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Clinical use of telomere
measurement in preventive and
personalized medicine
Dave Woynarowski, M.D.
22nd Annual World Cong...
Page 2
Clinical use of telomere measurement in preventive and
personalized medicine – Dr. Dave Woynarowski
The following p...
Page 3
What is all this fuss about Telomeres and where did it
come from?
• Nobel Laureates- telomeres and telomerase
• Pio...
Page 4
Nobel Laureates in Medicine
Elizabeth H. Blackburn
Congratulations to the pioneers in Telomere Biology for their 20...
Page 5
A Thorn among 2 Roses!
Page 6
2010 - Predictions came true!
Page 7
Bill Andrews, Ph.D., CEO at Sierra Sciences
Page 8
Maria Blasco, Ph.D., Director of Spanish National
Cancer Research Center, Madrid, Spain
Page 9
So what is a telomere and why is it important?
• Correlated with longevity
• Correlated with morbidity
• Correlated...
Page 11
And what do they look like?
Page 12
Most commonly seen graphic representation
Page 13
Functions of the telomere
1. Protect the chromosome and genes from loss of valuable genetic
material
2. Protect th...
Page 14
Protect the chromosome and genes from loss of
valuable genetic material
• The telomere segments were original thou...
Page 15
Cellular Fate and Telomerase Activation
Page 16
The Hayflick Limit
Page 17
Patterns of Gene Expression
The genes stay
the same
The pattern
alters
Page 18
There are numerous illnesses associated with aging
including but not limited to:
• WBC and tissue telomere length ...
Page 19
Short telomeres and age-related diseases
WEAK IMMUNITY
/ ASTHMA /
ALLERGIES
Senescence & Apoptosis
CANCER
WRINKLES...
Page 20
Longevity Studies
• Bodnar, Shay et al. Fibroblasts with telomerase + promoter= functional
immortality
• Blasco AA...
Page 21
0.25
0.20
0.15
0.10
0.05
0.00
120
100
80
60
40
20
0
150
100
50
0
8
6
4
2
0
100
80
60
40
20
0
100
75
50
25
0
Causal...
Page 22
Longevity Tied to Genes that Preserve Tips of
Chromosomes
• “Telomeres are one piece of the puzzle that accounts f...
Page 23
Human
skin on
a mouse
Young Telomerized
Skin Reconstitution:
Old Cells Become Young
Funk et al, Exp Cell Res, 2000...
Page 24
But what can you do with the number?
• Is mitigation possible?
• Lifestyle
• Diet
• Exercise
• Sleep
• Stress redu...
Page 25
Issues with measurement
• Populations sizes
• Confounding variables (twin studies and genetically similar populati...
Page 26
Source: Daniali et al., Nat.Commun., 2013
Telomeres shorten at equivalent rates in somatic
tissues of adults
4 6 8...
Page 27
For commercial clinical usage there are 3 basic ways to
measure
• Q-PCR- quantitative signal amplification
• FLO-F...
Page 28
Q-PCR Pro’s
1. Longstanding usage (decades) and data base- familiar technology.
2. Least costly of all technologie...
Page 29
Q-PCR Con’s
1. Better suited to smaller non repetitive gene sequences- telomeres are
relatively large (5-10Kb)
2. ...
Page 30
DNA is a double helix composed by two antisense strands. The strands have a direction (5´ to 3´ given by the succe...
Page 31
How does PCR work?
Below is a typical curve of amplification in a real-time PCR equipment. At the begining, and al...
Page 32
chromosome telomere
single copy gene
break
The whole DNA target cannot be amplified and so
there is no exponential...
Page 33
TTAGGG repetitions
There is still exponential amplification!
Although now you amplify two pieces
instead of one. H...
Page 34
FLO-FISH Pro’s
• Long standing use and reasonable data base size specifically for
telomere use.
• Primarily design...
Page 35
FLO-FISH Con’s
• Still rather costly
• Does not report short telomere data
• Accuracy and reproducibility are defi...
Page 36
HT Q-FISH Pro’s
1. Accurate reproducible scalable
2. Allows visualization of individual chromosomes
3. Defining fe...
Page 37
HT Q-FISH Con’s
• Expensive* and proprietary
• Logistics: Longer turn around time and live cell requirement
• Rece...
Page 38
Median TL
% Short telomeres
Blood uptake and PBMC purification PBMC plating and Q-FISH HT confocal microscopy
Capt...
Page 39
Advantages of HT Q-FISH histograms
> 20th Percentile < % short telomeres
Sample
with high %
short
telomeres
Sample...
