Silver bands in chronic granulocytic leukemia: I. Increased banding associated with blastic transformation

Silver Bands in Chronic Granulocytic Leukemia: I. Increased Banding Associated with Blastic Transformation Janice M. Brasch, Fran M. O'Malley, O. Margaret Garson, and D. R. Smyth

ABSTRACT:

We have tested the proposal that the level of silver banding in leukemic cells of Ph ~+ chronic granulocytic leukemia (CGL) patients increases as the disease progresses. Blood and/ or bone marrow cells fl'om 14 patients were cultured for 24 hr before banding. In all but one case, there were two populations of mitoses, those with silver bands on their nucleolar organ/zing regions (NORs) and those without. The percentage of cells that banded was higher, on average, in cultures from 7 patients in blastic transformation (80%) than in 8 chronic cases (36%) or in one accelerated phase (49%). Also, the mean number of NORs stained in banded cells was higher in blastic phase (6.9) compared with chronic phase cells (4.4). Hyperdiploid cell lines were present in four cases of myeloblastic transformation. All such cells were silver banded, and the mean fraction of NORs banded in them was relatively high. An increase in silver banding with time was shown in two of the patients. It seems that silver banding does increase in CGL cells as the disease progresses. This may arise either through an increase in the rate of ribosomal flNA synthesis in leukemic ceils present in the blastic phase or possibly by a decrease in the rate of degradation {or processing) of newly synthesized rBNA.

INTRODUCTION T h e n u c l e o l a r o r g a n i z i n g r e g i o n s of c h r o m o s o m e s (NORs) c a n b e i m p r e g n a t e d w i t h s i l v e r to p r o d u c e s i l v e r b a n d s . Data f r o m h u m a n r o d e n t h y b r i d cell l i n e s h a v e clearly demonstrated that only those NORs whose ribosomal RNA genes have been a c t i v e l y t r a n s c r i b e d w i l l s i l v e r b a n d ; s u p p r e s s e d g e n e s do n o t [1, 21. F u r t h e r , t h e N O R m u s t r e a c h a t h r e s h o l d of t r a n s c r i p t i o n a l a c t i v i t y b e f o r e it w i l l stain. S c h m i a d y et al. [3] p r o p o s e d t h i s to a c c o u n t for t h e p r e s e n c e of s o m e u n b a n d e d cells i n a r e l a t i v e l y u n i f o r m p o p u l a t i o n of c o n t a c t - i n h i b i t e d h u m a n fibroblasts. S u c h i n h i b ited cells h a d f e w e r s i l v e r b a n d s (a m e a n of 1.9 b a n d e d N O R s o u t of 10 in e a c h cell) c o m p a r e d w i t h e x p o n e n t i a l l y g r o w i n g f i b r o b l a s t s (4.0-4.8) or s t i m u l a t e d l y m p h o c y t e s (6.4) f r o m t h e s a m e i n d i v i d u a l . T h i s c a n b e a c c o u n t e d for if t h e n u m b e r of i u d i v i d u a l N O R s in a cell r e a c h i n g t h e b a n d i n g t h r e s h o l d i n c r e a s e s as t h e o v e r a l l level of r R N A t r a n s c r i p t i o n i n c r e a s e s . R e e v e s et al. [4] s t u d i e d b o n e m a r r o w cells f r o m c o n t r o l s u b j e c t s as w e l l as f r o m From the Department of Genetics, Monash University. Clayton. Victoria, Australia (J.M.B.. D.R,S.), and the Cytogenetics Unit, University of Melbourne Department of Medicine, St. Vincent's Hospital, Melbourne, Vict{)ria, Australia (F.M.O.. O,M.G,), Address requests for reprints to ]. M. Brasch, Department of Genetics, Monash University'. Clayton. Victoria 3168, Australia. Received December 27, 1982; accepted March 14, 1983.

61 a,, 1984 by Elsevier Science Publishing Co.. Inc. 52 Vanderbilt Ave., New York. NY 10017

Cancer Genetics and Cytogenetics 11, 61--68 (1984) 0165-4608/84/$03.00

62

I.M. Brasch et al. patients with various forms of leukemia [acute lymphocytic (ALL), acute n o n l y m phocytic (ANLL), and chronic granulocytic (CGL)]. They discovered that over 50% of all dividing cells had no silver bands, while those that were banded had fewer bands than stimulated lymphocytes. Among four CGL patients, Reeves et al. [4] found that the one patient in blastic phase had relatively more marrow cells banded [with a higher mean silver band number) than the three in the chronic phase. This may reflect increased NOR activity in the cells proliferating at the later phase. We have investigated this possibility by examining silver-banded NORs in leukemic cells from 14 p h i + CGL patients who were at various stages of the disease.

