Tumorigenicity of EJ-ras oncogene transformed NIH 3T3 cells and expression of plasminogen activators

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TUMORIGENICITY OF EJ-ras AND EXPRESSION Maninder

K.

Vol. 14, No. 6, June 1990

527

ONCOGENE TRANSFORMED NIH OF PLASMINOGEN ACTIVATORS

Sidhul Mark S. Russo', Louis &ala1 and Suriender

I Depa?Ttment of Biochemistry Department of Surgery, Newark,

and UMDNJ-New NJ, 07103

Karilyllah Kumar

Molecular Jersey Medical U.S.A.

32'3

A.

Biology, School,

CELLS

Zirvi?,

and

ABSTRACT

A number of clonal cell lines have been isolated from NIH 3T3 cells transfett2ed. with the plasmid, pSV2 gpt-EJ-m. The plasmid expresses Val and can be instead of Gly 12 In ~21 ras protein selected for the expression of E. & XGPRT gene in mammalian cells. Southern analyses of the & Rl and Barn Hl digests of copies of the plasmid are chromosomal DNA shows that multiple integrated in a tandem sequence in the clones used in this study. The transfectants showed refractile appearance and criss-crossed pattern of growth, exhibited elevated expression of ras mRNA and formed tumors in nude mice commensurate with the copy number of the integrated EJ-ras gene. The increased propensity to form tumors did not correlate with the expression of urinary or ti.ssue The cellular and secreted plasminogen activators (u-PA or t-PA). activity of u-PA in fact decreased as the ras gene expression increased. These data show that the enhanced tumorigenicity of transformed murine cells is related to the tandem integration and expression of human EJ-ras The overexpression of ras has very little effect on t-PA butappears to suppress u-PA activity. INTRODUCTION

The growth and metastatic dissemination of transformed cells requires local invasion of the adjacent tissue as well as the vascular wall or the lymphatic channels. These processes require the participation of proteolytic enzymes (Ossowski and Reich, 1983; Some of the proteases Liotta, 1986). implicated in this process are: t-PA and u-PA, collagenase type IV and cathepsin B. Most malignant tumors in animals and a variety of human neoplasms contain and secrete elevated levels of PA. A direct correlation between the expression of PA and tumor growth and metastasis has also been observed by Ossowski and Reich (1983) for human carcinoma line, HEp3. Additionally, the growth of mammary carcinoma line MCF7 shows a positive correlation between tumor growth and PA production (Butler et al., 1983). We have also recently demonstrated that human osteosarcoma cells transformed by Kirsten murine sarcoma virus N-methyl-N'-nitro-N(KiMSV) or nitrosoguanidine (MNNG) are metastatic and express a very high level of u-PA (Sidhu and Kumar,1989). The multi-stage process of carcinogenesis appears to involve oncogenes in several steps. Many types of human tumors exhibit high level of ras expression but whether this is sufficient to maintain 0 1990 Academic

Press Ltd.

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malignancy is not certain. Early studies by Land et al., (1983) and Ruley (1983) established the necessity of at least two dominant oncogene activating events in malignant transformation. However, recent studies by Spandidos and Wilkie (1984), Garbisa et al., (1987) and Bradley et al., (1987) on the transfection of 3T3 or rat embryo fibroblasts (REF) appear to be consistent with transformation potential of overexpressed normal or mutated u genes. A mutation affecting the 12th codon of the ras protooncogene expresses a modified p21 protein having valine for glycine at that position (Seeburg et al., 1984). The expression of modified p21 leads to a transformed phenotype which is highly tumorigenic in immunodeficient We have studied the pattern of animals. integration and overexpression of EJ-ras and its effect on the tumorigenic properties and the expression of PAS in NIH 3T3 cells. MATERIALS Cells

and

Growth

AND METHODS

Media

NIH 3T3 cells were grown in monolayer cultures in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% FBS in 95% air, 5% co2 atmosphere The cells were subcultured by at 37'. trypsinization prior to reaching confluency to minimize spontaneous mutations. Transfection

