Biomedical subjects

J M Horowitz

Publications and source records attributed to J M Horowitz.

At least 19 recordsLinked to original sources

PubMed1995

Functional interactions between the retinoblastoma (Rb) protein and Sp-family members: superactivation by Rb requires amino acids necessary for growth suppression.

The transient expression of the retinoblastoma protein (Rb) regulates the transcription of a variety of growth-control genes, including c-fos, c-myc, and the gene for transforming growth factor beta 1 via discrete promoter sequences termed retinoblastoma control elements (RCE). Previous analyses have shown that Sp1 is one of three RCE-binding proteins identified in nuclear extracts and that Rb functionally interacts with Sp1 in vivo, resulting in the "superactivation" of Sp1-mediated transcription. By immunochemical and biochemical criteria, we report that an Sp1-related transcription factor, Sp3, is a second RCE-binding protein. Furthermore, in transient cotransfection assays, we report that Rb "superactivates" Sp3-mediated RCE-dependent transcription in vivo and that levels of superactivation are dependent on the trans-activator (Sp1 or Sp3) studied. Using expression vectors carrying mutated Rb cDNAs, we have identified two portions of Rb required for superactivation: (i) a portion of the Rb "pocket" (amino acids 614-839) previously determined to be required for physical interactions between Rb and transcription factors such as E2F-1 and (ii) a novel amino-terminal region (amino acids 140-202). Since both of these regions of Rb are targets of mutation in human tumors, our data suggest that superactivation of Sp1/Sp3 may play a role in Rb-mediated growth suppression and/or the induction of differentiation.

Amino Acids↗

PubMed1995

Detection of a novel cell cycle-regulated kinase activity that associates with the amino terminus of the retinoblastoma protein in G2/M phases.

Recent genetic and functional evidence suggests that the amino terminus of the retinoblastoma (Rb) protein plays an important role in Rb-mediated growth suppression. To explore the mechanism(s) by which this portion of Rb may regulate cell growth, we have sought to characterize cellular proteins that associate with the Rb amino terminus using an in vitro protein-binding assay. Here we report that at least one such protein is a cell cycle-regulated Rb/histone H1 kinase (RbK) whose enzymatic and/or Rb association activity is most prevalent in G2/M phases of cells. In contrast to previously characterized cyclin-dependent and Rb-associated kinases, such as cdk1 (cdc2) and cdk2, G2/M RbK 1) is not depleted by incubation with p13suc-beads, 2) is not detected with antisera against several Rb-associated cyclins-cdks, and 3) associated with Rb via the Rb amino terminus, a region that is dispensable for interaction with other Rb-associated kinases. RbK is clearly distinct from previously characterized mitotic cdks since cyclin A-cdc2, cyclin A-cdk2, cyclin B-cdc2, and cyclin B-cdk2 did not associate with the Rb amino terminus. Coprecipitation experiments with Rb antisera confirmed the association of Rb with a RbK-like kinase in metaphase-arrested cells in vivo. Interestingly, G2/M RbK did not appreciably associate with an analogous portion of p107, a Rb-related protein. Taken together, these data indicate that the Rb amino terminus specifically associates with a novel cell cycle-regulated kinase in late cell cycle stages.

Amino Acid Sequence↗

PubMed1995

Retinoblastoma gene product as a downstream target for a ceramide-dependent pathway of growth arrest.

Ceramide, a lipid mediator, has been most closely associated with antiproliferative activities. In this study, we examine the mechanism by which ceramide induces growth suppression and the role of the retinoblastoma gene product (Rb) in this process. Withdrawal of serum from the serum-dependent MOLT-4 cells resulted in significant dephosphorylation of Rb, correlating with the induction of G0/G1 cell cycle arrest. Serum withdrawal resulted in marked elevation in the levels of endogenous ceramide (3-fold at 24 h and 8-fold at 96 h) with little changes in the endogenous levels of sphingosine. The addition of exogenous C6-ceramide resulted in a concentration- and time-dependent dephosphorylation of Rb. Exogenous ceramide was active at levels comparable to endogenous levels achieved with serum withdrawal. Peak activity of exogenous ceramide (at 6 h) correlated with the uptake of C6-ceramide by MOLT-4 cells. Next, a number of studies were conducted to determine whether Rb plays a role in ceramide-induced growth suppression. (i) C6-Ceramide was poorly active in growth suppression of retinoblastoma cells that lack Rb. (ii) Mink lung epithelial cells in which Rb had been sequestered by overexpression of large tumor antigen were resistant to the action of ceramide compared to cells transfected with large tumor antigen mutated in the Rb-binding pocket. (iii) Overexpression of the EIA adenoviral protein, which binds and sequesters Rb, resulted in protection from growth suppression and cell cycle arrest induced by ceramide. Thus, these studies demonstrate that Rb is a downstream target for ceramide and may function in a growth suppressor pathway resulting in cell cycle arrest.

