[Wikipedia, iHOP/hTERT defining information, iHOP/hTERT interaction information, iHOP/hTERT most recent information, Links, Books, LibCong, Books/hTERT insertion, Papers, Amazon/hTERT gene, Patents/hTERT, Patent Lens/Telomerase, LifeExtension].
The hTERT gene for the 127 kDa, 1132 amino acid catalytic protein portion of the telomerase holoenzyme, a reverse transcriptase, is located on the distal end of chromosome 5p at 5p15.33, probably the most distal gene on the chromosome, which contains 609 genes. The hTERT gene is located about 2 Mb away from the telomere. hTERT mRNA [Images] is transcribed toward the centromere. There are typically just 1 to 5 copies of hTERT mRNA per cell. (Cong, Wright, and Shay, 2002). The half-life of the associated molecule is up to 4 weeks. hTERT has an estimated size of about 40kb [Images], and is expressed in 16 exons and 15 introns. Alternative splicing gives rise to hTERT isoforms (hTERT splicing variants). The principal part of the hTERT promoter [Images] is located within 330 bp upstream of the the translation start site at -330. Promoter regulatory elements have been found all the way down to the 2nd exon of the gene at +228, while 59 bp in the region (-208 to -150) is required for maximal promoter activity. Regions further upstream of -330 may contain additional promoter elements or enhancers (J.-P.Liu, 2001). Alternatively spliced hTERT transcripts exist that may have physiological importance, but only the full-length hTERT transcript [Images] is associated with telomerease activity. "The hTERT mRNA is estimated at less than 1 to 5 copies per cell." (Cong, Wright, and Shay, 2002). The hTR sequence for the RNA part of telomerase is located on chromosome 3 (1,436 genes) at 3q26.3, and the associated RNA exhibits a half-life of about 5 days, and is highly expressed in all tissues, with 5-fold higher expression in cancer cells than in normal cells. There are indications that, in addition to lengthening telomeres, hTERT protein expression leads to otherwise improved DNA repair (10) and superior genomic stability. See Girdhar G Sharma, Arun Gupta, Huichen Wang, Harry Scherthan, Sonu Dhar, Varsha Gandhi, George Iliakis, Jerry W Shay, Charles S H Young and Tej K Pandita, (2003), hTERT associates with human telomeres and enhances genomic stability and DNA repair, Oncogene (2003) 22, 131–146. Also see Ki-Hyuk Shin, Mo K. Kang, Erica Dicterow, Ayako Kameta, Marcel A. Baluda and No-Hee Park, Introduction of Human Telomerase Reverse Transcriptase to Normal Human Fibroblasts Enhances DNA Repair Capacity, Clinical Cancer Research, December 1, 2009, 15 (23). If hTERT is deleted from the human genome, the result is the disorder Cri du chat. See Yu Sheng-Kong, Woodring E. Wright and Jerry W. Shay (2002), Human Telomerase and its Regulation, Microbiology and Molecular Biology Reviews, Sept. 2002, pp. 407-425, and associated papers. See also Xiaoming Yi, Jerry W. Shay, and Woodring E. Wright, Quantitation of telomerase components and hTERT mRNA splicing patterns in immortal human cells, Nucleic Acids Research, 2001 December 1; 29(23): 4818–4825. Expression of hTERT is sufficient for immortalizing human primary cells. (Bodnar et al. 1998, Vaziri and Benchimol 1998).
hTERT activation
[Wikipedia, Links, Books, Papers, Patents, Images, Amazon, LifeExtension, Biocarta Pathways]. See Telomerase Activation Therapies, Telomerase induction, Small Molecule Telomerase Activators, [81s, 81s/TELOMERASEACTIVATE], (7).
The topic "hTERT activation" arises most frequently in the discussion of cancer, although it is also mentioned occasionally in connection with aging or anti-aging treatment. Cancers frequently up-regulate telomerase as part of their program for undesirable cellular proliferation. On the other hand, hTERT is not an oncogene and up-regulation of telomerase does not induce cancer. For instance, the germ line cells are permanently telomerase up-regulated. As a matter of fact, telomerase up-regulation tends to prevent cancer where it occurs as a consequence of telomere shortening, as in carcinomas and adenocarcinomas based on the behavior of epithelial tissues. However, some compounds which activate telomerase are carcinogens. See Yu Sheng-Kong, Woodring E. Wright and Jerry W. Shay (2002), Human Telomerase and its Regulation, Microbiology and Molecular Biology Reviews, Sept. 2002, pp. 407-425, and associated papers. Also see Links/genes with enhancers. See hTERT activation pathways, Biocarta/Overview of telomerase protein component gene hTert Transcriptional Regulation, hTERT activation via the PI3K/Akt pathway and hTERT activation via the MAP Kinase pathway.
hTERT enhancer
[Links, Images, Papers, Books; Books/transcription enhancers, Patents/hTERT enhancer, Amazon/hTERT enhancer].
Gene transcription enhancers upstream or far downstream from the promoter of a gene and on the other side of the gene can sometimes enhance transcription very markedly via distant application of transcription factors acting on enhancers typically associated with gene looping promoting transcription. See activating hTERT transcription enhancers [Images, Papers, Books].
hTERT immortalization confers genomic stability
[Links, Papers, Patents, Books, Amazon].
hTERT insulator
[The hTERT gene is embedded within a Nuclease-resistant Chromatin Domain, Links, Books, Papers, Patents]
Usually, DNA methylation [Links] works to silence a gene, but in cases in which the gene is flanked by an insulator [Links, Books] and an enhancer [Links, Books], the enhancer is only turned on when the insulator is methylated at 5-cytosine in a CG-microsequence and covered with a silencing protein. Usually, a silencing protein binds to a similarly methylated promoter to silence a gene. I note that DNA methylation is primarily evident in placental mammals. In the mouse, gene silencing manages development from the embryonic stage, but only modifies about 100 genes.
hTERT Nuclear Transport
[Links, Images, Papers, Books].
