Chromosomes and causation of human cancer and leukemia. XXXVII. Nucleolus organizers on the Ph1 chromosome in chronic myelocytic leukemia

Chromosomes and Causation of Human Cancer and Leukemia. XXXVII. Nucleolus Organizers on the P h I Chromosome in Chronic Myelocytic Leukemia S. Kohno, S. Abe, S. Matsui, and A. A. Sandberg

ABSTRACT: In the light of reported evidence that Ag staining of nucleolar organizers (NOR) may reflect the transcriptionally active state of rDNA during the preceding interphase period, the present study addressed the question as to what kind of Ag-staining profiles are displayed by the secondary constrictions on Philadelphia (Ph1) chromosomes in chronic myelocytic leukemia (CIVIL). The average number of Ag-stained NOR regions (Ag-NOR) in bone marrow cells was 5.10-&--0.89per cell for CML patients and 4.84+0.30 per cell for controls, indicating that the active NOR in normal and CIVILbone marrow cells are equal in number. The distribution of Ag-NOB in the Ph~ chromosomes in CML was significantly lower (P < 0.005) than the expected value. There was no CML case where the Ph ~ chromosome showed a higher incidence of Ag-NOR than expected. The results indicate that the NOR (or rDNA) on the Ph~chromosomes may undergo transcriptionally inactive states. This phenomenon is further discussed in relation to other genetic mechanisms based on chromosome changes. INTRODUCTION Nucleolar organizers (NOR) are the small regions of m e t a p h a s e c h r o m o s o m e s containing structural ribosomal DNA (rDNA) [1]. In h u m a n cells NOR are located on the secondary constrictions or stalks of D- and G-group c h r o m o s o m e s [ 2 - 4 ] . The Nbanding technique, d e v e l o p e d by Matsui and Sasaki [5], m a d e possible the visualization of these regions on c h r o m o s o m e s of various mammals. A n o t h e r NOR staining method, called Ag-As, has been d e v e l o p e d by Goodpasture and Bloom [6,7]. Since AgAs-stained regions (Ag-NOR) do not a p p e a r clearly in cells w h e r e rDNA is transcriptionally inactive, several workers [8 - 10] have argued that Ag-NOR staining p r o b a b l y detects the NOR-associated r e m n a n t of ribonucleoproteins synthesized during the preceding interphase, w h i c h probably reflects the transcriptionally active state of rDNA. The results with Ag-NOR staining s h o w e d that the n u m b e r of NOR per cell varied from cell to cell, from tissue to tissue, and from person to person; thus, the variation in the n u m b e r of Ag-NOR may, indeed, account for the transcriptional state of the NOR (rDNA). Despite extensive studies on cells of normal subjects, there is scanty information on Ag-NOR of cancer or leukemic cells. Based on the results with h u m a n t u m o r cell

From the Roswell Park Memorial Institute, Buffalo, New York. Address reprint requests to: Dr. A. A. Sandberg, Boswell Park Memorial Institute, 666 Elm Street, Buffalo, NY 14263. Received January 10, 1979; accepted January 30, 1979.

© Elsevier North Holland, Inc., 1979 Cancer Genetics and Cytogenetics1, 15-20 (1979} 0165-4608/79/01001506502.25

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s. Kohno et al. lines, Hubbell and Hsu [11] demonstrated that despite an increase in the number of acrocentrics in these cells, the number of Ag-NOR remained at the control level. In a patient with chronic lymphocytic leukemia (CLL), Varley [12] found that the distribution pattern of Ag-NOR in CLL cells was distinctly different from that of normal cells. Since Ag-NOR may indeed relfect the genetic activity of rDNA, the characterization of Ag-NOR should be of particular importance in assigning a role to any specific chromosome in cancer cells. In the present study, we performed Ag-staining analyses on bone marrow cells of chronic myelocytic leukemia (CML) patients, and addressed the question as to whether the NOR on the Philadelphia (Ph') chromosome, a karyotypic change characteristic for this leukemia, are transcriptionally active.

