Immortalisasi sel dan tumorigenesis

I MMORTALISASI SEL DAN

TUMORIGENESIS

1



Dalam kultur sel/secara in vivo

 Ada sel yang masuk ke

stadium senescence sederhana/

replicative senescence



Sel senescence  metabolisme aktif tapi tidak dapat masuk kembali ke dalam siklus sel

2

Kapasitas pembelahan sel pada organisme menurun dengan bertambah- nya umur seseorang



Mekanisme penghitungan jumlah sel dalam tubuh : cell autonomous 



intrinsik sel dan tidak dipengaruhi oleh interaksi antar sel dengan lingkungan dan dengan tubuh secara keseluruhan



~ “generational clock” bergantung pada molekul intrasel :

Disintesis pada awal tahapan perkembangan (developmental stage) dan tidak disintesis sesudah stadium perkembangan

Terdapat dalam konsentrasi yang tinggi dalam sel embrio

Mengalami pengenceran dengan faktor 2 kali pada keturunannyahal ini yang mungkin menyebabkan senescence karena senyawa tersebut berada di bawah nilai ambang

P

EMBATAS REPLIKASI SEL

: S

TRESS FISIOLOGIS PADA SEL



Replikasi sel dipengaruhi :



Kadar oksigen

Kadar oksigen yang rendahpeningkatan replikasi sel

Kadar oksigen yang tinggiKemungkinan

terakumulasinya kerusakan oksidatifsenescence  bentuk guanin teroksidasi 4 x lipat ; ROS



Feeder layer (kultur sel)  akibat CDK inhibitor

Level CDK inhibitor : p16INK4A dan p21Cip1 meningkat pada kultur sel di atas plastiksenescence

5

P EMBATAS PROLIFERASI : TELOMER

6

 DNA linear tidak stabil

 DNA linear yang ditransfeksikan ke dalam selberfusi dengan DNA genom dengan bantuan nuklease dan ligase

 Telomer

 pada ujung kromosommemungkinkan DNA linear stabil dari kerja enzim

 Mencegah fusi ujung-ujung DNA dari kromosommencegah fusi kromosom

 Barbara McClintock : kromosom yang kehilangan telomerfusi

pembentukan megakromosom yang memiliki 2 atau lebih sentromer

7



Telomer :



Terdiri atas : hexanukleotida 5’-TTAGGG-3’, yang berulang sampai 1000 kali dan tersusun secara tandem



Memendek pada

keturunan sel

berikutnya dalam

siklus sel



jadi

tidak melindungi

kromosom lagi

T ELOMER



Terdiri dari untai kaya G & untai kaya C



Untai kaya G jauh lebih panjang  T-loop  bantu lindungi ujung DNA linear

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Struktur T-loop



DNA Telomer berikatan dengan protein-protein :



Pengontrol panjang telomer



Pelindung telomer

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Telomerase

3 komponen telomerase

• Telomerase reverse transcriptase (TERT)

• Telomerase-associated protein 1 (TEP1) – regulatory function (? not known for sure)

• Telomerase RNA subunit

Pemendekan Telomer  berkaitan dengan masalah replikasi ujung DNA

11

12



Beberapa sel immortal dapat memelihara telomer tanpa bantuan telomerase  10-15%



Gunakan mekanisme ALT (alternative lengthening of telomer



Terjadi pergantian telomer



tergantung pada mekanisme tipe interkromosom copy choice 

Gunakan polimerase biasa untuk perpanjang telomer

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F UNCTION OF TELOMERASE AND CONSEQUENCE OF ITS ACTIVATION

Function Consequence examples of

telomerase activation

Elongation of telomeres Elongation of cellular lifespan or immortalization

Maintenance of chromosomal

structure Telomerase is transiently expressed

in each S phase in normal cells Addition of malignant potential Tumor formation with

nontumorigenic ALT cells Promotion of stem cell proliferation e.g. increased hair growth

DNA repair? Required to form DNA damage foci

following irradiation

Self-renewal capacity Required to reprogram fibroblasts to iPS cells

14



Sel pada tubuh manusia mengalami mitosis 10

¹⁶

, mencit 10

¹¹

 resiko manusia terkena kanker lebih tinggi

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Regulation of telomerase in the hierarchy of the normal and leukemic 16

hematopoietic stem cell

Douglas Hanahan & Robert A. Weinberg, The hallmarks of cancer,

Cell 100:57–70, 2000.

Besides losing the ability to correctly determine if their environment is appropriate for division & the characteristics necessary for the immune system to remove them as damaged cells, cells on the path to tumor & cancer formation also gain several capacities: they evade apoptosis, produce their own growth factors, become insensitive to growth

suppressors & cell contact signals, gain telomerase activity

& overcome the Hayflick limit, &

express angiogenic factors &

molecules needed during metastasis.

lung fibroblast 55 times, heart 26, kidney 40, and skin 43

S EL KANKER IMMORTAL

Peningkatan jumlah sel pada kanker secara teoritis

Kultur sel – 50-60 doubling

60 cells doubling 10¹⁸sel = 10⁹cm³= 10⁶kg (theoretically)



Pada kanker, tidak semua sel survive, ada sel-sel apoptosis

T ELOMERASE

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Carcinogenesis and immortalization of human cells in vivo. Normal somatic cells, even stem cells or lymphocytes that have a capacity of telomerase activation upon proliferation, cannot be immortalized in vivo.