of

Cells

The cell lines used in this study were provided by Dr. R. S. Athwal, Department of Molecular Biology and Molecular Genetics, New Jersey Medical School, and were obtained using the standard transfection protocol. The plasmid used in this study is the shuttle vector (pSV2gpt) developed by Mulligan and Berg (1981) into which was subcloned a 6.6 kb Barn Hl fragment of T24 human bladder carcinoma containing m gene mutated at the 12th codon (pSV2gptEJ-m). Plasmid DNAs were isolated and purified from E. coli by alkaline lysis followed by centrifugation in cesium chlorideethidium bromide gradients according to Maniatis et al., (1982). For transfection, 1 X lo6 NIH 3T3 cells were seeded into 100 mm2 culture plates and transfected 24 hr later with 10 ug of plasmid DNA using the calcium phosphate precipitation method of Graham and van der Eb (1973). Eighteen hours after the addition of plasmid DNA, the cells were exposed to glycerol (15% v/v) for 2 min, washed with DMEM and refed with DMEM containing 10% FBS. After another 24 hr, the cells were placed in the Ecogpt selection medium (100 acid (25 ug/ml) and xanthine which contained mycophenolic 2-3 weeks in the selection medium, the cells were ug/ml > . After plated at low density (-200 cells per plate). Colonies arising presumably from single cells were isolated after 2-3 weeks with cloning cylinders and grown to mass culture. The clones designated as TRG 2, 5 and 10 were isolated based on their morphological Studies described here were appearance and growth characteristics. conducted with cells grown up to 20 passages.

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&&$ Extraction

&

Reports,

Restriction

Vol. 14, No. 6, June 1990

Enzvme

529

Analysis

High molecular weight DNA was prepared from cultures as described by Wigler et al., (1978). Cellular DNA (10 ug) each was digested with Eco Rl and Barn H7~ enzymes under conditions described (BRL). by the supplier DNA fragments were separated on 0.8% agarose gels and transferred to nitrocellulose filters. The filters were air dried and prehybridized overnight at 42'C in a mixture containing 5 X SSC, 10 X Denhardt's solution, 0.05 M Naphosphate, pH 6.7, 500 ug per ml sonicated denatured salmon sperm DNA and 50% (v/v) deionized formamide. Hybridization was carried out under similar conditions as prehybridization except for the addition of 32 P labelled DNA probe containing either 6.6 kb human m gene or linearized SV2gpt-EJ-m. The DNA was labelled to a specific activity of 10% cpm/ug using random primers supplied by Boehringer Manheim. After hybridization, the filters were washed for 30 min each at room temperature with 2 X SSC/O.l% SDS, 0.5 X SSC/O.l% SDS and 0.1 X SSC/O.l% SDS. Slot-Blot

Analysis

of

RNA

Total cellular RNA was extracted from semiconfluent cultures of isolated clones according to the procedure described by Maniatis et a1.,(1982). RNA samples were denatured with formaldehyde (final concentration, 7.4%) by incubating at 60' for 15 min and different amounts were then blotted onto a nitrocellulose filter. The filter was soaked in 2 X SSC and air dried. The protocols for prehybridization and hybridization have been described above. Assay

