Molecular and Cellular Probes (1991) 5, 207-213

Proto-oncogene expression in cultured synovial fibroblasts of patients with rheumatoid arthritis M . Anders,' K . Krohn,' H . Kroger,1 H . Huser,'* M. Sparmann? A. Meissner3 and W. Gombert° 'Abt . Biochemie, Robert Koch-lnstitut, Nordufer 20, 0-1000 Berlin 65, 2Orthopadische Klinik der FUB, Oskar-Helene-Heim, Clayallee 229, D-1000 Berlin 33, 3 Unfallchirurgie, Universitatsklinikum Steglitz, Hindenburgdamm 30, D-1000 Berlin 45 and 4Rittberg-Krankenhaus, Carstennstr . 58, D-1000 Berlin 45, Germany (Received 16 August 1990, Accepted 11 December 1990) Total RNA was isolated from cultured synovial fibroblasts of nine patients with rheumatoid arthritis and two controls (cruciate ligament ruptures) . RNA was dot-blotted and hybridized with nine different, cloned cellular or viral oncogene probes . None of the proto-oncogenes showed a significant difference of expression in cultured fibroblasts from patients with rheumatoid arthritis when compared to the expression of control fibroblasts .

KEYWORDS: Proto-oncogene expression, cultured synovial fibroblasts, rheumatoid arthritis . INTRODUCTION In rheumatoid arthritis patients, the synovial membrane lining a joint cavity grows excessively and penetrates into the joint's cavity . This tissue's weight may increase a thousand-fold .' The reasons why these lining cells proliferate so excessively are still unclear. In the last decade it has become apparent that in many different kinds of cancer there may be an increased expression, rearrangement or point mutations of proto-oncogenes ." These proto-oncogenes are coding for proteins involved in the cell's proliferation, differentiation, signal transduction or other vital functions .', ' Upon these changes these genes become oncogenic, that is they are able to transform NIH 3T3 mouse fibroblasts when the changed DNA is transfected into these cells . Some of the transfected cells are also able to cause cancer when injected into nude mice . It is not certain yet if this transforming ability alone is sufficient to cause transformation in normal cells .' Dayer et al.' have isolated synovial fibroblasts from synovectomized tissue of patients with rheumatoid arthritis and observed that rarely in some in vitro

cultured cell supernatants latent collagenase could be detected, in one case up to 1 year . Mostly, however, these cells ceased production of this enzyme after about 2 weeks . The secretion of procollagenases and other extracellular proteases in some forms of cancer and metastasis formation is by some authors considered to be important ." Some of these enzymes have also been measured to be present in higher concentration than normal in synovial fluids of some patients with rheumatoid arthritis .' We wanted to see if cultured synovial fibroblasts of synovectomized tissue of patients with rheumatoid arthritis showed higher expression of different protooncogenes as compared to analogously grown synovial fibroblasts of patients with cruciate ligament ruptures . MATERIALS AND METHODS The synovectomized tissue of patients who fulfilled the American Rheumatism Association criteria for

*Author to whom correspondence should be addressed . 0890-8508/91/030207 + 07 $03 .00/0

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rheumatoid arthritis was obtained 2-3 h after surgery and synovial fibroblasts were grown as described .' No trypsin was used to disintegrate tissue, however. The incubation time in presence of collagenase was extended to 18 h. The promyelotic cell line HL-60 was a gift of Dr Thierauch, Schering AG, Berlin . This cell line has a 15-30-fold genomic amplification of c-myc RNA ." Relatively high levels of c-src and c-myb and almost no c-fos are measured ." The HBL-100 cell line was obtained from Dr K . Bister, MPI for Molecular Genetics, Berlin . It is a human epithelial cell line, nontumorigenic in nude mice at least at low cell passages but able to form colonies in soft agar and contains 67 chromosomes ." All cells were cultivated in Dulbecco's minimal essential medium containing 10% of heat inactivated fetal calf serum and with 5% carbon dioxide . 'A total of 3-8 x 10' cells of synovial fibroblasts of rheumatoid arthritis patients were usually harvested at confluency at the third to fourth passage of cultivation . Due to the small amount of tissue obtained from the control patients, these cells were at the fifth passage when harvested for investigation . The plasmids used were gifts : c-erb B-2 in plasmid pCER 204 from Dr T . Yamamoto (Inst . Med . Science, Japan); human exons 3 + 4 of c-fos in plasmid pSP 64, human c-Ha-ras in pBR 322, v-src cloned in pBR 322, all from Dr R . Muller (EMBL, Germany) ; human c-int-2 in plasmid pBR 322 from Dr C . Dickson (Imperial Cancer Research Fund Laboratories, United Kingdom) ; v-myb in pBR 322 from Dr J . M . Bishop (University of California, USA); v-myc and v-mil cloned in pBR 322 from Dr K . Bister ; human c-sis containing plasmid pAO 13 in pBR 322 from Dr van den Ven (University of Nijmegen, Netherlands) . Transformations of bacteria with the above plasmids were performed as described ." Plasmids were isolated as published ." The oncogene probes were cut with commercial restriction enzymes . The inserts were isolated in low melting agarose gels 14 and purified . The following standard procedures were performed as described : nick translation," total RNA extraction, 16 dot-blot hybridization," The hybridized filters were exposed at - 70° C for varying periods of time and the X-ray films developed . The black spots on the films were evaluated densitometrically and recorded in arbitrary units on a Shimadzu dual wavelength TLC scanner CS-930 . Generally, the filters shown in the figures are the ones where the highest differences in expression between controls and patients' RNA were found .

