Volume 36, Issue 11, Pages 1403-1416 (November 2008)

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ABSTRACT

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Final checkup of neoplastic DNA replication: Evidence for failure in decision-making at the mitotic cell cycle checkpoint G1/S

Received 18 June 2008; received in revised form 29 July 2008; accepted 29 July 2008.

Objectives

Processing of epigenomic transcriptional information by cell cycle phase G1 and decision-making at checkpoint G1/S are the final organizational steps preceding gene replication in transcriptional reorientation programs (i.e., switches from proliferation to cycle arrest and neoplastic transformation). Further analyses of cycle progression will open up new approaches in antineoplastic therapy.

Materials and Methods

The following bibliographic databases were consulted: Central Medical Library Cologne, PubMed (English), the last search was done on April 23,2008 and key words searched were: cell cycle, cell memory, DNA methylation, embryonal/neoplastic stem cells, enzyme-modulated chromatin, G1-G1/S checkpoint, genomic/epigenomics, genomic viral DNA, histones, telomere/telomerases, transcription factors, neoplastic transformation, senescence.

Results

Gene transcription and epigenomic surveillance form a functional entity. In proliferation programs, transcriptional information is mediated by chromatin and DNA methylation, analyzed and processed in G1 phase, and converged on the parental checkpoint G1/S for final decision-making on DNA replication. Genomic reorientation appears to be associated with transcriptional instability, which normally is corrected, possibly during the G2/M phase, to new levels of epigenomic equilibria. We speculate that daughter stem cells inherit persistent neoplasm-specific transcriptional instabilities through failure of the parental G1/S checkpoint. Foreign, silenced, potentially oncogenic DNA sequences, i.e. regular components of the human genome such as endogenous retroviruses, could conceivably be activated for expression in neoplastic transformation by epigenomic histone deacetylase/acetyl transferase/histone methyltransferase-mixed lineage leukemia deregulations.

Conclusions

Failure of cell cycle G1/S decision-making for DNA replication is the final and possibly a major cause in neoplastic transformation. Therefore, further analysis of the dynamics of G1-G1/Sphases could provide new opportunities for therapeutic strategies.

Article Outline

• Abstract

• Clinical examples of epigenomic transcriptional instability

• Transcriptional instabilities in embryonal stem cells

• Epigenomics and cell memory

• Effects of telomeres/telomerases on G-G/S phases of the cell cycle

• Signals for G/S decision-making

• Transcription repressors/tumor suppressors

• Chromatin structuration through modifications by histone enzymes—Preprogramming of transcriptional information

• DNA CpG and non- CpG methylation

• Hypothesis: Decision-making at the G/S checkpoint—Epigenomic/genomic transcriptional instabilities of pre-/neoplastic stem cells

• Conclusions and speculations

• Acknowledgment

• References

• Copyright

Transcriptional stability is a basic requirement for maintenance of eukaryotic cell life. It has two components—genomic and epigenomic—which, together, form a functional entity. Epigenomic mechanisms secure stability of gene transcription by close guidance of the access of transcription factors to proliferation- and differentiation-bound genes employing fine-tuned adjustments in chromatin arrangements and DNA methylation 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. The preeminent role of epigenomic control in transcriptional stability has been well documented in nuclear transfer experiments 14, 15, 16, 17 where deregulated coordination between oocytic cytoplasmic factors and highly differentiated somatic cell nuclei frequently interfere with regular cell cycle progression. This results in failure to establish stabile transcriptional programs in attempts to generate pluripotent stem cell populations and may lead to cell death or neoplastic transformation 18, 19. Interestingly, in neoplasms, transcriptional deregulation does not affect single genes accidentally but rather involves entire sets of tumor-associated genes in a nonrandom manner 10, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29. This emphasizes the role of the cell cycle in genomic reorientation of both regular and neoplastic transcription programs. We decided to look at the dynamics of cell cycle phases G1-G1/S, which are the regular epigenomic control points that secure faithful DNA replication in S-phase 29, 30, 31, 32, 33, 34, 35, 36. We speculate that checkpoint G1/S and the preceding G1 phase are strategic steps in transcriptional decision-making, which might also be of major pathogenetic importance in neoplastic transcriptional gene reorientation 12, 13, perhaps as targets for therapeutic strategies.

