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Posts Tagged ‘Nrf2’


Sensors and Signaling in Oxidative Stress

Author and Curator: Larry H. Bernstein, MD, FCAP

This is article ELEVEN in the following series on Calcium Role in Cardiovascular Diseases

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton
Larry H Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-
that-are-related-to-the-actin-cytoskeleton/

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-
skeleton-and-lipid-structures-in-signaling-and-cell-motility/

Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease
Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD
and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-
exchange-mechanism-in-health-and-disease/

Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and
Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia,
Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-
involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-
post-ischemic-arrhythmia-similarities-and-differen/

Part V: Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Larry H Bernstein, MD, FCAP
and
Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocytosis/

Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary
Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-in-cardiac-gene-therapy-inhalable-gene-therapy-
for-pulmonary-arterial-hypertension-and-percutaneous-intra-coronary-artery-infusion-for-heart-failure-contributions-by-roger-j-hajjar/

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure –
Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-
and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells:
The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-
muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction
(Ryanopathy) and Calcium as Neurotransmitter Sensor
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-
and-calcium-release-related-contractile-dysfunction-ryanopathy/

Part X: Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of
vesicles with cell membranes during Neurotransmission
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-
regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

Part XI: Sensors and Signaling in Oxidative Stress
Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

This important article on oxidative stress was published in Free Radical Biol. and Med.

Nrf2:INrf2(Keap1) Signaling in Oxidative Stress

James W. Kaspar, Suresh K. Niture, and Anil K. Jaiswal*
Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD

Free Radic Biol Med. 2009 Nov; 47(9): 1304–1309.           http://dx.doi.org/10.1016/j.freeradbiomed.2009.07.035

Nrf2:INrf2(Keap1) are cellular sensors of chemical and radiation induced oxidative and electrophilic stress. Nrf2 is a nuclear transcription factor that controls the expression and coordinated induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins. This is a mechanism of critical importance for cellular protection and cell survival. Nrf2 is retained in the cytoplasm by an inhibitor INrf2. INrf2 functions as an adapter for Cul3/Rbx1 mediated degradation of Nrf2. In response to oxidative/electrophilic stress, Nrf2 is switched on and then off by distinct early and delayed mechanisms. Oxidative/electrophilic modification of INrf2cysteine151 and/or PKC phosphorylation of Nrf2serine40 results in

  • the escape or release of Nrf2 from INrf2.

Nrf2 is stabilized and

  • translocates to the nucleus,
  • forms heterodimers with unknown proteins, and
  • binds antioxidant response element (ARE) that
  • leads to coordinated activation of gene expression.
  • It takes less than fifteen minutes from the time of exposure to switch on nuclear import of Nrf2. This is followed by activation of a delayed mechanism that controls switching off of Nrf2 activation of gene expression. GSK3β phosphorylates Fyn at unknown threonine residue(s) leading to nuclear localization of Fyn. Fyn phosphorylates Nrf2tyrosine568
  • resulting in nuclear export of Nrf2, binding with INrf2 and
  • degradation of Nrf2.

The switching on and off of Nrf2 protects cells against free radical damage, prevents apoptosis and promotes cell survival.

Introduction

Oxidative stress is induced by a vast range of factors including xenobiotics, drugs, heavy metals and ionizing radiation. Oxidative stress leads to the generation of Reactive Oxygen Species (ROS) and electrophiles. ROS and electrophiles generated can have a profound impact on survival, growth development and evolution of all living organisms [1,2] ROS include

  • both free radicals, such as the superoxide anion and the hydroxyl radical, and
  • oxidants such as hydrogen peroxide [3].

ROS and electrophiles can cause diseases such as cancer, cardiovascular complications, acute and chronic inflammation, and neurodegenerative diseases [1]. Therefore, it is obvious that

  • cells must constantly labor to control levels of ROS, preventing them from accumulation.

Much of what we know about the mechanisms of protection against oxidative stress has come from the study of prokaryotic cells [4,5]. Prokaryotic cells utilize transcription factors OxyR and SoxRS to sense the redox state of the cell, and

  • during oxidative stress these factors induce the expression of nearly eighty defensive genes [5].

Eukaryotic cells have similar mechanisms to protect against oxidative stress [Fig. 1; ref. 3,6–9]. Initial effect of oxidative/electrophilic stress leads to activation of a battery of defensive gene expression that leads to detoxification of chemicals and ROS and prevention of free radical generation and cell survival [Fig. 1].

Fig 1.  Chemical and radiation exposure and coordinated induction of defensive genes.

Fig. 1. Chemical and radiation exposure and coordinated induction of defensive genes.

Of these genes, some are enzymes such as NAD(P)H:quinine oxidoreductase 1 (NQO1), NRH:quinone oxidoreductase 2 (NQO2), glutathione S-transferase Ya subunit (GST Ya Subunit), heme oxygenase 1 (HO-1), and γ-glutamylcysteine synthetase (γ-GCS), also known as glutamate cysteine ligase (GCL). Other genes have end products that regulate a wide variety of cellular activities including

  • signal transduction,
  • proliferation, and
  • immunologic defense reactions.

There is a wide variety of factors associated with the cellular response to oxidative stress. For example,

  • NF-E2 related factor 2 (Nrf2),
  • heat shock response activator protein 1, and
  • NF-kappaB promote cell survival,

whereas activation of c-jun, N-terminal kinases (JNK), p38 kinase and TP53 may lead to cell cycle arrest and apoptosis [10]. The Nrf2 pathway is regarded as the most important in the cell to protect against oxidative stress. [3,6–9]. It is noteworthy that accumulation of ROS and/or electrophiles leads to oxidative/electrophile stress,

  • membrane damage,
  • DNA adducts formation and
  • mutagenicity [Fig. 1].

These changes lead to degeneration of tissues and premature aging, apoptotic cell death, cellular transformation and cancer.

Antioxidant Response Element and Nrf2

Promoter analysis identified a cis-acting enhancer sequence designated as the antioxidant response element (ARE) that

  • controls the basal and inducible expression of antioxidant genes in response to xenobiotics, antioxidants, heavy metals and UV light [11].

The ARE sequence is responsive to a broad range of structurally diverse chemicals apart from β-nafthoflavone and phenolic antioxidants [12]. Mutational analysis revealed GTGACA***GC to be the core sequence of the ARE [11,13–14]. This core sequence is present in all Nrf2 downstream genes that respond to antioxidants and xenobiotics [3,6–9]. Nrf2 binds to the ARE and regulates ARE-mediated antioxidant enzyme genes expression and induction in response to a variety of stimuli including antioxidants, xenobiotics, metals, and UV irradiation [6,15–21].

Nrf2 is ubiquitously expressed in a wide range of tissue and cell types [22–24] and belongs to a subset of basic leucine zipper genes (bZIP) sharing a conserved structural domain designated as a cap’n’collar domain which is highly conserved in Drosphila transcription factor CNC (Fig. 2; ref. 25].

Fig. 2. Schematic Presentation of Various Domains of Nrf (Nrf1, Nrf2, Nrf3) and INrf2

Fig. 2. Schematic Presentation of Various Domains of Nrf (Nrf1, Nrf2, Nrf3) and INrf2

Nrf, NF-E2 Related Factor; INrf2, Inhibitor of Nrf2; NTR, N-Terminal Region; BTB, Broad complex, Tramtrack, Bric-a-brac; IVR, Intervening/linker Region; DGR, Kelch domain/ diglycine repeats; CTR, C-Terminal Region.

