Tumorigenicity, major histocompatibility antigens, and karyotypes of interspecific hybrids between mouse neuroblastoma and rat glioma or liver cells

Tumorigenicity, Major Histocompatibility Antigens, and Karyotypes of Interspecific Hybrids Between Mouse Neuroblastoma and Rat Glioma or Liver Cells Haruhiro Higashida, Kyoko Kano-Tanaka, Tatsuya Tanaka, Hiroko Fukami, and Shunnosuke Natsume-Sakai

Five interspecific hybrids of mouse neuroblastoma with rat glioma (NG108-15, 140-3, and 141-B) or with nontransformed rat liver cells (NBr-IOA and NBr-2OA) were examined for major histocompatibility (MHC) antigens and tumorigenicity in comparison with their karyotypes. Both mouse and rat MHC antigens were present in each hybrid population, as determined by a simple cytotoxicity test. All five hybrid cell lines produced tumors in athymic nude mice with varied take incidences. Four hybrid cells, NG108-15, 140-3, NBr-IOA, and NBr-2OA, were highly tumorigenic. Their karyotypes were characterized by a higher modal chromosome numbers than would be expected from the fusion of parent cells in which at least one parent contained an increased number of chromosomes. In contrast, 141-B cells, with massive loss of chromosomes from both malignant parents, were weakly tumorigenic. The results suggest that the retention of marker chromosomes as well as double minutes (DMs) or micrachromosomes of neuroblastoma origin may be required for expression of malignancy in these hybrid cells. The survival time of tumor-bearing mice also varied within the five cell lines, but it was significantly short in NG108-15, which yielded lung metastases in

ABSTRACT:

the host animals.

INTRODUCTION

A number of hybrid cell lines have been established to analyze the regulation of malignancy [1]. However, there is no general rule for estimating from chromosome constituents whether or not a given hybrid cell is malignant, unless it is determined by examining cellular tumorigenicity in athymic nude mice [2]. Although interspecific mouse neuroblastoma x glioma hybrid NG108-15 cells may serve as typical model cells with many neuronal properties [3-5], no precise analysis of their tumorigenic p r o p e r t i e s or p h e n o t y p i c e x p r e s s i o n of m o u s e or rat m a j o r h i s t o c o m p a -

From the Cancer Research Institute, (H.H., S.N.-S.), Kanazawa University, Kanazawa; the Aichi Cancer Center Research Institute (K.K.-T., T.T., H.F.), Nagoya; and the Division of Neurobiology (K,K.-T., T.T.)

Awazu Neuro-Sanatorium, Gamagohri, Japan. Address requests for reprints to Dr. Kyoko Kano-Tanaka, Awazu Neuro-Sanatorium, Division of Neurobiology, 54-3 Kitashinden, Gamagohri, Aiehi 443-01, Japan.

Received April 4, 1984; accepted July 9, 1984.

219 © 1985 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017

Cancer Genetics and Cytogenetics 16, 219-227 (1985) 0165-4608/85/$03.30

220

H. Higashida et al. tibility (MHC) antigens has been performed in this cell line. In addition to NG10815 cells, four interspecific hybrid cells derived from mouse neuroblastoma and rat glioma or liver cells have been cytogenetically analyzed [6]. These hybrid cells possessed characteristic karyotypes, with marker chromosomes originating from the mouse neuroblastoma cells. All hybrid cells also contained aberrant chromosomes, such as double minutes (DMs) and microchromosomes. The role of DMs in the biology of cancer cells still remains to be solved [7, 8]. Thus, it is important to examine tumorigenicity of hybrid cells in view of the concern about chromosome constituents and the significance of DMs or microchromosomes. We describe both mouse and rat MHC antigens expressed on cell surfaces of five hybrid cells tested; tumors were formed in athymic nude mice inoculated with the hybrid cells. Tumor take incidence of these hybrids was then analyzed in connection with their karyotypes.

