Chromosome 14 alteration is associated with increased collagenase expression and the metastatic potential of murine melanomas

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Chromosome 14 Alteration Is Associated with Increased Collagenase Expression and the Metastatic Potential of Murine Melanomas Bhavana J. Dave, Rakesh Singh, Isaiah J. Fidler, and Sen Pathak

ABSTRACT: The purpose of this study was to correlate abnormalities in chromosome 14 with the invasive metastatic phenotype of K-1 735 murine melanoma cells. Low metastatic K-1735 clone 10 and clone 23 cells were transfected with either basic fibroblast growth factor (bFGF), Kaposi's fibroblast growth factor (kFGF), or c-H-ras gene. A high number of bFGF- and H-ras-transfected cells exhibited chromosome i4 rearrangements. These cells also had increased expression of collagenase IV, The kFGF-transfected cells were highly metastatic but did not have increased expression of collagenase type IV, nor abnormalities in chromosome 14. The data imply that karyotypic changes in chromosome 14 are associated with increase expression of collagenase type IV. © Elsevier Science Inc., 1996

INTRODUCTION Multiple sequential steps are involved in the progression of p r i m a r y n e o p l a s m s to metastasis [1]. The genetic changes responsible for the d e v e l o p m e n t of metastatic p h e n o t y p e s are not well defined, due to the c o m p l e x nature of this process. In our earlier studies, we observed rearrangements of c h r o m o s o m e 14 in the metastatic clones of m e l a n o m a cell line K-173 5, but not in the nonmetastatic clones [2]. To determine w h e t h e r this is a c o m m o n phen o m e n o n existing in other metastatic neoplasms, we studied chromosomal alterations in B16 melanoma, a m u r i n e l y m p h o m a , a reconstituted prostate cancer, and CT26 colon carcinoma cells, and found alterations in chromosome 14 in each of these cancer cell lines [3]. Recent reports from our group suggest that stable transfection of growth factors and oncogenes can alter the metastatic capabilities of nonmetastatic K-1735 clones [4-6]. Transfection with cloned c-H-ras oncogenes has been shown to induce the metastatic p h e n o t y p e in some tumorigenic but nonmetastatic cells [6-9]. How the transfection of this oncogene regulates the conversion of a nonmetastatic cell

From the Department of Cell Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas. Address reprint requests to: Professor S. Pathak, Cellular Genetics Laboratory, Box 181, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, U.S.A. Received March 5, 1996; accepted May 13, 1996. Cancer Genet Cytogenet92:66-72 (1996) © Elsevier Science Inc., 1996 655 Avenue of the Americas. New York. NY 10010

to a metastatic one is not known. Whether these behavioral changes are due to different cytogenetic abnormalities that involve chromosome 14 is also not known. To investigate this, we designed experiments using two different low-metastatic clones (C-10 and C-23) of the K-1735 m u r i n e m e l a n o m a [10]. The cells were stably transfected w i t h either basic fibroblast growth factor (bFGF), Kaposi's fibroblast growth factor (kFGF), or c-H-ras (H-ras). We det e r m i n e d the relationship between alterations in chromosome 14 and expression of collagenase type IV. We show that alterations in c h r o m o s o m e 14 are associated with increased expression of collagenase type IV, but not necessarily with metastasis. MATERIALS AND METHODS Cells and Culture Conditions The K-1735 C-10 and C-23 m e l a n o m a cells were the gift of Dr. Margaret L. Kripke, The University of Texas M. D. A n d e r s o n Cancer Center [10]. The C-10 cells were tumorigenic but not metastatic, whereas C-23 cells were low metastatic [11]. To determine the role of expression of angiogenic factors bFGF and kFGF and oncogene H-ras, we transfected these cell lines. Briefly, for kFGF, cells were transfected with pCNECEB1 vector containing the kFGF gene. Control neo cells were transfected with pCNCEB8 (the gift of Dr. Marc E. Lippman, Lombardi Cancer Center) [12]. For bFGF, cells were t r a n s d u c e d w i t h Md-bFGF (the gift of Dr. Ruth Halaban, Yale University) [13]. For H-ras, cells were transfected w i t h pSV2neoEJ-ras m a m m a l i a n