CRM/exportin1 (112 kDa) binds to hTERT and transfers it into the cytoplasm. Antioxidants are sometimes used to suppress nuclear export of hTERT. The nuclear export signal at amino acids [Images] 969-981 has the amino acid sequence NMRRKLFGVLRLKC (W.Klapper et al, 2001).
hTERT Nuclear Import [Links, Images, Papers, Books].
14-3-3 protein (29-33 kDa) binds to hTERT for nuclear transport,
and also binds to CRM/exportin1 to suppress the nuclear export signal. The 14-3-3 proteins interact with cellular signaling molecules including Raf-1, Cbl, Bad, and IGF-1, and are molecular chaparones for cellular localization of several proteins. Phosphorylation of hTERT by Akt protein kinase or other kinases facilitates its import into the nucleus from the cytoplasm (W.Klapper et al, 2001).
hTERT plasmids
[Links/hTERT plasmid design; Links/hTERT plasmids, Images, Video, Papers, Patents, Books, Amazon; addgene, Links/hTERT plasmid vectors, Images, Video, Papers, Patents, Books]. See addgene/hTERT. See also Plasmids, Gene Therapy, Targeted Genome Editing, and zinc finger nucleases.
hTERT plasmids for Gene Therapy
[Links/hTERT plasmids for gene therapy; Images, Video, Papers, Patents, Books].
Transfection-Ready hTERT Plasmids
[Links/transfection-ready hTERT plasmids; Images, Video, Papers, Patents, Books].
See Origene, Origene NM_198253 with transfection-ready hTERT plasmids.
hTERT promoter
[Biocarta/hTERT_Pathway, Biocarta/Telomeres, Telomerase, Cellular Aging, and Immortality, Wikipedia/Promoter; Links/the hTERT promoter sequence, Images, Papers, Books; Links/hTERT Promoter, Books/hTERT Promoter, Patents/hTERT promoter, Patent Lens/Telomerase, Papers, Amazon, Books/human gene promoter and repressor design, Books/transcription promoters; hTERT promoter (-480 to +1), hTERT & mTERT Promoter Sequences (slide, article.PDF); Wikipedia/Nuclear receptor; hTERT Transcriptional Repressors].
See Morin, et.al, the Aug. 17, 2004 Geron patent Telomerase promoter driving expression of therapeutic gene sequences.
See also M Wick, D Zubov, G Hagen, 1999, Genomic organization and promoter characterization of the gene encoding the human telomerase reverse transcriptase (hTERT), Gene, Volume 232, Issue 1, 17 May 1999, Pages 97-106.
Furthermore, don't miss Cong, YS, The Human Telomerase Catalytic Subunit hTERT: organization of the gene and characterization of the promoter, Human Molecular Genetics, 1999, vol.8, no.1, 137-142.
See also Yu-Sheng Cong, Woodring E. Wright, and Jerry W. Shay, Human Telomerase and Its Regulation and Izumi Horikawa, P. LouAnn Cable, Cynthia Afshari and J. Carl Barrett, 1999: Cloning and Characterization of the Promoter Region of Human Telomerase Reverse Transcriptase Gene , Cancer Research, 59, 826-830, February 1, 1999 [Papers, Books].
See Takakura, M., Kanaya,T., et al, 1999, Cloning of human telomerase catalytic subunit (hTERT) gene promoter and identification of proximal core promoter sequences essential for transcriptional activation in immortalized and cancer cells, Cancer Research, 59, 551-557.
See also Telomerase, Aging and Disease (Advances in Cell Aging and Gerontology) by M.P. Mattson, Elsevier, 2001. See also Theodora R. Devereux, Izumi Horikawa, Colleen H. Anna, Lois A. Annab, Cynthia A. Afshari and J. Carl Barrett, 1999.
DNA Methylation Analysis of the Promoter Region of the Human Telomerase Reverse Transcriptase (hTERT) Gene, Cancer Research 59, 6087-6090, December 1, 1999; Wick M., Zubov D., and Hagan G. (1999),
Genomic organization and promoter characterization of the gene encoding the human telomerase reverse transcriptase (hTERT), Gene 232: 97-106; Wu, K.J., C. Grandori, M. Amacker, N. Simon-Vermot, A. Polack, J. Lingner, and R. Dalla-Favera, (1999),
Direct activation of TERT transcription via c-MYC, Nature Genetics. 21: 220-224. See also Joseph C. Poole, Lucy G. Andrews and Trygve O. Tollefsbol,
Activity, function, and gene regulation of the catalytic subunit of telomerase (hTERT), Gene, Volume 269, Issues 1-2, 16 May 2001, Pages 1-12. See Satoru Kyo, Masahiro Takakura, Toshiyoshi Fujiwara, Masaki Inoue (2008),
Understanding and exploiting hTERT promoter regulation for diagnosis and treatment of human cancers, Cancer Science, Volume 99, Issue 8, pages 1528–1538, August 2008. Don't miss Ralf Janknecht, ed. Varda Rotter (2004), On the road to immortality: hTERT upregulation in cancer cells, FEBS Letters, Volume 564, Issues 1-2, 23 April 2004, Pages 9-13. Also see Satoru Kyo and Masaki Inoue (2002), Complex regulatory mechanisms of telomerase activity in normal and cancer cells: How can we apply them for cancer therapy?, Oncogene, 21 January 2002, Volume 21, Number 4, Pages 688-697.
