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Methotrexate and bone marrow metaphases
Methotrexate and Bone Marrow Metaphases J. J. P. Cunningham, A. M. Potter, A. E. Watmore, and D. A. Winfield
ABSTRACT:
The efficacy of a methotrexate (MTX) block/thymidine release synchronization technique has been assessed in bone marrow cultures from patients with acute nonlymphocytic leukemia and myelodysplasia. In contrast to cultures of stimulated lymphocytes from normal individuals, no improvement in mitotic index (MI) or metaphase quality could be detected using this technique. Demonstration of an unchanged level of division in bone marrow cultures in the presence of MTX suggests that the technique is unsuitable for synchronization purposes in this tissue. The influence of preincubation prior to MTX exposure and duration of exposure to colcemid on MI and metaphase quality have also been examined.
INTRODUCTION
The importance of n o n r a n d o m karyotype changes in specific malignancies has been e m p h a s i z e d by the discovery that some cellular oncogenes are located at the sites of specific chromosome rearrangements. The deletion and precise identification of these c h r o m o s o m e changes has in the past been limited by the quality of preparations obtained. Technical i m p r o v e m e n t s to allow the discovery of further, perhaps more subtle, rearrangements are therefore of prime importance. Advances in h u m a n leukemia cytogenetics have been h a m p e r e d by the quality of preparations obtained. A n u m b e r of agents capable of interrupting the cell cycle have been used to synchronize cell cultures [1], and significant improvements in the number of high-quality metaphases obtained from stimulated l y m p h o c y t e preparations have been achieved using synchronization techniques. An advantage of synchronized cell culture is the ability to obtain an increased n u m b e r of divisions without extended exposure to colcemid, thereby maximizing the n u m b e r of cells seen in the early stages of metaphase when chromosomes are still long. Methotrexate (MTX) has been the most extensively e m p l o y e d synchronizing agent, and it acts by reducing the levels of folic acid available to cells during DNA synthesis [2]. This results in the i m p a i r m e n t of de novo purine and p y r i m i d i n e metabolism, leading to a block in nucleic acid synthesis. Hagemeijer et al. [3] reported a successful modification of the MTX synchronization technique for leukemia cultures, and this has been w i d e l y adopted. We have studied the role of MTX in leukemia bone marrow cultures and phytohemagglutinin
From the Centre for Human Genetics. Children's Hospital (J. J. P., A. M. P., A. E. W.) and the Department of Haematology,Royal Hallamshire Hospital (D. A. W.), Sheffield, England. Address requests for reprints to: Joan J, P. Cunningham, Centre for Human Genetics, 117 Manchester Road, Sheffield $10 5DN England. Received January 14, 1988; accepted February 23, 1988.
213 Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017 © 1988 Elsevier
Cancer Genet Cytogenet 33:213-224 (1988) 01654608/88/$03.50
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(PHA) s t i m u l a t e d b l o o d c u l t u r e s f r o m n o r m a l i n d i v i d u a l s . Its effects o n b o t h t h e q u a n t i t y a n d q u a l i t y of m e t a p h a s e s h a v e b e e n a s s e s s e d , a n d o t h e r e l e m e n t s of t h e synchronization technique have been examined.
MATERIALS AND METHODS I n v e s t i g a t i o n s w e r e c a r r i e d o u t o n b o n e m a r r o w s a m p l e s f r o m i n d i v i d u a l s w i t h hem a t o l o g i c d i s o r d e r s , a c u t e m y e l o i d l e u k e m i a (AML), m y e l o d y s p l a s i a (MDS), a n d c h r o n i c m y e l o i d l e u k e m i a (CML) (Table 1), a n d P H A - s t i m u l a t e d b l o o d f r o m o t h e r i n d i v i d u a l s w i t h o u t h e m a t o l o g i c d i s o r d e r s . B o n e m a r r o w (BM) w a s t r a n s p o r t e d to t h e l a b o r a t o r y i n H a m s F10 w i t h a n t i b i o t i c s a n d h e p a r i n . In o u r s t a n d a r d M T X s y n c h r o n i z a t i o n p r o c e d u r e for b o n e m a r r o w s , 4 - 6 x 106 n u c l e a t e d cells w e r e p r e i n c u b a t e d at 37°C for 2 h o u r s i n H a m ' s F10 w i t h 1 0 % p o o l e d h u m a n s e r u m . T h e M T X (Lederle) w a s t h e n a d d e d for 17 h o u r s at a final
Table 1
Individuals used in investigations
Individual
Sex
Age
Diagnosis and FAB type
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
F M F M M M M M M F M M M F F
46 29 60 53 19 50 47 52 81 68 16 97 34 91 56
MDS AML-M2 AML-M2 AML-M2 AMMoL-M4 AML-M2 AML-M2 AML-M2 AML-M2 AML-M4 AML-M1 AML-NK a AML-M2 MDS AML-M3
17 18 19
M F M
30 61 79
AML-NK AML-M3 AML-M5
20 21
M M
76 70
MDS MDS
22
F
40
AML-M2
23 24
F M
35 62
CML AML-NK
25 28 31
F F F
50 92 50
AML-M6 AML-M2 AML-M2
"NK, FAB type not known. bNAD, nothing abnormal demonstrated.
Karyotype 46,XX 46,XY/46,XY,inv(16) 46,XX 46,XY 46,XY 46,XY/47,XY, + 8 45,XY,- 17,inv(3)(q2 lq26) 46,XY 46,XY 45,X, - X,t(8;21)(q22;q22) 46,XY 45,X, - Y 46,XY,t(8;21)(q22;q22) 46,XX 46,XX/46,XX,t(15 ;17) (q22;q21),i(17q - ) 46,XY 46,XX 46,XY/47,XY, + 13/ 48,XY,+X,+ 13 47,XY, + 8 46,XY,2p - ,4q +, - 5, - 7, - 7,+ 8 , - 1 2 , - 15,16q+, + 4mar,t(1 ;3)(p13;p21), t(3;4)(p21;q21) 4 5 , X , - X,inv(16)(p13q22)/ 46,XX,inv(16)(p13q22) 46,XX,Ph/46,XX,Ph,inv(11) 46,XY,NAD b 46,XY,NAD 46,XX,NAD 4 6 , X X / 4 6 , X X , ? t ( 3 ;5 ), 7q - ,t(8;21)(q22;q22)
MTX and Bone Marrow Metaphases
215
concentration of 10-7 M. M e d i u m was then replaced with fresh m e d i u m containing 10% serum and additional t h y m i d i n e (final concentration of 10 s M), and cultures were reincubated for 5 hours and 45 minutes. Cells were then exposed to colcemid for 15 minutes at approximately 0.1 ixg/ml and harvested by conventional methods using 0.075 M KCI hypotonic treatment for 10 minutes at 37°C. Cells were fixed in at least four changes of 3:1 methanol:acetic acid. Fixed suspensions were spread on cold, wet, grease-free slides and air dried. Banding was by the trypsin-Giemsa method. The PHA-stimulated blood from normal i n d i v i d u a l s was treated in the same way except TC199 m e d i u m was used and the blood was given 48 hours incubation before the addition of MTX and only 1 m i n u t e hypotonic treatment (0.037 M KC1) at room temperature according to routine laboratory procedure. Single variables were investigated in the following experimental procedures. Procedure 1. Seven BMs and 10 PHA-stimulated bloods were cultured according to the standard synchronization procedure but omitting MTX. Procedure 2. The effectiveness of MTX in blocking cell division was investigated by examining cell division in the presence of the drug. Procedure 2A. Six BM cultures and ten PHA-stimulated blood cultures were processed by the standard synchronization method but harvested with 15 minutes colcemid at the end of 17 hours MTX treatment without block release. Procedure 2B. Control cultures were treated in the same way for the same time without MTX. Procedure 3. The length of time in culture prior to MTX treatment was varied to determine whether depletion of culture m e d i u m over a period of time was necessary for MTX to synchronize cells. Four BMs were cultured according to the standard synchronization procedure but with preincubation periods of 2, 24, or 48 hours. Procedure 4. U n s y n c h r o n i z e d cultures of five BMs and four PHA bloods were harvested after 10, 25, 40, 55, 70, or 85 m i n u t e s exposure to colcemid to assess the influence of colcemid alone.
