[Cell Cycle 5:8, 846-852, 15 April 2006]; ©2006 Landes Bioscience

Regulatory Molecular Biology Review

Original manuscript submitted: 02/26/06 Manuscript accepted: 02/27/06

INTRODUCTION

Numerous molecular mechanisms regulate normal and cancer cells’ biological machinery. These processes operate at multiple levels to produce coordinated and economically functioning biological activities and structures. The cells in a multi-cellular organism have essentially the same genes but differ in functions, and their genes are expressed differently. Thus the genotype does not alone determine phenotype, and life depends on both Nature and Nurture, interplay of heredity with environment, selecting expressions of hereditary information from genes and mRNAs, activities of enzymes, and specificity of membrane transport. These regulations act by different biochemistries and in different time frames. They control transit between cell quiescence and proliferation, and between stages of the cell cycle. The theme of this article is briefly to summarize innovative discoveries that continue to provide paradigms of regulatory processes. Mis-regulations of genetic and biochemical processes are at the root of cancer. And conversely, ‘the pathological illustrates the normal’. By way of background, a half century ago three molecular biological mechanisms for regulation of gene expression and function were discovered. One is enzyme induction, a mechanism by which production of a protein from a gene is controlled. The second is covalent modifications of proteins by phosphorylation, which can greatly increase or decrease their catalytic and binding activity and stability, especially in eukaryotes. Third is feedback inhibition by non-covalent binding of a small molecule to an enzyme’s non-catalytic but regulatory sites Thereby enzyme activities and the corresponding entire metabolic pathways are rapidly and sensitively regulated. Much of what we now take for granted was then unknown. Methods were comparatively primitive. Chromatography and spectrophotometry came in the early 1940’s. Radioactive organic compounds became available after World War II. There were no biochemical supply houses and no kits. Nucleic acids were not in the main picture; their status was like that of carbohydrates and fats. Around 1950 major interlocking developments of biochemistry with chemistry and genetics turned research from metabolism and enzymes toward macromolecules.1 The field now called Molecular Biology was born. Pinnacles are studies on organic structures and nature of the chemical bond by Linus Pauling, the first sequencing of a protein (insulin) by Frederick Sanger, and of 3-dimensional protein structures by Max Perutz and John Kendrew. The best known development by James Watson and Francis Crick is establishment of DNA’s double helical structure that provides the mechanism for

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Dedicated to Professor Van R. Potter (1911–2001), my postdoctoral advisor, who told me that “The Golden Age of Science is the decade after you got your Ph.D.” It is always a Golden Age for those who remain young in spirit and mind.

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molecular biology, feedback control, cell cycle, apoptosis, cancer, regulation, repression, allosteric

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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2634

Close regulations of molecular biological processes are essential for life. Defective controls cause diseases such as cancer and neurological malfunctions. We now are provided with a plethora of regulatory mechanisms exerted at many levels. Prominent are covalent protein modifications, non-covalent feedback inhibition that modifies enzyme activity, and enzyme induction. Non-covalent or covalent binding to them of either small molecules or proteins act on functional DNA, RNA, proteins and metabolites regulates their production and degradation rates, activities and intra-cell locations. Time frames differ greatly, from seconds to days or longer. A control at every level is balanced by an opposing mechanism: populations of organisms are balanced by birth vs. death, cell synthesis by apoptosis, mutation by DNA repair, macromolecular syntheses by their degradations, metabolite anabolism vs. catabolism, enzyme activation by inhibition, protein kinases by phosphatases. Any abnormal molecular condition is sensed when regulation is defective as in cancer, which leads to its rectification, to cell death, or to disease if this is not possible.

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Correspondence to: Arthur B. Pardee; Dana-Farber Cancer Institute; 44 Binney St., Boston, Massachusetts 02115 USA; Tel.: 617.632.3372; Email: [email protected]

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Arthur B. Pardee

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transfer of information to progeny. The enzymology of its synthesis, base sequencing and the genetic code by which DNA provides information via mRNAs for creation of proteins were worked out. Erwin Chargaff called these investigations “practicing biochemistry without a license”. Molecular-biochemical regulation is an enormous subject. It is summarized here historically as discoveries and functions, as I remember them in a scientific path that has led across unexplored terrain and along byways toward the goal of learning about the defects of molecular regulation that lie at the heart of cancer.2 References are limited to pioneering articles and germinal reviews to indicate thinking at the time, and to updating reviews. More can be readily found by searching the Internet (PubMed) for reviews on any topic.

MAJOR MECHANISMS OF REGULATION

Mutation. Genetic changes are now recognized to be the origins of cancer. Although genetics and biochemistry were separate disciplines in the 1950s, mutation was known to change enzyme activities dramatically, per the one gene-one enzyme model of George Beadle and Edward Tatum, And added genetic material changes metabolism; nine enzyme activities are quickly altered by additional genetic information provided by infection of Escherichia coli with a DNA bacteriophage. Strikingly, a completely novel enzyme involved in synthesizing hydroxymethyl-cytosine appears, discovered by Seymour Cohen in 1954. These include deoxyribonuclease, consistent with a role of DNA in virus replication, and many mutant progeny are produced after replacement of thymidine by bromodeoxyuridine in the phage DNA, as shown by Rose Litman in 1956. These experiments are forerunners of genetic engineering, involving introduction of normal or specifically modified DNAs. Control of metabolic pathways. The great achievement of biochemistry is to connect most metabolites into the now familiar pathways catalyzed by enzymes. Approaching its apex in the 1950s, most biochemists were very busy successfully creating this map. All its roads were of the same intensity, although traffic along some is far greater than on others. Questions about regulatory mechanisms were not posed. But it was noticed that metabolism is precisely regulated and is not wasteful; intermediary metabolites are not overproduced and do not accumulate in the medium.3,4 Living organisms usually produce their constituent molecules in amounts only sufficient to meet their needs, neither more nor less. It was also noticed that these balanced internal events respond to extracellular conditions. This tight control of metabolism is important for efficient and economical cell functioning. This focuses a cell’s resources. Feedback inhibition. A mechanism for adjustments to both environmental metabolites and to prevent excessive intracellular end products is by economically shutting down their synthesis when unneeded. A breakthrough that established a ‘Root’ of molecular biology was discovery of the general Feedback Inhibition mechanism. The end product of a biosynthetic pathway blocks production of an intermediate molecule in that pathway by inhibiting an enzyme’s activity, see ref. 5. Initial indications made in 1954 are rapid inhibition by added tryptophan of biosynthesis of an intermediate in its pathway, reported by Aaron Novick6 and Richard Yates and Arthur Pardee.7 stated “ added uracil blocks an enzyme step between aspartate and ureidosuccinate formation”; “this block may be an important regulatory mechanism in the cell” . Convincing demonstrations that an end-product can inhibit the initial enzymatic reaction was identified www.landesbioscience.com