MATERIALS AND METHODS

Materials used in this study were obtained from 25 CML patients who had a Ph I (due to a translocation between chromosomes No. 9 and No. 22) in 100% of their bone marrow cells and 10 patients with normal karyotypes. The ages of CML patients who were successfully analyzed by Ag staining ranged from 18 to 74 years (average : 43.9 yr). Eight of these CML patients were in remission of their disease (less than 3% myeloblasts in the bone marrow). In two patients (Cases 4 and 10), 6.8 and 14.0% of the bone marrow cells were myeloblasts, possibly indicative of imminent development of the blastic phase of CML. Bone marrows with normal chromosome constitutions and cytology were prepared from patients with malignant lymphoma, and the results used as "controls." As will be described, the Ag staining was successful in the cells of only 5 of the 10 patients, for 3 patients this occurred prior to chemotherapy. The cells from aspirates of iliac crest or sternum were cultured at 37°C for 24 hr in RPMI 1640 medium with 10% fetal calf serum, exposed to colcemide (1/~g/ml) for 1.5 hr, treated with 0.075 M KC1 for 30 min, and then fixed in Carnoy's. Chromosome preparations were made by the air-drying method. For Ag-NOR staining, we used the simplified method of Bloom and Goodpasture [6,7] as described by Lau and Arrighi [13] with some modifications, i.e., an incubation time of 2 4 - 50 hr at 60°C in 50% (W/V) AgNO s solution and a pretreatment time of 5 - 20 min in a borate buffer solution (0.1 M, pH 9.0). The distribution of Ag-NOR in metaphases were identified in the same metaphases by 3 different observers. RESULTS Unexpected difficulty was encountered by us in performing Ag staining on bone marrow chromosomes. First, the chromosomes in the 24-hr cultured bone marrow cells frequently exhibited fuzzy morphology with unclear contours and, therefore, the staining profile of Ag-NOR on such chromosomes was obscured. Secondly, the chromosomes sometimes showed C- or kinetochore-banding and no banding of AgNOR. For these reasons, only slides with a distinct morphology and unequivocal AgNOR profile were chosen for microscopic and statistical analysis (Fig. 1). Thus, NOR data based on the cells of successfully analyzed patients (10 out of a total of 25 CML patients and 5 out of 10 control patients) are presented here. A grand total of 1402 Ag-NOR were observed in 283 cells of the 10 CML bone marrows and 760 Ag-NOR in 157 cells of the 5 control ones. The distribution of Ag-NOR among chromosome groups (D, G, and Ph 1) is summarized in Table 1. The average numbers of Ag-NOR per cell in CML and normal bone marrows were 5.10 ± 0.89 and 4.84 ± 0.30, respectively, indicating no statistical difference between them. The modal numbers of Ag-NOR in the respective groups were 3 to 8 and 4 to 7.

17

Chromosomes a n d Causation of Cancer

\

i7"

I q

F i g u r e 1 Metaphase of a bone marrow cell from a CML patient (Case 7) showing 8 Ag-NOR (small arrows) and negative Ag staining on a Ph I chromosome (large arrow).

The control groups as a w h o l e d i s p l a y e d a r a n d o m distribution of Ag-NOR among the c h r o m o s o m e s of groups D and G (P > 0.1), although i n d i v i d u a l l y in 4 of the subjects (Table 1) there was some nonrandomness. In contrast, the CML group as a w h o l e s h o w e d a definite n o n r a n d o m distribution of the Ag-NOR among the D- and G-group c h r o m o s o m e s (P < 0.005), with only 2 (Cases 3 and 4) out of the 10 cases showing some randomness. In the CML group a higher frequency of Ag-NOR on D-group c h r o m o s o m e s and a lesser frequency on the Ph i c h r o m o s o m e were observed. Of particular interest was the fact that the Ph i c h r o m o s o m e d i s p l a y e d a lesser occurrence of Ag-NOR in 6 (Cases 5 - 1 0 ) out of 10 patients. Even with the data of the other 4 patients (Cases 1 - 4 ) , who d i d not show such a tendency, there was no case in w h i c h Phj chromosomes d i s p l a y e d a statistically higher occurrence of Ag-NOR than the expected value. Thus, t h e general impression, based on i n d i v i d u a l patient data as well as those of the 10 CML patients as a whole, was that the Ph 1 chromosomes have a lesser Ag-NOR frequency than expected. DISCUSSION The n u m b e r of NOR per cell, demonstrated by silver staining in h u m a n acrocentric chromosomes, varies from cell to cell, tissue to tissue, and person to person [11, 12,14 - 17]. The m o d a l numbers of Ag-NOR range from 4 to 10 per cell [11,12,14 - 17]. Since Ag staining of NOR m a y detect the transcriptionally active rDNA [ 8 - 1 0 ] and

