1 molecular biology of cancer eternal life: cell immortalization and tumorigenesis

1Molecular Biology of Molecular Biology of CancerCancer

Eternal Life: Cell Eternal Life: Cell Immortalization and Immortalization and

TumorigenesisTumorigenesis


Normal cell populations register the Normal cell populations register the number of cell generations separating number of cell generations separating them from their ancestors in the early them from their ancestors in the early embryoembryoNormal cells have a limited proliferative

potential.

Cancer cells need to gain the ability to proliferate indefinitely – immortal.The immortality is a critical component of the

neoplastic growth program.


““Hayflick limit” of Hayflick limit” of Normal human Normal human cells cells (Fibroblasts) in (Fibroblasts) in monolayer monolayer

culturecultureThey possess an

intrinsically programmed limit (now known as the ‘Hayflick limit’) to their capacity for proliferation even after a substantial

healthy period of cell division, they undergo a permanent growth arrest (replicative senescence).


Cells need to become immortal Cells need to become immortal in order to form cancersin order to form cancers

Two regulatory mechanisms to govern the replicative capacity of cells:

1.Senescence: Cumulative physiologic stress over extended periods

of time halts further proliferation. These cells enter into a state of senescence. Accumulation of oxidative damage contributes to

senescence, e.g., reactive oxygen species (ROS), DNA damage

2.crisis : Cells have used up the allowed “quota” of replicative

doublings. These cells enter into a state of crisis, which leads to apoptosis.


Replicative senescence Replicative senescence in vitroin vitro

Proliferating human Proliferating human fibroblasts

Senescent cells in culture:Senescent cells in culture:•“fried egg” morphology•Remain metabolically active, but lost the ability to re-enter into the active cell cycle•The downstream signaling pathways seem to be inactivated•Senescence associated β-galactosidase (lysosomal β-D-galactosidase)


Senescence-associated β-galactosidase (SA-β-gal)

Treatment of lung cancer with chemotherapeutic drugs appear to induce senescence in tumor cells

Cell senescence does occur Cell senescence does occur in in vivovivo


Young and old keratinocytes in the skin

Keratinocyte stem cells in the skin lose proliferative capacity with increasing age.


Cancer cells and embryonic stem cells Cancer cells and embryonic stem cells share some replicative propertiesshare some replicative properties

Embryonic stem (ES) cells show unlimited replicative potential in culture and are thus immortal.

The replicative behavior of cancer cells resembles that of ES cells.

Many types of cancer cells seem able to proliferate forever when provided with proper in vitro culture conditionsHeLa cells (Henrietta Lacks, 1951):

the 1st human cell line and 1st human cancer cell linen established in culture

derived from the tissue of cervical adenocarcinoma


cell cultures derived from human cancer tissues, once successfully established in vitro, are often immortal


Cell populations in crisis show Cell populations in crisis show widespread apoptosiswidespread apoptosis


The proliferation of cultured cells is The proliferation of cultured cells is limited by the telomeres of their limited by the telomeres of their

chromosomeschromosomesBarbara McClintoch discovered (1941)

specialized structures at the ends of chromosomes, the telomeres, that protected chromosomes from end-to-end fusions.

She also demonstrated movable genetic elements in the corn genome, later called transposons

Nobel prize in Physiology & Medicine in 1983


Telomeres detected by fluorescence in situ hybridization(FISH)

telomeric DNA


The telomeres lose their protective function in cells that have been deprived of TRF2, a key protein in maintaining normal telomere structure.

In an extreme form, all the chromosomes of the cell fused into one giant chromosome.

•Telomeric repeat-binding factor •Telomeric repeat-binding factor •Telomeric repeat-binding factor •Telomeric repeat-binding factor

TRF2: Telomeric repeat-binding factor 2


2 sister chromatids during the G2 phase of the cell cycle

Mechanisms of breakage-fusion-Mechanisms of breakage-fusion-bridge cyclesbridge cycles


truncationtranslocation

aneuploidy


telomere shortening chromosomes fuse apoptotic death

the end-replication problem:the end-replication problem:Telomeric DNA shortens progressively as cells Telomeric DNA shortens progressively as cells

dividedivideAn inevitable consequence of semi-

conservative DNA replication in eukaryotic cells The free DNA ends of each chromosome are not duplicated

completely by DNA polymerase. Consequently, the ends of human chromosomes can lose up to

200 bp of DNA per cell division.


this sequence is not replicated

Primers and the initiation of DNA Primers and the initiation of DNA synthesissynthesis


Telomeric DNA:

5’-TTAGGG-3’ hexanucleotide sequence, tandemly repeated thousands of times

Telomeres are complex molecular Telomeres are complex molecular structures that are not easily replicatedstructures that are not easily replicated


Structure of the T-loopStructure of the T-loop

• The 3' DNA end at each telomere is always longer than the 5’ end with which it is paired, leaving a protruding single-stranded

• This protruding end has been shown to loop back and tuck its single stranded terminus into the duplex DNA of the telomeric repeat sequence to form a t-loop


T-loops provide the normal ends of chromosomes with a unique structure, which protects them from degradative enzymes and clearly distinguishes them from the ends of the broken DNA molecules that the cell rapidly repairs


Multiple telomere-specific proteins Multiple telomere-specific proteins bound to telomeric DNAbound to telomeric DNA

TRF: Telomeric repeat-binding factor


Cancer cells can escape crisis by Cancer cells can escape crisis by expressing telomeraseexpressing telomerase

Telomerase activity (elongate telomeric DNA)

Clearly detectable in 85 to 90% of human tumor cell samples

Present at very low levels in most types of normal human cells.

