Boon and Bane of not Being Subject to the Hayflick Limit

Index

I. Introduction

II. Hayflick Limit
i. History of the Hayflick Limit
ii. The End Replication Problem
iii. Telomerase

III Cancer Cells
i. Telomeres in Age-Related Diseases
ii. Telomerase in Cancer
iii. Alternative Lengthening of Telomeres

IV Life Extension Science
i. Molecular Insights
ii. Telomerase Inhibitors
iii. Anti-Aging Industry

V Conclusion

VI. Bibliography

VII. References

I. Introduction

All living things have to die. This fundamental truth is held to apply even to the smallest unit of life – cells. However, there is a phenomenon that is sometimes called biological immortality. It refers to cells that live beyond their proclaimed life span, which is roughly set by the Hayflick limit. All cancer cells have acquired this property; they divide indefinitely, which is the essential problem with cancer cells. On the other hand, researchers are very much interested in the molecular mechanism behind this property to may be able to use it to extend life and rejuvenate cells. Cells that are not subject to the Hayflick limit are generally seen as a threat to the human body, however, they are interesting subjects of experiments and scientists have already learned a great deal of knowledge by studying these mutants and continue to gain more important insights into the functioning of any kind of human body cell. Immortal cells can be boon and bane for humankind. Certain aspects of this issue will be discussed.

II. Hayflick Limit

i. History of the Hayflick Limit

As mentioned above, normal cells are limited in their capacity to divide. This limit is called Hayflick limit, a term coined in 1974 by Sir Macfarlane Burnett^[1], for Leonard Hayflick was the one who demonstrated that cells are not able to divide indefinitely.

The idea was not new at the time; in 1881, the German biologist August Weismann proposed:

“Death takes place because a worn-out tissue cannot for ever renew itself, and because a capacity for increase by means of cell-division is not everlasting, but finite. […] Functional disturbances will appear soon as the rate at which the worn-out cells are renewed becomes slow and insufficient.”^[2]

Weismann could not prove his thesis though and it was soon to be almost entirely forgotten. By the time Hayflick performed research on cell culturing, Weismann’s concept had been overturned by Alexis Carrel’s hypothesis that every human cell in culture can proliferate indefinitely if provided with the correct medium and nutrition. He based this proposition on his experiments with chicken heart fibroblasts he supposedly grew in culture for more than 20 years^[3], which is far more than a normal chicken’s lifespan.

In 1958, Leonard Hayflick commenced research into the possible viral etiology of cancer. However, he soon came across certain difficulties. Initially, he intended to expose normal human body cells to cancer cell extracts, to hopefully observe cancer-like changes in the normal cells, but the normal cells died soon. According to the generally accepted thesis of Carrel, Hayflick sought the problem in his experimental procedure and set-up. He started a carefully conducted series of experiments over three years, which eventually convinced him that his experiments did not fail due to a technical mistake.1 In 1961, Hayflick and the cytogeneticist Paul Moorehead set up further experiments. They mixed equal amounts of normal human male fibroblasts at about their fortieth population doubling with normal human female fibroblasts at about their tenth population doubling. Unmixed controls of the two populations were kept. When the “old”, male control group stopped dividing, the mixed culture was examined. In fact, there were only female fibroblasts remained. This proved that the “old” cells stopped dividing even in the presence of “younger” cells, which continued to replicate and therefore a technical error or viral contamination were rather improbable explanations as to why only the male cells stopped dividing and died.^[4]

Other experiments showed that cells even “remembered” their age, after they had been frozen for some time, and stopped dividing after around 50 population doublings (human fibroblasts)5 regardless of the age they were at when they were frozen.1 The numbers differ greatly between species and individuals; most importantly, these observations indicate that there is some molecular mechanism which “counts” the divisions a cell completed and stops the replicative process as soon as the Hayflick limit is reached.^[5] This leads to another question: how does the Hayflick limit function on the molecular level?

ii. The End Replication Problem

Key to this question is the characteristic of the Hayflick limit to be determined by the number of divisions, not by time. Therefore the answer must be connected to the replication process of a cell, mitosis. The whole genome of the cell must be replicated before mitosis, so each new daughter cell can have the exact copy of genetic material.^[6] DNA is replicated in a semiconservative manner. Each of the two DNA-strands functions as a template for a new complementary strand, which leaves the cell with two exact copies of the same double-stranded DNA.7

DNA replication is a complex multistep process. Initially, initiation proteins locate particular points of the DNA known as origins of replication. An enzyme called helicase unwinds and splits the two strands at these sites and replication forks form in both directions. The forks consist each of a leading strand and a lagging strand. The leading strand is synthesized continuously. The enzyme primase assembles a short RNA primer to which a DNA polymerase attaches new nucleotides in 5’à3’ direction, whereat the existing DNA strand serves as a template for the new, complementary strand. Another enzyme named RNase H removes the RNA primers and DNA polymerase fills in the gap. Finally, all DNA fragments are joined together by the enzyme ligase to form a stable new strand.^[7]