Clinical examples of epigenomic transcriptional instability

Epigenomics are involved in the pathogenesis of a variety of human diseases 37, 38, 39, which are frequently associated with increased risks of tumorigenesis. Examples include functional restrictions of aging stem cells and diseases of accelerated aging 28, 40, 41; abnormal gestational environments of premature/small-for-date infants and in artificial fertilization both associated in later childhood with reduced life expectancies and increased morbidity rates, including neoplasms 39, 42, 43, 44, 45; embryos cloned by nuclear transfer causing severe pre- and postnatal morbidity/mortality 14, 15, 16, 17, 46, 47; discordant DNA transcription patterns between identical twins after prolonged separation in different environments 48, 49, 50; checkpoint adaptation of cells under DNA repair escaping epigenomic p53/Rb-mediated control 51, 52, 53 by resuming proliferation in spite of persisting DNA damage 35, 54; and classification of neoplasms as epigenomic diseases 21, 25, 55, 56, 57. In the latter, three steps have been suggested to set the molecular stage for neoplastic transformation in progenitor cells [58]: epigenomic control, gene mutations, and instabilities of “epigenomic gatekeepers” fostering tumor progression 25, 57. Because each type of neoplasm has its own stem cell signature [59], future therapeutic protocols will have to be adjusted for these specificities in every new cancer patient at diagnosis and, importantly, during disease progression.

Transcriptional instabilities in embryonal stem cells

Epigenomic transcriptional instability with innate potentialities for neoplastic transformation is particularly high in early embryonal developmental programs [15]. The majority of embryonal stem (ES) cells rapidly proceed to transcriptional gene reorientation and undergo polycomb-induced differentiation through mitotic divisions. Only a minority subpopulation of ES cells is maintained in a state of pluripotency 60, 61, 62. Transcriptional instabilities contributing to accelerated progression of ES cell cycles are promoted by shortened or absent cell cycle G1 phases, reduction of S phase to 2 hours’ duration, low levels of activated Rb and delayed cycle arrest in response to overexpression of p16INK4A 63, 64. Instabilities produce two waves of apoptotic scrutiny, both reflecting transcriptional reorientation: The first is seen in 4/8 cell human embryos that start transcribing their own genes after cessation of maternal mRNA functions 60, 61, 62, 65, 66, 67. A second apoptotic wave follows at gastrulation (around <10 weeks of gestation) when organ-specific gene transcription programs are introduced by DNA methyltransferase (DNMT)–mediated de novo DNA methylation 40, 68, 69. Additional transcriptional genomic/epigenomic instabilities occur in ES cells at checkpoint G2/M by uncoupling of apoptosis from anaphase 15, 27, 67, 70, 71 and may be associated with transitory instabilities in transcriptional reorientation programs, as discussed below. This corresponds to apoptotic uncoupling in neoplastic transformation of embryonal and postnatal stem cells 27, 72, 73, 74, 75, 76 and results in unequal distribution of genetic materials to daughter cells 26, 55, 70, 72, 74, 77.

Epigenomics and cell memory

Cell memory is a basic requirement for epigenomic stability. Memory mechanisms preserve parental developmental decisions over multiple cell generations as silenced, gene-specific transcriptional profiles stored in cyclin-dependent heterochromatin-engraved complexes which they communicate through mitotic cell cycles to daughter cells for later retrieval (Fig. 1) 25, 78, 79, 80, 81, 82. Before reaching the cell cycle at G1, transcriptional information is preprocessed by histone code-modified chromatin arrangements and by DNA methylation 78, 83, 84, 85. Key epigenomic chromatin modifiers are polycomb/trithorax (PcG/TrxG) proteins 86, 87, 88, 89, 90, transcription factors E2F 91, 92, and RNA interference 93, 94, 95. PcG/TRxG are evolutionary conserved methyltransferases containing a HMTs-SET domain with a CpG-binding CXXC finger protein. The core function of SET domains probably is associated with histone tail-binding and requires additional specializations for methyltransferase activity [87]. Two groups of PcG/TrxG, proto-oncogenes Ezh2 and bmi1, introduce long-term developmental decision-making for epigenomic memory inheritance 60, 62: Methyltransferase Ezh2 is involved in tumor-suppressive INK4A gene-silencing programs 96, 97 and connects specific chromatin-modified information to transcription-regulating complexes of de-novo methylated CpG sites 89, 98, 99, 100. bmi1s are zinc finger, E2F-dependent transcriptional repressor proteins that mediate deposition of information as heterodimers onto replicated DNA strands and mark INK4A/ARF and HOX genes for repressive signals of heritable transmission 87, 89, 101, 102. (For details on chromatin modifiers: see below, section: chromatin structuration).

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Figure 1 Transcriptional inheritance of cell memory (oversimplified). Parental cell: Transcriptional information on an earlier developmental decision is epigenomically preprocessed and inactivated. Mitosis: The inactivated information is stored in condensed heterochromatin. The parental cell divides into daughter cells both receiving a complete set of stored transcriptional information. Daughter cells: Stored information is either transcriptionally reactivated in decondensed chromatin by epigenomic factors and is ready to participate in the next cell cycle as transcribable signals (left branch). Alternatively, transcriptional signals remain postmitotically inactivated, stored in heterochromatin and will be presented as such to the next mitotic cell division (right branch).