0165-4608/96/$15.00 PII S0165-4608(96)00160-4

Chromosome 14 and Murine Metastatic Melanomas expression vector [6]. After transfection, all cells were selected in G418 sulfate containing media, and colonies were pooled. The cell lines were used in further studies. All cell lines were free of mycoplasma and murine viruses (M. A. Bioproducts, Walkersville, MD). All tumor cell lines were maintained in plastic flasks in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, sodium pyruvate, nonessential amino acids, L-glutamine, and 2-fold vitamin solution (GIBCO, Grand Island, NY), incubated in 5% CO2 and 95% air at 37°C. The cultures were free of mycoplasma and the following murine viruses: reovirus type 3, pneumonia virus, K virus, Theiler's encephalitis virus, Sendal virus, minute virus, mouse adenovirus, mouse hepatitis virus, lymphocytic choriomeningitis virus, ectromelia virus, and lactate dehydrogenase virus (as assayed by M. A. Bioproducts, Walkersville, MD). Cultures were maintained for no longer than 6 weeks after recovery from frozen stock. All in vivo studies and cytogenetic analyses were carried out with cultures at six to 10 passages to minimize problems of biologic stability [1].

Animals Specific pathogen-flee female mice of the inbred strains of syngeneic C3H/HeN were purchased from the Animal Production Area of the NCI-Frederick Cancer Research Facility (Frederick, MD). The mice were maintained under specific pathogen-flee conditions and used for experiments at 6-8 weeks of age. Animals were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the United States Department of Agriculture, Department of Health and Human Services, and National Institutes of Health.

Tumor Cell Injection Tumor cells were harvey;ted from subconfluent cultures by incubation with 0.25% l:rypsin and 0.02% EDTA solution for 1 min at 37°C. The cells were dislodged from the plastic flasks, washed in cold (4°C) 5% complete minimal essential medium (GIBCO), centrifuged at 4°C, and resuspended in Ca ++. and Mg+÷-free Hank's balanced salt solution (HBSS) at a concentration of 1 × 108 cells/ml. Only single cell suspensions of greater than 90% viability, as determined by trypan blue exclusion, were used for injection. A group of five C3H/HeN mice cell lines/site were injected subcutaneously (s.c.) or intraveneously (i.v.) with 2 x 105 cells in 0.2 ml cf HBSS. The mice were injected with either ceils of the control nontransfected cell line, control neo+--transfected cells, kFGF-transfected cells, bFGF-transfected cells, or H-ras transfected cells. The growth of s.c. tumors was monitored twice weekly by examination of the mice and measurement of tumors with calipers. The mice were killed 15 to 50 days after injection, depending on the cell line injected. The lungs were aseptically removed, and processed for in vitro culture for karyotypic analysis. To facilitate the counting of tumor colonies, lungs were rinsed in water and fixed in Bouin's solution for 24 h. The surface tumor colonies were

67

counted through a dissecting microscope. Sections of the lungs were stained with hematoxylin and eosin to confirm that the colonies were of melanoma and to monitor the presence of micrometastases. The in vitro established cultures were analyzed for cytogenetic abnormalities and collagenase production.

Chromosome Preparation and Banding Analysis Cultured cells were fed 24 hr prior to harvest. The cells were dislodged from the flask by trypsin-EDTA treatment and exposed to 0.4% KC1 for 20 rain at room temperature, followed by fixation in a methanol acetic acid mixture (3:1 by volume). Fixed cells were washed in fixative three times and dropped onto wet slides for air-dry preparation. Five- to seven-day-old slides were treated with trypsin solution for G-banding using Giemsa stain following the technique described elsewhere [14]. Forty to fifty G-banded metaphase spreads were evaluated from each clone and the parental cell lines to identify the marker chromosomes. Clonality was determined if the identical marker chromosome was present in at least two cells of the same sample. Mouse chromosomes were arranged following the recommendation of the Committee on Standardized Genetic Nomenclature for Mice [15].

Gelatin Zymography Aliquots of conditioned medium from tumor cells were harvested and subjected to substrate gel electrophoresis [16]. The samples were run under non-reducing conditions on a 7.5% polyacrylamide slab gel impregnated with 1 mg/ml gelatin (Sigma, St. Louis, MO). After electrophoresis, the gel was washed at room temperature for 30 min in washing buffer (50 mM Tris-HC1, pH 7.5, 5 mM CaCI2, 1 p~M ZnC1, 0.25% Triton X-100) and incubated overnight at 38°C with shaking in the same buffer, except using 1% Triton X-100. The gel was stained with a solution of 0.1% Coomassie Brilliant Blue R-250. Clear zones against blue background indicated the presence of genolytic activity.

