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The susceptibility to SV40 virus transformation of fibroblasts obtained from patients with Down's syndrome
Europ. 07. Cancer Vol. 7, pp. 337-339. Pergamon Press 1971. Printed in Great Britain
The Susceptibility t o SV40 Virus Transformation of Fibroblasts Obtained from Patients with Down's Syndrome DAVID Y O U N G M R C Clinical and Population Cytogenetics Unit, Western General Hospital, Crewe Road, Edinburgh, Great Britain
PATIENTS with Down's syndrome have been shown to be more prone to leukaemia (by a factor of 18) and to other solid tumours (by a factor of 2"6) [1]. Various workers have recently suggested that the increased risk of developing cancer associated with certain inherited h u m a n disorders, e.g. Fanconi's anaemia [2] and Down's syndrome [3, 4], may be reflected in the in vitro susceptibility to SV40 virus transformation of the cultured skin fibroblasts from these patients. The purpose of this report is to confirm and extend the observations [3, 4] that skin fibroblasts from patients with Down's syndrome are more easily transformed with SV40 virus than those from individuals with a normal chromosome constitution and to discuss the implications. The fibroblasts used in this work were derived from skin biopsies approximately 3 m m 2 taken from the deltoid region of the upper arm. Biopsies were obtained from four patients with Down's syndrome (two males and two females) who were all trisomic for a G-group autosome. Control biopsies were obtained from healthy, cytogenetically normal people (two males and one female) of similar ages to the patients with Down's syndrome. Cell cultures were initiated from skin biopsies held down by coverslips in 3 cm plastic dishes, sub-cultured into 5 cm dishes, and then infected with SV40 virus between their 5th and 15th sub-culture. Cells at these passage levels
still retain their original karyotypes, and do not show the chromosomal aberrations described by Saksela and Moorhead [5] which arise in aged cultures. The virus used was a strain of SV40 originally isolated by Pfizer and concentrated by dialysis to a final concentration of 1011.3 tissue culture infective doses (TCID/50) per ml - - determined by titration on secondary cultures of African Green Monkey kidney cells. This stock was diluted 1:10 using Dulbecco's saline and used to infect the fibroblasts, and assay the transformation rate of these cells, by the technique described by Todaro et al. [2]. Counts of the numbers of colonies of transformed cells in the cultures of normal and Down's syndrome cultures are shown in Table 1. The results reported here demonstrate a considerably higher number of SV40 transformed colonies in the cultures of fibroblasts from patients with Down's syndrome than in the cultures of normal fibroblasts. The multiplicity of infection was approximately 20,000 infectious units/cell, and this gave a transformation rate, in terms of colonies/cells infected, of approximately 0.008%. This compares with 1000 infectious units/cell and 0 . 0 3 % respectively, as reported by Todaro and Martin [3]. The difference between these two sets of results m a y be accounted for by the difference in the strain of SV40 virus used in these experiments, and also by the slightly different cultlaring techniques. The mean number of transformed colonies found in the cultures of fibroblasts from Down's patients, divided by the mean number of transformed colonies found in
Accepted 13 January 1971. 337
338
David Young
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The Susceptibility to SV40 Virus Transformation of Fibroblasts cultures o f n o r m a l cells, was 2 . 6 9 which is in good a g r e e m e n t with T o d a r o a n d M a r t i n ' s figure of 3.16. T h e small discrepancy b e t w e e n these two figures is well within the limits o f e x p e r i m e n t a l error using the techniques available. Consequently, these results substantiate the reports t h a t fibroblasts from skin biopsies o f patients with D o w n ' s s y n d r o m e are m o r e
susceptible to transformation with SV40 virus t h a n are n o r m a l fibroblasts. Aclmowledgements - - The author would like to express his thanks to the late Professor W. M. Court Brown, Professor D. G. Harnden, Professor H. J. Evans, Dr. Michael Faed and Dr. A. O. Langlands for their help and encouragement with this work. The technical assistance of Miss Kareen Livingstone is gratefully acknowledged. The author would also like to thank Pfizer Ltd. for providing the virus stock.
REFERENCES 1.
2. 3.
4. 5.
339
W . W . HOLLAND,R. DOLL and C. O. CARTER,Mortality for leukaemia and other cancers among patients with Down's syndrome (mongols) and among their parents. Brit. 07. Cancer 16, 177 (1962). G . J . TODARO, H. GREEN and M. R. SWIFT, Susceptibility of human diploid fibroblasts to transformation by SV40 virus. Science 153, 1252 (1966). G . J . TODARO and G. M. MARTIN,Down's syndrome: increased sensitivity of fibroblasts in cell culture to transformation by an oncogenic virus. Proe. Soc. exp. Biol. Med. 124, 1232 (1967). C . W . POTTER, A. M. POTTER and J. S. OXFORD, Comparison of T-antigen induction in human cell lines. 07. Virol. 5, 293 (1970). E. SAKSELA and P. S. MOORHEAD,Aneuploidy in the degenerative phase of serial cultivation of human cell strains. Proc. nat. Acad. Sd. (Wash.) 50, 390 (1963).