The basic region, just upstream of the leucine zipper region,

  • is responsible for DNA binding [3] and
  • the acidic region is required for transcriptional activation.

ARE-mediated transcriptional activation requires heterodimerization of Nrf2 with other bZIP proteins including Jun (c-Jun, Jun-D, and Jun-B) and small Maf (MafG, MafK, MafF) proteins [18– 20,26–27].

Initial evidence demonstrating the role of Nrf2 in antioxidant-induction of detoxifying enzymes came from studies on

  • the role of Nrf2 in ARE-mediated regulation of NQO1 gene expression [17].

Nrf2 was subsequently shown to be involved in

  • the transcriptional activation of other ARE-responsive genes such as
    • GST Ya, γ-GCS, HO-1, antioxidants, proteasomes, and drug transporters [3,6–9,28–33].

Overexpression of Nrf2 cDNA was shown to upregulate the expression and induction of the NQO1 gene in response to antioxidants and xenobiotics [17]. In addition, Nrf2-null mice exhibited a marked

  • decrease in the expression and induction of NQO1,
  • indicating that Nrf2 plays an essential role in the in vivo regulation of NQO1 in response to oxidative stress [26].

The importance of this transcription factor in upregulating ARE-mediated gene expression has been demonstrated by several in vivo and in vitro studies [reviewed in ref. 3]. The results indicate that Nrf2 is an important activator of phase II antioxidant genes [3,8].

Negative Regulation of Nrf2 mediated by INrf2

A cytosolic inhibitor (INrf2), also known as Keap1 (Kelch-like ECH-associating protein 1), of Nrf2 was identified and reported [Fig. 2; ref. 34–35]. INrf2, existing as a dimer [36], retains Nrf2 in the cytoplasm. Analysis of the INrf2 amino acid sequence and domain structure-function analyses have revealed that

  • INrf2 has a BTB (broad complex, tramtrack, bric-a-brac)/ POZ (poxvirus, zinc finger) domain and
  • a Kelch domain [34–35] also known as the DGR domain (Double glycine repeat) [37].

Keap1 has three additional domains/regions:

  1. the N-terminal region (NTR),
  2. the invervening region (IVR), and
  3. the C-terminal region (CTR) [8].

The BTB/POZ domain has been shown to be

  • a protein-protein interaction domain.

In the Drosophila Kelch protein, and in IPP,

  • the Kelch domain binds to actin [38–39]
  • allowing the scaffolding of INrf2 to the actin cytoskeleton
    • which plays an important role in Nrf2 retention in the cytosol [40].

The main function of INrf2 is to serve as

  • an adapter for the Cullin3/Ring Box 1 (Cul3/Rbx1) E3 ubiquitin ligase complex [41–43].

Cul3 serves as a scaffold protein that forms the E3 ligase complex with Rbx1 and recruits a cognate E2 enzyme [8].

INrf2

  1. via its N-terminal BTB/POZ domain binds to Cul3 [44] and
  2. via its C-terminal Kelch domain binds to the substrate Nrf2
  • leading to the ubiquitination and degradation of Nrf2 through the 26S proteasome [45–49].

Under normal cellular conditions, the cytosolic INrf2/Cul3-Rbx1 complex is constantly degrading Nrf2. When a cell is exposed to oxidative stress Nrf2 dissociates from the INrf2 complex, stabilizes and translocates into the nucleus leading to activation of ARE-mediated gene expression [3,6–9]. An alternative theory is that Nrf2 in response to oxidative stress escapes INrf2 degradation, stabilizes and translocates in the nucleus [49–50]. We suggested the theory of escape of Nrf2 from INrf2 [49] and similar suggestion was also made in another report [50]. However, the follow up studies in our laboratory could not support the escape theory. Escape theory is a possibility but has to be proven by experiments before it can be adapted. Therefore, we will use the release of Nrf2 from INrf2 in the rest of this review.

Numerous reports have suggested that

  • any mechanism that modifies INrf2 and/or Nrf2 disrupting the Nrf2:INrf2 interaction will result in the upregulation of ARE-mediated gene expression.

A model Nrf2:INrf2 signaling from antioxidant and xenobiotic to activation of ARE-mediated defensive gene expression is shown in Fig. 3.

Fig. 3. Nrf2 signaling in ARE-mediated coordinated activation of defensive genes

Fig. 3. Nrf2 signaling in ARE-mediated coordinated activation of defensive genes

Since the metabolism of antioxidants and xenobiotics results in the generation of ROS and electrophiles [51], it is thought that these molecules might act as second messengers, activating ARE-mediated gene expression. Several protein kinases including PKC, ERK, MAPK, p38, and PERK [49,52– 56] are known to modify Nrf2 and activate its release from INrf2. Among these mechanisms,

  1. oxidative/electrophilic stress mediated phosphorylation of Nrf2 at serine40 by PKC is necessary for Nrf2 release from INrf2, but
  2. is not required for Nrf2 accumulation in the nucleus [49,52–53].

In addition to post-translational modification in Nrf2, several crucial residues in INrf2 have also been proposed to be important for activation of Nrf2. Studies based on

  • the electrophile mediated modification,
  • location and
  • mutational analyses revealed
    • that three cysteine residues, Cys151, Cys273 and Cys288 are crucial for INrf2 activity [50].

INrf2 itself undergoes ubiquitination by the Cul3 complex, via a proteasomal independent pathway,

  • which was markedly increased in response to phase II inducers such as antioxidants [57].

It has been suggested that normally INrf2 targets Nrf2 for ubiquitin mediated degradation but

  • electrophiles may trigger a switch of Cul3 dependent ubiquitination from Nrf2 to INrf2 resulting in ARE gene induction.

The redox modulation of cysteines in INrf2

  • might be a mechanism redundant to the phosphorylation of Nrf2 by PKC, or that
  • the two mechanisms work in concert.

In addition to cysteine151 modification,

  • phosphorylation of Nrf2 has also been shown to play a role in INrf2 retention and release of Nrf2.

Serine104 of INrf2 is required for dimerization of INrf2, and

  • mutations of serine104 led to the disruption of the INrf2 dimer leading to the release of Nrf2 [36].

Recently, Eggler at al. demonstrated that modifying specific cysteines of the electrophile-sensing human INrf2 protein is insufficient to disrupt binding to the Nrf2 domain Neh2 (58). Upon introduction of electrophiles, modification of INrf2C151 leads to a change in the conformation of the BTB domain by means of perturbing the homodimerization site, disrupting Neh2 ubiquitination, and causing ubiquitination of INrf2. Modification of INrf2 cysteines by electrophiles does not lead to disruption of the INrf2–Nrf2 complex. Rather, the switch of ubiquitination from Nrf2 to INrf2 leads to Nrf2 nuclear accumulation.