of

Plasminoeen

Activator

Activity

PA activity of cell lysates as well as that secreted into the medium of the various cell lines was measured according to the following protocol. Cells were plated at a density of 5 X lo5 cells per 100 mm2 culture plate in complete growth medium. Twenty four hour later the growth medium was replaced with serum free DMEM. After 48 hr incubation, the medium was removed, centrifuged in a microcentrifuge at -14,000xg and tested for PA activity immediately or frozen at -8O'C for further analysis. Cell viability after 48 hr incubation with serum free DMEM was found to be greater than 95% by trypan blue exclusion. For measuring the cellular PA activity, the cell monolayer was washed twice with 2 ml of ice cold PBS and then lysed in 1 ml of 5 mM sodium phosphate, pH 8.0, containing 1% Triton-X 100. The lysate was centrifuged to remove nuclei and cellular debris and the amount of protein was determined by the method of Lowry et al., (1951). PA activity of the cell lysates and the medium was determined by an indirect spectrophotometric assay in which PA converts plasminogen to plasmin which in turn hydrolyzes a chromogenic substrate, D-ValLeu-Lys-p-nitroanilide (S-2251). Reaction mixture in a total volume of 0.5 ml contained: 0.05M Tris-HCl, pH 7.5; 0.1% Triton X100, 0.3 mM S-2251, 20 ug Lys-plasminogen and either 20 ug of the cell lysate or volume of the supernatant corresponding to 20 ug of cellular protein. Incubations were carried out at 30°C for a period of 3 hr and the absorbance of released p-nitroaniline was determined at 405 nm. The linearityof the assay was established

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using different protein concentrations and for different time intervals. Blanks corresponding to either the extracts or plasminogen alone were substracted to obtain the final activity values. For the sake of comparison, activities measured with cell extracts and supernatants obtained from 3T3 cells were arbitrarily fixed at 100%. Detection

of --

PA by

SDS-PAGE

Plasminogen activators were detected on 10% SDS non-reducing polyacrylamide gels copolymerized with Lys-plasminogen (10 ug/ml) and casein (0.1%; w/v) (Roche et al., 1983). This assay procedure is based on the fact that plasminogen and casein are trapped and retained in the gel matrix and thus serve as --in situ substrates for the localization of protease bands by negative staining. Gels lacking plasminogen were used as controls for the indication of protease activities other than that of PAS. Each well of the gel contained 50 ug of cellular protein (for cellular activity) or volume of the serum-free medium corresponding to the above amounts of the cellular protein (for secreted activity). Electrophoresis was run overnight at 4OC at a constant current of 10 mA per gel according to Laemmli (1970). After electrophoresis, the gels were washed three times in 100 ml of 50 mM Tris-HCl, pH 7.5 and 2% Triton-X 100 with gentle shaking to remove SDS. Following the wash, the casein-plasminogen gels were incubated in 50 mM Tris-HCl, pH 7.5 for 3 hr at 37'C, stained in 0.1% coomasie blue and destained in acetic acid:methanol:H20 (1:3:6, v/v). Nude Mouse

Assay

of

Tumoriaenesis

Monolayer cultures of -80% confluent cells were washed with cold PBS and harvested with 0.025% trypsin-0.25% EDTA. The cells were washed once with DMEM containing 10% FBS and then three times with ice-cold PBS. 3T3 and transformed clones (0.1 ml; lo6 cells) were injected into the flanks of 4-5 week old athymic nude mice (6 mice for each line). Primary tumor growth was monitored on a weekly basis by caliper measurements of two perpendicular diameters, and the tumor size was estimated by the formula: W= a x b2/2; where W is the volume of the tumor in mm3, "a" is the length of in mm, and "b" is the width in mm. This is an approximation the volume of an ellipsoid, and it can be considered as a valid (Zirvi et al., estimation of tumor mass assuming unit density 1983). We followed tumor growth up to 6-8 week after injection of the cells; however, the tumor became necrotic after 4-5 weeks and under these conditions, the application of the above formula for The tumor volume was the volume of an ellipsoid is not tenable. calculated and plotted for periods up to 3 weeks and represents the mean + sem mass for 6 animals. RESULTS Morpholonv

of

the

Transformed

Cells

When NIH 3T3 cells were transfected with the recombinant pSV2gpt-EJ-m, plasmid, a number of individual mycophenolic acid resistant colonies became apparent within 2-3 weeks. A number of colonies by limit dilution and clones were isolated from these

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531

selected clones were further propagated in the selection medium for 2-3 weeks and then grown in DMEM containing 10% FBS. The clones were chosen because of their used in this study, TRG 2, 5, 10 morphological appearance and growth characteristics, The difference in morphology between 3T3 and selected clones, TRG 2, TRG 5 and TRG 10 is shown in Fig. 1. The parent 3T3 cells show a flat refractile

Fig.