Adenovirus E1A Proteins↗

PubMed1994

Negative regulation of Sp1 trans-activation is correlated with the binding of cellular proteins to the amino terminus of the Sp1 trans-activation domain.

Sp1 is a well characterized and ubiquitously expressed transcription factor that regulates the constitutive and induced expression of a variety of mammalian genes. It is unclear whether Sp1 activity is regulated in vivo; the mechanism by which Sp1 interacts with the basal transcription complex has not been firmly established. We report the identification of a ubiquitously expressed and evolutionarily conserved nuclear protein, p74, that specifically binds Sp1 in vivo and in vitro. p74 interacts with several portions of the Sp1 trans-activation domain in vitro, and we correlate the binding of p74 to the amino-terminal serine/threonine-rich subdomain of Sp1 with the inhibition of Sp1-mediated transcription in vivo.

Binding Sites↗

PubMed1993

Sp-1 binds promoter elements regulated by the RB protein and Sp-1-mediated transcription is stimulated by RB coexpression.

The retinoblastoma (RB) protein is implicated in transcriptional regulation of at least five cellular genes, including c-fos, c-myc, and transforming growth factor beta 1. Cotransfection of RB and truncated promoter constructs has defined a discrete element (retinoblastoma control element; RCE) within the promoters of each of these genes as being necessary for RB-mediated transcription control. Previously, we have shown that RCEs form protein-DNA complexes in vitro with three heretofore unidentified nuclear proteins and mutation of their DNA-binding site within the c-fos RCE results in an abrogation of RCE-dependent transcription in vivo. Here, we demonstrate that one of the nuclear proteins that binds the c-fos, c-myc, and transforming growth factor beta 1 RCEs in vitro is Sp-1 and that Sp-1 stimulates RCE-dependent transcription in vivo. Moreover, we show that Sp-1-mediated transcription is stimulated by the transient coexpression of RB protein. We conclude from these observations that RB may regulate transcription in part by virtue of its ability to functionally interact with Sp-1.

3T3 Cells↗

PubMed1993

Regulation of transcription by the retinoblastoma protein.

The product of the retinoblastoma gene (RB1) is believed to function as a negative regulator of cell growth. Recent experimental results suggest that RB1 may exert its growth-suppressing activity by regulating the transcription of a variety of growth-related genes, including FOS, MYC, and TGFBI. A series of biochemical and molecular analyses suggest that RB1 indirectly affects gene expression via cell-cycle-regulated interactions with transcription factors, such as E2F and SPI. Determination of the mechanisms regulating such protein-protein interactions and the identification of additional targets of RB1 function will provide vital insights into the role of this tumor-suppressor gene in mammalian cell proliferation.

Adenovirus E1A Proteins↗

PubMed1992

Functional analysis at the Cys706 residue of the retinoblastoma protein.

A missense mutation at cysteine 706, resulting in a retinoblastoma (RB) protein defective in phosphorylation and oncoprotein binding, has been isolated from a human tumor cell line. Since this residue is conserved in murine RB and in the related p107 protein, we studied the activity of in vitro mutants flanking this position. These experiments demonstrated that the thiol atom at codon 706 does not possess intrinsic functional activity as small polar or nonpolar residues could substitute at either codons 706 or 707, while bulkier R-group changes in these positions interfered with in vitro oncoprotein binding or in vivo protein phosphorylation. A series of missense mutants in an adjacent leucine repeat domain also demonstrated a loss of oncoprotein binding that was proportional to the magnitude of amino acid substitutions. To determine whether the cysteine 706 --> phenylalanine RB mutant retained any protein binding activity, we examined its ability to precipitate MYC, which was recently identified as a potential RB-associated protein. These experiments demonstrated that the mutant RB product is capable of binding in vitro to c-myc and L-myc proteins with comparable affinity as wild-type RB. These findings raise questions about the functional role of the RB:MYC interactions and emphasize important differences in the binding patterns between MYC and the other RB-associated proteins.

