Normal Tissue and Tumors – Assays and Response to Radiation Flashcards
the only event in the cell cycle that can be identified with a light microscope
The condensation of chromosomes during M phase
G0
cells stop progressing through the cell cycle
G1 and G2
the gaps in activity,
Radiation will cause primary cells to arrest in
both G1 and G2, while tumor cells would show only a G2 arrest point
most sensitive cell types
that divide
Cells are most sensitive during
G2 and M
Cells in ____ phase are most resistant
S
Mitotic Index (MI):
proportion of cells in mitosis
MI = TM(time for mitosis)/TC (total cycle time)
Labeling Index (LI):
the proportion of cells in S phase
LI = TS (time for DNA synthesis)/TC(total cell cycle time)
Labeling Index can be determined by
pulse labeling (10-30 min)cells with DNA precursors (3H-thymidine or with BrdUrd), fixing, staining as appropriate and counting labeled cells.
Cell cycle time frame
10h to 10 days
G1 phase length
150 h
S phase length
6-10 h
G2 phase length
1-2 h
M phase length
1h
The “percent-labeled mitoses technique” entails:
the feeding of a population of cells in the S-phase and the subsequent observation of the stained cells in the M phase.
Growth fraction
proliferating cells/total #cells = P/(Q+P) Q-quiescent cells
Potential Doubling Time (Tpot)
the cell cycle time/fraction of cycling cells: Tpot = TC/GF=(TS/LI). Does not take into account cell loss
Tumor growth
net result of cell growth and cell loss:• inadequate nutrition (necrosis, hypoxia, inability of vascular supply to keep up with growth) • apoptosis • immunological surveillance • metastasis • exfoliation
Cell loss factor
φ=1–(Tpot/TD) or TD (tumor volme doubling time)= Tpot/(1-φ).
If TC = 22h, GF = 0.6, and φ = 0.9, the TD = 366 hrs
In experimental animal tumors, cell loss factors range from 0 to 90%.
Three factors determine tumor growth
Cell cycle time of proliferating cells
• Growth fraction
• Cell loss fraction
Metastases appear to grow
more rapidly than the primaries in the same individual.
determine response to chemotherapy
GF, LI, cell loss factor
tumors with high LI
cured by chemotherapy
TCD50
Tumor Control Dose - dose to control 50% of tumor cells. Compare treated and untreated cells number
Hewitt Dilution Assay
Withdraw a few tumor cells from donor mouse (lymphocytic leukemia). Irradiate withdrawn cells in vitro. Inject cells into target mouse
LD50
mean lethal dose -dose required to kill 50% of the animals in a given time period,
e.g., 30 days, LD50/30
Lung Colony Assay
Tumor irradiated in situ, then excised and made into single cell suspension. Injected into animal and in 18-21 days lung cancer
In Vitro-In Vivo Assays
tumor extracted from mouse, suspension prepared and plate tumor cells-> produce clones. The fastest and cheapest and most accurate assay but assume that there is no difference whether the tumor in vivo or in vitro
spheroids
3-D spheres formed by cells aggregated in soft agar or suspension. Show in vitro tumor model
Three basic types Dose-response assays for normal tissues
direct clonogenic assay;
functional assay;
multi fraction experiments used to assemble dose-response relationship or α/β ratios
Spleen Colony Assay
normal bone marrow cells, blood, and spleen transferred to the lethally irradiated animal. Check spleen colonies
Human A-T cells
one of the most radiation-sensitive human cell lines.
Multifraction Experiments
- log (S/nd) = 𝜶 + 𝜷d
differences in G1,
the difference in cell cycle between fast- and slow growing cells
Dose-response curves for functional endpoints, distinct from cell
survival, can be obtained for:
pig skin and rodent skin by measuring skin reactions
early and late response of the lung by measuring breathing rate
spinal cord by observing myelopathy