(B), cells

A

B

C

D

Light micrographs (magnification 100X) of 3T3 (A), TRG 2 were taken when the TRG 10 (C) and TRG 5 (D). Photographs were -75% confluent.

1.

appearance (Fig. 1A) but the transformed cells are less refractile, occupy less surface area, have many processes, show a criss-crossed growth pattern (Fig. lC), and grow somewhat loosely attached to the tissue culture plates. The TRG 10 clone which has the highest number of mutant ras copies also exhibited a number of floating cells prior to reaching confluency. Analyses_

of

the

Pattern

of

Inteeration

by Restriction

Enzvmes

The copy number and pattern of integration of the mutated ras gene was assessed by slot-blot hybridization and restriction enzyme Slot-blot hybridization of genomic DNA analysis of genomic DNA. isolated from the parent and transformed lines is consistent with the presence of multiple mutated ras in the copies of the transformed clones (data not shown). The pattern of integration of the plasmid in genomic DNA was studied using two restriction enzymes, Eco Rl, and Barn Hl. Eco Rl cleaves the 12.1 kb Ecogpt-EJras plasmid at a single site. Thus if single copies of the plasmid were to be integrated at multiple sites one could see different

532

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sized DNA fragments hybridizing with the labelled probe. On the copies of the plasmid were to be integrated other hand, if multiple one would expect to get mainly a single band in a tandem sequence, whose intensity would depend upon the number of plasmid copies. The data of Fig. 2 shows a major 12.1 kb band for each of the three The intensity of this band reflects transformed lines. qualitatively at least the copy number of the mutated ras in

3

12.l kb 6,.6 kb

4

5

6 Fig.2. Southern analysis of the Eco Rl digests of genomic DNA probed with 32P labelled linearized pSV2gptw. Lane 1 Hind III digest of l&%-DNA; 12.1 kb lane 2, pSV2gpt-EJ-ras plasmid; lane 3, TRG 10; TRG lane 4, 5; lane 5, TRG 2; lane 6, 3T3.

Higher molecular weight DNA hybridizing with the different clones. 2,1ane 3) probably represents probe in the TRG 10 line (Fig. the parent 3T3 lacks the 12.1 kb band. undigested DNA. As expected, The Southern analysis of restriction fragments obtained using Barn Since 6.6 kb mutated ras is cloned into Hl is shown in Fig. 3. Ecogpt plasmid at the Barn Hl restriction site, cleavage of genomic A 6.6 kb fragment DNA should release this 6.6 kb ras insert. corresponding to the m gene is present in the parent and the that all transformants possess the transformed lines suggesting lower molecular weight fragments entire EJ-ras DNA. The additional seen in this figure may be either the vector Ecogpt DNA (5.5 kb), It is plus flanking cellular DNA sequences. or a segment of it, also possible that the additional smaller fragments represent a

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mutated or rearranged form of the integrated DNA. However, this in view of the results obtained using Eco Rl appears unlikely restriction digestion where small fragments are apparently absent. a faint 3.4 kb fragment seen for all the cell lines Additionally, most probably represents part of a endogenous murine ras gene and this has been observed in previous studies (Pulciani et al., 1985; Sistonen et al., 1987). An assessment of the ras gene expression in the parent and transformed clones was made from slot-blot hybridization of the

Fig.3. Bam Hl digests of genomic DNA probed with labelled plasmid. Lane 1, pSV2gpt-ras plasmid; lane 2,3T3; lane 3, TRG 2; lane TRG 5; lane 5, 4, TRG 10.