mRNA Analysis mRNA was extracted from 1 x 107 tumor cells growing in culture using the FastTrack mRNA isolation system (Invitrogen, Inc., San Diego, CA). For Northern blot analyses, polyadenylated RNA was fractionated on 1% denaturing formaldehyde/agarose gels, electrotransferred at 0.6 amp to GeneScreen nylon membranes (DuPont Co., Boston, MA), and UV cross-linked with 120,000 uJ/cm 2 using a UV Stratalinker 1800 (Stratagene, La Jolla, CA) [17]. Filters were washed two to three times at 60°C with 30 mM NaC1/3 mM sodium citrate (pH 7.2) 0.1 SDS (wv). The cDNA probes used were a 1.3-kb PstI restriction endonuclease cDNA fragment corresponding to the rat GAPDHgene [18] and a 1.1-kb EcoRI gene fragment corresponding to human collagenase IV (courtesy of Dr. W. Stetler-Stevenson, National Cancer Institute, NIH, Bethesda, MD). Each DNA fragment was purified by agarose gel electrophoresis, recovered using GeneClean (BI0 101, Inc., LaJolla, CA), and radiolabeled with the random

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B.J. Dave et al.

Table 1

Biological Characteristics of the Murine Melanoma Cell Lines

Identification

Cell Lines

Cells with chromosome 14 aberrations (%)

SP-2554 SP-2559

K-1735C-10 K-1735 C-10-neo

SP-2555 SP-2556 SP-2557

K-1735C-IO-kFGF K-1735 C-IO-kFGF/LM K-1735 C-1O-bFGF

--

SP-2558 SP-2560 SP-2631 SP-2577

K-1735C-IO-bFGF/LM K-1735C-10-bFGF/SC K-1735 C-23-bFGF K-1735C-23-bFGF/LM

100 100 5 5

SP-2570

K-1735 C-23-c-H-ras

100

+ = present; -

Collagenase activity 72 kD 92 kD

Type of aberration

2

t(14;18)

--

--

5 5

= absent; LM = lung metastasis; SC = subcutaneous

primer technique using (x-32P) deoxyribonucleotide triphosphate [19]. RESULTS The cell lines had modal chromosome n u m b e r s ranging from 38 to 42. Trisomy of chromosomes 3 a n d 15 was observed in almost all cell lines analyzed. Along with aberrations in chromosome 14, other clonal structural abnormalities involved chromosomes 4, 8, 9, 10, 11, 12, 16, and 18. Such chromosomal abnormalities formed variant markers in different cell lines analyzed in this report. Table 1 presents the details of the cell line transfection and gives the data regarding aberrations in chromosome 14 and the expression of collagenase IV activity. The parental clone C-10 (without transfection) did show 2% of the cells with an aberration in chromosome 14. However, the neo-transfected cells did not show any cell with an aberration in chromosome 14 (SP-2559). After transfection with bFGF (SP-2557), 5% of the cells showed abnormalities of chromosome 14 and other clonal markers involving chromosome 12 in C-10. W h e n the bFGF-transfected C-10 cells were injected i.v. or s.c. in mice, the resultant tumors showed 100% of the metaphases with chromosome 14 rearrangement. Coincidentally, the marker was the same, i.e., a Robertsonian translocation between two homologs of chromosome 14, t(14;14) (SP-2558, SP-2560). However, these two cell lines possessed other u n i q u e markers. Apart from t(14;14), SP-2558 showed t(1;10), t(12;?), and t(11;?); SP-2560 showed t(4;?), t(8;?), t(12;12), and t(18;?) as distinct clonal structural abnormalities. Although the metastatic potential of kFGF-transfected C-10 cell lines (SP1555, SP-2556) was high, we did not find clonal structural alterations of chromosome 14 i n the kFGF transfectants. However, they did contain other clonal structural alterations, viz., t(9;16) and t(10;10). The cells, w h e n injected into mice, produced a tumor that did contain a translocation involving chromosomes 5 and 14 and other clonal markers such as t(9;16) t(10;19), and t(18;?).

-t(5;14) loss of one chromosome 14 t(14;14) t(14;14) t(14;19) loss of one chromosome 14 t(14;14)

+

+

+

+

+ + ++

Metastatic potential Nonmetastatic Nonmetastatic High High High

-

+ +++ ++++ + +

+ (n.d.)

