Telomerase, Cancer and Aging

Group no. 6

MED 1 -B2

Jamee Dela Rosa

Haiezel dela Cruz

Lalaine dela Cruz

Maureen dela Cruz

Erika dela Fuente

Mariel dela Cruz

OBJECTIVES

1. Describe the physical structure of a chromosome and describe what makes up the centromere and telomeres of a chromosome

2. Briefly review the process of replication of a linear chromosome and explain what accounts for the natural shortening of the lagging strand

3. Explain Cellular Senescence, some mechanisms that accounts for it and how is it related to cancer

4. Describe the enzyme Telomerase and its structure.

5. Identify specific cell populations that express the enzyme h.

6. Discuss the mechanism involve in Telometric

lengthening and of sealing the telometric ends.

7. Explain the role of telomere binding proteins in the

regulation of telomerase function.

8. Explain the relationship between telomeres in regards

to aging and cancer.

9. Discuss the potential advantages of using anti-

telomerase agents in treating cancer cells.

CHROMOSOME

Tightly coiled DNA

Carriers of gene Unit of Heredity

2 pairs of 23 filamentous rod shape body

Present in the nucleus

Chromosomes are not visible in active nucleus, but are clearly seen during cell division

PHYSICAL STRUCTURE Parts:

CHROMATIDS

Double Stranded DNA

Long arm (q)

Short arm (p)

CENTROMERE

Joins chromatids together

TELOMERE

Found at each end of Chromatid

CENTROMERE

Part of a chromosome that links sister chromatids

Rich in adenine-thymine base pairs

Condensed regions within the chromosome that are responsible for the accurate segregation of the replicated

chromosome during mitosis and meiosis

TELOMERE

Telos - END'

Mers -PART

Short thymine-guanine sequence

5-TTAGGG-3 Sequence

Found at each end of a chromatid, which protects the end of the

chromosome from deterioration or

from fusion with neighbouring

chromosomes

DNA Replication: Review

KEY POINTS

ORIGIN is the beginning

REPLICATION FORKS are the sites at which DNA

synthesis is occurring

New chains grow 5-3

Bidirectional

Semi-conservative

ORDER OF ACTION

Unwinding proteins

Preventing the Reannealing

Primase makes RNA primer

DNA polymerase makes DNA

RNAse H removes RNA primer

DNA polymerase fills in gaps

DNA ligase joins gaps

UNWINDING

OF PROTEINS

HELICASE

Made up of 6 proteins

arranged in a ring shape

Motor proteins

Unpackage an organisms gene

PREVENTING THE

REANNEALING

SINGLE STRAND BINDING

PROTEINS

Tetramers

Attached to the post-replication fork single

strands of DNA,

preventing their

"reannealing

PRIMASE MAKES

RNA PRIMER PRIMASE

A type of RNA Polymerase

Creates a RNA Primer

Key importance in DNA Replication

DNA POLYMERASE MAKES DNA

DNA POLYMERASE

Creates DNA Molecules by assembling

nucleotides

RNAse H

REMOVES RNA

PRIMER

RNAse H

Non- Specific Endonuclease that

catalyzes the cleavage

of RNA

Removing the RNA primer

DNA POLYMERASE

FILLS IN THE GAPS

DNA LIGASE JOINS

GAPS

DNA LIGASE

A ligase that facilitates the joining of DNA strand

together by catalyzing the

formation of a

phosphodiester bond.

WHAT ACCOUNTS FOR THE NATURAL SHORTENING OF LAGGING STRAND?

During chromosome replication, the enzymes that duplicate the DNA cannot continue duplicating all the way to the end of the chromosome.

