Effect of differentiating agents on nucleolar organizer region activity in human melanoma cells

Effect of Differentiating Agents on Nucleolar Organizer Region Activity in Human Melanoma Cells Yan Yongshan and Wayne S. Stanley

ABSTRACT: A human cell line established from a metastatic melanoma had both multiple numerical and structural chromosome aberrations including one to two copies of a submetacentric marker chromosome with an insertion of an active nucleolar organizer region (NOR). Treatment of this cell line with retinoic acid (RA) induced morphologic differentiation and reduced the cellular saturation density concomitant with a significant decrease in Ag-NOR activity. RA-treated cells grown in the absence of this differentiating agent, however, displayed a re~ turn to normal Ag-NOR activity, indicating the effect of this chemical on ribosomal genes is reversible.

INTRODUCTION Normal cellular proliferation and differentiation depend on the coordinated regulation of gene expression [1]. Gene mutation or transposition resulting from chrom o s o m e rearrangements may serve to disrupt normal regulatory m e c h a n i s m s and lead to neoplastic growth [2]. In Burkitt l y m p h o m a with the t(8;14), for example, the c-myc oncogene, w h i c h is normally quiescent in B lymphocytes, is activated as a result of its transposition into the transcriptionally active Ig gene co m p l ex [3]. Because ribosomal RNA (rRNA) synthesis is required for cell proliferation, structural c h r o m o s o m e rearrangements involving nucloelar organizer regions (NORs) could result in the activation of genes placed in juxtaposition to NORs and be biologically important in determining tumorigenic properties. Recently, Takahashi et al. (4) reported that the c-abl oncogene in a rat leukemia cell line appeared to be activated as a result of the translocation of a NOR. The activation of genes by c h r o m o s o m e rearrangement, however, may not be irreversible. Many agents have been demonstrated to induce cellular differentiation and to modulate gene expression including rRNA synthesis in tumor cells [5]. In a m e l a n o m a cell line established in our laboratory, we found an insertion of an NOR to a marker chromosome. Because NOR activity, which can be cytologically determ i n ed with silver staining (Ag-NOR), is d e p e n d e n t on cell type and stage of differ-

From the Department of Pathology and Laboratory Medicine, Division of Cytogenetics~Medical University of South Carolina, Charleston. Permanent address of Y. Y. is Institute of Genetics. Academia Sinica, Beijing, China. Address reprint requests to: Dr. Wayne S. Stanley, Division of Cytogenetics, Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston. SC 29425. Received July 15, 1987; accepted September 28, 1987.

253 ¢) 1988 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017

Cancer Genet Cytogenet 31:253 262 (19881 0165-4608/88/$03.50

254

Y. Yongshan and W. S. Stanley

entiation in normal cells [6], we investigated the Ag-NOR activity in this cell line following treatment with differentiating agents.

MATERIALS AND METHODS Cells H u m a n cell line HXG-2 was established in this laboratory from an athymic n u d e mouse heterotransplant of a m e l a n o m a from a male with metastases to the leg. Cells were grown in Dulbecco's modified Eagle's m i n i m u m essential m e d i u m (DMEM) s u p p l e m e n t e d with 15% fetal calf serum (FCS) and 50 ~tg/ml gentamycin (complete medium). Cultures were incubated at 37°C in a CO2-enriched air atmosphere.

Cytogenetic Analysis Cell cultures were treated with 0.01 p,g/ml Colcemid for 45 minutes prior to harvesting for cytogenetic analysis. Trypsin-released cells were hypotonically treated with 0.075 M KCL for 25 minutes and then fixed three times in methanol/acetic acid (3:1). Slides were prepared by routine air-drying methods and heated overnight at 50°C before treatment with trypsin and staining with Wright stain for Gbanding. For C-banding, 3-10-day-old slides were treated for 40 minutes in 0.1 N HC1, 30-60 seconds in saturated barium hydroxide at 58°C, and then 2 hours in 3X SSC at 58°C as previously described [7]. Slides were dried through alcohol and stained in 10% Giemsa. For Ag-NOR staining, 2-6-day-old slides were treated with five to six drops of 50% Ag-NO3 and one drop of 3% formalin. Slides were coverslipped and incubated in a moisture chamber at 37°C for 18-26 hours before washing in distilled water and staining with Giemsa [8]. The n u m b e r of Ag-NORs per cell and the chromosomes bearing Ag-NORs were recorded and statistically analyzed by the Student's t-test.