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Table I

S. K o h n o et al.

F r e q u e n c i e s of A g - N O R grains in c h r o m o s o m e g r o u p s of b o n e m a r r o w cells f r o m 10 CML p a t i e n t s and 5 p e r s o n s w i t h n o r m a l b o n e m a r r o w ~' Chromosome group G D

Case CML

1' 2" 3 4 5c 6~ 7c 8c 9c 10 c

total ~ Control

total ~

117 (99.6) 150b(110.4) 106 (96.0) 92 (94.2) 81 (69.0) 61 (76.2) 154°(129.0) 78 (88.2) 81 (72.6) 35 (42.0) 955°(877.2)

1~ T' 3c 4 5c

107 b 76 b 122 b 100 72

(81.6) (97.8) (95.4) (94.8) (86.4)

477 (456.0)

G(-Ph')

Ph'(22q-)

30° 12 ° 38 53 28 63 ° 59 67 ° 39 33 b

19 22 16 12 6 3~ 2° 2b I° 2b

(49.8) (55.2) (48.0) (47.1) (34.5) (38.1) (64.5) (44.1) (36.3) (21.0)

Total

No. of Ag(+) cells observed

No. of Ag grains per cell

Mode of Ag grains

5.19 5.75 5.00 5.81 5.23 4.38 6.72 4.90 4.48 2.91

6 5.5 4 6 4 3 8 4 3 4

(16.6 (18.4 (16.0 (15.7 (11.5 (12.7 (21.5 (14.7 (12.1 (7.0)

166 184 160 157 115 127 215 147 121 70

32 32 32 27 22 29 32 30 27 20

85°(146.2)

1462

283

(54.4 (65.2 (63.6 (63.2 (57.6

136 163 159 158 144

3O 32 32 31 32

283 (304.0

760

157

422 (438.6) 29 b 87 b 37 b 58b 72

a v e . 5.10_+0.89 4.53 5.09 4.97 5.10 4.50

4 6 5 7 5

ave. 4.84÷0.30

"Numbers in parentheses represent the expected frequencies, bStatistically significant (P<0.05) compared with the expected values. ~Statistically significant distributions (P<0.05). dStatistically significant distributions (P<0.005); when calculated for 2 groups, (D and G including Ph ~chromosomes), P
s i n c e a h i g h p r o d u c t i o n of r R N A g e n e r a l l y a c c o m p a n i e s an u n c o n t r o l l e d g r o w t h of c a n c e r ceils [1], the q u e s t i o n arises as to w h a t k i n d of A g - N O R profile w o u l d be disp l a y e d by c a n c e r or l e u k e m i c cells. W o r k i n g o n 9 c u l t u r e d cell l i n e s d e r i v e d f r o m h u m a n tissues of v a r i o u s p a t h o l o g i c a l origin, H u b b e l l a n d H s u [11] d e m o n s t r a t e d that d e s p i t e t h e i n c r e a s e in the n u m b e r of a c r o c e n t r i c s in t h e s e cell lines, the n u m bers of A g - N O R r e m a i n e d at c o n t r o l levels. Based o n the statistical a n a l y s i s of the AgN O R d i s t r i b u t i o n in t h e P H A - s t i m u l a t e d l y m p h o c y t e s of 20 n o r m a l subjects a n d on t h e c e l l s of a case w i t h CLL, V a r l e y [12] a r g u e d that the d i s t r i b u t i o n of A g - N O R a m o n g t h e c h r o m o s o m e s of D a n d G g r o u p s d i f f e r e d d i s t i n c t l y f r o m that s e e n in the controls. H o w e v e r , it is n o t c e r t a i n f r o m a single s a m p l i n g of CLL, p a r t i c u l a r l y w h e n b a s e d on a p p a r e n t l y P H A - s t i m u l a t e d c e i l s (probably n o r m a l T cells?), if s u c h a differe n c e is d u e to t h e i n d i v i d u a l v a r i a t i o n or the n a t u r e of A g - N O R in CLL. In the p r e s e n t study, t h e v a r i a t i o n in t h e n u m b e r of A g - N O R was s i m i l a r l y obs e r v e d in t h e b o n e m a r r o w cells of b o t h c o n t r o l a n d CML patients. Thus, it s e e m s v e r y d i f f i c u l t to assign a A g - N O R p r o f i l e to a n y single p a t h o l o g i c a l c o n d i t i o n . N e v e r theless, w h i l e w e do n o t k n o w w h e t h e r t h e o b s e r v e d v a r i a t i o n a c c o u n t s for i n d i v i d ual or cell specificity, an i n t e r e s t i n g f i n d i n g is that t h e P h ' c h r o m o s o m e r a t h e r cons i s t e n t l y d i s p l a y e d a lesser i n c i d e n c e of A g - N O R t h a n t h e e x p e c t e d v a l u e (Table 1). T h e r e w a s no case w h e r e the P h 1 c h r o m o s o m e d i s p l a y e d a h i g h e r A g - N O R i n c i d e n c e than expected.