OKAZAKI FRAGMENTS

RNA Primers attached ahead on the lagging strand

CELLULAR SENESCENCE

Latin: Senescere Grow Old

It is the phase or stage in which normal cells cease to divide

Associated with a loss of telomerase activity

As human telomeres grow shorter, eventually cells reach the limit of their replicative capacity and progress into senescence

PROPERTIES OF CELLS IN CELLULAR SENESCENCE

Increase synthesis of proteases

Decrease in synthesis of pro-collagen and tissue inhibitors of metalloproteases

Irreversible growth arrest

Dramatic change in morphology

-lose original shape

-acquire distinct flattened cytoplasm

-change in nuclear structure, gene expression, protein processing and metabolism

CELLULAR SENESCENCE Today it is known that somatic cells derived from

human newborns will usually divide 80 to 90 times in culture

Whereas those from a 70-year-old are likely to divide only 20 to 30 times

When human cells that are normally capable of dividing stop reproducingor, in Hay- flick's words, become "senescent"they look different and function less efficiently than they did in youth, and after a while they die.

CELLULAR SENESCENCE: TELOMERE SHORTENING

Telomeres shorten because of the lagging strand phenomenon.

A section of telomeres is lost during each cycle of replication.

Progressively lose approximately 50-200 nucleotides during each mitotic replication.

Cellular senescence is triggered when cells acquire one or a few critically short telomeres

CELLULAR SENESCENCE: ACCUMULATION OF DAMAGE

Major cause of aging which is due to highly reactive substances containing oxygen (oxygen free radicals)

Oxygen free radicals damages the DNA

Damaged DNA accumulate mutations with fewer proliferation

CELLULAR SENESCENCE: GLYCATION

Glycation is the result of a sugar reducing molecule, such as fructose or glucose, bonding to a protein or Lipid molecule without the controlling action of an enzyme.

This reaction products [AGEs or advanced glycation end products] are irreversible and detrimental for extracellular protein function.

CELLULAR SENESCENCE: MITOCHONDRIAL DNA DAMAGE

The mitochondria have their own genetic material (mtDNA), which is distinct from the nuclear DNA in the cell.

Mitochondria produce ATP (energy) and free radical (byproducts of respiration)

Aerobic condition 4% of oxygen are metabolize by mitochondria.

In normal condition, the oxygen is reduced to produce water.

However, the oxygen is instead prematurely and incompletely reduced to give the superoxide ion.

The superoxide ion hydrogen peroxide hydroxyl radical Reactive oxygen species (ROS)

ROS leads to damage the cell membrane, structural protein and mitochondrial and nuclear DNA.

CELLULAR SENESCENCE: MITOCHONDRIAL DNA DAMAGE

CELLULAR SENESCENCE SUMMARY

TELOMERE

SHORTENING MITOCHONDRIA

ROS

CELLULAR

SENESCENCE

OXIDATIVE STRESS

OXYGEN FREE RADICALS GLYCATION

AGEs

CELLULAR SENESCENCE AND CANCER

Senescence involves p53 and pRb pathways and leads to the arrest of cell proliferation plays an important role in suppression of

emergence of cancer, although inheriting shorter telomeres probably does not protect against cancer. Why?

Because with critically shortened telomeres, further cell proliferation can be achieved by inactivation of p53 and pRb pathways.

Cells entering proliferation after inactivation of p53 and pRb pathways undergo crisis.

CELLULAR SENESCENCE AND CANCER

Usually almost all cells die when it enters crisis but rare cells emerge from crisis and immortalized through telomere elongation by either activated telomerase (becomes cancer cells)

WHAT IS TELOMERASE?

TELOMERASE

Background

Telomerase is an enzyme, discovered by Elizabeth Helen Blackburn (professor of biochemistry and biophysics at the University of California, San Francisco School of Medicine) and Carol Greider (professor of molecular biology and genetics at the Johns Hopkins University School of Medicine ) in 1985.

TELOMERASE

A ribonucleoprotein reverse transcriptase (enzyme) that synthezises telomeric DNA.

Tetrahymena in which the enzyme was first discovered.

Tetrahymena 5 TTGGGG 3

In humans 5 TTAGGG 3

TELOMERASE

is an enzyme that adds telomeric sequences to the ends of each chromosome. Unlike most enzymes, which consist entirely of protein, telomerase is a combination of a protein and an RNA.