G1 phase and the G1/S checkpoint are the final steps for the detection and elimination of transcriptional abnormalities before DNA replication is initiated. In regular cell cycles, premitotic scrutiny during G1 depends, for example, on epigenomic differentiation-associated tumor suppressors, e.g. PcG/TRxG proteins 60, 62, 103 from non-/antineoplastic memory information, which eliminate transcription-deregulating factors, such as ectopic leukemic cell fusion products 22, 59, 104. However, in the pathogenesis of certain tumors, pretranscriptional failure at G1-G1/S will allow nonneoplastic daughter stem cells and stromal cells [104] to inherit neoplastic potentialities by mitotic acquisition: In contrast to regular cell cycles, these cells appear to activate parental neoplastic memory information in successive steps during repeated cycle passages 21, 24, 25, 36, 55, 56, 57 that will eventually lead to replication of neoplasia-associated genes during S phase 1, 20, 59, 105, 106. Alternatively, certain pediatric leukemias appear to undergo neoplastic transformation as a primary event in early ES cells 75, 76.

Effects of telomeres/telomerases on G1-G1/S phases of the cell cycle

Telomeres/telomerases play important roles in the control of cell proliferation, not only in senescence but also in neoplastic growth [107]. Telomeres shortened down to a critical length of 4 to 7kb (from a normal of 15–20 kb in humans) profoundly affect the processing of transcriptional information by cycle G1 and G1/S 108, 109, 110, 111, 112. Shortening is involved in genomic instability of senescence, impaired cell viability 113, 114, age-accelerating diseases [115] and neoplastic transformation [109]. Short telomeres induce irreversible cycle arrest and apoptosis by DNA-CpG methylation, activation of p53/pRb, and CDKIs p21, p16, p14, p15. They inhibit G1 progression [116] and modulate chromatin histone codes (e.g., at H3Lys4,9 and H4Lys20) [108]. Disruption of gene-specific, histone deacetylase (HDAC)–mediated chromatin deacetylation inhibits binding of mixed lineage leukemia (MLL) (a histone H3Lys4 methyltransferase) to telomeric complexes. This denies access of transcription factors to telomerase genes 117, 118, 119, 120 and results in net losses of telomere repeats 121, 122, 123, abrogation of cycle-dependent telomerase-catalyzed resynthesis of telomere nucleotides in senescence programs 124, 125, and of tumor suppressor functions 117, 126. On the other hand, high telomerase activity in conjunction with genomic instability promotes neoplastic transformation 109, 127, 128, 129, 130. Synthetic telomerase-inhibitors, such as azidothymidine block elongation of telomeres by phosphorylating DNA sequences and accelerate losses of telomere repeats in pre-/neoplastic stem cells 131, 132, 133, 134.

Signals for G1/S decision-making

Accessibility of transcription factors to the cell cycle is determined by preprocessing of transcriptional information in G0 through DNA methylation and chromatin stucturation 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145. During passage through G1 phase, which may vary considerably in duration, a wealth of transcription-relevant information is scrutinized, condensed, and presented to checkpoint G1/S for signal identification and final decision-making on whether or not the cell is to be admitted to S-phase for DNA replication (Fig. 2). For example, signals from neoplasm-associated genes activate during G1 the ATM/ATR (ataxia telangiectasia mutated/ataxia telangiectasia related proteins, i.e. DNA damage response PIKK protein kinases)-regulated G1/S checkpoint as an antineoplastic barrier in attempts to safeguard genomic integrity 36, 80, 146, 147. Cell cycle dynamics are regulated by bidirectional equilibria of cyclins, their cyclin-dependent kinases (CDKs), and their inhibitors (CDKI), the levels of which undulate during cycle progression as a result of degradation by the ubiquitin-proteasome system [139]. Cyclins D-CDK4/6 and E-CDK2 drive G1 and G1/S transitions. Cyclin levels are low in early G1, and rise to full activity in late G1 and G1/S [148]. Preparatory steps for passage of the G1/S checkpoint include disruption of nucleosome structures through SWI/SNF (an adenosine triphosphate–dependent chromatin-remodeling complex) complexes 149, 150, and the assembly of transcriptional polypeptides by RNA polymerase II (RNA-Poly II) at every promotor gene 151, 152. Activation of RNA-Poly II is, indeed, of particular functional significance since gene regulation is executed predominantly at the level of DNA templates and RNA-Poly II is responsible for all mRNA synthesis [153]. When CDKI p16 and p27 are downregulated and MLL methyltransferases are degraded (together with inactivation of pRb) cyclin E/CDK2 levels increase again and marshal cell transit through G1/S to enter S-phase 154, 155.