The process of synthesizing a complementary strand to the lagging strand is a little more complicated. DNA polymerase can add nucleotides to a nucleic acid only to its 3’-end. Therefore the lagging strand must be synthesized discontinuously. Primase has to synthesize primers within short distances. The DNA polymerase starts adding nucleotides to the first primer until it reaches the next primer. Doing so, it creates fragments of complementary DNA, called Okazaki fragments, between two primers. Afterwards the enzyme skips ahead to the next primer upstream and assembles a new Okazaki fragment. The lagging strand is synthesized fragment by fragment and again RNase H degrades the primers, DNA polymerase fills in the gaps and ligase joins all fragments together. This process works flawlessly with circular DNA;^[8] however, eukaryotic cells have linear chromosomes. This offers an advantage in recombination and enhances genetic diversity, but it also poses a new problem to the cell. Since DNA polymerase can add nucleotides only to existing nucleic acids and only in 5’à3’ direction, it cannot replicate the very ends of linear chromosomes. Even if the last primer would line up perfectly with the chromosome end, the primer would eventually be degraded and, again, leave a single-stranded 3’-end exposed. This problem is termed the end replication problem.^[9]

This is an issue concerning chromosome stability and DNA erosion.44 The terminal overhang resembles a double-strand break and could be recognized by the cell’s repair mechanisms as such, which could lead to chromosome aberration and eventually to apoptosis.8 The unstable ends might fuse with other ends, amid another chromosome or even with the nuclear membrane and form miscellaneous, unfavorable chromosome forms. Besides, the end replication problem causes the DNA to shorten with every replication cycle, since not all of it can be replicated. This erosion might lead to the loss of gene function or mutations.^[10]

To prevent these problems, linear chromosomes have repetitive non-coding sequences at every end. In human beings, the sequence is 5’-TTAGGG-3’.7 The existence of such non-coding sequences at the tips of chromosomes had been known since 1938; however no one could plausibly explain what they were good for. One of the involved geneticists, Hermann J. Muller, coined the term telomeres for these terminal regions from the Greek telos “end” and menos “part”. These “end parts” have varying lengths in different species and individuals.1 Human beings’ telomeres are on average about 5,000-15,000 bp long and are shortened by approximately 100 bp with every cell cycle.5 These non-coding regions can safely be shortened during every cell replication without functional losses of the genome. Also, it has been shown that telomeres are different from normal linear nucleic acid ends. Most of the telomere region is double-stranded, but it ends in a long 3’ one-stranded overhang, which forms a loop resembling a knot, which is called t-loop. This part may also develop four-stranded formations called G quadruplexes. They make telomeres distinguishable from double-strand breaks.8

The functions telomeres fulfill are vital to the cell and repetitive sequences of DNA alone are not enough to maintain chromosomal stability. In fact, telomeres are nucleoprotein complexes consisting of the mentioned repetitive sequence and associated proteins. The telomeric protein complex and its associating molecules have not yet been fully deciphered.9 In humans, a few proteins like TRF1/TRF2, POT1/POT2, and TIN1/TIN2 are known to be sequence specific proteins binding to 5’-TTAGGG-3’. They are referred to as shelterin complex, because their functions include protecting the protrusions from fusion, chemical modifications and nucleases, holding the t-loops and quadruplexes together, as well as regulating telomere length.^[11]

Even if telomeres fulfill their tasks perfectly, the cell will at some point reach senescence, because telomeres are not a remedy for the end replication problem, but only a deferment.1 When the telomeres are too short to sustain chromosomal stability, a cryptic splice site is activated leading to a mutant form of the lamin A protein called progerin.^[12] This means that the RNA transcript for the protein is spliced in an alternative way, which is also actively involved in the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Lamin A is a structural protein in the nucleus. Progerin is a pared version of lamin A and thus cannot fulfill its tasks properly, which leads to a deformed nucleus. Short telomeres are thought to yield further alternative splicing in multiple other genes collectively causing the cell to stop dividing.12 This state is referred to as senescence and leads eventually to cell death. However, there remains a slight chance for the cell to overcome this crisis, if it manages to elongate its telomeres and overcome the Hayflick limit.^[13]

iii. Telomerase

Telomeres are found in every human body cell. However, they are longest in germ line cells.7 This is necessary, because human beings develop from one single zygote, which is subject to numerous cell divisions to form a new body. This would not be possible, if its telomeres shortened with every cell cycle and the cell soon reached the Hayflick limit. The natural remedy to this problem was discovered by Carol W. Greider, Elizabeth H. Blackburn and Jack W. Szostak, who received the 2009 Nobel Prize in Physiology or Medicine for their findings. They proved the existence of an enzyme called telomerase.^[14] It is a ribonucleoprotein reverse transcriptase, which is able to elongate telomeres.8