Amino Acid Sequence↗

PubMed1992

The interaction of RB with E2F coincides with an inhibition of the transcriptional activity of E2F.

Recent experiments have shown that the E2F transcription factor is in a complex with the RB1 gene product. The E2F-pRB complex can be reconstituted in an in vitro assay using a GST-RB fusion protein isolated from Escherichia coli. This interaction is dependent on pRB sequences involved in E1A/T-antigen binding as well as carboxy-terminal pRB sequences that are not necessary for E1A/T binding. Moreover, reconstitution assays reveal a requirement for an accessory factor, in addition to E2F and pRB, for formation of the E2F-pRB complex. Assays of transcription from the adenovirus E2 promoter in transfection experiments demonstrate that formation of the complex containing pRB and E2F coincides with an inhibition of E2F-dependent transcriptional activity. A mutant pRB protein that does not associate with E2F does not inhibit transcription. We conclude that as a consequence of its interaction with E2F, pRB may regulate the transcriptional function of the E2F factor.

Adenovirus Early Proteins↗

PubMed1992

A common set of nuclear factors bind to promoter elements regulated by the retinoblastoma protein.

A 30-base pair element within the c-fos promoter, termed the RCE (retinoblastoma control element), has previously been shown to be the target of transcriptional regulation by the product of the retinoblastoma (Rb) gene. We have identified three nuclear proteins [retinoblastoma control proteins (RCPs)] that complex with this promoter element in vitro. The Rb gene does not appear to encode the RCPs as the expression of Rb in vivo does not correlate with RCE-RCP complex formation in vitro. A single binding site for the RCPs within the c-fos RCE was identified, and the nucleotides required for protein-DNA complex formation were defined. Similar sequences are found in the promoters of two additional genes that are regulated by Rb (c-myc and TGF-beta 1), and binding assays demonstrate that the RCPs also interact with these elements. Linkage of the c-fos RCE to the herpes simplex virus thymidine kinase promoter led to a 4-fold stimulation of expression in transient transfection assays. Mutations within the RCP binding site that abrogate stable interaction of the RCPs with the RCE in vitro block RCE transcriptional activity in vivo. Our results suggest a role for the RCPs in RCE-dependent transcription and the regulation of transcription by the Rb protein.

Base Sequence↗

PubMed1991

The E2F transcription factor is a cellular target for the RB protein.

Although it is generally believed that the product of the retinoblastoma susceptibility gene (RB1) is an important regulator of cell proliferation, the biochemical mechanism for its action is unclear. We now show that the RB protein is found in a complex with the E2F transcription factor and that only the under phosphorylated form of RB is in the E2F complex. Moreover, the adenovirus E1A protein can dissociate the E2F-RB complex, dependent on E1A sequence also critical for E1A to bind to RB. These sequences are also critical for E1A to immortalize primary cell cultures and to transform in conjunction with other oncogenes. Taken together, these results suggest that the interaction of RB with E2F is an important event in the control of cellular proliferation and that the dissociation of the complex is part of the mechanism by which E1A inactivates RB function.

Adenovirus Early Proteins↗

PubMed1991

Diurnal responses of mammals to acute exposure to a hyperdynamic environment.

