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Papers by Paul Epstein

Cellular signalling, Dec 26, 2017

The levels of cAMP are regulated by phosphodiesterase enzymes (PDEs), which are targets for the t... more The levels of cAMP are regulated by phosphodiesterase enzymes (PDEs), which are targets for the treatment of inflammatory disorders. We have previously shown that PDE8 regulates T cell motility. Here, for the first time, we report that PDE8A exerts part of its control of T cell function through the V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) kinase signaling pathway. To examine T cell motility under physiologic conditions, we analyzed T cell interactions with endothelial cells and ligands in flow assays. The highly PDE8-selective enzymatic inhibitor PF-04957325 suppresses adhesion of in vivo myelin oligodendrocyte glycoprotein (MOG) activated inflammatory CD4(+) T effector (Teff) cells to brain endothelial cells under shear stress. Recently, PDE8A was shown to associate with Raf-1 creating a compartment of low cAMP levels around Raf-1 thereby protecting it from protein kinase A (PKA) mediated inhibitory phosphorylation. To test the function of this complex in Teff cells...

Frontiers in pharmacology, 2016

Abolishing the inhibitory signal of intracellular cAMP is a prerequisite for effector T (Teff) ce... more Abolishing the inhibitory signal of intracellular cAMP is a prerequisite for effector T (Teff) cell function. The regulation of cAMP within leukocytes critically depends on its degradation by cyclic nucleotide phosphodiesterases (PDEs). We have previously shown that PDE8A, a PDE isoform with 40-100-fold greater affinity for cAMP than PDE4, is selectively expressed in Teff vs. regulatory T (Treg) cells and controls CD4(+) Teff cell adhesion and chemotaxis. Here, we determined PDE8A expression and function in CD4(+) Teff cell populations in vivo. Using magnetic bead separation to purify leukocyte populations from the lung draining hilar lymph node (HLN) in a mouse model of ovalbumin-induced allergic airway disease (AAD), we found by Western immunoblot and quantitative (q)RT-PCR that PDE8A protein and gene expression are enhanced in the CD4(+) T cell fraction over the course of the acute inflammatory disease and recede at the late tolerant non-inflammatory stage. To evaluate PDE8A as a...

The present invention discloses a method for in certain embodiments, the treatment of MIF- mediat... more The present invention discloses a method for in certain embodiments, the treatment of MIF- mediated condition. In some embodiments, the method (i) binding of MIF to the CXCR2 and / or CXCR4; (Ii) CXCR2 and / or activation of CXCR4 MIF-; (Iii) the ability of MIF to form a homogeneous oligomer; (Iv) binding of CD74 to the MIF; Or it comprises the step of administering an active agent to inhibit the combination thereof.

Biochemical Journal, 1987

Frontiers in pharmacology, 2015

Stimulation of cAMP signaling induces apoptosis in glucocorticoid-sensitive and resistant CEM leu... more Stimulation of cAMP signaling induces apoptosis in glucocorticoid-sensitive and resistant CEM leukemic and MM.1 multiple myeloma cell lines, and this effect is enhanced by dexamethasone in both glucocorticoid-sensitive cell types and in glucocorticoid-resistant CEM cells. Expression of the mRNA for the glucocorticoid receptor alpha (GR) promoters 1A3, 1B and 1C, expression of mRNA and protein for GR, and the BH3-only proapoptotic proteins, Bim and Bad, and the phosphorylation state of Bad were examined following stimulation of the cAMP and glucocorticoid signaling pathways. Expression levels of GR promoters were increased by cAMP and glucocorticoid signaling, but GR protein expression was little changed in CEM and decreased in MM.1 cells. Stimulation of these two signaling pathways induced Bim in CEM cells, induced Bad in MM.1 cells, and activated Bad, as indicated by its dephosphorylation on ser112, in both cell types. This study shows that leukemic and multiple myeloma cells, incl...

Breast Cancer Research and Treatment, 2015

Almost all deaths from breast cancer arise from metastasis of the transformed cells to other site... more Almost all deaths from breast cancer arise from metastasis of the transformed cells to other sites in the body. Hence, uncovering a means of inhibiting breast cancer cell migration would provide a significant advance in the treatment of this disease. Stimulation of the cAMP signaling pathway has been shown to inhibit migration and motility of a number of cell types. A very effective way of selectively stimulating cAMP signaling is through inhibition of cyclic nucleotide phosphodiesterases (PDEs). Therefore, we examined full expression profiles of all known PDE genes at the mRNA and protein levels in four human breast cancer cell lines and eight patients' breast cancer tissues. By these analyses, expression of almost all PDE genes was seen in both cell lines and tissues. In the cell lines, appreciable expression was seen for PDEs 1C, 2A, 3B, 4A, 4B, 4D, 5A, 6B, 6C, 7A, 7B, 8A, 9A, 10A, and 11A. In patients' tissues, appreciable expression was seen for PDEs 1A, 3B, 4A, 4B, 4C, 4D, 5A, 6B, 6C, 7A, 7B, 8A, 8B, and 9A. PDE8A mRNA in particular is prominently expressed in all cell lines and patients' tissue samples examined. We show here that stimulation of cAMP signaling with cAMP analogs, forskolin, and PDE inhibitors, including selective inhibitors of PDE3, PDE4, PDE7, and PDE8, inhibit aggressive triple negative MDA-MB-231 breast cancer cell migration. Under the same conditions, these agents had little effect on breast cancer cell proliferation. This study demonstrates that PDE inhibitors inhibit breast cancer cell migration, and thus may be valuable therapeutic targets for inhibition of breast cancer metastasis. Since PDE8A is expressed in all breast cancer samples, and since dipyridamole, which inhibits PDE8, and PF-04957325, a selective PDE8 inhibitor, both inhibit migration, it suggests that PDE8A may be a valuable novel target for treatment of this disease.

