Biomedical subjects
Publications and source records attributed to B D Price.
Congenital pseudoarthrosis of the clavicle (CPC) is a rare condition of unresolved etiology and pathogenesis. Familial occurrence of this anomaly has been documented but the pattern of genetic transmission remains obscure. Two cases involving a father and daughter are presented here with a review of the literature. These cases add support to the genetic basis for CPC.
Suramin is an antineoplastic agent which has a cytostatic effect on both normal and tumor-derived cells. We have investigated whether the induction of growth arrest by suramin requires the p53 protein, a tumor suppressor gene product involved in the initiation of growth arrest following DNA damage. Activation of the p53 protein by genotoxic agents causes increased p53 protein levels and p53-dependent transcription of the p21 gene. The p21 protein then inhibits cyclin-dependent kinases, initiating G1 arrest. Exposure of NIH-3T3 cells to suramin caused a rapid (1-2 h) increase in the level of p53-DNA-binding activity. Flow cytometric analysis indicated that suramin arrested NIH-3T3 cells in G0-G1. However, suramin did not increase the p53-dependent transcription of the p21 gene or inhibit cyclin-dependent kinase 2 kinase activity. If NIH-3T3 cells were exposed to radiation or suramin plus radiation, p21 mRNA levels were increased and cyclin-dependent kinase 2 kinase activity was inhibited, indicating that suramin does not block the cells' ability to increase p21 levels. To determine whether the G0-G1 arrest induced by suramin required p53, NIH-3T3 cells transfected with a dominant negative mutant p53 gene to eliminate wild-type p53 function (NMP cells) were exposed to suramin. NMP cells still exhibited G0-G1 arrest after suramin treatment. Suramin increases p53 protein levels, but fails to increase p21 mRNA levels or to activate the G1 checkpoint. These data suggest that suramin induces growth arrest in NIH-3T3 cells by a mechanism that is independent of cellular p53 status.
A range of DNA-damaging agents has been shown to increase cellular levels of the nuclear phosphoprotein p53 and to induce p53-dependent processes. We examined the ability of three microtubule-active agents, taxol, vinblastine, and nocodazole, to increase p53 levels and activate p53-dependent processes. When tested using a p53 DNA-binding assay, all three agents induced p53 in a dose-dependent manner. To varying degrees, these agents also induced p21WAF1/CIP1 mRNA and transcription in a chloramphenicol acetyl transferase reporter system. These data suggest there is an additional pathway for activating p53 and subsequent p53-dependent processes.
DNA damage increases p53 protein levels and activates transcription of the p21 gene. The p21 protein binds to and inhibits cdk2 kinase, causing G1 arrest. Here, we have investigated if a p53 fusion protein is a substrate for cdk2 kinase in vitro. Cdk2 kinase was immunoprecipitated from NIH3T3 cells and allowed to phosphorylate a human p53-GST (glutathione-s-transferase) fusion protein. Cdk2 and cyclin E-cdk2 efficiently phosphorylated both wild-type (wt) and mutant p53-GST. Cdk2 immunoprecipitated from cells in Go and early G1 exhibited minimal p53 kinase activity, whereas cells in S-phase displayed high levels of p53 kinase activity. If NIH3T3 cells were X-ray irradiated to induce DNA damage, cdk2 p53 kinase activity was rapidly inhibited within 1 h, but had recovered by 4 h post irradiation. Mutation of serine 315 of p53 to alanine (p53-S315A) abolished phosphorylation by cdk2 kinase. However, wtp53 and p53-S315A were equally effective at activating transcription when cotransfected with a p53 reporter construct. The results demonstrate that ser 315 of p53 is phosphorylated by cdk2 in vitro. However, ser 315 of wtp53 is not required for transcriptional activity in vivo, suggesting that cdk2 phosphorylation of p53 may be involved in regulating other cellular functions of wtp53.