Mitotic Index Slides were coded and randomized prior to scoring for mitotic index (MI) using the percentage metaphases in a m i n i m u m of 2000 cells. Three slides from each of two replicate cultures were scored. Slides and replicate cultures were pooled after ttesting, and heterogeneity of variance was estimated using ×2. Mean MI for groups of individuals was the total n u m b e r of metaphases/total cells scored. Band Count and Band Count Distribution The n u m b e r of discernible bands on chromosome 1 was used as an objective measurement of chromosome length and quality. Consecutive cells on each of two slides per replicate were scored whether or not they were suitable for full analysis. Results were pooled and band counts tested as a normally distributed variable by a mixed model analysis of variance (ANOVA). The proportion of cells with chromosome 1 band counts in the ranges less than 9, 9-12, 13-16, 17-20, 21-24, 25-28,
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29-32, and more than 32 was also noted. These are referred to as ranges 1-8, respectively. The mean band count for groups of i n d i v i d u al s was the total n u m b e r of bands/total cells scored.
RESULTS Effect of MTX Results on leukemic bone marrows s h o w e d no significant difference between the mean MI using the standard synchronization procedure and that achieved in cultures wh er e MTX was omitted (Procedure 1). By contrast, the overall MI in PHAstimulated lymphocytes from ten individuals w i t h o u t hematologic disorders was significantly higher in the standard MTX procedure than w h e n MTX was omitted (Table 2). The i n d i v i d u a l responses of the seven BMs are sh o w n in Table 3. None showed a significant difference in MI w h e n MTX was used. However, the MI (0.111.25%) seen in these experiments compared favorably with reports of i m p r o v e d MI after synchronization (0.02-0.4%) [4]. The mean n u m b e r of bands on c h r o m o s o m e 1 was not significantly different w h e n MTX was present or absent in 10/11 BMs (Table 4). Increases were seen in 2
Table 2
Mean mitotic index (%) for seven bone marrows and ten stimulated l y m p h o c y t e preparations comparing standard MTX technique with same procedure omitting MTX Mitotic index (%) Standard MTX technique
Omitting MTX
p°
0.31 3.6
0.27 2.65
NS ***
BM Stimulated lymphocytes
"NS, p > 0.05; ***, p < 0.0Ol.
Table 3
Mitotic indexes for seven bone marrows summarized in Table 2 Mitotic index (%)
Individual
Standard MTX technique
Omitting MTX
p°
1 2 3 4 5 6 7
0.216 0.375 0.6 0.4 1.25 0.15 0.25
0.233 0.325 0.75 0.35 0.75 0.11 0.15
NS NS NS NS NS NS NS
°NS, p > 0.05.
217
MTX a n d B o n e M a r r o w M e t a p h a s e s
Table 4
M e a n b a n d c o u n t s o n c h r o m o s o m e 1 in 11 b o n e m a r r o w s c o m p a r i n g s t a n d a r d MTX t e c h n i q u e w i t h t h e s a m e p r o c e d u r e o m i t t i n g MTX Mean no. 1 band count
Individual
Standard MTX technique
No. of cells
Omitting MTX
No. of cells
pa
1 2 3 5 7 15 21 22 22 24 25
11.06 11.19 11.85 11.19 12.0 12.17 9.55 14.69 10.94 11.00 10.89
111 99 203 134 29 47 58 78 147 125 48
10.71 10.9 11.12 11.81 14.39 12.45 9.5 14.08 11.6 11.97 10.00
116 99 220 167 46 44 34 132 211 104 9
NS NS NS NS * NS NS NS NS NS NS
Mean
11.47
11.68
NS
"NS, p > 0.05; *, p < 0.05.
Table 5
M e a n b a n d c o u n t s o n c h r o m o s o m e 1 in s t i m u l a t e d l y m p h o c y t e s f r o m f o u r individuals comparing standard MTX technique with the same procedure omitting MTX Mean no. 1 band count
Individual
Standard MTX technique
No. of cells
Omitting MTX
No. af cells
E G H K
22.2 16.6 19.46 15.27
100 101 98 100
20.34 17.28 17.28 15.75
102 101 107 87
Mean
18.39
17.73
p°
NS NS NS
°NS, p > 0.05; *, p < 0.05; **, p < 0.01.
of 4 s t i m u l a t e d l y m p h o c y t e p r e p a r a t i o n s (Table 5). T h e p r o p o r t i o n of c e l l s w i t h c h r o m o s o m e 1 b a n d c o u n t s in t h e r~inge 1 - 8 is s h o w n in F i g u r e s 1A a n d lB. No c o n s i s t e n t d i f f e r e n c e s w e r e o b s e r v e d b e t w e e n c u l t u r e s w i t h or w i t h o u t M T X in eit h e r t e n BMs or P H A b l o o d f r o m f o u r i n d i v i d u a l s . It is i m p o r t a n t to n o t e h e r e t h a t for t h e s t i m u l a t e d b l o o d , t h e A N O V A of t h i s data s h o w e d s i g n i f i c a n t v a r i a t i o n in m e a n b a n d c o u n t s f r o m r e p l i c a t e c u l t u r e s a n d e v e n b e t w e e n d u p l i c a t e s l i d e s of t h e s a m e c u l t u r e . T h i s v a r i a t i o n c o u l d h a v e b e e n s u f f i c i e n t to m a s k a l t e r a t i o n d u e to t h e MTX t r e a t m e n t itself. H o w e v e r , s i g n i f i c a n t v a r i a t i o n w a s n o t e n c o u n t e r e d in b o n e m a r r o w s at e i t h e r t h e r e p l i c a t e c u l t u r e or d u p l i c a t e s l i d e level, w h i c h i n d i cates t h a t t h e r e s u l t s in T a b l e 4 a n d F i g u r e 1A are g e n u i n e l y r e p r e s e n t a t i v e . Five of t h e six BMs c u l t u r e d for 17 h o u r s w i t h M T X a n d h a r v e s t e d without s u b sequent block release ( P r o c e d u r e 2A) d i d n o t s h o w a s i g n i f i c a n t d e c r e a s e in MI
2 18
J . J . P . C u n n i n g h a m et al.
Individual
Individual %
Band Range No.of Bands
80
• + MTX [] - M T X
1
~
40
e
20
21
2 ~ 4°~
l 2 3 4 5
<9 9-12 13-16 17-20 >20
z2
•.~ 20
3 "~ 20 7o
23
,
,,, 4 0
"
15
'°I 00~
40
25
I
n
20 ] 2345
l 2 34 5 Band Range
F i g u r e 1A Proportion of cells with chromosome I band counts in ranges 1-5 (less than 9 bands to more than 20 bands) in 10 bone marrows with the standard MTX technique and the same procedure omitting MTX.
