Tgp chi Cong ngh? Sinh hoc 1 (l{ 2) 197-206. 2012
USE OF YEAST SACCHAROMYCES CEREMSUli AS A \ KRSATILE. MODr.l. FOR STUDY OF BIOCHEMICAL PROCESSES. DRIC; DISCOVERY. A \ D CANCER RESEARCH IN HIGHER EUKARYOTES
Bui Van Ngoc, Ngliicm Ngoc Minh
Institute of Biotechnulogy: Vielinnii Acadeim of Science ami Technology
SUMMARY
During cell growth and prolilenHion eukaiyniic cells arc L-\]H>sed to many kinds ofadvcisc agcnls, including exogenous agcnls such as chemical and physical agenis. as well .is lo endogenous agents like rcaai\c oxygen species (ROS) liom oxidative metabolism or electron transport diam that can all result in damage to DNA In yeast, high intracellular ROS levels pose a significant threat lo cellular intcgiii\ .md can lead to mitochondrial DNA damage, impainnent ol'mitochondiial resinralion, reduction of oxygen consumption inhibition of iw<i key glycoKiic cn/\incs. GAPDH and P\ K The metabolic llux does not shunt to the late stage of glycolysis, Krebs cycle, and througli electron transport chain where a large amount of ATP are generated per glucose molecule, but diverts to the early stage of glycolysis, namely to the pentose phosphate paih\\a>, hcxosamme biosynthesis, and finally to glycogen and trehalose synthesis 01>cogcn and trehalose act as readily mobilized storage m the form of glucose for survival of cells and maintenance of cellular energy. In handle such kinds of DNA damage, \cast cells ha\e e\olved a number ol cellular mechanisms or icsponses including DNA damage checkpoints, cell cycle arrest, transcnptional program acii\ation, stimulation of DNA repair, tolerance of DNA damage and initiation of apoptosis These cellular defense mechanisms are regulated by specific genes in
\\hich their role and fijncuon are similar to those found in mammanlians Thus, in this review, we discuss DNA mutating agent, DNA damage repair and cellular defense mechanisms, and the homology of ftinclion between human- and yeast genes, as well as the use of yeast cell as a model organism for cell cycle and biochemical pathway stud>, new drug disco\ery, and cancer research.
Keywords: Appotosis (programmed cell death), DNA damage. PYK (pyruvate kinase). GAPDH (glyceraldehyde-3-phosphafe dehydrogenase). G6PDH (glucose-6-phosphote dehydrogenase). ROS (oxygen reactive species)
ROS GENERATION motive force Through the electron transport chain, the large amount of energy released is conserved in the . . ^ „ form of ATP by a process called oxidative Cells can obtain energy m the form of ATP p|,osph„^,3„„„ (Nelson, Cox. 2005).
either by anaerobic fermentation m which glucose is
broken dowTi into some fermentation products like However, in the course of the electron transport lactate, ethanol, etc or by aerobic cellular respiration chain, about 1 - 2% of oxygen, rather than being where glucose is completely oxidized to C O : and reduced to H^O, is partially reduced to the H2O. This oxidation occurs inside mitochondria superoxide anion radical {'O2) or hydrogen peroxide called the tricarboxylic acid cycle (TCA cycle, also (H2O2). During oxidative phosphorylation, electrons known as the citric acid cycle or Krebs cycle), (Berg are delivered through the mitochondrial electron et al, 2002). The TCA cycle is the final common transport chain (mtETC) and a proton gradient is pathway for the oxidation of ftiel molecules. Of the established across the inner mitochondrial products, the reduced coenzymes i.e, electron carriers membrane. When an electron escapes from the NADH and FADHi are themselves oxidized, giving mtETC. it may react with a molecular oxygen to up protons ( l O a"** electrons, the electrons are form O i ' that combines with a hydrogen atom transferred to O2 - the final electron acceptor via the yielding hydroperoxyl radical H O . ' (pK^ = 4.88), electron transport chain known as respiration chain thus at physiological pH HO;' exists as H2O2 wiiich reduces O2 to H2O and generates a proton- Furthermore, generation of ROS also occurs through
Bui Van Ngoc & Nghiem Ngoc Minh exposure to numerous exogenous agents including
ionizing radiation (IR), UV. and cnvironmcnial toxins (Salmon el al.. 2004). Thus, ROS ('O: , 'Ol 1.
H:02.,.) are geneniled through both c,\ngciunis and endogenous routes and pose a .signitiLinit thieal lo cellular inlcgiil\. chemically modify DNA. lipids, proteins, and other macromolcculc.s (Cooke et al..
2003), Oxidative dainago generated \i\ inliacellular ROS can lead to DNA base modillcalmn, single- and double-strand hiciks, or formation of apurinic/apyrimidinic lesions (Salmon ct al. 2004).
In S. icievisiae ROS cause cellular oxidative stress and high accumulated ROS le\els will damage mitochondrial DNA (mtDNA), reduce mitochondrial actnity. and consequently decreasing n.wgcn consumption and ATP synthesis (Bui. 2010). In following SCI.II0I1S. the homology of cellular defense mechanisms, regulation genes, and biochemical pathways in response lo DNA damage between yeast and inantmalian cells will be elueidaied,
CELLULAR DEFENCE MECHANISMS IN RESPONSE TO DNA DAMAGE
To handle such kinds of DNA damage as mentioned above, yeast cells have evolved a number of cellular defense mechanisms or responses including DNA damage checkpoints, cell cycle arrest, transcriptional program activation, stimulation of DNA repair (Caldecott, 2004). tolerance of DNA damage (Salmon et al. 2004), and initiation of apoptosis(Wissing^;o/. 2004; Almeida*rM/,. 2008), DNA damage checkpoints
The DNA damage checkpoints are signal transduction pathways that sense the presence of DNA damage and transmit a signal lo downstream effectors to execute the various cellular responses to DNA damage DNA damage checkpoints attenuate mutagenicity, genomic instability, and cell lethality by delaying cell cycle progression, increasing the transcription of genes involved in DNA repair, as well as directly modulating DNA repair enzyme activity (Caldecott, 2004).