The Susceptibility t o SV40 Virus Transformation of Fibroblasts Obtained from Patients with Down's Syndrome DAVID Y O U N G M R C Clinical and Population Cytogenetics Unit, Western General Hospital, Crewe Road, Edinburgh, Great Britain
PATIENTS with Down's syndrome have been shown to be more prone to leukaemia (by a factor of 18) and to other solid tumours (by a factor of 2"6) [1]. Various workers have recently suggested that the increased risk of developing cancer associated with certain inherited h u m a n disorders, e.g. Fanconi's anaemia [2] and Down's syndrome [3, 4], may be reflected in the in vitro susceptibility to SV40 virus transformation of the cultured skin fibroblasts from these patients. The purpose of this report is to confirm and extend the observations [3, 4] that skin fibroblasts from patients with Down's syndrome are more easily transformed with SV40 virus than those from individuals with a normal chromosome constitution and to discuss the implications. The fibroblasts used in this work were derived from skin biopsies approximately 3 m m 2 taken from the deltoid region of the upper arm. Biopsies were obtained from four patients with Down's syndrome (two males and two females) who were all trisomic for a G-group autosome. Control biopsies were obtained from healthy, cytogenetically normal people (two males and one female) of similar ages to the patients with Down's syndrome. Cell cultures were initiated from skin biopsies held down by coverslips in 3 cm plastic dishes, sub-cultured into 5 cm dishes, and then infected with SV40 virus between their 5th and 15th sub-culture. Cells at these passage levels
still retain their original karyotypes, and do not show the chromosomal aberrations described by Saksela and Moorhead [5] which arise in aged cultures. The virus used was a strain of SV40 originally isolated by Pfizer and concentrated by dialysis to a final concentration of 1011.3 tissue culture infective doses (TCID/50) per ml - - determined by titration on secondary cultures of African Green Monkey kidney cells. This stock was diluted 1:10 using Dulbecco's saline and used to infect the fibroblasts, and assay the transformation rate of these cells, by the technique described by Todaro et al. [2]. Counts of the numbers of colonies of transformed cells in the cultures of normal and Down's syndrome cultures are shown in Table 1. The results reported here demonstrate a considerably higher number of SV40 transformed colonies in the cultures of fibroblasts from patients with Down's syndrome than in the cultures of normal fibroblasts. The multiplicity of infection was approximately 20,000 infectious units/cell, and this gave a transformation rate, in terms of colonies/cells infected, of approximately 0.008%. This compares with 1000 infectious units/cell and 0 . 0 3 % respectively, as reported by Todaro and Martin [3]. The difference between these two sets of results m a y be accounted for by the difference in the strain of SV40 virus used in these experiments, and also by the slightly different cultlaring techniques. The mean number of transformed colonies found in the cultures of fibroblasts from Down's patients, divided by the mean number of transformed colonies found in
Accepted 13 January 1971. 337
338
David Young
t~ C~4 r~ e~
H
0
0
0
o
r~
N
,a=
x v
~eb 0
v
,+
-
g.
•
o
×~ ~b
% g~
&
y,
w~
~8 z
The Susceptibility to SV40 Virus Transformation of Fibroblasts cultures o f n o r m a l cells, was 2 . 6 9 which is in good a g r e e m e n t with T o d a r o a n d M a r t i n ' s figure of 3.16. T h e small discrepancy b e t w e e n these two figures is well within the limits o f e x p e r i m e n t a l error using the techniques available. Consequently, these results substantiate the reports t h a t fibroblasts from skin biopsies o f patients with D o w n ' s s y n d r o m e are m o r e
susceptible to transformation with SV40 virus t h a n are n o r m a l fibroblasts. Aclmowledgements - - The author would like to express his thanks to the late Professor W. M. Court Brown, Professor D. G. Harnden, Professor H. J. Evans, Dr. Michael Faed and Dr. A. O. Langlands for their help and encouragement with this work. The technical assistance of Miss Kareen Livingstone is gratefully acknowledged. The author would also like to thank Pfizer Ltd. for providing the virus stock.
REFERENCES 1.
2. 3.
4. 5.
339
W . W . HOLLAND,R. DOLL and C. O. CARTER,Mortality for leukaemia and other cancers among patients with Down's syndrome (mongols) and among their parents. Brit. 07. Cancer 16, 177 (1962). G . J . TODARO, H. GREEN and M. R. SWIFT, Susceptibility of human diploid fibroblasts to transformation by SV40 virus. Science 153, 1252 (1966). G . J . TODARO and G. M. MARTIN,Down's syndrome: increased sensitivity of fibroblasts in cell culture to transformation by an oncogenic virus. Proe. Soc. exp. Biol. Med. 124, 1232 (1967). C . W . POTTER, A. M. POTTER and J. S. OXFORD, Comparison of T-antigen induction in human cell lines. 07. Virol. 5, 293 (1970). E. SAKSELA and P. S. MOORHEAD,Aneuploidy in the degenerative phase of serial cultivation of human cell strains. Proc. nat. Acad. Sd. (Wash.) 50, 390 (1963).