MATERIALS AND METHODS

The patients had been referred to the Cytogenetics Laboratory at St. Vincent's Hospital, Melbourne, for chromosomal assessment. The Philadelphia [Ph 1) chromosome was present in all cases. Bone marrow (BM) and/or blood samples (BL) were incubated at 37°C for 24 hr in RPMI 1640 m e d i u m s u p p l e m e n t e d with 20% fetal calf serum. Colcemid was added to a concentration of 0.02 ~,g/ml 2 hr before harvest. All cultures were harvested in the conventional way and slides were air-dried. Silver staining was performed by a modification of the methods of Bloom and Goodpasture [5]. Slides were treated at 65°C with 50% AgNOa for 16-20 hr or with 50% A g N Q plus 0.03% formalin for 2 hr. Cells were then counterstained with 2% Giemsa buffered to pH 6.8 for 30 sec. To score silver bands, the slides were scanned systematically to locate all metaphase cells. Occasionally, small areas of the slides were unaffected by silver nitrate, and these could be distinguished by their pale blue color and the lack of any silver deposit on interphase nuclei. Metaphase spreads in such areas were not scored.

RESULTS

Eight patients in the chronic phase were examined (Table 1). In four subjects, parallel cultures of peripheral blood and bone marrow cells gave similar results, allowing data from the two origins to be considered together. Preparations from all eight cases revealed that not all mitoses were banded, e.g., only 19% in patient S.E. The highest percentage was 70% in patient M.D. In this patient, there was a large n u m ber of cells with only one silver band. This was always a D chromosome, which had an u n u s u a l l y large band. The other variable of interest is the mean n u m b e r of bands per banded cell. This ranged from 3 . 1 / c e l l in S.E. to 6.2 in F.E. Such values are lower than we usually obtain in stimulated lymphocytes. An increase in NOR activity during the progression of CGL would be apparent by an increase in the percentage of banded cells, and possibly an increase in the mean n u m b e r of bands in banded cells. To test this, seven patients, either in an accelerated phase or blastic transformation, were examined (Table 1). The patient in accelerated phase (W.R.) had a fairly high percentage of banded cells [approximately 50%), but the mean n u m b e r of bands per banded cell was quite low {3.8). Values for both these variables overlap with those of the eight subjects in chronic phase (Table 1). More revealing data came from S.E., a patient who developed lymphoblastic transformation. She was studied during both the chronic and the transformed phase (Table l a and c; Fig. 1). Quite clearly, there were more banded cells, and more bands per banded cell, in the blastic phase. Data from five patients in myeloblastic transformation also support the hypoth-

I

Age

Sex

Silver band

46

45 56

M.N.

S.K. N.A.

F M

M

M

13 34 12 26 18 13 12 ~ 0

78 82 61 50 57 70 42

48 70 31 16 64 73 21 23 32 12 11 21

30 63

59 87 40 20 91 91 32 32 54 30 30 71

4 6

2

2 4

3 2 3 1 1 2 10

1

1

4 1 1

5

1 3

4 1 5 1 6 2 1 1

4

2

i

2 3

3

1 7

2 1 1 6

1

4 3 3 1

3

5 4 4

4

3 8

2 1 1 2 6 4 1 10

2 1

4

11 14 1 1 1

11

5 7

2 6 4 3 21

4 2 3 2 1

5

5 10 5 5 2 2

14

4

1 3 6 2

2

2

1 3 2

6

10 5 3 7 8 3

22

1

2 3

2 6 1 1

2

7

N o . of A g b a n d s p e r c e l l

6 8 17 2

28

5

1 2

11 2

3 1

8

4 15 30 10

2

3 3

9

5 2 22

1

lo

2

3

11

68% 70% 74% 79% 87% 100%

85%

'iExalnined 9/t~2.

' Examined 6/81.

'Examined 6/82.

"Examined 9/81.

6.6 4.4 6.0 8.2 8.3 9.2

5,6

3,8

3.7 7O%

49%

5.0

3.6 62%

3.3 41%

6.2

4.5

3.1 5,8

Mean no. bands per banded cell

31%

25%

22%

19% 2O%

Percent cells banded

Bone marrow and blood ~:.lls were cultured for 24 hr without PHA. All cells carried a P h i l a d e l p h i a (;hroinosonle. A space indicates that no cells were observed.

BM BL {c) L Y M P H O B L A S T I C T R A N S F O R M A T I O N S,E. 58 F B M ~' (d] M Y E L O B L A S T I C T R A N S F O R M A T I O N S.L. 36 F BL G.Y. 24 M BL P,A. 32 F BM': S.H. 45 M BL H.R. 56 F BM P.A. 33 F BL a

B M ~' BL BM BL BM BL BM BL BL BM BL BL

0

cells of 14 CGL patients

N o of cells

in leukemic

Bone marrow or b l o o d

patterns

M.D. 32 F {b) A C C E L E R A T E D P H A S E W.R. 69 F

38

F.E.

