Cells Abilities of Oncogene-transformed Human ...

Expression of Type IV Collagenase and Procollagen Genes and Its Correlation with the Tumorigenic, Invasive, and Metastatic Abilities of Oncogene-transformed Human Bronchial Epithelial Cells Hitoshi Ura, R. Daniel Bonfil, Reuven Reich, et al. Cancer Res 1989;49:4615-4621. Published online August 1, 1989.

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[CANCER RESEARCH 49. 4615-4621, August 15. 1989]

Expression of Type IV Collagenase and Procollagen Genes and Its Correlation with the Tumorigenic, Invasive, and Metastatic Abilities of Oncogene-transformed Human Bronchial Epithelial Cells1 Hitoshi Ura, R. Daniel Bonfil, Reuven Reich, Roger Reddel, Andrea Pfeifer, Curtis C. Harris, and Andres J. P. Klein-Szanto2 Department of Pathology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19! ! 1 ///. V., R. D. B., A. J. P. K-S.], and Â¡Mboratoryof Human Carcinogenesis, National Cancer Institute, [R. R., A. P., C. C. H.], and Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, Â¡Re.R.], National Institutes of Health, Bethesda, Maryland 20892

year after s.c. injection of 5 x 10'1passage 18 cells. However,

ABSTRACT In a series of immortalized human bronchial epithelial cell lines confining various oncogenes, the transcriptional levels of the type IV collagenase and the type IV procollagen genes were compared with the properties of invasiveness in vitro and tumorigenicity and metastatic ability in athymic nude mice. \-Ha-ras greatly enhanced invasion and metastasis, whereas v-Ki-ras, Ã§-myc,and c-raf had lesser effects on these malignant phenotypes. In addition, cell lines derived from tumors ob tained by injecting the original immortalized human bronchial epithelial cell lines into nude mice exhibited enhanced invasive and metastatic abilities and increased level of type IV collagenase mRNA when com pared with the original immortalized human bronchial epithelial cell lines. Invasiveness and metastatic capacity correlated positively with expression of the type IV collagenase gene and negatively with the expression of the type IV procollagen gene, suggesting that these phe notypes are associated both with decreased production and increased dissolution of extracellular matrix.

INTRODUCTION Tumor progression is characterized by many progressive genetic and phenotypic changes that culminate in the metastatic phenotype. This phenotype is characterized partly by the ability of the cells to invade the extracellular matrix of surrounding normal tissues, including blood and lymphatic endothelial cells (1-3). Changes in cell surface properties permit cell detachment and implantation at distant sites (4), resulting in the formation of metastatic colonies. Secretion of proteolytic enzymes, in cluding plasminogen activators (5), lysosomal hydrolases (6), and collagenases (1, 2, 7-15), plays an essential role in the degradation of the basement membrane that constitutes one of the first steps of the metastatic cascade (3, 16). In the process of progression from benign epithelial tumor to metastatic car cinoma, a decrease of basement membrane material including type IV collagen around tumor cells has been observed in many types of tumors (17, 18). Whether basement membrane loss is due to decreased synthesis or increased degradation of basement membrane by proteolytic enzymes or both is still controversial (2). We have been studying oncogene-induced malignant progres sion in an in vitro human bronchial epithelial cell system. Cell culture conditions have been defined for NHBE' cells obtained from expiants of autopsy tissue (19), and SV40-positive im mortalized clonally derived cell lines have been established (20). One of these IHBE cells, BEAS-2B, had formed no tumors 1 Received 2/22/89; revised 5/8/89; accepted 5/12/89. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported in part by NIH Grants CA 44981 and CA 06927. 2To whom requests for reprints should be addressed. 'The abbreviations used are: NHBE. normal human bronchial epithelial: IHBE. immortalized human bronchial epithelial: cDNA. complementar) DNA; CNase. collagenase; TIMP. tissue inhibitor of metalloproteinases.