More recently, our laboratory demonstrated that phosphorylation and de-phosphorylation of tyrosine141 in INrf2 regulates its stability and degradation, respectively [59]. The de-phosphorylation of tyrosine141 caused destabilization and degradation of INrf2 leading to the release of Nrf2. Furthermore, we showed that prothymosin-α mediates nuclear import of the INrf2/Cul3-Rbx1 complex [60]. The INrf2/Cul3-Rbx1 complex inside the nucleus exchanges prothymosin-α with Nrf2 resulting in degradation of Nrf2. These results led to the conclusion that prothymosin-α mediated nuclear import of INrf2/Cul3-Rbx1 complex leads to ubiquitination and degradation of nuclear Nrf2 presumably to regulate nuclear level of Nrf2 and rapidly switch off the activation of Nrf2 downstream gene expression. An auto-regulatory loop also exists within the Nrf2 pathway [61]. An ARE was identified in the INrf2 promoter that facilitates Nrf2 binding causing induction of the INrf2 gene. Nrf2 regulates INrf2 by controlling its transcription, and INrf2 controls Nrf2 by serving as an adaptor for degradation.

Other Regulatory Mediators of Nrf2

Bach1 (BTB and CNC homology 1, basic leucine zipper transcription factor 1) is a transcription repressor [62] that is ubiquitously expressed in tissues [63–64] and distantly related to Nrf2 [8]. In the absence of cellular stress, Bach1 heterodimers with small Maf proteins [65] that bind to the (ARE) [66] repressing gene expression. In the presence of oxidative stress, Bach1 releases from the ARE and is replaced by Nrf2. Bach1 competes with Nrf2 for binding to the ARE leading to suppression of Nrf2 downstream genes [66].

Nuclear import of Nrf2, from time of exposure to stabilization, takes roughly two hours [67]. This is followed by activation of a delayed mechanism involving Glycogen synthase kinase 3 beta (GSK3f3) that controls switching off of Nrf2 activation of gene expression (Fig. 3). GSK3f3 is a multifunctional serine/threonine kinase, which plays a major role in various signaling pathways [68]. GSK3f3 phosphorylates Fyn, a tyrosine kinase, at unknown threonine residue(s) leading to nuclear localization of Fyn [69]. Fyn phosphorylates Nrf2 tyrosine 568 resulting in nuclear export of Nrf2, binding with INrf2 and degradation of Nrf2 [70].

The negative regulation of Nrf2 by Bach1 and GSK3f3/Fyn are important in repressing Nrf2 downstream genes that were induced in response to oxidative/electrophilic stress. The tight control of Nrf2 is vital for the cells against free radical damage, prevention of apoptosis and cell survival [3,6–9,70].

Nrf2 in Cytoprotection, Cancer and Drug Resistance

Nrf2 is a major protective mechanism against xenobiotics capable of damaging DNA and initiating carcinogenesis [71]. Inducers of Nrf2 function as blocking agents that prevents carcinogens from reaching target sites, inhibits parent molecules undergoing metabolic activation, or subsequently preventing carcinogenic species from interacting with crucial cellular macromolecules, such as DNA, RNA, and proteins [72]. A plausible mechanism by which blocking agents impart their chemopreventive activity is the induction of detoxification and antioxidant enzymes [73]. Oltipraz, 3H-1,2,-dithiole-3-thione (D3T), Sulforaphane, and Curcumin can be considered potential chemopreventive agents because

  • these compounds have all been shown to induce Nrf2 [74–81].

Studies have shown a role of Nrf2 in protection against cadmium and manganese toxicity [82]. Nrf2 also plays an important role in reduction of methyl mercury toxicity [83]. Methylmercury activates Nrf2 and the activation of Nrf2 is essential for reduction of methylmercury by facilitating its excretion into extracellular space. In vitro and in vivo studies have shown a role of Nrf2 in neuroprotection and protection against Parkinson’s disease [84– 86]. Disruption of Nrf2 impairs the resolution of hyperoxia-induced acute lung injury and inflammation in mice [87]. Nrf2-knockout mice were more prone to

  • tumor growth when exposed to carcinogens such as benzo[a]pyrene, diesel exhaust, and N-nitrosobutyl (4-hydroxybutyl) amine [88–90].

INrf2/Nrf2 signaling is also shown to regulate oxidative stress tolerance and lifespan in Drosophila [91].

A role of Nrf2 in drug resistance is suggested based on its property to induce detoxifying and antioxidant enzymes (92–97). The loss of INrf2 (Keap1) function is shown to

  • lead to nuclear accumulation of Nrf2, activation of metabolizing enzymes and drug resistance (95).

Studies have reported mutations resulting in dysfunctional INrf2 in lung, breast and bladder cancers (96–100). A recent study reported that somatic mutations also occur in the coding region of Nrf2, especially in cancer patients with a history of smoking or suffering from squamous cell carcinoma (101). These mutations abrogate its interaction with INrf2 and nuclear accumulation of Nrf2. This gives advantage to

  • cancer cell survival and
  • undue protection from anti-cancer treatments.

However, the understanding of the mechanism of Nrf2 induced drug resistance remains in its infancy. In addition, the studies on Nrf2 regulated downstream pathways that contribute to drug resistance remain limited.

Future Perspectives

Nrf2 creates a new paradigm in cytoprotection, cancer prevention and drug resistance. Considerable progress has been made to better understand all mechanisms involved within the intracellular pathways regulating Nrf2 and its downstream genes. Preliminary studies demonstrate that

  • deactivation of Nrf2 is as important as activation of Nrf2.

Further studies are needed to better understand the negative regulation of Nrf2. Also better understanding of the negative regulation of Nrf2 could help design a new class of effective chemopreventive compounds not only targeting Nrf2 activation, but also

  • targeting the negative regulators of Nrf2.

Abbreviations: 

Nrf2    NF-E2 related factor 2;  INrf2   Inhibitor of Nrf2 also known as Keap1;   ROS    Reactive oxygen species.

References (1-15 of 101)

1. Breimer LH. Molecular Mechanisms of oxygen radical carcinogenesis and mutagenesis: the role of DNA base damage. Mol Carcinog 1990;3:188–197. [PubMed: 2206282]

2. Meneghini R. Iron homeostasis, oxidative stress, and DNA damage. Free Radic Biol Med 1997;23:783– 792. [PubMed: 9296456]

3. Jaiswal AK. Nrf2 signaling in coordinated activation of antioxidant gene expression. Free Radic Biol Med 2004;36:1199–1207. [PubMed: 15110384]

4. Bauer CE, Elsen S, Bird TH. Mechanisms for redox control of gene expression. Annu Rev Microbiol 1999;53:495–523. [PubMed: 10547699]

5. Zheng M, Storz G. Redox sensing by prokaryotic transcription factors. Biochem Pharm 2000;59:1–6. [PubMed: 10605928]

6. Dhakshinamoorthy S, Long DJ II, Jaiswal AK. Antioxidant regulation of genes encoding enzymes that detoxify xenobiotics and carcinogens. Current Topics in Cellular Regulation 2000;36:201–206. [PubMed: 10842753]

7. Zhang DD. Mechanistic studies of the Nrf2-Keap1 signaling pathway. Drug Metab Rev 2006;38:769– 789. [PubMed: 17145701]

8. Kobayashi M, Yamamoto M. Nrf2-Keap1 regulation of cellular defense mechanisms against electrophiles and reactive oxygen species. Adv Enzyme Regul 2006;46:113–140. [PubMed: 16887173]

9. Copple IM, Goldring CE, Kitteringham NR, Park BK. The Nrf2-Keap1 defense pathway: role in protection against drug-induced toxicity. Toxicology 2008;246:24–33. [PubMed: 18083283]