Telomerase holoenzyme:1. hTERT catalytic subunit2. hTR RNA subunit (At least 8 other subunits may exist in the

holoenzyme but have not been characterized.)


human telomerase reverse transcriptase

human telomerase-associated RNA

(template for hTERT)


Oncoproteins and tumor suppressor Oncoproteins and tumor suppressor proteins play critical roles in governingproteins play critical roles in governing

hTERThTERT expression expressionThe mechanisms that lead to the de-

repression of hTERT transcription during tumor progression in humans are complex and still quite obscure.Multiple transcription factors appear to collaborate

to activate the hTERT promoter.

For example, the Myc protein and Menin (the product of the MEN1 tumor suppressor gene), deregulate the cell clock.


Prevention of crisis by expression of telomerase

HEK: human embryonic kidney cells


The role of telomeres in The role of telomeres in replicative senescencereplicative senescence

In cultured human fibroblasts, senescence can be postponed by expressing hTERT prior to the expected time for entering replicative senescence.

However, senescence is also observed in cells that still possess quite long telomeres.

Why?


Possible explanations:Possible explanations:

When cells encounter cell-physiologic stress or the stress of tissue culture, telomeric DNA loses many of the single-stranded overhangs at the ends.

The resulting degraded telomeric ends may release a DNA damage signal, thereby provoking a p53-mediated halt in cell proliferation that is manifested as the senescent growth state


Replicative senescence and the actions of telomerase

This is a still-speculative mechanistic model of how and why telomerase expression can prevent human cells from entering into replicative senescence.


Telomerase plays a key role in Telomerase plays a key role in the proliferation of human the proliferation of human

cancer cellcancer cellExpression of antisense RNA in the

telomerase (+) HeLa cellsThey stop growing 23 to 26 days.

Expression of the dominant negative hTERT subunit in telomerase (+) human tumor cell lines:They lose all detectable telomerase activity with some delay, they enter crisis.


Suppression of telomerase results in the loss of the neoplastic growth in 4 different human cancer

cell lines(length of telomeric DNA at the onset of the experiment)


Some immortalized cells can Some immortalized cells can maintain telomeres without maintain telomeres without

telomerasetelomerase85 to 90% of human tumors have been found

to be telomerase-positive.The remaining 10 to 15% lack detectable

telomerase activity, yet they need to maintain their telomeres above some minimum length in order to proliferate indefinitely.

These cells obtain the ability to maintain their telomeric DNA using a mechanism that does not depend on the actions of telomerase.


- the vast majority of the yeast Saccharomyces cervisiae cells enter a state of crisis and die following inactivation of genes encoding subunits of the telomerase holoenzyme.

- Rare variants emerged from these populations of dying cells that used the alternative lengthening of telomerase (ALT) mechanism to construct and maintain their telomeres.

- This ALT mechanism is also used by the minority of human tumor cells that lack significant telomerase activity, e.g., 50% osteosarcomas and soft-tissue sarcomas and many glioblastomas.


The ALT (alternative lengthening of telomerase ) mechanism (or copy-choice mechanism)


neomycin-resistant gene was introduced into the midst of the telomeric DNA

Exchange of sequence information between the telomeres of different chromosomes


Telomeres play different roles in the Telomeres play different roles in the cells of laboratory mice and in cells of laboratory mice and in

human cellshuman cellsRodent cells, especially those of the laboratory

mouse strains, express significant levels of telomerase throughout life.

The double-stranded region of mouse telomeric DNA is as much as 30 to 40 kb long (~ 5 times longer than corresponding human telomeric DNA).Therefore, laboratory mice do not rely on telomere

length to limit the replicative capacity of their normal cell lineages and that telomere erosion cannot serve as a mechanism for constraining tumor development in these rodents.


Long telomeres (in mice) do not Long telomeres (in mice) do not suffice for tumor formation suffice for tumor formation

Transgenic mice expressing mTERT (mouse homolog of telomerase reverse transcriptase) contributes to tumorigenesis even though the mouse cells in which this enzyme acts already possess very long (>30 kb) telomeres.

Thus, the mTERT enzyme aids tumorigenesis through mechanisms other than simple telomere extension.


- Mouse cells can be immortalized relatively easily following extended propagation in culture.

- Human cells require, instead, the introduction of both the SV40 large T oncogene (to avoid senescence) and the hTERT gene (to avoid crisis).


SV40 and T antigensSV40 and T antigens

If the SV40 large T oncoprotein is expressed in human fibroblasts, these cells will continue to replicate another 10 to 20 cell generations and then enter crisis.

On rare occasion, a small propotion of cells (1 out of 106 cells) will proceed to proliferate and continue to do indefinitely → becoming immortalized.


SV40: the 40th simian virus in a series of isolates

papovavirus: papilloma, polyoma & vacuolating agent


SV40 large T antigen can circumvent senescence

HEK: human embryonic kidney cells

1 molecular biology of cancer eternal life: cell immortalization and tumorigenesis

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types of cancer cells

tumor cells cell senescence

replicative capacity

human cancer cell linen

human cancer tissues

embryonic stem cells

immortal slide

galactosidase slide