passage 39 cells were found subsequently to occasionally pro duce cystic tumors after latency periods of at least 16 weeks, and a tumor cell line, B39-TL, was established from one of these tumors.4 BEAS-2B has been used as a recipient for a variety of oncogenes, including v-Ha-ras (21), v-Ki-ras (22), and a combination of c-myc and c-ra/(23). In a previous report (24) we have analyzed the capacity of a set of three IHBE cell lines to repopulate deepithelialized trachÃ©alxenotransplants, as well as their tumorigenicity, inva siveness, and metastatic properties. The three cell lines were: BEAS-2B, BZR (BEAS-2B cells infected with a recombinant retrovirus, containing the v-Ha-ras oncogene) (21), and a tumor cell line (BZR-T33) derived from a tumor formed by BZR cells injected s.c. into an athymic nude mouse. It was found that the BEAS-2B cell line had some preneoplastic properties and that v-Ha-ras caused the acquisition of a highly malignant and metastatic phenotype. The BZR-T33 cell line, which has am plification and increased expression of the V-Ha-ras oncogene (21), was even more malignant. Increasing malignancy in this small series of cell lines correlated with increasing type IV collagenase enzyme activity and mRNA expression (24). In the present paper we have extended some of these obser vations to other IHBE cell lines, including some containing oncogenes other than v-Ha-ras. Further, paired tumor cell lines derived by animal passage of the original IHBE cell lines were also analyzed. For these pairs of cell lines, we have analyzed and correlated in vivo and in vitro invasive and metastatic potential with collagen mRNA expression and type IV colla genase activity and mRNA expression. MATERIALS AND METHODS Cell Lines. IHBE cells containing various oncogenes were obtained by viral infection and/or oncogene transfection, as described previously (20-23). Briefly, NHBE cells infected with an adenovirus 12-SV40 hybrid virus produced the BEAS-2B cell line. BEAS-2B cells are nonvirus-producers that contain the SV40 large-T antigen (20). B39-TL cells were derived from a slow growing cystic tumor produced in an athymic nude mouse which received 5x10* BEAS-2B cells (passage 39).4 BEAS-2B cells infected with Kirsten sarcoma virus (pseudotyped with baboon endogenous virus) and mass culture propagated after infection are referred to as BVK cells (22). BVK-T11 is a tumor cell line derived from an athymic nude mouse treated s.c. with BVK cells (22). BEAS-2B cells were infected with Zip NEOSV(X) recombinant retrovirus in which the v-Ha-ras oncogene has been cloned and the population consisting of G418-resistant cells was designated BZR. The BZR-T33 cell line is a tumor-derived line obtained from a nude mouse that received BZR cells s.c. (21). BEAS-2B/ra///Â«yc originated from BEAS-2B cells into which human c-raf and murine c-myc had been transferred in Zip NEOSV(X) vectors and selected for G418 resistance (23). RMT-2 cell line originated from a tumor produced after s.c. 4 R. Reddel et ai., unpublished data.

4615


TYPE IV CNASE, INVASION, AND METASTASIS OF IMMORTALIZED

inoculation of a nude mouse with BEAS-2B/raf/myc. NHBE cells, BEAS-2B cells, and oncogene-containing cell lines derived from BEAS-2B, including tumor cell lines, were cultured in LHC-9 medium (19), with the exception of RMT-2 cells, which were grown in LHC-9 supplemented with 1% fetal bovine serum. Histology and Cytochemistry. All tumors and lungs of experimental animals were fixed in neutral buffered formalin. Routine paraffin embedding and hematoxylin-eosin staining of sections was used to characterize the histolÃ³gica! structure of tumors and mÃ©tastases.Immunocytochemistry of cell smears was done to determine the presence of type IV collagen in all cell lines. Indirect immunofluorescence with a mouse monoclonal anti-collagen type IV antibody (ICN Immunobiological, Lisle, IL) and a fluorescein isothiocyanate-labeled goat antimouse IgG as secondary antibody was used. Controls without the primary antibody were carried out for each cell line. DNA Probes. DNA probes for RNA blotting were the 2.7-kilobase Smal-ffindlll fragment of plasmid Gel 186.2 (human type IV specific collagenase cDNA) (25), the 1.7-kilobase Pstl fragment of plasmid KK4 (human Â«1type IV procollagen cDNA) (26), and the 1.4-kilobase Pstl fragment of plasmid Tl (chicken a-tubulin cDNA) (27). Northern Blot Analysis. Total RNA was extracted from each cell line (28) and was electrophoresed in 1% agarose gels that contained 2 M formaldehyde, 20 mM 3-[7V-morpholino]propanesulfonic acid (pH 7.0), 5 mM sodium acetate, and 1 mM EDTA. RNA marker lanes were excised from the gel and stained with ethidium bromide. RNA was transferred to nitrocellulose as described elsewhere (29). The filters were baked at 80 Â°Cfor 2 h and then prehybridized in a solution containing 50% formamide, 4.2x SSC, 4.2x Denharts, 42 mM sodium phosphate, 0.83% glycine, and 125 Mg/ml denatured salmon sperm DNA, for 2-4 h, followed by hybridization with a solution of 50% formamide, 3.2x SSC, 26 mM sodium phosphate, 118 Mg/ml salmon sperm DNA, 10.5% dextran sulfate, and IO6 cpm/ml oligolabeled denatured probe, for 16-20 h. Blots were subsequently washed with 2x SSC, 0.5% sodium dodecyl sulfate, and 0.1% sodium pyrophosphate for 1 h, then with 0.5% sodium dodecyl sulfate and 0.1% sodium pyrophosphate for 1 h. Filters were then autoradiographed using Kodak XAR-5 X-ray film at â€”70Â°C. The relative amounts of type IV collagen ase, procollagen, and a-tubulin mRNA were determined from Northern blots by scanning densitometry using a spectrophotometer (DU-7; Beckman Instruments, Inc., Palo Alto, CA). Tumorigenicity, Spontaneous Metastasis, and Lung Colonization Eval uation. Athymic nude mice of BALB/c and C57 backgrounds, 3 to 4 weeks old and sex matched, were obtained from the breeding unit of the Fox Chase Cancer Center Laboratory Animal Facility. The mice were maintained at all times in microisolator cages in a HEPA filtered laminar flow containment system (Lab Products, Maywood, NJ). Tu morigenicity of IHBE cells was evaluated by inoculating s.c. with IO6 cells. Occasionally IO7 cells were also inoculated. The presence of