Page 40
Sample with normal telomere length variability
Why Median Length alone misses the whole picture!
TL dispersion
Mut...
Page 41
How to use the numbers
• Need to repeat testing at intervals no matter what technology you
choose (accuracy and re...
Page 42
Walking the walk - Do you?
Page 43
My telomeres as of November 2013
Page 44
Results report
Page 45
Results report
Page 46
Correlation with Flo-FISH
Page 47
Q-PCR Comparisons sample 1
Page 48
QPCR comparison sample #2 same time same date
Page 49
New applications of telomere length as a biomarker
Genotoxic chemotherapy drugs,
targeting preferentially on telom...
Page 50
Imetelstat response time is based on telomere length
Source: (Robin Frink, John Minna)
H1703
H661
H1299
H1975
HCC4...
Page 51
The question of “dilution” be careful interpreting studies,
especially if the accuracy and reproducibility are sus...
Page 52
David Woynarowski, MD, CPT
Photos Copyright © 2009, www.telonauts.com
THANK YOU!
Page 53
Essential References
1. Deng Y, Chan SS, Chang S. Nat Rev Cancer. 2008 Jun; 8(6):450-8. 8:450-458.
2. Harley CB.Mu...
Page 54
General references
• High-throughput telomere length quantification by FISH and its application to human populatio...
Page 55
Oncology
• A prospective study of relative telomere length and postmenopausal breast cancer risk. De Vivo et al.
C...
Page 56
Oncology (cont.)
• Mean telomere length and risk of incident colorectal carcinoma: a prospective, nested case-cont...
Page 57
Aging
• Telomeres and human disease: ageing, cancer and beyond. Blasco, M.A., Nat Rev Genet, 2005. 6(8): p. 611-22...
Page 58
Infertility
• Characterization of telomerase activity in the human oocyte and preimplantation
embryo. Wright et al...
Page 59
Cardiology
• White cell telomere length and risk of premature myocardial infarction. Brouilette, S., et al., Arter...
Page 60
Neurology
• Shorter telomeres are associated with mortality in those with APOE epsilon4 and dementia. Honig et al....
Clinical use of telomere measurement in preventive and personalized medicine   dr. dave woynarowski - a4 m may 2014
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Clinical use of telomere measurement in preventive and personalized medicine dr. dave woynarowski - a4 m may 2014

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Learn more about the value of telomere measurement, and how to use it in the field of preventive and personalized medicine.

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Clinical use of telomere measurement in preventive and personalized medicine dr. dave woynarowski - a4 m may 2014

  1. 1. Clinical use of telomere measurement in preventive and personalized medicine Dave Woynarowski, M.D. 22nd Annual World Congress on Anti-Aging May 2014 – Orlando, Florida
  2. 2. Page 2 Clinical use of telomere measurement in preventive and personalized medicine – Dr. Dave Woynarowski The following potential conflict of interest relationships are germane to my presentation. • Equipment: none • Speakers Bureau: none • Stock Shareholder: none • Grant/Research Support: none • Consultant: Life Length* • Status of FDA devices used for the material being presented • Enter Device Name or state NA/Non-Clinical • Status of off-label use of devices, drugs or other materials that constitute the subject of this presentation • Enter Device or Drug Name or state NA/Non-Clinical
  3. 3. Page 3 What is all this fuss about Telomeres and where did it come from? • Nobel Laureates- telomeres and telomerase • Pioneers in the field - discovery of the human telomerase gene (Htert) Dr. Bill Andrews at Geron • Discovery of telomerase activating natural substances Cal Harley at Geron • Prediction of the whole thing in 1997 by Mike Fossel in the book Reversing Human Aging • Confluence of information - The Immortality Edge 2010
  4. 4. Page 4 Nobel Laureates in Medicine Elizabeth H. Blackburn Congratulations to the pioneers in Telomere Biology for their 2009 Award! Jack W. Szostak Carol W. Greider
  5. 5. Page 5 A Thorn among 2 Roses!
  6. 6. Page 6 2010 - Predictions came true!
  7. 7. Page 7 Bill Andrews, Ph.D., CEO at Sierra Sciences
  8. 8. Page 8 Maria Blasco, Ph.D., Director of Spanish National Cancer Research Center, Madrid, Spain
  9. 9. Page 9 So what is a telomere and why is it important? • Correlated with longevity • Correlated with morbidity • Correlated with specific “Diseases of Aging” • A major cause of the Diseases of Aging! • Potential for predictive value • Potential for intervention • A lie detector for interventions!!!! • Applications for drug testing, supplement testing • Potential to identify at risk individuals who otherwise might be missed (HT-QFISH)
  10. 10. Page 11 And what do they look like?