High High Low Low

+++ +

+

High

tumors; n,d. = not done

In the bFGF-transfected C-23 cells (SP-2631) as well as in lung metastasis (SP-2577), we found only 5% of the cells with chromosome 14 aberrations. The H-ras-transfected C-23 line (SP-2570) had 100% of the cells with a Robertsonian translocation between two homologs of chromosome 14. Cytogenetically, the marker appeared similar to that found in the metastatic tumors SP-2558 and

Figure I Giemsa-banded normal chromosome 14 and the altered chromosome 14 from parental C-10 and C-10 cell lines transfected in different ways. A and B, from SP-2554, showing a normal pair of chromosomes 14, and a translocation between chromosomes 14 and 18, respectively. C and D, from C-10 SP2556 and C-23 SP-2631, showing t(5;14) and t(14;19), respectively. A normal homolog of chromosome 14 is arranged on the right hand side of these markers. E, F, and G, from C-23 SP-2570, C-10 SP-2558, and C-10 SP-2560, respectively, showing Robertsonian translocations between two homologs of chromosome 14. An idiogram of the mnrine chromosome 14 showing a cluster of breaks (arrow) in the 14B1 region.

it A

h B

A:A, C

D

I i I E

F

G

L1

1~'

El.31

"

Chromosome 14 a n d Mu rine Metastatic Melanomas

69

A

B

C

D

E

F

G

H

I

J

K

~ - - 9 2 kd " ~ ' 7 2 kd

Figure 3 Expression of gelatinase/collagenase IV in murine K-1735 melanoma cells. Culture supernatants from transfected or untransfected cells were analyzed by gelatin zymography as described in "Materials and Methods."

levels of both 72-kD and 92-kD collagenase protein. It is noteworthy that bFGF-transfected C-10 cells expressed very high levels of the 72-kD collagenase (Fig. 3). There was no increase in 92-kD gelatinase activity. C-23 bFGFtransfected cells d i d not show an increase in any collage° nase activity, whereas H-ras-transfected C-23 cells expressed very high levels of 72-kD collagenase activity (Fig. 3). C-10 bFGF subcutaneous (s.c.) and lung metastasis (1.m.) cells also s h o w e d increased 72-kD collagenase activity, whereas

Figure 2 Two C-banded partial metaphase spreads from the C-10 SP-2558 cell line showing a Robertsonian translocation between two chromosomes 14 (arrows). Note the presence of minimal C-banded material in the centromeric regions of these marker chromosomes as compared to other intact chromosomes (A and B).

SP-2560 after bFGF transfection. The cell line SP-2570 had other distinct markers involving c h r o m o s o m e 1. Interestingly, the aberration in c h r o m o s o m e 14 involved the same breakpoint every time. Figure 1 illustrates some of the different alterations involving chromosome 14, with an idiogram showing the breakpoint (14B1). A normal pair of chromosomes 14 is also shown for comparison with the parental C-10 cell. Figure 2 illustrates two C-banded partial metaphase spreads from the SP-2558 cell line, which shows no distinct heterochromatin in the t(14;14) chromosomes. Expression of Collagenase IV In the next set of experiments, we a n a l y z e d the expression of the extracellular degradative enzyme collagenase IV (72 kD and 92 kD) by gelatin z y m o g r a p h y (Table 1, Figs. 3 and 4). Parental C-10 and neo-transfected cells expressed low

Figure 4 mRNA analysis of transfected cells of murine K-1735 ceils, Five micrograms of mRNA/lane was used. The probes used were a 1.1-kb PstI fragment corresponding to the collagenase IV gene and a 1.3-kb PstI fragment corresponding to rat GAPDH gene.

UU AB

U U U U C D E F

~--

~--

3.1 kb Collagenase IV

1.3 kb GAPDH

70 C-10 kFGF s.c. and 1.m. cells did not (Fig. 4). The expression of collagenase activity correlated with increased metastatic capability and translocation t(14:14), suggesting a role for this chromosomal aberration in metastasis. Similar to gelatinase activity, the C-10 bFGF-, C-10 bFGF s.c.-, and C-10 bFGF 1.m.-transfected cells showed enhanced expression of 3.1-kD collagenase IV-specific mRNA transcripts (Fig. 4). In contrast, the C-10 parental and neo-transfected cells expressed low levels of collagenase IV-specific mRNA transcripts. C-23 H-ras-transfected ceils expressed high levels of collagenase IV (72-kD)-specific mRNA transcripts (data not shown). DISCUSSION