for pathways synthesizing isoleucine-valine8 by Edwin Umbarger in 1956, and for pyrimidines by Yates and Pardee where the first enzyme in the seven successive enzyme-catalyzed reactions (aspartate transcarbamylase, ATCase) is feedback inhibited by added uracil.9 As expected mutant bacteria could accumulate the metabolite prior to their missing reaction, but only when the end product of the pathway was not provided. This observation led to perhaps the first review on regulation of metabolism.10 Feedback mechanisms have now been verified for numerous pathways.11 Some of these are complex, involving metabolic branching as investigated by Earl Stadtman.12 A remarkable example is ribonucleotide reductase, whose feedback regulation Peter Reichard demonstrated balances production of the four deoxynucleotide building blocks of DNA from alternative substrates.13 Feedback inhibition immediately created the problem if its molecular basis. How can ATCase be inhibited by uracil that is structurally very dissimilar from the substrates aspartate and carbamyl phosphate? Enzyme catalysis was described in the 1940’s as a three-step process in which substrate(s) specifically bind to the catalytic site of an enzyme, are then converted to product(s), and are released. The enzyme is then free for another catalysis. Inhibitors were seen to compete specifically for the enzyme’s catalytic site, thereby excluding substrate. This was quantitatively described in 1913 by the equation of Lenor Michaelis and Maude Menten: the velocity of the reaction (v) depends on the maximal rate (VM), concentrations of enzyme (E), substrate (S), and inhibitor (I), and their affinities (KM) and (KI). v= VM E S______ S + [KM (I + KI) ]/ KI. Also, in comparison to these classical asymptotic enzyme kinetics of rate vs. substrate concentration, kinetics are cooperatively S-shaped, for the isoleucine synthetic enzyme14 and for ATCase.15 This suggests that these enzymes are composed of interacting subunits. A pure enzyme was required to understand these discrepancies, to eliminate uncertainties posed by effects and explanations based on complexities of properties and metabolism in crude extracts. For starting material, ATCase was de-repressed 1,000 fold, and then purified and crystallized by Margaret Shepherdson. With this pure enzyme the feedback inhibitor was demonstrated to be not uridine but cytidine triphosphate (CTP). But this compound has no apparent structural similarity to the pathway’s initial substrates.16 This dilemma was solved by the discovery of enzyme’s regulatory sites distinct from catalytic sites. An initial demonstration of a regulatory site was that ATP (which is not a substrate) increases ATPase activity, in contrast to inhibitory CTP.15 This rules out the mechanism of an interaction at the catalytic site, since binding of ATP to the active site would have to be inhibitory. It must therefore bind to a different, regulatory site. Such binding of a regulatory molecule can change the protein’s structure and thereby its catalytic site’s activity, per the induced fit model proposed for enzymes by Daniel Koshland.17 The key demonstration by John Gerhart and Pardee of independent catalytic and regulatory sites came from an unexpected observation made to establish the basis for the feedback control of activity. Variable results of inhibition of the pure enzyme by CTP were repeatedly obtained. Frozen enzyme thawed at the beginning of a week was strongly inhibited. But thereafter, inhibition was lost during storage in the refrigerator. Furthermore the activity actually increased, and kinetics changed from the subunit-cooperative S-shape to the classical Michaelis-Menten shape. Hypothesizing that ATCase

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must change, even at zero degrees, systematic warming showed that five minute exposure to 65˚ C abolishes its inhibition by CTP but not its catalytic activity. Thus, catalytic and regulatory functions were separated, and are distinct. The enzyme was concluded to be a complex made of two proteins, one designed for catalysis and the other for regulation.15,16,18 This is in principle similar to adding a thermostat to regulative a furnace’s heat production. Regulatory sites and catalytic activities of ATCase were physically separated by heating, or by treatments with urea or heavy metals. Properties of its regulatory and catalytic subunits were investigated in detail by enzymology and physical chemistry.19 The complete structure determined by X-ray diffraction shows that there are six catalytic and six regulatory subunits.20 In agreement with these findings, Jean-Pierre Changeux in Jacques Monod’s laboratory investigated the mechanism of the feedback inhibition of L-threonine deaminase discovered by Edward Adelberg and Umbarger , and from kinetic studies with extracts proposed that this enzyme has inhibitory sites in addition to catalytic ones.21 These several findings revealed a mechanism for the classical non-competitive inhibitions, as involving interactions at regulatory sites. That enzymes are often complexes rather than single proteins, as was then the general biochemical concept is major development arising from feedback inhibition, now well established. Hemoglobin and the b-galactosidase repressor are tetramers of identical subunits, ribonucleotide reductase has catalytic and regulatory subunits, and there are many other multi-protein complexes. Examples are cyclins that activate cdks (see below). And more than a dozen B proteins differently control properties of the pleiotropically functioning and ubiquitous PP2A phosphatase; one regulates degradation of oncogenic myc.22 An early extreme example is the ribosome, a multi-protein complex that catalyzes protein synthesis. And DNA synthesis is catalyzed by a Replitase complex that contains enzymes for both precursor synthesis and polymerase, as found by Prem Reddy.23 It contains among other proteins retinoblastoma-related pRb, cdc2 kinase and E2F-1. This finding initially met with considerable opposition from biochemists who favored a monomeric enzyme, but it is now confirmed. Associated proteins have widespread importance in controlling activities, locations and specificities of enzymes and other proteins.