  11. 11. Page 12 Most commonly seen graphic representation
  12. 12. Page 13 Functions of the telomere 1. Protect the chromosome and genes from loss of valuable genetic material 2. Protect the chromosome from recombination certain mutations and other aberrations 3. Signal the end of the cells life (via apoptosis, etc.) or replicative senescence 4. Confer longevity to certain cells
  13. 13. Page 14 Protect the chromosome and genes from loss of valuable genetic material • The telomere segments were original thought to be junk DNA. • The end replication problem obligates the loss of DNA because RNA primers are used as templates by at the ends of the replication process. When those primers and their attendant fragments are removed the chromosome is shorter. • This happens EVERY TIME the cell replicates and leads to the loss of DNA Since the DNA of the telomere does not contain “genes” it can be sacrificed. • Average base pair (bp) loss is 30 to 250 bp per year and varies from cell line to cell line
  14. 14. Page 15 Cellular Fate and Telomerase Activation
  15. 15. Page 16 The Hayflick Limit
  16. 16. Page 17 Patterns of Gene Expression The genes stay the same The pattern alters
  17. 17. Page 18 There are numerous illnesses associated with aging including but not limited to: • WBC and tissue telomere length is shortened in: • Cancer’s of most types – JAMA Dec. 2010 – Typical AMI – CHF* – COPD – Diabetes – Arthritis – Alzheimer’s – Obesity and high stress situations • Progeriod syndromes with Werner’s syndrome the prototypical telomere dysfunction/shortening results in premature aging phenotype and increased susceptibility to cancer. Sub types of Pulmonary Fibrosis and Aplastic Anemia
  18. 18. Page 19 Short telomeres and age-related diseases WEAK IMMUNITY / ASTHMA / ALLERGIES Senescence & Apoptosis CANCER WRINKLES, ARTHRITIS, OSTEOPOROSIS REPRODUCTIVE ORGANS INFERTILITY, MENOPAUSE DIABETES CARDIOVASCULAR DISEASE, HYPERTENSION & ATHEROSCLEROSIS DNA Damage Senescence & Apoptosis BRAIN CNS (CENTRAL NERVOUS SYSTEM) DISEASES ALZHEIMER’S, PARKINSONS, DEMENTIA RETINA MACULAR DEGENERATION DISEASE LEVEL ORGAN LEVEL CELLULAR LEVEL STEM CELLS DNA Damage Source: Life Length and Recharge Biomedical Clinic SHORT TELOMERES
  19. 19. Page 20 Longevity Studies • Bodnar, Shay et al. Fibroblasts with telomerase + promoter= functional immortality • Blasco AAV- virus x 1 injection, 25% extension in middle aged mice 13% extension in old mice • DePinho et. al.- tamoxifen trigger “reversed the phenotype of aging” • Gil Aztmon- Increased longevity associated with mutated telomerase
  20. 20. Page 21 0.25 0.20 0.15 0.10 0.05 0.00 120 100 80 60 40 20 0 150 100 50 0 8 6 4 2 0 100 80 60 40 20 0 100 75 50 25 0 Causal relationship between telomere length and aging (in vivo) Source: Jaskelioff et al., Nature, 2011 (edited) Testes Spleen Intestinal crypts Vehicle 4-OHT Terc-/- mice bearing knocked-in ER-Telomerase rejuvenate upon telomerase reactivation with 4-hydroxytamoxifen Vehicle 4-OHT Vehicle 4-OHTa b c G0 Testesweight(g) G4 d e f G0 G4 G0 G4 *** *** ** * *** *** 53BP1fociper 100cells 53BP1+fociper 100crypts G0 Pulseperlitter G4 G0 G4 G0 G4 ** * * * ** *** *** Spleenweight (mg) Apoptoticcellsper 100crypts g h i G4 / OHT Vehicle 4-OHT Vehicle 4-OHT G0 G4 a b a c 500 400 300 200 100 0 Brainweight(mg) *** * G0 G4 100 80 60 40 20 0 Spherediameterʯm * *
  21. 21. Page 22 Longevity Tied to Genes that Preserve Tips of Chromosomes • “Telomeres are one piece of the puzzle that accounts for why some people can live so long," says Gil Atzmon, Ph.D., Assistant Professor of Medicine and of Genetics at Einstein, Genetic Core Leader for The Longevity Project at Einstein's Institute for Aging Research, and a lead author of the paper. "Our research was meant to answer two questions: Do people who live long lives tend to have long telomeres? And if so, could variations in their genes that code for telomerase account for their long telomeres?” The answer to both questions was “yes.” • Genetic Variation in Human Telomerase is Associated with Telomere Length in Ashkenazi Centenarians, appears in the November 9th on-line issue of the Proceedings of the National Academy of Sciences.