10. Halliwell, B.; Gutteridge, JMC. Free radicals in biology and medicine. Vol. 4. Oxford University Press; 2007.

  • 11. Rushmore TH, Morton MR, Pickett CB. The antioxidant responsive element. Actiavtion by oxidative stress and identification of the DNA consensus sequence required for functional activity. J Biol Chem 1991;266:11632–11639. [PubMed: 1646813]
  • 12. Rushmore TH, King RG, Paulson KE, Pickett CB. Regulation of glutathione S-transferase Ya subunit gene expression: identification of a unique xenobiotics-responsive element controlling inducible expression by planar aromatic compounds. Proc Natl Acad Sci USA 1990;87:3826–3830. [PubMed: 2160079]
  • 13. Xie T, Belinsky M, Xu Y, Jaiswal AK. ARE and TRE-mediated regulation of gene expression: response to xenobiotics and antioxidants. J Biol Chem 1995;270:6894–6900. [PubMed: 7896838]
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Rushmore TH, Pickett CB. Glutathione S-transferases, structures, regulation, and therapeutic implications. J Biol Chem 1993;268:11475–11478. [PubMed: 8505281]

15. Jaiswal AK. Regulation of genes encoding NAD(P)H:quinone oxidoreductases. Free Radic Biol Med 2000;29:254–252. [PubMed: 11035254]

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Larry H Bernstein, MD, Reporter & Curator
https://pharmaceuticalintelligence.com/2013/06/21/Nrf2 Role in Blocking DNA Damage/lhbern

 

DNA damage has been a central focus of carcinogenesis.  The following is of great interest in this respect.

 

 

 

Nrf2 as a novel molecular target for chemoprevention.

 

Lee JS, Surh YJ.

 

Cancer Lett. 2005 Jun 28;224(2):171-84. Epub 2004 Nov 11.

 

Source

 

National Research Laboratory of Molecular Carcinogenesis and Chemoprevention, College of Pharmacy, Seoul National University, Shinlim-dong, Kwanak-ku, Seoul 151-742, South Korea.

 

 

Abstract

 

One of the rational and effective strategies for chemoprevention is the blockade of DNA damage caused by carcinogenic insult. This can be achieved either

 

  • by reducing the formation of reactive carcinogenic species
  • or stimulating their detoxification.

 

A wide spectrum of xenobiotic metabolizing enzymes catalyze both phase I (oxidation and reduction) and phase II biotransformation (conjugation) reactions involved in carcinogen activation and/or deactivation. Several antioxidant-response element (ARE)-regulated gene products such as

 

  • glutathione S-transferase,
  • NAD(P)H:quinone oxidoreductase 1,
  • UDP-glucuronosyltransferase,
  • gamma-glutamate cysteine ligase, and
  • hemeoxygenase-1

 

are known to mediate detoxification and/or to exert antioxidant functions thereby protecting cells from genotoxic damage.

 

The transcription of ARE-driven genes is regulated, at least in part,

 

  • by nuclear transcription factor erythroid 2p45 (NF-E2)-related factor 2 (Nrf2),
  • which is sequestered in cytoplasm by Kelch-like ECH-associated protein 1 (Keap1).

 

Exposure of cells to ARE inducers results in

 

  1. the dissociation of Nrf2 from Keap1 and
  2. facilitates translocation of Nrf2 to the nucleus,
  3. where it heterodimerizes with small Maf protein, and
  4. binds to ARE,

 

eventually resulting in the transcriptional regulation of target genes.

 

The Nrf2-Keap1-ARE signaling pathway can be modulated by several upstream kinases including

 

  • phosphatidylinositol 3-kinase,
  • protein kinase C, and
  • mitogen-activated protein kinases.

 

Selected Nrf2-Keap1-ARE activators, such as

 

  • oltipraz,
  • anethole dithiolethione,
  • sulforaphane,
  • 6-methylsulphinylhexyl isothiocyanate,
  • curcumin,
  • caffeic acid phenethyl ester,
  • 4′-bromoflavone, etc.

 

are potential chemopreventive agents. This mini-review will focus on a chemopreventive strategy directed towards

 

  • protection of DNA and other important cellular molecules by
  • inducing de novo synthesis of phase II detoxifying or antioxidant genes via the Nrf2-ARE core signaling pathway.

 

 

PMID: 15914268 [PubMed – indexed for MEDLINE]

 

 

 

 

 

English: Graph of Nrf2 publications (pubmed se...

English: Graph of Nrf2 publications (pubmed search) by year (Photo credit: Wikipedia)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Most lifespan influencing genes affect the rat...

Most lifespan influencing genes affect the rate of DNA damage (Photo credit: Wikipedia)

 

Single-strand and double-strand DNA damage

Single-strand and double-strand DNA damage (Photo credit: Wikipedia)

 

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CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease – Part IIC


CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease – Part IIC

Author: Larry H. Bernstein, MD, FCAP, Triplex Medical Science

Part I: The Initiation and Growth of Molecular Biology and Genomics – Part I From Molecular Biology to Translational Medicine: How Far Have We Come, and Where Does It Lead Us?

https://pharmaceuticalintelligence.wordpress.com/wp-admin/post.php?post=8634&action=edit&message=1

Part II: CRACKING THE CODE OF HUMAN LIFE is divided into a three part series.

Part IIA. “CRACKING THE CODE OF HUMAN LIFE: Milestones along the Way” reviews the Human Genome Project and the decade beyond.

https://pharmaceuticalintelligence.com/2013/02/12/cracking-the-code-of-human-life-milestones-along-the-way/

Part IIB. “CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics” lays the manifold multivariate systems analytical tools that has moved the science forward to a groung that ensures clinical application.

https://pharmaceuticalintelligence.com/2013/02/13/cracking-the-code-of-human-life-the-birth-of-bioinformatics-and-computational-genomics/

Part IIC. “CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease “ will extend the discussion to advances in the management of patients as well as providing a roadmap for pharmaceutical drug targeting.

https://pharmaceuticalintelligence.com/2013/02/14/cracking-the-code-of-human-life-recent-advances-in-genomic-analysis-and-disease/

To be followed by:
Part III will conclude with Ubiquitin, it’s role in Signaling and Regulatory Control.

Part IIC of series on CODE OF HUMAN LIFE
CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease

This final paper of Part II concludes a thorough review of the scientific events leading to the discovery of the human genome, the purification and identification of the components of the chromosome and the DNA structure and role in regulation of embryogenesis, and potential targets for cancer.

The first two articles, Part IIA, Part IIB,  go into some depth to elucidate the problems and breakthoughs encountered in the Human Genome Project, and the construction of a 3-D model necessary to explain interactions at a distance.

Part IIC, the final article, is entirely concerned with clinical application of this treasure trove of knowledge to resolving diseases of epigenetic nature in the young and the old, chronic inflammatory diseases, autoimmune diseases, infectious disease, gastrointestinal disorders, neurological and neurodegenerative diseases, and cancer.

CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease – Part IIC

1. Gene Links to Heart Disease

 

Recently, large studies have identified some of the genetic basis for important common diseases such as heart disease and diabetes, but most of the genetic contribution to them remains undiscovered. Now researchers at the University of Massachusetts Amherst led by biostatistician Andrea Foulkes have applied sophisticated statistical tools to existing large databases to reveal substantial new information about genes that cause such conditions as high cholesterol linked to heart disease.