Journal of bacteriology, 1978

Extracts of vegetative cells of Blastocladiella emersonii contain 5% or less of the cyclic AMP ph... more Extracts of vegetative cells of Blastocladiella emersonii contain 5% or less of the cyclic AMP phosphodiesterase activity in zoospore extracts. This difference in activity could be accounted for entirely by an increase in the differential rate of phosphodiesterase synthesis during sporulation, beginning after a lag period of about 60 min and extending for at least an additional 90 min into the 4-h sporulation process. To examine the relation between enzyme synthesis and cyclic nucleotide metabolicm, we determined the substrate specificity of phosphodiesterase synthesized during sporulation and partially purified from zoospores. Zoospore extracts contain two components, separable by gel filtration chromatography, with cyclic AMP phosphodiesterase activity. The larger component accounts for 20% of the total activity and the smaller component for 80%. Both components show essentially an absolute substrate specificity for cyclic AMP among several cyclic purine and cyclic pyrimidine nucl...

The Biochemical journal, Jan 15, 1987

The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotido... more The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and calmodulin content during development of chick ventricular myocardium were determined. The specific activity of cyclic AMP phosphodiesterase was relatively low in early embryos, increased during embryogenesis by about 4-fold to reach highest values just before hatching, and then decreased by approx. 30% within 1 week after hatching. In contrast, adenylate cyclase did not change during embryonic development, but increased by approx. 50% within 1 week after hatching. Calmodulin content remained constant at 9 micrograms/g wet wt. during embryonic development and decreased to 6 micrograms/g wet wt. by 1 week after hatching. DEAE-Sephacel chromatography of chick ventricular supernatant revealed a single major form of cyclic nucleotide phosphodiesterase activity in early embryonic (9-day E) and ...

Biochimica et biophysica acta, Jan 8, 1978

BHK fibroblasts contain two forms of cyclic AMP phosphodiesterase 3':5'-cyclic nucleotide... more BHK fibroblasts contain two forms of cyclic AMP phosphodiesterase 3':5'-cyclic nucleotide 5'-nucleotidohydrolase EC 3.1.4.17) as analyzed by linear sucrose gradient fractionation; a 3.6-S form (peak I) and a 6.7-S form (peak II). Peak I is specific for cyclic AMP as substrate and displays Michaelis-Menten kinetics with an apparent Km of 2--3 micrometer. Peak II hydrolyzes cyclic GMP and displays anomalous kinetics for cyclic AMP hydrolysis. The activity of isolated peak II for cyclic AMP is increased by storage at 4 degrees C, treatment with trypsin, or treatment with rat brain and BHK fibroblast activator proteins. The activity of isolated peak I is unaffected by these conditions. Linear sucrose gradient fractionation demonstrates that activation of peak II by trypsin leads to the formation of a 3.6-S cyclic AMP-specific enzyme form, possibly peak I. In contrast to BHK fibroblasts (and most other mammalian tissues), rat uterus contains only one form of cyclic nucleotide...

SpringerPlus, 2013

Considerable epidemiological evidence demonstrates a positive association between artificial ligh... more Considerable epidemiological evidence demonstrates a positive association between artificial light at night (LAN) levels and incidence rates of breast cancer, suggesting that exposure to LAN is a risk factor for breast cancer. There is a 30-50% higher risk of breast cancer in the highest LAN exposed countries compared to the lowest LAN countries, and studies showing higher incidence of breast cancer among shift workers exposed to more LAN have led the International Agency for Research on Cancer to classify shift work as a probable human carcinogen. Nevertheless, the means by which light can affect breast cancer is still unknown. In this study we examined established human breast cancer cell lines and patients' primary breast cancer tissues for expression of genetic components of phosphodiesterase 6 (PDE6), a cGMP-specific PDE involved in transduction of the light signal, and previously thought to be selectively expressed in photoreceptors. By microarray analysis we find highly significant expression of mRNA for the PDE6B, PDE6C, and PDE6D genes in both the cell lines and patients' tissues, minimal expression of PDE6A and PDE6G and no expression of PDE6H. Using antibody specific for PDE6β, we find expression of PDE6B protein in a wide range of patients' tissues by immunohistochemistry, and in MCF-7 breast cancer cells by immunofluorescence and Western blot analysis. Considerable expression of key circadian genes, PERIOD 2, CLOCK, TIMELESS, CRYPTOCHROME 1, and CRYPTOCHROME 2 was also seen in all breast cancer cell lines and all patients' breast cancer tissues. These studies indicate that genes for PDE6 and control of circadian rhythm are expressed in human breast cancer cells and tissues and may play a role in transducing the effects of light on breast cancer.