(a) C H R O N I C P H A S E S.E. 58 F A.S. 40 M L.S. 23 M

Patient

Table

64

J.M. Brasch et al.

-50

SE

9/a

SE

6/82

40 30

b9 d Iii

20 I0

LL 0

0

-30

Z

"20

o 0,1

2

rl

3,L,5,6,7,8,9

NO. OF AG BANDS

I ,10,

PER

CELL

Figure 1 Patient S.E. Frequency distributions of cells classified according to the number of silver-banded NORs they contain during chronic phase (upper graph) and 9 months later during lymphoblastic transformation (lower graph). esis (Table ld). All were found to have relatively high numbers of banded cells. The overall mean percentage of cells banded (75%, excluding the repeat sample from P.A.) was significantly higher than the 36% averaged in the 8 patients in chronic phase {Table la) (t~l - 4.10; p <1%). Likewise, the overall mean n u m b e r of bands in banded cells was higher in myeloblastic cases (6.7) than in chronic phase patients (4.4) (tit - 2.98; p <2%). In four cases (S.L., S.H., H.R., and P.A.), myeloblastic transformation had led to hyperdiploid cell lines. Significantly, all hyperdiploid cells were banded {Figs. 2 and 3). Some diploid p h i + cells were also usually present, but these were mostly unbanded. A progressive increase in silver b a n d i n g was shown in patient P.A., whose cells were examined on two occasions in myeloblastic transformation (Table ld). Initially, the proportion of banded cells {74%) and their mean band n u m b e r ( 6 . 0 / c e l l ) were relatively high. However, 15 months later, all cells were banded, and there were 9.2 b a n d s / c e l l on average. Karyotype analysis of P.A.'s cells {Fig. 3) showed that hyperdiploid cells with 47 chromosonms had more bands per cell on average than banded diploid cells (7.2 versus 5.8}, and that the 48 and 49 chromosome cell lines had an even higher mean n u m b e r {9.3). We interpret these findings in S.L., S.H., H.R., and P.A. to indicate that the hvperdiploid cells that developed later have more NOR activity than the leukemic diploid cells from which they are derived.

65

Silver Bands in CGL

SL

.30 -20 -10

1

-0 bO

0

-F1

2!:

SH 10

LL

dZ

HR

-30 -20 -10 "0

.0,1

.2,3.

NO. OF AG

4,5

,6

, 7,8

, 9 ,10,11

,

BANDS PER CELL

Figure 2 Frequei/cy distribtdions of cells classified by their munber of silver bands (in patients S.L.. S.H., and H,R.), showing di[ferem:es in each case in the number of bands per (:ell in hyperdiploid cells (shaded) and diploid ceils (open},

DISCUSSION

We have presented two lines of evidence suggesting that the natural progression of changes in C(;L cells is associated with increased silver banding. First, in two patients, sampled at intervals during their disease, both the percentage of leukemic cells banded and the n u m b e r of bands per (:ell increased (Figs. 1 and 3). Second, in four patients showing myeloblastic transformation, all of the hyperdiploid cells were banded, and they had a relatively high band n u m b e r per cell (Figs. 2 and 3). Tile high n u m b e r of bands in hyperdiploid cells cannot be fully accounted for by the presence of additional NOR chromosomes. For example, in S.L. there were two of these, lint the mean n u m b e r of bands per hyperdiploid cell was 7.7 compared with only 4.1 in banded diploid (:ells of the same culture (Fig. 2). Likewise, the additional NOR chromosomes in hyperdiploid cells of S.H. {two), H.R. {three}, and P.A. (one in the 48 49 cell line} are insufficient to account for their high silver band n u m b e r relative to the diploid cells of the same patients (Figs. 2 and 3) and of those patients in chronic phase {Table 1). Our results are similar to those of Reeves et al. 14] who found that a higher percentage of (:ells were bdnded in one patient in blastic phase (83%) than in three

66

t.M. BraNch , I ,

20 if] ._] d LIJ C_) LL 0

PA

6/81

PA

9/82

10

0 30 20

Z 10

-0 I

0 1 2 3 z, 5 6 7 8 9 0,11

NO. O F

I

I

AG

I

I

I

I

BANDS

I

I

I

PER

I

CELL

Figure 3

l:requency distributions of cells classified by their number of silver bands in patient P. A. sampled on two occasions in blastic phase. Diploid cells (open) and hyperdiploid cells with 47 chromosomes [dark shading) were present on first sampling. The latter cells and cell lines with 48 and 49 chromosomes (light shading) were observed 15 months later.

