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Eldad Zacksenhaus

Publications and source records attributed to Eldad Zacksenhaus.

12 recordsLinked to original sources
PubMed2005

The retinoblastoma gene pathway regulates the postmitotic state of hair cells of the mouse inner ear.

Precursors of cochlear and vestibular hair cells of the inner ear exit the cell cycle at midgestation. Hair cells are mitotically quiescent during late-embryonic differentiation stages and postnatally. We show here that the retinoblastoma gene Rb and the encoded protein pRb are expressed in differentiating and mature hair cells. In addition to Rb, the cyclin dependent kinase inhibitor (CKI) p21 is expressed in developing hair cells, suggesting that p21 is an upstream effector of pRb activity. p21 apparently cooperates with other CKIs, as p21-null mice exhibited an unaltered inner ear phenotype. By contrast, Rb inactivation led to aberrant hair cell proliferation, as analysed at birth in a loss-of-function/transgenic mouse model. Supernumerary hair cells expressed various cell type-specific differentiation markers, including components of stereocilia. The extent of alterations in stereociliary bundle morphology ranged from near-normal to severe disorganization. Apoptosis contributed to the mutant phenotype, but did not compensate for the production of supernumerary hair cells, resulting in hyperplastic sensory epithelia. The Rb-null-mediated proliferation led to a distinct pathological phenotype, including multinucleated and enlarged hair cells, and infiltration of hair cells into the mesenchyme. Our findings demonstrate that the pRb pathway is required for hair cell quiescence and that manipulation of the cell cycle machinery disrupts the coordinated development within the inner ear sensory epithelia.

Animals↗
PubMed2005

Conserved and specific functions of mammalian ssu72.

We describe the cloning and characterization of a human homolog of the yeast transcription/RNA-processing factor Ssu72, following a yeast two-hybrid screen for pRb-binding factors in the prostate gland. Interaction between hSsu72 and pRb was observed in transfected mammalian cells and involved multiple domains in pRb; however, so far, mutual effects of these two factors could not be demonstrated. Like the yeast counterpart, mammalian Ssu72 associates with TFIIB and the yeast cleavage/polyadenylation factor Pta1, and exhibits intrinsic phosphatase activity. Mammals contain a single ssu72 gene and a few pseudogenes. During mouse embryogenesis, ssu72 was highly expressed in the nervous system and intestine; high expression in the nervous system persisted in adult mice and was also readily observed in multiple human tumor cell lines. Both endogenous and ectopically expressed mammalian Ssu72 proteins resided primarily in the cytoplasm and only partly in the nucleus. Interestingly, fusion to a strong nuclear localization signal conferred nuclear localization only in a fraction of transfected cells, suggesting active tethering in the cytoplasm. Suppression of ssu72 expression in mammalian cells by siRNA did not reduce proliferation/survival, and its over-expression did not affect transcription of candidate genes in transient reporter assays. Despite high conservation, hssu72 was unable to rescue an ssu72 lethal mutation in yeast. Together, our results highlight conserved and mammalian specific characteristics of mammalian ssu72.

Amino Acid Sequence↗
PubMed2004

Splitting the apoptosome.

Assembly of the apoptosome in response to mitochondrial permeabilization, the hallmark of the intrinsic apoptotic pathway, involves binding of cytochrome c to Apaf1, recruitment and auto-processing of the apical/signaling pro-caspase-9, and coupled activation of downstream/executioner caspases like caspase 3. Evidence now indicates that certain apoptotic cascades can bypass the apoptosome and activate caspase-9 independent of the mitochondria. Recently, we have demonstrated that caspase-9 can be activated in Apaf1-mutant primary myoblasts, but not fibroblasts, in response to stimuli that are known to act via the mitochondria. Thus, apoptosomal activation of caspase-9 seems to represent only one of the routes for its activation; other pathways, some of which are yet to be discovered, can bypass the requirement for Apaf1 and activate caspase-9 in a tissue and context specific manner.

Animals↗
PubMed2004

Coupling of caspase-9 to Apaf1 in response to loss of pRb or cytotoxic drugs is cell-type-specific.

Inactivation of the tumor suppressor Rb in the mouse induces cell death, which depends entirely (in lens, CNS) and only partly (PNS, skeletal muscles) on Apaf1/Ced4, an apoptosomal factor thought to be required for processing procaspase-9 following mitochondrial permeabilization. Here, we report that in response to cytotoxic drugs, Apaf1(-/-) primary myoblasts but not fibroblasts undergo bona fide apoptosis. Cell demise was associated with disruption of mitochondria but not endoplasmic reticulum. Processing of procaspase-9 occurred in Apaf1(-/-) myoblasts but not fibroblasts, and ablation of Casp9 prevented drug-induced apoptosis in both cell types. Deregulation of the Rb pathway by overexpression of E2F1 also induced caspase-9-dependent, Apaf1-independent apoptosis in myoblasts. Despite its requirement for apoptosis in vitro, mutation in Casp9 abrogated cell death in the nervous system and lens but only partly in skeletal muscles of Rb-deficient embryos. In addition, developmental cell death in fetal liver and PNS was not inhibited in Casp9(-/-) embryos. Therefore, loss of pRb elicits apoptosome-dependent and apoptosome-independent cell death, and the requirement and coupling of caspase-9 to Apaf1 are both context-dependent.