Damage to chromosomal DNA increases the levels of the transcriptional regulatory protein p53. We have investigated how the MDM2 protein, which binds to p53 and inactivates its transcriptional activity, may be controlled following DNA damage. Irradiation of human GM2149 fibroblast cells causes an increase in MDM2 mRNA levels within 1 h, and levels remain elevated for at least 8 h. The induction of MDM2 mRNA following irradiation is not blocked by inhibitors of protein synthesis and can be detected after doses of 2-5 Gy. In ataxia telangiectasia cells or cells where p53 is mutated/deleted, MDM2 mRNA levels are not increased after DNA damage. This suggests that p53 is required for transcription of the MDM2 gene following DNA damage.
We have investigated the effect of chemotherapeutic and DNA damaging agents on binding of the tumor suppressor phosphoprotein p53 to its consensus DNA sequence. Activation of p53-DNA binding was seen for treatment with radiation, hydrogen peroxide, actinomycin D, Adriamycin, etoposide, camptothecin, 5-fluorouracil, mitomycin C, and cisplatin. These results showed that DNA strand breaks were sufficient to lead to increased levels of p53. The protein synthesis inhibitor cycloheximide blocks the increase in p53 following DNA damage. The increase in p53 activation in camptothecin treated cells may result, at least in part, from an increased half-life of the protein and consequent increases in intracellular protein concentration.
Mammalian NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase-5,10-methenyltetrahydrofolate cyclohydrolase is a bifunctional mitochondrial enzyme expressed in most established cell lines but only in developing normal tissues. We report the cloning and molecular characterization of a Drosophila gene (DNMDMC) that encodes a protein with 56% identity to the mammalian bifunctional protein. Like the mammalian bifunctional proteins, the Drosophila protein contains a putative mitochondrial targeting sequence and its transcripts are expressed in developing tissues. Unlike its mammalian homologs, DNMDMC is expressed at high levels in adult tissues. DNMDMC maps to polytene chromosome band 85C, is encoded in three exons, and is closely flanked by two additional genes.
When cells are exposed to heat shock, heavy metals, amino acid analogues, and other stresses, the heat shock transcription factor (HSF) is activated. The HSF then binds to the promoter of the heat shock genes, stimulating transcription of the heat shock proteins. Here, we demonstrate that exposure of NIH-3T3 cells to oxidants (H2O2 or menadione) also causes activation of the HSF. This activation is not blocked by inhibitors of protein synthesis (cycloheximide) or by inhibitors of protein kinases (2-aminopurine or genistein). In addition, the oxidant activated HSF is located in the nucleus of the cells. However, oxidant activation of the HSF does not result in the accumulation of hsp70 mRNA or of heat shock proteins. This is in contrast to the accumulation of heat shock proteins seen after heat shock activation of the HSF. This suggests that oxidant induced activation of HSF binding may have a function different from that of heat induced activation of HSF binding.
The neurogenic genes of Drosophila are required for cell-cell communication that determines the choice between neuronal and epidermal cell fate. Here we report the molecular characterization of the neurogenic gene neuralized (neu) and show that it encodes a protein containing a C3HC4 zinc finger DNA-binding motif. This motif has been previously characterized in a variety of regulatory proteins, including transcription factors, locus-specific Drosophila chromosomal proteins, and oncoproteins. These results suggest a nuclear function for neu in the cell-cell signalling process responsible for inhibiting neuronal determination.
The heat shock response in mammals consists of a complex array of intracellular reactions initiated by stress, although its regulation is poorly understood. We have investigated the role of transmembrane signal transduction in the response, examining mechanisms involved in the activation of phospholipase C (PLC) by heat shock. In rodent fibroblasts permeabilized with digitonin, heat shock and receptor-mediated PLC activity exhibited a strict GTP analog dependency. This indicates that heat shock-mediated phospholipase activation, in common with receptor mediated stimulation, does not involve direct effects on the phospholipases and suggests the participation of GTP binding (G) proteins in the activation process. When cells were treated with the inhibitor pertussis toxin (PTX), the phospholipases retained their inducibility by heat shock, but became refractory to thrombin treatment, indicating that heat shock may influence PLC activity through a distinct population of G proteins compared to thrombin. The data seem to exclude a role for PTX sensitive G proteins in the production of IP3 after heating and suggest a pathway involving the direct thermal activation of the Gq class of G proteins, which are coupled to the PLC beta 1 isoform.