ACTIVATION BY COVALENT MODIFICATION.

After a protein is synthesized it may not have enzymatic activity, which can be produced by a subsequent covalent modification. A major mode of changing activity (plus or minus) in higher organisms is produced by covalent phosphorylation of proteins by the kinases, discovered by Eugene Kennedy in 1954, which can be reversed by phosphatases. Edwin Krebs and Edward Fisher in the 1950s discovered that this covalent protein phosphorylation is a mechanism for enzyme activity regulation; ATP level controls glycogen phosphorylase which provides metabolic energy.24 The human genome contains 518 kinases (the kinome), each of which is regulated to phosphorylate a distinct set of substrates.25 Kinases, and also proteases, are often organized into sequentially activating cascades that catalyze rapid, exponential-like amplifications of downstream activity. Examples are the kinase cascades activated by binding of growth factors to their receptors on the mammalian cell’s surface (see below).

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CONTROL OF GENE ACTIVITY The rate of a reaction depends upon the amount of its enzyme as well as upon its activity, as seen in the Michealis-Menten equation. Amounts (maximal activities) of some enzymes in bacteria known before 1950 to be change by environmental molecules. They “adapted” as a function of extracellular nutrients, dramatically increasing in amount when their substrate is provided. Jacques Monod, the outstanding investigator of this problem, performed elegant experiments on the dependence of b-galactosidase production in E. coli as a function of availability of β-galactoside sugars which were proposed to act as ‘inducers ‘ of the gene.26 This control of gene expression acts relatively slowly as compared to feedback inhibition of metabolic reactions. As an early molecular-genetic mechanism by which gene expression is activated to produce enzymes, Roger Stanier early made the hypothesis that bacterial catabolism successively produces inducing molecules that cause as set of enzyme to appear. A breakthrough was made in 1959, the PaJaMa experiment, so named for the authors27 and because it depends on bacterial mating. In brief, mutants of inducible E. coli were made that constitutively produce β-galactoside, independent of an added inducer. The key experiment consisted of mixing these genes by mating wild-type β-galactosidase inducible bacteria with a constitutive mutant that also has an inactive β-galactosidase gene. Then which gene dominates—whether the recipient mated cells are inducible or constitutive was determined. The enzyme activity, in absence of inducer, increased within minutes after mating. And it stopped after two hours unless inducer was added. Also, the reciprocal transfer of the constitutive gene into inducible cells did not cause constitutive enzyme production. Furthermore, Monica Riley, et al. in 1962 demonstrated that synthesis of β-galactosidase by the mated cells requires integrity of the introduced DNA, because destruction of the introduced gene by radioactive decay of 32P incorporated into it caused cessation of enzyme synthesis.28 The constitutive cells was concluded to lack a repressor protein that is present in inducible bacteria and is gradually produced in the mated cells after its gene is introduced. This means that the repressor specifically blocks gene expression; coding DNA is shut down when repressor protein binds to an upstream DNA repressor sequence.27 The repressor is released when its other site binds a low molecular weight inducer molecule. Specifically, expression of β-galactosidase (and two adjacent genes) is inhibited when a lac repressor protein binds to its upstream DNA operator region. and mutant bacteria that cannot make repressor produce the enzyme constitutively. The lac repressor protein was isolated in 1966 by Walter Gilbert and Benno Muller-Hill. Several sequentially located genes (an operon) can be co-repressed. Evidence is weak for operons in higher organisms, but all the genes in one of the two entire X chromosomes in female mammalian cells are silent. A mammalian unit of epigenetic co-regulation has been suggested (which could be named a “chromeron”), as based upon chromosomal co-localization of sets of genes involved in apoptosis and in responsiveness to retinoids.29 Repression has been comprehensively reviewed.30 Major developments from PaJaMa. i) Primarily, this experiment is the foundation of transcriptional control of gene expression by both bacteria and eukaryotes. ii) Enzymes in synthetic pathways can be repressed by low molecular weight compounds, as well as those involved in catabolism; a metabolite can repress transcription of its biosynthetic pathway. Examples are the pathway of pyrimidine biosythesis by Richard Yates and Arthur Pardee, and for arginine by Luigi Gorini and Werner Maas (for a review see 30). iii) The broad

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biological roles of functional sites interacting with separate regulatory sites depends upon these concepts of repressor and regulatory DNA promoter sequences (see allostery below). iv) One basis for messenger RNA which transfers information from genes to proteins was appearance in bacteria after phage-infection of a new RNA whose base composition corresponds to that of the virus DNA, by Elliot Volkin et al.31 Elucidation of repression provided the second basis. A requirement of RNA synthesis for protein production had been shown; mutants of E. coli deprived of purines or pyrimidines stopped making proteins as well as nucleic acids, reported by Pardee in 1954 as “…. continuous formation of RNA is essential for protein formation.”32 An unstable intermediate between gene and enzyme was proposed in 1958: “DNA does make an intermediate carrier of information, perhaps RNA,”33 this is in accord with an unstable intermediate.28 Pardee and Louise Prestidge in 1961 found that on/off kinetics of b-galactosidase induction are very rapid, with a 3 min half-life.34 mRNA was identified in 1961 by two laboratories.28,35 Allostery. The two types of binding sites of proteins, one functional and the other regulatory, permit many types of biological reactions to be controlled by a molecule that has no structural similarity to the molecules acted upon. Jacques Monod combined three lines of research to create this allosteric concept,30 which he called “the second secret of life”: i) feedback inhibition with its catalytic and regulatory sites (see above), ii) a site for binding galactosides to the lac repressor modifies another functional site that binds it to a DNA sequence, and iii) cooperative binding of oxygen to the four protein subunits of hemoglobin and which are modified by their interactions with CO2. For an historical review see ref. 36. Reports of feedback inhibition, regulatory subunits, allosteric sites, and multi-protein complexes abound in the literature and it remains a subject of active investigation. Active sites are regulated by three main allosteric bindings i) non-covalently of functional proteins to other proteins such as activators and repressors; ii) of metabolites, cyclic nucleotides, GTP37 and other small molecules non-covalently to catalytic or regulatory proteins; iii) covalent attachment of phosphate-, methyl-, acetyl-, etc. Mathematics of multi-subunit interaction. Allosteric activity depends on functional regulation by alternative structures of multiprotein complexes. An early example is the Hill equation which mathematically describes interactions of the four subunits of hemoglobin upon binding of O2. General allosteric equations have been described by two mathematical models, based upon alternative active and inactive conformations of subunits controlled by regulator binding. In one, the subunits conformations change in a concerted, all-or-none, manner.38 In the other, each binding sequentially alters the protein’s structure and changes the next binding affinity; technical methods, such as 3D protein structure determinations, are resolving this question of allosteric changes.39