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Figure 2 Signal presentation to G1/S transition of the cell cycle: Hypothesis. G0 phase: Generation of transcriptional signals—Epigenomic processing of transcriptional information through chromatin modifiers, DNA methylation status, etc. G0/G1 transition: Entry of the cell cycle by transcriptional signals. G1 phase: Signals are identified, filtered, and condensed to a single command (or to a limited number of commands) for presentation to G1/S transition. G1/S transition: Final decision-making whether the cell should be allowed to enter S-phase for DNA replication or, alternatively, be rejected for cycle arrest, senescence, or neoplastic transformation.

Inhibition and arrest of G1 progression are important in transcriptional safety. They are induced by CDKI proteins p16INK4A, p14/p19ARF, p15INK4B, and p21Sdi1,Cip1,Waf1, p27Kip1, respectively, of suppressor genes INK4A/ARF and Cip/Kip 148, 156, 157, 158, 159. p16/p19 form complexes with transcription repressors E2F4,5, establish links to gene-specific tumor suppressor pathways pRb and p53, induce cell cycle arrest, and are also involved in overlapping pathways in cell immortalization 159, 160, 161, 162, 163. Loss of p16 induces transcriptional instabilities and proliferation of pre-/neoplastic cells 97, 164, 165 identifying p16 is a major tumor suppressor protein. p15 links cyclin D-CDK4/6 inhibition to telomere functions [166]. Because abnormalities of gene transcription are of major pathogenetic importance in numerous human diseases, including genetic instabilities of neoplasms 26, 138, 167, 168, 169, 170, arrest of the cell cycle should occur prior to DNA replication, i.e., at G1-G1/S, to be effective as a protective measure against faulty gene expression. Arrest may be irreversible in response to major DNA abnormalities, or temporary to allow sufficient time for the completion of reparative steps of damaged DNA. Inadequate repair, e.g. in checkpoint adaptation, can result in premature release from cycle arrest and, through overexpression of cyclin E-CDK2, in transcriptional deregulation and neoplastic DNA replication 35, 54. Synthetic anticancer CDKI-drugs block abnormal cell cycle progression 171, 172, 173 or reactivate p53-dependent senescence by inhibition of telomeres/telomerase activity, and increase expression of CDKI proteins 131, 132, 133, 134.

Transcription repressors/tumor suppressors

Major tumor suppressors are pRb, p53, E2F, and TIS21/BTG2/PC3 that operate in synergy with CDKI. They are integral components of an elaborate network for transcriptional decision-making at G1-G1/S checkpoint 174, 175, 176, 177. Expression of pRb is initiated early in G1 (by hypophosphorylation at position Ser 780) [169]. It directly activates suppressor genes INK4A/ARF 151, 152 and E2F4,5-repressive promotors [137]; inactivates catalytic activities of cyclins E-CDK2 and D1-CDK4/6, and temporarily blocks G1 progression 175, 176, 177, 178, 179. pRb also regulates polycomb-mediated DNA hypermethylation and suppresses proliferation-promoting E2F1-3-responsive genes 180, 181. Arrest of cycle progression is relinquished by inactivation of pRb late in G1, so that repression of E2F1-3 complexes is alleviated and cycles progress to S-phase [137]. Importantly, pRb also affects cell cycles of auxiliary genes.

p53 pathways are multifunctional in maintaining transcriptional genomic/epigenomic stability and likewise control auxiliary genes: They target cycle phase-controlling genes, in functional balances with their inhibitor MDM2, and reduce cyclin/CDKs levels 98, 182, 183, 184, 185, 186. With its downstream effector p21, p53 initiates cell cycle arrest at G1, even prior to expression of the key regulator of cell cycle arrest, p16 63, 152, 182. Together with p15, p53 prolongs G1 phase, represses DNA methylation by DNMT1 through specific DNA binding 187, 188, reacts to telomere attrition by promoting telomere-associated replicative stem cell senescence, and contributes to DNA damage repair 34, 121, 147, 168, 187. Furthermore, p53 mediates transforming growth factor–β1-induced apoptotic growth arrest downstream of the pRb/E2F pathway [183] and regulates epigenomic chromatin surveillance through suppression of histone methyltransferase polycomb Ezh2 [98]. (For additional information on chromatin arrangements and DNA methylation in early stages of the cell cycle, see below, section: chromatin structuration).