The hTERT promoter has relatively high GC content and is lacking both TATA and CAAT boxes, unlike the hTR promoter (C.J. Cairney and W.N. Keith, 2007), which has both TATA and CAAT boxes and is transcribed by RNA polymerase II. Note that "overexpression of tumor suppressor p53 can decrease the expression of hTERT mRNA, as well as directly repress transcriptional activation of the hTERT promoter construct." HDACs (histone deacetylases) that condense chromatin can repress transcriptional activation of hTERT, and conversely sometimes HDAC inhibitors such as Tricostatin A which expand chromatin can activate hTERT mRNA transcription. Transcription factors acting on the hTERT promoter [Images, Papers, Books] and kinases for hTERT activation via hTERT catalytic component phosphorylation include:
Transcription Factors with Sites on the hTERT Promoter [Links, Images, Papers, Books; Links/Transcriptional Regulation of the hTERT promoter, Images, Papers, Patents, Books]. "Several transcription factor and repressor binding sites have been identified in the hTERT promoter... Notable among these are two E-boxes for the Myc oncogene, several GC-boxes as Sp1 binding sites, and cis-regulatory elements [definition] that may recruit AP1, AP2, AP4, ATF, CREB, ER, PR, GC, IK2, MYMOD, NF1, T3Ralpha and USF." (Jun-Ping Liu, 2001).
(1) c-Myc activates hTERT promoter [Notes/(59), Wu, Nature Genetics, 1999, List/c-Myc, Links, Books, Papers]. The c-Myc protein produced by the c-Myc gene is a transcription factor [Links, Papers, Books; Wikipedia/C-myc]. Note that the c-Myc (Myc) gene is an oncogene [Links/Oncogene, Wikipedia/Oncogene], which can cause cancer if it is overexpressed, mutated, or translocated. c-Myc is a basic helix–loop–helix leucine zipper protein that dimerizes with Max to bind the DNA sequence 5'-CACGTG-3', known as an E-box, and activates transcription. The C-Myc transcription factor is regulated by it binding proteins including Max, Mad, TFII.1, YY1 and Mitz. "c-Myc-induced increase in hTERT transcription may occur through the local retrieval of c-Myc to the hTERT gene promoter, since inhibition of protein synthesis by cycloheximide does not block c-Myc activity in hTERT gene transcription." (Jun-Ping Liu, 2001). There are two E-boxes for c-Myc on the hTERT promoter. "Studies have shown that binding of c-Myc to the E-box can activate transcription of the hTERT gene, and that this induction depends on Sp1 binding to its sites in the hTERT promoter". The hTERT promoter and the hTR promoter both feature Hypoxia Response Element (HRE) sites for binding the transcription factor HIF-1 which overlap the E-boxes for binding c-Myc (C.J. Cairney and W.N. Keith, 2007). Conversely, c-Myc dimerizes with Mad (a transcriptional repressor) to repress activation of hTERT by c-Myc. The two E-box sites to which c-Myc binds for activation are also essential to hTERT transcriptional repression by Mad and SMAD3 (C.J. Cairney and W.N. Keith, 2007). C-myc sites at the 2 E-boxes overlap sites used by HIF-1 to activate hTERT transcription in hypoxia. Dr. Michael Fossel suggested using c-Myc plasmids (perhaps with supplemental controls, such as telomerase inhibitors, of course) to activate hTERT in human cells for life extension in Cells, Aging, and Human Disease. "The hTERT promoter lacks TATA and CAAT boxes and is in a CpG island with an E-box (CACGTG) binding site and (GGGCGG) sites for Sp1 and several for c-Myc (CACGTG) ...hTERT and c-Myc are expressed in actively dividing cells, but down-regulated in non-dividing cells. ... C-Myc rapidly and directly induces hTERT expression." (M.Fossel, p.286). Search for binding sites for human genome transcription factors. See Wu KJ, Grandori C, Amacker M, et al, 1999, Direct activation of TERT transcription by c-Myc, Nature Genetics, 21, 220-224. See [Links/C-myc activates telomerase, Papers/Direct activation of hTERT by C-myc]. C-myc [Index] is upregulated by Epidermal Growth Factor (EGF), which may be a lymphoma hazard if Bcl-2 is simultaneously upregulated. EGF is found in Colostrum. C-myc plasmids may have short lifetimes on the order of weeks or days when inserted into cells using cationic liposome technology or electroporation, so that plasmid transfections may have to be done on a periodic basis (say bi-weekly), to be effective. Note that PDGF (Platelet-Derived Growth Factor) activates c-Myc transcription to promote hTERT transcription.