REGULATION OF MEMBRANE FUNCTIONS.

Control by molecular location is seen at three levels, whose amounts and activities are regulated both genetically and environmentally First is extra-cellular vs. intra-cellular location. Many molecules generally must pass into a cell to metabolized. They cross the cell membrane via specific transport mechanisms that permit either passive entry or catalyze enzyme-like energy-dependent accumulation. Second are systems that move molecules between cytoplasm and organelles. For example, enzymes involved in DNA synthesis accumulate in the nucleus before S-phase. Third, enzymes are often assembled, onto protein scaffolds, into multi-protein complexes that www.landesbioscience.com

perform cooperative functions.40 As an example of such interactions, compounds that specifically inhibit an isolated enzyme also inhibit others that are in the replitase complex.41 Individual mRNAs similarly have been localized in cells.42 The extracellular membrane selectively filters a cell’s environment, actively or passively transmitting some molecules into the cell, responding to others, and excluding many. And it retains the cell’s contents. Environment and heredity are thus connected by the membrane. “The true secret of life lies in understanding the elegantly simple biological mechanisms of the magical membrane—the mechanisms by which your body translates environmental signals into behavior”. And “It is a single cell’s “awareness” of the environment, not its genes, that sets into motion the mechanisms of life.”43 Active transport of a molecule into the cell is a first step in many metabolic pathways. Kinetics of substrate uptake and enzymes are similar. It is therefore not surprising that trans-membrane transport is regulated and inhibited similarly to enzyme activity. Transport of galactosides across the membrane of E. coli is inducible;44 adjacent genes for β-galactosidase and galactoside transport (permease) are co-induced by β-galactosides, per the operon model proposed by Jacob and Monod.30 Molecules catalyzing transport were unknown in the 1950’s. One of the first transport-related molecules to be purified is a regulatory factor for sulfate transport.45 A transport system was demonstrated for uptake of sulfate ion into Salmonella typhimurium. Mutants that could not grow on sulfate were isolated by applying toxic chromate ion; they were defective in transport. But a very small amount of sulfate was noticed, located between their cell wall and membrane and firmly bound to a small protein. It and transport are repressible by cysteine. Following de-repression and a selective technique for release of periplasmic proteins, crystals were obtained after an only four-fold purification. This ‘binding protein’, is the first of several identified. They are also involved in chemotaxis, as shown by Julius Adler.46 Cell surface membrane and transport are very important in eukaryote metabolism and regulation, e.g., the coupled transport of hydrogen ions across the mitochondrial membrane that produces ATP discovered by Peter Mitchell, or control of neuronal transmission by regulated receptor-mediated transport of ions. Density-dependent contact inhibition of cell growth involves surface proteins such as cadherins, integrins, etc. that make connections to other cells and to the extracellular matrix. Membranes of eukaryotic cells contain proteins with extra-cellular binding sites that are specific receptors for protein growth factors. These regulate these receptors’ intracellular tyrosine kinase activity, as shown by Joseph Schlessinger.47 In accord with Gordon Tompkins’ concept, this provides another example of interacting regulatory and catalytic sites, in this case operating across space. These regulatory mechanisms are not mutually exclusive; pyrimidine biosynthesis is controlled by both feedback and repression,15 and cholesterol regulates its availability by mechanisms operating at the levels of gene, enzyme, and intercell transport as discovered by Michael Brown and Joseph Goldstein. Relocation of molecules between subcellular compartments in eukaryoteic cells provides yet another mechanism of regulation. For example, several enzymes involved in DNA synthesis are produced in the cytoplasm at the G1/S-interface and translocate into the nucleus where they form the Replitase multi-protein complex for DNA synthesis mentioned above.23

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MORE MECHANISMS Epigenetic controls can permit a cell to express only a subset of its genes, for example differently in liver than skin. Pioneering experiments by Werner Arber demonstrated DNA methylation protects bacterial DNA from hydrolysis of by restriction endonucleases, and by Ruth Sager who found that methylation is the basis of non-Mendelian inheritance of organelle genes in the eukaryotic alga Chlamydomonas. The effect of methylation then shifted from elimination of DNA to blocking gene expression in higher organisms. Methylation of DNA attracts enzymes that catalyze acetylation of histones and thereby changes of chromatin structure and activity. Mechanisms of histone modifications and their effects on gene expression are under vigorous investigation.48 Information about the classes and functions of RNAs are increasing dramatically. Mechanisms are newly discovered. that on the one hand regulate amounts and functions of mRNA, and on the other regulations by RNAs For an overview see ref. 49. Transcriptional production of pre-mRNA is followed by its processing and splicing, which produces hundreds of mRNAs and then their corresponding proteins.50 mRNA production is also controlled by complex reactions such as trans-splicing to their 5’ ends of short synthesis-regulating leader RNAs in some organisms.51 About 13,000 target relationships have been identified as complimentary seed sequences.52 Ribozymes catalyze molecular reactions. siRNAs can block translational activity, and importantly they activate specific mRNA degradation,53 which takes place in cellularly localized P-bodies. Activity of an mRNA can be regulated at a new level, by by feedback, binding to it of a small molecule metabolically related to the transcribed protein.54 A short RNA has been synthesized that selectively binds theophylline, whereby its structure is modified so that it specifically inhibits an in vivo translation. But caffeine is inactive though it differs by only a methyl group.55