Bone-Metabolic Functions and Modulators, 2012

This chapter will review the evidence that stimulation of cAMP signaling through inhibition of cy... more This chapter will review the evidence that stimulation of cAMP signaling through inhibition of cyclic nucleotide phosphodiesterases (PDEs) may provide a novel means to build bone and thus form the basis of a new therapeutic treatment for osteoporosis. The maintenance of normal bone mass depends on a balance between osteoblastic bone formation and osteoclastic bone destruction [8, 10, 24]. Osteoclasts are highly motile cells, and bone resorption involves active motility of osteoclasts on bone surfaces to be resorbed [13]. Hence, agents that inhibit osteoclast motility should inhibit bone resorption. Rho GTPases, small GTP-binding proteins that belong to the Ras superfamily, represent a group of proteins that play a pivotal role in regulating cell motility and migration [69]. RhoA, a major isoform of Rho, promotes stress fiber formation in cells through activation of a downstream kinase effector termed ROCK (Rho-associated kinase). Once activated by RhoA, ROCK promotes phosphorylation of myosin light chain (MLC), producing increased contractility and stress fiber formation, necessary for motility [25]. In osteoclasts, RhoA is activated by engagement of the αV/β3 integrin receptor and the hyaluronan receptor, CD44, by osteopontin, and this activation of RhoA in osteoclasts is critical for its motility and function [13–15, 58]. RhoA can be directly phosphorylated and inactivated by cAMP-dependent protein kinase (PKA) [25, 45]. Hence, agents that stimulate the cAMP signaling pathway should inactivate RhoA, inhibit osteoclast motility, and inhibit bone resorption. Inhibitors of cyclic nucleotide phosphodiesterases (PDEs), the enzymes that normally degrade cAMP, would be excellent candidates for increasing cAMP and inhibiting motility in osteoclasts. PDEs are encoded by 21 different genes grouped into 11 different gene families based on sequence similarity, mode of regulation, and specificity for cAMP or cGMP as substrate [17]. With alternative splicing as well as the existence of multiple transcription initiation sites, at least 100 different forms of PDE have been cloned and many are expressed in a cell and tissue selective manner. As depicted in Fig. 16.1, PDEs play a central role in controlling cAMP signaling in that they regulate the steady-state levels of cAMP as well as the temporal and spatial components of cAMP within the cell, thus affecting a host of cell processes through both posttranslational modification of proteins, as well as changes in gene transcription. Pharmacological inhibitors of PDEs are under intense development and are rapidly becoming available for clinical use. Moreover, recent studies have shown that inhibitors of the fourth gene family of PDE (PDE4) profoundly inhibit the migration of several types of cells, including fibroblasts [25, 92], endothelial cells [60], leukocytes [21, 34], and several types of cancer cells [23, 57, 62, 80]. Little work has been done on modulation of RhoA activity in osteoclasts, by PDE inhibitors and the cAMP signaling pathway, but studies have demonstrated effects of PDE4 inhibitors to increase bone density. Although the mechanism(s) by which PDE4 inhibitors achieve this is still not known, we hypothesize that they may act in part through inhibition of RhoA, to inhibit osteoclast motility and function, thus inhibiting bone resorption and increasing bone density. Additionally, PDE inhibitors appear to have direct effects on osteoblasts, as well, to stimulate bone formation [37–39, 44, 53, 93, 97, 100]. This chapter will examine current evidence that PDE inhibitors may be beneficial as therapeutic agents to build bone density and treat osteoporosis.

Proceedings of the National Academy of Sciences, 1975

Cyclic GMP and cyclic AMP levels during growth and differentiation of B. emersonii were measured ... more Cyclic GMP and cyclic AMP levels during growth and differentiation of B. emersonii were measured by use of chemical, biochemical, and immunologic assays. Of particular interest was the finding that net synthesis of cyclic GMP occurred during a single stage of its life cycle, sporulation, when intracellular levels increased 50to 100-fold in a process requiring protein and RNA synthesis.

Proceedings of the National Academy of Sciences, 1996

Cytosolic extracts from a human lymphoblastoid B-cell line, RPMI-8392, established from a patient... more Cytosolic extracts from a human lymphoblastoid B-cell line, RPMI-8392, established from a patient with acute lymphocytic leukemia, contain two major forms of cyclic nucleotide phosphodiesterase (PDE): Ca2+-calmodulin dependent PDE (PDE1) and cAMP-specific PDE (PDE4). In contrast, normal quiescent human peripheral blood lymphocytes (HPBL) are devoid of PDE1 activity [Epstein, P. M.,

Neuroscience Letters, 2004

Adenosine appears to be an endogenous somnogen. The lateral dorsal tegmental/pedunculopontine nuc... more Adenosine appears to be an endogenous somnogen. The lateral dorsal tegmental/pedunculopontine nucleus (LDT/PPT) located in the mesopontine tegmentum is important in the regulation of arousal. Neurons in this nucleus are strongly hyperpolarized by adenosine and express neuronal nitric oxide synthase. Zaprinast is a cyclic nucleotide phosphodiesterase inhibitor, and has been shown in the hippocampal slice to inhibit the field excitatory postsynaptic potential. This action could be blocked by an adenosine receptor antagonist, and therefore is presumably due to adenosine release stimulated by zaprinast. In the present study we tested the effect of zaprinast on extracellular adenosine accumulation in pontine slices containing the LDT. Zaprinast at 10 M evoked an increase in extracellular adenosine concentration. This effect was blocked by impermeant inhibitors of 5-nucleotidase, indicating that the extracellular adenosine was derived from extracellular AMP. However, inhibitors of cAMP degradation had little or no effect on zaprinast-evoked adenosine accumulation, suggesting that extracellular cAMP was not the source. Removal of extracellular calcium inhibited the effect of zaprinast. These results demonstrate that a pathway exists by which zaprinast stimulates extracellular adenosine accumulation, and the presence of this pathway in the pontine slice suggests the possibility that it may be relevant for the regulation of behavioral state.