Members of the E2F family are transcription regulators that affect the cell cycle in opposing manners. They have coordinating functions on G1-G1/S cycle progression, integrate DNA replication and repair 91, 92, 189, connect chromatin-modifying histone enzymes to cycle progression, and regulate access of transcription factors to DNA replication 92, 190, 191. E2F1-3 are constitutive transcription activators of proliferation-bound genes. They trigger promotor genes for cell entry into G1 and transition of checkpoint G1/S 91, 92, 181. When unbalanced by pRb, overexpressed E2F1 synergizes with oncogenes, triggers quiescent cells to enter proliferative cycles and to participate in neoplastic invasion, metastasis, and angiogenesis 181, 191. On the other hand, E2F4,5 are transcription repressors 137, 189. E2F4,5 silence E2F1-3 targeted genes 152, 180, 181, form complexes with p53/pRb and p16/p19 to prevent cell entry into S-phase 162, 192, 193, 194, induce cell differentiation at G1/S 91, 92, 149, 189, 190, and inhibit uncoupling of proliferative from tumor-suppressive pathways 162, 163, 189, 195 in senescent pre-neoplastic cells 33, 34.

Among other transcription regulating factors, TPA-inducible early growth response gene TIS21/BTG2/PC3 is noteworthy as a pan-cell cycle tumor suppressor and endogenous cell death molecule that acts at both G1/S and G2/M checkpoints. It represses cyclin D1, regulates proliferation-suppressive pRb/p16 pathways, and inhibits early phase neoplastic transformation at G1/S. Even in tumor cells in which pRb/p53 is inactivated, TIS21 still inhibits degradation of cyclins A/B1, binds directly to CDK2 and induces tumor cell senescence/apoptosis [177]. Thus, therapeutically induced overexpression of TIS21 might shift neoplastic transformation processes towards innate cell senescence.

Chromatin structuration through modifications by histone enzymes—Preprogramming of transcriptional information

Incoming transcriptional signals at G1 originate from chromatin arrangements 141, 142, 144, 145 and from DNA methylation 135, 136, 143. (For the latter, see section: DNA CpG and non-CpG methylation). Chromatin signals control access of transcription factors to gene promotors 28, 140, 141, 142, 143, 144, 145, 196, 197, and are further processed during G1 cell passage. Indeed, a multitude of chromatin signals reach G1 where, most likely, they are processed, identified, selected, filtered, condensed, and converged on to the G1/S checkpoint (Fig. 2). Based on this information, the checkpoint then makes the decision whether the cell should be allowed to proceed in cycle to S phase for regular DNA replication, or to undergo differentiation, cycle arrest, senescence, or, possibly, suffer neoplastic transformation (Fig. 3). Thus, G1-G1/S collaborate in key positions in regular and neoplastic cell fate. The other major control mechanisms against neoplastic transformation, namely G2 phase and G2/M checkpoint of the cell cycle, are not considered here.

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Figure 3 Transcriptional instability: transitory in cell senescence/cycle arrest vs permanent in neoplastic transformation. Incoming epigenomic signals from histone-mediated chromatin arrangements and DNA methylation are processed during G1 passage and presented as transcriptional information to parental checkpoint G1/S for decision-making on DNA replication, see Figure 2. Messages to regular transcriptional reorientation programs (differentiation, cycle arrest, senescence) to nonneoplastic daughter cells seem to be associated with transient transcriptional instabilities (symbolized by the red crescent in the left daughter cell); new levels of transcriptional stability are rapidly established, possibly during G2/M phase. In a few stem cells, however, failure or abnormal G1/S decision-making leads to persistence of epigenomic/genomic transcriptional instabilities/deregulations involving entire sets of pathogenetic neoplasm-associated genes (right daughter cell) and may initiate the expression of neoplastic transcription programs, probably over several cell cycles.

Chromatin transcriptional signals are gene-specific. Specificity is established predominantly by enzymatic acetylation, methylation, phosphorylation, and ubiquitination of nucleosomal histones H2A/B, H3, and H4. Preferential sites for fine-tuning of transcriptional factor accessibility are lysine residues at defined positions 9, 88, 198, 199 on NH2-terminals of protruding histone tail domains 86, 95, 142, 200, 201. Phosphorylation of histone operates mostly in anaphase, apoptosis, DNA repair 145, 202 and in tumorigenesis [203] but is of lesser importance in this discussion. Chromatin-modifying enzymes are histone deacetylases (HDACs) 144, 203, 204, 205, acetyltransferases (HATs) [145] and methyltransferases (HMTs) 206, 207. HDACs exert major epigenomic transcription-repressing control functions on stabilization of DNA replication in collaboration with proliferating cell nuclear antigen and adenosine triphosphate–dependent complexes [52]. Repressing HDACs 93, 174, 208, 209 and transcription-promoting HATs [210] operate in shifting bidirectional equilibria, respectively, through transcription-inhibitory heterochromatin and transcription-permissive euchromatin. Gene replication modified by HDAC/HAT chromatin structurization also establish direct synergistic links to DNMT-mediated DNA transcription and thus stabilize E2F-targeted promotor- and tumor suppressor–genes. In fact, HDAC/HAT balances also affect auxiliary transcription-controller genes, such as telomere/telomerases, which, for example, in recollection of earlier memory information, supervise tumor suppressor genes and cycle arrest 58, 119, 120, 211, 212. Frequent pathogenetic constellations seen in neoplastic stem cells are inactivation of tumor suppressor genes by chromatin deacetylation (in particular of H3Lys9 and H4Lys16), deregulated histone acetylation 203, 204, 205 and methylation especially by MLL 165, 203, and concurrent disruption of the DNA methylation status by CpG hypermethylation/non-CpG hypomethylation of DNA promotor sequences 140, 143, 200, 213, 214. Not surprisingly, a wide range of synthetic HDAC transcription inhibitors are presently explored in clinical trials for their antineoplastic properties in attempts to restabilize epigenomic surveillance at G1 and G1/S, and to prevent transcription of abnormal genes 204, 215, 216, 217, 218, 219, 220, 221.

