Telomere Stabilizing Mechanisms

(1)

Dr.Klaus Holzmann 1

Telomere Stabilizing Mechanisms

Cell Immortalization and Tumorigenesis

Basic Seminar Malignant Diseases I

N094 PhD Program Malignant Diseases

N790 Dr Program Clinical Experimental Oncology N090 Dr Program Tumorbiology - Oncology

Content

Normal cell populations register the number of cell generations

separating them from their ancestors in the early embryo

Cancer cells need to become immortal in order to form tumors Cell-physiologic stresses impose a limitation on replication The proliferation of cultured cells is also limited by the telomeres

of their chromosomes

Telomeres are complex molecular structures that are not easily

replicated

Incipient cancer cells can escape crisis by expressing telomerase Telomerase plays a key role in the proliferation of human cancer

cells

Some immortalized cells can maintain telomeres without

telomerase

Telomeres play different roles in the cells of laboratory mice and

in human cells

The mechanisms underlying cancer pathogenesis in

telomerase-negative mice may also operate during the development of human tumors

(2)

Dr.Klaus Holzmann 3

Eternal Life: Cell Immortalization and

Tumorigenesis

Biologist August Weissmann (1881):

Death takes place because a worn-out tissue cannot forever renew itself, and because a capacity for increase by means of cell division is not everlasting but infinite.

-> Cancer cells must break the barrier that normally limits cell proliferative potential, so that they can successfully complete the multiple steps of tumor development.

Literature: The Biology of Cancer: RA Weinberg (2007) Chapter 10

Serial passaging of living tissue cells in

culture in vitro

(3)

Dr.Klaus Holzmann 5

Hayflick Limit

Nat Rev Mol Cell Biol. 2000 Oct;1(1):72-76.

Hayflick, his limit, and cellular ageing. Shay JW, Wright WE.

STOP after 30-60 „population doublings“ (PDs)

log(number cells harvested)-log(number cells seeded) log 2

Lifespan calculated in PDs PD =

Figure 10.4 The Biology of Cancer(© Garland Science 2007)

Loss of proliferative capacity with age

same color = inter-individual variability

-> Keratinocyte stem cells lose proliferative capacity with age

Consequences observed in vivo:

- thinning of the keratinocyte layer of the skin

- loss of the ridge architecture

(4)

Dr.Klaus Holzmann 7

Senescent cells in vitro

Pre-sensecent human fibroblast cells

Phase II – PD 20 Phase III - PD 55

Sensecent human fibroblast cells ->cease proliferation but remain viable

->express senescent-associated acidic ß-galactosidase enzyme (blue)

(5)

Dr.Klaus Holzmann 9

Cancer cells need to become immortal in

order to form tumors

Why normal cells lack immortalized growth properties?

-> anti-cancer defense mechanism model

based on facts:

- human tumors are clonal (mean all the neoplastic cells in the tumor mass descend from a common ancestral cell that underwent transformation at one point in time)

- required tumor cell population doublings to make a clinically detectable human tumor can be calculated

Figure 10.5a The Biology of Cancer(© Garland Science 2007)

Some types of normal human cells are known to pass

through 50 or 60 cycles of growth and division

before they become senescent and stop growing

Cell numbers are fare more than required to constitute

a life-threatening tumor mass

2

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(6)

Dr.Klaus Holzmann 11

->cells do not only exponentially growth but also die off

A number of defense mechanisms in the body‘s

tissues exist to make life very difficult for cancer

cells, e.g.:

-withdrawal of growth factors

-withdrawal of adequate oxygen

-ability to eliminate metabolic waste via the

vasculature

In reality 2-3 times more PD may be required

Generation-counting device?

Properties:

Cell-autonomous (intrinsic to the cell)

Must be biochemically stable over extended period of time as it

stores the past history of a cell lineage

A “counting” molecule can not work over a concentration range

of 250_{or 2}60

two regulatory mechanism were defined:

1. One measures the cumulative physiologic stress that lineages of cells experience over extended period of time and halts further proliferation once that damage exceeds a certain threshold (phase termed senescence)

2. The second mechanism measures how many replicative generations a cell lineage has passed through and alarms if the allowed quota of replicative doublings has been used up (phase termed crisis)

(7)

Induction of tumor suppressor proteins

during in vitro culture

Adult human endometrial fibroblasts

PD3+ectopic p16

PD 3 PD 43

yellow: focal contacts orange: actin stress fibers

INK4a/ARF Locus

Curr Opin Genet Dev. 2002 Feb;12(1):98-104.