The enzyme is a protein and RNA complex called

telomere terminal transferase, or telomerase. Telomerase is present in most fetal tissues, normal

adult male germ cells, stem cells, in proliferative cells of renewal tissues, and in most tumor cells.

TELOMERASE STRUCTURE

TELOMERASE STRUCTURE

Human telomerase is composed of at least two sub-units:

human Telomerase Reverse Transcriptase (hTERT) - protein component

human Telomerase RNA (hTR or hTERC)

- RNA Component

human Telomerase Reverse Transcriptase (hTERT)

Protein Component

The coding region of the hTERT gene is 3396bp, and translates to a protein of 1131 amino acids

The hTERT gene maps to chromosome band 5p15.33.

RNA component

referred to as the RNA guide

template region of hTR is

3'-CAAUCCCAAUC-5'

sequence complementary to telomere repeat [TTAGGG]

serve as template for telomere synthesis and elongation

human Telomerase RNA (hTR or hTERC)

MECHANISM OF TELOMERIC LENGTHENING

CAAUCCCAAUC

3

5

hRNA

hTERT

GGTTAGGGTTAGGG

CCAAUCC

3

3 5

5

TELOMERE

TELOMERASE

CAAUCCCAAUC

3

5

GGTTAGGGTTAGGG

CCAAUCC

3

3 5

5

STEP 1: BINDING

CAAUCCCAAUC

3

5

GGTTAGGGTTAGGG

CCAAUCC 3 5

5 TTAGGG TTAG

STEP 2: ELONGATION

GGTTAGGGTTAGGG

CCAAUCC 3 5

5

CAAUCCCAAUC

3

5

TTAGGG TTAG

STEP 3: TRANSLOCATION

GGTTAGGGTTAGGG

CCAAUCC 3 5

5

CAAUCCCAAUC

3

5

TTAGGG TTAG TTAGGG TTAG

STEP 4: ELONGATION

MECHANISM OF SEALING THE TELOMERIC ENDS

T-loop

HETERO-DUPLEX OR T-LOOP

It was proposed that the t-loop is formed by strand invasion of the 3' G-rich overhang into the preceding telomeric tract to form a lariat with a D-loop at the looptail junction .

Importance of T-loop

stabilizes the telomere

prevents the telomere ends from being recognized as break points by the DNA repair machinery thus it gives protection from exonucleases.

preventing the telomere from eliciting a DNA damage response manifested as cell-cycle arrest or apoptosis

Importance of T-loop

prevention of chromosome end fusions or non homologous end joining (NHEJ)

prevention of homologous recombination between telomeric regions

regulation of telomere length homeostasis.

What prevents the telomerase from over-extending the ends of a linear chromosome?

Shelterin Complex

The T-loop is held together by six known proteins:

TRF1, TRF2, POT1, TIN2, RAP 1 and TPP1,

Shelterin Complex 1. TRF 1 (Telomeric Repeat

binding Factor 1)

It binds along the length of the T-loop.

along with TRF2, it normally prevents telomerase from adding more telomere units to telomeres.

But when telomere lengthening is required, TRF1 recruits helicases to facilitate the process.

Shelterin Complex 2. TRF2 (Telomeric Repeat binding Factor 2)

It appears to promote formation of D-loop

prevents ataxia telangiectasia mutated (ATM) activation, which is a DNA damage response (DDR) to DNA double strand breaks.

But when DNA repair of telomeres is required, TRF2 recruits DNA repair proteins.

Shelterin Complex 3. TIN 2 (TRF1 Interacting

Nuclear factor 2)

links TRF1 with TRF2, and connects both to TPP1 .

TIN2 is believed to facilitate recruitment oft single-stranded telomere-binding proteins to telomeres.

TIN2 interacts with TRF1 and has been suggested to stabilize the T-loop.

Shelterin Complex 4. POT 1 (Protector Of

Telomeres 1)

only binds to the single-stranded 3-end DNA overhang.