HUMAN CELLS

resuspended in minimum essential medium with 0.1% bovine serum albumin. The chambers were incubated for 6 h at 37Â°Cin a humidified atmosphere with 5% CO2. After aspiration of the remaining fluid of the upper compartment, the upper surface of the filter was wiped off with a cotton swab and fixed in neutral buffered formaldehyde. The filters were then stained with Giemsa and mounted on glass slides. The invasive ability of the different IHBE cell lines was determined by counting the number of cells on the under-surface of the filters, using a light microscope. Type IV Collagenase Assay. Measurment of type IV collagenase was done as previously described (31). Briefly, a solution of the '"I-labeled type IV collagen (~2 x IO4cpm) from Englebreth-Holm-Swarm tumors was applied to microtiter plates and media from invasion assay cham bers were applied to the wells for 24 h at 37Â°C.The amount of labeled collagen released from the well under this condition was measured.

RESULTS Ability of IHBE Cells to Form Tumors and Metastasis in Nude Mice. IHBE cells were inoculated either s.c. or i.v. into nude mice in order to assess their tumorigenicity as well as their spontaneous metastatic and lung-colonizing abilities, re spectively. Different tumor incidences as well as spontaneous metastasis and lung-colonization potentials were observed (Ta ble 1). No tumors appeared 16 weeks after s.c. inoculation of IO6 BEAS-2B cells; however, five of six animals developed tumors at this time point when IO7 BEAS-2B cells were s.c. injected. These tumors were slow growing cystoadenomas or low grade cystoadenocarcinomas and no spontaneuos mÃ©tastaseswere seen (Fig. IA). B39-TL, the BEAS-2B tumor-derived cell, was more tumorigenic, i.e., inoculation of IO6 cells produced s.c. tumors in most nude mice. These tumors were very similar to the cystoadenomas observed after s.c. injection of IO7 BEAS-