  22. 22. Page 23 Human skin on a mouse Young Telomerized Skin Reconstitution: Old Cells Become Young Funk et al, Exp Cell Res, 2000 Slide courtesy of Bill Andrews Old
  23. 23. Page 24 But what can you do with the number? • Is mitigation possible? • Lifestyle • Diet • Exercise • Sleep • Stress reduction meditation • Supplements • Use of Telomere length to follow anti-aging programs and choices • Drugs and therapies
  24. 24. Page 25 Issues with measurement • Populations sizes • Confounding variables (twin studies and genetically similar populations) • Test variability and accuracy- the subject of the rest of this talk! • Tissue vs. PMC’s • Stem cell aging versus circulating cells • Telomapping - HT Q-FISH (TAT)
  25. 25. Page 26 Source: Daniali et al., Nat.Commun., 2013 Telomeres shorten at equivalent rates in somatic tissues of adults 4 6 8 10 12 Leukocytetelomerelength(kb) Muscle telomere length (kb) y = 0.77x + 0.45 R2 = 0.71; P<0.0001 Men Women 4 6 8 10 12Leukocytetelomerelength(kb) Muscle telomere length (kb) y = 0.77x + 0.72 R2 = 0.69; P<0.0001 Men Women 4 6 8 10 12 Leukocytetelomerelength(kb) Muscle telomere length (kb) y = 0.79x + 1.27 R2 = 0.69; P<0.0001 Men Women 4 6 8 10 12 12 10 8 6 4 Leukocytetelomerelength(kb) Muscle telomere length (kb) y = 0.76x + 1.72 R2 = 0.59; P<0.0001 Men Women 4 6 8 10 12 12 10 8 6 4 Leukocytetelomerelength(kb) Muscle telomere length (kb) y = 0.82x + 0.59 R2 = 0.71; P<0.0001 Men Women 4 6 8 10 12 12 10 8 6 4 Leukocytetelomerelength(kb) Muscle telomere length (kb) y = 0.69x + 1.14 R2 = 0.51; P<0.0001 Men Women 12 10 8 6 4 12 10 8 6 4 12 10 8 6 4
  26. 26. Page 27 For commercial clinical usage there are 3 basic ways to measure • Q-PCR- quantitative signal amplification • FLO-FISH -cytometry • HT Q-FISH-confocal microscopy
  27. 27. Page 28 Q-PCR Pro’s 1. Longstanding usage (decades) and data base- familiar technology. 2. Least costly of all technologies. 3. Not labor or knowledge intensive. 4. Fast turn around. 5. Has been “gold standard” of telomere measurements in the past
  28. 28. Page 29 Q-PCR Con’s 1. Better suited to smaller non repetitive gene sequences- telomeres are relatively large (5-10Kb) 2. Subject to errors of “DNA Quality” 3. Accuracy and Reproducibility of telomere measurements 4. Gives only Average telomere length- cannot supply median or short telomere percentages
  29. 29. Page 30 DNA is a double helix composed by two antisense strands. The strands have a direction (5´ to 3´ given by the succesion of sugars and phospates). Never mind the details, the point is that a new DNA synthesis can only occur in the 5´ to 3´ direction as depicted in the cartoon. To amplify DNA, you need DNA polymerase, nucleotides and a short piece of DNA called a “primer” to initiate the DNA polymerization. By cycling between heating (to separate the DNA strands) and cooling (to allow the join of the DNA and primers) it is possible to duplicate the original DNA material. Theoretically at least, in every cycle you duplicate the number of molecules present in the previous one. Therefore, the amplification is exponential. 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ Original DNA molecule Heat, strands separation. Then, cool down and primer annealing. DNA synthesis in the presence of DNA polymerase DNA duplication Cyclingaround30times Primer Primer How does PCR work?
  30. 30. Page 31 How does PCR work? Below is a typical curve of amplification in a real-time PCR equipment. At the begining, and although there is amplification, the detector cannot detect any meaninful increase of DNA. However, after some cycles (10 in the example) there is an exponential increase in the amount of DNA. Later on, the reaction reaches a plateau due to the lack of reagents and saturation of the detector. So scientists compare the reaction at the region pointed by the arrow. If you have two samples, one with more DNA target than the other, the first one will get into the exponential phase sooner. This allows the quantitative comparison of samples (look at the picture to the right).