Methods, 1998

been grouped into seven broad gene families based Cyclic nucleotide phosphodiesterases (PDEs) are... more been grouped into seven broad gene families based Cyclic nucleotide phosphodiesterases (PDEs) are repreon similar structural and functional relationships: sented by a superfamily of structurally and functionally re-Ca 2/-calmodulin-dependent (PDE1), cGMP-stimulated enzymes of which more than 30 different forms have lated (PDE2), cGMP-inhibited (PDE3), cAMP-speso far been identified and grouped into seven broad gene cific (PDE4), cGMP-specific (PDE5), photoreceptor families, some of which contain multiple genes and many (PDE6), and higher-affinity, drug-resistant, cAMPsplice variants, within a given gene family. Since all of the specific (PDE7) PDEs (2). A number of excellent reforms of PDE have the potential to regulate levels of the views have been written recently that describe the second messenger, cAMP or cGMP, and some of the forms characteristics of these different PDE forms, their appear to be tissue specific in their expression and differenregulation, potential physiological function, and tially regulated, it would be useful to be able to selectively progress in development of pharmacological inhibiinhibit a given form of PDE, to study the physiological consetors of PDE as therapeutic agents (2-11). Gene famquences of this inhibition, with the intent of possible theraily-specific inhibitors have been found for all but the peutic application. While gene family-specific pharmacologi-PDE7 gene family, but no pharmacological inhibitor cal inhibitors exist for six of the seven gene families, none of is yet capable of selectively inhibiting a specific PDE these inhibitors is yet capable of distinguishing PDE members isoform within a given gene family. Selective inhibiwithin a given gene family in its inhibition. One approach to tion of the expression of specific PDE isoforms could, selectively inhibit a specific form of PDE, without affecting however, be achieved through use of antisense techothers, is through use of antisense oligonucleotides to block nology, thus providing a means to examine the physthe expression of a given PDE form. This article describes iological function of specific PDE isozymes, as well ways to optimally develop and test antisense oligonucleoas providing potential therapeutic applications. tides to inhibit expression of PDE. ᭧ 1998 Academic Press The goal of antisense technology is to develop small oligonucleotides or oligodeoxynucleotides (ODNs), plasmids, or retroviral vectors that can be introduced easily into viable cells in order to inhibit gene products specifically (12). Although retroviral delivery of reverse-oriented cDNAs expressing anti-Since the first report, in 1958, of an enzymatic sense RNA have been used successfully in some exactivity capable of hydrolyzing cAMP (1), it has beperimental systems (12), this article concentrates on come clear that this enzymatic activity, termed cyclic the development of ODNs, with the object in mind nucleotide phosphodiesterase (PDE), consists of a of using these ODNs for selective inhibition of the complex isozymic superfamily represented by differexpression of PDE isoforms. In designing antisense ent forms more than 30 of which have been identified and cloned so far. These isozymic PDE forms have ODNs (aODNs) for use in biological systems, one 21

Mammalian Genome, 2000

Abstract. CDC25A is a member of a group of highly related, dual-specificity phosphatases that pro... more Abstract. CDC25A is a member of a group of highly related, dual-specificity phosphatases that promote cell cycle phase tran-sitions by regulating the activity of cyclin-dependent kinases. Here we report the cloning and genomic sequence of 21,067 nucleotides encompassing the ...

Life Sciences, 1991

Using O-phosphotyrosine as a substrate, human platelets were shown to contain a highly active pho... more Using O-phosphotyrosine as a substrate, human platelets were shown to contain a highly active phosphotyrosine phosphatase (PTPase) activity. This activity was potently inhibited by vanadate, molybdate, and HgCl2. About 80% of the PTPase activity was particulate. When Triton-solubilized PTPase activity from whole platelets was applied to a DEAE Sephacel column about 40% came through unbound. The activity that bound was eluted by a NaCl gradient as a broad, heterogeneous peak. The possibility is raised for the existence of multiple forms of phosphotyrosine phosphatases in human platelets. That one or more of these forms may be regulated by activators of platelet aggregation and secretion, such as thrombin and collagen, is discussed.

Journal of Cellular Physiology, 1979

The activities of myometrial cyclic nucleotide phosphodiesterases (PDEs) and the sensitivity of t... more The activities of myometrial cyclic nucleotide phosphodiesterases (PDEs) and the sensitivity of these enzymes to the effector molecules, cGMP and cAMP, were determined in the 100,000 g supernatant of homogenates from pregnant and spayed rhesus monkeys. The specific activities (per mg nitrogen) of the myometrial cyclic nucleotide PDEs in the supernatant from spayed monkeys were higher than those from pregnant monkeys at all substrate levels studied. However, when calculated on the basis of the DNA content of the myometrium, which was 8 times higher in the spayed than in the pregnant animals, the specific activities were lower in the tissue from spayed animals. At substrate levels of 2 . 5 micron-cAMP, low levels of cGMP (0 . 1-1 . 0 micron) caused the same percentage increase in cGMP-PDE activity in both tissues. At high substrate levels of 100 micron-cAMP, 1 micron-cGMP inhibited only the cAMP-PDE from spayed monkeys, and the enzyme from spayed monkeys was more effectively inhibited by 10 and 40 micron-cGMP than was the enzyme from pregnant animals. The cGMP-PDE activity was inhibited by cAMP (1 . 0-50 . 0 micron), and the percentage inhibition with increasing levels of cAMP appeared to be similar in the two series. The levels of cGMP and cAMP that modify the rate of hydrolysis of the other nucleotide in rhesus myometrium seem to be within the physiological range for these compounds in situ. It therefore appears possible that cAMP and cGMP are each involved in regulating the degradation of the other nucleotide in rhesus myometrium.