219
MTX and Bone Marrow Metaphases
~G 60 1 Individual E
~
50~
Band Range
• +MTX~22.27 []- MTX R 20.34
l 2 3 4 5
,~
40
~
30
~
20
6
10
7 8
ii
No.of Bands
<9 9-12 13-16 17-20 21 - 24 25 - 28 29 - 32 >32
R 17.28
~ 19.46 17.28
u~
% Individual m 60
50 _~ 40 30 0 20 10
~l |i
K m+MTX R15.27 []_MTXR15.75
L 12345678 BandRange F i g u r e 1B Proportion of cells with chromosome 1 band counts in ranges 1-8 (less than 9 bands to more than 32 bands) in stimulated lymphocytes from four individuals with the standard MTX technique and the same procedure omitting MTX.
220
J . J . P . C u n n i n g h a m et al.
Table 6
Mitotic indexes t h e p r e s e n c e of equivalent time 2B i n m a t e r i a l s
for six b o n e m a r r o w s h a r v e s t e d i n a M T X b l o c k or h a r v e s t e d after a n w i t h o u t M T X ( p r o c e d u r e s 2A a n d and methods) Mitotic index
Individual
MTX block present (procedure 2A)
No MTX block present (procedure 2B)
8 9 10 11 14 31
O.2 0.78 0.087 0.35 1.06 0.375
0.27 0.41 0.025 0.65 1.42 0.437
NS ** NS NS * NS
NS, p > 0.05; *, p < 0.05; **, p < 0.01.
w h e n c o m p a r e d to s i m i l a r 1 7 - h o u r c u l t u r e s w h e r e M T X h a d n o t b e e n a d d e d (Proc e d u r e 2B), s u g g e s t i n g n o b l o c k i n g a c t i o n . By c o n t r a s t 9 of 10 P H A b l o o d c u l t u r e s s h o w e d s i g n i f i c a n t l y l o w e r MIs i n t h e p r e s e n c e of M T X after 22 h o u r s (Tables 6 a n d 7).
Effect of Preincubation Period T a b l e 8 s h o w s t h a t e x t e n d e d c u l t u r e of b o n e m a r r o w p r i o r to a d d i t i o n of M T X ( P r o c e d u r e 3) d i d n o t i n c r e a s e t h e MI after MTX. In fact, s i g n i f i c a n t l y l o w e r MIs w e r e s e e n after 48 h o u r s p r e i n c u b a t i o n i n t w o o u t of f o u r p a t i e n t s .
Table 7
M i t o t i c i n d e x e s for s t i m u l a t e d l y m p h o c y t e s i n t e n i n d i v i d u a l s h a r v e s t e d i n t h e p r e s e n c e of a M T X b l o c k or h a r v e s t e d after a n e q u i v a l e n t t i m e w i t h o u t M T X ( p r o c e d u r e s 2 A a n d 2B i n m a t e r i a l s a n d m e t h o d s ) Mitotic: index
Individual A B C D E F G H I
l
MTX block present (procedure 2A)
No MTX block present (procedure 2B)
O.2 0.2 0.0 1.3 O.2 0.0 O.2 0.7 1.3 0.4
NS, p > 0.05; *, p ~ 0.05; **, p ~ 0.01; ***, p < 0.001.
1.3 0.4 O.2 3.5 1.1 0.6 2.0 2.O 2.0 1.7
NS
221
MTX and Bone Marrow Metaphases
Table 8
M i t o t i c i n d e x e s i n four b o n e m a r r o w s u s i n g s t a n d a r d M T X t e c h n i q u e after 2, 24, or 48 h o u r s p r e i n c u b a t i o n Mitotic indexes with standard MTX technique
Individual
2 hours preincubation
24 hours preincubation
48 hours preincubation
p
12 13 20 22
0.35 0.6 0.2 0.25
-0.65 0.1 0.6
0.125 -0.15 0.05
*** NS NS ***
NS, p > 0.05; ***, p < 0.001.
Table 9
M e a n b a n d c o u n t i n five b o n e m a r r o w s after 1 0 - 7 0 m i n u t e s e x p o s u r e to c o l c e m i d Mean no. 1 band count: duration of colcemid treatment in minutes
Individual
70
55
40
25
10
p
17 18 19 20 28
10.69 6.40 11.50 9.83 12.84
13.54 7.85 12.40 10.50 14.36
12.82 6.72 12.89 11.14 15.06
13.21 7.80 12.50 12.50 15.06
14.35 7.70 12.50 12.50 16.95
*** NS NS NS * **
NS, p ~ 0.05; ***, p ( 0.001.
Effects of Colcemid Duration T h e m e a n b a n d c o u n t s o n five b o n e m a r r o w s w i t h d e c r e a s i n g c o l c e m i d e x p o s u r e t i m e s are s h o w n i n T a b l e 9. A n A N O V A s u g g e s t e d t h a t i n d i v i d u a l s w e r e s h o w i n g a s i g n i f i c a n t r e s p o n s e to c o l c e m i d e x p o s u r e (F = 18.5; P ( 0 . 0 0 1 ) . H o w e v e r , t h e i n t e r a c t i o n t e r m b e t w e e n i n d i v i d u a l s a n d c o l c e m i d e x p o s u r e t i m e s w a s also signifi c a n t (F ~ 2.1; p ( 0 . 0 0 1 ) , i n d i c a t i n g t h a t i n d i v i d u a l s w e r e r e s p o n d i n g d i f f e r e n t l y to d e c r e a s i n g c o l c e m i d e x p o s u r e . To f u r t h e r a n a l y z e this, i n d i v i d u a l r e g r e s s i o n a n a l y s i s w a s c a r r i e d out. O n l y t w o i n d i v i d u a l s (17 a n d 28) s h o w e d s i g n i f i c a n t inc r e a s e s i n b a n d c o u n t w i t h r e d u c e d c o l c e m i d e x p o s u r e (a m e a n of 11.7 b a n d s at 70 m i n u t e s to 15.6 b a n d s at 10 m i n u t e s ) . In t h r e e o u t of four s t i m u l a t e d b l o o d s , i n c r e a s e s i n m e a n b a n d c o u n t w e r e obs e r v e d w i t h d e c r e a s i n g c o l c e m i d e x p o s u r e ( P r o c e d u r e 4). A m e a n of 19 b a n d s w a s s e e n after 85 m i n u t e s a n d 23 b a n d s after 10 m i n u t e s (Table 10). H o w e v e r , as i n t h e MTX experiment, significant interreplicate variation was observed. T h e p r o p o r t i o n s of c e l l s w i t h b a n d c o u n t s i n t h e r a n g e 1 - 8 in five B M s a n d four s t i m u l a t e d b l o o d s are s h o w n i n F i g u r e s 2A a n d 2B, r e s p e c t i v e l y . A s h i f t i n b a n d r a n g e s i n B M s 17 a n d 28 t o w a r d r e d u c e d b a n d n u m b e r w a s o b s e r v e d , t h e r e b y c o n f i r m i n g t h e r e s u l t s of t h e r e g r e s s i o n a n a l y s i s . In t h r e e of f o u r s t i m u l a t e d b l o o d s , a s h i f t f r o m b a n d r a n g e 5 to 4 w a s o b s e r v e d w i t h i n c r e a s e d e x p o s u r e to c o l c e m i d . DISCUSSION S i n c e t h e first r e p o r t of t h e b e n e f i c i a l effect of M T X s y n c h r o n i z a t i o n o n l e u k e m i a b o n e m a r r o w c u l t u r e s , m a n y w o r k e r s h a v e r e p o r t e d a n i n c r e a s e d MI or i m p r o v e m e n t of c h r o m o s o m e q u a l i t y u s i n g t h i s t e c h n i q u e [1, 5, 6]. W e h a v e e x a m i n e d M T X
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Table 10
Mean band count in stimulated lymphocytes from four individuals after 10-85 minutes exposure to colcemid Mean no. 1 band count: duration of co|cemid treatment in minutes
Individual
85
70
55
40
25
10
p
L M N O
24.92 19.80 19.29 18.17
24.69 20.60 19.12 19.36
23.08 21.70 20.17 19.68
26.54 21.90 18.85 17.59
26.24 20.20 19.64 19.57
25.56 25.20 21.87 21.97
NS * * *
NS, p > 0.05: *, p < 0.05.
