Differential silver-positive nucleolus organizer region activity in normal and malignant murine tissues

Differential Silver-Positive Nucleolus Organizer Region Activity in Normal and Malignant Murine Tissues K. C. Arden, S. Pathak, S. Stettner, and E. Ritchie

ABSTRACT: Transcriptionally active nueleolus organizer regions (NORs) were analyzed using a silver-staining technique. The levels of silver-positive NOR activity for normal murine bone marrow and thymus cells were established, and significant differences in ribosomal RNA gene activity were observed. When tumor cells originating from these two tissue types were also studied, significant differences were seen not only between the normal and malignant tissues, but between the two tumor types as well. These differences in ribosomal RNA gene activity with respect to cell type and malignancy may be useful diagnostically.

INTRODUCTION

N u c l e o l u s o r g a n i z e r regions (NORs) r e p r e s e n t the c h r o m o s o m a l l o c a t i o n s of the genes c o d i n g for 18S and 28S r i b o s o m a l R N A in e v e r y o r g a n i s m s t u d i e d t h u s far [1, 2]. A l t h o u g h t h e s e regions c a n n o t be stained w i t h c o n v e n t i o n a l c h r o m o s o m e stains, t h e y can be v i s u a l i z e d by u s i n g a silver(Ag)-staining t e c h n i q u e [3, 4[. H o w e v e r , the n u m b e r of r i b o s o m a l D N A sites identified by in situ h y b r i d i z a t i o n rarely equals the n u m b e r of A g - p o s i t i v e N O R s (Ag-NORs); in most cases, the total n u m b e r of NORs is h i g h e r t h a n the n u m b e r of Ag-NORs. O n l y those NORs c o n t a i n i n g r i b o s o m a l R N A (rRNA) genes that w e r e t r a n s c r i p t i o n a l l y active in the p r e c e d i n g i n t e r p h a s e are c o u n t e d as stained u n d e r the specific c o n d i t i o n s s t i p u l a t e d for the t e c h n i q u e [5-7]. Specifically, this s i l v e r - s t a i n i n g t e c h n i q u e marks a protein i m p o r t a n t in the s y n t h e s i s of r R N A [8-10]. B e c a u s e this t e c h n i q u e stains o n l y t r a n s c r i p t i o n a l l y active NORs, it can be u s e d to m o n i t o r the a c t i v i t y of r R N A genes. Reports conflict as to w h e t h e r N O R a c t i v i t y differs in different tissues. T h e o n l y s t u d y d e s i g n e d to a d d r e s s this q u e s t i o n specifically r e p o r t e d no d i f f e r e n c e in N O R a c t i v i t y w h e n fibroblasts and p h y t o h e m a g g l u t i n i n - s t i m u l a t e d b l o o d from the s a m e i n d i v i d u a l s w e r e c o m p a r e d [11]. H o w e v e r , other investigators h a v e r e p o r t e d differe n c e s a m o n g tissues w i t h respect to n u m b e r s of A g - p o s i t i v e NORs [12-14]. T h e r e h a v e b e e n reports of t e c h n i c a l difficulty w i t h this t e c h n i q u e w h e n it has b e e n a p p l i e d

From the Department of Cell Biology (K. C. A., S. P.}, The University of Texas M.D. Anderson Cancer Center, Houston; the Department of Carcinogenesis, Science Park, M. D. Anderson Cancer Center (S. S., E. R.), Smithville; and The University of Texas Graduate School of Biomedical Sciences at Houston (K. C. A). Current address of K. C. A. is Ludwig Institute for Cancer Research, 687 Avenue des Pins Ouest, Montreal, Quebec H3A 1A1, Canada. Address reprint requests to: Dr. S. Pathak, Cellular Genetics Laboratory, Box 181, M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Received July 8, 1988; accepted September 7, 1988.

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to direct bone marrow preparations. However, the low incidence of silver staining in bone marrow metaphases that has been attributed to technical difficulty may be a normal characteristic of bone marrow cells. Increased rRNA synthesis and nucleolar changes associated with certain tumors also have been reported [15, 16]. In a previous study from this laboratory, an increase in NOR activity was observed in bone marrow cells from patients with acute lymphocytic leukemia w h e n compared with the NOR activity in bone marrow cells from healthy individuals [17]. We have studied silver staining in the normal and malignant tissues of AKR/J mice. The AKR/J strain of mice provides a readily available source of both normal and tumor tissue. This strain is prone to develop leukemia. Normal tissue can be obtained before the onset of disease, and tumor tissue can be obtained once tumorigenesis occurs. Because the AKR strain of mouse is inbred, comparisons can be made between tissue samples obtained from different animals. Normal and malignant tissues from AKR/J mice were processed, and the NOR activity in the tissue samples was compared. Significant differences in NOR activity were found between the two types of normal tissues as well as between the normal tissues and their malignant counterparts.