HATs, the opposing partners of HDACs in eu-/heterochromatic equilibria [164], provide access of transcription factors by decondensing regional chromatin and lysine-specific acetylation of H3/H4 histones 145, 210. HATs regulate levels of cyclin E/CDK2 complexes for cell entry of S-phase and trigger transcription of both proliferation- and differentiation-associated genes 9, 222, 223. HATs synergize with HMT-protein Ezh2 in antisenescent, proliferation-promoting and chromatin-remodeling effects on stem cells. Overexpression and aberrant acetylation by HATs of certain histone lysines operate as transcriptional cofactors for oncoproteins in the pathogenesis for some leukemias and solid tumors with a poor prognosis [224]. For example, in acute monocytic t(8;22)(p11;q13) leukemias, fusions of enzymes HAT-MOZ (monocytic leukemia zinc finger protein) and HAT-p300/CBP (CREB-binding protein) induce characteristic losses of histone acetylation and inhibition of cell differentiation 224, 225. On the other hand, HAT-p300/CBP enzymes may reinforce p53/p16 mediated tumor suppression 164, 204.

Methylation of chromatin-modifying enzymes is catalyzed by gene-specific HMTs 200, 206, 207, which either activate or repress gene transcription during G1-G1/S-processing depending on the position of lysines on histone molecules 141, 196, 197, 199, 207, 226, 227. HMTs include four MLL-H3Lys4 methyltransferases. MLLs complexes promote, coordinate and ensure proper cell cycle phase transitions and are of prime importance in the epigenomic “switch-on/off” control mechanisms of the cell cycle, but functional details are not fully understood 165, 203, 227. MLLs have C-terminal SET1 domains consisting of genes SUV39H1; Ezh1,2; and TRxG/PcG 197, 206, 207 that connect MLL to both, promotive and suppressive epigenomic regulators in mid- and late-phase G1 respectively 165, 228, 229, 230. MLLs engage in cross-talks with HATs [231], activate cyclin E/CDK2 complexes, mark CDKI p27 for degradation through phosphorylation, and have links to gene silencing by DNA-CpG methylation (via DNMTs) 163, 230. MLLs interact with tumor-suppressor Menin that promotes expression of CDKI p27Kip and p18INK4c [232]. In promotor genes lacking standard CpG islands [233], MLL-mediated gene silencing is accomplished by chromatin hypermethylation and upregulated polycomb transcription repressors 21, 52, 161. Deregulations of MLL genes are associated with clinically aggressive leukemic cell immortalization 203, 206, 229, 234, 235. MLL fusion proteins enforce high-level expression of genes HOX and HOAX co-factor MEIS1 which is pivotal for leukemigenesis [236]. In 10% of pediatric leukemias with 11q23 fusions, the DNA methylation status at chromosomal breakpoints is deregulated by HMT-mediated transcriptional repressors 161, 206, 237.