Chromosomes and Causation of Cancer

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The question arises as to whether the observed phenomenon represents a fortuitous observation or reflects, indeed, the genetic activity of NOR in the deleted Ggroup chromosome, i.e., the PhL Taking into consideration the available evidence (see below), as well as the present findings, the meaning of this phenomenon is discussed below by raising a number of questions and attempting to answer them. 1. Is there any possibility that chromosome No. 22, an ancestor of the Ph ~ chromosome, is involved less frequently in Ag staining? Except for one study [15], which suggested a statistically nonsignificant lower frequency of Ag-NOR in chromosome No. 22, other reports clearly indicate a random distribution of Ag-NOR among D- and G-group chromosomes [6,7,12,13,18,19]. MIoreover, since the N bands appear uniformally in all members of acrocentric chromosomes [20], it appears unlikely that chromosome No. 22 alone has a small NOR. 2. Is the lesser occurrence of Ag-NOR a phenomenon peculiar to deleted acrocentric chromosomes? It is well known that more consistent gene inactivation takes place in translocated or deleted X chromosomes than in intact ones. Based on studies of lymphocytes of 175 patients, Orye [21] suggested that the inactivation mechanism, comparable to that for X-chromosome genes, is operative in the nucleolarorganizing activity of G-group, and probably D-group, chromosomes in man. We have recently observed bone marrow cells of a CIVILpatient where a Ph ~and 13qcoexisted, both displaying a satistically lesser involvement by Ag staining {Kohno, unpublished data). Thus, the possibility should not be ruled out that the lesser occurrence of Ag-NOR may be related to the altered genetic activity as a consequence of chromosomal rearrangement (see also quest/on 4 below). 3. Does the Ph ~chromosome contain rDNA? As yet, we have not tried in situ DNArRNA hybridization on CIVILchromosomes. Based on a N-banding analysis of the Oxford factor in Xenopus chromosomes, Funaki et al. [22] unequivocally demonstrated that only the stalk or secondary constrictions contain rDNA or NOR. Inasmuch as the stalk or secondary constrictions are readily recognized in substantial numbers of Ph ~ chromosomes (Kohno, unpublished data), it seems unlikely that rDNA is missing from the Ph I chromosome. We do not rule out, however, the possibility that the Ph 1has less rDNA compared to other G-group chromosomes, since Warburton et al. showed that No. 22 chromosomes hybridize less to rDNA and participate less frequently in satellite association [23]. 4. Does the Ph ~ chromosome have less transcriptionally active rDNA? Based on Agstaining analyses of chromosomes and nuclei during the early embryogenesis in the mouse, Engel et al. [8] indicated that Ag stains transcriptionally active NOR on the chromosomes and in interphase nuclei. Similar indications have been obtained by MIiller et al. [10] and Schwarzacher et al. [9]. Of special interest was the finding of MMiller et al. [10] that in h u m a n - m o u s e somatic hybrid cells, where human rDNA is not expressed, only the mouse NOR was stained with silver. In the light of these reports, we surmise that the lesser occurrence of Ag-NOR on Ph ~ chromosomes may, indeed, indicate their lesser transcriptional activity. The patients with CIVILstudied by us were essentially in the chronic phase of the disease, at which time the leukemic cells do not show blastic morphology, as in the blastic phase of CIVIL.Accordingly, it should be worthwhile to ascertain whether the Ag-NOR profile in Phi-positive bone marrow cells changes in relation to the various stages of this disease. Such a study is in progress. The authors wish to thank Mr. Yun-Fai Chris Lau and Dr. Francis Arrighi, Section of Cell Biology, M.D. Anderson Hospital and Tumor Institute, Houston, Texas, for providing them their unpublished data. This research was supported in part by grant CA-14555 from the National Cancer Institute.

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s. K o h n o et al.

