Radiobiology
(HTI 27103)
Cellular effects
Department of Health Technology and Informatics
Liang-Ting Lin
Intended learning outcomes
• After this class, you should be able to
‐ Describe the DNA damage events
‐ Understand the different processes of DNA damage repair
‐ Tell the consequence of mis-repaired DNA damage
2
Hereditary concerns - radiation
1. It is a commonly held view that radiation produces bizarre mutants and monsters.
2. Radiation does not result hereditary effects that are new or unique but rather increases the
frequencies of the same mutations that already occur spontaneously
Cell cycle – The life of cells
Quiescent (Q) stage
Proliferative (P) stage
M: mitotic phase
I: interphase
Cancer biology – cell doubling time (CDT)
• Time required for a group of tumor cells to double the number =
around the time for one cell cycle
• The CDT of cancer cells are ~ 24 hours
• Somatic cells may not re-enter cell cycle
but stay in G0 phase eternally.
E.g., skin cells or neuron cells
• G1 always takes the largest part
https://www.thermofisher.com/hk/en/home/life-science/cell-analysis/flow-cytometry/flow-cytometry-assays-reagents/cell-cycle-assays-flow-cytometry.html
Cell cycle – checkpoints secure cell integrity
• Checkpoints
‐ Cell cycle progression is regulated by several proteins as being the Go/no-Go criteria.
‐ The stoppage of cell cycle is to assure the cell contents are intact to enter mitosis
‐ G1/S checkpoint
❑ Clear to initiate DNA synthesis
‐ G2/M checkpoint
❑ DNA is correctly synthesized
❑ Clear to enter mitosis
‐ Mitotic checkpoint
❑ Spindle fibers well-attached
❑ Chromosome can be separated
Cell cycle - visualization
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Cell 132, 487–498, February 8, 2008
DNA damage – DNA structural damage
• Crosslinking with materials and subsequently interrupting the DNA unwinds
and replication
• DNA-Protein Crosslinking
• DNA inter-/intra-strand crosslinking
• No mutation or mismatch
DNA damage – DNA-protein crosslinking
• DNA-protein crosslinking (DPC)
• Caused by UV, chemotherapy
and ionizing radiation
• Can be removed by DPC-PR
(DNA-protein crosslink
proteolysis repair)
Trends in Biochemical Sciences 2017 42, 483-495DOI: (10.1016/j.tibs.2017.03.005)
DNA damage – Inter-strand crosslinking (ICL)
The ICL (Inter-strand crosslinking)
interrupt the unwinding of DNA helices,
and thus inhibits DNA replication.
The repair and recognition can be done by
FA core protein complex.
DNA damage – DNA strand breaks
• Breaks of DNA strand
‐ Single strand break (SSB)
▪ Quickly repaired; no or little biological effect
▪ Base/nucleotide excision repair (BER/NER)
‐ Double strand break (DSB)
▪
▪
▪
▪
Primary lesion with ionizing radiation
Lead to cell death, mutation, and carcinogenesis
Homologous recombination repair (HRR)
Non-homologous end joining (NHEJ)
‐ DSB events are only 4% of SSB
• Incident rate of DSB is proportional to the radiation dose
DNA damage repair – Base excision repair
For single or limited range damage on single strand DNA
This repair happens when a loss-of-base occurred.
http://www.atdbio.com/content/15/Mutagenesis-and-DNA-repair
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DNA damage repair – Nucleotide excision repair
For multiple or structural damage on single strand DNA
The length of fragment excised is about 12-16 (E. Coli) or 24-32 (mammals) nucleotides
The damage can be multiple mismatches of DNA paring or dimer-forming distortion.
http://www.atdbio.com/content/15/Mutagenesis-and-DNA-repair
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DNA damage repair – Double strand break
• Homologous recombination
‐
‐
‐
‐
HR (or HRR for repair)
Error-free
Sister chromatid referenced
Time-consuming
• Non-homologous end joining
‐
‐
‐
‐
NHEJ
Error-prone
More efficient
Much more than HRR
(Over 80% of damages are
repaired through NHEJ)
Francisco J Fernandez, Miguel Lopez-Estepa and M. Cristina Vega (2011). Nucleases of Metallo-Beta-Lactamase and Protein Phosphatase Families in DNA Repair, DNA
Repair - On the Pathways to Fixing DNA Damage and Errors, Dr. Francesca Storici (Ed.), InTech, DOI: 10.5772/20884.