Immortalisation and transformation revisited. Drayton S, Peters G.

(8)

Cell-physiologic stresses impose a

limitation on replication

Human diploid fibroblasts Human epithelial cells

(foreskin keratinocytes)

Role of large T antigen in circumventing

senescence

(9)

Evidence of senescent cells in living

tissues

Figure 10.9c and suppl. Sidebar 14 The Biology of Cancer(© Garland Science 2007)

Acidic β-galactosidase (SAβ-gal) enzyme staining (blue) of lung carcinoma tissue from patient treated with the drugs carboplatin and taxol

2 covalent alterations of histones exist in senescent cells but not in cells in the G0, quiescent state:

1. SAHF(senescence-associated

heterochromatic foci) detected by antibodies against histone H3 methylated on lysine 9 residue

2. γ-H2AX foci(greenspots) specific for dsDNA breaks after X-irradiation (disappear after repair) and senescence cells (persist)

% of human fibroblast cells β-gal-positive

2 8 58 95

17 24 30 32 passage number

The proliferation of cultured cells is also limited

by the telomeres of their chromosomes

Telomeres (green) detected by fluorescence in situ hybridisation (FISH) in human cells trapped in metaphase (using a microtubule antagonist), chromatids (red)

(10)

Telomere function

Figure 10.11b and 10.14a The Biology of Cancer(© Garland Science 2007)

Chromosomes that have lost functional telomeres at their ends soon fuse, end-to-end, with one another.

One key protein in

maintaining normal telomere structure, TRF2, was removed and resulted in formation of cells with virtually all the chromosomes fused into one giant one

Shortening of telomeric DNA in concert with

cell proliferation

each passage represented 3-6 PDs, loss of telomeric DNA can be calculated to be between 50 and 100 base pairs per cell generation

(11)

Shortening of telomeric DNA in concert with

cell proliferation

Figure 10.13b The Biology of Cancer(© Garland Science 2007)

Telomeres - Structure

(12)

Lassos at the ends of chromosomal DNA

Structure of the T-Loop

(13)

Structure of the T-Loop

Figure 10.17c The Biology of Cancer(© Garland Science 2007)

Multiple telomer-specific proteins bound to telomeric DNA

(14)

How telomeres are replicated?

All DNA synthesis occurs in 5’-to-3’ direction

RNA primers will be degraded -> leading strand is under-replicated (8-12 nt) Why lose of 50-100bp per cell generation? Exonucleases involved

(8-12 nt)

End-replication problem = generation-counting device!

Incipient cancer cells can escape crisis by

expressing the enzyme telomerase

Nat Rev Cancer. 2002 May;2(5):331-41.

Modelling the molecular circuitry of cancer. Hahn WC, Weinberg RA.

(15)

Signals from short telomeres

J Clin Oncol. 2003 May 15;21(10):2034-43.

Role of telomeres and telomerase in the pathogenesis of human cancer. Hahn WC.

Telomerase Activity – TRAP Assay

(16)

The catalytic subunit of telomerase

Enzyme first described 1985 by Carol W. Greiderand Elizabeth H. Blackburn Identification of a specific telomere terminal transferase activity in tetrahymena extracts.In: Cell (1985) 43:405.

Cloning History:

1996 p123from the ciliate E.aediculatus(PNAS 93:10712)

1997 Est2(ever-shorter telomeres) from S.cerevisiae(PNAS 94:9202) 1997 hTERTfrom H.sapiens(Science 277:955 and Cell 90:785)

Structure and function of the human telomerase

holoenzyme

hTERT 127kDa hTR 451nt

(17)

Telomerase gene expression

Nucleic Acids Res. 2002 Feb 15;30(4):839-65.

Natural and pharmacological regulation of telomerase. Mergny JL et al.

Prevention of crisis by expression of telomerase

(18)

Suppression of telomerase activity -> Restoration of Crisis

DNhTERT expression results in loss of the neoplastic growth program in 4 different human cancer cell lines

DNhTERT expressing cells continue proliferation until the reach crisis

Telomerase activity and the prognosis of

pediatric tumors

(19)

Some immortalized cells can maintain

telomeres without telomerase

85-90% of human tumors are telomerase positive, so 10-15% lack detectable telomerase activity but need to maintain their telomeres above some minimum length in order to proliferate indefinitely.