Differentiating Agents Dimethylsulfoxide (DMSO), retinol (Rt), and retinoic acid (RA) were all purchased from Sigma Chemical Co. A 1.0 X 10 3 M stock solution of Rt was prepared by first dissolving retinol in 0.5 ml ethanol and then diluting with complete medium. A 1.0 X 10 :~ M stock solution of RA was prepared by dissolving retinoic acid in two drops of DMSO followed by 0.5 ml ethanol and diluting in complete medium. Stock solutions were stored at -20°C.

Growth Curves To obtain growth curves, 5.8 X 10 ~' cells were seeded into 35-mm diameter tissue culture dishes containing 1.0 ml complete m e d i u m with or without RA. The cell number in each of three dishes was determined by hemocytometer counts of trypsinized cells at different time intervals. In the RA-treated group, HXG-2 cells were grown in RA-containing m e d i u m for 27 days prior to seeding for growth measurements.

NORs in H u m a n Melanoma

255

RESULTS

Cytogenetic Analysis of Cell Line HXG-2 A modal c h r o m o s o m e number of 69 was determined from an analysis of 360 metaphase cells. Fifteen cells were karyotyped. As illustrated in Figure 1, this cell line had multiple numerical and structural aberrations. The transposition of a NOR into a submetacentric marker c h r o m o s o m e was identified by sequential staining methods (Fig. 2) and all cells had at least one copy of this marker.

Morphologic and Cellular Response of HXG-2 Cells to Differentiating Agents Distinct morphologic changes were i n d u ced in HXG-2 cells following 3-6 days treatment with either RA, Rt, or DMSO. As sh o w n in Figure 3, these cells have a more flattened appearance and display extensive bipolar and branched dendritic processes characteristic of normal melanocytes. The growth cycle of untreated and RA-treated HXG-2 cells was determined and is illustrated in Figure 4. Although the population-doubling kinetics during the log phase of the growth curve were paral-

Figure 1

G-banded karyotype of a modal HXG-2 cell illustrating numerical and structural aberrations. Arrows indicate p arm deletions on chromosome 2 and Robertsonian translocalions involving chromosome 13.

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lel, the saturation d e n s i t i e s d u r i n g the plateau phase w e r e m a r k e d l y different. The cell d e n s i t y for u n t r e a t e d cells was 1.87 X 10 ~ cel]s/cm 2, w h i l e RA-treated cells had a d e n s i t y of 1.33 X 10" cells/cm 2, r e p r e s e n t i n g a 29% decrease.

Effect of Differentiating Agents on A g - N O R A c t i v i t y A m e t a p h a s e cell illustrating A g - N O R activity on a c r o c e n t r i c c h r o m o s o m e s as w e l l as on the long arm of a s u b i n e t a c e n t r i c marker c h r o m o s o m e is s h o w n in Figure 5. U n t r e a t e d HXG-2 cells h a v e all average of 6.22 Ag-NORs per cell, with 1.78 AgNORs identified oll c o p i e s of the marker c h r o m o s o m e with a NOR insertion. Foll o w i n g t r e a t m e n t with 1.0 x 10 "M RA for 72 hours, there was a significant decrease in total A g - N O R activity, but e s s e n t i a l l y no d i f f e r e n c e in the marker chrom o s o m e A g - N O R activity. After 17 days of treatment, total Ag-NOR activity was decreased by 73% and that on the marker c h r o I n o s o m e by 65%. T r e a t m e n t with 1.0 X 10 ~M RA for 14 days d e c r e a s e d total Ag-NOR a c t i v i t y by 86% and that on the marker c h r o m o s o m e by 85%. T r e a t m e n t of HXG-2 cells with 0.1% D M S O alone, 1.0

NORs in H u m a n M e l a n o m a

257

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Figure 3 Morphologic changes in HXG-2 cells induced by differentiating agents. (A) tintreated cells,(B) ].OX]0 ~MRt for 27 days,(C) 1.0X 10 ~I'v1RAfor27 days,[D) 0.1% DMS() for 15 days.