synchronization i n d e p e n d e n t l y of other elements of the technique. Our results show that, in contrast to stimulated lymphocytes, MTX does not significantly alter MI in acute n o n l y m p h o c y t i c leukemic bone marrow cultures. Methotrexate may be an ineffective blocking agent in leukemic bone marrow because of the different metabolic requirements of the cells. It reduces the availability of free folic acid required for DNA synthesis, thus blocking the cell cycle and producing synchronized division w h e n the block is released. There is evidence that in malignant and immature bone marrow cells purine synthesis is less dependent on the MTX-sensitive de novo pathway, and these cells can more readily utilize alternative biochemical (salvage) pathways not affected by MTX [7, 8]. In these cells DNA synthesis will c o n t i n u e even w h e n free folic acid is available only at low levels; hence, effective blocking may not be possible. The time cells were incubated prior to MTX treatment is a point of difference between the procedures for bone marrow and stimulated lymphocytes that may influence response to MTX. Preincubation for 48 hours may deplete the level of free folic acid in the culture m e d i u m , and there is evidence that low folate conditions can slow the cell cycle [9]. Cultured lymphocytes may cycle slowly and therefore be more susceptible to the synchronizing action of MTX than u n c u l t u r e d bone marrow cells. However, extending the i n c u b a t i o n period in bone marrows to 48 hours before MTX treatment did not improve synchronization (Table 8). This could mean that intracellular folate was not significantly depleted for MTX to act, or that such depletion is not limiting in malignant cells with more effective salvage pathways. Increased chromosome length has been reported in MTX-synchronized stimulated lymphocyte cultures [2]. However, we were unable to demonstrate any increase in band count using MTX in bone marrow cultures or stimulated blood cultures. In the case of stimulated blood, variation between replicate cultures attributed to slide making may have masked any treatment response. This p h e n o m enon was not encountered in bone marrow cultures, where chromosomes are shorter (mean band count = 11, compared to 18 in bloods), and perhaps the influences of slide making are less readily measureable. Thus in bone marrows the intrinsic levels of variation were considered insufficient to obscure the possible effects of MTX. Reduced exposure to colcemid is an integral part of synchronization procedures, and we have therefore examined the effects of colcemid in isolation. Over the period examined (10-70 minutes) there was evidence that colcemid did not consistently influence mean b a n d count in leukaemic bone marrows. Effects were seen, however, in two patients whose marrow yielded above-average band counts. It is interesting to note that after 70 m i n u t e s exposure to colcemid their band counts were reduced to levels similar to those of the other three individuals at 10 minutes.
223
MTX and Bone Marrow Metaphases
Individual l l % lO0
19
18
20
28
80 ~
10 Bins
60
c
20
=
%
~
lOO
.'2_
I ....
No.of Bands
I 2 3 4 5
<9 9-12 13- 16 17 - 20 21 - 24 25- 28
6
8O
E
70
8
%
40
Band Range
mins
60 40
&
2O 0
135 246
135 246
46
35
35 46
46
Band Range Figure 2A Proportion of cells with chromosome 1 band counts in ranges 1-6 (less than 9 bands to more than 25 bands) in five bone marrows after exposure to colcemid for 10-70 minutes. It is possible that in more contracted chromosomes any differences are too subtle to measure accurately. As a result of slide-making variation, the effect of colcemid on stimulated blood cultures could not be determined. Frequently, reports of i m p r o v e m e n t in bone marrow cultures attributed to MTX synchronization have compared results from MTX-synchronized cultures to direct or short-term cultures with many points of technical difference. By examining single elements of the te c h n i q u e u n d e r controlled conditions we can suggest that the use of MTX for synchronization is inappropriate in the quest for i m p r o v e d bone marrow c h r o m o s o m e preparations, and that other technical elements may be more important. The reduction of colcemid exposure time does not seem to be critical Figure 2,1] Proportion of cells with chromosome 1 band counts in ranges 1-8 (less than 9 bands to more than 32 bands) in stimulated lymphocytes from four individuals after exposure to colcemid for 10-85 minutes.
Individual H %
L
N
0 Band Range No.ofBands
IO ~--~ 30 I , - ~ mins ~
g~
g
2l
<99-12 13-16
lO %
4
5
17-20 21 - 24
Bins "~ 30 •~ 10
7 8
29-32 >32
2468
3
2468
1 357 2468
Band Range
2468
224
J.J.P. C u n n i n g h a m et el.
and, in fact, longer exposure times may be advantageous in giving a higher MI. As the variation between replicate slides, at least in lymphocytes, is greater than any variation resulting from MTX or colcemid manipulation, we suggest that slide making and perhaps other aspects of the technique significantly affect the final quality of preparations and therefore merit further investigation. It may be that this and alternative approaches, such as the use of intercalating agents [10, 11], will lead to more consistent improvements. Supported by Yorkshire Cancer Research Campaign Grant No 40913.
REFERENCES 1. Gallo JH, Ordanez JV, Brown GE, Testa JR (1984): Synchronization of human leukemia cells. Relevance for high resolution chromosome banding. Hum Genet 66:220-224. 2. Yunis JJ (1976): High resolution of human chromosomes. Science 191:1268-1270. 3. Hagemeijer A, Smit EME, Bootsma D (1979)'. Improved identification of chromosomes of leukemia cells in methotrexate treated cultures. Cytogenet Cell Genet 23:208-212. 4. Yunis JJ (1982): Comparative analysis of high-resolution chromosome techniques for leukemic bone marrows. Cancer Genet Cytogenet 7:43-50. 5. Taniwaki M, Edagawa J, Sonada Y, Ide T, Misawa S, Abe T, Takino T (1981): Synchronization of leukaemic cells by MTX for the high resolution analysis of chromosome banding structure. Acta Haematol Jpn 44:161-164. 6. Morris CM, Fitzgerald PN (1985): An evaluation of high resolution chromosome banding of hematologic cells by methotrexate synchronization and thymidine release. Cancer Genet Cytogenet 14:275-284. 7. Howell SB, Mansfield SJ, Raymond T (1981): Thymidine and hypoxanthine requirements of normal and malignant cells for protection against methotrexate cytotoxicity. Cancer Res 41:945-950. 8. Jackson RC, Harkrader RJ (1981): The contributions of de-novo and salvage pathways of nucleotide biosynthesis in normal and malignant cells. In: Nucleosides and Cancer Treatment, MHN Tattersall, RM Fox, eds. Academic Press, Sydney, pp. 18-31. 9. Stevenson AC (1978): Effects of twelve folate analogues on human lymphocytes in vitro. In: Mutagen Induced Chromosome Damage in Man. HJ Evans, DC Lloyd, eds. Yale University Press, New Haven, CT, pp. 227-238. 10. Ikeuchi T (1984): Inhibitory effect of ethidium bromide on mitotic chromosome condensation and its application to high resolution chromosome banding. Cytogenet Cell Genet 38:56-61. 11. Dewald GW, Broderick DJ, Tom WW, Hagstrom JE, Pierre RV (1985): The efficiency of direct, 24 hour culture, and mitotic synchronization methods for cytogenetic analysis of bone marrow in neoplastic hematologic disorders: Cancer Genet Cytogenet 18:1-10.