Foulkes says, “This new approach to data analysis provides opportunities for developing new treatments.” It also advances approaches

  • to identifying people at greatest risk for heart disease. Another important point is that our method is straightforward to use with freely
  • available computer software and can be applied broadly to advance genetic knowledge of many diseases.

The new analytical approach she developed with cardiologist Dr. Muredach Reilly at the University of Pennsylvania and others is called “Mixed modeling of Meta-Analysis P-values” or MixMAP. Because it makes use of existing public databases, the powerful new method

  • represents a low-cost tool for investigators.
  • MixMAP draws on a principled statistical modeling framework and the vast array of summary data now available from genetic association
  • studies to formally test at a new, locus-level, association.

While that traditional statistical method looks for one unusual “needle in a haystack” as a possible disease signal, Foulkes and colleagues’

  • new method uses knowledge of DNA regions in the genome that are likely to
  • contain several genetic signals for disease variation clumped together in one region.
  • Thus, it is able to detect groups of unusual variants rather than just single SNPs, offering a way to “call out” gene
  • regions that have a consistent signal above normal variation.

http://Science.com/Science News/Identify Genes Linked to Heart Disease/

2. Apolipoprotein(a) Genetic Sequence Variants

The LPA gene codes for apolipoprotein(a), which, when linked with low-density lipoprotein particles, forms lipoprotein(a) [Lp(a)] —

  • a well-studied molecule associated with coronary artery disease (CAD). The Lp(a) molecule has both atherogenic and thrombogenic effects in vitro , but the extent to which these translate to differences in how atherothrombotic disease presents is unknown.

LPA contains many single-nucleotide polymorphisms, and 2 have been identified by previous groups as being strongly associated with

  • levels of Lp(a) and, as a consequence, strongly associated with CAD.

However, because atherosclerosis is thought to be a systemic disease, it is unclear to what extent Lp(a) leads to atherosclerosis in other arterial beds (eg, carotid, abdominal aorta, and lower extremity),

  • as well as to other thrombotic disorders (eg, ischemic/cardioembolic stroke and venous thromboembolism).

Such distinctions are important, because therapies that might lower Lp(a) could potentially reduce forms of atherosclerosis beyond the coronary tree.

To answer this question, Helgadottir and colleagues compiled clinical and genetic data on the LPA gene from thousands of previous

  • participants in genetic research studies from across the world. They did not have access to Lp(a) levels, but by knowing the genotypes for
  • 2 LPA variants, they inferred the levels of Lp(a) on the basis of prior associations between these variants and Lp(a) levels. [1]

Their studies included not only individuals of white European descent but also a significant proportion of black persons, in order to

  • widen the generalizability of their results.

Their main findings are that LPA variants (and, by proxy, Lp(a) levels) are associated with

  • CAD,
  • peripheral arterial disease,
  • abdominal aortic aneurysm,
  • number of CAD vessels,
  • age at onset of CAD diagnosis, and
  • large-artery atherosclerosis-type stroke.

They did not find an association with

  • cardioembolic or small-vessel disease-type stroke;
  • intracranial aneurysm;
  • venous thrombosis;
  • carotid intima thickness; or,
  • in a small subset of individuals, myocardial infarction.

Apolipoprotein(a) Genetic Sequence Variants Associated With Systemic Atherosclerosis and Coronary Atherosclerotic Burden but Not With Venous Thromboembolism. Helgadottir A, Gretarsdottir S, Thorleifsson G, et al.    J Am Coll Cardiol. 2012;60:722-729

English: Structure of the LPA protein. Based o...

English: Structure of the LPA protein. Based on PyMOL rendering of PDB 1i71. (Photo credit: Wikipedia)

Micrograph of an artery that supplies the hear...

Micrograph of an artery that supplies the heart with significant atherosclerosis and marked luminal narrowing. Tissue has been stained using Masson’s trichrome. (Photo credit: Wikipedia)

Genomic Blueprint of the Heart

Scientists at the Gladstone Institutes have revealed the precise order and timing of hundreds of genetic “switches” required to construct a fully

  • functional heart from embryonic heart cells — providing new clues into the genetic basis for some forms of congenital heart disease.

In a study being published online today in the journal Cell, researchers in the laboratory of Gladstone Senior Investigator Benoit Bruneau, PhD,

  • employed stem cell technology, next-generation DNA sequencing and computing tools to piece together the instruction manual, or “genomic
  • blueprint” for how a heart becomes a heart. These findings offer renewed hope for combating life-threatening heart defects such as arrhythmias (irregular heart beat) and ventricular septal defects (“holes in the heart”).

ScienceDaily (Sep. 13, 2012)

They approach heart formation with a wide-angle lens by

  • looking at the entirety of the genetic material that gives heart cells their unique identity.

The news comes at a time of emerging importance for the biological process called “epigenetics,” in which a non-genetic factor impacts a cell’s genetic

  • makeup early during development — but sometimes with longer-term consequences. All of the cells in an organism contain the same DNA, but the
  • epigenetic instructions encoded in specific DNA sequences give the cell its identity. Epigenetics is of particular interest in heart formation, as the
  • incorrect on-and-off switching of genes during fetal development can lead to congenital heart disease — some forms of which may not be apparent until adulthood.

the scientists took embryonic stem cells from mice and reprogrammed them into beating heart cells by mimicking embryonic development in a petri dish. Next, they extracted the DNA from developing and mature heart cells, using an advanced gene-sequencing technique called ChIP-seq that lets scientists “see” the epigenetic signatures written in the DNA.

Map of Heart Disease Death Rates in US White M...

Map of Heart Disease Death Rates in US White Males from 2000-2004 (Photo credit: Wikipedia)

Estimated propability of death or non-fatal my...

Estimated propability of death or non-fatal myocardial-infarction over one year corresponding ti selectet values of the individual scores. Ordinate: individual score, abscissa: Propability of death or non-fatal myocardial infarction in 1 year (in %) (Photo credit: Wikipedia)

simply finding these signatures was only half the battle — we next had to decipher which aspects of heart formation they encoded

To do that, we harnessed the computing power of the Gladstone Bioinformatics Core. This allowed us to take the mountains of data collected from

  • gene sequencing and organize it into a readable, meaningful blueprint for how a heart becomes a heart.”

http://ScienceDaily.org/Scientists Map the Genomic Blueprint of the Heart.  ScienceDaily.

Performance of transcription factor identification tools from differential gene expression data

A three step process is a clear way to establish belief in the performance of transcription factor identification tools

  • from differential gene expression data.
  • identify several types of differential gene expression data sets where the stimulus or trigger is clearly know
  • identify the transcription factors most likely associated with the sets expression data.
  • perform an upstream analysis from the identified transcription factor.

If the transcription factor and upstream analysis tools can trace the signal cascade back to the stimulus, the tools are

  • clearly producing relevant results, and belief in the performance of the analysis tools is established.

At this point, the tools can be directed with confidence to more challenging analyses such as

  • developed resistance or pathway elucidation.

