Telomere, Telomerase, and Senescence

Telomeres mark ends

to prevent DS breaks

Cells that divide rapidly

Keeps telomerase fully active, eg bone marrow or gut lining

How telomerase adds telomeres

Bind to template strands in direction of DNA synthesis at 3’ end and adds additional telomere repeat

p arm

petite, small arm

q arm

longer arm on chromosomes

Telomerase is a

RNA dependent DNA polymerase

Telomerase ribonucleoprotein contains

TERT and TERC

Telomerase decreases

Telomeres shorten

Senescence cell staining

Sa-Beta-Gal — blue staining

Cellular senescence marker

p16lnk4a, related to Baker

Senescence bypassed by

TERT expression in fibroblast, stabilizes telomeres

TERT

catalytic subunit of telomerase

TERC

ncRNA that serves as RNA template for telomere replication. Reverse transcription

hTERT activity

Usually silenced in almost all somatic cells, significantly expressed in 90% of human cancers

hTERT (Human telomere reverse transcriptase)

Hahn found it was involved with human tumor; more difficult to convert human normal genes into tumorigenic with just oncogenes

hTERT increases

Telomerase activity increases

Large T and Ras protein

Seen throuh immunoblotting

hTERT expression by

RT-PCR

Telomere length

Southern blotting

Telomerase activity measured using

TRAP assay (Telomeric Repeated Amplification Protocol)

Blackburn, Greider, Szostak

How chromosomes are protected by telomeres

Telomerase necessary for cancer

Prevents shortening of telomeres — uncontrolled cell proliferation

Spaceflight conditions

Confinement, isolation

Exposure to microgravity, infrared radiation, noise

Garett Bakelman study on Nasa twins

Telomere length assessed by qRT-PCR for HR and TW

Increased number of short telomeres

Increased telomere length in CD8, CD4, LD, but not CD19

PBMC

Mononuclear blood cells: lymphocytes (T, B, NK cells), monocytes, dendritic cells

NOT WBCS (granulated leukocytes)

Polymorphonuclears WBCs

Granulocytes like neutrophils

Buffy coat

Contains platelets and WBCs

PBMC isolation

Uses density gradient separation (Ficoll or Histopaque)

Macrophages

Monocytes that have migrated from blood stream to body tissue

CD4+ Cells

Helper T cells, 25-60% of PBMCs, Cd3+CD4+

CD8+

Killer T cells, 5-30%, CD3+CD8+

B cells

Antibodies secreting, 5-10%, CD19+

NK cells

Trigger lysis or apoptosis, 10-30%, CD56+CD3-

Monocytes

Phagocytosis, antigen presentation, produce cytokines; 5-10% CD14+

Dendritic cells

1-2%, process and present antigen material to T cells

Density gradient centrifugation

Isolates circulating tumor cell

CTCs density

lower than blood cells