Animals↗
PubMed2004

Dynamic regulation of the Stra13/Sharp/Dec bHLH repressors in mammary epithelium.

Mammary gland development is a dynamic process involving cyclical proliferation, cellular differentiation, and cell death. In this study, we have determined that expression of the Stra13/Sharp/Dec basic helix-loop-helix (bHLH) family is dynamically regulated in mammary epithelium. In cultured HC11 cells, epidermal growth factor (EGF) treatment rapidly induces Stra13 protein accumulation, which is blocked by the synthetic glucocorticoid, dexamethasone. Neither the induction of Stra13 by EGF nor its repression by dexamethasone correlates with changes in Stra13 mRNA levels. During mouse mammary gland development in vivo, Stra13 is highly expressed in epithelial ducts during puberty, and strongly induced in both ducts and alveoli during early involution, while the related Sharp-1 gene is highly expressed only during late stages of involution. Together, these data indicate that Stra13/Dec/Sharp-family bHLH repressors are dynamically regulated during mammary gland development and may function to regulate apoptosis in this tissue.

Animals↗
PubMed2004

High expression during neurogenesis but not mammogenesis of a murine homologue of the Deleted in Breast Cancer2/Rhobtb2 tumor suppressor.

Deleted in Breast Cancer-2, Dbc2, identified as a candidate tumor suppressor gene for breast cancer and other human malignancies, is a member of a novel gene family encoding putative small GTPases designated as Rhobtb. In this report, we describe the spatio-temporal expression of mDbc2/Rhobtb2 mRNA during mammary gland development and embryogenesis in the mouse. We have first determined that mDbc2/Rhobtb2 transcripts are expressed at low, but seemingly constant levels during mammopoiesis. Secondly, we have found that mDbc2/Rhobtb2 is expressed highly and specifically in the central and peripheral nervous systems during mouse embryogenesis. While our results do not reveal any connection between elevated expression of mDbc2/Rhobtb2 and a specific stage of mammary gland development, they strongly support a role for this gene during development of the nervous system.

Animals↗
PubMed2003

The tyrphostin AG1024 accelerates the degradation of phosphorylated forms of retinoblastoma protein (pRb) and restores pRb tumor suppressive function in melanoma cells.

Constitutive cell surface receptor kinase signaling and persistent phosphorylation/inactivation of the retinoblastoma (pRb) family of proteins (pRb, p107 and p130, known as pocket proteins) have been implicated in conferring uncontrolled growth to melanoma cells. However, the signals linking receptor kinase activity to neutralization of pocket proteins have not yet been fully elucidated. We therefore used specific chemical inhibitors to examine pRb regulation in melanoma cells. The most efficient agent, AG1024, known as an inhibitor of insulin-like growth factor 1 receptor and insulin receptor, arrested melanoma cell growth in vitro at nanomolar concentrations within 24 h of application. AG1024 inhibited the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and restored pRb tumor suppressive function. The latter was observed by the reduction in the phosphorylated forms of pRb, p107 and p130, and the formation of growth suppressive DNA binding complexes consisting of pRb and E2F1 or E2F3. The loss of phosphorylated forms of pRb at early time points after AG1024 application was not associated with suppression of cyclin-dependent kinases 2 and 4 activity but rather with proteasomal and nonproteasomal degradation. Thus, inhibition of melanoma cell proliferation by AG1024 is mediated by inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase 2 signaling and activation of pRb by a mechanism involving protein degradation.

Animals↗
PubMed2002

Stra13 homodimers repress transcription through class B E-box elements.

A mammalian basic helix-loop-helix protein known variably as Stra13, Sharp2, and Dec1 has been implicated in cell activation, proliferation, and differentiation. Indeed, Stra13 null mice develop age-induced autoimmunity as a result of impaired T-lymphocyte activation, leading ultimately to the accumulation of autoreactive T-cells and B-cells. Stra13 is expressed in embryonic as well as adult tissues derived from neuroectoderm, mesoderm, and endoderm and has been associated with response to hypoxia, suggesting a complex role for this protein and the highly related Sharp1/Dec2 protein in homeostatic regulation. Whereas Stra13 is known to regulate many important cellular functions and is known to cross-regulate biological responses to other basic helix-loop-helix containing transcription factors, including c-Myc and USF, it is unclear if this protein binds directly to DNA. Indeed, the basic domain of Stra13 contains a proline residue at an unprecedented position. Herein, we have determined that Stra13 binds with high affinity to CACGTG class B E-box elements as a homodimer with preference for elements preceded by T and/or followed by A residues. In addition, transient transfection experiments reveal that Stra13 represses transcription when bound to these and related sites. Our data suggest that Stra13 regulates cellular functions through antagonism of E-box activator proteins and also through active repression from E-box elements.