On the contrary, once telomerase activation occurs in cancer cells, it is usually irreversible and such cancer cells are easily immortalized. Whereas key genes responsible for cellular transformation are heterogenous among the individuals, those for cellular immortalization are considered to be relatively monotonous, mostly ‘‘telomerase’’ except for ALT (alternative lengthening of telomeres) or other rare events

22

T

ELOMERASE AND POSSIBLE CAUSES TO ITS ACTIVITY IN CANCER



p53 inactivation



C-myc expression



Steroid hormones

www.biocarta.com/pathfiles/h_tertPathway.gif



Sel kanker mengekspresikan telomerase  crisis dapat diatasi



Sel embrionik  diferensiasi

 telomerase menurun

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Telomere biology in CML model: Biological properties of CML cells in three hematologically different stages (normal, chronic phase, and blastic phase) are shown in the triangle. The peak telomere lengths in each stage are shown in the lower part of the figure, showing the telomere attrition and disease progression in LSC.

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T C

 Normal Cells

 little to no Telomerase activity

 Limited life span

 Exception: highly proliferative tissues

 Cancer Cells

 High telomerase Activity

 Immortalized

X Yuan, et al. 1999

H AYFLICK L IMIT AND C RISIS

William C. Hahn, 2003

Clinical significance: Cancer

or absent telomere due to progressive shortening with DNA replication

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PEMATAHAN

-

FUSI KROMOSOM

(

BREAKAGE

-

FUSION

-

BRIDGE

/ BFB

CYCLE

)

Kromosom disentrik



Pada manusia

terjadi pula BFB

cycles pada saat

fungsi p53 hilang

CLINICAL SIGNIFICANCE: CANCER

SELF-RENEWAL OF EPITHELIAL CELL POPULATION BY REPEATED CELL DIVISION Telomeres shorten and uncap

Normal p53 cell cycle checkpoint control

Normal senescent cells stop dividing

Loss of p53 and cell cycle checkpoint control

Mutant cell survives and proliferates Chromosome

fusion

Chromosome bridge Chromosome

breakage Chromosome

translocation

CHROMOSOME BREAKGE-FUSION- BRIDGE CYCLE

Massive chromosomal damage Cell dies due to

catastrophic genomic instability and

DNA damage Telomerase reactivated

Chromosomes are partially stabilized and cell survives

with many mutations CANCER



Pada sel kanker, telomerase ~ onkogen

34

35



Tidak ada telomerase  bisa menurunkan dan sekaligus meningkatkan kerentanan terhadap kanker

36

37

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Hypothetical model of telomeres and telomerase in primary and metastatic lesions of human cancer. Human cancers may be developed from

telomerase-negative normal cells (upper) and telomerase-positive normal cells, typically from normal stem cells through cancer stem cells (lower). In the former mechanism, the population of cancer cells that have activated telomerase in mutational manner increases according to tumor development through clonal selections, while in the latter, cancer cells have high

telomerase activity from an early stage

39

Transformation and immortalization of human cells in vitro. TERT alone transfection sometimes immortalizes normal fibroblasts but not normal epithelial cells. SV40 early region (SV40ER) immortalizes neither.

Although cotransfection of TERT and SV40ER can immortalize both, they do not have tumorigenicity. Addition of oncogenic ras, mutated H-ras or K-ras, makes

them genuine immortal cancer cells with tumorigenicity

40

41

42 a, Breast cancer, G5 mTERC-/- p53+/-mouse; b, squamous cell carcinoma, G6 mTERC-/-p53+/- mouse. c, Gross view of caeca from mTERC+/- p53+/- (left), G6 mTERC -/- p53+/- (middle), and G5 mTERC-/- p53+/- (right). d, Histologyof normal caecum, mTERC +/- p53+/-, shows typical colonic villi and ordered nuclei. . e, Adenomatous polyp in the caecum, G5mTERC-/- p53 +/-. Inset, glands remain round with basal nuclei. f, Caecal adenocarcinoma, G5 mTERC-/- p53+/-. Inset, disordered glands and pleiomorphic nuclei. . g,Invasive adenocarcinoma of colon, G5 mTERC-/- p53 +/-. Inset, tumour cells (t) with

a, p53-/- mice. The number of tumours identified (t) and the total number of mice (n) ineach cohort is indicated. Hatched line, G1–G2 mTERC-/-; triangles, G5–G6 mTERC-/-;

circles, G7–G8 mTERC-/-. b, p53+/- mice.

Hatched line, mTERC+/+, mTERC+/- or G1–

G2 mTERC-/-; triangles, G5–G6 mTERC-/-;

circles, G7–G8 mTERC-/- .

Karyotype chaos in cancer cells