Like other signal transduction pathways, DNA damage checkpoint pathways involve sensors, transducers, and effectors. Sensors monitor the DNA for the presence of abnormalities, by interacting indirectly or directly with DNA lesions and then initiating transduction of the checkpoint signal. The transducers further transmit and amplify this signal.
while the el'fectors control the biological consequences nf triggering (he pathway. The pi ;ici icjil distinction between "sensors" and
••(laii.sdiicers" is not apparent, since such
"iransducer" kinases as Mecl (in S. cerevisiae), Rad3 (.V potiihe). and ATM (in humans) are capable of sensing certain types of DNA damage indcpciulenlly (Caldecott, 2004)
DNA repair complexes may not only have direct rules in signaling the presence of lesions to the checkpoint machinery, but may also facilitate signaling by modifying different types of primary lesions to common structures such as through processing by specific repair pathways or as a result of DNA replication. Recognition of such structures might then trigger a checkpoint response (Caldecott.
20(14) The damage-sensing, checkpoint and repair pathways arc illu.slraled in Figure I,
Initial detection of DNA damage by specific repair or replication-associated protein complexes recruited lo the lesion. Most lesions are repaired quickly, slower repair results m lesion processing/
modification. Initial recruitment of Mecl damage sites allows it to phosphorylate local targets such as Rad9. The PCNA (proliferating cell nuclear antigen)-like complex may be independently recruited. If the repair is successful, activation of the Mec 1-dependent damage signaling cascade may be avoided. Checkpoint and repair factors including PCNA-like complex accumulates at difficult-to- repair lesions, contributing to a sustained and elevated level of Mec I-dependent checkpoint signaling. This results in activation of the transducer kinases Rad53 and Chkl, and phosphorylation of damage checkpoint effectors, and leads to dovmstream consequences, including cell cycle arrest, transcription, and increased repair.
Cell cycle checkpoints
Cell cycle checkpoints recognize damage and arrest the cell cycle, OT regulate transcriptional induction and progression of DNA replication. In humans, cell cycle checkpoints are regulatory pathways that govern the order and timing of cell cycle transitions to ensure completion of one cellular event prior to initiation of another. The key regulators of the checkpoint pathways in tlje mammalian DNA damage response are ATM and ATR. Both ATM and ATR appear to phosphorylate many of the same cellular substrates, but they generally respond to distinct types of DNA damage. ATM is the primary 198
Tap chi Cong ngh? Sinh hgc 10(2): 197-206. 2012 mediator of the response to DNA double-strand breaks (DSBs) which can arise by exposure to ionizing radiation (IR), while ATR plays only a backup role in DSB response, but directs the principle response to UV damage and stalls in DNA replication.
Cell cycle transitions are regulated by cyclin- dependent kinases (CDKs) that promote ceil
proliferation (Lodish, 2008), CDK activity is also required in human cells to maintain function of the Chkl pathway, a key component of the response lo DNA damage or stalled replication (Maude and Enders. 2005), In .S' ccicvisiae. single CDK encoded by (.'!K'2<S interacts with several dilVerent cyclins during dilTcrent phases of the cell cycle (Figure 2).
Figure 1 Sensing, signaling and DNA damage repair in S cerevisiae (Caldecott, 2004)
Each group of cyclins directs the S cerevisiae CDK to specific functions associated with various cell cycle phases. Cln3-CDK induces expression of Clnl.
Chi2, and other proteins m mid-late G, by phosphorylating and activating the SEP and MBF transCTiption factors, the Gi CInl-CDK and Cln2-CDK inhibit die APC, allowmg B-type cyclins to accumulate (Clb6, Clb5, Clb4,..). These Gi cyclin-CDKs also activate degradaticai of S-phase inhibitor Sicl.
Subsequently, the S-phase Cln6-, Cln5-, Cln4-, Cln3- CDK complexes trigger DNA synthesis, hi Cln4- and Cln3-CDK complexes also fiuiction as mitotic cyclins by tnggering formation of mitotic spindles. The
function of the last two cyclin-forming complexes with CDK (Cln I - and Cln2-CDK) is to trigger chromosome segregation and nuclear division (Lodish, 2008).
When certain cell cycle checkpoints are nonflinctional, the cells form abnormal numbers of chromosomes, consequently chromosome aberrations abound in tumor cells. If DNA sequence changes are left uncorrected, both proliferating and somatic cells might accumulate mutations.
Additionally, germ cells may accrue too many mutations for viable offspring to be formed.
Therefore, the prevention of DNA sequence errors in all types of cells is important for survival, and
Bui Van Ngoc & Nghiem Ngoc Minh several cellular mechanisms for icpainng DNA
damage and correcting DNA sequences have evolved, called DNA repair pathways. These pathways (not described in detail) include direct repair or reversal (DR). base e\cisioii repaii (BER).
nucleotide excision repair (NER). mismatch repair (MR), double-strand breaks repair (DSBs repair) including homologous recombination (MR) and non- homologous end-joining (NHE,I). And if unrepaired DNA or mutations are reinaiiied. activation of apoptosis would be induced.