(2) Sp1 [Notes/(43), Links, Papers, Books]. There are 5 Sp1 sites on the hTERT promoter. (Cong, Wright, and Shay, 2002). (Note that that the transcription factor Sp1 binds to a GC box with the sequence GGGCGG, and represses transcription by recruiting histone deacetylase, which compacts chromatin. Sp1 has been described as working with C-myc to activate telomerase. Sp1 mutation retards c-Myc stimulated hTERT gene transcriptional activity. A transcriptional derepressor such as CGK 1026 might work by filling the GC box pocket used by Sp1 for repression, although I find that E2F pocket protein complexes assembled on the hTERT promoter inhibited by CGK 1026 instead.) SP1 is a zinc finger transcription factor. (C.J. Cairney and W.N. Keith, 2007) pictures Sp1 as a transcriptional activator of hTERT and Sp3 as a transcriptional repressor. According to them, the 5 Sp1 binding sites are used for transcriptional repression of hTERT by transcription factors CtBP, p53, p73, and TGF-beta. See transcriptional regulators mediating their effects through Sp1 and Sp3. Sp1 overexpression upregulates hTERT and Sp3 overexpression down-regulates hTERT (Jun-Ping Liu, 2001). "...Sp1 is ubiquitously expressed and implicated in activating genes involved in various cellular processes including cell cycle regulation, chromatin remodeling and the propagation of methylation-free islands...". According to another source, "Sp1 is also a key molecule that binds to GC-rich sites on the core promoter and activates hTERT transcription (Kyo et al., 2000)" - from Satoru Kyo and Masaki Inoue (2002), Complex regulatory mechanisms of telomerase activity in normal and cancer cells: How can we apply them for cancer therapy? Oncogene, 21 January 2002, Volume 21, Number 4, Pages 688-697.
(3) estrogen receptor (ER) [Estrogen receptor pathway, Notes/(10), Index, Links, Wikipedia/Estrogen Receptor]. Estrogen (17-beta-estradiol), like many other nuclear receptor superfamily targeting molecules (nuclear receptor superfamily ligands including the steroid hormones, vitamin D3, thryoid hormone, and retinoic acid), can penetrate the cell membrane. Estrogen penetrates to bind with estrogen receptor in the cytosol. Two Estrogen receptors, each coupled with an estrogen molecule, then forms a dimer that penetrates through the nuclear pore into the nucleus and is there capped with HAT (histone acetyltransferase). This is the transcription factor complex that activates target DNA gene promoters in the cell nucleus at the estrogen response element sequence (ERE sequence: article; more than one ERE exists: the vitellogenin Estrogen Response Element (ERE) sequence is: GGTCA CAG TGACC. See the hTERT promoter estrogen receptor binding site). (Until the estrogen receptor is coupled to estrogen from cell membrane penetration, it is coupled with the heat shock protein HSP90.) Note (C.J. Cairney and W.N. Keith, 2007) pictures Estradiol as interacting with the hTERT promoter via the transcription factor ESR-1. "In addition to direct action on the hTERT promoter, estrogen and progesterone also regulate telomerase activity indirectly... E2 (17-beta-estradiol) stimulates c-Myc gene expression and induces an additive c-Myc interaction with the E-box of the hTERT promoter in human breast cancer cells..." (Jun-Ping Liu, 2001).
(4) AP-1 transcription factor activator protein: [Wikipedia, Links, Images, Papers, Books]. "The transcription factor activator protein 1 (AP-1) is involved in cellular proliferation, differentiation, carcinogenesis, and apoptosis and is expressed broadly in both cancer and normal cells. There are several putative AP-1 sites in the hTERT promoter, but their functions are unknown. " (from Masahiro Takakura, Satoru Kyo, Masaki Inoue, Woodring E. Wright, and Jerry W. Shay, (2005), Function of AP-1 in Transcription of the Telomerase Reverse Transcriptase Gene (TERT) in Human and Mouse Cells, Molecular and Cellular Biology, September 2005, p. 8037-8043, Vol. 25, No. 18. AP-1 is sometimes termed "the early response transcription factor", and is formed from a combination of c-Jun and c-Fos.
(5) Id-1 helix-loop-helix protein immortalizes human keratinocytes by activating hTERT and can be produced by nerve growth factor (NGF), which can be generated by application of Rosemary (carnosic acid), acteyl-L-carnitine, or platelet activating factor. Like AP-1 above, Id-1 is a helix-loop-helix transcription factor.
(6) Hypoxia-inducible factor 1 (HIF-1) [Wikipedia/Hypoxia-inducible factors, Links, Images, Video, Papers, Books, WikiGenes/HIF-1]. HIF-1 (hypoxia-inducible factor 1) activates telomerase [Article, Links]. "Hypoxia activates telomerase via transcriptional activation of hTERT, and HIF-1 plays a critical role as a transcription factor." See [Wikipedia/Hypoxia-induced factors, Images/HIF-1, Links/HIF-1 molecule]. HIF-1 activates transcription from hTR. In fact, the hTERT promoter and the hTR promoter both feature Hypoxia Response Element (HRE) sites for binding the transcription factor HIF-1 which overlap the E-boxes for binding c-Myc. "HIF-1 is known to bind to HRE sites in the promoters of a variety of genes where it recruits the basal transcriptional machinery and transcriptional coactivators such as P300/CBP to induce transcription." (C.J. Cairney and W.N. Keith, 2007).
(7) Tankyrase [List/Tankyrase, Wikipedia/Tankyrase, Links, Images, Papers, Books, WikiGenes/Tankyrase]. Tankyrase is a poly(ADP-ribose) polymerase associated with an hTERT poly(ADP-ribose) binding site. Tankyrase is a positive regulator of telomere enlongation in vivo, apparently by inhibiting TRF1, which binds the trailing single strand in the telomere loop. The hTERT poly(ADP-ribose) binding site is at amino acids 962-983 and has the amino acid sequence RGFKAGRNMRRKLFGVLRLKCH (W.Klapper et al, 2001).
(8) Ets [List/Ets, Links/transcription factor Ets, Images, Papers, Books] is an hTERT transcriptional activator (C.J. Cairney and W.N. Keith, 2007).