DNA CpG and non- CpG methylation

DNMT are transcription repressors of gene promotors. They catalyze DNA methylation through interaction with chromatin proteins TRxG/Ezh2 and, thus, are likely to also provide signals for processing of G1-G1/S cycle transitions 100, 102, 136, 230, 235. Indeed, DNMT1 regulates replication and maintenance of CpG-methylation patterns by repressing E2F1-responsive promotors through complex formation with pRb and HDAC1 181, 238. Its functions are repressed by losses or mutations of p53/pRb pathways, by overexpression of the p53-antagonist MDM2, or deregulation of CDKs and cycle arrest 26, 68, 135, 182, 188. DNMT3a,b affect the accessibility of transcription factors to genes and thus suppress gene transcription 40, 68, 81. They hypermethylate CpG islands including de novo methylation of premarked unmethylated CpG 57, 239, 240, 241 and hypomethylate non-CpG (i.e., CpA and CpT) sites 21, 25, 242, 243. In most human neoplasms, tumor suppressor genes are silenced by DNMT3a,b. This leads to transcriptional instability 244, 245, 246 (especially in aging cells in which abnormally methylated DNA sequences accumulate 81, 247, 248), and to expression of leukemia-associated oncogenes 25, 26, 105, 203, 249. Of pathogenetic importance is the association of abnormal DNA methylation with transcription repressor HDAC 144, 213, 244, with GATA-2-binding transcription factor PU.1 250, 251, and with regional communicative complexes to gene-specific heterochromatin 11, 38, 88, 201, 239, 247. Synthetic DNMT-inhibitors aim at selectively restoring activation of tumor suppressor functions by downregulating tumor-promotive cyclins, reactivating silenced CDKI, and dephosphorylating pRb [249].

Hypothesis: Decision-making at the G1/S checkpoint—Epigenomic/genomic transcriptional instabilities of pre-/neoplastic stem cells

Reorientation of transcriptional genomic programs by mitotic divisions (e.g. in switches from regular proliferation to differentiation/cycle arrest) requires epigenomically controlled decision-making for stabile DNA replication at the G1/S checkpoint, i.e. whether or not to allow a cell to enter S-phase 12, 13. Expression of neoplastic markers during aging processes of normal stem cells [30] suggests that genomic reorientation is associated with transcriptional instability. Although instabilities in regular switches appear to be momentary and are rapidly corrected to new stability levels, probably in G2/M phase, transcriptional genomic reorientation does entail the possibility of neoplastic transformation. Apparently the biology of transcriptional decision-making occasionally allows individual stem cells normally doomed for senescence, to avoid regular differentiation/apoptotic programs and instead divert to neoplastic proliferation (Fig. 3). In these instances, transcriptional epigenomic/genomic stability is not restored. Rather, abnormal neoplastic DNA replication due to deregulations and failure in scrutiny of the G1/S checkpoint directly activates CDKs [36] and repress CDK inhibitors 97, 161, 164, 252. In addition, chromatin-modifying enzymes HDAC/HAT/HMT-MLL 145, 205, 206, 213, 224, histone phosphorylation 145, 202, 203, PcG/TRxG/Ez2h 100, 102, 240, and DNMTs-mediated DNA methylation 240, 253, 254 also become involved in abnormal transcription. As a result, tumor suppressor proteins p15INK4B/p16INK4A are not activated, chromatin signaling does not allow access of differentiation-associated genes to S-phase for replication, and mutated genes are not silenced by de novo CpG hypermethylation 93, 240/non-CpG DNA hypomethylation 245, 246. In short, decision-making at the G1/S checkpoint is severely disturbed. However, it may not be completely abrogated 102, 161, 252 because transcriptional deregulations/instabilities in neoplastic transformation are nonrandom [255]. Rather, deregulations are tumor-specific involving entire sets of pathogenetic tumor-associated genes [29] in a wide spectrum of pre-/neoplastic human lesions 35, 147, 168. This agrees with concepts that each type of neoplasm has its own genomic transcription program [59].

Because epigenomic deregulation from chromatin-modifying enzymes and transcriptional instabilities during G1 interfere with regular G1/S decision-making in genomic reorientation programs of neoplasms, the possibility exists that developmentally silenced genes are activated for expression 243, 249, 251. These could even include components of the normal human genome that are nonhuman, infectious, and potentially tumorigenic DNA sequences. In this connection, it is important to remember that a sizable portion of the human genome normally is prevented from expression by silencing [256]. About half are mobile, transposable elements of nonhuman origin, transposons and retrotransposons, poorly characterized or even unidentified viral and parasitic DNA sequences 256, 257, 258. Retrotransposons, the most frequent variety, are capable of disrupting regular cell cycle proceedings, inhibit mRNA and protein expression, and even serve as fine-tuners for the human transcriptome. They establish new epigenomic balances and replicate in the host genome in response to telomeric erosion 259, 260, 261. To prevent transcription and clone formation of such potentially pathogenetic sequences, the human host regularly inactivates accumulating transposable elements by epigenomic silencing through DNA methylation 241, 257, 262, 263. In abnormal mitotic cycles with G1-G1/S deregulations, however, defense mechanisms will be severely disturbed. Of special concern are endogenous, vertebrate-specific retrovirus-like sequences (hERVs), which account for about 8% of the entire human genome [256]. hERV retroviruses are associated with human diseases, including neoplasms, but their infectivity and tumorigenicity in man has not been firmly established 264, 265, 266, 267, 268, 269. Thus, the possibility has not been excluded that they become activated during transcriptional reorientation/deregulation by reverse transcriptases and cause disease 270, 271, 272, 273.