Apoptosis
Apoptosis (programmed cell death) plays a crucial role in the normal development and differentiation of multicellulai organisms and is essential for einbryogenesis and metamorphosis (Kenetal. 1972) Apoptosis may be triggered by an external signal, acting at a receptor in the plasm.i membrane, or b> internal events, e.g. viral infection (Nelson and Cox. 2005). Apoptosis is also induced by a variety of agents such as heat shock (Ghibeili el al, 1992) and various cytotoxic substances (McGowan el al., 1996). Under either physiological or pathological conditions, apoptosis is mostly driven by interactions among several families of proteins, i.e. caspases, Bcl-2 family proteins, and inhibitOT of apoptosis proteins (lAP proteins)
In mammalian cells, n is reported that mitochondria are involved in the regulation of apoptosis. In healthy cells, Bcl-2 family proteins located mainly on the outer membrane of mitochondria inhibit apoptosis by preventing the release of cytochrome c into cytosol which activates caspases leading to apoptosis. while lAP proteins suppress apoptosis by binding to activated caspases that carry out this process (Wright and Duckett, 2005).
In cancer cells, when the cell receives the signal for apoptosis, one consequence is an increase in permeability of the outer mitochondrial membrane, allowing cytochrome c to be released from mitochondria (along with other proteins such as apoptosis-inducing factor - AIF and pro-caspase-9) and binds to the protein Apaf-1 (apoptotic protease activating factor 1), These complexes aggregate to form apoptosomes which then bind to and activate caspase-9; this activated protease activates other caspases e.g. caspases-3 and -7. Consequentiy, these executioner caspases induce an expanding cascade of
proteolytic activity which leads to digestion of .struclural proleins in the cytoplasm, degradation of chromosomal DNA and cell phagocytosis (Desagher,
1999),
Like mammalian cells, yeast cells can undergo cell death accompanied by cellular markers of apoptosis. Cell death with apoptosis-like features has also been reported in yeast after treatment with acetic acid, UV-irradialion, glulathione-depleting chemicals and H^O^d-iidovico (•/«/., 2001; Del Carraloree/o/., 2002). The common dcnnminatur in most of these ccll-dealh models that involve yeast seems lo be an accumulation of ROS. A caspase-like protein with homology lo mammalian caspases has been identified in S. cerevisiae (Yorl97w) and implicated in cell death that is induced by H ; 0 ; , acetic acid and ageing (Madeo el al.. 2002). This protein is called yeast caspa.se-1 (YCAI) and a member of the metacaspase family - putative proteases that have a caspase-hke folding. Overexprcssion of YCAI enhances apoptosis-like death of yci.st that is induced by H : 0 : or acetic acid, whereai excision of the YCAl- encoding gene reduces cell death (Jin and Reed, 2002) Furthermore, the YCAI protein also seems to undergo proteolytic processing in a active-site cysteine dependent-manner, ss^ich is similar to mammalian caspases (Madeo et al.. 2002).
THE ACTIVATION OF IMPORTANT GENES IN RESPONSE TO DNA DAMAGE
The role of some crucial genes in D.N.A damage repair, cell cycle arrest, a n d apoptosis
Genes required for cell cycle arrest h a \ e been identified and reported in budding fission yeasts, and exert al least two functions: as sensors that associate with DNA and as signal transducers that mediate dovmstream events by phosphorylatiwi (Nickoloff, Hoekslra, 1998), of which RAD9 encodes Rad9, a DNA damage-dependent checkpoint protein, Rad9 is an adaptor protein required for cell cycle checkpoint function m S. cerevisiae and is a mediator of cell cycle arrest at the G2/M cell cycle checkpoint induced by double-strand breaks (Bennett el al.
1993). It IS required throughout the cell cycle and has been shown to function at the G|/S. intra-S, and G2/M phases (Weinen. Hartwell, 1988, Toh, Lowndes. 2003). Furthermore, Rad9 is required for activation of both Rad53 and Chkl by the DNA damage checkpoint pathway (Sanchez el al, 1999), Additionally, Rad9 is a component of the
Tap chi Cong nghe Sinh hpc 10(2): 197-206, 2012
Mecl/Rad9/Rad53 DNA damage repair patliway that controls mtDNA copying and mitochondrial stability (Taylor el al.. 2005). Thus. Rad9 plays a role in controlling inh-acellular ROS levels (Loegering el
al. 2004),However, mitochondria, which .ue major consumers of oxygen and responsible for ATP synthesis by oxidative phosphorylation, are the chief source of cellular ROS (Peiicano f/(;/,, 2003).
Mitochondrial DNA is a critical target foi such oxidants generated by the niilochondnal electron transport chain (Van Houten et al.. 2006). The
HAP4 gene encodes Hap4 that is a subunit of theheme-activated, glucose-repressed Hap2p/3p/4p/5p complex This complex acts as a transcriptional activator and global regulator of respiratory gene expression and plays a role in transcription of genes involved in the TCA cycle, the mitochondrial electron transport chain. ATP production and mitochmdria biogenesis (Raghevendran el al.
2006). Thus, Hap4 plays a crucial role in controlling oxygen consumption and mitochondrial activity; and deletion of HAP4 should result in reduction of mitochondrial activity and oxygen consumption.