REGULATION OF CELL PROLIFERATION

Research shifted from bacteria toward higher organisms in the 1960s, along with the rise of molecular biology. Techniques had progressed sufficiently to make in vitro culture of mammalian cells generally feasible. Functions in eukaryotic cells are controlled by interplay of genetic and environmental factors, and as with bacteria these can regulate DNA and protein functions. Processes that involve an entire cell homeostasis take us into a new realm of regulation; these are at least an order of magnitude more complex than is gene expression or a metabolic pathway. The mechanisms that control gene expression and enzyme activity are applied in regulation of cell proliferation. Cell cycle control. Regulation in eukaryotic cells is at a slower pace than controls in bacteria; completing the cycle can require a day or longer vs. an hour. The sequentially organized processes of cell proliferation are described as the cell cycle. Early research on the eukaryotic cycle is summarized,56 and has since often been reviewed.57 To produce two cells from one requires that all molecules, large and small, must be duplicated precisely. These syntheses take place at specific times, the most prominent example being duplication of DNA in mid-cycle S-phase, shown by Howard and Pelc in 1951.58 The cell cycle of bacteria had begun to be investigated by 1960. Its duration as measured with synchronized E. coli depends on carbon source, requiring over an hour on acetate and as short as 15 min on glucose. Several waves of DNA initiation must take place between 850

the successive divisions of rapidly growing bacteria, because DNA replication in mid-cycle requires about an hour.59 Activities of enzymes increase periodically during the bacterial cycle.60 Controls are of course essential to regulate reactions both on and within the cell membrane. Formation of ATP by both glycolysis and by the Krebs citric acid cycle is closely coordinated by feedbacks of enzyme production and activity.61 As an early example, Krebs cycle intermediates do not accumulate as they are oxidized and smoothly pass through these many reactions. There must therefore be coordinating mechanisms. An early clue in 1948 suggests that a feedback from strong inhibition of succinic dehydrogenase by oxalacetate produced three reactions downstream. Emergence from quiescence and transit through G1 is inhibited by density dependent physical-chemical interactions between adjacent cell surfaces. It is activated by proteins in serum, such as insulinderived growth factor and epidermal growth factor. These bind to external receptors on the cell membrane, which activates intracellular auto-phosphorylation by the receptor’s tyrosine kinase. Alternatively, estrogen and androgen initiate proliferation of female and male sexrelated cells, respectively, and these relatively small molecules bind to receptors located to the nucleus. Both activations initiate kinase cascades that activate transcriptions. The cyclin proteins increase and then decrease in a specific sequence during the cycle, as discovered by Tim Hunt and colleagues.62 They complex with and regulate several cyclin-dependent kinases (cdks), discovered by Paul Nurse.63 These complexes are the keys to cycle progression. These interactions provide yet another example of catalytic-regulatory protein complexes. Furthermore, activities of cdks depend on their phosphorylationdephosphorylation, and on binding of inhibitory proteins to them. Other complicated mechanisms limit DNA replication to only once per cycle, and yet others control mitosis and daughter cell separation. Toward the end of G1 phase cyclin/cdk activities phosphorylate the retinoblastoma protein, causing its inactivation and release and activation of E2F-1, a transcription factor for many enzymes required for DNA synthesis. E2F-1 is also the (autocatalytic) factor for its own transcription, and therefore it increases dramatically at the G1/S boundary. But excessive E2F-1 is apoptotic, and so a feedback control must exist; perhaps inhibition by the end product dNTPs is responsible. Based upon this idea, a novel chemotherapeutic principle for action of agents that deplete dNTP pools has been suggested.64 Feedback loops between plus and minus balancing controls are indicated, such that excess of one activates the opposite. A major question that determines detailed investigations of molecular mechanisms was at what point in the cell cycle growth is regulated. The prior belief was that growth control is exerted at the end of a cycle. In contrast, two major researches on this question showed it to be exerted in late G1 phase This process, at about two hours prior to initiation of DNA synthesis in mammalian cells at, the Restriction (R) Point discovered by Pardee in 1974.65 Lee Hartwell at this time demonstrated that growth of cycle-regulating yeast mutants that he isolated is similarly regulated at “START” in G1.66 R and START are the first demonstrations of what were later named “checkpoints”. Thus, a wholly different set of events came into consideration, and it involved molecules soon identified. Numerous intracellular mechanisms regulate eukaryotic cell cycling. Protein synthesis is one such essential process.67 Rapid protein synthesis is needed to enter S-phase, which suggests the requirement for a growth regulating protein with a short half-life. A sensor for protein synthesis and cell growth is the kinase mTor; locking its activity with rapamycin stops cells in G1.68 This led to discovery