Here we report the discovery and characterization of the Drosophila tartan gene. tartan is transcribed in an unusual embryonic pattern of intersecting stripes which are generated in response to the anterior-posterior and dorsal-ventral regulatory systems. tartan encodes a putative transmembrane protein containing extracellular leucine-rich repeats characteristic of numerous cell surface receptors and adhesion proteins. Its expression is correlated with aspects of segmentation and neurogenesis, including the formation of neuroblasts, sensory mother cells, and peripheral nerves. Mutants homozygous for a recessive lethal tartan loss-function allele exhibit defects in the position and number of cells within peripheral sense organs, the routing of peripheral nerves, and the organization of commissures within the central nervous system. Mutants are also defective in muscle organization. These results suggest that tartan is required for cell surface interactions important for normal organization of epidermal and subepidermal structures.
Damage to cellular DNA greatly increases the levels of the tumor-suppressor gene p53 and induces cell cycle arrest in G1. A critical function of wild-type p53 is its ability to bind to specific DNA sequences. The effect of DNA damage on the sequence-specific DNA-binding properties of cellular p53 was investigated using DNA gel mobility-shift assays with nuclear extracts from NIH-3T3 cells. DNA damage (initiated by radiation) induced a rapid, cycloheximide-sensitive increase in the levels of nuclear p53-DNA binding activity and an increase in the half-life of the p53 protein. Increased p53-DNA binding activity could be detected at low (0.2 Gy), non-lethal doses of radiation. The tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) attenuated the DNA damage-induced increase in p53-DNA binding activity by decreasing the half-life of the p53 protein. The tumor promoter properties of TPA may therefore be mediated by interfering with the cellular p53 response to DNA damage. The increased levels of p53 bound to specific DNA sequences following DNA damage may induce cell cycle arrest. p53-mediated growth arrest could occur by inhibition of DNA replication and/or alterations in transcription of cell cycle genes.
We have investigated overlapping activation pathways for two families of stress genes that are expressed in cells exposed to hypoxia. The growth arrest and DNA damage (gadd) genes are induced by DNA damage and irradiation, and their expression is associated with growth arrest. The glucose-regulated proteins (GRPs) are induced by chemical agents that disrupt protein trafficking in the endoplasmic reticulum such as tunicamycin and A23187 and by hypoxia. Here, we demonstrate that the treatment of NIH-3T3 cells with chemical inducers of GRPs results in increased levels of gadd45 and gadd153 mRNA as well as GRP78 mRNA. In addition, hypoxia was also able to increase gadd45, gadd153, and GRP78 mRNA. Therefore the GRP and gadd genes can be activated by similar stimuli (e.g., hypoxia and chemical inducers). However, the mechanisms leading to increased levels of GRP78 and gadd gene mRNA are different and may involve distinct protein kinases. Increased expression of GRPs after treatment with chemical inducers is sensitive to cycloheximide and the protein kinase inhibitors genistein, 2-aminopurine, and H7, whereas the increase in gadd gene mRNA could be blocked by the protein kinase inhibitors H7 and 2-aminopurine but not by genistein or cycloheximide. GRP78 induction occurs by a pathway that requires protein synthesis and is sensitive to genistein, H7, and 2-aminopurine, whereas gadd gene induction is independent of protein synthesis and is inhibited by H7 and 2-aminopurine only.
After cells have been exposed to a nonlethal heat shock, they develop an enhanced resistance to subsequent prolonged heat shock. This process, termed thermotolerance, correlates with the expression of a group of proteins called the heat shock proteins. When cells are exposed to heat, protein synthesis is rapidly turned off and takes 5-6 hr to recover. In thermotolerant cells, protein synthesis is not blocked by heat. The heat shock proteins are thought to be responsible for the development of thermotolerance and the protection of the protein synthesis machinery from heat inactivation. To test the hypothesis that the heat shock proteins are involved in the heat shock response, we used two inhibitors to block their transcription and expression during heating and then monitored the effect on the development of thermotolerance and on protein synthesis. Camptothecin inhibits DNA topoisomerase I and blocks transcription of all actively transcribed genes, whereas dichloro-D-ribofuranosylbenzimidazole (DRB) inhibits only those genes transcribed by RNA polymerase II. Both DRB and camptothecin blocked the heat-induced expression of the heat shock proteins, but the absence of these proteins did not block either the development of thermotolerance or the protection of protein synthesis after heating. The data indicate that thermotolerance can develop in the absence of new protein synthesis.