The performance of IPA‘s new Transcription Factor and Upstream analysis tools was evaluated on the following datasets (processing details below):

  • TGFb stimulation, 1 hour, A549 lung adenocarcinoma cell line
  • BMP2 stimulation, 1 hour, Mouse Embryonic Stem Cell E14Tg2A.4
  • TNFa stimulation, 1 hour primary murine hepatocytes

For each of the above datasets, an upstream analysis from the identified transcription factors correctly identified the stimulus. IPA’s tools were very

  • easy to use and the
  • analysis time for the above experiments was less than one minute.

The performance, speed, and ease of use can only be characterized as very good, perhaps leading to breakthroughs when extended and used creatively. Ingenuity’s new transcription factor analysis tool in IPA, coupled with Ingenuity’s established upstream grow tools,  should be strongly considered for every lab analyzing differential expression data.

http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17896

http://www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSE2639

http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE19272

Differential expression data was obtained from CEL files using the Matlab functions:

affyrma, genelowvalfilter, genevarfilter, mattest, and mavolcanoplot.

Rick Stanton, Pathway Analysis Consultant Ingenuity.com

3. miR-200a regulates Nrf2 activation by targeting Keap1 mRNA in breast cancer cells.

Eades G, Yang M, Yao Y, Zhang Y, Zhou Q. J Biol Chem. 2011 Nov 25;286(47):40725-33. Epub 2011 Sep 16.
http://JBiolChem.com/miR-200a regulates Nrf2 activation by targeting Keap1 mRNA in breast cancer cells.

NF-E2-related factor 2 (Nrf2) is an important transcription factor that

  • activates the expression of cellular detoxifying enzymes.

Nrf2 expression is largely regulated through the association of Nrf2 with Kelch-like ECH-associated protein 1 (Keap1), which

  • results in cytoplasmic Nrf2 degradation.

Conversely, little is known concerning the regulation of Keap1 expression. Until now, a regulatory role for microRNAs (miRs) in controlling Keap1 gene expression had not been characterized. By using miR array-

  • based screening, we observed miR-200a silencing in breast cancer cells and
  • demonstrated that upon re-expression, miR-200a
  • targets the Keap1 3′-untranslated region (3′-UTR), leading to Keap1 mRNA degradation. Loss of this regulatory mechanism may
  • contribute to the dysregulation of Nrf2 activity in breast cancer. Previously, we have identified epigenetic repression of miR-200a

in breast cancer cells. Here, we find that treatment with epigenetic therapy, the histone deacetylase inhibitor suberoylanilide hydroxamic acid, restored miR-200a expression and reduced Keap1 levels. This reduction in Keap1 levels corresponded with

  • Nrf2 nuclear translocation
  • and activation of Nrf2-dependent NAD(P)H-quinone oxidoreductase 1 (NQO1) gene transcription.

Moreover, we found that Nrf2 activation inhibited the anchorage-independent growth of breast cancer cells. Finally, our in vitro observations were confirmed in a model of carcinogen-induced mammary hyperplasia in vivo. In conclusion, our study demonstrates

  • that miR-200a regulates the Keap1/Nrf2 pathway in mammary epithelium, and we find that epigenetic therapy can restore miR-200a
  • regulation of Keap1 expression,
  • reactivating the Nrf2-dependent antioxidant pathway in breast cancer.

Nuclear factor-like 2  (erythroid-derived 2, also known as NFE2L2 or Nrf2, is a transcription factor that in humans is encoded by the NFE2L2 gene.[1])  NFE2L2 induces the expression of various genes including those that encode for several antioxidant enzymes, and it may play a physiological role in the regulation of oxidative stress. Investigational drugs that target NFE2L2 are of interest as potential therapeutic interventions for

  • oxidative-stress related pathologies.

4. Highly active zinc finger nucleases by extended modular assembly

MS Bhakta, IM Henry, DG Ousterout, KT Das, et al.  Corresponding author; email: djsegal@ucdavis.edu
http://CSHNLpress.com/Highly active zinc finger nucleases by extended modular assembly

Zinc finger nucleases (ZFNs) are important tools for genome engineering. Despite intense interest by many academic groups,

  • the lack of robust non-commercial methods has hindered their widespread use. The modular assembly (MA) of ZFNs from
  • publicly-available one-finger archives provides a rapid method to create proteins that can recognize a very broad spectrum of DNA sequences.

However, three- and four-finger arrays often fail to produce active nucleases. Efforts to improve the specificity of the one-finger archives have not increased the success rate above 25%, suggesting that the MA method might

  • be inherently inefficient due to its insensitivity to context-dependent effects.

Here we present the first systematic study on the effect of array length on ZFN activity.  ZFNs composed of six-finger MA arrays produced mutations at 15 of 21 (71%) targeted

  • loci in human and mouse cells. A novel Drop-Out Linker scheme was used to rapidly assess three- to six-finger combinations,
  • demonstrating that shorter arrays could improve activity in some cases. Analysis of 268 array variants revealed that half of

MA ZFNs of any array composition that exceed an ab initio

  • B-score cut-off of 15 were active.
  • MA ZFNs are able to target more DNA sequences with higher success rates than other methods.

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date http://genome.cshlp.org/site/misc/terms.xhtml
After six months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported License), as described at
http://creativecommons.org/licenses/by-nc/3.0/Highly_active_zinc_finger_nucleases_by_extended_ modular_assembly/

PERSONALIZED MEDICINE in the Pipeline

These insightful reviews are based on the strategic data and insights from Thomson Reuters Cortellis™ for Competitive Intelligence.  (A Review of April-June 2012).

http://ThomsonReuters.com/DIFFERENTIATED INNOVATION: PERSONALIZED MEDICINE IN THE PIPELINE/ Cortellis™ for Competitive Intelligence/APRIL-JUNE 2012

The majority of diseases are complex and multi-factorial, involving multiple genes interacting with environmental factors. At the genetic level,

  • information from genome-wide association studies that elucidate common patterns of genetic variation across various human populations,
  • in addition to profiling, technologies can be utilized in discovery research to provide snapshots of genes and expression profiles that are controlled
  • by the same regulatory mechanism and are altered between healthy and diseased states.

The characterization of genes that are abnormally expressed in disease tissues could further be employed as

  • diagnostic markers,
  • prognostic indicators of efficacy and/or toxicity, or as
  • targets for therapeutic intervention.

As the defining catalyst that exponentially paved the way for personalized medicine, information from the published genome sequence revealed that much of the genetic variations in humans are concentrated in about 0.1 percent of the over 3 billion base pairs in the haploid DNA. Most of these variations involve substitution of a single nucleotide for another at a given location in the genetic sequence, known as single nucleotide polymorphism (SNP).

  • Combinations of linked SNPs aggregate together to form haplotypes and
  • together these serve as markers for locating genetic variations in DNA sequences.

SNPs located within the protein-coding region of a gene or within the control regions of DNA that regulate a gene’s activity could

  • have a substantial effect on the encoded protein and thus influence phenotypic outcomes.

Analyzing SNPs between patient population cohorts could highlight specific genotypic variations which can be correlated with specific phenotypic variations in disease predisposition and drug responses.

Prior to the genomic revolution, many of the established therapies were directed against less than 500 drug targets, with many of the top selling drugs acting on well defined protein pathways. However, the sequencing of the human genome has massively expanded the pool of molecular targets that could be exploited in unmet medical needs and currently, of the approximately 22,300 protein-coding genes in the human code, it has been estimated that up to 3000 are druggable. Furthermore, genomic technologies such as

  • high-throughput sequencing
  • and transcription profiling,

can be used to identify and validate biologically relevant target molecules, or can be applied to cell-based and mice disease models or directly to in vivo human tissues,

  • helping to correlate gene targets with phenotypic traits of complex diseases.