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of control of the R protein by its synthetic vs. degradation rates. Only one protein (p68) of the many detected on 2-D gels had three characteristics of R. It increases in G1, has a short half-life, and is elevated in cancer cells.69 R protein might be cyclin E, one of the cyclin family of regulatory proteins that are central to cell cycle control. Several proteins including structural actin also increase after cells enter their proliferation cycle. Production and removal are balanced at every biological level. Specific multi-protein enzymatic machineries label and then degrade metabolites, RNAs, proteins, and cells. Such a major regulatory mechanism is proteolysis, the most dramatic alteration of a protein’s structure. It was discovered early to convert extracellular inactive proteins (zymogens) to active enzymes; trypsinogen to trypsin is an example. But proteolysis usually eliminates intracellular activity,70 and the protein is in a steady state determined by balance with its synthesis. This is especially so for regulatory proteins including cyclins that are produced transiently and are degraded when their roles are complete. Another prominent example is removal of proapoptotic P53, activated by its ubiquitination involving Mdm271 and then degraded by proteasomes.72 This control is a feedback loop because P53 induces Mdm2. Another level of regulation by liability is the removal of mRNAs, balanced by their transcriptions. For cells also, proliferation is balanced by loss which can be caused by an organized series of molecular steps termed Programmed Cell Death (apoptosis), often reviewed see ref. 57. A key to this complex process is the apoptosis activating tumor suppressor p53 protein. It is very frequently inactivated in cancers. Another antiapoptotic molecule is the transcription factor NF-κB, constitutively elevated in many cancers. Its activation provides an example of the complexity of regulation. NF-κB is usually as an inactive protein dimer combined with an inhibitory protein IκB, and is located in the cytoplasm. An activated kinase cascade causes phosphorylation of IκB and releases it from NF-κB, followed by proteolytic degradation. The freed NF-κB relocalizes, moving into the nucleus where it itself is phosphorylated and binds to DNA motifs to activate over 100 transcriptions. Furthermore a feedback control is brought into play by NF-vB’s transcription of IκB, the inhibitor of its activation.73 Cancer and mis-regulation. For regulatory mechanisms, ‘The pathological illuminates the normal’. Defective controls created by mutations and altering gene expressions are causal of cancer. Genetic material introduced by viruses can also cause cancer. These genetic level changes can produce either gain of an activity of an oncogene such as ras discovered by Ed Skolnik and shown by Robert Weinberg and Geoffery Cooper to be mutated in cancers. Mutational loss of a tumor suppressor gene such p53 or pRb was proposed by Ruth Sager.74 The latter are more frequent, and the more probable because the normal phenotype is dominant in fused normal plus cancer cells, as shown by Henry Harris and Boris Ephrussi. Cancer and normal cells differ in their surface chemistry and functions. Proliferation is improperly regulated by cell-cell contacts, serum factors, etc. Hormone and growth factor receptors are quantitatively and qualitatively modified. Membrane carbohydrates are altered75 and phospholipid turnover increases.76 Rates of transport of small molecules into cancer cells and into normal cells differ. Again, one finds roots in genetic—environmental miscommunication, here due to imperfect signaling between cells of a multi-cell organism.77 Many intracellular regulations also are changed. The first proposal for a biochemical basis of cancer made by Otto Warburg was misregulated production of energy (ATP) by glycolysis vs. respiration.78 Cancer cells require more oxygen, more energy, and more active metabolism than do normal cells, which are usually quiescent. www.landesbioscience.com

A hallmark of cancer is deregulated cell proliferation, and changes of controls through the cell cycle are reported, particularly in G1 phase. Numerous changes of kinases and phosphorylations have been reported. Cyclin E is over expressed and modified in advanced cancers, and it provides a clinical marker.79 The tumor suppressing retinoblastoma protein is very frequently inactive or absent, and the E2F-1 protein that it negatively regulates is released to activate S phase transcriptions. The critical Restriction point control is relaxed or absent in cancers,80 the R protein can be more stable, a difference that provides a molecular basis for greater proliferative capacity.81 Furthermore, cancer cells are protected from apoptotic activity, due to mutations such as of p53 or NF-κB. Developments of this enormous subject are reviewed in ref. 57. Killing cancer cells is easy; selectivity is the problem—to kill them but not normal cells.82 The discovery of differences of regulation between normal and cancer cells provides potential targets for detection, prognosis and therapy.83 An emergency exit mechanism that leads to apoptosis may be activated if a normal cell overexpresses a proliferation-related gene. Such a mechanism may have become inactive in a cancer, which might provide the needed difference. Studies of regulation have practical aspects. Drugs such as Gleevec, an inhibitor of an over expressed proliferation-related kinase, are being applied clinically, as are proteasome inhibitors such as PS341. Many other targeted applications are under development, of which a potential anti-cancer therapy initiated in this laboratory provides an example. b-Lapachone, a natural product, increases E2F-1,84 decreases ATP and NAD,85 and selectively causes apoptosis of cancer cells. It is remarkably and specifically effective against tumors implanted in mice, and is now in clinical trials.

CONCLUSION

Of fundamental importance are the dynamic steady states between production and removal. These are active at all levels—molecular, cellular, and biological. Regulatory interactions create off-on switches between alternative pathways, negative feedback loops for limiting pathways, positive feedback loops that convert transient into sustained signals, feed-forward loops and successive activation pathways that amplify signals, as by MAP kinases.86 Systems biology—mathematical computer models are being developed to grasp these interactions in large genetic and metabolic networks.87 References 1. Judson HF. The Eighth Day of Creation. Expanded Edition. Cold Spring Harbor: Cold Spring Harbor Lab., 1996. 2. Pardee AB. Regulation, restriction and reminiscences. J Biol Chem 2002; 277:26709-16. 3. Davis BD. Introduction. Cold Spring Harbor Symp. Quant Biol 1961; 26:1-10. 4. Roberts RB, Cowie DB, Abelson PH, Bolton ET, Britten RJ. Studies on biosynthesis in Escherichia coli. Vol 607. Washinton, DC:Carnegie Inst. of Washington Publ., 1955. 5. Pardee AB. Molecular basis of biological regulation: Origins from feedback inhibition and allostery. BioEssays 1985; 2:37-40. 6. Novick A, Szilard L. In: Boell EJ, ed. Dynamics of Growth Processes. Princeton:Princeton Univ. Press, 1954:21-31. 7. Yates RA, Pardee AB. Studies on pyrimidine synthesis in E. coli. Science 1954; 120:903. 8. Umbarger HE. Evidence for a negative-feedback mechanism in the biosynthesis of isoleucine. Science 1956; 123:848. 9. Yates RA, Pardee AB. Control of pyrimidine biosynthesis in E. coli by a feed-back mechanism. J Biol Chem 1956; 221:757-70. 10. Pardee AB. In: Colowick SP, Kaplan NO, eds. The Enzymes. 2nd Ed. New York:Academic Press, 1959:681-716. 11. Atkinson DE. Regulation of enzyme function. Annu Rev Microbiol 1969; 23:47-68. 12. Stadtman ER. Allosteric regulation of enzyme activity. Adv Enzymol 1966; 28:41-154. 13. Thelander L, Reichard P. Reduction of ribonucleotides. Ann Rev Biochem 1979; 48:133-58. 14. Umbarger HE. Feedback control by endproduct inhibition. Cold Spring Harbor Symp Quant Biol 1961; 26:301-12.