ABSTRACT:
The efficacy of a methotrexate (MTX) block/thymidine release synchronization technique has been assessed in bone marrow cultures from patients with acute nonlymphocytic leukemia and myelodysplasia. In contrast to cultures of stimulated lymphocytes from normal individuals, no improvement in mitotic index (MI) or metaphase quality could be detected using this technique. Demonstration of an unchanged level of division in bone marrow cultures in the presence of MTX suggests that the technique is unsuitable for synchronization purposes in this tissue. The influence of preincubation prior to MTX exposure and duration of exposure to colcemid on MI and metaphase quality have also been examined.
INTRODUCTION
The importance of n o n r a n d o m karyotype changes in specific malignancies has been e m p h a s i z e d by the discovery that some cellular oncogenes are located at the sites of specific chromosome rearrangements. The deletion and precise identification of these c h r o m o s o m e changes has in the past been limited by the quality of preparations obtained. Technical i m p r o v e m e n t s to allow the discovery of further, perhaps more subtle, rearrangements are therefore of prime importance. Advances in h u m a n leukemia cytogenetics have been h a m p e r e d by the quality of preparations obtained. A n u m b e r of agents capable of interrupting the cell cycle have been used to synchronize cell cultures [1], and significant improvements in the number of high-quality metaphases obtained from stimulated l y m p h o c y t e preparations have been achieved using synchronization techniques. An advantage of synchronized cell culture is the ability to obtain an increased n u m b e r of divisions without extended exposure to colcemid, thereby maximizing the n u m b e r of cells seen in the early stages of metaphase when chromosomes are still long. Methotrexate (MTX) has been the most extensively e m p l o y e d synchronizing agent, and it acts by reducing the levels of folic acid available to cells during DNA synthesis [2]. This results in the i m p a i r m e n t of de novo purine and p y r i m i d i n e metabolism, leading to a block in nucleic acid synthesis. Hagemeijer et al. [3] reported a successful modification of the MTX synchronization technique for leukemia cultures, and this has been w i d e l y adopted. We have studied the role of MTX in leukemia bone marrow cultures and phytohemagglutinin
From the Centre for Human Genetics. Children's Hospital (J. J. P., A. M. P., A. E. W.) and the Department of Haematology,Royal Hallamshire Hospital (D. A. W.), Sheffield, England. Address requests for reprints to: Joan J, P. Cunningham, Centre for Human Genetics, 117 Manchester Road, Sheffield $10 5DN England. Received January 14, 1988; accepted February 23, 1988.
213 Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017 © 1988 Elsevier
Cancer Genet Cytogenet 33:213-224 (1988) 01654608/88/$03.50
214
J . J . P . C u n n i n g h a m et al.
(PHA) s t i m u l a t e d b l o o d c u l t u r e s f r o m n o r m a l i n d i v i d u a l s . Its effects o n b o t h t h e q u a n t i t y a n d q u a l i t y of m e t a p h a s e s h a v e b e e n a s s e s s e d , a n d o t h e r e l e m e n t s of t h e synchronization technique have been examined.
MATERIALS AND METHODS I n v e s t i g a t i o n s w e r e c a r r i e d o u t o n b o n e m a r r o w s a m p l e s f r o m i n d i v i d u a l s w i t h hem a t o l o g i c d i s o r d e r s , a c u t e m y e l o i d l e u k e m i a (AML), m y e l o d y s p l a s i a (MDS), a n d c h r o n i c m y e l o i d l e u k e m i a (CML) (Table 1), a n d P H A - s t i m u l a t e d b l o o d f r o m o t h e r i n d i v i d u a l s w i t h o u t h e m a t o l o g i c d i s o r d e r s . B o n e m a r r o w (BM) w a s t r a n s p o r t e d to t h e l a b o r a t o r y i n H a m s F10 w i t h a n t i b i o t i c s a n d h e p a r i n . In o u r s t a n d a r d M T X s y n c h r o n i z a t i o n p r o c e d u r e for b o n e m a r r o w s , 4 - 6 x 106 n u c l e a t e d cells w e r e p r e i n c u b a t e d at 37°C for 2 h o u r s i n H a m ' s F10 w i t h 1 0 % p o o l e d h u m a n s e r u m . T h e M T X (Lederle) w a s t h e n a d d e d for 17 h o u r s at a final
Table 1
Individuals used in investigations
Individual
Sex
Age
Diagnosis and FAB type
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
F M F M M M M M M F M M M F F
46 29 60 53 19 50 47 52 81 68 16 97 34 91 56
MDS AML-M2 AML-M2 AML-M2 AMMoL-M4 AML-M2 AML-M2 AML-M2 AML-M2 AML-M4 AML-M1 AML-NK a AML-M2 MDS AML-M3
17 18 19
M F M
30 61 79
AML-NK AML-M3 AML-M5
20 21
M M
76 70
MDS MDS
22
F
40
AML-M2
23 24
F M
35 62
CML AML-NK
25 28 31
F F F
50 92 50
AML-M6 AML-M2 AML-M2
"NK, FAB type not known. bNAD, nothing abnormal demonstrated.
Karyotype 46,XX 46,XY/46,XY,inv(16) 46,XX 46,XY 46,XY 46,XY/47,XY, + 8 45,XY,- 17,inv(3)(q2 lq26) 46,XY 46,XY 45,X, - X,t(8;21)(q22;q22) 46,XY 45,X, - Y 46,XY,t(8;21)(q22;q22) 46,XX 46,XX/46,XX,t(15 ;17) (q22;q21),i(17q - ) 46,XY 46,XX 46,XY/47,XY, + 13/ 48,XY,+X,+ 13 47,XY, + 8 46,XY,2p - ,4q +, - 5, - 7, - 7,+ 8 , - 1 2 , - 15,16q+, + 4mar,t(1 ;3)(p13;p21), t(3;4)(p21;q21) 4 5 , X , - X,inv(16)(p13q22)/ 46,XX,inv(16)(p13q22) 46,XX,Ph/46,XX,Ph,inv(11) 46,XY,NAD b 46,XY,NAD 46,XX,NAD 4 6 , X X / 4 6 , X X , ? t ( 3 ;5 ), 7q - ,t(8;21)(q22;q22)
MTX and Bone Marrow Metaphases
215
concentration of 10-7 M. M e d i u m was then replaced with fresh m e d i u m containing 10% serum and additional t h y m i d i n e (final concentration of 10 s M), and cultures were reincubated for 5 hours and 45 minutes. Cells were then exposed to colcemid for 15 minutes at approximately 0.1 ixg/ml and harvested by conventional methods using 0.075 M KCI hypotonic treatment for 10 minutes at 37°C. Cells were fixed in at least four changes of 3:1 methanol:acetic acid. Fixed suspensions were spread on cold, wet, grease-free slides and air dried. Banding was by the trypsin-Giemsa method. The PHA-stimulated blood from normal i n d i v i d u a l s was treated in the same way except TC199 m e d i u m was used and the blood was given 48 hours incubation before the addition of MTX and only 1 m i n u t e hypotonic treatment (0.037 M KC1) at room temperature according to routine laboratory procedure. Single variables were investigated in the following experimental procedures. Procedure 1. Seven BMs and 10 PHA-stimulated bloods were cultured according to the standard synchronization procedure but omitting MTX. Procedure 2. The effectiveness of MTX in blocking cell division was investigated by examining cell division in the presence of the drug. Procedure 2A. Six BM cultures and ten PHA-stimulated blood cultures were processed by the standard synchronization method but harvested with 15 minutes colcemid at the end of 17 hours MTX treatment without block release. Procedure 2B. Control cultures were treated in the same way for the same time without MTX. Procedure 3. The length of time in culture prior to MTX treatment was varied to determine whether depletion of culture m e d i u m over a period of time was necessary for MTX to synchronize cells. Four BMs were cultured according to the standard synchronization procedure but with preincubation periods of 2, 24, or 48 hours. Procedure 4. U n s y n c h r o n i z e d cultures of five BMs and four PHA bloods were harvested after 10, 25, 40, 55, 70, or 85 m i n u t e s exposure to colcemid to assess the influence of colcemid alone.