Telomerase consists of a RNA subunit and a protein subunit. The protein compound called TERT surrounds the RNA subunit called TR.^[15] TR is 11 bp long and complementary to the telomeric sequence: 3’-CAAUCCCAAUC-5’. It serves as a template to add new repeats to the existing telomeric DNA strand.8 The single-stranded end of a telomere ends in 5’-TTAGGGTTAG-3’. hTR (human telomerase RNA) bonds to the existing DNA beginning from the last G of the last complete TTAGGG sequence and then adds six new nucleotides, 5’-GGTTAG-3’, by serving as a template for DNA-polymerase. TERT acts as a catalyst in this reaction and shields it from chemical disturbances.15

Telomere lengthening through telomerase is highly regulated.^[16] First, the constituting components have to be assembled and brought together. The latter happens in Cajal bodies, sub-organelles, which shelter and transport telomerase to telomeres. These telomerase-containing Cajal bodies can be recruited to the telomere sites during S phase of the cell cycle, when the whole genome is replicated and telomeres have to be uncapped.^[17] The process of how telomeres and the telomerase in the Cajal bodies are brought together is incompletely understood. Recent studies suggest a participation of the Homeobox binding protein 1 (HOT1). HOT1 binds to double-stranded telomere regions as well as to the outer layer of Cajal bodies and brings these two into close proximity. Furthermore, experiments with mice showed that HOT1 is most likely necessary for Cajal bodies to bind to chromatin.17 Still, this model needs further testing and refinement to fully understand this complex regulatory pathway.

Telomerase is not only expressed in germ line cells, but also in tissues with high turnover.15 These types of cells are referred to as telomerase positive cells and include white blood cells, testis tissue, intestinal villi, bone marrow, skin cells, pulmonary tissue and lymph nodes.8 The activity of telomerase in these cells is considerably lower than in germ line cells, thus it is not sufficient to elongate telomeres, but it slows the degradation.16 Obviously, telomerase is crucial to life, but reactivation of telomerase in somatic cells could enable them to circumvent the Hayflick limit with disastrous consequences.

III Cancer Cells

i. Telomeres in Age-Related Diseases

Just as Weismann suggested in 1881, age-related diseases suggest themselves to be associated with shortened telomeres, which cause cells to become senescent so “worn-out tissue cannot […] renew itself”.2 Prof. Carol Greider et al. performed further experiments with mice to show the correlation of age-related diseases and the absence of telomerase which furthermore implies the correlation with shortened telomeres, too.^[18] They knocked out the genes for telomerase in a generation of mice. They called this generation G1 and continued to breed the mice for a couple of generations. Analyses made evident that short telomeres accumulated continuously with progressive generations. Also, the telomeres seemed to lose their functions and chromosome fusions became common. Strikingly, cellular senescence and apoptosis became only from the G4 generation on a wide spread phenomenon that lead to tissue deformation. Therefore it must truly be the shortened telomeres, not the absence of telomerase in general that lead to malformation.15 The hypothesis is that too short telomeres lose their protection and appear like a double-strand break to the cell, which triggers a signal transduction pathway leading to either senescence or cell death. The genotypic observations were reflected in the phenotypes, too.^[19] The knock-out mice were more prone to age-related degenerative diseases in general, more often infertile, showed reduced cellularity in e.g. bone marrow and spleen tissue, had less B and T lymphocytes, and experienced high rates of cancer.15 The later the generation, the shorter was the life expectancy. Prof. Greider and Cal Harley also analyzed the telomeres of white blood cells from people of different age and found a progressive decline of telomere length with years.^[20] This confirms that even telomerase positive cells can show telomere shortening, if the loss of telomeric DNA is greater than the expression of telomerase.

Extremely short telomeres may also lead to the loss of tumor suppressor mechanisms, because they forfeit their protecting and stabilizing functions.15 This in effect promotes disorder in the cell45 and abets the worst case scenario: cancer.

ii. Telomerase in Cancer

Cancer is a group of diseases marked by uncontrollable cell growth, most of the time in forms of malignant tumors.10 In 2000, two cancer researchers, Douglas Hanahan and Robert Weinberg, attempted to define distinct capabilities of cancerous cells. They found six hallmarks of malignant tumors.