This is particularly important, as

  • insufficient validation of target gene/proteins in complex diseases may be a contributing factor in the decline in R&D productivity.

Personalized medicine no doubt is already having a tremendous impact on drug development pipelines. According to a study conducted by the Tufts Center for the Study of Drug Development, more than 90 percent of biopharmaceutical companies now utilize at least some

  • genomics-derived targets in their drug discovery programs.

However, pipeline analysis from Cortellis for Competitive Intelligence suggests that there is still a scientific gap that has resulted in difficulty optimizing these novel genomic targets into the clinical R&D portfolios of major pharmaceutical companies, particularly outside the oncology field. Selected examples of personalized medicine product candidates in clinical development include (see TABLE 4).

Table 4: Selected Personalized Medicines in Clinical Development
(DATA are Derived from Cortellis for Competitive Intelligence & Thomson Reuters IntegritySM)
http://Thomson Reuters.com/Cortellis for Competitive Intelligence/IntegritySM/Table_4_Selected_Personalized_Medicines_in_Clinical_Development/

PHARMA MATTERS | SPOTLIGHT ON… PERSONALIZED MEDICINE

The paucity of actual targeted therapy examples, especially outside oncology, suggest

  • that integration of the personalized medicine paradigm into biopharmaceutical R&D is still fraught with challenges.

Despite the fact that the Human genome Project has been completed for over ten years, the broader application of genomics with drug development

  • still remains unrealized, and is hampered by a number of scientific challenges. One of the major obstacles stems from
  • incomplete association of genomic alterations with complex disease pathways and the phenotypic consequences.

As the modality of most complex diseases are multi-factorial, understanding how each genomic driver event plays a role in disease and the

  • interaction/interdependence with other genetic and environmental factors is important for
  • determining the rationale for targeted prevention or treatment of the disease.

Mutations found in Melanomas may shed light on Cancer Growth

Gina Kolata. New York Times.
http://NewYorkTimes.com/mutations_found_in_melanomas_may_shed-light_on_how_cancers_grow/

Mutations in Melanoma are in regions that control genes, not in the genes themselves. The mutations are exactly the type caused by exposure to ultraviolet light.  The findings are reported in two papers in http://Science.com/ScienceExpress/

The findings do not suggest new treatments, but they help explain how melanomas – and possibly – other cancers – develop and what drives their growth. This is a modification found in the “dark matter”, according to Dr. Levi A. Garraway,  the 99 percent of DNA in a region that regulates genes. A small control region was mutated in 7 out of 10 of the tumors, commonly of one or two tiny changes.
A German Team led by Rajiv Kumar (Heidelberg) and Dirk Schadendorf (Essen) looked at a family whose members tended to get melanomas.  Their findings indicate that those inherited with the mutations might be born with cells that have taken the first step toward cancer.
The mutations spur cells to make telomerase, that keeps the cells immortal by preventing them from losing the ends of their chromosome, the telomere. Abundant telomerase occurs in 90 percent of cancers, according to Immaculata De Vivo at Harvard Medical School.
The importance of the findings is that the mechanism of telomerase involvement in cancer is now within view. But it is not clear how to block the telomerase production in cancer cells.
A slight mutation in the matched nucleotides c...

A slight mutation in the matched nucleotides can lead to chromosomal aberrations and unintentional genetic rearrangement. (Photo credit: Wikipedia)

Comment

This discussion addresses the issues raised about the direction to follow in personalized medicine. Despite the amount of work necessary to bring the clarity that is sought after, the experiments and experimental design is most essential.

  • The arrest of ciliogenesis in ovarian cancer cell lines compared to wild type (WT) ovarian epithelial cells, and
  •  The link to suppressing ciliogenesis by AURA protein and CHFR at the base of the cilium, which disappears at mitosis or with proliferation.
  •  There is no accumulation by upregulation of PDGF under starvation by the cancer cells compared to the effect in WT OSE.

Here we have a systematic combination of signaling events tied to changes in putative biomarkers that occur synchronously in Ov cancer cell lines.

These changes are identified with changes in

  • proliferation,
  • loss of ciliary structure, and
  • proliferation.

In this described scenario,

  • WT OSE cells would be arrested, and
  • it appears that they would take the path to apoptosis (under starvation).

Even without more information, this cluster is what one wants to have in a “syndromic classification”. The information used to form the classification entails the identification of strong ‘signaling-related’ biomarkers. The Gli2 peptide has to be part of this.

In principle, a syndromic classification would be ideally expected to have no less than 64 classes. If the classification is “weak”, then the class frequencies would be close to what one would expect in the WT OSE. In this case, in reality,

  • several combinatorial classes would have low frequency, and
  • others would be quite high.

This obeys the classification rules established by feature identification, and the information gain described by Solomon Kullback and extended by Akaike.

Does this have to be the case for all different cancer types? I don’t think so. The cells are different in ontogenesis.  In this case, even the WT OSE have mesenchymal features and so, are not fully directed to epithelial expression.  This happens to be the case in actual anatomic expression of the ovary.  On the other hand, one would expect shared features of the

  • ovary,
  • testes,
  • thyroid,
  • adrenals, and
  • pituitary.

There is biochemical expression in terms of their synthetic function – TPN organs. I would have to put the liver into that broad class. Other organs – skeletal muscle & heart – transform substrate into energy or work.  (Where you might also put intestinal smooth muscle).

They have to have different biomarker expressions, even though they much less often don’t form neoplasms. (Bone is not just a bioenergetic force. It is maintained by muscle action. It forms sarcomas. But there has to be a balance between bone removal by osteoclasts and refill by osteoblasts.)

Viewpoint: What we have learned

  1. The Watson-Crick model proposed in 1953 is limited for explaining fully genome effects
  2. The Pauling triplex model may have been prescient because of a more full anticipation of molecular bonding variants
  3. A more adequate triple-helix model has been proposed and is consistent with a compact genome in the nucleus

The structure of the genome is not as we assumed – based on the application of Fractal Geometry.  Current body of evidence is building that can reveal a more complete view of genome function.

  • transcription
  • cell regulation
  • mutations

Summary

I have just completed a most comprehensive review of the Human Genome Project. There are key research collaborations, problems in deciphering the underlying structure of the genome, and there are also both obstacles and insights to elucidating the complexity of the final model.

This is because of frequent observations of molecular problems in folding and other interactions between nucleotides that challenge the sufficiency of the original DNA model proposed by Watson and Crick. This has come about because of breakthrough innovation in technology and in computational methods.

Radoslav Bozov •

Molecular biology and growth was primarily initiated on biochemical structural paradigms aiming to define functional spatial dynamics of molecules via assignation of various types of bondings – covalent and non-covalent – hydrogen, ionic , dipole-dipole, hydrophobic interactions.