Emerging Therapeutic Targets, 1998

Cellular signalling, Dec 26, 2017

The levels of cAMP are regulated by phosphodiesterase enzymes (PDEs), which are targets for the t... more The levels of cAMP are regulated by phosphodiesterase enzymes (PDEs), which are targets for the treatment of inflammatory disorders. We have previously shown that PDE8 regulates T cell motility. Here, for the first time, we report that PDE8A exerts part of its control of T cell function through the V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) kinase signaling pathway. To examine T cell motility under physiologic conditions, we analyzed T cell interactions with endothelial cells and ligands in flow assays. The highly PDE8-selective enzymatic inhibitor PF-04957325 suppresses adhesion of in vivo myelin oligodendrocyte glycoprotein (MOG) activated inflammatory CD4(+) T effector (Teff) cells to brain endothelial cells under shear stress. Recently, PDE8A was shown to associate with Raf-1 creating a compartment of low cAMP levels around Raf-1 thereby protecting it from protein kinase A (PKA) mediated inhibitory phosphorylation. To test the function of this complex in Teff cells...

Frontiers in pharmacology, 2016

Abolishing the inhibitory signal of intracellular cAMP is a prerequisite for effector T (Teff) ce... more Abolishing the inhibitory signal of intracellular cAMP is a prerequisite for effector T (Teff) cell function. The regulation of cAMP within leukocytes critically depends on its degradation by cyclic nucleotide phosphodiesterases (PDEs). We have previously shown that PDE8A, a PDE isoform with 40-100-fold greater affinity for cAMP than PDE4, is selectively expressed in Teff vs. regulatory T (Treg) cells and controls CD4(+) Teff cell adhesion and chemotaxis. Here, we determined PDE8A expression and function in CD4(+) Teff cell populations in vivo. Using magnetic bead separation to purify leukocyte populations from the lung draining hilar lymph node (HLN) in a mouse model of ovalbumin-induced allergic airway disease (AAD), we found by Western immunoblot and quantitative (q)RT-PCR that PDE8A protein and gene expression are enhanced in the CD4(+) T cell fraction over the course of the acute inflammatory disease and recede at the late tolerant non-inflammatory stage. To evaluate PDE8A as a...

The present invention discloses a method for in certain embodiments, the treatment of MIF- mediat... more The present invention discloses a method for in certain embodiments, the treatment of MIF- mediated condition. In some embodiments, the method (i) binding of MIF to the CXCR2 and / or CXCR4; (Ii) CXCR2 and / or activation of CXCR4 MIF-; (Iii) the ability of MIF to form a homogeneous oligomer; (Iv) binding of CD74 to the MIF; Or it comprises the step of administering an active agent to inhibit the combination thereof.

Biochemical Journal, 1987

Frontiers in pharmacology, 2015

Stimulation of cAMP signaling induces apoptosis in glucocorticoid-sensitive and resistant CEM leu... more Stimulation of cAMP signaling induces apoptosis in glucocorticoid-sensitive and resistant CEM leukemic and MM.1 multiple myeloma cell lines, and this effect is enhanced by dexamethasone in both glucocorticoid-sensitive cell types and in glucocorticoid-resistant CEM cells. Expression of the mRNA for the glucocorticoid receptor alpha (GR) promoters 1A3, 1B and 1C, expression of mRNA and protein for GR, and the BH3-only proapoptotic proteins, Bim and Bad, and the phosphorylation state of Bad were examined following stimulation of the cAMP and glucocorticoid signaling pathways. Expression levels of GR promoters were increased by cAMP and glucocorticoid signaling, but GR protein expression was little changed in CEM and decreased in MM.1 cells. Stimulation of these two signaling pathways induced Bim in CEM cells, induced Bad in MM.1 cells, and activated Bad, as indicated by its dephosphorylation on ser112, in both cell types. This study shows that leukemic and multiple myeloma cells, incl...

Breast Cancer Research and Treatment, 2015

Almost all deaths from breast cancer arise from metastasis of the transformed cells to other site... more Almost all deaths from breast cancer arise from metastasis of the transformed cells to other sites in the body. Hence, uncovering a means of inhibiting breast cancer cell migration would provide a significant advance in the treatment of this disease. Stimulation of the cAMP signaling pathway has been shown to inhibit migration and motility of a number of cell types. A very effective way of selectively stimulating cAMP signaling is through inhibition of cyclic nucleotide phosphodiesterases (PDEs). Therefore, we examined full expression profiles of all known PDE genes at the mRNA and protein levels in four human breast cancer cell lines and eight patients' breast cancer tissues. By these analyses, expression of almost all PDE genes was seen in both cell lines and tissues. In the cell lines, appreciable expression was seen for PDEs 1C, 2A, 3B, 4A, 4B, 4D, 5A, 6B, 6C, 7A, 7B, 8A, 9A, 10A, and 11A. In patients' tissues, appreciable expression was seen for PDEs 1A, 3B, 4A, 4B, 4C, 4D, 5A, 6B, 6C, 7A, 7B, 8A, 8B, and 9A. PDE8A mRNA in particular is prominently expressed in all cell lines and patients' tissue samples examined. We show here that stimulation of cAMP signaling with cAMP analogs, forskolin, and PDE inhibitors, including selective inhibitors of PDE3, PDE4, PDE7, and PDE8, inhibit aggressive triple negative MDA-MB-231 breast cancer cell migration. Under the same conditions, these agents had little effect on breast cancer cell proliferation. This study demonstrates that PDE inhibitors inhibit breast cancer cell migration, and thus may be valuable therapeutic targets for inhibition of breast cancer metastasis. Since PDE8A is expressed in all breast cancer samples, and since dipyridamole, which inhibits PDE8, and PF-04957325, a selective PDE8 inhibitor, both inhibit migration, it suggests that PDE8A may be a valuable novel target for treatment of this disease.