Mitotic Index Slides were coded and randomized prior to scoring for mitotic index (MI) using the percentage metaphases in a m i n i m u m of 2000 cells. Three slides from each of two replicate cultures were scored. Slides and replicate cultures were pooled after ttesting, and heterogeneity of variance was estimated using ×2. Mean MI for groups of individuals was the total n u m b e r of metaphases/total cells scored. Band Count and Band Count Distribution The n u m b e r of discernible bands on chromosome 1 was used as an objective measurement of chromosome length and quality. Consecutive cells on each of two slides per replicate were scored whether or not they were suitable for full analysis. Results were pooled and band counts tested as a normally distributed variable by a mixed model analysis of variance (ANOVA). The proportion of cells with chromosome 1 band counts in the ranges less than 9, 9-12, 13-16, 17-20, 21-24, 25-28,
216
J . J . P . C u n n i n g h a m et al.
29-32, and more than 32 was also noted. These are referred to as ranges 1-8, respectively. The mean band count for groups of i n d i v i d u al s was the total n u m b e r of bands/total cells scored.
RESULTS Effect of MTX Results on leukemic bone marrows s h o w e d no significant difference between the mean MI using the standard synchronization procedure and that achieved in cultures wh er e MTX was omitted (Procedure 1). By contrast, the overall MI in PHAstimulated lymphocytes from ten individuals w i t h o u t hematologic disorders was significantly higher in the standard MTX procedure than w h e n MTX was omitted (Table 2). The i n d i v i d u a l responses of the seven BMs are sh o w n in Table 3. None showed a significant difference in MI w h e n MTX was used. However, the MI (0.111.25%) seen in these experiments compared favorably with reports of i m p r o v e d MI after synchronization (0.02-0.4%) [4]. The mean n u m b e r of bands on c h r o m o s o m e 1 was not significantly different w h e n MTX was present or absent in 10/11 BMs (Table 4). Increases were seen in 2
Table 2
Mean mitotic index (%) for seven bone marrows and ten stimulated l y m p h o c y t e preparations comparing standard MTX technique with same procedure omitting MTX Mitotic index (%) Standard MTX technique
Omitting MTX
p°
0.31 3.6
0.27 2.65
NS ***
BM Stimulated lymphocytes
"NS, p > 0.05; ***, p < 0.0Ol.
Table 3
Mitotic indexes for seven bone marrows summarized in Table 2 Mitotic index (%)
Individual
Standard MTX technique
Omitting MTX
p°
1 2 3 4 5 6 7
0.216 0.375 0.6 0.4 1.25 0.15 0.25
0.233 0.325 0.75 0.35 0.75 0.11 0.15
NS NS NS NS NS NS NS
°NS, p > 0.05.
217
MTX a n d B o n e M a r r o w M e t a p h a s e s
Table 4
M e a n b a n d c o u n t s o n c h r o m o s o m e 1 in 11 b o n e m a r r o w s c o m p a r i n g s t a n d a r d MTX t e c h n i q u e w i t h t h e s a m e p r o c e d u r e o m i t t i n g MTX Mean no. 1 band count
Individual
Standard MTX technique
No. of cells
Omitting MTX
No. of cells
pa
1 2 3 5 7 15 21 22 22 24 25
11.06 11.19 11.85 11.19 12.0 12.17 9.55 14.69 10.94 11.00 10.89
111 99 203 134 29 47 58 78 147 125 48
10.71 10.9 11.12 11.81 14.39 12.45 9.5 14.08 11.6 11.97 10.00
116 99 220 167 46 44 34 132 211 104 9
NS NS NS NS * NS NS NS NS NS NS
Mean
11.47
11.68
NS
"NS, p > 0.05; *, p < 0.05.
Table 5
M e a n b a n d c o u n t s o n c h r o m o s o m e 1 in s t i m u l a t e d l y m p h o c y t e s f r o m f o u r individuals comparing standard MTX technique with the same procedure omitting MTX Mean no. 1 band count
Individual
Standard MTX technique
No. of cells
Omitting MTX
No. af cells
E G H K
22.2 16.6 19.46 15.27
100 101 98 100
20.34 17.28 17.28 15.75
102 101 107 87
Mean
18.39
17.73
p°
NS NS NS
°NS, p > 0.05; *, p < 0.05; **, p < 0.01.
of 4 s t i m u l a t e d l y m p h o c y t e p r e p a r a t i o n s (Table 5). T h e p r o p o r t i o n of c e l l s w i t h c h r o m o s o m e 1 b a n d c o u n t s in t h e r~inge 1 - 8 is s h o w n in F i g u r e s 1A a n d lB. No c o n s i s t e n t d i f f e r e n c e s w e r e o b s e r v e d b e t w e e n c u l t u r e s w i t h or w i t h o u t M T X in eit h e r t e n BMs or P H A b l o o d f r o m f o u r i n d i v i d u a l s . It is i m p o r t a n t to n o t e h e r e t h a t for t h e s t i m u l a t e d b l o o d , t h e A N O V A of t h i s data s h o w e d s i g n i f i c a n t v a r i a t i o n in m e a n b a n d c o u n t s f r o m r e p l i c a t e c u l t u r e s a n d e v e n b e t w e e n d u p l i c a t e s l i d e s of t h e s a m e c u l t u r e . T h i s v a r i a t i o n c o u l d h a v e b e e n s u f f i c i e n t to m a s k a l t e r a t i o n d u e to t h e MTX t r e a t m e n t itself. H o w e v e r , s i g n i f i c a n t v a r i a t i o n w a s n o t e n c o u n t e r e d in b o n e m a r r o w s at e i t h e r t h e r e p l i c a t e c u l t u r e or d u p l i c a t e s l i d e level, w h i c h i n d i cates t h a t t h e r e s u l t s in T a b l e 4 a n d F i g u r e 1A are g e n u i n e l y r e p r e s e n t a t i v e . Five of t h e six BMs c u l t u r e d for 17 h o u r s w i t h M T X a n d h a r v e s t e d without s u b sequent block release ( P r o c e d u r e 2A) d i d n o t s h o w a s i g n i f i c a n t d e c r e a s e in MI
2 18
J . J . P . C u n n i n g h a m et al.
Individual
Individual %
Band Range No.of Bands
80
• + MTX [] - M T X
1
~
40
e
20
21
2 ~ 4°~
l 2 3 4 5
<9 9-12 13-16 17-20 >20
z2
•.~ 20
3 "~ 20 7o
23
,
,,, 4 0
"
15
'°I 00~
40
25
I
n
20 ] 2345
l 2 34 5 Band Range
F i g u r e 1A Proportion of cells with chromosome I band counts in ranges 1-5 (less than 9 bands to more than 20 bands) in 10 bone marrows with the standard MTX technique and the same procedure omitting MTX.
