changes that occur at the gene and protein level during differentiation. Using the sensitive technique of proteomics [two-dimensional gel electrophoresis (2DGE)], changes in protein profile were evaluated in the control and forskolin-induced differentiated cells of trophoblastic choriocarcinoma BeWo cell line. Several proteins were differentially expressed in control and differentiated cells. Four major proteins were up-regulated as assessed by silver staining, and were further characterized as c-h-ras p 21 (phosphorylated), retinoblastoma susceptibility protein 1 and integrase interactor protein 1. These proteins are known to play an important role in growth arrest of cells, and thus may play a role in initiating the process of differentiation.

Separation of Anti-Proliferation and Anti-Apoptotic Functions of Retinoblastoma Protein through Targeted Mutations of Its A/B Domain

BackgroundThe human retinoblastoma susceptibility gene encodes a nuclear phosphoprotein RB, which is a negative regulator of cell proliferation. The growth suppression function of RB requires an evolutionarily conserved A/B domain that contains two distinct peptide-binding pockets. At the A/B interface is a binding site for the C-terminal trans-activation domain of E2F. Within the B-domain is a binding site for proteins containing the LxCxE peptide motif.Methodology/Principle FindingsBased on the crystal structure of the A/B domain, we have constructed an RB-K530A/N757F (KN) mutant to disrupt the E2F- and LxCxE-binding pockets. The RB-K530A (K) mutant is sufficient to inactivate the E2F-binding pocket, whereas the RB-N757F (N) mutant is sufficient to inactivate the LxCxE-binding pocket. Each single mutant inhibits cell proliferation, but the RB-KN double mutant is defective in growth suppression. Nevertheless, the RB-KN mutant is capable of reducing etoposide-induced apoptosis.Conclusion/SignificancePrevious studies have established that RB-dependent G1-arrest can confer resistance to DNA damage-induced apoptosis. Results from this study demonstrate that RB can also inhibit apoptosis independent of growth suppression.

RB, the conductor that orchestrates life, death and differentiation

The retinoblastoma susceptibility gene was the first tumor suppressor gene identified in humans and the first tumor suppressor gene knocked out by targeted deletion in mice. RB serves as a transducer between the cell cycle machinery and promoter-specific transcription factors, its most documented activity being the repression of the E2F family of transcription factors, which regulate the expression of genes involved in cell proliferation and survival. Recent investigations of RB function suggest that it works as a fundamental regulator to coordinate pathways of cellular growth and differentiation. In this review, we unravel the novel role of an equally important aspect of RB in downregulating the differentiation inhibitor EID-1 during cellular differentiation by teasing apart the signal, which elicit differentiation and limit cell cycle progression, since the molecular mechanisms relating to RB activation of differentiation is much less understood. We review the various roles for RB in differentiation of neurons, muscle, adipose tissue, and the retina. In addition, we provide an update for the current models of the role of RB in cell cycle to entry and exit, extending the view toward chromatin remodeling and expose the dichotomies in the regulation of RB family members. We conclude with a discussion of a novel RB regulatory network, incorporating the dynamic contribution of EID family proteins.

The first knockout mouse model of retinoblastoma

The retinoblastoma susceptibility gene (RB1) was the first tumor suppressor gene identified in humans and the first tumor suppressor gene knocked out by targeted deletion in mice. Children with a germline mutation in one of their RB1 alleles are likely to experience bilateral multifocal retinoblastoma; however, mice with a similar disruption of RB1 do not develop retinoblastoma. The absence of a knock-out mouse model of retinoblastoma has slowed the progress toward developing new therapies and identifying secondary genetic lesions that occur after disruption of the RB signaling pathway.

A retinoblastoma ortholog controls stalk/spore preference inDictyostelium

We describe rblA, the Dictyostelium ortholog of the retinoblastoma susceptibility gene Rb. In the growth phase, rblA expression is correlated with several factors that lead to 'preference' for the spore pathway. During multicellular development, expression increases 200-fold in differentiating spores. rblA-null strains differentiate stalk cells and spores normally, but in chimeras with wild type, the mutant shows a strong preference for the stalk pathway. rblA-null cells are hypersensitive to the stalk morphogen DIF, suggesting that rblA normally suppresses the DIF response in cells destined for the spore pathway. rblA overexpression during growth leads to G1 arrest, but as growing Dictyostelium are overwhelmingly in G2 phase, rblA does not seem to be important in the normal cell cycle. rblA-null cells show reduced cell size and a premature growth-development transition; the latter appears anomalous but may reflect selection pressures acting on social ameba.