  31. 31. Page 32 chromosome telomere single copy gene break The whole DNA target cannot be amplified and so there is no exponential duplication of the entire sequence. How does PCR work? Now, to correctly quantify the copy number of a certain DNA sequence (such as a telomere) you also need to quantify an internal control (which is single copy to assure the normalization). Because the single copy gene has a unique sequence (no repetitive sequence) any damage to the sequence target inhibits PCR amplification:
  32. 32. Page 33 TTAGGG repetitions There is still exponential amplification! Although now you amplify two pieces instead of one. However, what matters is the total amplification not the length. CONCLUSION: Telomere sequence amplification is more robust than the internal control sequence. That means that telomere sequences will amplify regardless of the DNA quality. However, you need a the ratio between the quantification of both telomere and control. Thus, the quantification is not reliable. How does PCR work? In contrast, telomere sequence is highly repetitive. Therefore, even if the DNA sequence is damaged, there is always chances to still amplify full pieces of telomeres
  33. 33. Page 34 FLO-FISH Pro’s • Long standing use and reasonable data base size specifically for telomere use. • Primarily designed to cater to institutions • More costly than QPCR but less costly than HT-QFISH • Allows differentiation between granulocytes and lymphocytes (may have some importance in viral infections) • Reports Median Telomere Length instead of average
  34. 34. Page 35 FLO-FISH Con’s • Still rather costly • Does not report short telomere data • Accuracy and reproducibility are defined and probably better than QPCR and similar to HT-QFISH • FACS technology is well defined and automated
  35. 35. Page 36 HT Q-FISH Pro’s 1. Accurate reproducible scalable 2. Allows visualization of individual chromosomes 3. Defining feature: Allows determination of critically important short telomeres and of “signal free ends” 4. Expanding data base and increasing scientific usage 5. Ideal for individual clinical evaluations and repeated determinations 6. Telomapping possible
  36. 36. Page 37 HT Q-FISH Con’s • Expensive* and proprietary • Logistics: Longer turn around time and live cell requirement • Recent technology so less familiarity in scientific community • Requires higher levels of expertise * Expected cost reductions in the near future demand for latest technology replaces obsolete methods
  37. 37. Page 38 Median TL % Short telomeres Blood uptake and PBMC purification PBMC plating and Q-FISH HT confocal microscopy Capture of individual telomere signalsData processingData analysis HT Q-FISH (TAT) technology overview 21 3 56 4
  38. 38. Page 39 Advantages of HT Q-FISH histograms > 20th Percentile < % short telomeres Sample with high % short telomeres Sample with low % short telomeres As there are not many short telomeres, we need to move toward longer telomeres to cover 20% of the total
  39. 39. Page 40 Sample with normal telomere length variability Why Median Length alone misses the whole picture! TL dispersion Mutation in telomerase? Sample with wide telomere length variability TL dispersion
  40. 40. Page 41 How to use the numbers • Need to repeat testing at intervals no matter what technology you choose (accuracy and reproducibility will now become a huge factor for the individual) • Patient doctor as allies in the quest for better health span and longer life span • Review of history and physical including family history for factors that can be modified • Again follow up!
  41. 41. Page 42 Walking the walk - Do you?
  42. 42. Page 43 My telomeres as of November 2013
  43. 43. Page 44 Results report
  44. 44. Page 45 Results report
  45. 45. Page 46 Correlation with Flo-FISH
  46. 46. Page 47 Q-PCR Comparisons sample 1
  47. 47. Page 48 QPCR comparison sample #2 same time same date
  48. 48. Page 49 New applications of telomere length as a biomarker Genotoxic chemotherapy drugs, targeting preferentially on telomeres (e.g. topoisomerase inhibitors, cisplatin, HU) Targeting specifically on telomeres(e.g. RHPS4) Genotoxic chemotherapy (e.g. anthracyclin, oxalipatin) Source: Lu et al., Front. Med., 2013; Gramatges et al., Clin. Cancer Res., 2013 • Telomere length can be a surrogate marker of chemotherapy sensitivity and toxicity. • If PBMC telomeres from cancer survivors who underwent chemotherapy are shorter than those from healthy normal persons, these patients may be at high risk for developing secondary cancer disorders. • In fact, Non-Hodgkin’s Lymphoma patients have a significant risk for developing secondary malignant neoplasms such as solid tumors, melanoma, Hodgkin’s disease, and cancer of the lung, brain, kidney and bladder.