219
MTX and Bone Marrow Metaphases
~G 60 1 Individual E
~
50~
Band Range
• +MTX~22.27 []- MTX R 20.34
l 2 3 4 5
,~
40
~
30
~
20
6
10
7 8
ii
No.of Bands
<9 9-12 13-16 17-20 21 - 24 25 - 28 29 - 32 >32
R 17.28
~ 19.46 17.28
u~
% Individual m 60
50 _~ 40 30 0 20 10
~l |i
K m+MTX R15.27 []_MTXR15.75
L 12345678 BandRange F i g u r e 1B Proportion of cells with chromosome 1 band counts in ranges 1-8 (less than 9 bands to more than 32 bands) in stimulated lymphocytes from four individuals with the standard MTX technique and the same procedure omitting MTX.
220
J . J . P . C u n n i n g h a m et al.
Table 6
Mitotic indexes t h e p r e s e n c e of equivalent time 2B i n m a t e r i a l s
for six b o n e m a r r o w s h a r v e s t e d i n a M T X b l o c k or h a r v e s t e d after a n w i t h o u t M T X ( p r o c e d u r e s 2A a n d and methods) Mitotic index
Individual
MTX block present (procedure 2A)
No MTX block present (procedure 2B)
8 9 10 11 14 31
O.2 0.78 0.087 0.35 1.06 0.375
0.27 0.41 0.025 0.65 1.42 0.437
NS ** NS NS * NS
NS, p > 0.05; *, p < 0.05; **, p < 0.01.
w h e n c o m p a r e d to s i m i l a r 1 7 - h o u r c u l t u r e s w h e r e M T X h a d n o t b e e n a d d e d (Proc e d u r e 2B), s u g g e s t i n g n o b l o c k i n g a c t i o n . By c o n t r a s t 9 of 10 P H A b l o o d c u l t u r e s s h o w e d s i g n i f i c a n t l y l o w e r MIs i n t h e p r e s e n c e of M T X after 22 h o u r s (Tables 6 a n d 7).
Effect of Preincubation Period T a b l e 8 s h o w s t h a t e x t e n d e d c u l t u r e of b o n e m a r r o w p r i o r to a d d i t i o n of M T X ( P r o c e d u r e 3) d i d n o t i n c r e a s e t h e MI after MTX. In fact, s i g n i f i c a n t l y l o w e r MIs w e r e s e e n after 48 h o u r s p r e i n c u b a t i o n i n t w o o u t of f o u r p a t i e n t s .
Table 7
M i t o t i c i n d e x e s for s t i m u l a t e d l y m p h o c y t e s i n t e n i n d i v i d u a l s h a r v e s t e d i n t h e p r e s e n c e of a M T X b l o c k or h a r v e s t e d after a n e q u i v a l e n t t i m e w i t h o u t M T X ( p r o c e d u r e s 2 A a n d 2B i n m a t e r i a l s a n d m e t h o d s ) Mitotic: index
Individual A B C D E F G H I
l
MTX block present (procedure 2A)
No MTX block present (procedure 2B)
O.2 0.2 0.0 1.3 O.2 0.0 O.2 0.7 1.3 0.4
NS, p > 0.05; *, p ~ 0.05; **, p ~ 0.01; ***, p < 0.001.
1.3 0.4 O.2 3.5 1.1 0.6 2.0 2.O 2.0 1.7
NS
221
MTX and Bone Marrow Metaphases
Table 8
M i t o t i c i n d e x e s i n four b o n e m a r r o w s u s i n g s t a n d a r d M T X t e c h n i q u e after 2, 24, or 48 h o u r s p r e i n c u b a t i o n Mitotic indexes with standard MTX technique
Individual
2 hours preincubation
24 hours preincubation
48 hours preincubation
p
12 13 20 22
0.35 0.6 0.2 0.25
-0.65 0.1 0.6
0.125 -0.15 0.05
*** NS NS ***
NS, p > 0.05; ***, p < 0.001.
Table 9
M e a n b a n d c o u n t i n five b o n e m a r r o w s after 1 0 - 7 0 m i n u t e s e x p o s u r e to c o l c e m i d Mean no. 1 band count: duration of colcemid treatment in minutes
Individual
70
55
40
25
10
p
17 18 19 20 28
10.69 6.40 11.50 9.83 12.84
13.54 7.85 12.40 10.50 14.36
12.82 6.72 12.89 11.14 15.06
13.21 7.80 12.50 12.50 15.06
14.35 7.70 12.50 12.50 16.95
*** NS NS NS * **
NS, p ~ 0.05; ***, p ( 0.001.
Effects of Colcemid Duration T h e m e a n b a n d c o u n t s o n five b o n e m a r r o w s w i t h d e c r e a s i n g c o l c e m i d e x p o s u r e t i m e s are s h o w n i n T a b l e 9. A n A N O V A s u g g e s t e d t h a t i n d i v i d u a l s w e r e s h o w i n g a s i g n i f i c a n t r e s p o n s e to c o l c e m i d e x p o s u r e (F = 18.5; P ( 0 . 0 0 1 ) . H o w e v e r , t h e i n t e r a c t i o n t e r m b e t w e e n i n d i v i d u a l s a n d c o l c e m i d e x p o s u r e t i m e s w a s also signifi c a n t (F ~ 2.1; p ( 0 . 0 0 1 ) , i n d i c a t i n g t h a t i n d i v i d u a l s w e r e r e s p o n d i n g d i f f e r e n t l y to d e c r e a s i n g c o l c e m i d e x p o s u r e . To f u r t h e r a n a l y z e this, i n d i v i d u a l r e g r e s s i o n a n a l y s i s w a s c a r r i e d out. O n l y t w o i n d i v i d u a l s (17 a n d 28) s h o w e d s i g n i f i c a n t inc r e a s e s i n b a n d c o u n t w i t h r e d u c e d c o l c e m i d e x p o s u r e (a m e a n of 11.7 b a n d s at 70 m i n u t e s to 15.6 b a n d s at 10 m i n u t e s ) . In t h r e e o u t of four s t i m u l a t e d b l o o d s , i n c r e a s e s i n m e a n b a n d c o u n t w e r e obs e r v e d w i t h d e c r e a s i n g c o l c e m i d e x p o s u r e ( P r o c e d u r e 4). A m e a n of 19 b a n d s w a s s e e n after 85 m i n u t e s a n d 23 b a n d s after 10 m i n u t e s (Table 10). H o w e v e r , as i n t h e MTX experiment, significant interreplicate variation was observed. T h e p r o p o r t i o n s of c e l l s w i t h b a n d c o u n t s i n t h e r a n g e 1 - 8 in five B M s a n d four s t i m u l a t e d b l o o d s are s h o w n i n F i g u r e s 2A a n d 2B, r e s p e c t i v e l y . A s h i f t i n b a n d r a n g e s i n B M s 17 a n d 28 t o w a r d r e d u c e d b a n d n u m b e r w a s o b s e r v e d , t h e r e b y c o n f i r m i n g t h e r e s u l t s of t h e r e g r e s s i o n a n a l y s i s . In t h r e e of f o u r s t i m u l a t e d b l o o d s , a s h i f t f r o m b a n d r a n g e 5 to 4 w a s o b s e r v e d w i t h i n c r e a s e d e x p o s u r e to c o l c e m i d . DISCUSSION S i n c e t h e first r e p o r t of t h e b e n e f i c i a l effect of M T X s y n c h r o n i z a t i o n o n l e u k e m i a b o n e m a r r o w c u l t u r e s , m a n y w o r k e r s h a v e r e p o r t e d a n i n c r e a s e d MI or i m p r o v e m e n t of c h r o m o s o m e q u a l i t y u s i n g t h i s t e c h n i q u e [1, 5, 6]. W e h a v e e x a m i n e d M T X
222
J.J.P. C u n n i n g h a m et el.