CGA codons multiplex PCR in rapid diagnosis of retinoblastoma

Background: Multiplex polymerase chain reaction allows amplification of multiple target sequences of a genome under identical conditions in a single tube. This one-shot polymerase chain reaction detection is time and cost effective when large or multiple genes, with many target fragments are investigated. This is applicable for retinoblastoma susceptibility gene having 27 exons with recurrent mutations reported at most of the 12 CGA codons. Materials and Methods: Multiplex polymerase chain reaction assay for the amplification of 12 CGA codons, which constitutes about 50 % of retinoblastoma susceptibility gene mutations has been designed. The time and cost (includes only reagent cost) involved in both multiplex and uniplex polymerase chain reaction was also calculated. Results: Twelve CGA codons were multiplexed in 5 instead of 12 uniplex polymerase chain reactions, which took 36 hours and 9.78 US$ whereas multiplex polymerase chain reaction took 15 hours and 6.88 US$. Multiplex polymerase chain reaction method saved 58.3% of time and 29.6% of cost over uniplex polymerase chain reaction. Conclusion: Saving time by more than half and cost by nearly a third would help clinicians and geneticists while counseling retinoblastoma patients.

Screening for RB1 Mutations in Tumor Tissue Using Denaturing High Performance Liquid Chromatography, Multiplex Ligation-Dependent Probe Amplification, and Loss of Heterozygosity Analysis

Retinoblastoma is a malignant retinal neoplasm arising in infancy as a result of inactivating mutations in both alleles of the retinoblastoma susceptibility gene, RB1. Identification of the causative RB1 mutations in a patient assists in the clinical management of the affected patient and risk assessment of family members, principally on the basis of whether there is a germline mutation. In this paper, we describe our experience with molecular analysis of RB1 mutations in tumor and nontumor samples from 18 retinoblastoma patients, using multiplex ligation dependent probe amplification (MLPA) to detect large deletions or duplications, microsatellite analysis to detect loss of heterozygosity (LOH), and denaturing high performance liquid chromatography (D-HPLC) analysis to detect point mutations and small insertions or deletions. We found LOH in 71% of all cases, and 83% of these were due to acquired isodisomy rather than chromosomal deletions. Small mutations identified by D-HPLC accounted for 78% of the non-LOH mutations, and large deletions/duplications detected by MLPA accounted for the remaining 22%. We give the first report of a large, multiexon duplication in RB1 of exons 8 to 18.

Animal models for the study of squamous cell carcinoma of the upper aerodigestive tract: A historical perspective with review of their utility and limitations part B. Transgenic mouse models

Animal models for the study of squamous cell carcinoma of the upper aerodigestive tract: A historical perspective with review of their utility and limitations part B. Transgenic mouse models

Pocket proteins p107 and p130 exhibit increased expression in macrophages during SIV encephalitis

Progression of HIV encephalitis (HIVE) is associated with neuronal damage and loss because of infiltration of infected and/or activated macrophages into the CNS. We have previously observed increased inactivation of the retinoblastoma susceptibility gene product (pRb) by phosphorylation in neurons and glia of HIVE and the simian model of HIVE (SIVE). To determine if other pRb family members are altered in response to increased macrophage-secreted factors, we investigated expression of pRb family members p107 and p130 in SIVE. Both p130 and p107 exhibited increased staining in macrophages, but not neurons, astrocytes or T-cells in SIVE. Increased p130 and p107 immunostaining was not limited to virally infected or PCNA-expressing macrophages. Most p107-positive staining was observed in perivascular macrophages, suggesting p107 may indicate macrophages at a specific stage of differentiation soon after migration. In contrast, cytoplasmic p130 was found in the majority of macrophages present in SIVE cases and may indicate activation as it was not seen in microglia in control CNS. These findings suggest that p107 and p130 are differentially expressed in CNS macrophage populations which may have multiple derivations and/or roles in lentiviral encephalitis.

Achieving pathogenesis understanding of ocular diseases by deciphering the underlying molecular pathways

The field of ophthalmology research has experienced a revolution since the 1970's, when molecular biology techniques were gradually and widely adopted. Many of the developments generated impact that went far beyond the field of ophthalmology. A classic case was the identification and characterization of the Retinoblastoma susceptibility gene (Rb), whose impact went far beyond the rare and obscure disease, as it provides key evidence for the concept of tumor suppressor gene and the "two hit" theory of tumor formation. The identification of scores of genes involved in retinitis pigmentosum (pigmentosa), on the other hand, show cases the complexity of multi-factorial diseases. Ophthalmology researchers in China have been quick in integrating these novel tools into their research. However, the field still lags behind in the effective use of these technologies to carry out in-depth inquiries into key disease mechanisms. The advent of "omics" technologies heralded a new era in biomedical research that allows for the global and rapid survey of genetic and biochemical profiles. Effective integration of these novel technologies into ophthalmology research will have far-reaching impact for the whole field.