POT1 prevents ataxia telangiectasia and Rad3 related (ATR) activation, which is a DNA damage response (DDR) to DNA double strand breaks.

Humans only have a single POT1, whereas mice have POT1a and POT1b.

Shelterin Complex 5. RAP1 (Repressor/Activator Protein 1)

binds to TRF2, and facilitates TRF2 function.

RAP1 protects telomeres from non homologous end joining (NHEJ).

Unlike the other shelterin proteins, RAP1 has functions independent of its function within the shelterin complex: RAP1 regulates transcription and affects NF- kb signaling.

Shelterin Complex

6. TPP1 (TINT1, PTOP, PIP1 POT1-TIN2 organizing protein)

interacts with POT1 and regulates its function.

When telomeres are to be lengthened, TPP1 is a central factor in recruiting telomerase to telomeres.

Deletion of TPP1 from shelterin elicits an ATR-mediated DDR.

AGING

What is aging?

Aging is a degenerative process that is

associated with progressive accumulation of

deleterious changes with time, reduction of

physiological function and increase in the

chance of disease and death.

Telomeres alone do not reduce lifespan but there are some factors that also plays an important role in aging.

According to geneticist Richard Cawthon and

the colleagues at the University of Utah, Shorter telomeres are associated with shorter lives. Among people older than 60, those with shorter telomeres were three times more likely to die from heart disease and eight times more likely to die from infectious disease.

Telomere shortening

When people are divided into two groups based on telomere length, the half with longer telomeres lives an average of five years longer than those with shorter telomeres.

Chronological Age After age 60, the risk of death doubles every 8 years. So a

68-year-old has twice the chance of dying within a year compared with a 60-year-old.

Oxidative Stress Oxidative stress is the damage to DNA, proteins, and

lipids (fats) caused by oxidants, which are highly reactive substances containing oxygen

Glycation Glycation happens when glucose, the main sugar we use

as energy, binds to some of our DNA, proteins, and lipids, leaving them unable to do their jobs.

Is it possible to revert old cells into young cells again?

Is it possible to revert old cells into young cells again?

No. Why?

Using that in humans will be more difficult because Mice make telomerase throughout their lives but the enzyme is switched off in adult humans.

Telomerase and Cancer

Telomerase is expressed in almost all human cancers but is inactive in most normal cells.

Cancer cells are malignant cells which multiply until they form a tumor that grows uncontrollably.

Telomerase is a good biomarker for cancer detection because most human cancers cells express high levels of telomerase.

Telomerase as an Agent to Extend Cellular Life Span

Telomere shortening has been observed in most dividing somatic cells, eventually leading to cell senescence when critically short telomeres are reached.

Telomerase has been identified as a ribonucleoprotein enzyme that can synthesize telomeric repeats onto chromosomes.

Telomerase can be an agent to extend cellular life span especially in cancer cells.

Telomerase as an Agent to Extend Cellular Life Span

ADVANTAGES DISADVANTAGES

If telomerase are activated in somatic cell, human cells will not

age.

If telomerase are inactivated, telomeres in cancer cells would

shorten, just like they do in normal

body cells

It will be able to mass produce cells for transplantation

If telomerase are inactivated, telomere shortening can be

anticipated.

Telomerase has been detected in human cancer cells and is found

to be 10-20 times more active

than in normal body cells

Blocking telomerase could impair fertility, wound healing and

production of blood cells and

immune cells

Anti-telomerase Agents to Treat Cancer Cells

Harley & Greider

inhibitory agent could

cause the telomeres of

cultured tumor cells to

shrink

Apoptosis

Blackburn

cells sometimes

compensate for the loss

of telomerase, repair

shorter ends for

Recombination

Potential advantages of anti-telomerase agents

SPECIFICITY

Anti-telomerase treatment would be very selective in that only cells with an activated telomerase would be affected (most normal adult tissues have NO telomerase activity).

THANK YOU!