DNA damage repair – Double strand break
The presence of sister chromatid / duplicated template is the key issue
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DNA damage repair – Double strand break
Proteins involved
Common:
NBS1/MRE11/Rad50 complex
NHEJ:
Ku70, Ku80, DNA-PKcs
HRR:
Rad51, BRCA1, BRCA2
DNA damage repair – Wrap-up
1. DNA in cells is more resistant to IR than in free status – the repair
mechanism
2. IR (1-2Gy) may cause DNA damage events in mammalian cells :
1.
2.
3.
4.
5.
6.
Base damage > 1000
Sugar damage 800 ~ 1000
SSBs 1000
DSBs 40
DNA-DNA crosslink 30
DNA-protein crosslink 150
3. DSBs lead to chromosomal aberrations
Normal Human Chromosomes
From DNA to chromosome
DNA damage – chromosome aberrations
• After chromosome replication (1n → 2n), the replica will be torn
apart making two individuals.
• Centromeres are attached by spindle fibers for the division of
duplicated chromosomes.
Centromere
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http://ib.bioninja.com.au/standard-level/topic-1-cell-biology/16-cell-division/mitosis.html
DNA damage – chromosome aberrations
• Chromosomes are made up of DNA strands
• Results:
‐
‐
‐
‐
Cell death
Malfunction of cell
Genetic mutation → abnormal daughter cells
Lost of reproductive ability
• Chromosomal aberrations are mainly caused by:
‐ double strand break of DNA strands
‐ mis-repair of the broken DNA chain
Chromosomal aberrations – replication matters
Post-replication scenario
Pre-replication scenario (mutant before duplication)
Chromosomal aberrations – the karyotyping
Normal
Chromosome aberrations (lethal)
Acentric
Centric ring
Dicentric
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Chromosomal aberrations – non-lethal repairs
Symmetric translocation
Interstitial deletion
Interphase rearrangement
Although the cells are not dying, the mutation has occurred.
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Chromosomal aberrations – the aftermath
• The fates of the broken chromosomes
‐
‐
‐
‐
Just broken (without repair)
Break reconstitutes (error?)
Deletion
Mismatch
• The fates of the cells
‐ Aberrations and rearrangement affect
‐ Lethal:
▪ Dicentric
▪ Centric Ring
▪ Anaphase Bridge
‐ Non-lethal
▪ Symmetric translocations
▪ Small deletions (interstitial and/or terminal)
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Chromosomal aberrations – summary
• The fates of the cells
‐ Aberrations and rearrangement affects
‐ Lethal:
▪ Dicentric
▪ Centric Ring
▪ Anaphase Bridge
‐ Non-lethal
▪ Symmetric translocations
▪ Small deletions (interstitial and/or terminal)
‐ Non-lethal and physiological normal
▪ Robertsonian translocation
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Chromosomal aberrations – Robertsonian translocation
• Chromosome 13, 14, 15, 21, and 22 are “acrocentric,” meaning they
have very short p arm
• Two of the chromosomes fused by the centromere
http://uvmgg.wikia.com/wiki/Translocation
Robertsonian translocation – Down syndrome
Robertsonian translocation – Down syndrome
Parent A
Robertsonian!!!!!
c13PA
c13PA
c13MA
c13MA
+
c21PA
c21PA
c21MA
c21MA
Male
c13PA
c13MB
c21MA
c21MB
c13PB
c13MB
c21PB
c21MB
Parent B
Female
c13PB
c13MA
+
c21PA
c21MA
c21MB
DOWN