REFERENCES 1. Busch H, Smetana K (1970): The Nucleolus, Academic Press, New York, pp. 116-159, 448-471. 2. Evans HJ, Buckland RA, Pardue ML (1974): Location of the genes coding for 18S and 28S ribosomal RNA in the human genome. Chromosoma 48,405 -426. 3. Henderson AS, Warburton D, Atwood KC (1972): Location of ribosomal DNA in the human chromosome complement. Proc Natl Acad Sci USA 69, 3394- 3398. 4. Ohno S, Trujillo JM, Kaplan WD, Kinoshita R (1961): Nucleolus-organisers in the causation of chromosomal anomalies in man. Lancet 2, 123 - 126. 5. Matsui S, Sasaki M (1973): Differential staining of nucleolus organizers in mammalian chromosomes. Nature 246, 148-150. 6. Goodpasture C, Bloom SE (1975): Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining. Chromosoma 53, 37 - 50. 7. Bloom SE, Goodpasture C (1976): An improved technique for selective silver staining of nucleolar organizer regions in human chromosomes. Hum Genet 34, 199 - 206. 8. Engel W, Zenzes MT, Schmid M (1977): Activation of mouse ribosomal RNA genes at the 2cell stage. Hum Genet 38, 57 - 63. 9. Schwarzacher HG, Mikelsaar A-V, Schnedl W (1978): The nature of the Ag-staining of nucleolar organizer regions. Electron- and light-microscopic studies on human cells in interphase, mitosis, and meiosis. Cytogenet Cell Genet 20, 2 4 - 39. 10. Miller OJ, Dev GV, Tantravahi R, Miller OJ (1976): Suppression of human nucleolus organizer activity in mouse-human somatic cell hybrid cells. Exp Cell Res 101,235 - 243. 11. Hubbell HR, Hsu TC (1977): Identification of nucleolus organizer regions (NORs) in normal and neoplastic human cells by the silver-staining technique. Cytogenet Cell Genet 19, 185-196. 12. Varley JM (1977): Patterns of silver staining of human chromosomes. Chromosoma 61, 207-214. 13. Lau Y-F, Arrighi FE (1979): Comparative studies of N-banding and silver staining of NORs in human chromosomes. Monograph concerning Seminar-Workshop held in Montevideo, Uruguay. 14. Miller DA, Tantravahi R, Dev VG, Miller OJ (1977): Frequency of satellite assocation of human chromosomes is correlated with amount of Ag-staining of the nucleolus organizer region. Am J Hum Genet 29,490 - 502. 15. Mikelsaar A-V, Schmid M, Krone W, Schwarzacher HG, Schnedl W (1977): Frequency of Ag-stained nucleolus organizer regions in the acrocentric chromosomes of man. Hum Gellet 37, 7 3 - 7 7 . 16. Mikelsaar A-V, Schwarzacher HG, Schnedl W, Wagenbichler, P (1977): Inheritance of Agstainability of nucleolus organizer regions. Investigations in 7 families with trisomy 21. Hum Genet 38, 183-188. 17. Heneen WK (1978): Silver staining and nucleolar patterns in human heteroploid and measles-carrier cells. Hereditas 88,213 - 227. 18. de Capoa A, Ferraro M, Menendez F, Mostacci C, Pelliccia F, Rocchi A (1978): Ag staining of the nucleolus organizer (NO) and its relationship to satellite association. Hum Genet 44, 71 - 77. 19. de Capoa A, Ferraro M, Archidacono N, Pelliccia F, Rocchi M, Rocchi A (1976): Nucleolus organizer and satellite association in a variant D-group chromosome. Hum Genet 34, 13 - 16. 20. Hayata I, Oshimura M, Sandberg AA (1977): N-band polymorphism of human acrocentric chromosomes and its relevance to satellite association. Hum Genet 36, 55 - 61. 21. Orye E (1974): Relative activation and inactivation phenomena between homologous and nonhomologous nucleolus organizer on the normal human G chromosomes. Cytogenet Cell Genet 13,352-368. 22. Funaki K, Matsui S, Sasaki M (1975): Location of nucleolar organizers in animal and plant chromosomes by means of an improved N-banding technique. Chromosoma 4 9 , 3 5 7 - 370. 23. Warburton D, Atwood KC, Henderson AS (1976): Variation in the number of genes for rRNA among human acrocentric chromosomes: correlation with frequency of satellite association. Cytogenet Cell Genet 17,221 - 230.