(9) Glucocorticoids. [List/Glucocorticoids, Links/glucocorticoid activation of hTERT, Images, Papers, Books] Several "putative" binding sites for the glucocorticoid, progesterone, and androgen steroid hormones have been found in the 5' flanking region of the hTR gene and may also be present in the hTERT gene (C.J. Cairney and W.N. Keith, 2007).
Factors Influencing Telomerase Activity
[List Activators, List Inhibitors; Links, Images, Papers, Books].
(10) E1A [TAList/E1A, Links/E1A protein, Images, Papers, Books] increased luciferase assay reports of hTERT promoter activity and hTR promoter activity by up to a factor of 3, and exon 2 of E1A alone was sufficient to introduce a 2-fold increase. E1A was shown to interact with Sp1 sites on the hTR promoter to produce transcriptional activation, (and no doubt similarly on the hTERT promoter). - after (C.J. Cairney and W.N. Keith, 2007).
(11) NF-kappa-B: [Index, TA/NF-kappa-B, Wikipedia, Links, Images, Papers, Books; Links/NF-kappa-B as a telomerase activator, Links/NF-kappa-B hTERT activation pathway]. See M Natarajan, S Mohan, R Konopinski, R A Otto, and T S Herman (2008), Induced telomerase activity in primary aortic endothelial cells by low-LET gamma-radiation is mediated through NF-kappa-B activation, British Journal of Radiology, (2008) 81, 711-720.
(12) progesterone [Notes/(60), Index, Links, Wikipedia/Progesterone receptor]. It is believed by some experimenters that progesterone, which is antagonistic to estrogen, acts primarily through the Map Kinase pathway to activate hTERT mRNA transcription, although some crosstalk may enable it to act through the estrogen receptor. See human progesterone receptor (hPR), a member of the steroid-receptor superfamily of nuclear receptors. Note that "the hTERT promoter lacks a canonical progesterone-responsive element." "Progesterone significantly induced hTERT mRNA expression within 3 hours after exposure. This transient effect peaked at 12 hours and then decreased." - Zhuo Wang, Satoru Kyo, et al, (2000), Progesterone Regulates Human Telomerase Reverse Transcriptase Gene Expression via Activation of Mitogen-activated Protein Kinase Signaling Pathway, Cancer Research, Oct 1, 2000, 60, 5376. That is, progesterone modulates hTERT gene expression by activating the Mitogen-Activated Protein Kinase (MAPK) pathway. "Progesterone usually antagonizes estrogen action and inhibits estrogen-induced cell proliferation in reproductive tissue such as the endometrium. Consequenty, progesterone is therapeutically applied to inhibit estrogen-dependent cancers." Progesterone-stimulated hTERT mRNA expression rises after 3 hours and is inhibited after 12 hours by a mechanism involving p21WAF1/Cip1, a cyclin-dependent kinase inhibitor.
Phosphorylating Kinases Influencing hTERT Location and Telomerase Activity
[Links, Images, Papers, Books; hTERT Protein Phosphorylation].
(13) Protein kinase C [Notes/(27), Links, Books, Books/protein kinase C and telomerase] is involved in the regulation of telomerase activity. Note that kinases phosphorylate proteins, so that Protein Kinase Cα phosphorylates the hTERT catalytic component of telomerase, enabling its transport into the nucleus from the cytoplasm.
(14) Akt Protein Kinase [Notes/(58), Index, Links; Links/Akt Protein Kinase and telomerase, Books] is associated with two receptor sites on the hTERT promoter. See Sang Sun Kang, Taegun Kwon, Do Yoon Kwon, and Su Il Do, Akt Protein Kinase Enhances Human Telomerase Activity through Phosphorylation of Telomerase Reverse Transcriptase Subunit, J Biol Chem, Vol. 274, Issue 19, 13085-13090, May 7, 1999. Note that resveratrol activates telomerase via Akt1, although it also activates SIRT1, which deacetylates DNA with HDAC effect, compacting chromatin and generally repressing transcription with gene silencing. Ordinarily, this represses transcription of hTERT. Conversely, HDAC inhibitors like Tricostatin A often activate hTERT. It seems that several growth factors such IGF-1 activate telomerase via Akt1. See also the index entry for Promoters (Gene Promoters and Transcription Factors). See hTERT activation via the PI3K/Akt pathway. By now we realize that phosphorylation of the hTERT catalytic component of telomerase can "activate" existing hTERT molecules (so that they can migrate from cytoplasm into the nucleus) without supplying a transcription factor that increases the number of hTERT mRNA transcripts. Usually "telomerase activators" increase the number of hTERT mRNA transcripts. It will be useful to keep this difference in mind while analyzing "telomerase activators". The AKT protein kinase phosphorylates hTERT in the cytoplasm to enable its import into the nucleus. AKT is not a transcription factor, but a kinase with a mission to phosphorylate.
(15) the MAP Kinase pathway: hTERT activation via the MAP Kinase pathway. EGF activates hTERT via the MAP Kinase pathway. See Yoshiko Maida, Satoru Kyo, et.al, (2002), Direct activation of telomerase by EGF through Ets-mediated transactivation of TERT via MAP kinase signaling pathway, Oncogene, 13 June 2002, Vol 21, No.26, Pages 4071-4079. TAT2 (cycloastragenol) has been identified as hTERT activating via the MAP Kinase pathway, so that the astragalosides probably probably primarily activate hTERT via the MAP Kinase pathway. "...investigation showed that TAT2 enhances telomerase activity by activation of the ERK/MAPK pathway", from Fauce, Steven Russell, PhD, Univ.Calif., LA, (2007), Telomerase modulation and its effect on antiviral activity of CD8+ T lymphocytes, PhD thesis dissertation, advisor, Rita B. Effros. See Wikipedia/MAPK/ERK pathway.