X 10 ~M Rt alone, and 1.0 X 10 "M Rt or 1.0 X 10 :'M Rt with DMSO significantly decreased total Ag-NOR activity but not to the level observed with RA. These results indicate that the decrease ill the number of Ag-NORs per celt was dependent oi1 the type of differentiating agent, dose, and length of treatment (Table 1). However, as shown in Figure 6, RA treatment does not induce an irreversible suppression of NOR activity. When treated cells were grown in RA-free medium, NOR activity remained low for a short period of time but then increased with continued cultivation in the absence of the differentiating agent. DISCUSSION

Cell line HXG-2 w a s established from a metastatic lesion. In addition to several numerical and structural c h r o m o s o m e aberrations, it had one to two copies of a

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submetacentric marker chromosome with a NOR insertion. In Giemsa-stained preparations, a copy of this chromosome had a n o n s t a i n i n g region subterminal to the qarm telomere. Sequential Giemsa and Ag-NOR staining demonstrated this segment to contain an active NOR. In normal D and G group chromosomes, NORs are flanked by short-arm and satellite heterochromatin. In this marker chromosome, however, there were no C-band positive bands on either side of the NOR, suggesting that the transposition of this material resulted from a chromosome insertion of just the NOR rather than the translocation of an acrocentric short arm. In the h u m a n melanoma cell line MeWo, Holden et al. [9] recently described homogeneously staining regions (HSRs) that were derived from rearrangements and duplications of NORs. They suggested these regions may provide a selective advantage to cells containing them. Other studies with this cell line [10] have demonstrated that HSR-containing cells are more tumorigenic and metastatic when transplanted into athymic n u d e mice. Because actively dividing cells require rRNA synthesis, amplification or enhanced expression of rDNA might provide a selective growth advantage in tumor cells. However, most investigations of leukemias and solid tumor cells have shown equal or reduced Ag-NOR staining [11-17], indicating that the level of NOR activity alone may be of secondary importance. Other studies with leukemias and lymphomas [18], sarcomas and carcinomas [19], testicular cancers [171, and meningiomas [20] have demonstrated structural chromosome rearrangements resulting in the transposition of NORs. These, in addition to the NOR rearrangements in melanoma

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Table 1

NOR activity in untreated and treated HXG-2 m e la nom a

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0 RA(10 6M) 72 hr RA{10 6M) 17 day RA(10 5M) 75 hr RA(10 5M) 14 day DMSO (0.1%) 15 day Rt[10 6M) 72 hr Rt(10 6M) + 0.1% DMSO 72 hr Rt(10 6M) + 0.1% DMSO 15 day Rt(10 5M)+1% DMSO 46 hr

63 68 65 66 66 62 63 77

1.78 1.71 0.63 1.70 0.27 1.65 1.77 1.52

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NORs in H u m a n Melanoma

261

described by Holden et al. [9] and in the present paper, indicate this type of aberration is not u n c o m m o n . Activation of the c-abl oncogene following the translocation of a NOR in a rat leukemia cell line [4] and the enhanced tumorigenic potential of a subline of MeWo m e l a n o m a cells containing rearranged and amplified NORs [10] suggest that c h r o m o s o m e aberrations involving NORs could be biologically important in tumor initiation and/or progression. The modulation of c - m y c expression in Burkitt's l y m p h o m a by a genetic position effect [3] could be a paradigm of a more c o m m o n and general p h e n o m e n o n of gene activation by transposition of genes into or adjacent to transcriptionally active loci. In our case. the NOR on the marker c h r o m o s o m e may activate a gene(s) and thereby effect tumorigenic or metastatic properties. If so, these phenotypes might be altered by modulating gene expression in the controlling locus. Because NOR activity can be determined cytologically, we first ascertained the effect of differentiating agents on these rDNA loci. RA, Rt, and DMSO were all able to significantly reduce AgNOR activity on all c h r o m o s o m e s in a time- and dose-dependent manner. Additional studies with RA demonstrated that the decrease in Ag-NOR activity was, however, not permanent. The removal of RA from the culture m e d i u m of treated cells led to a return of normal Ag-NOR activity. In the promyelocytic leukemia cell line HL-60, Reeves et al. [211 demonstrated that DMSO-induced differentiation was a c c o m p a n i e d by a suppression of rDNA transcription and suggested from this that Ag-NOR activity in these cells is an indicator of the differentiation/maturation state. In our studies with m e l a n o m a cells, the differentiating agents used i n d u ced morphologic changes and reduced the cellular saturation density, a characteristic of a more differentiated state [22], in parallel with a decrease in Ag-NOR activity. These studies with cell line HXG-2, then, suggest that Ag-NOR activity may provide a cytologic marker for i n d u c e d differentiation and facilitate further studies on the effect of gene m o d u l a t i o n on the tumorigenic and/or metastatic p h en o t y p e in human m e l a n o m a cells. Supported by a grant from the Elsa U. Pardee Foundation. The authors thank Tasneem Khan for technical and Judy Futral for typing assistance.

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