MATERIALS AND METHODS

Cells and Culture Methods The five hybrid cell lines used here were described previously [4-6]. The cells were cultured as described previously (5,9). The passage numbers of these hybrid cells are listed in Table 3. The C-banding technique (formamide-SSC) [10], applied for scoring metaphases of rat-mouse hybrid cells [11], was used to distinguish chromosomes of mouse and rat origin in all cell lines involved, as reported [11].

Tumorigenicity The ability of N18TG-2, C6BU-1, and NG108-15 hybrid cells to form tumors was determined after inoculating them subcutaneously into newborns (3-5 days of age) of the A/J mouse or the Wistar-King-Aptekman (WKA) rat. Congenital athymic nude mice (nu/nu BALB/c) were used for tumorigenicity testing of five hybrid and two parental cells, and were obtained from the Institute for Medical Science, University of Tokyo. Nude mice of either sex were used and were 8-9 weeks of age at the time of subcutaneous inoculation with 106 cells of each cell line [12]. Mice were observed every other day for tumor development until the 80th day. Selected mice from each group were autopsied. For light microscopic examinations, various organs fixed in 10% buffered formalin were routinely processed by paraffin embedding and sectioning. Sections of 10 ~ thickness were stained with hematoxylineosin.

Antisera A potent alloantiserum for H-2Kk was obtained from the B10D2 strain of mice, as described [13]. A xenogeneic antiserum specific for rat spleen cells was raised in C3H/He mice. C3H/He mice were hyperimmunized by repeated injections of 107 spleen cells from WKA rats. Mice were bled by heart puncture at 10 days after the seventh immunization. The sera were pooled and heat-inactivated at 56°C for 30 min. The antiserum was preabsorbed with an excess of spleen cells of ACA rats to remove antibody against minor histocompatibility antigens. Another xenogeneic antiserum to mouse spleen cells was raised in rats hyperimmunized with spleen cells of C3H/He mice by the procedure described above, and treated with C3H.SW spleen cells.

2 21

Tumorigenicity of Neuroblastoma Hybrids

Cytotoxic Test About 108 spleen or h y b r i d cells in 0.1 ml Dulbecco's modified Eagle's m e d i u m (DMEM) were incubated at 37°C for 1 hr with an equal a m o u n t of serially d i l u t e d antiserum and then w a s h e d 3 times at 4°C with DMEM. The w a s h e d cells were collected by centrifugation (300 g for 5 min), and were r e s u s p e n d e d with 100 ~.1 of DMEM and replaced w i t h an equal volume of fivefold diluted rat serum (complement) at 37°C for 30 min. Rat serum was freshly obtained and preabsorbed w i t h spleen cells of either C3H/He mice or WKA rats. After incubation, cell suspensions were placed on ice and the viable cells were counted with a h e m o c y t o m e t e r by the trypan blue dye exclusion method. RESULTS Five hybrid cell lines grew vigorously in vitro, with an average doubling time between 24 and 48 hr. All hybrid cells d i d not show contact inhibition in culture, though the final cell density at the late phase of cultures {7-10 days after plating) did not exceed 5 x 1 0 4 cells/cm 2.

MHC Antigen Analysis The expression of species-specific MHC antigens in five h y b r i d cell lines was measured by the cytotoxicity test. Figure 1A shows that NG108-15 and B10A mouse spleen cells were lysed by alloantiserum to B10A spleen cells, whereas spleen cells of WKA rats were not. Conversely, mouse anti-rat (WKA) spleen serum was cytotoxic to either rat spleen or NG108-15 cells, but not to spleen cells of B10A (Fig. 1B). This demonstrates that NG108-15 h y b r i d cells express MHC gene products of both mice and rats. The other four hybrid cell lines were also susceptible to cytotoxic actions of mouse anti-rat serum and alloantiserum (anti H-2K k) (Table 1). Differentiated NG108-15 hybrid cells that had been treated with 1 mM dibutyryl cyclic adenosine m o n o p h o s p h a t e (cAMP) for 5 days were also cytotoxic with either antiserum. The results indicate that all hybrid cell lines tested express MHC antigens of both mice and rats on their cell surface membranes.