Knowledge regarding specific chromosome alterations that could be playing a crucial role in acquisition of the invasive and metastatic phenotypes can increase the understanding of the complex process of metastasis. Our earlier report [2] suggested that mouse chromosome 14 is associated with the metastatic phenotypes. We also noted similar chromosome 14 aberrations in other metastatic murine tumors with different histopathologies, viz., B16 melanoma clone 10, colon carcinoma CT-26 line, a murine mammary tumor, and an in vitro-constituted murine prostate cancer [3]. Interestingly, all of these relate to a specific region of mouse chromosome 14, which includes the centromere. A number of genes related to tumor progression and metastasis have been mapped on mouse chromosome 14 [20]. These include the Rb gene [21], a pseudogene of transforming protein 53 [22], a retinoic acid receptorrelated gene (Hap), and genes for extracellular degradative enzymes such as urokinase plasminogen activator (Plan) [23]. Collagenase is one of the extracellular degradative enzymes which plays a role in tumor invasion and metastasis [24]. The expression of type IV collagenase in tumor correlates with tumor invasion and metastasis [25]. We have analyzed the expression of collagenase type IV enzyme in the various cell lines transfected with growth factors and oncogenes which alter the metastatic capabilities of nonmetastatic K-1735 clones. We also correlated the changes in chromosome 14 and the expression of col]agenase type IV with the metastatic potential of the transfected cell lines. The expression of 72-kD type IV collagenase was found greatly increased in the cell lines which showed aberrations of chromosome 14 (Table 1, Figs. 3 and 4). Brown et al. [26] have reported independent expression of 72-kD type IV collagenase in human tumorigenic cell lines. It was interesting to note that three cell lines (SP 2558, SP 2560, SP 2750) which shared the same rearrangement of chromosome 14 (Table 1, Fig. 1) showed high expression of 72-kD collagenase type IV and possessed high metastatic potential. These cell lines also possessed other marker chromosomes that were distinct, thus making each cell line cytogenetically unique. It should be noted, however, that cells isolated from lung metastases produced by kFGF-transfected cells showed very little increase in cells with chromosome 14 abnormalities and no increase in the expression of 72-kD collage-

B.J. Dave et al. nase (SP 2556, Table 1). These cells contained two distinct markers originating from a t(10;10) and a t(9;16). These markers were also found in C-10 cells transfected with kFGF (SP 2555). Both of these cell lines had high metastatic potential, a minor increase in percentage of cells with chromosome 14 aberrations, and no increase in 72kD type IV collagenase expression (Table 1), indicating the possibility of genes on other chromosomes playing a role in conferring metastatic capacity. The possibility of point mutation in chromosome 14 cannot be ruled out. These subtle changes cannot be deciphered by the classical cytogenetic analysis used in the present investigation. The high correlation of t(14;14) and expression of 72-kD collagenase leads us to speculate that the proximal region of chromosome 14 which is lost by the Robertsonian translocation of 14;14 might possess the genes that govern the expression of 72-kD collagenase type IV. It has been hypothesized that metastasis is a nonrandom selective process [27], and lung metastases are of clonal origin [28-30]; hence, stable metastatic phenotype(s) should be present in the parental tumor cell population. Cytogenetic analysis showed that the parental clone K-1735 C-10 contained a marker and the marker chromosome involved a translocation of chromosomes 14 and 18. Also, in the metastatic cells, the origin of the marker chromosome always remained chromosome 14, consistently involving breakage and loss of the proximal q arm of this chromosome (Table 1; Fig. 1). This leads us to speculate that fragility in chromosome 14 plays a crucial role in conferring the invasive phenotype, or at least has a close association with progression. We have reported previously that different chromosomes show fragility in different types of human cancers [31-35] in nonrandom fashions, and that this fragility can also be expressed in premalignant conditions [36-38]. In this report and in our earlier findings [2, 3], we have observed the association of chromosome 14 with the metastatic potential of different types of murine neoplasms. The proximal q arm of chromosome 14 might contain a metastasis suppressor gene(s), and deletion/mutation of this gene(s) might render the tumor cells highly metastatic [2, 3]. This region contains some genes for extracellular degradative enzymes [20]. The genetic linkage map of mouse chromosome 14 shows an homology with human chromosomes 10, 13, and :14 [20]. Structural abnormalities of these human chromosomes have been reported in advanced renal cell carcinomas, as well as in other epithelial and hematologic malignancies [31, 32, 39, 40]. Other experiments in our laboratory have shown that treating the nonmetastatic C-]O cells with mitomycin-c resulted in an altered chromosome 14, and that these cells were highly tumorigenic and metastatic in nude mice (Nemeth et al., under preparation). To determine the possible subtle alterations in region B:1 of mouse chromosome 14 and to decipher whether these changes are casual or the result of metastatic process, we have initiated studies on micro-dissecting this region of murine chromosome 14. These will be followed by amplifying the DNA by polymerase chain reaction, coupled with transfer of intact murine chromosome 14 into highly metastatic tumors.

C h r o m o s o m e 14 a n d M u r i n e Metastatic M e l a n o m a s

This work was supported in part by the John S. Dunn Research Foundation of Houston, Texas, NIH grants RRO 4999-01 and CA 55769. We thank Sharon Y. Hogan for secretarial assistance and Leslie Wildrick for editorial comments.

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