ALT (alternative lengthening of telomeres) is telomerase independent; associated preferentially with ostesarcomas, soft-tissue sarcomas and glioblastomas

FISH:in yellow telomere specific probe TRF: ALT cell line

crises

Nat Rev Cancer. 2002 Nov;2(11):879-84.Telomere maintenance and cancer -- look, no telomerase. Neumann AA, Reddel RR.

ALT -> heterogeneous telomere length

The ALT mechanism

Telomere sequence information exchanged between chromosomes in ALT cells -> interchromosomal recombination involved

(20)

ALT model – copy choice mechanism

Nat Rev Cancer. 2002 Nov;2(11):879-84. Telomere maintenance and cancer -- look, no telomerase. Neumann AA, Reddel RR.

Der DNA Strang eines kurzen Telomers dient als Primer für die DNA Synthese bei einem anderen Telomer.

->Nettogewinn an Telomerlänge

Gleiches Telomer, funktioniert über die T-Loop-Struktur

„rolling circle“ wurde in der Hefe funktionell gezeigt, Möglichkeit zur fast unbegrenzte Verlängerung der Telomere

Extrachromosomale DNA Elemente (circle, linear) existieren in ALT-Zellen,

Telomeres play different roles in the cells of

laboratory mice and in human cells

Erosion of telomeres over multiple generations in populations of mTR-/- mice

The onset of human genetic disease with telomeres involved (e.g. X-linked form of dyskeratosis congenita ) is happen already in the first generation

(21)

Telomerase-negative mice show both decreased

and increased cancer susceptibility

Rate of tumor formation in cancer-prone mTR-/- p53-/- mice mTR-/- p16INK4A_/p19ARF_{-/- mice: reduced}_{rate of cancer} mTR-/- p53-/- mice: increasedrate of cancer

Mechanistic model of how breakage-fusion-bridge

(BFB) cycles promote human carcinoma formation

(22)

Main concepts – Take Home Message

Two barriers prevent cultured cells from replicating indefinitely in culture

(senescenceand crisis)

Senescence involves the long term residence of cells in a non growing but

viable state

Crisis involves the apoptotic death of cells

Senescence is provoked by physiological stresses that cells experience in vitro

(in vivo not yet clear)

Crisis is provoked by the erosion of telomeres, which result in widespread

end-to-end chromosomal fusions, karyotypic chaos, and cell death

Most pre-malignant cells escape from crisis by activating of telomerase (hTERT)

and elongation of telomeric DNA

Some cancer cells escape crisis by regenerating their telomeric DNA through

the ALT mechanism

Cells that have stabilized their telomeres through hTERT or ALT can than

proliferate indefinitely and are therefore said to be immortalized

Cell immortalization is a step that appears to govern the development of all

human cancers

The end-to-end chromosmal fusionsthat accompany crisis appear in turn to be

responsible for much of the aneuploidyassociated with the karytypes of many kinds of solid human tumors

Hahn, W. C. J Clin Oncol; 21:2034-2043 2003

(23)

Recent Findings

The TERT–β-catenin connection. a, The telomerase complex functions in progenitor cells to repair chromosome ends, known as telomeres, during cell division. TERT provides reverse transcriptase activity to the complex, and uses TERC, the RNA component of telomerase, as a template. b, Park et al.1 find that TERT also increases the transcriptional activity of β-catenin/TCF complexes through interaction with BRG1, a factor that binds the Wnt signalling molecule β-catenin and alters the conformation of chromatin. These two separate functions of TERT may simultaneously prevent cellular senescence and increase proliferation of progenitor cells, permitting embryonic development and renewal of adult tissues. (from Millar 2009 The not-so-odd couple. Nature, 460:44)

TERRA/TelRNAs associate to telomeric chromatin and may be involved in regulation of telomere length.Model for a role of telomeric RNAs in the regulation of telomere length. TelRNAs are potent inhibitors of telomerase activity in vitro, possibly by forming of RNA:RNA hybrids with the template region of the telomerase RNA component (Terc). (from Schoeftner and Blasco 2009 Chromatin regulation and non-coding RNAs at mammalian telomeres. Seminars in Cell & Developmental Biology. Online)