生物醫學暨環境生物系 助理教授 張學偉http://genomed.dlearn.kmu.edu.tw

Survival curve= radiation dose & cell survival fraction

Reproductive Integrity

Cell survival cell death

For differentiated cells that do not proliferate e.g., nerve, muscle lose of specific function (death)

For proliferating cells e.g., hematopoietic stem cells, culture cells lose for sustained proliferation (death) lose of reproductive integrity (reproductive death)

Definition of survival for radiobiology

Proliferate indefinitely to produce colony (a large clone = colony = clonogenic)

Retain reproductive integrity

The in vitro survival curve

Cell culture

Established cell lines

Tissue trypsin cell culture in vitro

Dose-survival curve

PE = plating efficiency

Serial dilution

The shape of the survival curve

log

Linear –quadratic function

Width of shoulder= Dq or n=semi-threshold dose

Multi-target model

Initial slope single-event killing

final slope multiple-event killing

= D1

= D0

D1 & D0 = the dose required to reduce the fraction of surviving cells

to 37% of it previous value.

D0 (straight at high dose)

= the average dose required to deliver one inactivating event (one hit) per cell.= the dose required to reduce survival from 0.1 to 0.037 or 0.01 to 0.0037. = D0 (37% survival)

dose required to reduce survival to e-1 (0.37)

Dq (quasithreshold dose)

= Width of shoulder =semi-threshold dose (almost the threshold dose)

Threshold dose = the dose below which there is no effect.

Logen = Dq/D0

n = extrapolation number

D0/0.37(e-1) = Dq/n

D0/e-1 = Dq/n n= e-1xDq/D0 ln n = -Dq/D0

Linear –quadratic model

Two components to cell killing by radiation

One that is proportional to doseOne that is proportional to square of dose (dual-radiation action; two separate breaks)

S = e-D-D2

S = e-D-D2

S = the fraction of surviving a dose D, = constant

When D = D2 D = /Linear & quandratic contribution equally to cell killing at same dose

Target : DNA (nucleus), nuclear membrane

The mechanism of cell killing

Chromosome, specifically the DNA, as the primary target for radiation-induced lethality.

Apoptotic and mitotic death: AD

(falling off)

http://www-micro.msb.le.ac.uk/3035/kalmakoff/baculo/baculohostinteract.html

http://www.niaaa.nih.gov/publications/arh25-3/image01.gif

http://www.copewithcytokines.de/cope.cgi?000638

http://www.ucihs.uci.edu/anatomy/histo/corenotes/celldeath2004.pdf

•Apoptosis: Dsb in linker DNA between nucleosome 185bp [“ladder” in Gel]

•nercosis [“smear” in Gel]

Fig.3-9

The detection of this DNA ladder is still currently used to distinguish at the molecular level apoptosis from necrosis.

Radiation-induced apoptosis is highly cell type dependent.

Hemopoietic cells & lymphoid cells

prone to rapid radiation-induced apoptosis.

Most tumors mitotic cell death & apoptosis.

or mitotic cell death only.

• The most common form of cell death from radiation is mitotic death (MD).

Cell die in attempting to divide because of damaged chromosomes.

Apoptotic and mitotic death: MD

http://www.nature.com/cgi-taf/DynaPage.taf?file=/onc/journal/v23/n16/abs/1207528a.html&dynoptions=doi1097246946

Review

Cell death by mitotic catastrophe: a molecular definition

Asymmetric exchange-type chromosome aberrations (i.e., dicentrics and rings )Loss reproductive integrity Unable to proliferatedeath

Asymmetric exchange-type chromosome aberrations represent the principle mechanism for radiation-induced mitotic death in mammalian cells.

Cell surviving & cell without visible aberration correlation

No apoptosis

The probability of an interaction between the two breaks is related to D (low dose) or D2 (high dose).

Exchange type aberrations require two chromosome breaks.

Chromosome aberrations in human lymphocytes

Ch 2

Survival curves for various mammalian cells in culture

All mammalian cells, normal & malignant, exhibit similar x-ray survival curve (initial shoulder but size vary)

First in vitro survival curve

The Do of X-ray survival curves for most culture cells range from 1 to 2 Gy (100-200 rad or cGy). (page 41)

radiosensitivity

Survival-curve shape and mechanism of cell death

mitotic death

Most cells fall between apopototic & mitotic death

Note! Shoulder

Mitotic death results (principally) from exchange-type chromosomal aberration. log-linear plot with broad shoulder Characterized by subsequently dose-rate effect (page 74).

Apoptotic death result unknow mechanism. straight line on log-linear plot. Characterized by expotential function of dose. little or no dose-rate effect.

S = e-(MAD-MD2

A = cell killing from apoptotic death (vary linear)M = cell killing from mitotic death (vary linear)M = cell killing from mitotic death (vary square)

S = e-D-D2 Linear –quadratic model

apoptotic death mitotic death

Oncogenes and radioresistance

Transfection of activated oncogenes to culture cells Increase radioresistance

Genetic control of radiosensitivity

ATM (AT-mutated) gene

Intrinsic Radiosensitivity and predictive assay

Semisolid agar gel with growth factor

Nonclonogenic assay

Cell growth in multi-wells plate, e.g. MTT assay or chapter 15

Courtenary assay

Surviving cell number is then determined indirectly by MTT dye reduction. (Fig23.4)

The amount of MTT-formazan produced can be determined spectrophotometrically once and solublilized it in a suitable solvent.

補充

The effective survival curve for a multifraction regimen

multifraction regimen are most often used in clinical radiotherapy

What is multifraction regimen?

sublethal damage & time for repair Shoulder

Dilute dose to fraction at time intervals

The effective survival curve

For human, effective D0 (37% survival) = 300cGy = 3Gy

D10 (the dose required to kill 90% = 10 % survival) = one decade of cell killing = 2.3 X D0

Ps: equal Slope Logen = Dq/D0

(F3.3 & page 37)

•Natural log 10= 2.3•equal Slope

The Do of effecitive survival curve (slope) has a value close to 300cGy for cells of human origin.

This is an avarage value and can differ significantly for different tumors.

Calculations of tumor cell kill

109

The radiosensitivity of mammalian cells compared with microorganisms