Journal of bacteriology, 1978

Extracts of vegetative cells of Blastocladiella emersonii contain 5% or less of the cyclic AMP ph... more Extracts of vegetative cells of Blastocladiella emersonii contain 5% or less of the cyclic AMP phosphodiesterase activity in zoospore extracts. This difference in activity could be accounted for entirely by an increase in the differential rate of phosphodiesterase synthesis during sporulation, beginning after a lag period of about 60 min and extending for at least an additional 90 min into the 4-h sporulation process. To examine the relation between enzyme synthesis and cyclic nucleotide metabolicm, we determined the substrate specificity of phosphodiesterase synthesized during sporulation and partially purified from zoospores. Zoospore extracts contain two components, separable by gel filtration chromatography, with cyclic AMP phosphodiesterase activity. The larger component accounts for 20% of the total activity and the smaller component for 80%. Both components show essentially an absolute substrate specificity for cyclic AMP among several cyclic purine and cyclic pyrimidine nucl...

The Biochemical journal, Jan 15, 1987

The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotido... more The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and calmodulin content during development of chick ventricular myocardium were determined. The specific activity of cyclic AMP phosphodiesterase was relatively low in early embryos, increased during embryogenesis by about 4-fold to reach highest values just before hatching, and then decreased by approx. 30% within 1 week after hatching. In contrast, adenylate cyclase did not change during embryonic development, but increased by approx. 50% within 1 week after hatching. Calmodulin content remained constant at 9 micrograms/g wet wt. during embryonic development and decreased to 6 micrograms/g wet wt. by 1 week after hatching. DEAE-Sephacel chromatography of chick ventricular supernatant revealed a single major form of cyclic nucleotide phosphodiesterase activity in early embryonic (9-day E) and ...

Biochimica et biophysica acta, Jan 8, 1978

BHK fibroblasts contain two forms of cyclic AMP phosphodiesterase 3':5'-cyclic nucleotide... more BHK fibroblasts contain two forms of cyclic AMP phosphodiesterase 3':5'-cyclic nucleotide 5'-nucleotidohydrolase EC 3.1.4.17) as analyzed by linear sucrose gradient fractionation; a 3.6-S form (peak I) and a 6.7-S form (peak II). Peak I is specific for cyclic AMP as substrate and displays Michaelis-Menten kinetics with an apparent Km of 2--3 micrometer. Peak II hydrolyzes cyclic GMP and displays anomalous kinetics for cyclic AMP hydrolysis. The activity of isolated peak II for cyclic AMP is increased by storage at 4 degrees C, treatment with trypsin, or treatment with rat brain and BHK fibroblast activator proteins. The activity of isolated peak I is unaffected by these conditions. Linear sucrose gradient fractionation demonstrates that activation of peak II by trypsin leads to the formation of a 3.6-S cyclic AMP-specific enzyme form, possibly peak I. In contrast to BHK fibroblasts (and most other mammalian tissues), rat uterus contains only one form of cyclic nucleotide...

SpringerPlus, 2013

Considerable epidemiological evidence demonstrates a positive association between artificial ligh... more Considerable epidemiological evidence demonstrates a positive association between artificial light at night (LAN) levels and incidence rates of breast cancer, suggesting that exposure to LAN is a risk factor for breast cancer. There is a 30-50% higher risk of breast cancer in the highest LAN exposed countries compared to the lowest LAN countries, and studies showing higher incidence of breast cancer among shift workers exposed to more LAN have led the International Agency for Research on Cancer to classify shift work as a probable human carcinogen. Nevertheless, the means by which light can affect breast cancer is still unknown. In this study we examined established human breast cancer cell lines and patients' primary breast cancer tissues for expression of genetic components of phosphodiesterase 6 (PDE6), a cGMP-specific PDE involved in transduction of the light signal, and previously thought to be selectively expressed in photoreceptors. By microarray analysis we find highly significant expression of mRNA for the PDE6B, PDE6C, and PDE6D genes in both the cell lines and patients' tissues, minimal expression of PDE6A and PDE6G and no expression of PDE6H. Using antibody specific for PDE6β, we find expression of PDE6B protein in a wide range of patients' tissues by immunohistochemistry, and in MCF-7 breast cancer cells by immunofluorescence and Western blot analysis. Considerable expression of key circadian genes, PERIOD 2, CLOCK, TIMELESS, CRYPTOCHROME 1, and CRYPTOCHROME 2 was also seen in all breast cancer cell lines and all patients' breast cancer tissues. These studies indicate that genes for PDE6 and control of circadian rhythm are expressed in human breast cancer cells and tissues and may play a role in transducing the effects of light on breast cancer.