In order to become a malignant cancer, a cell has to be able to:

1. divide independently from external growth factors
2. ignore external tumor suppressor signals
3. avoid apoptosis
4. divide indefinitely without senescence
5. stimulate stained angiogenesis
6. invade tissue and establish distant secondary tumors

[...]

^[1] Shay, Jerry W., and Woodring E. Wright. "Hayflick, His Limit, and Cellular Ageing." Nature Oct. 2000. 72-76. Web. 01 Oct. 2013.http://67-20-95-176.bluehost.com/Hayflick.NatureNotImmortal.pdf.

^[2] Weismann, August. Trans. Arthur E Shipley. "The Duration of Life, 1881." Essays upon Heredity and Kindered Biological Problems. Trans. Arthur E Shipley. Ed. Edward B Poulton and Selmar Schönland. Oxford: Clarendon Press, 1889.

^[3] Skloot, Rebecca. The Immortal Life of Henrietta Lacks. New York: Broadway Paperbacks, 2010. Print.

^[4] De Magalhães, João P. "The Hayflick Limit." Senescence.info. N.p., 1997. Web. 19 Oct. 2013. http://www.senescence.info/cell_aging.html.

^[5] Ganten, Detlev, Klaus Ruckpaul, and Antonio Ruiz-Torres. Molekularmedizinische Grundlagen Von Altersspezifischen Krankheiten. Berlin, Heidelberg: Springer Verlag, 2004. Print

^[6] Grundmann, Ekkehard. Das Ist Krebs. München: W. Zuckschwerdt Verlag GmbH, 2007. Print

^[7] Campbell, Neil A., and Jane B. Reece. "Die Molekularen Grundlagen Der Vererbung." Biologie. München: Pearson Studium, 2009. 409-434. Print.

^[8] Pecorino, Lauren. Molecular Biology of Cancer. Oxford, UK: Oxford University Press, 2012. Print.

^[9] Read, Andrew, and Dian Donnai. New Clinical Genetics. Banbury, UK: Scion Publishing Ltd, 2011. Print.

^[10] Bronchud, Miguel H., Mary Ann Foote, William P. Peters, and Murray O. Robinson, eds. Principles of Molecular Oncology. Totowa, New Jersey: Humana Press, 2000. Print.

^[11] Xing, Huawei, Dan Liu, and Zhou Songyang. "The Telosome/Shelterin Complex and Its Functions." Genome Biology 9.9 (2008): N. pag. Web. 16 Nov. 2013. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2592706/pdf/gb-2008-9-9-232.pdf.

^[12] Cao, Kan, Cecilia D. Blair, Dina A. Faddah, Julia E. Kieckhaefer, Michelle Olive, Michael R. Erdos, Elizabeth G. Nabel, and Francis S. Collins. "Progerin and Telomere Dysfunction Collaborate to Trigger Cellular Senescence in Normal Human Fibroblasts." The Journal of Clinical Investigation 121.7 (2011): 2833-2844. Print.

^[13] Halvorsen, Tanya L., Gil Leibowitz, and Fred Levine. "Telomerase Activity Is Sufficient to Allow Transformed Cells to Escape from Crisis." N.p., American Society For Microbiology, 23 Nov. 1998. Web. 29 Oct. 2013. http://mcb.asm.org/content/19/3/1864.full.

^[14] "The Nobel Prize in Physiology or Medicine 2009". Nobelprize.org. Nobel Media AB 2013. Web. 28 Dec 2013. http://www.nobelprize.org/nobel_prizes/medicine/laureates/2009/>

^[15] Nihvcast. “Telomerase and the Consequences of Telomere Dysfunction”. Youtube. Youtube, LLC. 07 Dec 2010. Web. 14 Sep 2013. http://www.youtube.com/watch?v=fIelXeAFIO0.

^[16] Meyerson, Matthew. "Role of Telomerase in Normal and Cancer Cells." Journal Of Clinical Oncology. American Society Of Clinical Oncology, 1 July 2000. Web. 29 Nov. 2013. http://jco.ascopubs.org/content/18/13/2626.full.

^[17] Tarsounas, Magdalena. "It's Getting HOT at Telomeres." The EMBO Journal 32.12 (2013): 1655-1657. Print.

^[18] Wynford-Thomas, David, and David Kipling. "Cancer and the Knockout Mouse." Nature 389(1997): 551f. Print.

^[19] "Video Player". Nobelprize.org. Nobel Media AB 2013. Web. 28 Dec 2013. http://www.nobelprize.org/mediaplayer/index.php?id=1214.

^[20] Krupp, Guido, and Reza Parwaresch. Telomerases, Telomeres and Cancer. New York City, New York, USA: Kluwer Academic/ Plenum Publishers, 2002. Print.