The expression of Hap4 is also repressed via the Migl pathway. Migl encoded by MIGl is a transcription factor involved in glucose repression.
In high glucose condition, the Hxk2/Snfl complex binds to Migl to form a new complex that translocates from the cytoplasm to the nucleus to repress transcription of target genes encoding the utilization of sugars (Moreno et al. 2005).
Thereby, the activation of respiration is prevented in high glucose conditions (Bitterman et al, 2003).
In low glucose conditions, Migl is phosphorylated by Snfl kinase and exported from the nucleus to the cytoplasm leading to derepression of target genes (Moreno et al.. 2005), in this case respiration is activated. Therefore, the Migl repressor inhibits genes involved in respiration, gluconeogenesis, and alternative carbon source utilization (Bitterman et
al, 2003) and deficiency in MIGl should lead to adecrease of mitochondrial respiration and loss of control in glucose utilization.
Besides the central role of mitochondria in energy metabolism, mitochondria also play an important role in apoptosis. In yeast, Ycal (yeast caspase 1) and Aifl (apoptosis inducing factor) encoded by the YCA1 and AIFI genes are important -components in yeast apoptosis in response to DNA
damage. Ycal. a member of the metacaspase family, plays a crucial role in the regulation of yeast apoptosis When cells are exposed to oxidative stress (ROS) or DNA damaging agents, cylochrome c is released from mitochondria to the c\losol (along with other proteins such as Aifl), which aclivates Ycal to induce apoptosis (Almeida
el al. 2008). Aifl is a flavoprotein withoxidoreductase activity, localized in the mitochondrial intermembrane space. Upon apoptosis induction. Aifl translocates from the mitochondrial intermembrane space lo the nucleus where it causes chromatin condensation and DNA degradation (Wissiiig ci al. 2004); it also controls a caspase-independent pathway of apoptosis (Cregan (•/ al. 2002), Thus, cells defective in YCAI or AIFI should result in induction of cell death or apoptosis Additionally, Pex6 is a peroxin encoded by PEX genes and a peroxisomal membrane protein involved in peroxisomal protein import Pex6 acts downstream of the receptor docking in the terminal steps of peroxisomal matrix protein import.
Pex6 is supposed to control ROS accumulation and cell viability in early stationary phase, and it does not depend on the apoptotic activators Ycal and Aifl in apoptosis, but triggers necrotic cell death (Jungwqrth el al. 2008), Thus, deficiency in PEX6 should lead lo loss of cell viability and cause necrosis. The role of these genes controlling cellular defense mechanisms in response to DNA damage is summarized and outlined in Figure 3
In mammalian cells, several proteins of damage checkpoint pathways also function as tumor suppressors, e.g. p53. Defects in checkpoint signaling pathways are frequently associated with cancer (Lodish el al.. 2008), Cellular responses to DNA damage, including cell cycle arrest, are a common feature of all eukaryotic cells and genes that mediate these responses are highly conserved (Caldecott, 2004), Many of the genes that regulate cell division in yeast were also shown to work similarly in humans (Hartwell, 2002), The budding yeast S. cerevisiae has long been recognized as a versatile model system for drug discovery and studying of eukaryotic cells, since many of the basic cellular processes of both yeast and humans are also' highly conserved. To date, no detailed analysis has been made on energy metabolism and the metabolic alterations in cellular response to DNA damage induced by DNA damaging agents; also needed is an investigation of key genes that are crucial m DN.^
201
Bui Van Ngoc & Nghiem Ngoc Minh damage repair, cell cycle arrest, lespiralion, and
apoptosis.
The metabolic alteriHions In cellular response lu l > \ A damage
A research work was carried out to investigate the metabolic clianiics in cellular lesponsc to DNA damage by Ireatmeni with a gciioloMC agent, methyl methanesulfonale (MMS). And by using specific knock-out \casi strains, the role of some important genes controlling the cellular defen.se mechanisms (as described above) in such condition would be elucidated (Figure 3) (Bui. 2010).
The findings indicate thai the specific response of the individual niulanl strain {AraiI9. \hap4.
Amigl. Meal. AaiJI. and Apc.v6) elucidates the role of the corresponding gene deputed by the respective pathway in respon.sc to DNA damage. Indeed, fully functional DNA damage repair and cell cycle checkpoint {RAD9) significantly reduces intracellular ROS accumulation, defects of RAD9 (Aia<i9) results in high ROS accumulation, thereby increasing damage to mtDNA leading to inhibition of mitochondrial activity and oxygen consumption.
Similarly, fully mitochondrial function and activity (HAP4) also attenuates ROS accumulation and enables efficient electron transport, lack of HAP4 {Ahap4) causes blockage of mtETC, persistent inhibition of mitochondrial activity, also leading to reduced oxygen consumption. Taken together, high mitochondrial activity reduces development of oxidative stress and acts as a protective mechanism against oxidative stress, low mitochondrial activity can lead to enhanced cytotoxicity and cellular sensitivity to DNA damaging agents. Cells defective in peroxisomal protein import (Apex6), yeast caspase 1 {Aycal), and apoptosis inducing factor (Aaifl) which have an extremely low mitochondrial activity and strong repression of oxygen consumption, consequently cause loss of cell viability {Apex6) and induction of cell death or apoptosis (Aycal, Aaifl).