  • Lab techniques based on z/m paradigm allowed separation, isolation and identification of bio substances with a general marker identity finding correlation between physiological/cellular states.
  • The development of electronic/x-ray technologies allowed zooming in nano space without capturing time.
  • NMR technology identified the existence of space topology of initial and final atomic states giving a highly limited light on time – energy axis of atomic interactions.
  • Sequence technology and genomic perturbations shed light on uncertainty of genomic dynamics and regulators of functional ever expanding networks.
  • Transition state theory coupled to structural complexity identification and enzymatic mechanisms ran up parallel to work on various phenomena of strings of nucleotides (oligomers and polymers) – illusion/observation of constructing models on the dynamics of protein-dna-rna interference.
  • The physical energetic constrains of biochemistry were inapplicable in open biological systems. Biologists have accepted observation as a sole driver towards re-evaluating models.
  • The separation of matter and time constrains emerged as deviation of energy and space constrains transforming into the full acceptance of code theory of life. One simple thing was left unnoticed over time –
  • the amount of information of quantum matter within a single codon is larger than that of a single amino acid. This violated all physical laws/principles known to work with a limited degree of certainty.
  • The limited amount of information analyzed by conventional sequence identity led to the notion of applicability of statistical measures of and PCR technology. Mutations were identified over larger scale of data.
  • Quantum chemistry itself is being limited due discrete space/energy constrains, thus it transformed into concepts/principles in biology that possess highly limited physical values whatsoever.
  • The central dogma is partially broken as a result of
  1. regulatory constrains
  2. epigenetic phenomena and
  3. iRNA.

Large scale code computational data run into uncertainty of the processes of evolution and its consequence of signaling transformation. All drugs were ‘lucky based’ applicability and/or discovery with largely unpredictable side effect over time.

Other Related articles on this Open Access Online Sceintific Journal include the following:

Big Data in Genomic Medicine  lhb

https://pharmaceuticalintelligence.com/2012/12/17/big-data-in-genomic-medicine/

BRCA1 a tumour suppressor in breast and ovarian cancer – functions in transcription, ubiquitination and DNA repair S Saha    https://pharmaceuticalintelligence.com/2012/12/04/brca1-a-tumour-suppressor-in-breast-and-ovarian-cancer-functions-in-transcription-ubiquitination-and-dna-repair/

Computational Genomics Center: New Unification of Computational Technologies at Stanford A Lev-Ari  https://pharmaceuticalintelligence.com/2012/12/03/computational-genomics-center-new-unification-of-computational-technologies-at-stanford/

Personalized medicine gearing up to tackle cancer ritu saxena     https://pharmaceuticalintelligence.com/2013/01/07/personalized-medicine-gearing-up-to-tackle-cancer/

Differentiation Therapy – Epigenetics Tackles Solid Tumors sj Williams     https://pharmaceuticalintelligence.com/2013/01/03/differentiation-therapy-epigenetics-tackles-solid-tumors/

Mechanism involved in Breast Cancer Cell Growth: Function in Early Detection & Treatment A Lev-Ari   https://pharmaceuticalintelligence.com/2013/01/17/mechanism-involved-in-breast-cancer-cell-growth-function-in-early-detection-treatment/

The Molecular pathology of Breast Cancer Progression tilde barliya      https://pharmaceuticalintelligence.com/2013/01/10/the-molecular-pathology-of-breast-cancer-progression/

Gastric Cancer: Whole-genome reconstruction and mutational signatures A Lev-Ari     https://pharmaceuticalintelligence.com/2012/12/24/gastric-cancer-whole-genome-reconstruction-and-mutational-signatures-2/

Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1 (pharmaceuticalintelligence.com) A Lev-Ari                  https://pharmaceuticalintelligence.com/2013/01/13/paradigm-shift-in-human-genomics-predictive-biomarkers-and-personalized-medicine-part-1/

LEADERS in Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in Cancer Personalized Treatment: Part 2 A Lev-Ari
https://pharmaceuticalintelligence.com/2013/01/13/leaders-in-genome-sequencing-of-genetic-mutations-for-therapeutic-drug-selection-in-cancer-personalized-treatment-part-2/

Personalized Medicine: An Institute Profile – Coriell Institute for Medical Research: Part 3 A Lev-Ari   https://pharmaceuticalintelligence.com/2013/01/13/personalized-medicine-an-institute-profile-coriell-institute-for-medical-research-part-3/

Harnessing Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of Cancer Scientific Leaders @ http://pharmaceuticalintelligence.com ALA    https://pharmaceuticalintelligence.com/2013/01/13/7000/Harnessing Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of Cancer Scientific Leaders/

GSK for Personalized Medicine using Cancer Drugs needs Alacris systems biology model to determine the in silico effect of the inhibitor in its “virtual clinical trial” A Lev-Ari     https://pharmaceuticalintelligence.com/2012/11/14/gsk-for-personalized-medicine-using-cancer-drugs-needs-alacris-systems-biology-model-to-determine-the-in-silico-effect-of-the-inhibitor-in-its-virtual-clinical-trial/

Recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes in serous endometrial tumors S Saha   https://pharmaceuticalintelligence.com/2012/11/19/recurrent-somatic-mutations-in-chromatin-remodeling-and-ubiquitin-ligase-complex-genes-in-serous-endometrial-tumors/

Personalized medicine-based cure for cancer might not be far away ritu saxena   https://pharmaceuticalintelligence.com/2012/11/20/personalized-medicine-based-cure-for-cancer-might-not-be-far-away/

Human Variome Project: encyclopedic catalog of sequence variants indexed to the human genome sequence A Lev-Ari
https://pharmaceuticalintelligence.com/2012/11/24/human-variome-project-encyclopedic-catalog-of-sequence-variants-indexed-to-the-human-genome-sequence/

Prostate Cancer Cells: Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition sjwilliams
https://pharmaceuticalintelligence.com/2012/11/30/histone-deacetylase-inhibitors-induce-epithelial-to-mesenchymal-transition-in-prostate-cancer-cells/

Inspiration From Dr. Maureen Cronin’s Achievements in Applying Genomic Sequencing to Cancer Diagnostics A Lev-Ari
https://pharmaceuticalintelligence.com/2013/01/10/inspiration-from-dr-maureen-cronins-achievements-in-applying-genomic-sequencing-to-cancer-diagnostics/

The “Cancer establishments” examined by James Watson, co-discoverer of DNA w/Crick, 4/1953 A Lev-Ari
https://pharmaceuticalintelligence.com/2013/01/09/the-cancer-establishments-examined-by-james-watson-co-discover-of-dna-wcrick-41953/

Directions for genomics in personalized medicine lhb    https://pharmaceuticalintelligence.com/2013/01/27/directions-for-genomics-in-personalized-medicine/

How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis. Sjwilliams
https://pharmaceuticalintelligence.com/2012/10/31/how-mobile-elements-in-junk-dna-prote-cancer-part1-transposon-mediated-tumorigenesis/

Mitochondria: More than just the “powerhouse of the cell” eritu saxena   https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

Mitochondrial fission and fusion: potential therapeutic targets? Ritu saxena    https://pharmaceuticalintelligence.com/2012/10/31/mitochondrial-fission-and-fusion-potential-therapeutic-target/

Mitochondrial mutation analysis might be “1-step” away ritu saxena     https://pharmaceuticalintelligence.com/2012/08/14/mitochondrial-mutation-analysis-might-be-1-step-away/

mRNA interference with cancer expression lhb    https://pharmaceuticalintelligence.com/2012/10/26/mrna-interference-with-cancer-expression/

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