Figure 1 Cytotoxic responses of NG108-15 and BIOA mouse or WKA rat spleen cells to mouse anti-H-2Kk (A) or mouse anti-rat (B) antiserum. A quantity of 107 cells was incubated with antiserum diluted as described in "Materials and Methods," and viable cells were counted. ( • , O ) Percent dead cells incubated with Fab' of each antiserum alone and cornplement alone (&,A). ~100

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H. H i g a s h i d a et al.

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MHC antigen expression in mouse

x rat h y b r i d cells a Antisera BIOD2 antiB10A (antiH-2K k)

C3H/He anti-WKA rat Hybrid cell NG108-15 140-3 141-B NBr-10A NBr-20A

Origin

x 10

N18TG-2 b × C6BU-1 c Differentiated d N4TG-3 b x C6BU-1

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Tumorigenicity Test T u m o r i g e n i c i t y w a s first e x a m i n e d w i t h N G 1 0 8 - 1 5 h y b r i d a n d p a r e n t a l c e l l l i n e s i n n e w b o r n s y n g e n e i c a n i m a l s ( T a b l e 2). A/J m i c e i n o c u l a t e d s u b c u t a n e o u s l y w i t h 106 N18TG-2 cells on the back produced tum ors in 100% a nim a ls , with a s urviva l tim e of 36 d a y s i n 5 0 % of t h e a n i m a l s , w h e r e a s t h e m i c e w h o r e c e i v e d 4 x 106 N G 1 0 8 15 h y b r i d cell i n o c u l u m ,

Table

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T u m o r i g e n i c i t y a s s a y of h y b r i d s a n d p a r e n t a l c e l l s i n n e w b o r n m i c e a n d r a t s o r in n u d e mice A/J mice

Cell line

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N u d e mice b

No. of cells inoculated ~

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x × × x x × x

106 106 107 106 106 106 106

aCells, in a volume of 0.1 ml, were injected intraperitoneally into newborn animals and subcutaneously into nude mice. bData are plotted in Fig. 2, and survival time is calculated from the curves. CTumor incidence is expressed as no. of tumors/no, of mice inoculated. dND, not determined.

223

Tumorigenicity of Neuroblastoma Hybrids

survival time of 39 days. C6BU-1 rat glioma cells gave a complete take incidence in syngeneic newborn WKA rats, with a survival time of 25 days. No tumor formation was observed in newborn rats during the 50 days after inoculation with NG108-15 cells, though temporary small nodules were observed in the first 2-3 weeks in several rats. Tumor incidence was examined in congenic athymic nude mice of BALB/c background by inoculating with 108 hybrid and parental cells (Fig. 2). All hybrid cell lines produced tumors in athymic hosts with varied take incidences from 100% with NBr-10A and NG108-15 to 44% with 141-B cells. Fifty percent survival of nude mice inoculated with NG108-15 hybrid cells (16 days) was significantly shorther than the half-life of 42-51 days with the three other cell lines (NBr-2OA, 140-3, and NBr-10A). The half-life of 141-B recipients was 78 days, a value much longer than that of others.

Karyologic Examination The karyologic analyses in all hybrid cell lines concerned here have been reported [6, 14]. It revealed that their karyotypes are characterized by a complex heteroploid nature with distinct marker chromosomes of neuroblastoma origin. DMs and microchromosomes observed in neuroblastoma cells were also demonstrated in all hybrid cells. Further analysis by C-banding was attempted to express the chromosome contributions of the mouse or rat (Fig. 3). Table 3 shows the chromosome constitutions at the time tumorigenicity was tested (2-5 passages after the previous chromosome examination). Chromosome numbers, both in mode and range, of the five hybrid cells appeared to be fairly stable throughout these passages. The modal number of NG108-15, 140-3, NBr-10A, and NBr-20A cells ranged from 124 to 158, which represents 100%-128% of the expected number of fused cells, whereas the chromosome number for 141-B was 98 (77%), indicating chromosome segregation by fusion. Although the chromosome constitution of these hybrids was cell-line-specific, as seen from the contribution patterns of both parents, the former four hybrid cells showed an increase of chromosome numbers in either partner which is malignant in three cell lines. NBr-IOA cells may be exceptional, as the dominant contribution Figure 2 Survival curves of nude mice with NG108-15 (~), NBr-20A (O), N18TG-2 (O), 140-3 (11), NBr-IOA ( • ), C6BU-1 (0), and 141-B ([~) cells. Ten to 26 mice of each group shown in table 2 were inoculated subcutaneously on the back with 108 cells. 10011