Cell Cycle

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Regulatory Molecular Biology

15. Gerhart JC, Pardee AB. The effect of the feedback inhibitor, CTP, on subunit interactions in aspartate transcarbamylase. Cold Spring Harbor Symp Quant Biol 1963; 28:491-6. 16. Gerhart JC, Pardee AB. The enzymology of control by feedback inhibition. J Biol Chem 1962; 237:891-6. 17. Koshland DE, Jr. Enzyme flexibility and enzyme action. J Cell Comp Physiol 1959; 54:245-58. 18. Gerhart JC, Pardee AB. Separation of feedback inhibition from activity of aspartate transcarbamylase. Fed Proc 1961; 20:249. 19. Schachman HK. Anatomy and physiology of a regulatory enzyme-aspartate transcarabamylase. Harvey Lecture 1974; 68:67-113. 20. Kosman RP, Gouaux JE, Lipscomb WN. Crystal structure of CTP-ligated T state aspartate transcarbamylase at 2.5A resolution: implications for ATCase mutants and the mechanism of negative cooperativity. Proteins 1993; 15:147-76. 21. Changeux JP. The feedback control mechanisms of biosynthetic L-threonine deaminase by L-isoleucine. Cold Spring Harbor Symp Quant Biol 1961; 26:313-8. 22. Yeh E, Cunningham M, Arnold H, Chasse D, Monteith T, Ivaldi G, Hahn WC, Stukenberg PT, Shenolikar S, Uchida T, Counter CM, Nevins JR, Means AR, Sears R. A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells. Nat Cell Biol 2004; 6:308-18. 23. Reddy GPV, Pardee AB. Multienzyme complex for metabolic channeling in mammalian DNA replication Proc Natl Acad Sci USA 1980; 77:3312-6. 24. Krebs EG, Fischer EH. Phosphorylase and related enzymes of glycogen metabolism. Vitam Horm 1964; 22:399-410. 25. Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. Science 2002; 298:1912-34. 26. Cohen M, Monod J. The induced biosynthesis of enzymes. Adv Enzymol 1952; 8:67-119. 27. Pardee AB, Jacob F, Monod J. The genetic control and cytoplasmic expression of “Inducibility” in the synthesis of β-galactosidase by E. coli. J Mol Biol 1959; 1:165-78. 28. Riley M, Pardee AB, Jacob F, Monod J. On the expression of a structural gene. J Mol Biol 1960; 2:216-25. 29. Goodman AB, Pardee AB. Meeting report: Molecular Neurobiological Mechanisms in Schizophrenia: Seeking a Synthesis. Biol Psychiatry 2000; 48:173-83. 30. Jacob F, Monod J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol 1963; 3:318-56. 31. Volkin E, Astrachan L, Countryman X. Metabolism of RNA phosphorus in Escherichia coli infected with bacteriophage T7. Virology 1958; 6:545-55. 32. Pardee AB. Nucleic acid precursors and protein synthesis. Proc Natl Acad Sci USA 1954; 40:263-70. 33. Pardee AB. Experiments on the transfer of information from DNA to enzymes. Exp Cell Res, Suppl. 1958; 6:142-51. 34. Pardee AB, Prestidge LS. The initial kinetics of enzyme induction. Biochim Biophys Acta 1961; 49:77-88. 35. Brenner S, Jacob F, Meselson M. An unstable intermediate carrying information from genes to ribosomes for protein synthesis. Nature 1961; 190:76. 36. Creager ANH, Gaudilliere P-P. Meanings in search of experiments and vice-versa: the invention of allosteric regulation in Paris and Berkeley, 1958-1968.Historical Studies in the Physical and Biological Sciences 1996; 27:1-89. 37. Wettschureck N, Offermanns S. Mammalian G proteins and their cell type specific functions. Physiol Rev 2005; 85:1159-1204. 38. Monod J, Wyman J, Changeux JP. On the nature of allosteric transitions: a plausible model. J Mol Biol 1965; 12:88-118. 39. Koshland DE Jr, Hamadani K. Proteomics and models for enzyme cooperativity. J Biol Chem 2002; 277:46841-4. 40. Burack WR, Cheng AM, Shaw AS. Scaffolds, adaptors and linkers for TCR signaling: Theory and practice. Curr Opin Immuol 2002; 14:312-6. 41. Reddy GPV, Pardee AB. Inhibitor evidence for allosteric interaction in the replitase multienzyme complex. Nature 1983; 303:86-8. 42. Braasted CD, et al. Architectural organization of the regulatory machinery for transcription, replication, and repair: dynamic temporal -spatial parameters of cell cycle control. In: Stein GS, Pardee AB, eds. Cell Cycle and Growth Control: Biomolecular Regulation and Cancer. Hoboken:John Wiley & Sons, Inc., 2004:15-92. 43. Lipton BH. Biology of Belief, Mountain of Love. Santa Rosa:Elite Books, 2005. 44. Cohen GN, Monod J. Bacterial permeases. Bacteriol Rev 1957; 21:169-94. 45. Pardee AB. Membrane transport proteins. Science 1968; 162:632-7. 46. Baker MD, Wolanin PM, Stock JB. Signal transduction in bacterial chemotaxis. Bioessays 2006; 28:9-22. 47. Schlessinger J. Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell 2002; 110:669-72. 48. Grace Goll M, Bestor TH. Eukaryotic cytosine methyltransferases. Annu Re Biochem 2005; 74:481-514. 49. Riddihough G. In the forest of RNA dark matter. Science 2005; 309:1507-90. 50. Sharp PA. The discovery of split genes and RNA splicing. Trends Biochem Sci 2005; 279-81. 51. Pouchkina-Stantcheva NN, Tunnacliffe A. Spliced leader RNA-mediated trans-splicing in phylum Rotifera. Mol Biol Evol 2005; 22:1482-9. 52. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005; 120:15-20. 53. Southheimer EJ, Carew RW. Silence from within: endogenous siRNAs and miRNAs. Cell 2005; 122:9-11.