The role of the retinoblastoma protein (Rb) in the nuclear localization of BAG-1: implications for colorectal tumour cell survival

Although the retinoblastoma susceptibility gene RB1 is inactivated in a wide variety of human cancers, the retinoblastoma protein (Rb) has been shown to be overexpressed in colon cancers, which is linked to the anti-apoptotic function of the protein. However, the mechanisms by which Rb regulates apoptosis are yet to be fully elucidated. We have established that Rb interacts with the anti-apoptotic BAG-1 (Bcl-2 associated athanogene-1) protein, and that a decrease in nuclear localization of BAG-1 is detectable when the interaction between Rb and BAG-1 is disrupted by expression of the E7 viral oncoprotein. Interestingly, although reported as deregulated in colorectal cancers, we have found that BAG-1 expression is also altered in small adenomas, where its localization was found to be predominantly nuclear. In addition, we have established that maintenance of high nuclear BAG-1 in vitro increases the resistance of adenoma-derived cells to gamma-radiation-induced apoptosis. Our work suggests a novel function for Rb, involving modulation of the subcellular localization of BAG-1. We have found predominant nuclear BAG-1 localization in small adenomas, and suggest that BAG-1 may promote colorectal tumour cell survival by making colonic epithelial cells less sensitive to DNA damage.

Molecular cloning of cDNA for SPase, a monkey cathepsin L orthologue

SPase is a cysteine protease isolated from an African green monkey kidney cell line, CV-1, and has selective cleavage activity toward transcription factor SP-1 and retinoblastoma susceptibility gene product RB. In this study, a cDNA encoding SPase was cloned from a cDNA library prepared from CV-1 cells. The cDNA clone encodes 333 amino acids and is 96.5% identical to human cathepsin L at the nucleotide and amino acid sequence levels. SPase appears to be translated as a preproenzyme based on the comparison between the deduced amino acid sequence and the N-terminal sequence of the purified enzyme. Northern blot analysis exhibited the considerably higher expression of SPase in CV-1 cells compared with COS-1 cells, showing a good correlation with enzymatic activity in these cell lines. Bacterially expressed SPase protein exhibited proteolytic activity toward SP-1 and RB proteins. These observations suggest that SPase is a monkey cathepsin L orthologue.

Activation of CREB/ATF Sites by Polyomavirus Large T Antigen

Polyomavirus large T antigen (LT) has a direct role in viral replication and a profound effect on cell phenotype. It promotes cell cycle progression, immortalizes primary cells, blocks differentiation, and causes apoptosis. While much of large T function is related to its effects on tumor suppressors of the retinoblastoma susceptibility (Rb) gene family, we have previously shown that activation of the cyclin A promoter can occur through a non-Rb-dependent mechanism. Here we show that activation occurs via an ATF/CREB site. Investigation of the mechanism indicates that large T can synergize with CREB family members to activate transcription. Experiments with Gal4-CREB constructs show that synergy is independent of CREB phosphorylation by protein kinase A. Examination of synergy with Gal4-CREB deletion constructs indicates that large T acts on the constitutive activation domain of CREB. Large T can bind to CREB in vivo. Genetic analysis shows that the DNA-binding domain (residues 264 to 420) is sufficient to activate transcription when it is localized to the nucleus. Further analysis of the DNA-binding domain shows that while site-specific DNA binding is not required, non-site-specific DNA binding is important for the activation. Thus, CREB binding and DNA binding are both important for large T activation of CREB/ATF sites. In contrast to previous models where large T transactivation occurred indirectly, these results also suggest that large T can act directly at promoters to activate transcription.

Chemokine- and neurotrophic factor-induced changes in E2F1 localization and phosphorylation of the retinoblastoma susceptibility gene product (pRb) occur by distinct mechanisms in murine cortical cultures

The retinoblastoma susceptibility gene product (pRb) and E2F1 have been found to exhibit altered localization and increased staining in several neurodegenerative diseases. We have observed similar localization in primary murine cortical cultures treated with neurotrophic factors (NTF) or chemokines. In untreated cultures, E2F1 exhibited minimal immunostaining using the KH95 antibody, which recognizes the pRb interaction domain. In primary E16 murine cortical cultures, NTF- or chemokine-treated neurons, KH95 E2F1 staining was increased in the cytoplasm. However, an antibody recognizing the amino-terminus of E2F1 (KH20) stained the cytoplasm of both untreated and treated neurons. Taken together these results suggest that the change seen in E2F1 using the KH95 antibody is due to antigen unmasking of a carboxy-terminal epitope in response to NTF and chemokines. When we assessed staining for the hyperphosphorylated, inactive form of pRb (ppRb) in untreated cultures, ppRb was predominantly cytoplasmic. In response to NTF or chemokine treatment, staining for ppRb was observed predominantly in nuclei of neurons indicating a change in subcellular distribution. Immunoblot analysis demonstrated increased levels of ppRb in response to NTF and chemokines. Inhibitors of translation, nuclear export, and phoshpatidylinositol-3-kinase blocked NTF- and chemokine-induced nuclear ppRb localization while having no effect on E2F1 staining. Instead increased cytoplasmic KH95 E2F1 staining was dependent on cytoskeletal destabilization which did not influence ppRb localization. These findings demonstrate that alterations in ppRb distribution and E2F1 antigen availability by NTF and chemokines occur by distinct mechanisms suggesting that E2F1 function may be independent of pRb regulation in post-mitotic neurons.