See also hTERT Transcriptional Repressors. "The minimum sequence of promoter activity is contained within 330 bp upstream of the ATG (the translation start site)... The GC-rich region forms a large CpG island arund the ATG, suggesting methylation may be involved in the regulation of hTERT expression." - Yu Sheng-Kong, Woodring E. Wright and Jerry W. Shay (2002), Human Telomerase and its Regulation, Microbiology and Molecular Biology Reviews, Sept. 2002, pp. 407-425, and associated papers. See also Analysis of the hTERT promoter occupancy in vivo using chromatin immunoprecipitation assays, chromatin immunoprecipitation assays, Wikipedia/Chromatin Immunoprecipitation (ChIP) [Links, Images, Video, Books], and Wikipedia/ChIP-on-chip [Links, Images, Video, Books, Publications].
Note that the hTERT C-site repressor region was discovered by Sierra Sciences, which has developed an inhibitor for the C-site repressor inside the hTERT promoter that can highly activate telomerase. See Sierra Sciences (WO/2002/101010) and METHODS AND COMPOSITIONS FOR MODULATING TELOMERASE REVERSE TRANSCRIPTASE (TERT) EXPRESSION, which explain the location of the repressor regions and the inhibitor for promoting telomerase activation. Also see the Sierra Sciences patent 7795416, Telomerase expression repressor proteins and methods of using the same. The Sierra Sciences telomerase activator would probably work better in a short telomerase activation/telomerase inhibition cycle than in the six-month long cycle of the Patton Protocol. High doses may be dangerous, as it can massively activate telomerase, in which case it may seem to behave like a tumor promoter.
hTERT Promoter methylation [Links, Images, Papers, Books]. The hTERT promoter is in a CpG island regulated in part by DNA methylation. CpG islands are often found in the vicinity of gene promoters functioning as potential targets for gene repression via DNA methylation. ..."In telomerase-positive samples, a methylation of all the CpG sites was observed for the hTERT promoter region (-500 to +1), whereas the exonic part (+1 to +450) revealed an unstable methylation pattern. Incomplete methylation of the proximal exon region could be necessary for, at least, a low level of hTERT transcription." See Isabelle Guilleret1, a and Jean Benhatta (2004), Unusual distribution of DNA methylation within the hTERT CpG island in tissues and cell lines, Biochemical and Biophysical Research Communications, Volume 325, Issue 3, 17 December 2004, Pages 1037-1043. Demethylation of DNA could contribute to aging by interfering with the hTERT promoter. See Cheng Liu, Xiaolei Fang, Zheng Ge, Marit Jalink1, Satoru Kyo, Magnus Björkholm, Astrid Gruber, Jan Sjöberg, and Dawei Xu (2007), The Telomerase Reverse Transcriptase (hTERT) Gene Is a Direct Target of the Histone Methyltransferase SMYD3, Cancer Research, March 15, 2007 67; 2626. Writers vary somewhat in their descriptions of CpG island methylation in hTERT: "The data show that the hTERT CpG island is not methylated in primary tissues and cultured cells, suggesting that CpG island methylation is not responsible for hTERT repression in telomerase-negative cells." In some tumors, however, demethylation of methylated CpG islands with 5-azacytidine upregulated hTERT expression. In normal human cells, histone deacetylation is primary in silencing hTERT transcription, as shown by upregulation of hTERT after application of Tricostatin A, a histone deaceylase inhibitor. - (Jun-Ping Liu, 2001).
hTERT protein phosphorylation [Links, Images, Papers, Books; Links/the role of hTERT phosphorylation in the regulation of telomerase activity, Images, Papers, Books]. The phosphorylation of hTERT protein (the catalytic component of telomerase) is associated with translocation of hTERT from the cytoplasm to the nucleus during CD4+ T-cell activation [Images]. Telomerase activity in resting T-cells [Images] is not dependent on net hTERT protein increase [Images], but on hTERT protein phosphorylation associated with its import into the nucleus from the cytoplasm [Images]. See Kebin Liu, Richard J. Hodes and Nan-ping Weng (2001), Cutting Edge: Telomerase Actiation in Human T-Lymphocytes Does not Require increase in Telomerase Reverse Transcriptase (hTERT) Protein but is Associated with hTERT phosphorylation and Nuclear Translocation, Journal of Immunology, 2001; 166; 4826-4830. Sang Sun Kang, Taegun Kwon, Do Yoon Kwon and Su I Do (1999), Akt Protein Kinase Enhances Human Telomerase Activity through Phosphorylation of Telomerase Reverse Transcriptase Subunit, The Journal of Biological Chemistry, May 7, 1999, 274, 13085-13090. Kinases phosphorylate proteins.
(1) AKT protein kinase
[Links,
Images,
Papers,
Books]
phosphorylates
hTERT in the cytoplasm
for import
into the nucleus,
promoting
telomerase activity. There are two AKT phosphorylation sites on hTERT,
one at amino acids 220-229 with
the sequence GARRRGGSAS and the other at
amino acids 817-826 with
the sequence AVRIRGKSYV.
(W.Klapper et al, 2001).