6.6 kb 5.5 kb

3 kb

total RNA (Fig. 4). High level of ras expression is seen in clones TRG 2, TRG 5 and TRG 10 as compared to 3T3. TRG 10 has the most tandemly integrated copies of mutated m. and also exibits the highest level of ras expression. TRG 2 and 5 appear to have somewhat similar message levels even though Barn Hl digestion appears to show a somewhat higher number of mutated ras copies in TRG 5. Since murine ras mRNA does not hybridize very efficiently with human u probe, the enhanced expression observed in the transformed cells is most likely due to mutated B. Tumoriprenicity

of

Overexpression

Transformed

of normal

Cells

ras

or mutated

ras

has been reported

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to be sufficient for converting 3T3 cells to the tumorigenic phenotype. Therefore, we tested our clones to determine if tumor growth could be correlated with the expression of EJ-m. For this purpose, lo6 3T3 or transformed cells were injected into two flanks of 4-5 week old athymic nude mice and primary tumor growth was monitored on a weekly basis by caliper measurements. Tumor growth was not observed in animals injected with the parent 3T3 cells. However, it is clear from the data of Fig 5. that the relative increase in tumor volume after the tumors became palpable follows the ras gene expression in the transformed cells. After 6 weeks the tumors became quite necrotic and the tumor volume calculations could not be accurately determined. However, we did not find any of any of the animals metastasis in the lungs or other organs sacrificed after 8 weeks.

TRG-2 4. Slot-Blot Fig. analysis of total RNA for the expression of m gene. Lanes 1 and 2 contain 5 and 10 ug respectively of the isolated RNA. The l~t;;s 35ere probed P labelled 6.6 kb EJ-u.

TRG-5 TRG-10

3T3

Cellular

&

Secreted

PA Activity

of

the

Transformed

Cells

Since elevated levels of PA are secreted by most if not all to determine if the transformed cells and tumors, we wanted overexpression of mutated _ras and enhanced tumorigenic properties of the transformed cells are reflected in the enhanced expression of PAS. The results of this study are shown in Fig. 6 where all the activity values are normalized to 3T3. The cellular PA activity

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of TRG 2 is almost double, whereas the secreted PA level is only about 50% of the parent 3T3 cells. Thus the overall total activity does not appear to be very different for 3T3 and TRG 2. On the other hand TRG 10, the most tumorigenic clone, containing the highest ras copy number has nearly similar cellular but only about 25% of the secreted PA activity. Therefore, it is apparent that the enhanced expression of EJ-ras in 3T3 cells is not accompanied by the enhanced synthesis of PA and actually results in the altered secretion of PA. Since normal and transformed cells are known to secrete inhibitors of PA, we wanted to establish whether the measured low activity of secreted PA in TRG 2, 5 and 10 lines is due to the presence of an inhibitor of PA in the medium (Saksela et al., 1984). Treatment with SDS or maintenance of low pH for a period of

TUMOR GROWTH IN NUDE MICE a. 7

--A--TRG-10 0 . TRG-5

-U-

TRG-2

1 /Y

6-

,/

‘E4LE OV

E

3-

c’ 2-

1

3 TIME2

(weeks)

Fig. 5. Six mice were injected with lo6 cells for each of the transformed cell lines and tumor volume was determined as described in the Methods section. Values represent mean +sem for at least 5 animals. 30 min is known to dissociate PA from its bound inhibitor (Sprengers et al., 1985). Therefore, we carried out electrophoresis in polyacrylamide gels copolymerized with casein and plasminogen. When the gels are run in SDS, PA is not active and is therefore, unable to activate plasminogen to plasmin. On renaturation in the presence of 2% Triton-X 100, plasminogen trapped in polyacrylamide is activated by PA at a position to which it migrates in the gel. Plasmin formed and trapped in the gel cleaves casein to its

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proteolytic products leaving a zone of clearance after staining. Such a gel analysis is shown in Fig. 7. Two clearance zones are observed for all the cell lines. The higher molecular weight species corresponds to the 68 kDa human recombinant t-PA (lane 10) whereas the faster migrating species has a molecular weight which is less than the 55 kDa human u-PA (lane 9). The murine u-PA has been shown to be a 48 kDa protein and our lower molecular weight clearance zones are nearly this size. When PA levels are compared for cell lysates (lanes l-4, Fig.-/), the intensity of the clearance zone corresponding to t-PA appears to be similar for 3T3 and for the transformed cells. On the other hand, u-PA levels appear to decrease as the ras gene copy number increases in clones TRG 2, 5 and 10 (lanes l-3). When secreted PA activity is estimated from the gels (lanes 5-8, Fig. 7), the results are essentially the same.