The glucose regulated proteins (GRPs) are major structural components of the endoplasmic reticulum (ER) and are involved in the import, folding, and processing of ER proteins. Expression of the glucose regulated proteins (GRP78 and GRP94) is greatly increased after cells are exposed to stress agents (including A23187 and tunicamycin) which inhibit ER function. Here, we demonstrate that three novel inhibitors of ER function, thapsigargin (which inhibits the ER Ca(2+)-ATPase), brefeldin A (an inhibitor of vesicle transport between the ER and Golgi) and AIF4-, (which inhibits trimeric G-proteins), can increase the expression of both GRP78 and 94. The common characteristic shared by activators of GRP expression is that they disrupt some function of the ER. The increased levels of GRPs may be a response to the accumulation of aberrant proteins in the ER or they may be increased in response to structural/functional damage to the ER. The increased accumulation of GRP78 mRNA after exposure of cells to either thapsigargin, brefeldin A, AIF4-, A23187, or tunicamycin can be blocked by pre-incubation in cycloheximide. In contrast, accumulation of GRPs after exposure to hypoxia was independent of cycloheximide. In addition, the protein kinase inhibitor genistein blocked the thapsigargin induced accumulation of GRP78 mRNA, whereas the protein phosphatase inhibitor okadaic acid caused increased accumulation of GRP78 mRNA. The data indicates that there are at least 2 mechanisms for induced expression of GRPs, one of which involves a phosphorylation step and requires new protein synthesis (e.g., thapsigargin, A23187) and one which is independent of both these steps (hypoxia).
Heat shock leads to co-ordinate increases in transcription of a family of heat shock genes, including the mouse hsp70.1 and B2 genes. Activation of the heat shock transcription factor (HSF) by heat shock stimulates transcription of the murine hsp70.1 gene (by RNA polymerase II). B2 genes are short, repetitive sequences whose transcription (by RNA polymerase III) are also increased after heat shock. We have studied whether heat-induced transcription is auto-regulated by the products of the heat shock genes. The results indicate: (1) after an initial heat shock, transcription of the heat shock genes by RNA polymerases II and III becomes desensitized to further heat shock, and the heat-induced DNA binding activity of the HSF is lost, (2) if accumulation of heat shock gene products is inhibited, the desensitizing effect of a prior heat shock is removed, and (3) transcription of the hsp70.1 and B2 genes apparently involves different mechanisms, with hsp70.1 employing the HSF and the B2 gene using a separate, heat-activated transcriptional mechanism. However, the level of transcription from the hsp70.1 and B2 genes and the stability of their respective RNAs are co-ordinately regulated by the level of heat shock protein in the cell. The data indicate that auto-regulation of the level of mouse heat shock gene products is mediated by RNA polymerase II transcripts but that the regulatory mechanism can control transcription from RNA polymerase III genes as well.
The endoplasmic reticulum (ER) is a membrane-bound organelle responsible for the synthesis, assembly and post-translational modification of proteins destined for the lysosomes, Golgi and for secretion. The processes which occur in the lumen of the ER are vital to the correct functioning of the cell, and mechanisms must exist to enable the cell to monitor events within the lumen of the ER. How the cell is able to do this is not known, but it would apparently require the passage of signals from the lumen of the ER to the cytosol, from where signals can be sent to, for example, the nucleus to effect changes in transcription. Here, it is suggested that the membrane of the ER may contain the components (i.e. receptors, kinases, etc.) required for transmembrane signalling in much the same way as the plasma membrane does. This hypothesis will be discussed in relation to known ER proteins which might act as signalling proteins.
We have developed a novel permeabilized-cell system to study transcription mechanisms. In permeabilized cells, heat-induced activation of the heat shock factor and transcription of the hsp70 gene require Ca2+. Activation involves at least two steps: Ca(2+)- and heat-dependent activation of heat shock factor binding and a second step, prior to transcription of hsp70, that requires ATP and is sensitive to genistein, a protein kinase inhibitor.