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F i g u r e 3 C-banded metaphases of two hybrid cell lines (140-3 and 141oB) derived from mouse neuroblastoma N4TG-3 and rat glioma C6BU-1. The 140-3 cell has 141 chromosomes and consists of 22 rat (R) chromosomes and 119 mouse (M) chromosomes. Extra 4 microchromosomes (C-band positive; marked by A) are present. Three DMs (C-band negative) are not shown. The 141-B cell has 88 chromosomes and consists of 28 rat and 60 mouse chromosomes, plus one microchromosome (A}. Mouse chromosomes have strong C-bands, whereas weak or no bands are seen in rat chromosomes. of c h r o m o s o m e s w a s f r o m n o r m a l rat c e l l s a n d m a n y c h r o m o s o m e s f r o m t h e m a l i g n a n t m o u s e c e l l s w e r e lost. N e v e r t h e l e s s , t h i s cell l i n e a p p e a r e d to r e t a i n t h e full n u m b e r of m a r k e r c h r o m o s o m e s as w e l l as D M s a n d m i c r o c h r o m o s o m e s [6]. W h e n t h e c h r o m o s o m e c o n s t i t u t i o n s w e r e c o m p a r e d w i t h t h e take i n c i d e n c e i n t h e n u d e m i c e , t h e s e f o u r h y b r i d s p r o d u c e d t u m o r s w i t h h i g h take i n c i d e n c e . In c o n t r a s t , t h e

Table 3

A n a l y s i s of c h r o m o s o m e c o n t r i b u t i o n s of h y b r i d c e l l s b y the C-banding technique a Chromosome contribution

Cell line

Passage number

Hybrids NG108-15 140-3 141-B NBr-10A NBr-20A

24 12 16 14 11

Total chromosome n u m b e r mode (range) 139 128 98 124 158

(129-144) (120-136) (85-125) (104-156) (148-167)

Rat origin 41 29 29 81 41

(30- 47) c (24- 33) (21- 39) (58-112) (36- 50)

98 99 69 43 117

MCs b

DMs

Mouse origin

Mouse origin

Mouse origin

(85-110) (92-109) ( 5 8 - 90) ( 3 6 - 46) (108-130)

3 4 2 5 1

3 4 1 1 1

(0-4) (1-6) (0-4) (1-7) (0-2)

(0-5) (0-6) (0-2) (0-2) (0-2)

°Average number of 10 hybrid cells observed. See references 6 and 11 for chromosome number of parental cell lines. bMicrochromosomes. CAverage (range).

Tumorigenicity of Neuroblastoma Hybrids

225

141-B cells showed significant loss of chromosome complements from both malignant parents and were weakly tumorigenic.

Histologic Examinations Preparations were made from solid tumors, lung, liver, spleen, kidney, heart, and brain in tumor-bearing nude mice at 21 days after inoculation with NG108-15 hybrid cells. Generally, the tumor cells at the primary inoculation site were ovoid in shape, with a compact cytoplasm and large nucleus (Fig. 4A). The metastatic tumor cells were found in lungs and appeared diffusely over all areas (Fig. 4B). Although a large colony of tumor cells was not formed, there were many small (3-20 cells/ colony) colonies at a density of about 20 colonies/mm 2. No invasion was detectable in other organs.