2B cells (Fig. 1Ã„).These tumors did not metastasize sponta neously and were unable to colonize in the lung after i.v. inoculation (Table 1). BVK cells (containing v-Ki-ros) were not tumorigenic under the conditions of the assay and did not exhibit lung-colonizing ability. Conversely, their tumor-derived counterpart BVK-T11 was markedly tumorigenic, exhibiting both spontaneous and experimental metastatic abilities. Tumors produced by BVKTll were poorly differentiated carcinomas that showed local invasive behavior (Fig. 1, C and D). BZR cells (containing vHa-ras) exhibited a high spontaneous and experimental metas palpable nodules larger than 5 mm in diameter was recorded and tasis incidence. The cell line derived from a tumor produced by histologically confirmed. The existence of spontaneous lung mÃ©tastases s.c. inoculation of BZR cells, i.e., BZR-T33, exhibited a still was investigated during autopsy and in step histological sections of the higher metastatic ability with a much shorter latency period lungs. To evaluate experimentally induced metastasis, a lung coloniza (24). Tumors produced by BZR and BZR-T33 were very simi tion assay was performed. Single-cell suspensions of IHBE cells were lar, namely poorly differentiated carcinomas that showed rapid injected i.v. into the retroorbital plexus of nude mice (5 x 10s cells/ local invasive behavior. BEA.S-2B/raf/myc was marginally tu mouse). Four and 16 weeks later, the lungs were removed and examined morigenic after s.c. inoculation. Nevertheless, in the lung col with a stereo-microscope to detect pulmonary colonies, that were onization assay most animals presented with micrometastases subsequently confirmed by histological studies. 16 weeks after i.v. inoculation (Fig. IE). RMT-2, the BEASChemoinvasion and Chemotaxis Assays. The chemoinvasion and 2B/raf/myc tumor-derived cell line, was markedly tumorigenic chemotaxis assay was performed as previously described (30). Briefly, polyvinylpyrrolidone-free polycarbonate filters, 8-ÃŸtnpore size (Nuclebut did not exhibit an increased metastatic ability. BEAS-2B/ pore, Pleasanton, CA), were coated with basement membrane Matrigel raf/myc tumors were histologically similar to B39-TL or BEAS(50 Mg/filter) prepared as described previously (24) for the chemoinva 2B tumors, i.e., a glandular and cystic pattern was evident, sion assay and with type IV collagen alone (5 jug/filter) (Collaborative conferring to the tumors the characteristics of a moderately Research, Lexington, MA) for the chemotaxis assay. Matrigel thickness differentiated adenocarcinoma (Fig. 1Â£);occasionally nests of and even distribution was checked in each batch in cross-sections and less well differentiated epithelial cells could be seen (Fig. IF). by scanning electron microscopy. In both cases the filters were placed It was the latter feature that predominated in the RMT-2 in a modified Boyden chamber containing fibronectin (Collaborative tumors and mÃ©tastases(Fig. l, G and H), thus producing a Research, Lexington, MA), 5 Mg/5 ml minimum essential medium with poorly differentiated carcinoma with local invasive behavior 0.1% bovine serum albumin, in the lower compartment of the chamber. The upper compartment was loaded with 3 x IO5 cells that were (Fig. IG). 4616


TYPE IV CNASE. INVASION. AND METASTASIS OF IMMOKTAI.I/.ED

HUMAN CELLS

Table 1 Tumorigenicity unti spontaneous and experimental melastalif potentials oflHBE cells The number of cells injected into each animal was IO6 cells s.c. and 5 x 10* cells i.v. MÃ©tastasesin lung were determined macroscopically and confirmed in histological sections. colonizainjectionCell s.c. ability''4 tion lunj l metastasis inci dence"0/0 wk0/15 wk0/15 wk0/15 originAdeno wk0/7 wk0/9 linesBEAS-2B' 12-SV40 infection of NHBE B39-TL From tumor of BEAS-2B cells 0/4 0/4 3/4 (9 Â±if 0/3 0/50/57/10160/5 BEAS-2B plus v-Ki-roi BVK 0/4 0/4 0/4 0/0 0/54/5 BVK-T11 From tumor of BVK cells 1/11 6/6 ( 13 Â±if 4/6 4/6 BZR'' Â±3)'1/5(13)''2/6 8/10 (15 BEAS-2B plus v-Ha-rai 0/10 4/56/73/5 3/80/51/616 6/8 BZR-T33' From tumor of BZR cells 8/8 (24 Â±9f 8/8 BEAS-2B raf/myc BEAS-2B plus c-ra/plus c-myc 0/5 0/1 (30 Â±4)JSpontaneous 0/0Lung From tumor of BEAS-2B/ra///n>T4 RMT-2Cell 0/68

No. of mice with spontaneous lung metastasis Total no. of animals with s.c. tumor

Spontaneous lung metastasis incidence =

No. of mice with lung nodules No. of mice treated i.v.

Lung colonization ability

c Data from Bonfil et al. (24). 11Larger diameter of s.c. tumor (mean Â±SD) expressed in mm. ' Except for s.c. injection and spontaneous metastasis at 16 weeks, the data are from Bonfil et al. (24).