Acute exposure to hyperdynamic environments elicits significant depressions in core temperature in both diurnal squirrel monkeys and nocturnal rats. This study describes time of day variations in core temperature responses in squirrel monkeys and rats exposed for 70 min to a hyperdynamic environment (2 g) produced via centrifugation. Experiments were performed during the middle of the light or dark phase. After a 70-min control period, squirrel monkey resting core temperature was 38.6 +/- 0.2 (SE) and 36.8 +/- 0.2 degrees C during the day and night, respectively. At the end of a 7-min exposure to 2 g, squirrel monkey core temperature decreased 1.3 +/- 0.2 degree C during the day but remained a constant 36.8 +/- 0.4 degrees C at night. Core temperature of rats exposed to an identical 70-min 2 g profile decreased 1.5 +/- 0.4 degree C from a resting 37.1 +/- 0.6 degrees C during the day and 2.3 +/- 0.2 degrees C from a resting 37.8 +/- 0.3 degrees C at night. Thus, in both species, there were clear day-night differences in response to hyperdynamic environments, with the greatest fall in core temperature occurring during the animal's active period.

Adipose Tissue, Brown↗

PubMed1990

Tetanus during a high extracellular calcium pulse overrides the block of long-term potentiation seen at 20 degrees C in the hamster hippocampal slice.

Extracellular CA1 pyramidal cell activity was measured at different temperatures in hamster hippocampal slices with bath Ca2+ concentration set at either 2.0 mM or 4.5 mM. Records taken before and after tetanic stimulation of Schaffer collateral/commissural pathways were compared to determine if long-term potentiation (LTP) developed. LTP, which cannot be elicited at 20 degrees C with 2.0 mM calcium in the bath, was elicited at 20 degrees C when a tetanus was administered during a high calcium (4.5 mM) pulse. This LTP was limited to the tetanized pathway and was blocked by APV. Moreover, to elicit LTP at 20 degrees C, high (4.5 mM) extracellular calcium was needed both during and for several minutes following the tetanus. We conclude that mechanisms responsible for a thermal block of LTP are sensitive to calcium and that the thermal block can be overcome by increasing the amount of calcium that enters a cell during a tetanus.

2-Amino-5-phosphonovalerate↗

PubMed1990

Negative regulation of human c-fos expression by the retinoblastoma gene product.

Inactivation of the retinoblastoma susceptibility gene (RB-1) has been associated with the aetiology of many types of human cancers, leading to the classification of RB-1 as an anti-oncogene or tumour suppressor gene. Given that the protein product of RB-1 (Rb) has a nuclear localization and DNA-binding activity in vitro, it is possible that Rb regulates transcription of certain genes. The promoter of the c-fos gene might be a target for regulation by Rb, because both v-fos and RB-1 are associated with the induction of osteosarcomas in mice and humans, respectively. Also, fos expression is thought to be required for quiescent cells to enter the cell cycle, making the fos promoter an attractive target for suppressors of cell growth. Here we report that Rb can repress c-fos expression and AP-1 transcriptional activity in both serum-induced and cycling 3T3 cells. We have mapped a cis-acting element in the human c-fos promoter that can confer repression by Rb to a heterologous promoter. We have the termed the cis-acting sequence regulated by Rb the retinoblastoma control element.

Animals↗

PubMed1990

Thermal effects on long-term potentiation in the hamster hippocampus.

Extracellular CA1 pyramidal cell activity was measured at different temperatures in hippocampal slices from the Syrian hamster (Mesocricetus auratus), a hibernator. Control records taken before and after tetanic stimulation of Schaffer collateral/commissural pathways were compared to determine if long-term potentiation (LTP) was established. LTP (an enhancement of the population spike amplitude or population synaptic response following tetanus) was elicited in slices at temperatures above 22 degrees C, but not in slices at temperatures of 20 degrees C. When LTP was established at temperatures above 24 degrees C, however, lowering the temperature to 20 degrees C did not abolish the LTP. Furthermore, when a tetanus was delivered at 20 degrees C and the bath temperature was then raised above 22 degrees C, LTP was established. These results for step changes in temperature suggest that the sequence of cellular mechanisms leading to LTP is activated, but then arrested in slices maintained at a constant temperature of 20 degrees C. Assuming this type of activity in the slice parallels in vivo hippocampal activity, it follows that the ability to elicit LTP in CA1 hippocampal pyramidal cells is lost when the core temperature of an animal entering hibernation falls to 20 degrees C.

Animals↗

PubMed1990

Thermal dependence of serotonergic modulation of neural activity in the hamster.