DISCUSSION There have been many studies on tumorigenicity of interspecific or intraspecific hybrid cells derived from fusion of malignant cells and either malignant or nonmalignant cells [15-26]. However, these results were complex, indicating supression of malignancy in some instances [15-20] and expression of malignancy in others [15-26]. It is, therefore, probable that the expression or suppression of malignancy in hybrids may not exist as a general phenomenon but rather as a specific characteristic of the parent cells, as suggested by Carney et al. [20]. The present results clearly showed that five different hybrid cells derived from mouse neuroblastoma and either tumorigenic rat glioma or nontumorigenic rat liver cell lines were all tumorigenic in athymic nude mice. MHC antigens of both mice and rats were coexpressed in all five hybrid cells, whose chromosome constituents varied between cell lines. This suggests that both species of chromosomes are actively transcribed in the hybrid cells and that their cell surface membranes are a mosaic of the mouse and rat. Tumorigenicity of the present hybrid system was demonstrated to be related to expression of MHC antigens with the use of the athymic nude mouse [2]. When the tumorigenicity was tested in syngeneic animals to reFigure 4 Photomicrograph of a section of solid tumor in the primary site (A) and lungs {B) of nude mice 21 days after subcutaneous inoculation of NG108-15 cells. Arrows indicate metastatic NG108-15 cells (hematoxylin-eosin, × 200}.

226

H. Higashida et al. spective parental cells, NG108-15 was resistant. Although this is only one cell line tested, the presence of the heterologous antigen to the host species was s u p p o s e d to be responsible for this resistance [25]. Furthermore, it was obvious that the high tumor take incidence was observed in hybrid cells that contained a total number of chromosomes more than 100% of the expected n u m b e r by cell fusion. The case of NBr-10A was remarkable, showing a high tumorigenicity despite a great loss of malignant chromosomes. Thus, the retention of the full n u m b e r of marker chromosomes in this hybrid suggests that marker chromosomes m a y play a significant role in tumorigenic expression. However, the possibility that specific chromosome(s) associated with tumorigenicity, as described in h u m a n × mouse hybrid c e l l s Y m a y also be involved in the present h y b r i d system, could not be ruled out. The functional significance of DMs and h o m o g e n e o u s l y staining regions (HSRs) is not presently u n d e r s t o o d [7]. However, in some mouse tumors, DMs were found to be responsible for the tumorigenic capacity of cells that are grown in vivo and in vitro [28]. Quite recently, HSRs and DMs were found to contain amplified copies of cellular oncogenes (c-Ki-ras, c-myc, and N-myc) in m u r i n e and h u m a n tumors [29-33]. Although the amplification of N-myc found in h u m a n neuroblastomas [3133] has not yet been related to mouse neuroblastoma, it w o u l d be of particular interest to determine whether a correlation does exists. A n y positive correlation between the presence of DMs and tumorigenic potentials in the hybrid cells tested could not be demonstrated, but the significance of DMs for the expression of malignancy in these cells might still be considerable. A m a i n reason w h y take incidence was low and lifespan of the tumor-bearing mice was extended for 141-B cells a p p e a r e d to be due to a massive loss of chromosomes of both malignant parents. Because m y c o p l a s m a c o n t a m i n a t i o n causes delayed tumorigenicity [34], we examined 141-B cells by transparent electron microscope and found t h e m to be m y c o p l a s m a free. The very short survival of NG108-15-bearing n u d e mice is probably due to metastases of cells to the lungs (Fig. 4). As metastatic cell clones can occur as a result of acquired genetic alterations [35], the metastatic properties of NG108-15 cells w o u l d be a n e w phenotype, observed in neither parental nor other h y b r i d cells derived from neuroblastoma cells. NG108-15 hybrid cells with metastatic properties may provide a potential m o d e l for h u m a n neuroblastoma, if some of the disadvantages of karyotypic instability are carefully controlled.

REFERENCES

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