A further consequence of the cellular metabolic response to DNA damage is the inhibition of two key glycolytic enzymes, GAPDH and PYK, that leads to reduced ATP production. The metabolic flux diverts to the PPP reflected in high G6PDH activity and to die HBP resulting in high UDP-NacGlu level, and finally to glycogen and trehalose synthesis. High glycogen and trehalose accumulation reflects the inactivation of MIGI that controls the glucose
repression and gluconeogenesis in mutant Amigl, and it preferentially acts as a readily mobilized storage Ibrm of glucose for survival of cells with low energy production like mutants Aycal. Apex6, and A , » / / ( l - i g u i e 3 ) ( B u i . 2010).
These findings also fit to the observation that tumor cells have an increased rate of glycolysis compared with normal cells, and support that this increase is becau.se of impaired respiratory capacity of tumor cells i.e, tumor cells totally rely on glycolysis (Warburg, l'''^6). In recent years two human genes, p!'i AXXAAIM. have been studied to set up the relationship between checkpoints and cancer (Nickoloff and Hoekslra, 1998). The checkpoint defects in cancer cells may be usellit in designing new L.inccr therapy siralegies (Hartwell, 2002).
Inlereslingly, many of the genes that regulate cell division in yeast also act similarly in humans e.g.
cell division cycle {CliC). cyclin-dependenl kinase 1 iCDKI), and DNA repair and checkpoint genes iRAD9) in S. cerevisiae (Hartwell, 2002). Indeed, regulation of the cell cycle is impaired in mutant AratJ9 upon radiation, and Arad9 showed a 20-fold increase of chromosome loss r<ite even in the absence of any extrinsic DNA damage (Hartwell.
2002). In addition, checkpoint defects in yeast cells lead to increased sensiti\ity to DNA damaging agents, for example, deletion of RAD9 {Arad9) is sensitive to even 0.00125% MMS (Kitanovic. 2006).
This suggests that the checkpoint fijnction is needed in those cells to assure correct repair of the damage and such defects may be manipulated in mammalian cells as well, and suppiffts the prospect that yeast cells have been used as versatile model organisms for cancer research and drug discovery.
Treatment of DNA damage agents (MMS) strongly inhibits mitochondrial electron transport chain - mtETC (perhaps because of damaging mtDNA) and decreases oxygen consumption of Arad9. Ahap4, Amigl. particularly Aycal. Apex6, Aaifl resulting in toss of cell viability of Apex6, a mutant defective in peroxisomal protein import machinery, and inhibition of cell growth of Aycal.
Aatfl. mutants defective in regulation of yeast apoptosis. MMS treatment induced high ROS accumulation, especially in mutant defective in cell cycle arrest and control of mtDNA copying, Arad9.
ROS directly inhibit GAPDH activity, this effect downstream reduces PYTC activity leading to initiation of cell cycle arrest. Further effect is the inhibition of ATP synthesis. Glucose flux thus does
202
Tap chi C<h,gngh?Smh hoc 10(2): 197-206, 2012
not shunt to the lower part of glycolysis, namely to the TCA cycle and mtETC where a large amount of ATP molecules is produced per glucose molecule, but is re-routed to upper part of glycolysis, namely to the PPP as a result of increasing G6PDH acliviK, through the PPP an excessive amount of iinlio\idaiU cofactor NADPH and nucleotides is generated for cellular antioxidative defense system and DNA repair. Also, glucose is diverted to the HBP yielding high UDP-NacGlu level as a signal of cellular signaling in response to DN.\ damage. Finally, glucose IS shunt to reser\c carbohydrates through glycogen and trehalose s\iithesis Mutant A/mgl accumulates high gKcogen and trehalose levels. This mutant lacks Migl, a transcription factor involved in glucose repression and a componenl of H\k2 SntT/Migl complex wheie Migl acts as a repressor to repress genes involved in respiration.
gluconeogenesis. and alternative carbon source utilization, that leads to persistent activation of glucos; utilization and gluconeogenesis in mutant
Amigl While very high accumulated glycogen andtrehalose levels of Avcal, Apex6. Aaifl mutants result from more severe impairment of mitochondrial respiration leading to redirection of all glucose to
glycogen and trehalose and rapid accumulation of these reserve carbohydrates for their energy maintenance
Figure 2. Activity of S cerevisiae cyclin-CDK complexes through the course of the cell cycle {Lodish, 2008) The vanous cyclins, which are expressed dunng different phases of the cell cycle, control single cycl in-dependent kinase (CDK)
[amigl. /iycot
^a6,aoifii
t
{&rad9, ihop^, amigl, aycal,
i'"*- -<S>'
Figure 3. Schematic representation of the rote of some important genes controlling the cellular defence mechanisms by using specific knocl<-out yeast strains and cellular metabolic alterations in response to DNA damage (Bui, 2010).
Bui Van Ngoc & Nghiem Ngoc Minh YEAST IN APOPTOSIS AND CANCER
RESEARCH
The yeasi S. eerevi.Miie is .1 simple eukaryotic organism with jusi approxuiiaicly 6000 genes. More than 60% ol" the genes have an assigned function, while more than 40"o shaie conseived sequences with at least one known 01 predicted human gene (Alves-Rixlrigues el al. 2(l(l()). Thus, due lo the high consei\';iiioii of IlindainciUal biochemical pathways, yeast has Iwen used as a versatile model for drug discovery and for stud>ing biological processes in higher eukaryoles.
i-\cn ilmiigh yeast lacks much of the molecular machinery involved in ;ipop1osis of mel;i/oiiiis, they can be a powcrfijl tool in apoptosis research, Tlie ectopic expression of several animal apoptosis proteins in \e.ist can help to discover new genes and chemical compounds thai modulate the cell death pathways of higher eukaryotes (Jin, Reed, 2002), As described above, apoptosis in human cells is controlled by many protein families that serve to either promote or suppress caspase activation leading to trigger or inhibit apoptosis. Of which are caspases, inhibitor of apoptosis (lAP), B-cell lymphoma-2 (Bcl-2). caspases-3. -7, -9 (caspase-recruitment domain, CARD), apoptotic protease activating factor 1 (Apaf-1).