Table 10
Mean band count in stimulated lymphocytes from four individuals after 10-85 minutes exposure to colcemid Mean no. 1 band count: duration of co|cemid treatment in minutes
Individual
85
70
55
40
25
10
p
L M N O
24.92 19.80 19.29 18.17
24.69 20.60 19.12 19.36
23.08 21.70 20.17 19.68
26.54 21.90 18.85 17.59
26.24 20.20 19.64 19.57
25.56 25.20 21.87 21.97
NS * * *
NS, p > 0.05: *, p < 0.05.
synchronization i n d e p e n d e n t l y of other elements of the technique. Our results show that, in contrast to stimulated lymphocytes, MTX does not significantly alter MI in acute n o n l y m p h o c y t i c leukemic bone marrow cultures. Methotrexate may be an ineffective blocking agent in leukemic bone marrow because of the different metabolic requirements of the cells. It reduces the availability of free folic acid required for DNA synthesis, thus blocking the cell cycle and producing synchronized division w h e n the block is released. There is evidence that in malignant and immature bone marrow cells purine synthesis is less dependent on the MTX-sensitive de novo pathway, and these cells can more readily utilize alternative biochemical (salvage) pathways not affected by MTX [7, 8]. In these cells DNA synthesis will c o n t i n u e even w h e n free folic acid is available only at low levels; hence, effective blocking may not be possible. The time cells were incubated prior to MTX treatment is a point of difference between the procedures for bone marrow and stimulated lymphocytes that may influence response to MTX. Preincubation for 48 hours may deplete the level of free folic acid in the culture m e d i u m , and there is evidence that low folate conditions can slow the cell cycle [9]. Cultured lymphocytes may cycle slowly and therefore be more susceptible to the synchronizing action of MTX than u n c u l t u r e d bone marrow cells. However, extending the i n c u b a t i o n period in bone marrows to 48 hours before MTX treatment did not improve synchronization (Table 8). This could mean that intracellular folate was not significantly depleted for MTX to act, or that such depletion is not limiting in malignant cells with more effective salvage pathways. Increased chromosome length has been reported in MTX-synchronized stimulated lymphocyte cultures [2]. However, we were unable to demonstrate any increase in band count using MTX in bone marrow cultures or stimulated blood cultures. In the case of stimulated blood, variation between replicate cultures attributed to slide making may have masked any treatment response. This p h e n o m enon was not encountered in bone marrow cultures, where chromosomes are shorter (mean band count = 11, compared to 18 in bloods), and perhaps the influences of slide making are less readily measureable. Thus in bone marrows the intrinsic levels of variation were considered insufficient to obscure the possible effects of MTX. Reduced exposure to colcemid is an integral part of synchronization procedures, and we have therefore examined the effects of colcemid in isolation. Over the period examined (10-70 minutes) there was evidence that colcemid did not consistently influence mean b a n d count in leukaemic bone marrows. Effects were seen, however, in two patients whose marrow yielded above-average band counts. It is interesting to note that after 70 m i n u t e s exposure to colcemid their band counts were reduced to levels similar to those of the other three individuals at 10 minutes.
223
MTX and Bone Marrow Metaphases
Individual l l % lO0
19
18
20
28
80 ~
10 Bins
60
c
20
=
%
~
lOO
.'2_
I ....
No.of Bands
I 2 3 4 5
<9 9-12 13- 16 17 - 20 21 - 24 25- 28
6
8O
E
70
8
%
40
Band Range
mins
60 40
&
2O 0
135 246
135 246
46
35
35 46
46
Band Range Figure 2A Proportion of cells with chromosome 1 band counts in ranges 1-6 (less than 9 bands to more than 25 bands) in five bone marrows after exposure to colcemid for 10-70 minutes. It is possible that in more contracted chromosomes any differences are too subtle to measure accurately. As a result of slide-making variation, the effect of colcemid on stimulated blood cultures could not be determined. Frequently, reports of i m p r o v e m e n t in bone marrow cultures attributed to MTX synchronization have compared results from MTX-synchronized cultures to direct or short-term cultures with many points of technical difference. By examining single elements of the te c h n i q u e u n d e r controlled conditions we can suggest that the use of MTX for synchronization is inappropriate in the quest for i m p r o v e d bone marrow c h r o m o s o m e preparations, and that other technical elements may be more important. The reduction of colcemid exposure time does not seem to be critical Figure 2,1] Proportion of cells with chromosome 1 band counts in ranges 1-8 (less than 9 bands to more than 32 bands) in stimulated lymphocytes from four individuals after exposure to colcemid for 10-85 minutes.
Individual H %
L
N
0 Band Range No.ofBands
IO ~--~ 30 I , - ~ mins ~
g~
g
2l
<99-12 13-16
lO %
4
5
17-20 21 - 24
Bins "~ 30 •~ 10
7 8
29-32 >32
2468
3
2468
1 357 2468
Band Range
2468
224
J.J.P. C u n n i n g h a m et el.
and, in fact, longer exposure times may be advantageous in giving a higher MI. As the variation between replicate slides, at least in lymphocytes, is greater than any variation resulting from MTX or colcemid manipulation, we suggest that slide making and perhaps other aspects of the technique significantly affect the final quality of preparations and therefore merit further investigation. It may be that this and alternative approaches, such as the use of intercalating agents [10, 11], will lead to more consistent improvements. Supported by Yorkshire Cancer Research Campaign Grant No 40913.
REFERENCES 1. Gallo JH, Ordanez JV, Brown GE, Testa JR (1984): Synchronization of human leukemia cells. Relevance for high resolution chromosome banding. Hum Genet 66:220-224. 2. Yunis JJ (1976): High resolution of human chromosomes. Science 191:1268-1270. 3. Hagemeijer A, Smit EME, Bootsma D (1979)'. Improved identification of chromosomes of leukemia cells in methotrexate treated cultures. Cytogenet Cell Genet 23:208-212. 4. Yunis JJ (1982): Comparative analysis of high-resolution chromosome techniques for leukemic bone marrows. Cancer Genet Cytogenet 7:43-50. 5. Taniwaki M, Edagawa J, Sonada Y, Ide T, Misawa S, Abe T, Takino T (1981): Synchronization of leukaemic cells by MTX for the high resolution analysis of chromosome banding structure. Acta Haematol Jpn 44:161-164. 6. Morris CM, Fitzgerald PN (1985): An evaluation of high resolution chromosome banding of hematologic cells by methotrexate synchronization and thymidine release. Cancer Genet Cytogenet 14:275-284. 7. Howell SB, Mansfield SJ, Raymond T (1981): Thymidine and hypoxanthine requirements of normal and malignant cells for protection against methotrexate cytotoxicity. Cancer Res 41:945-950. 8. Jackson RC, Harkrader RJ (1981): The contributions of de-novo and salvage pathways of nucleotide biosynthesis in normal and malignant cells. In: Nucleosides and Cancer Treatment, MHN Tattersall, RM Fox, eds. Academic Press, Sydney, pp. 18-31. 9. Stevenson AC (1978): Effects of twelve folate analogues on human lymphocytes in vitro. In: Mutagen Induced Chromosome Damage in Man. HJ Evans, DC Lloyd, eds. Yale University Press, New Haven, CT, pp. 227-238. 10. Ikeuchi T (1984): Inhibitory effect of ethidium bromide on mitotic chromosome condensation and its application to high resolution chromosome banding. Cytogenet Cell Genet 38:56-61. 11. Dewald GW, Broderick DJ, Tom WW, Hagstrom JE, Pierre RV (1985): The efficiency of direct, 24 hour culture, and mitotic synchronization methods for cytogenetic analysis of bone marrow in neoplastic hematologic disorders: Cancer Genet Cytogenet 18:1-10.