(2) Protein Kinase C [Index, Links, Images, Papers, Books] activates telomerase. (3) C-Abl tyrosine kinase [Images, Papers, Books] inactivates telomerase. (4) Protein Phosphatase 2A [Images, Papers, Books] switches off telomerase activity. (after Mouldy Sioud, Methods in Molecular Biology, Vol.2, Vol. 361, p.241.) |
hTERT repressor [Links, Papers, Patents/hTERT repressor, Books, Amazon, LifeExtension1, LifeExtension2, Biocarta Pathways]. See also William H. Andrews, C.A.Foster, S. Fraser, Hamid Mohammedpour, of Sierra Sciences, 2004: Methods and Compositions for Modulating Telomerase Reverse Transcriptase (TERT) Expression, United States Patent No. 6,686,159 B2, Andrews, et al., Feb 3, 2004. Explains the hTERT repressor part of the hTERT promotor and means of interfering with hTERT repression. "In 2001, Sierra Sciences discovered a repressor binding site (dubbed "Site C") that blocks the expression of telomerase reverse transcriptase ("TERT"). For this discovery, Sierra Sciences was issued U.S. patent #6,686,159 in 2004. Sierra Sciences discovered another repressor binding site, "GC-Box 5," in 2004, for which it was issued patent #7,279,328 in 2007." - Wikipedia/Sierra Sciences. See US Patent 7,279,328 (2007) Methods and Compositions for Modulating Reverse Transcriptase (TERT) Expression (2007), which refers the reader to (Nozawa K., et al., 2001, Horikawa et al., 2005, Oh, S., et al., 1991, and Oh, S., et al., 2000). See also (Won, Yim and Kim, 2002, Won, Chang, Oh, and Kim, 2004). Transcriptional repressors of hTERT include Sp3, AP-1, MZF, WT1 and E2F (C.J. Cairney and W.N. Keith, 2007).
hTERT Transcriptional Repression [Telomerase inhibitors, Links/hTERT transcriptional repression, Images, Papers, Books]. Notes (Cong, Wright, and Shay, 2002): "hTERT is repressed in most tissues prior to birth. P53 transcriptionally represses hTERT. This takes place within hours, before cell cycle arrest or apoptosis takes place. Histone deacetylases (HDACs) are involved in hTERT transcriptional repression. Interferon-alpha represses hTERT activity within 4 hours in malignant and non-malignant human hematopoietic stem cell lines, primary leukemic cells and normal T-lymphocytes. The hTERT gene is thought to be a direct transcriptional target of the interferon-alpha signaling pathway. The new vitamin D3 analog 5,6-trans-16-ene-vitamin D3 represses hTERT transcription. Retinoic acid and dimethyl sulfoxide repress hTERT transcription. Treatment with the DNA methylation inhibitor 5-azacytidine induced hTERT transcription in 2 telomerase-negative ALT cell lines. (Showing methylation is an important factor in hTERT transcriptional repression.) Transcriptionally repressed genes tend to be associated with hypoacetylated histones, whereas transcriptionally active genes tend to be associated with hyperacetylated histones. (Acetylation expands chromatin, making it available for transcription.) Accumulating evidence exists that telomerase activity can be regulated by hTERT phosphorylation. Won, Chang, Oh, and Kim, 2004). Transcriptional repressors of hTERT include Sp3, AP-1, MZF, WT1 and E2F (C.J. Cairney and W.N. Keith, 2007).
hTERT Transcriptional Repressors
[Links, Images, Papers, Books]. hTERT transcriptional repressors exist in several chromosomes. MAD/MAX heterodimers bound to E-boxes repress hTERT transcription. Repressors of hTERT transcription include:
| (1)The MAD1/MAX heterodimer
[Links/MAD1/MAX heterodimer,
Images,
Papers,
Books].
(2) Mad [Links, Images, Papers, Books], (2b) Mad dimerizes with c-Myc to repress activation of hTERT by c-Myc. ___[Links/the c-Myc/Mad dimer, Images, Papers, Books]. ___C-Myc inhibits the effect of Mad in a concentration-dependent manner and conversely, ___Mad inhibits the effect of c-Myc in a concentration-dependent manner, and ___Mad competes with c-Myc for binding to Max. C-myc/Max activates, Mad/Max inhibits. ___Mad may also recruit histone deacetylases to repress hTERT transcription. (3) The tumor suppressor p53 [Index/p53, Links, Images, Papers, Books]. ___P53 interacts with Sp1 to inhibit hTERT transcription. (4) MZF-2 [Links, Images, Papers, Books]. (K.Fujimoto & M.Takahashi, 1997). ___Multiple sites exist on the hTERT promoter for MZF-2 binding. ___Mutations of the MZF-2 (Myeloid-specific Zinc Finger Protein 2) gene ___promote hTERT transcription. (5) Wilms tumor 1 suppressor gene [Index/p53, Links, Images, Papers, Books]. ___WT1 binds on the hTERT promoter at -307 to -423. (6) p21WAF1/Cip1 overexpression downregulates hTERT transcription ___in glioma cell lines and in immortalized keratinocytes. (Jun-Ping Liu, 2001). |
hTERT transfection [Links, Books, Papers, Patents, Amazon, LifeExtension2].