Invasive and Chemotactic Abilities of IHBE Cells. This study confirmed that BEAS-2B exhibited a very high chemotactic response, although no increase in the invasive capacity as compared to NHBE cells (Table 2). In addition, parental cell lines containing the v-Ha-rai oncogene showed the highest values in the invasion assay. Parental lines containing raf/myc (BEAS-2B/'raf/myc) were more invasive than cells containing v-Ki-ras oncogene (BVK). These lines were 1 to 2 orders of magnitude less invasive that the v-Ha-ras-containing cell lines. An enhancement of the in vitro invasive ability could be ob served in most tumor cell lines when compared to their respec tive parental lines. This was not the case for B39-TL cells, which did not change their invasive behavior with respect to BEAS-2B. Expression of Type IV Collagenase Gene. The transcriptional level of the type IV collage-mise gene was analyzed by Northern blot hybridization of total RNA from NHBE and IHBE cell lines (Fig. 2). A lower transcriptional level was observed in BEAS-2B cells. BVK cells showed similarly low transcriptional level; conversely, BZR showed high gene expression. Cells containing c-ra/and c-myc genes also exhibited little change. All tumor-derived cell lines had higher levels of mRNA than their corresponding original IHBE cell lines. The correlation of the transcriptional level of type IV collagenase with invasive ability and spontaneous metastatic ability were examined. The correlation coefficient of mRNA message level and invasive ability in vitro was 0.622. The correlation coefficient of mRNA message and spontaneous metastasis incidence was 0.70. The correlation between the mRNA message and tumorigenicity and lung-colonizing ability, respectively, was less than 0.40, indicating a positive although poor association. Measurement of type IV collagenase enzyme activities of the different cell lines showed a good correlation with the mRNA levels (Table 3). Expression of al Type IV Procollagen Gene. The less malig nant cell lines, BEAS-2B, B39-TL, and BVK, expressed high levels of al(IV) procollagen mRNA. Except for BVK-T11, the highly tumorigenic cell lines expressed similar or lower tran scriptional levels than NHBE (Fig. 2; Table 3). Of the tumorderived cell lines, only BZR-T33 and RMT-2 had slightly higher mRNA levels than their respective parental cell lines.

Correlation coefficients between the transcriptional level of ttl(IV) procollagen and invasive ability, tumorigenicity after s.c. inoculation, spontaneous metastatic ability, and lung colony colony-forming ability were -0.59, -0.38, -0.29, and -0.60, respectively. Cytochemical Evaluation of Collagen Type IV. Immunocytochemistry using anti-collagen type IV monoclonal antibody showed the presence of marked immunofluorescence in most cells of lines BEAS-2B (Fig. 3/4), B39-TL, BVK, and BVKTl 1. Less fluorescence in fewer cells was seen in the cell lines BEAS-2B/ra///n>'c, RMT-2, and BZR. Weak or absent immu nofluorescence was noted in most cells of the malignant cell line BZR-T33 (Fig. 3Ã„). DISCUSSION Our results show that: (a) many IHBE cells expressed the metastatic phenotype in nude mice; (b) cells containing both vHa-ras and large-T antigen (BZR and BZR-T33) showed higher invasive and metastatic potential than cells containing other oncogenes (v-K.i-ras,raf/myc); (c) the transcriptional level of type IV CNase gene correlated with the invasive, and to a lesser degree with metastatic, potential; and these changes of type IV CNase mRNA level are probably related to the presence of oncogenes, especially ras; (a") an inverse relationship between the expression of type IV CNase and type IV procollagen mRNA was noticed; and (c) tumor-derived cells obtained by animal passaging of the original cell lines showed an enhanced metastatic potential when compared to the respective parental line. Recent investigations on the mechanisms of metastasis have supported the concept of metastatic cascade (2) and emphasized the role of proteolytic enzymes that degrade the basement membrane components (1, 32, 33). Except for RMT-2, which showed high invasiveness and elevated type IV CNase values but was moderately invasive and metastatic in vivo, the corre lation between the in vivo behavior, in vitro invasiveness, and type IV CNase gene expression was very significant. Although there are many indications that type IV CNase is important in the degradation of basement membranes by metastatic cells, other investigators have found that some nonmetastatic neoplastic populations can secrete very high levels of type IV CNase

4617


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Fig. 1. Histology of s.c. tumors and mÃ©tastasesproduced by inoculation of immortalized human bronchial epithelial cells. A, slow growing adenocarcinoma produced by s.c. injection of 1(T BEAS-2B cells. Note the clear glandular differentiation and the expansive local growth as seen by the compression without local invasion of the surrounding muscle tissue (A/). B, similar tumor produced by s.c. inoculation of IO6 B39-TL cells. Note slightly more atypical glandular structures and a tendency to local invasion. C, poorly differentiated carcinoma developing s.c. after the injection of IO6 BVK-T11 cells. Note clear invasion of muscle (M). D, the same type of tumor developing in the lung after i.v. inoculation of 5 x 10' BVK-T11 cells. E, lung colony produced by i.v. injection of 5 x IO5 BEAS-2B/ra//m>'