However, yeast does not have the same apoptotic machinery as human cells, while apoptosis-like features have been reported in yeast CDC48 mutants (Madeo et al., 1997, 1999). The Cdc48 protein of 5 cerevisiae is a putative ATPase and is normally involved in membrane-fusion events that are associated with cell division and secretion.
The common denominator in most of cell-death models that involve yeast seems to be ROS accumulation (Madeo et al. 2002). Yeast caspase-l (YCAI) is a caspase-like protein with homology to mammalian caspases and YCAI has been implicated in cell death that is induced by HiOi, acetic acid and ageing (Madeo et al., 2004),
Many studies suggest that yeast may be useful for studying proleins involved in apoptosis in animals. For example, the ectopic expression of the anti-apoptotic Bcl-2 protein rescued SOD (superoxide dismutase)-deficient yeast strains And the ectopic expression of Bax (a pro-apoptotic Bcl-2 family member) in yeast produced a lethal phenotype. Moreover, Bax-induced death of budding yeast is specifically suppressible by Bcl-2 and other
anti-apoptotic Bcl-2 family members, but not by mulanl versions of Bcl-2 that fail to protect mammalian cells from apoptosis (Jin, Reed, 2002), Additionally, ectopic expression of caspases in yeast has also been u.sed lo study the mechanism of lAP family proteins and iheir antagonists, and co- cvpression ol lAPs and certain caspases also rescue yeast (Hawkins el al. 1999; Jin, Reed. 2002)
Yeast has been serving as a model organism in cancer I cscarch, since the same genes that control the cell cycle in yeast or that malfunction in tumor cells exist in more or less the same capacity in human cells. In ycasl, these include the CDC (cell division cycle) and CDKI (cyclin-dcpendent kinase I, Figure 2) genes Many of the same genes that regulate cell division in yeast also act similarly in humans. In S.
cerevisiae there arc three types of genes that regulate the cell cycle: CDCK l)K. checkpoint genes, and DNA repair genes (Hartwell. 2002).
Aeknowlcdgemcnl: / wish lo express in\ sincere graliliicle to my profcs.\or. Prof Dr Stefan iVdlfl (Institiile of Pharmacy and Molecular Biotechnology - Universily of Heidelberg - Germany), who gave me an opportunity to do research works of biochemistry and pharmui ciitu al biology when I was studying at University of Heidelberg and offered an interesting idea for using yeast cells to study on biochemical pathway and cancer therapy in higher eukaryotic cells when I returned Vietnam. I also sincerely thank lo all ojficials and staff members of Department of Environmental Biotechnology -IBT- VAST who rendered their support, help, and kind co- operation for completion of this work.
REFERENCES
Almeida B, Sitva A. Mesquila A, Sampaio-Marques B.
Rodrigues F, Ludovico P (2008) Drug-induced apoptosis in yeast Biochim Biophys Acta 1783(7): 1436-1448.
Alves-Rodrigues I, Gallo RP, Meyerhans A, Diez J (2006) S.
cerevisiae: a uselul model host to study fundamental biology of viral replication. VinisRes 120(1-2) 49-56.
Bennett CB, Lewis AL, Baldwin KK, Resnick MA (1993) Lethality induced by a single site-specific double-strand break in a dispensable yeast ptasmid. Proc Natl Acad Sci t/5-4 90(12): 5613-5617,
Bennett RA (1999) The Saccharomyces cerevisiae ETHl gene, an inducible homolog of exonuclease III that provides resistance to DNA-damaging agents and limits spontaneous mutagenesis, Mol Cell Biol 19: 1800-1809.
204
Tap chi Cong ngh? Sinh hQC 10(2); 197-206, 2012 Berg JM, Tymoczko JL, Stryer L (2002) Biochemistry, 5th Edition, W H, Freeman and Company, New York; 392- 560,
Bitterman KJ, Medvedik O, Sinclair DA (2003) Longcvily regulation in 5 cerevisiae linking metabolism, genome stability, and heterochromatin Murobiol \hl Biol Rev 67(3): 376-399.
Bui VN (2010) Modulation of cellular metabolism in response to DNA damiige. Doctoral Tliesis. Inslitule of Pharmarcy and Molecular Biolecbnology. Universily of Heidelberg, Germanv.
Caldecott KW (2004) Etikannlic DNA damage surveillance and repair. Kluwer Acadennc/ Plenuiii Pulishers, New York USA' 1-20.
Cooke MS. Evans MD. Dizdaroglu M, Lunec J (2003) 0\idati\c DNA damage: mechanisms, mutiUion. and disease FASEBJMOO). 1195-1214
Cregan SP, Fortin A. MacLaurin JG, Callaghan SM, Cecconi F, Yu SW. Davraon TM, Dawson VL, Park OS.
kroemer G, Slack RS (2002) Apoptosis-mducing factor is involve i in the regulation ofcaspase-independenl neuronal cellde&lh J Cell Biol 158(3): 507-517.