Finally, it has been suggested that, in spite of uncoupling of proliferative from suppressive transcriptional pathways during tumorigenesis, “. . . the underlying tumour-suppressor programmes remain intact” [33]. If correct, therapeutic reactivation of tumor suppressor programs theoretically could coax neoplastic stem cells into transcriptional reorientation, i.e., to revert back to stabile senescent programs with innate mechanisms for physiological senescence, quasi as legacy of their former “friendly” behavior as physiological stem cells. Reorientation of entire transcription programs through epigenomic switches is, in fact, a not unusual biological phenomenon. It occurs in epithelial-mesenchymal transition 10, 23, 274, 275, in oncogene-induced expression of tumor suppressors 33, 192, 276, 277, in uncoupling of neoplastic transcription programs from apoptosis in mitotic spindle checkpoint abnormalities 27, 70, 72, 74, and in neoplastic transformation 25, 278. Numerous current pre-/clinical therapeutic trials with chromatin enzyme-modifying drugs 28, 204, 216, 279, 280, 281 address alterations in the CpG methylation status of aberrantly silenced genes [255], recoupling of proliferative with tumor suppressive pathways 21, 27, 282, 283, 284, and premarking for transcriptional repression by polycomb factors 103, 240.

Conclusions and speculations

Gene transcription and epigenomic control form a physiological entity that together governs the mitotic cell cycle. G1 phase and G1/S checkpoint are the final control points in gene reorientation programs before DNA replication, both regular and neoplastic, is implemented in S-phase: A wealth of incoming gene-specific information, mediated by precycle epigenomic chromatin arrangements and the DNA methylation status, is processed during G1 phase, which may vary considerably in duration. Indeed, transcriptional information is analyzed, selected, filtered, condensed, and then converged on the G1/S checkpoint for decision-making, i.e., whether the cell is admitted to S-phase for regular DNA replication, alternatively triggered into differentiation and/or cycle arrest or, occasionally, into neoplastic transformation (Fig. 2). Although little information exists on details of G1-G1/S processing for transcription of individual genes, it seems that epigenomic signals are transmitted from parental cells to their daughter cells for mitotic inheritance of genomic programs. This information may include neoplastic memory data. Of particular interest are questions why and how individual non- or preneoplastic daughter stem cells instead of undergoing senescence and/or cycle arrest, acquire neoplastic properties in genomic reorientation programs. A plausible explanation could be a failure of control functions of the G1/S checkpoint to exclude cells with transcriptional instabilities from DNA replication. But then, immediately, additional questions arise: By what mechanisms does G1/S decision-making fail? Or is it failure of signal processing and presentation during G1 passage? Is faulty decision-making of G1/S reversible at any point? We hypothesize that momentary transcriptional instabilities exist in genomic rearrangements during regular mitosis that are rapidly corrected to new levels of stability by epigenomic signals, probably during G2/M phase. In preneoplastic stem cells, however, abnormal transcriptional information transmitted by deregulated HDAC/HAT/HMT-MLL chromatin-modifying enzymes in combination with abnormal DNA methylation, are not corrected during signal processing in G1 phase. This leads to failure in G1/S decision-making, propagation of transcriptional instabilities, and replication of abnormal DNA sequences in S-phase (Fig. 3). It has recently been suggested that transcriptional instabilities in neoplastic transformation are tumor-specific involving entire sets of previously silenced pathogenetic genes so that eventually, during subsequent cell cycles, full neoplastic transformation is implemented. At this point it is worth remembering that the possibility has not been excluded that regular, but normally silenced components of the human genome, e.g., nonhuman infective and even potentially oncogenic genomic components such as hERV-Ks (endogenous retroviral) sequences, could be transcribed in abnormal DNA replication and cause disease.

In conclusion, we believe that abnormalities in processing of transcriptional information during G1 phase and/or failure of G1/S checkpoint of the mitotic cell cycle are the final, and possibly decisive steps that trigger neoplastic DNA transformation and replication of daughter stem cells. Thus, further studies of the dynamics of pre–S-phase processing of transcriptional information by the cell cycle will open up new therapeutic strategies.

Acknowledgments

There are no potential author conflicts of interest that relate to this manuscript.

We thank Drs. Robert J. Arceci, Baltimore, and Zina Ben-Ishay, Jerusalem, for valuable comments.

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Department of Pediatrics, University of Göttingen, Göttingen, Germany

Offprint requests to: Gregor Prindull, M.D., University of Göttingen, Department of Pediatrics, Robert-Koch-Strasse 4, Göttingen, 37075, Germany

PII: S0301-472X(08)00374-3

doi:10.1016/j.exphem.2008.07.009

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