MATERIALS A N D METHODS

After the mice were killed, bone marrow was obtained by removing the femurs and tibias and t r i m m i n g off the ends of the bones. The bones were then flushed using a syringe filled with 0.075 M KC1, forcing the bone marrow into a centrifuge tube. The thymus was removed, minced, and suspended in 0.075 M KC1. The tumor cells were cultured in RPMI-1640 complete medium. All samples were incubated in the hypotonic solution for 25 minutes at 37°C and fixed in a methanol : glacial acetic acid (3 : 1) mixture. Cells were dropped on glass slides, and air-dried preparations were made for silver staining. Silver staining was performed by modifying the technique developed by Goodpasture and Bloom [4] as described by Pathak and Elder [18]. All slides were coded before data collection and decoded after analysis. In each sample, 50 metaphase spreads were analyzed, and the n u m b e r of Ag-NORs was determined. Only those metaphases with adjacent silver-stained interphase nuclei were scored. A metaphase spread with one or more Ag-NORs was considered positively stained. Because the data for the bone marrow samples were not distributed normally, we used the M a n n - W h i t n e y test to analyze differences between sets of data. This statistical test is a nonparametric analogue to the Student's t test. Because there were ties between observations and sample sizes exceeded 40, the modified normal approximation was used.

RESULTS

There are six NOR-bearing chromosomes in the normal mouse karyotype. Silverstained metaphase spreads from normal bone marrow and thymus, and their respective B- and T-cell tumors are shown in Figure 1. Because AKR-J mice are prone to develop B- and T-cell leukemias, we chose to study two normal types of tissue from w h i c h these tumors develop, along with the corresponding types of tumor cells. The frequency of Ag-NOR staining for the normal and malignant cells is s h o w n in Table 1. The lowest frequency of active NORs was in normal bone marrow. This was reflected in the n u m b e r of silver-positive NORs, which range from 0 - 3 , with all but two cells showing 2 or fewer Ag-NORs. The mean n u m b e r of Ag-NORs per metaphase

57

Ag-NOR Activity in Murine Tissues *v

f

4 ~i~i

¸

t

Q L

4

O

e

V 4 •i

~iii

P

'5

6

i Silver-stained chromosome preparations from AKR mouse: a) normal bone marrow; b) normal thymus; c) B-cell leukemia clone; and d) T-cell leukemia clone. Silver-positive NORs are marked by arrowheads. Note the presence of staining in the heterochromatic regions. Overstaining was deliberately performed to identify very small silver-positive regions. Figure 1

was 1.00. Contributing to this relatively low mean value was the absence of silver staining in 64% of the metaphases studied. This lack of silver staining could not be attributed to technical difficulties because only those metaphases s u r r o u n d e d by silver-stained nuclei were scored. Compared with the results obtained from normal bone marrow, the cells from the normal thymus showed marked NOR activity. In these cells, the range of NORs per cell was 4 to 6, and the average n u m b e r of Ag-NORs per metaphase was 5.00. In addition, silver staining was apparent in 100% of the metaphases. For both tissue types, the tumor cells showed a higher degree of silver staining than did their normal counterparts. Two clones (A and B) from both the thymus-derived and bone marrow derived tumors were studied. Both tumor clones from the

58

Table 1

K . C . A r d e n et al.

F r e q u e n c y of active NORs in n o r m a l b o n e m a r r o w , t h y m u s , and their t u m o r - d e r i v e d m e t a p h a s e s in AKR/J mice. No. of cells analyzed

Range of Ag-NORs

Ag-NORs per metaphase

Ag-NORs per chromosome

Percent metaphases with Ag-NORs

Thymus-derived tumor Clone A Clone B Normal thymus

50 50 50

6-8 6-9 4 6

7.66 7.42 5.00

0.95 0.93 0.83

100 100 100

Bone marrow derived tumor Clone A Clone B Normal bone marrow

50 50 50

4-6 4-6 0-3"

5.10 5.36 1.00

0.85 0.89 0.16

100 100 36

Cell type

" OnLy two metaphases showed three NOR-positive chromosomes.