  49. 49. Page 50 Imetelstat response time is based on telomere length Source: (Robin Frink, John Minna) H1703 H661 H1299 H1975 HCC4006 H1993 H1568 HCC1359 H1819 H2882 H2009 H1693 H226 H838 H2126 H2347 H1355 HOP62 H460 H1155 H157 HCC2429 A549 H322 H1395 H596 H1792 H3122 H1666 H2122 H727 H650 HCC78 H2228 H358 H2291 H3255 H1373 H441 HCC1438 HCC44 H2073 H2087 HCC95 HCC827 H522 H1437 H1838 HCC4019 H23 HCC2279 H1650 H920 HCC193 HCC1833 Calu-6 H1648 Calu-1 H1944 HCC515 H820 H2887 Calu-3 TelomereLength(kb) TotalPopulationDoublings TotalPopulationDoublings
  50. 50. Page 51 The question of “dilution” be careful interpreting studies, especially if the accuracy and reproducibility are suspect!
  51. 51. Page 52 David Woynarowski, MD, CPT Photos Copyright © 2009, www.telonauts.com THANK YOU!
  52. 52. Page 53 Essential References 1. Deng Y, Chan SS, Chang S. Nat Rev Cancer. 2008 Jun; 8(6):450-8. 8:450-458. 2. Harley CB.Mutat Res. 1991 Mar-Nov;256(2-6):271-82. 3. Bollmann, F. M. (2007) Cancer Treat. Rev., 33, 704-709. 4. Valdes AM, Andrew T, Gardner JP, Kimura M, Oelsner E, Cherkas LF, Aviv A, Spector TD.Lancet. 2005 Aug 20- 26;366(9486):662-4. 5. Du M, Prescott J, Kraft P, Han J, Giovannucci E, Hankinson SE, De Vivo I. Am J Epidemiol. 2012 Mar 1;175(5):414-22. 6. Epel ES, Blackburn EH, Lin J, Dhabhar FS, Adler NE, Morrow JD, Cawthon RM. Proc Natl Acad Sci U S A. 2004 Dec 7;101(49):17312-5. 7. Drury SS, Theall K, Gleason MM, Smyke AT, De Vivo I, Wong JY, Fox NA, Zeanah CH, Nelson CA.Mol Psychiatry. 2012 Jul;17(7):719-27. 8. Paul L.J Nutr Biochem. 2011 Oct;22(10):895-901. 9. Farzaneh-Far R, Lin J, Epel ES, Harris WS, Blackburn EH, Whooley MA. JAMA. 2010 Jan 20;303(3):250-7. 10. Song Z, von Figura G, Liu Y, Kraus JM, Torrice C, Dillon P, Rudolph-Watabe M, Ju Z, Kestler HA, Sanoff H, Lenhard Rudolph. Aging Cell. 2010 Aug;9(4):607-15 11. Vera E, Bernardes de Jesus B, Foronda M, Flores JM, Blasco MA. PLoS One.2013;8(1):e53760. 12. Shin YA, Lee JH, Song W, Jun TW. Mech Ageing Dev. 2008 May;129(5):254-60. 13. Kadi F, Ponsot E, Piehl-Aulin K, Mackey A, Kjaer M, Oskarsson E, Holm L. Med Sci Sports Exerc. 2008 Jan;40(1):82-7. 14. Smith DL Jr, Mattison JA, Desmond RA, Gardner JP, Kimura M, Roth GS, Ingram DK, Allison DB, Aviv A. J Gerontol A Biol Sci Med Sci. 2011 Nov;66(11):1163-8. 15. Möller P, Mayer S, Mattfeldt T, Müller K, Wiegand P, Brüderlein S. Aging (Albany NY). 2009 Jul 14;1(8):733-9. 16. Canela A, Vera E, Klatt P, Blasco MA.Proc Natl Acad Sci U S A. 2007 Mar 27;104(13):5300-5.