Del Carratore R, Delia Croce C, Simili M, Taccini E, Scavuzzo M. Sbrana S (2002) Cell cycle and morphological alterauons as indieaUve of apoptosis promoted by UV irradiation in S cerevisiae Mulat Res 513(1-2): 183-191
Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S. Lauper S, Maundrell K, Antonsson B, MarUnou JC (1999) Bid-induced conformational change of Bax is responsible for mitochondnal cytochrome c release during apoptosis JCellBiol 144(5): 891-901.
Ghibeili L, Nosseri C, Oliveno S, Piacentini M, Autuon F (1992) Cyclohexamide can rescue heat-shocked L cells from death by blocking stress-induced apoptosis. Exp Cell Acs 201(2); 436-443.
Hartwell LH (2002) Nobel Lecture. Yeast and cancer Biosci Rep 22(3-4)- 373-394.
Hawkins C, Wang S, Hay B (1999) A cloning method to identify caspases and their regulators in yeasf identification of Drosophila LAPl as an inhibitor of the Drosophila caspase DCP-I, Proc Natl Acad Sci USA 96(6)' 2885-2890.
Jin C, Reed JC (2002) Yeast and apoptosis. Nat Rev Mol Cell Biol 3(6): 453A59.
Jungwirth H, Ring J, Mayer T, Schauer A, BUttner S, Eisenberg T, Carmona-Gutienez D, Kuchler K, Madeo F, (2008) Loss of peroxisome function triggers necrosis, FEBSLetl 582(19): 2882-2886.
Kerr JFR, Willie AH, Cume AR (1972) Apoptosis: a basic biological phenomenon with wideranging implications in
tissue kinetics. Br J Cancer 26' 239-257,
Kilanovic A (2006) Regulation of I'lMM expression and changes in glucose metabolism are crucial for ;in appropriale response to DNA damage and aging in S cerevisiae Disserlalioiv 75-76,
Lodish H, Berk A. Kaiser C, Krieger M, Scott M, Bretscher A, Ploegh II. Matsudaira P (2008) Molecular Cell Biology.
6'^ Edn W II Freeman and Coinpjny, New York: 305, 878- 966
Loegering D, Arlandei SJ, Haekbarth J, Vmman BT, Roos-Matljus P, Hopkins KM, Lieherman HB, Kamilz LM, Kaulinann SH, (2004) Rad9 protects cells from topoi sonic rase poison-in due ed cell death J Biol Cheiii 279(18), I864I-I8647,
Ludovico P. Sousa MJ. Silva MT. Leao C. Corte-Real M (2001) Saccharomyces cerevisiae commits lo a programmed cell death process in response to acetic acid.
Microbiology 147(Pl 9); 2409-2415
Madeo F, Frihlich E, Frohlich KU (1997) A yeast mutant showing diagnostic markers of early and late apoptosis J Ce//BIO/139, 729-734
Madeo F, FrShlich E, Ligr M, Grey M, Sigrist SJ, Wolf DH, Frfthlieh KU (1999) Oxygen stress' a regulator of apoptosis in yeast J Cell Biol 145(4), 757-767, Madeo F, Herker E, Maldener C, Wissing S, Lachelt S, Herlan M, Fehr M, Lauber K, Sigrist SJ, Wesselborg S, Frohlich KU (2002) A caspase-relaled protease regulates apoptosis in yeast Mol Cell 9(4): 911-917
Madeo F, Herker E, Wissing S, Jungwirth H, Eisenberg T, Frohlich KU (2004) Apoptosis in yeast. Ctirr Opin Mcraftio/7(6): 655-660.
Maude SL, Enders GH (2005) Cdk inhibition in human cells compromises Chkl function and activates a DNA damage response. Cancer Res 65(3): 780-786 McGowan AJ, Femandes RS, Samali A, Cotter TG (1996) Antioxidants and apoptosis Bioch Soc Trans 24(1): 229- 233.
Moreno F, Ahuatzi D, Riera A, Palomino CA, Henero P (2005) Glucose sensing through the Hxk2-dependent signalling pathway, Biochem Soc Trans 33(Pt 1): 265-268, Nelson DL, Cox MM (2005) Lehninger Principles of Biochemistry, S"' Ed. W.H. Freeman and Company, New York.
Nickoloff JA, Hoekstra MF (1998) DNA Damage and Repair HUmana Press, Totowa, New Jersey: 305-376.
Pelicano H, Feng L, Zhou Y, Carew JS, Hileman EO, Plunkett W, Keating MJ, Huang P (2003) hihibition of mitochondrial respiration: a novel strategy to enhance drug-induced apoptosis in human leukemia cells by a 205
Bui Van Ngoc & Nghiem Ngoc Minh reactive oxygen species-medialed niechamsin / Biol
(ViLVH 278(39)-37832-37839.
Raghevendran V, Patil KR, OKson I, Nielsen I (2006) llap4 IS iiiii es.scnlial for iicliviiliim of respiration al low speeillc giimih rates in Sm rhuriiinvce.^ i'cirviM<ie .1 Biol r/;<7j, 2SI(18): 1 21flN-| 2114,
S.ilmou IH, Ivcil HA. Son;: U, D.K-t^ch PW (2004) Biological consequences ol oMd.iUvc slicss-induied DNA d.iniage in Saccliaioimre^ cerevisiae. \iiilcu ,(i/<A Re.', 32(12), 3712-172.'