b o n e m a r r o w had 4 - 6 Ag-NORs per cell and m e a n s of 5.10 and 5.36 Ag-NORs per m e t a p h a s e , r e s p e c t i v e l y . In each clone, 100% of the m e t a p h a s e s w e r e stained w i t h silver. T h e t w o c l o n e s d e r i v e d from the T-cell t u m o r averaged 7.66 and 7.42 Ag-NORs per m e t a p h a s e , w i t h ranges of 6 - 8 and 6 - 9 Ag-NORs per m e t a p h a s e , and 100% of the m e t a p h a s e s p r e a d s w e r e silver stained. As the n u m b e r of N O R - b e a r i n g c h r o m o s o m e s in a n o r m a l m o u s e cell is 6, the h i g h e r n u m b e r s of Ag-NORs in the two T-cell c l o n e s can be attributed to the p r e s e n c e of a h y p e r d i p l o i d c h r o m o s o m e c o m p l e m e n t , i n c l u d i n g a d d i t i o n a l c o p i e s of the N O R - b e a r i n g c h r o m o s o m e s (Pathak, in preparation). W h e n the data w e r e statistically a n a l y z e d , significant differences w e r e a p p a r e n t (Table 2). T h e two n o r m a l tissues s h o w e d v e r y different levels of N O R activity. T h e l e v e l of a c t i v i t y in the b o n e m a r r o w was c o n s i d e r a b l y l o w e r t h a n that in the n o r m a l t h y m u s tissue. For b o n e m a r r o w and t h y m u s cells, the i n c r e a s e in N O R a c t i v i t y in the m a l i g n a n t cells was statistically significant. The level for the B-cell t u m o r was l o w e r t h a n that for the T-cell tumor, p a r a l l e l i n g our finding in the n o r m a l tissues.

DISCUSSION A c o m p a r i s o n of s i l v e r - s t a i n e d m e t a p h a s e s in two n o r m a l m u r i n e tissues and t u m o r s d e r i v e d f r o m t h e s e tissues r e v e a l e d significant differences. First, silver s t a i n i n g of n o r m a l b o n e m a r r o w cells r e v e a l e d l o w n u m b e r s of Ag-NORs per m e t a p h a s e as w e l l as a c o n s i d e r a b l e fraction of n o n s t a i n i n g m e t a p h a s e s . It s e e m s u n l i k e l y that the n o n s t a i n i n g cells w e r e not p r o d u c i n g rRNA. It is possible that the level of a c t i v i t y of the r R N A genes was b e l o w the detectable t h r e s h o l d for this t e c h n i q u e . The meta-

Table 2

Results of statistical analysis u s i n g the M a n n - W h i t n e y test

Tissues compared

Z value ¢~

Thymus vs. bone marrow T-cell tumor vs. thymus B-cell tumor vs. bone marrow T-cell tumor vs. B-cell tumor

17.12 ~' 9.19 ~' 8.86 ~ 8.75 b

'~Z a nonparametric analogue to the Student's t-test. ~'Highly significant.

Ag-NOR Activity in Murine Tissues

59

phases from the t h y m u s had comparatively high numbers of Ag-NORs per cell, indicating that tissue-specific differences in rRNA gene activity do exist. W h e n the NOR activity of the tumor cells was compared with that of the normal cells, NOR activity was significantly higher for both the thymus-derived T-cell tumor cells and the bone marrow-derived B-cell tumor cells than in the normal cells. It is possible that this apparent increase in 18S and 28S rRNA production is a general property of tumor cells. Kano-Tanaka and Tanaka [19] found changes in the pattern of the distribution of silver-stained NORs in transformed rat glial cells compared with normal glioblasts, indicating that changes in silver-stained NORs may be related to malignancy. Leukemia is characterized by errors in cell maturation. It has been suggested that rather than being a disease of aberrant differentiation, leukemia may reflect a clonal selection of cell types that are normally infrequent and transitory [20]. It is possible that the increase in silver-stained NORs seen in leukemic bone marrow reflects an increase in a cell type found infrequently in normal bone marrow, as evidenced by the presence of a few normal bone marrow cells with higher numbers of NORs. It is also possible that the increase in silver staining seen in the leukemic bone marrow is a characteristic of malignancy in general. Although the m e c h a n i s m for the differential activity of rRNA genes remains u n k n o w n , the differences in silver staining in relation to malignancy point toward the technique's potential usefulness as a diagnostic tool. Differences in silver staining may prove particularly useful in detecting malignant cells that appear normal cytogenetically. These cells can be distinguished from normal cells on the basis of differential rRNA gene activity. Recent interest in this technique is evidenced by an editorial supporting our original suggestion that silver staining may prove useful diagnostically and r e c o m m e n d i n g that pathologists explore this possibility [21]. Silver staining has been used on paraffin-embedded tissues from patients with Hodgkin's disease to detect malignant cells [22] and to stage non-Hodgkin's lymphoma [23]. We have recently used computer-assisted image analysis to quantitate the a m o u n t of silver staining in interphase cells of normal and leukemic h u m a n bone marrow samples and found that the data from the interphase cells parallels that obtained from metaphases [24]. This finding indicates that this technique could be easily evaluated by noncytogenetics and could be made available for more general application by clinicians using bone marrow transplants and treating leukemic patients.

This work was supported in part by grants from the John S. Dunn Research Foundation and the M. D. Anderson Annual Campaign Fund. K. C. A. was a recipient of an American Legion Auxiliary predoctoral fellowship during the course of this investigation. REFERENCES

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