BL22, a Recombinant Anti-CD22 Immunotoxin, Induces Cell Cycle Arrest and Apoptosis in B-Cell Lymphoma.

A recombinant immunotoxin, BL22 has shown clinical efficacy against hairy cell leukemia and other B-cell non-Hodgkin's lymphomas. BL22 contains an antibody-derived domain that recognizes CD22, and PE38, a truncated Pseudomonas exotoxin A domain, for inhibition of protein synthesis. Previous studies have shown that Pseudomonas exotoxin A-based immunotoxins exert their cytotoxicity by apoptosis and additional, yet uncharacterized mechanisms. We investigated the cytotoxicity of BL22 in two mantle cell lymphoma lines, NCEB-1 and Granta-519, and in the diffuse, large-cell lymphoma line SU-DHL-4. Presence of the CD22 target was confirmed by flow cytometry. Treatment of NCEB-1 cells to 1 μg/ml BL22 for 24-32h reduced the numbers of viable cells in comparison to cells exposed to a non-binding immunotoxin. Induction of apoptosis, determined by activation of caspase-3 and exposure of phosphatidylserine, was not detected before 48h of exposure. Incorporation of bromo-deoxyuridine into DNA was, however, almost completely inhibited after 24h. Correspondingly, the numbers of G1 phase cells were increased upon treatment with BL22 as determined by flow cytometry. G1-arrest resulted from decreased enzyme activities of cyclin-dependent kinases (cdk)-2 and 4 as assessed in vitro with respective substrates, histone H1 and the retinoblastoma susceptibility (RB) protein. Immunoblotting revealed markedly reduced amounts of cyclins A, E, D1 and D3 whereas cdk2 and cdk4 protein expression levels remained unchanged. Expression of the RB protein was decreased, and the protein was hypophosphorylated in immunotoxin-treated cells. Pulse-labeling with [35S]-labeled amino acids, followed by immunoprecitation of cyclins E and D1 confirmed that BL22 inhibited synthesis of these cyclins. Thus, immunotoxin-induced cell cycle arrest results from insufficient synthesis of regulatory cyclins required for progression through G1 into S-phase. Similar results were obtained in Granta-519 cells. SU-DHL-4 diffuse, large-cell lymphoma cells were highly sensitive to BL22, and G1 arrest was observed as early as after 8–24h in these cells. Expression of cyclins A, D3 and E was decreased while cyclin D1 was not expressed in SU-DHL-4 cells. Interestingly, cell cycle arrest did not prevent from subsequent induction of apoptosis in lymphoma cells continuously exposed to the immunotoxin. In conclusion, we have characterized cell cycle arrest as a previously unknown mechanism that contributes to the cytotoxicity of Pseudomonas exotoxin-based immunotoxins.

Transforming growth factor-beta antagonizes alveolar type II cell proliferation induced by keratinocyte growth factor.

Keratinocyte growth factor (KGF) is a mitogen for rat type II cells and also stimulates differentiation in vitro. Administration of KGF also protects the lung from a variety of injuries and subsequent development of fibrosis. Because transforming growth factor (TGF)-beta has been shown to inhibit epithelial cell proliferation and surfactant protein gene expression in other systems and is thought to be a major effector in pulmonary fibrosis, we sought to determine if TGF-beta would antagonize the effects of KGF in primary cultures of alveolar type II cells. Type II cells were cultured on a matrix of type I collagen and Matrigel in the presence or absence of KGF and/or TGF-beta. KGF alone greatly stimulated proliferation and increased cyclin-dependent kinase (cdk) 2 kinase activity and Retinoblastoma susceptibility gene product (Rb) phosphorylation. Cyclin D1, cdk2, and cdc25A protein levels were increased, and p15(Ink4b) and p27(Kip1) protein levels were decreased. TGF-beta markedly inhibited alveolar epithelial cell proliferation induced by KGF. TGF-beta inhibited cdk2 enzyme activity and Rb phosphorylation and increased p15(Ink4b) protein levels. TGF-beta also inhibited differentiation induced by KGF as measured by secretion of surfactant protein-A into the apical media. In summary, TGF-beta inhibits the proliferative effect of KGF in vitro and may be a biologic antagonist of KGF.