S.mche/ 'i', Bachanl J. W.mj: H. Hu 1-. I.iu t), Teli-LiK M.
Elledge SJ (I9')9) Control ol ilic DNA damage checkpoint by chkl and rjd53 piolein kiii.isfs iliioii(;h dislmil inechaniMiis .V/OKV 2S(t(S44:i Il(»h-ll7l.
hivlni SI). Zhang II, Ealon JS, Rodchel'ler MS, U-hedeva MA. OVourke T W Sicde W. Shadel (iS (2005) The conseived ML-Ll/Rad53 nuclear checkpoint pathway regulates mitochondrial DNA copy number in 5 cerevLsuie. Mol Biol Cell 16(6)' 3010-3018.
Toh (iW, Lowndes NK (2003) Role of the Saccharomyces cerevisiae Kad9 protein in sensing ;ind responding to DNA damage niachemSoc Trans 3\(V\ I), 242-246.
Van lloulen H. Woshner V, Snnlos JH (2006) Role of milochondrial UNA in loxie icspon.ses lo oxidative stress.
DNA Repair 5(2): 145-152, Warburg (). (1956) On the origin of cancer cells, .Science 123' 309-3 14.
Weiiierl'IA, Ilartweli LM (1988) The KAD9 gene controls the cell cycle icsponse lo DNA damage in Saccharomyces
<ereviM,ic Science 14\{4V,b'i) 117-322.
Wissiny S, I udi'vico P, Herker E. BUUner S. Engelhardt SM. Decker I. Link A, Proksch A, Kodrigues F, Corie- RLMI M , l-r<Shlieh KU, Manns J. Cande C, Sigrist SJ, Kroemer (I, Madeo F (2004) An AIF orthologue regulales apoptosis 111 yeasI, JC<'///fWlf.f.(7), 969-974, Wiiyhl CW, Duckett CS (2005) Reawakening the cellular dealb piograin in neoplasia through the therapeutic blockade of lAP function J Clin Inve.it 115(10)- 2673- 2678.
SI in\C. TV BAO NAM M\:\ S.4CCH.\ROMYCES CEREVISIAE LAM MO HrNH
TU()N(; N ( ; H I K \ C I i < . u Q I \ T R I N H S I M I H O A T E B A O , P H A T MIEN T H U O C\ i 6 i \ \ N ( ; H I F N C I U I : N ( ; T H U O S I N H \ A T N H A N C H U A N B A C C A O BiJi \ an Nsoc, Nghiem Ngoc Minh'
I 'icn Cong nghe sinh hoc. Vien Khoa hpc va Cong ngh^ Vi^l Nam TOM T A T
Trong qua trinh phat trien te bao nhan that se phai doi mat voi nhieu tac nhan bat Igi khac nhau, bao gom lac nhan ngoai bao tac nhan vat ly, hoa hpc, dac biet lae nhan npi bao, cac goc oxy gay phan ung (ROS) duoe sinh ra trong qua trinh van ehuyen di?n tu cua chuoi ho hap, Ngoat ra, ROS cung dugc hinh thanh gian tiep do lac nhan ngoai bao. 0 nam men, khi muc ROS npi bao lang cao sfi de dpa tinh on djnh trong moi Inrong noi bao, gay ton thuong DNA, d^c bi?t DNA ty the, dan den lie che hogt dpng ho hap ciia ty the, kirn ham hoat do cua cae enzyme GAPDH va PYK trong pha sau cua chu trinli dirong phan. Khi do dong carbonhydrate se khong Hep tuc di vao chu trinh Krebs, qua chu6i v^n ehuyen di?n tu noi ma nhieu phan tir ATP duoc tao ra tren mgt phan ttr glucose, ma ehuyen huong len phia Iren cua chu tfinh duong phan va bj phan nhanh \'ao cac con duong pentose phosphate, long hqp hexosamme, euoi eung dan den smh long hgp glycogen va trehalose.
Glycogen va trehalose dong vai tro la ngudnn^glugngdM-lri^ducri d^n^ glucose giiip te bao duy tri hoat dong song khi gua trinh long hpp ATP da b] ngung U-e De doi pho v6i cac bat loi ke tren, nam men da hinh thanh cac CO che bao ve te bao, sua sai DNA, lam cham chu trinh \k bao, kiem soat hoat dpng ho hap, kich ihich sir tir ehel te bao (appotosis) Cac ca che nay dugc dieu khien boi c^c gen ehuyen bi?t, cac gen nay lai c6 su tirong dong cao ve vai tro va chirc niing voi mpl so gen dugc tim thay 6 dpng vat c6 vii b^c cao. Chinh vi the, bai long quan nay se gidi thieu ve lac nhan gay dpt bien gen, co che sua sai DNA va bao ve te bao, s\f tuong dong
\k chuc nSng cua cac gen dieu khien co chi nay giOa ngudi va nam men, cijng nhu vi^c sir dyng ndm men trong nghien cihi chu trinh tl bao, cac qua trinh sinh h6a le bao 6 dgng vat c6 vu bac cao, nghien ciru ung thu.
Keywords. Appotosis (programmed cell death). DNA damage. PYK (pyruvate kinase). GAPDH (glyceraldehyde-3-phosphale dehydrogenase). G6PDH (glucose-6-phosphaie dehydrogenase). ROS (oxygen I eactive species)
' Author for correspondence. Tel: +84-4-37917975; E-mail: nghiemminh(^,ibt.ac.v 206