in the chronic stage (46%-54%). Also, the mode and mean number of silver bands per cell was higher in their patient in blastic phase even after compensating for the two extra NORs that were frequently present. However, we have extended their results through the study of a larger sample and demonstrated that the only unhanded Ph ~ cells in the blastic phase were those with the less modified diploid c o m p l e m e n t - - a l l the h y p e r d i p l o i d cells were banded (Figs. 2 and 3). Kohno e t a ] . [6] have also silver-stained bone marrow cells of CGL patients in the chronic phase. They reported " u n e x p e c t e d difficulty" in silver staining and presented no data concerning unhanded cells. However, they were successful in banding the cells of 10 of the 25 patients examined. They found the mean number ot bands in banded cells to be 5.1, ranging from 2.9 to 6.7. These values correspond closely to ours for newly diagnosed patients in chronic phase (Table i). One property likely to lead to increased celhdar rRNA production, and consequently to increased silver banding, is an increased growth rate [3]. However, this does not seem to be the explanation here, as there is sufficient evidence that CGI, cells proliferate more slowly than normal marrow cells [7]. Also, cells with additional chromosoine abnormalities from patients in blast phase have been shown to have an even slower cell cycle time, at least in culture [8]. Biochemical studies in myeloma and neuroblastoma (:ell lines have clearly shown that growth rate alone does not determine the rate of rRNA transcription [9]. Alternatively, increased rRNA production may occur in order to provide increased numbers of ribosomes per cell or to replace ribosomes that are inore rapidl3 turned over. The dividing cells analyzed in patients in blast phase are at an earlier stage of differentiation, where perhaps more ribosomes are needed to acco m m o d at e higher mRNA translation. Another possibility is thai rRNA production in leukemic blasts is not controlled at th.e level of transcription, but bv selective degradation of the new transcript. In

Silver Bands in CGL

67

this case, h i g h e r r R N A t r a n s c r i p t i o n w o u l d not n e c e s s a r i l y lead to the p r o d u c t i o n of m o r e ribosomes. C o o p e r [10] has p r o p o s e d that, in resting h u m a n l y m p h o c y t e s , s u c h " w a s t a g e " of 18S r R N A occurs before it can be t r a n s p o r t e d to the c y t o p l a s m . H o w e v e r , in resting l y m p h o c y t e s , the level of t r a n s c r i p t i o n is l o w [10] and w h e n c h r o m o s o m e s of such cells are p r e m a t u r e l y c o n d e n s e d , t h e y do not silver b a n d I3l. Of course, it c o u l d be argued that the i n c r e a s e d b a n d i n g does not solely reflect h i g h e r levels of rRNA synthesis. C y t o c h e m i c a l e v i d e n c e i n d i c a t e s that silver stains the specific p r o t e i n s a s s o c i a t e d w i t h n e w p r e - r R N A transcripts, rather t h a n the R N A itself [11, 12]. T h e r e is a l r e a d y e v i d e n c e for a s l o w e r rate of p r o c e s s i n g of the prer R N A in blast cells of acute m y e l o i d l e u k e m i a [13]. Thus, it is possible that relatively m o r e a r g e n t o p h i l i c p r o t e i n c o u l d be associated w i t h the a c c u m u l a t e d , u n p r o cessed p r e - r R N A of NORs of CGL blast cells. This, and the " s e l e c t i v e d e g r a d a t i o n " proposal, are both c o n s i s t e n t w i t h a u t o r a d i o g r a p h s of l e u k e m i c cells from CGL patients in w h i c h h i g h levels of n u c l e a r R N A synthesis, but r e l a t i v e l y l o w c y t o p l a s m i c labeling, has b e e n r e v e a l e d [14]. W h a t e v e r the b i o l o g i c e x p l a n a t i o n for the i n c r e a s e d silver staining of l e u k e m i c cells d u r i n g the c o u r s e of CGL, silver b a n d i n g m i g h t p r o v i d e a useful g u i d e to the progression of the disease in i n d i v i d u a l cases. We are c u r r e n t l y e x a m i n i n g this possibility. This work was supported in part by grants from the Anti-Cancer Council of Victoria to D.R.S. and O.M.G. We wish to thank the hematologists who referred their patients for study.

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nucleolus organizer regions. Electron- and light-microscopic studies on human cells i~ interphase, mitosis, and meiosis. Cytogenet Cell Genet 20, 24-39. 13. Seeber S, K&ding J, Brucksch KP, Schmidt CG (1974): Defective rRNA synti}esis in [roman leukaemic blast cells? Nature 248, 673-675. 14. Hyman GA, Fingerhut B, Tiburcio AC (1968): Possible defect in RNA metabolism in leukemic cells. Cancer 21, 357-367.