1234

5678

910

Cellular and secreted PA activities of 3T3 and EJ-ras Fig. 6. transformed 3T3 cells. For each cell line, 20 ug of the cellular protein or volume of the conditioned medium corresponding to 20 ug of the cellular protein was assayed using S-2251 synthetic in the Methods. Values and plasminogen as described substrate obtained for 3T3 cells were fixed at 100%. The extent of u-PA secretion decreases as the lla~ copy number unusual relationship between a increases establishing a rather The results obtained expression and u-PA synthesis and secretion. from SDS-PAGE (Fig. 7) show that low cellular and secreted level of u-PA in TRG 10 clone is not due to the simultaneous overexpression of the inhibitors of PA.

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DISCUSSION Carcinogenesis is considered to be a multi-step process Early studies requiring possible activation of a number of genes. by Land et al., (1983), Ruley (1983) and Newbold and Overall (1983) showed that the transformation of rat embryo fibroblasts, baby rat the kidney cells or hamster fibroblasts by T24 Ha-m requires c-myc or chemical establishment functions provided by Ad-Ela, agents. Subsequently studies by Spandidos and Wilkie (1984), Pulciani et al., (1985) and more recently by others (Bradley et 1987; Garbisa et al., 1987; Hill et al., 1988; Suzuki et al., al., 1989) have shown that the amplification and overexpression of either the ras protooncogene or the T24 Ha-m brought about morphological transformation and tumorigenic conversion of rodent cells. Induction of proteases has been postulated to be an important step in carcinogenesis. Thus protease activity has been implicated

PA Activity

in rat-Transformed

TRG-2

TRG-5

Cell

Cells

TRG-10

line

SDS-PAGE of cellular extracts (50 ug protein) and volume Fig. 7. of the conditioned media corresponding to 50 ug cellular protein. The gels were copolymerized with casein and plasminogen as described in the Methods. Lanes l-4, cell extracts,lanes 5-8, conditioned media and lanes 9 and 10 represent 0.1 unit each of high molecular weight human u-PA (55 kDa) and human t-PA (68 kDa) respectively. Lanes 1 and 5, TRG 2; lanes, 2 and 6, TRG 5; lanes 3 and 7, TRG 10; and lanes 4 and 8 3T3. in