hTERT vectors [Patents/hTERT vectors, Books/hTERT vectors, Amazon/hTERT vectors, LibCong/DNA viral vectors LifeExtension2]. See Mogfield JE, Liu WR, Reid R, 2006: Adenoviral human telomerase reverse transcriptase dramatically improves ischemic wound healing without detrimental immune response in aged rabbit model., Human Gene Therapy, 2006; 17(6): 651-660. This shows how telomerase may be activated via gene therapy using a viral vector. See Origene, Genetiq or Vector Biolabs for hTERT vectors [Links/hTERT viral vectors, Images; Links/hTERT vectors, Images]. See also [Links/targeted insertion of the hTERT gene, Images, Books, Papers, Amazon], targeted gene insertion [Links, Images, Books, Papers, Amazon] and gene insertion [Links, Images, Books, Papers, Amazon]. Also see Anti-Aging Insertion of the hTERT gene [Links, Papers] with anti-aging gene insertion [Links, Papers, Amazon]. See also targeted genome editing with zinc finger nucleases.
hTR (hTERC) [Biocarta/Overview of telomerase RNA component gene hTerc Transcriptional Regulation, Wikipedia/Telomerase RNA component, Links/hTR gene, Images, Links/hTR plasmid, Links/hTR transfection]. hTR (hTERC) - The gene encoding the RNA component of human telomerase corresponding to the telomeric DNA 5'-TTAGGG-3' minisatellite tandem repeat. The hTR sequence for the RNA part of telomerase is located on chromosome 3 (1,436 genes) at 3q26.3, and the associated RNA exhibits a half-life of about 5 days, having a size of 451 nt. Telomerase activators that work via activation of hTERT may be limited in their rate of telomerase generation by the availability of hTR RNA, the 2nd component of telomerase, limiting the rejuvenation rate. See Links/hTR activators; Links/hTERC activators, Papers/hTERC activators. Expression of hTR is readily detected in many tissues, including testes, ovary, brain, liver, small intestine, thymus, kidney, and prostate. On the other hand expression of hTERT is difficult to detect in most somatic tissues with TRAP assay PCR except in the testes and endometrium, although it is also seen weakly in the skin, spleen, stomach, and small intestine (C.J. Cairney and W.N. Keith, 2007). See the impact of histone acetylation [Links, Books] on hTR transcription [Links, Books]. It may be useful to take HDAC inhibitors [Links, Books] such as sodium butyrate [Links, Wikipedia] or trichostatin A [Links] to improve transcription of the hTR RNA component of the telomerase telomeric DNA repair enzyme. For the 3D structure of hTR [Links, Wikipedia], see Gerald Gavory, Martyn F. Symmons, Yamuna Krishnan Ghosh, David Klenerman, and Shankar Balasubramanian, 2006, Structural Analysis of the Catalytic Core of Human Telomerase RNA by FRET and Molecular Modeling, Biochemistry, 2006. "hTR is highly expressed in all tissues regardless of telomerase activity, with cancer cells generally having a five-fold higher expression than normal cells. In contrast, mRNA for hTERT is estimated at less than 1 to 5 copies per cell." (Cong, Wright, and Shay, 2002).
The hTR (hTERC) Promoter
[Links/The hTR Promoter, Images, Papers, Books; Links/The hTERC Promoter, Images, Papers, Books]. The hTR promoter contains a TATA box-like element and a consensus CCAAT box, so that it is probably transcribed by RNA polymerase II. Deletion of the CAAT box eliminates hTR transcription in luciferase assays. See (C.J. Cairney and W.N. Keith, 2007). Binding of the transcriptional activator NF-Y to the CCAAT box is essential for activation of transcription from hTR.
Promoting hTR (hTERC) Transcription
[Links, Images, Papers, Books]. Endogenous levels of hTR (hTERC) can be increased by application of the JNK inhibitor SP600125. This has been demonstrated in several cell lines. Chromatin immunoprecipitation (ChiP) demonstrated that SP600125 caused a switch in the ratio of Sp1/Sp3 binding to the endogenous hTR promoter (Bilsland et al. 2006, W.N.Keith and A.E.Bilsland, Therapeutic Options for Cancer Treatment in K.Lenhard Rudolph, 2008, p.266).
Sp1 (binding at GC boxes) and HIF-1 are positive regulators of hTR transcription (C.J. Cairney and W.N. Keith, 2007). The 4 Sp1 binding sites in the hTR promoter are not required for its transcriptional activity, although mutation of the sites somewhat reduces hTR transcription. Binding of the transcriptional activator NF-Y to the hTR promoter CCAAT box is essential for activation of hTR transcription. It has been noted that upregulation of either hTR alone, hTERT alone, or both together can produce more telomerase molecules, although hTERT molecules are typically least numerous (C.J. Cairney and W.N. Keith, 2007). Rb protein is thought to reduce repression of hTR transcription due to MDM2 binding with Sp1, by sequestering MDM2 from Sp1. Several "putative" binding sites for the glucocorticoid, progesterone, and androgen steroid hormones have been found in the 5' flanking region of the hTR gene C.J. Cairney and W.N. Keith, 2007). Binding all three subunits of NF-Y to the hTR promoter CCAAT box maintains basal levels of hTR transcription. A dominant-negative subunit of NF-YA exists capable of reducing hTR transcription by a factor of 5-7 (op cit).
Inhibiting hTR (hTERC) Transcription
[Links, Images, Papers, Books]. Transcription from hTR is sometimes inhibited in cancer therapy, to inhibit telomerase activity. In other words. hTR inhibitors (hTERC inhibitors) are used as telomerase inhibitors [List]. Promoter activity in hTR can be repressed with Sp3 (binding at GC-boxes) (op cit, p.266). GRN163L from Geron is an hTR inhibitor (P Phatak and A M Burger, 2007). MDM2 acts to repress hTR transcription by binding with Sp1, preventing hTR transcription activation via Sp1. The transcriptional repressor CtBP also works by binding to Sp1.