  53. 53. Page 54 General references • High-throughput telomere length quantification by FISH and its application to human population studies. Canela, A., et al., Proc Natl Acad Sci, 2007. 104(13): p. 5300-5. • Telomerase in the human organism. Collins et al. Oncogene 21(4): 564-579 • The longest telomeres: a general signature of adult stem cell compartments. Flores, I., et al., Genes Dev, 2008. 22(5): p. 654-67. • The shortest telomere, not average telomere length is critical for cell viability and chromosome stability. Hemann et al. Cell 107(1): 67-77 • Obesity, cigarette smoking and telomere length in women. Valdes et al. Lancet 366(9486): 662-664 • Telomere Length predicts replicative capacity of human fibroblasts. Allsopp et al. Proc Natl Acad Sci 89(21) 10114-10118 • Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Blasco, M.A., et al., Cell, 1997. 91(1): p. 25-34. • Oxidative stress shortens telomeres, von Zglinicki. Trends Biochem Sci 27(7): 339-344 • Hepatocyte telomere shortening and senescence are general markers of human liver cirrhosis. Wiemann et al. FASEB J 16(9): 935-942 • Cellular senescence in cancer and aging. Collado et al. Cell 130(2) 223-233 • Human diseases of telomerase dysfunction: insights into tissue aging. García et al. Nucleic Acid Res. 35(22): 7406-7416
  54. 54. Page 55 Oncology • A prospective study of relative telomere length and postmenopausal breast cancer risk. De Vivo et al. Cancer Epidemiol Biomarkers Prev 18(4) 1152-1156 • Telomere dysfunction and tumour suppression: the senescence connection. Nat Rev Cancer 8(6):450- 458 • Mean leukocyte telomere length and risk of incident colorectal carcinoma in women: a prospective, nested case-control study. Lee et al. Clin Chem Lab Med 48(2): 259-262 • Telomere length, cigarette smoking and bladder cancer risk in men and women. McGrath et al. Cancer Epidemiol Biomarkers Prev 16(4): 815-819 • Telomere length in prospective and retrospective cancer case-control studies. Pooley et al. Cancer Res 70 (8): 3170-3176 • Short telomere length and breast cancer risk: a study in sister sets. Shen et al. Cancer Res 67(11): 5538-5544 • Breast cancer survival is associated with telomere length in peripheral blood cells. Svenson et al. Cancer Res 68(10): 3618-3623 • Absence of a telomere maintenance mechanism as a favourable prognostic factor in patients with osteosarcoma. Ulaner et al. Cancer Res 58(18): 4168-4172 • Telomere dysfunction: a potential cancer predisposition factor. Wu, X., et al., J Natl Cancer Inst, 2003. 95(16): p. 1211-8.
  55. 55. Page 56 Oncology (cont.) • Mean telomere length and risk of incident colorectal carcinoma: a prospective, nested case-control approach. Zee et al. Cancer Epidemiol Biomarkers Prev 18(8): 7423-7428 • Telomere dysfunction and evolution of intestinal carcinoma in mice and humans. Rudolph KL, Millard M, Bosenberg MW, DePinho RA (2001) Nat Genet 28(2): 155-159 • Telomere length is a prognostic factor in neuroblastoma. Ohali A, Avigad S, Ash S, Goshen Y, Luria D, Feinmesser M, Zaizov R, Yaniv I (2006) Cancer 107(6): 1391-1399 • Telomerase is active in normal gastrointestinal mucosa ns not up-regulated in precancerous lesions. Bachor et al. J Cancer Res Clin Oncol 125(8-9) 453-460 • Telomerase and cancer therapeutics. Harley. Nat Rev Cancer 8(3): 167-179 • Screening for bladder cancer: a perspective. Lotan et al. World J Urol 26(1): 13-18 • Telomerase therapeutics for cancer: challenges and new directions. Shay et al. Nat Rev Drug Discov 5(7):577-584 • Expression of human telomerase subunits and correlation with telomerase activity in cervical cancer. Cancer Res 58(7): 1558-1561
  56. 56. Page 57 Aging • Telomeres and human disease: ageing, cancer and beyond. Blasco, M.A., Nat Rev Genet, 2005. 6(8): p. 611-22. • Telomere length, stem cells and aging. Blasco, M.A., Nat Chem Biol, 2007. 3(10): p. 640-9. • Extension of life-span by introduction of telomerase into normal human cells. Bodnar et al. Science 279 (5349) 349-352 • Association between telomere length in blood and mortality in people aged 60 years or older. Cawthon, R.M., et al., Lancet, 2003. 361(9355): p. 393-5. • Telomerase induction in T-Cells: a cure for aging and disease? Effros. Exp Gerontol 42(5):416-420 • Accelerated telomere shortening in response to life stress. Epel et al. Proc Natl Acad Sci 101(49):17312-17315 • The rate of leukocyte telomere shortening predicts mortality from cardiovascular disease in elderly men. Epel, E.S., et al., Aging 2009. 1(1): p. 81-8. • The association between telomere length physical health, cognitive aging and mortality in non-demented older people. Harris et al. Neurosci Lett 406(3): 260-264 • The power of exercise: buffering the effect of chronic stress on telomere length. Puterman, E., et al., PLoS One. 5(5): p. e10837. • Telomeres as biomarkers for ageing and age-related diseases.Von Zglinikci et al. Curr Mol Med 5(2):197-203 • Telomerase reverse transcriptase delays aging in cancer-resistant mice. Tomas-Loba A, Flores I, Fernandez-Marcos PJ, Cayuela ML, Maraver A, Tejera A, Borras C, Matheu A, Klatt P, Flores JM, Vina J, Serrano M, Blasco MA (2008) Cell 135(4): 609-622 • Telomeres shorten during ageing of human fibroblasts. Harley CB, Futcher AB, Greider CW (1990). Nature 345(6274): 458-460 • Telomeric Length and telomerase activity vary with age in peripheral blood cells obtained from normal individuals. Iwama et al. Hum Genet 102 (4): 397-402 • Disease anticipation is associated with progressive telomere shortening in families with dyskeratosis congenita due to mutations in TERC. Vulliamy, T., et al., Nat Genet, 2004. 36(5): p. 447-9.