Bone-Metabolic Functions and Modulators, 2012

This chapter will review the evidence that stimulation of cAMP signaling through inhibition of cy... more This chapter will review the evidence that stimulation of cAMP signaling through inhibition of cyclic nucleotide phosphodiesterases (PDEs) may provide a novel means to build bone and thus form the basis of a new therapeutic treatment for osteoporosis. The maintenance of normal bone mass depends on a balance between osteoblastic bone formation and osteoclastic bone destruction [8, 10, 24]. Osteoclasts are highly motile cells, and bone resorption involves active motility of osteoclasts on bone surfaces to be resorbed [13]. Hence, agents that inhibit osteoclast motility should inhibit bone resorption. Rho GTPases, small GTP-binding proteins that belong to the Ras superfamily, represent a group of proteins that play a pivotal role in regulating cell motility and migration [69]. RhoA, a major isoform of Rho, promotes stress fiber formation in cells through activation of a downstream kinase effector termed ROCK (Rho-associated kinase). Once activated by RhoA, ROCK promotes phosphorylation of myosin light chain (MLC), producing increased contractility and stress fiber formation, necessary for motility [25]. In osteoclasts, RhoA is activated by engagement of the αV/β3 integrin receptor and the hyaluronan receptor, CD44, by osteopontin, and this activation of RhoA in osteoclasts is critical for its motility and function [13–15, 58]. RhoA can be directly phosphorylated and inactivated by cAMP-dependent protein kinase (PKA) [25, 45]. Hence, agents that stimulate the cAMP signaling pathway should inactivate RhoA, inhibit osteoclast motility, and inhibit bone resorption. Inhibitors of cyclic nucleotide phosphodiesterases (PDEs), the enzymes that normally degrade cAMP, would be excellent candidates for increasing cAMP and inhibiting motility in osteoclasts. PDEs are encoded by 21 different genes grouped into 11 different gene families based on sequence similarity, mode of regulation, and specificity for cAMP or cGMP as substrate [17]. With alternative splicing as well as the existence of multiple transcription initiation sites, at least 100 different forms of PDE have been cloned and many are expressed in a cell and tissue selective manner. As depicted in Fig. 16.1, PDEs play a central role in controlling cAMP signaling in that they regulate the steady-state levels of cAMP as well as the temporal and spatial components of cAMP within the cell, thus affecting a host of cell processes through both posttranslational modification of proteins, as well as changes in gene transcription. Pharmacological inhibitors of PDEs are under intense development and are rapidly becoming available for clinical use. Moreover, recent studies have shown that inhibitors of the fourth gene family of PDE (PDE4) profoundly inhibit the migration of several types of cells, including fibroblasts [25, 92], endothelial cells [60], leukocytes [21, 34], and several types of cancer cells [23, 57, 62, 80]. Little work has been done on modulation of RhoA activity in osteoclasts, by PDE inhibitors and the cAMP signaling pathway, but studies have demonstrated effects of PDE4 inhibitors to increase bone density. Although the mechanism(s) by which PDE4 inhibitors achieve this is still not known, we hypothesize that they may act in part through inhibition of RhoA, to inhibit osteoclast motility and function, thus inhibiting bone resorption and increasing bone density. Additionally, PDE inhibitors appear to have direct effects on osteoblasts, as well, to stimulate bone formation [37–39, 44, 53, 93, 97, 100]. This chapter will examine current evidence that PDE inhibitors may be beneficial as therapeutic agents to build bone density and treat osteoporosis.

Proceedings of the National Academy of Sciences, 1975

Cyclic GMP and cyclic AMP levels during growth and differentiation of B. emersonii were measured ... more Cyclic GMP and cyclic AMP levels during growth and differentiation of B. emersonii were measured by use of chemical, biochemical, and immunologic assays. Of particular interest was the finding that net synthesis of cyclic GMP occurred during a single stage of its life cycle, sporulation, when intracellular levels increased 50to 100-fold in a process requiring protein and RNA synthesis.

Proceedings of the National Academy of Sciences, 1996

Cytosolic extracts from a human lymphoblastoid B-cell line, RPMI-8392, established from a patient... more Cytosolic extracts from a human lymphoblastoid B-cell line, RPMI-8392, established from a patient with acute lymphocytic leukemia, contain two major forms of cyclic nucleotide phosphodiesterase (PDE): Ca2+-calmodulin dependent PDE (PDE1) and cAMP-specific PDE (PDE4). In contrast, normal quiescent human peripheral blood lymphocytes (HPBL) are devoid of PDE1 activity [Epstein, P. M.,

Neuroscience Letters, 2004

Adenosine appears to be an endogenous somnogen. The lateral dorsal tegmental/pedunculopontine nuc... more Adenosine appears to be an endogenous somnogen. The lateral dorsal tegmental/pedunculopontine nucleus (LDT/PPT) located in the mesopontine tegmentum is important in the regulation of arousal. Neurons in this nucleus are strongly hyperpolarized by adenosine and express neuronal nitric oxide synthase. Zaprinast is a cyclic nucleotide phosphodiesterase inhibitor, and has been shown in the hippocampal slice to inhibit the field excitatory postsynaptic potential. This action could be blocked by an adenosine receptor antagonist, and therefore is presumably due to adenosine release stimulated by zaprinast. In the present study we tested the effect of zaprinast on extracellular adenosine accumulation in pontine slices containing the LDT. Zaprinast at 10 M evoked an increase in extracellular adenosine concentration. This effect was blocked by impermeant inhibitors of 5-nucleotidase, indicating that the extracellular adenosine was derived from extracellular AMP. However, inhibitors of cAMP degradation had little or no effect on zaprinast-evoked adenosine accumulation, suggesting that extracellular cAMP was not the source. Removal of extracellular calcium inhibited the effect of zaprinast. These results demonstrate that a pathway exists by which zaprinast stimulates extracellular adenosine accumulation, and the presence of this pathway in the pontine slice suggests the possibility that it may be relevant for the regulation of behavioral state.