1. The modulatory effect of serotonin on CA1 pyramidal cells in the hamster (Mesocricetus auratus) hippocampus was examined over a range of temperatures. 2. Following repetitive Schaffer collateral/commissural stimulation, changes in the amplitude of population spikes (the synchronous firing of CA1 pyramidal cells) were recorded in the hamster, a hibernator. Amplitudes were measured after 10 microM serotonin was added to and then withdrawn from the perfusing medium with the temperature of the bath fixed at different temperatures. 3. Between 35 degrees C and 15 degrees C a depression in population spike amplitude of at least 10% was seen in 36 of 43 trials, with an average depression of 68%. No significant temperature dependence of the depressive effect was seen. 4. Following the removal of serotonin from the perfusate, the spike amplitude was enhanced over the same range of temperatures, averaging 33% higher than control values. The enhancement was most pronounced at 35 degrees C and 15 degrees C and smallest at 25 degrees C. 5. Thus, over the entire temperature range of 35 degrees C to 15 degrees C, serotonin exerted a dual modulatory effect on the spike amplitude, a depression followed by an enhancement. Serotonin's modulatory effects on pyramidal cell excitation persist over temperatures encountered as the hamster enters hibernation.

Action Potentials↗

PubMed1990

A single amino acid substitution results in a retinoblastoma protein defective in phosphorylation and oncoprotein binding.

We have previously identified a small-cell lung cancer cell line (NCI-H209) that expresses an aberrant, underphosphorylated form of the retinoblastoma protein RB1. Molecular analysis of RB1 mRNA from this cell line revealed a single point mutation within exon 21 that resulted in a nonconservative amino acid substitution (cysteine to phenylalanine) at codon 706. Stable expression of this mutant RB1 cDNA in a human cell line lacking endogenous RB1 demonstrated that this amino acid change was sufficient to inhibit phosphorylation. In addition, this cysteine-to-phenylalanine substitution also resulted in loss of RB1 binding to the simian virus 40 large tumor and adenovirus E1A transforming proteins. These results confirm the importance of exon 21 coding sequences and suggest that the cysteine residue at codon 706 may play a role in achieving a specific protein conformation essential for protein-protein interactions.

Amino Acid Sequence↗

PubMed1990

Frequent inactivation of the retinoblastoma anti-oncogene is restricted to a subset of human tumor cells.

We have used polyclonal anti-synthetic peptide serum to study the role of retinoblastoma gene (RB) inactivation in a variety of human tumor cell lines. Our analysis indicates that inactivation of the RB protein, p105-Rb, is universal in retinoblastoma cells, vindicating the predictions of the Knudson "two-hit" hypothesis. In addition, our analysis has shown that inactivations of the RB gene are nearly as frequent in a more common human tumor, small cell lung carcinoma. One-third of bladder carcinomas surveyed also carry altered or absent p105-Rb. Other human tumors by contrast demonstrate only infrequent inactivation of the RB gene. These results suggest that inactivation of the RB gene is a critical step in the pathogenesis of a subset of human tumors.

Base Sequence↗

PubMed1990

Effects of temperature and acid-base state on hippocampal population spikes in hamsters.

Previous studies have shown that changes in temperature, within the range encountered by hamsters entering hibernation, alter the evoked response of hippocampal pyramidal cells to stimulation of an afferent pathway. The present study was designed to determine whether these alterations are due to changes in the acid-base status of the neural tissue brought about by changes in temperature. Extracellular-evoked responses were recorded from hamster hippocampal slices after Schaffer collateral stimulation. The pH was changed by varying the concentration of CO2 aerating the bathing medium. Buffers contained either 26 or 40 mM bicarbonate ion. The width of the population spike (the synchronous firing of pyramidal cells) was measured as pH was varied between 7.5 and 7.1, with slice temperature set at either 25 or 20 degrees C. There was a significant increase in spike width as temperature was lowered to 20 degrees C, but no significant change in spike width or amplitude as pH or bicarbonate was varied. The effect of temperature (20 degrees C for half-maximal stimulation, and from 20 to 25 degrees C for just maximal stimulation) on spike width and amplitude thus does not appear to be due to pH- or bicarbonate-induced changes.

Acid-Base Equilibrium↗