  57. 57. Page 58 Infertility • Characterization of telomerase activity in the human oocyte and preimplantation embryo. Wright et al. Mol Hum Reprod 7(10): 947-955 • Telomere length predicts embryo fragmentation after in vitro fertilization in women-toward a telomere theory of reproductive aging in women. Keefe DL, Franco S, Liu L, Trimarchi J, Cao B, Weitzen S, Agarwal S, Blasco MA (2005). Am J Obstet Gynecol 192(4): 1256-1260; discussion 1260-1251 • The telomere theory of reproductive senescence in women. Keefe DL, Marquard K, Liu L (2006) Curr Opin Obstet Gynecol 18(3): 280-285 • Telomere length in the new born. Okuda et al. Pediatr Res 52(3): 377-381
  58. 58. Page 59 Cardiology • White cell telomere length and risk of premature myocardial infarction. Brouilette, S., et al., Arterioscler Thromb Vasc Biol, 2003. 23(5): p. 842-6. • Telomere length inversely correlates with pulse pressure and is highly familiar. Jeanclos et al. Hypertension 36(2): 195-200 • Telomere shortening in human coronary artery diseases. Ogami et al. Arterioscler Thromb Vasc Boil 24(3):546- 550 • Telomere shortening in atherosclerosis. Samani, N.J., et al., Lancet, 2001.358(9280): p. 472-3. • Telomere length as an indicator of biological aging: the gender effect and relations with pulse pressure and pulse wave velocity. Benetos et al. Hypertension 37 (2 part 2): 381-385 Immunology • Genetic Manipulation of telomerase in HIV-specific CD8+ T Cells: enhanced antiviral functions accompany the increased proliferative potential and telomere length stabilization. Dagarag et al. J Inmmunol 173(10): 6303- 6311 • Telomerase-based pharmacologic enhancement of antiviral function of human CD8+ T lymphocytes. Fauce et al. J Immunol 181(10): 7400-7406 • Natural Product Telomerase Activator As Part of a Health Maintenance Program. Harley CB, Liu W, Blasco M, Vera E, Andrews WH, Briggs LA, Raffaele JM A Rejuvenation Res • Telomerase-based pharmacologic enhancement of antiviral function of human CD8+ T lymphocytes.Fauce SR, Jamieson BD, Chin AC, Mitsuyasu RT, Parish ST, Ng HL, Kitchen CM, Yang OO, Harley CB, Effros RB (2008) J Immunol 181(10): 7400-7406 stroke mortality, dementia and cognitive decline. Ann Neurol 60(2): 174-180
  59. 59. Page 60 Neurology • Shorter telomeres are associated with mortality in those with APOE epsilon4 and dementia. Honig et al. Ann Neurol 60(2):181-187 • Telomere shortening in T cells correlates with Alzheimer´s disease status. Panossian et al. Neurobiol Aging 24(1):77-84 • Telomere shortening and mood disorders: preliminary support for a chronic stress model of accelerated aging. Simon et al. Biol Psychiatry 60(5): 432-435 • The association between telomere length, physical health, cognitive aging and mortality in non-demented older people. Harris et al. Neurosci Lett 406(3): 260-264 Respiratory • Telomerase mutations in families with idiopathic pulmonary fibrosis. Armanios et al.New England Journal of Medicine 356 (13) 1317-1326 • Telomere shortening in smokers with and without COPD. Morla et al. Eur Respir J 27 (3): 525-528 • Adult-onset pulmonary fibrosis caused by mutations in telomerase. Tsakiri et al. Proc Natl Acad Sci 104(18): 7552-7557

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