Animals↗
PubMed2002

Activation of retinoblastoma protein in mammary gland leads to ductal growth suppression, precocious differentiation, and adenocarcinoma.

The retinoblastoma (Rb) tumor suppressor controls cellular proliferation, survival, and differentiation and is functionally inactivated by mutations or hyperphosphorylation in most human cancers. Although activation of endogenous Rb is thought to provide an effective approach to suppress cell proliferation, long-term inhibition of apoptosis by active Rb may have detrimental consequences in vivo. To directly test these paradigms, we targeted phosphorylation-resistant constitutively active Rb alleles, Rb Delta Ks, to the mouse mammary gland. Pubescent transgenic females displayed reduced ductal elongation and cell proliferation at the endbuds. Post-puberty transgenic mice exhibited precocious cellular differentiation and beta-casein expression and extended survival of the mammary epithelium with a moderate but specific effect on the expression of E2F1, IGF1R alpha, and phospho-protein kinase B/AKT. Remarkably, approximately 30% Rb Delta K transgenic females developed focal hyperplastic nodules, and approximately 7% exhibited full-blown mammary adenocarcinomas within 15 mo. Expression of the Rb Delta K transgene in these mammary tumors was reduced greatly. Our results suggest that transient activation of Rb induces cancer by extending cell survival and that the dual effects of Rb on cell proliferation and apoptosis impose an inherent caveat to the use of the Rb pathway for long-term cancer therapy.

Adenocarcinoma↗
PubMed2002

Coordinated expression of Rb gene family in the mammary gland.

We have previously demonstrated that the retinoblastoma gene family, Rb, p107 and p130, is differentially expressed during mouse embryogenesis. Here we show that this gene family is coordinately regulated in the mammary luminal epithelium. Expression of Rb, p107 and p130 in the epithelial compartment is low in nulliparous female mice and early stages of pregnancy but is induced at mid-pregnancy and peaks at lactation. During involution p107 expression is lost whereas expression pRb and p130 persist. The induction of this gene family at mid-pregnancy accompanies the expression of beta-casein. However, whereas beta-casein transcripts are confined to the lobuloalveolar compartment, the Rb gene family is expressed both in lobuloalveoli and ducts. The co-expression of the Rb family in the mammary gland may allow functional compensation among these family members. This in turn may explain the recent observations that loss of Rb alone in the mammary gland is inconsequential, whereas overexpression of cyclin D1 or SV40 large T antigen, which can abrogate all members of the pRb protein family, induces mammary gland carcinogenesis.

Animals↗
PubMed2002

Coordinated expression of Rb gene family in the mammary gland.

We have previously demonstrated that the retinoblastoma gene family, Rb, p107 and p130, is differentially expressed during mouse embryogenesis. Here we show that this gene family is coordinately regulated in the mammary luminal epithelium. Expression of Rb, p107 and p130 in the epithelial compartment is low in nulliparous female mice and early stages of pregnancy but is induced at mid-pregnancy and peaks at lactation. During involution p107 expression is lost whereas expression pRb and p130 persist. The induction of this gene family at mid-pregnancy accompanies the expression of beta-casein. However, whereas beta-casein transcripts are confined to the lobuloalveolar compartment, the Rb gene family is expressed both in lobuloalveoli and ducts. The co-expression of the Rb family in the mammary gland may allow functional compensation among these family members. This in turn may explain the recent observations that loss of Rb alone in the mammary gland is inconsequential, whereas overexpression of cyclin D1 or SV40 large T antigen, which can abrogate all members of the pRb protein family, induces mammary gland carcinogenesis.

Animals↗
PubMedDate not supplied

Alternative reading frame supports an alternative model for retinoblastoma.

The recent observation by Tyler Jacks' group that loss of ARF accelerates pituitary tumors in Rb(+/-):Arf(-/-) mice by inducing proliferation without affecting apoptosis allows one to speculate about an alternative model for retinoblastoma. Retinoblasts in human and pituitary gland melanotrophs in the mouse might be uniquely susceptible to bi-allelic inactivation of Rb because upon loss-of-heterozygosity, these tissues are able to slowly proliferate without effectively activating the cell death machinery, and respond to an additional oncogenic signal. But what is the nature of this proliferation-permissive oncogenic alteration?

Animals↗