Methods, 1998

been grouped into seven broad gene families based Cyclic nucleotide phosphodiesterases (PDEs) are... more been grouped into seven broad gene families based Cyclic nucleotide phosphodiesterases (PDEs) are repreon similar structural and functional relationships: sented by a superfamily of structurally and functionally re-Ca 2/-calmodulin-dependent (PDE1), cGMP-stimulated enzymes of which more than 30 different forms have lated (PDE2), cGMP-inhibited (PDE3), cAMP-speso far been identified and grouped into seven broad gene cific (PDE4), cGMP-specific (PDE5), photoreceptor families, some of which contain multiple genes and many (PDE6), and higher-affinity, drug-resistant, cAMPsplice variants, within a given gene family. Since all of the specific (PDE7) PDEs (2). A number of excellent reforms of PDE have the potential to regulate levels of the views have been written recently that describe the second messenger, cAMP or cGMP, and some of the forms characteristics of these different PDE forms, their appear to be tissue specific in their expression and differenregulation, potential physiological function, and tially regulated, it would be useful to be able to selectively progress in development of pharmacological inhibiinhibit a given form of PDE, to study the physiological consetors of PDE as therapeutic agents (2-11). Gene famquences of this inhibition, with the intent of possible theraily-specific inhibitors have been found for all but the peutic application. While gene family-specific pharmacologi-PDE7 gene family, but no pharmacological inhibitor cal inhibitors exist for six of the seven gene families, none of is yet capable of selectively inhibiting a specific PDE these inhibitors is yet capable of distinguishing PDE members isoform within a given gene family. Selective inhibiwithin a given gene family in its inhibition. One approach to tion of the expression of specific PDE isoforms could, selectively inhibit a specific form of PDE, without affecting however, be achieved through use of antisense techothers, is through use of antisense oligonucleotides to block nology, thus providing a means to examine the physthe expression of a given PDE form. This article describes iological function of specific PDE isozymes, as well ways to optimally develop and test antisense oligonucleoas providing potential therapeutic applications. tides to inhibit expression of PDE. ᭧ 1998 Academic Press The goal of antisense technology is to develop small oligonucleotides or oligodeoxynucleotides (ODNs), plasmids, or retroviral vectors that can be introduced easily into viable cells in order to inhibit gene products specifically (12). Although retroviral delivery of reverse-oriented cDNAs expressing anti-Since the first report, in 1958, of an enzymatic sense RNA have been used successfully in some exactivity capable of hydrolyzing cAMP (1), it has beperimental systems (12), this article concentrates on come clear that this enzymatic activity, termed cyclic the development of ODNs, with the object in mind nucleotide phosphodiesterase (PDE), consists of a of using these ODNs for selective inhibition of the complex isozymic superfamily represented by differexpression of PDE isoforms. In designing antisense ent forms more than 30 of which have been identified and cloned so far. These isozymic PDE forms have ODNs (aODNs) for use in biological systems, one 21

Mammalian Genome, 2000

Abstract. CDC25A is a member of a group of highly related, dual-specificity phosphatases that pro... more Abstract. CDC25A is a member of a group of highly related, dual-specificity phosphatases that promote cell cycle phase tran-sitions by regulating the activity of cyclin-dependent kinases. Here we report the cloning and genomic sequence of 21,067 nucleotides encompassing the ...

Life Sciences, 1991

Using O-phosphotyrosine as a substrate, human platelets were shown to contain a highly active pho... more Using O-phosphotyrosine as a substrate, human platelets were shown to contain a highly active phosphotyrosine phosphatase (PTPase) activity. This activity was potently inhibited by vanadate, molybdate, and HgCl2. About 80% of the PTPase activity was particulate. When Triton-solubilized PTPase activity from whole platelets was applied to a DEAE Sephacel column about 40% came through unbound. The activity that bound was eluted by a NaCl gradient as a broad, heterogeneous peak. The possibility is raised for the existence of multiple forms of phosphotyrosine phosphatases in human platelets. That one or more of these forms may be regulated by activators of platelet aggregation and secretion, such as thrombin and collagen, is discussed.

Journal of Cellular Physiology, 1979

The activities of myometrial cyclic nucleotide phosphodiesterases (PDEs) and the sensitivity of t... more The activities of myometrial cyclic nucleotide phosphodiesterases (PDEs) and the sensitivity of these enzymes to the effector molecules, cGMP and cAMP, were determined in the 100,000 g supernatant of homogenates from pregnant and spayed rhesus monkeys. The specific activities (per mg nitrogen) of the myometrial cyclic nucleotide PDEs in the supernatant from spayed monkeys were higher than those from pregnant monkeys at all substrate levels studied. However, when calculated on the basis of the DNA content of the myometrium, which was 8 times higher in the spayed than in the pregnant animals, the specific activities were lower in the tissue from spayed animals. At substrate levels of 2 . 5 micron-cAMP, low levels of cGMP (0 . 1-1 . 0 micron) caused the same percentage increase in cGMP-PDE activity in both tissues. At high substrate levels of 100 micron-cAMP, 1 micron-cGMP inhibited only the cAMP-PDE from spayed monkeys, and the enzyme from spayed monkeys was more effectively inhibited by 10 and 40 micron-cGMP than was the enzyme from pregnant animals. The cGMP-PDE activity was inhibited by cAMP (1 . 0-50 . 0 micron), and the percentage inhibition with increasing levels of cAMP appeared to be similar in the two series. The levels of cGMP and cAMP that modify the rate of hydrolysis of the other nucleotide in rhesus myometrium seem to be within the physiological range for these compounds in situ. It therefore appears possible that cAMP and cGMP are each involved in regulating the degradation of the other nucleotide in rhesus myometrium.

Emerging Therapeutic Targets, 1998