852

54. Breaker RR. Natural and engineered nucleic acids as tools to explore biology. Nature 2004; 432:838-45. 55. Bayer TS, Smolke CD. Programmable ligand-controlled riboregulators for eukaryotic gene expression. Nature Biotechnol 2005; 23:337-43. 56. Baserga R. The Biology of Cell Reproduction. Cambridge:Harvard University Press, 1985. 57. Stein GS, Pardee AB, eds. Cell Cycle and Growth Control: Biomolecular Regulation and Cancer. Hoboken:John Wiley & Sons, Inc., 2004. 58. Howard A, Pelc SR. Synthesis of nucleoprotein in bean root cells. Nature 1951; 167:599-600. 59. Cooper S, Helmstetter CE. Chromosome replication and the division cycle of Escherichia coli B/r. J Mol Biol 1968; 31:519-40. 60. Masters M, Pardee AB. Sequence of enzyme synthesis and gene replication during the cell cycle of Bacillus subtilis. Proc Natl Acad Sci USA 1965; 54:64-70. 61. Williamson JR, Cooper RH. Regulation of the citric acid cycle in mammalian systems. FEBS Lett 1980; 25:117 Suppl:K73-85. 62. Minshull J, Pines J, Golsteyn R, Standart N, Mackie S, Colman A, Blow J, Ruderman JV, Wu M, Hunt T. The role of cyclin synthesis, modification and destruction in the control of cell division. J Cell Sci 19889; Suppl 12:77-97. 63. Nurse P. The central role of a CDK in controlling the fission yeast cell cycle. Harvey Lecture 1996-1997; 92:55-64. 64. Wang AJ, Li CJ, Reddy PV, Pardee AB. Cancer chemotherapy by deoxynucleotide depletion and E2F-1 elevation. Cancer Res 2005; 65:7809-14. 65. Pardee AB. A restriction point for control of normal animal cell proliferation. Proc Natl Acad Sci USA 1974; 71:1286-90. 66. Hartwell L, Culotti J, Pringle J, Reed BJ. Genetic control of the cell division cycle in yeast. Science 1974; 183:46-51. 67. Rossow PW, Riddle VGF, Pardee AB. Synthesis of labile, serum-dependent protein in early Gl controls animal cell growth. Proc Natl Acad Sci USA 1979; 76:4446-50. 68. Richardson CJ, Schalm SS, Blenis J. PI3-kinase and TOR: PIKTORing cell growth. Sem Cell Dev Biol 2004; 15:147-59. 69. Croy RG, Pardee AB. Enhanced synthesis and stabilization of a Mr 68,000 protein in transformed Balb/c-3T3 cells: candidate for restriction point control of cell growth. Proc Natl Acad Sci USA 1983; 80:4699-703. 70. Schimke RT, Doyle D. Control of enzyme levels in animal tissues. Annu Rev Biochem 1970; 39:929-76. 71. Hershko A. The ubiquitin system for protein degradation and some of its roles in the control of the cell division cycle. Cell Death Differ 2005; 12:1191-7. 72. Goldberg AL. Protein degradation and protection against misfolded or damaged proteins. Nature 2003; 426:895-9. 73. Ghosh S, Karin M. Missing pieces in the NF-κB puzzle. Cell 2002; Suppl. 109:S81-96. 74. Sager R. Genetic suppression of tumor formation. Adv Cancer Res 1985; 44:43-68. 75. Burger MM, Goldberg AR. Identification of a tumor-specific determinant on neoplastic cell surfaces. Proc Natl Acad Sci USA 1966; 57:359-66. 76. Cunningham DD. Changes in phospholipid turnover following growth of 3T3 mouse cells to confluency. J Biol Chem 1972; 247:2464-70. 77. Pardee AB. Cell division and a hypothesis of cancer. Natl Cancer Inst Monograph 1964; 14:7-20. 78. Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? Nat Cancer 2004; 4:891-9. 79. Akli S, Keyomarsi K. Low-molecular-weight cyclin E: the missing link between biology and clinical outcome. Breast Cancer Res 2004; 6:188-91. 80. David-Pfeuty T. The flexible anchorage-dependent Pardee’s restriction point of mammalian cells. How its deregulation may lead to cancer. Biochim Biophys Acta 2006; 1765:38-66. 81. Pardee AB. G1 events and regulation of cell proliferation. Science 1989; 246:603-8. 82. Blagosklonny MV, Pardee AB. Exploiting cancer cell cycling for selective protection of normal cells. Cancer Res 2001; 61:4301-5. 83. Blagosklonny MV, Pardee AB. The restriction point of the cell cycle. Cell Cycle 2002; 1:103-10. 84. Li Y, Sun X, LaMont JT, Pardee AB, Li CJ. Selective killing of cancer cells by β-lapachone: Direct checkpoint activation as a strategy against cancer. Proc Natl Acad Sci USA 2003; 100:2674-8. 85. Pink JJ, Planchon SM, Tagliarino C, Varnes ME, Siegel D, Boothman DA. NAD(P)H:Quinone oxidoreductase activity is the principal determinant of β-lapachone cytotoxicity. J Biol Chem 2000; 275:5416-24. 86. Bornholdt S. Less is more in modeling large genetic networks. Science 2005; 310:449-51. 87. Jasny BR, Ray LB. Life and art of networks. Science 2003; 301:1863-77.

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