Status Report: “Characterization of a panel of cell lines to be used in in the framework of a

Treatment Planning System”

Caterina TanzarellaAntonio AntocciaAntonella SguraFrancesco Berardinelli

Dipartimento di Biologia, Università “Roma Tre” INFN – Sezione di “Roma Tre”

b. First priority measures in collaboration with the LNL group

ENDPOINTS TO BE ANALYZED

1) -H2AX assay for quantitation of tracks at t=30 min2) Cytogenetic analysis to evaluate the clastogen effect in IR-exposed cells.

c. Additional measures

2) Cell inactivation to evaluate the surviving fraction in IR-exposed cells

a. Characterization of cell lines 1) Nuclear area 2) Nuclear thickness

In V79, AG01522D, CCD18Lu, HSG, T98G

1) Cell growth curves

Measure of nuclear area

-Cells were seeded at a final concentration of 12000/cm2 in chamber slides successively fixed in absolute methanol and stained with 0,5µg/ml DAPI

- Images were captured at 63X magnification and nuclear area evaluated using the ISIS sofware by Metasystems

~ 0,38 µm

Thickness (µm) = (number of slices -1) * thickness of a single slice (µm)

BF

BF

5 6 71

BF

42 3

12 13 148 119 10

Each cell was analysed indipendently and at least 100 cells were scored for each cell line

Measure of nuclear thickness

Analysis performed in vivo using DRAQ5 dye in confocal microscopy

0

1

2

3

4

5

6

7

8

9

1 51 101 151 201 251 301 351

Nuclear Area (um2)

Fre

qu

ency

Nuclear area and thickness in AG01522 D

0

5

10

15

20

25

30

1 11 21 31 41 51 61 71 81

Nuclear thickness (um*10-1)

Fre

qu

ency

24 hrs 48 hrs total

Nuclei scored 235 115 350Mean area (µm^2) 167,90 164,40 167,15St Dev 54,63 43,56 51,31

24 hrs

Nuclei scored 165Mean thickness (µm) 4,335St Dev 0,719

0

1

2

3

4

5

6

7

8

9

10

1 11 21 31 41 51 61 71 81 91 101 111 121

Nuclear thickness (um*10-1)

Fre

qu

ency

0

1

2

3

4

5

6

7

1 51 101 151 201 251 301

Nuclear area (um2)

Fre

qu

enc

yNuclear area and thickness in V79

24 hrs

Nuclei scored 118Mean area (µm^2) 115,6822St Dev 23,64919

24 hrs

Nuclei scored 134Mean thickness (µm) 7,163St Dev 1,675

Cell growth of AG01522 D

Doubling time: ~ 22 hrs T =T = tt log2 /log2 / ( (log N -log N0)log N -log N0)

0

20

40

60

80

100

120

0 24 48 72 96 120

Time (hours)

Cel

ls (

*104 )

Cells were seeded at a final concentration of 16*104 in plastic Petri dishes and counted in triplicate every 24 hours for 5 days.