increasing

the

rate

of

cell

proliferation

in

certain

tumor

538

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cells (Scher et al., 1982). In addition, the transformation of NIH 3T3 cells by Ha-m oncogene is reported to be inhibited by plasmin and trypsin inhibitors (Garte et al., 1987) and Bowman-Birk soybean protease inhibitor suppresses X-ray induced in vitro transformation of C3H lOT1/2 cells (Yavelow et al., 1983). The role of PAS (u-PA and t-PA) has been extensviely studied in rodent and human malignancies (Marcus, 1988; Quax et al., 1990). Oncogenic viruses and tumor promoters induce the synthesis and release of PAS in a variety of cell lines and in primary cell cultures (Wigler and Weinstein, 1976; Ossowski and Reich, 1983; Sidhu and Kumar, 1989). u-PA has been localized immunohistochemically in the areas of invasive growth and in the adjacent degrading tissue in an invading and metastasizing murine lung tumor (Skriver et al., 1984). Recently, Sappino et al., (1987) showed 4-20 fold higher content of u-PA mRNA in 43 out of 56 human lung and breast carcinomas. These and other studies are consistent with the overexpression of u-PA as one of the indices of malignancy. We have isolated transformed clones of 3T3 containing multiple copies of human EJ-u. While integration and expression of mutated ras aa tumorigenic potential has been y& demonstrated previously, very little information is available as to how the exogenous human x is integrated into the mouse genome. Results of Southern analysis of Eco Rl digests of genomic DNAs isolated from our clones are consistent with the integration of EJin the multiple-copy ras containing plasmid as tandem repeats clones. Additionally, to be little evidence of there appears integration of fragmented plasmid. If this were so, it would have become evident from Eco Rl digests of genomic DNA probed with full length plasmid DNA which would have shown hybrid bands of less than 12.1 kb size for either the integrated plasmid alone or the EJ-ras gene alone. Southern analysis using Barn Hl digested DNAs show a signal of increasing intensity at 6.6 kb for TRG 2, 5 and 10 (Fig. that the major portion of integrated plasmid 3). This indicates contains untruncated 6.6 kb m gene. In addition, we observed the hybridization of the 5.5 kb plasmid fragment and the expected increase in the intensity of this band with increasing copies of tandemly integrated plasmid (Fig. 3). The choice of NIH 3T3 cells for transfection experiments could be problematic because these cells are highly aneuploidal and have high rate of spontaneous transformation (Van Roy et al., 1986). The latter can be controlled by not allowing the culture to become confluent. 3T3 cells used in our study were never grown to above did not form colonies in semi-solid medium and 70-80X cofluency, did not form tumors in nude mice at 8-10 weeks. However, our isolated clones are highly tumorigenic but show no spontaneous lung metastasis when injected subcutaneously into nude mice and have lower expression of u-PA. This observation differs from the reports and Bradley et (1985), Garbisa et al., (1987) by Muschel et al., (1987). Muschel et al., (1985) and Garbisa et al., (1987) al., reported that the transfection of NIH 3T3 cells by overexpressed metastatic tumorigenic but not Ha-m protooncogene produced by T24-Ha-u produced cells which cells whereas transfection and tumorigenic expressed elevated levels of PAS and were

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spontaneously metastatic. Bradley et al., (1987) reported that the of normal cellular or bladder tumor m genes, overexpression viral-cellular recombinants or yeast-mammalain ras recombinants all transform 3T3 cells. These authors did not isolate individual clones and study the pattern of integration of ras and did not determine PA activity, but the mixed population of cells did form spontaneous lung metastasis in beige mice. The reasons for the differences between our results and the above reports is not clear at present. It could be that the site and the manner in which the plasmid is integrated is important in the induction of other cellular complementing genes essential for the expression of metastatic potential. Our data is consistent with previous observations on the overexpression of EJ-ras and transformation of rodent cells to the tumorigenic phenotype. The possibility that low PA activities might result from enhanced synthesis or release of the inhibitors of PA does not appear likely as judged from the SDS-PAGE gel studies. The low PA activities could also result from the suppression of PA synthesis by growth factors synthesized and secreted at elevated levels by the transformed cells. Thus Saksela et al., (1987) have recently demonstrated the inhibitory effect of TGFB on u-PA synthesis in bovine capillary endothelial cells. A possible explanation of earlier studies might be that random integration of EJ-ras results in the activation of complementing cellular genes which directly or indirectly induce the transcription of the gene for PA. Our isolated clones are interesting in that they can be further transfected with other possible complementing oncogenes to identify the steps leading to the induction of the proteolytic cascade in the multi-stage process of carcinogenesis. Acknowledeements

These studies were supported by a postdoctoral fellowship from New Jersey Commission on Cancer Research to M.K.S. and research grants from UMDNJ and Pardee Foundations to S.K. These studies represent thesis research of M.S.R. towards the partial fulfillment of the requirements for the degree of Doctor of Philosophy at UMDNJ. REFERENCES

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Paper received

29.12.89.

Revised

paper

accepted

01.03.90.