the unlabelled Sal 1-Bam H1 fragment of v-myc with the same radioactively labelled v-myc fragment . The absorption values of the 3 spots at 440 nm are recorded in Fig . 1(b) . They can also be expressed in percent of the total as depicted in Fig . 1(c). The drawn line shows the approximate linear relationship between the applied amount of DNA and light absorption . Similar hybridizations with other oncogenes showed similar correlations (data not shown) . Some of these filters were also hybridized with nonhomologous probes . In none of these was there any spot found on the films . Figure 2 shows the results of dot-blotted hybridizations of different amounts of total RNA of seven patients synovial fibroblasts, two controls (*1,*2, synovial fibroblasts of two patients with cruciate ligament ruptures) and total RNA of cell lines HL-60, HBL100 and rRNA with c-Ha-ras . The densitometric evaluation of the dots (Fig . 2(b), 4 pg of RNA) shows no distinct differences in expression between controls and patients . Quantitation of the row where 10 µg of total RNA was applied yielded in similar relative percentage of absorption . No homology is detected between ribosomal RNA (rRNA) and the human c-Haras probe . On other filters more total RNAs of further patients and controls were hybridized with similar results (data not shown) . The differences in expression of c-myc between control fibroblasts (*1 and *2) and the same synovial fibroblasts of patients with v-myc were also investigated (Fig . 3). The intensity of the HL-60 dot is about the same as in the case of control *2 . HL-60 cells are known to have a 15-30-fold genomic amplification of c-myc as compared to normal cells ." This causes a high level of steady state c-myc mRNA . Control cells *1 show a 3 . 5-fold lower amount of c-myc mRNA than *2 . The densitometric evaluation of the spots on several filters showed a specific increase of 3 . 7 when the value of patient 33 was compared to the one of control *1 . However, this value dropped to 1 when compared to control *2 on the same filter (data not shown) . These differences are therefore not considered to be significant . They were also the most pronounced found with any of the oncogenes investigated . A weight of 200 pg of v-myc gives a quite

RESULTS

good signal (Fig . 3a) . C-src gene expression also shows no significant differences between control fibroblasts and those of the arthritic patients (Fig . 4) . A good positive signal is given by 200 pg of v-src DNA . The expression of the proto-oncogene c-fos is also investigated (Fig . 5) . Scarcely any c-fos mRNA is

Figure 1 shows the sensitivity of the hybridization of

present in the HL-60 total RNA as reported in the literature ." Quite a bit of it occurs in the controls as

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well as in some patients' RNA. No significant differences can be seen upon quantitation of the intensities of the spots between controls and patients . rRNA was applied as a negative control . The results of the c-myb mRNA concentrations in the total RNA of the same patients' and control synovial fibroblasts are reported in Table 1 . They are taken from several filters . The differences of expres(a)

sion evaluated densitometrically are smaller than with c-myc (Table 1) . A dark spot of diameter of 7 mm was yielded by 200 pg of v-myb . The following proto-oncogenes c-mil, c-erbB-2, int2 and c-sis were neither expressed in control synovial fibroblasts nor the rheumatoid patients' ones nor in HL-60 cells .

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400 P9 200 v-myc-DNA-dots Fig. 1 . (a) Autoradiography of the dot-blot filter hybridization of the v-myc gene . Varying weights, 50, 200, and 400 pg of the Sal 1-Bam H1 fragment of the v-myc gene were bound to nitrocellulose filters . The filter was then hybridized with the same radioactively labelled v-myc fragment . Specific activity of the probe: 1 x 108 dpm µg - ' DNA; washing conditions : 0. 5 x SSC, 40°C; filter exposure at -80° C for 12 h. (b) Densitometric evaluation of the dots of (a) at 440 nm . (c) Integrated absorption values of the spots expressed in percentage of the total absorption value . 50

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Fig. 2. (a) Autoradiography of the dot-blot filter hybridized with the c-Ha-ras gene . Varying weights, 10 µg, 4 lag and 1 gg of RNA isolated from seven rheumatoid arthritic patients synovial fibroblasts (patients 21, 24, 27, 34, 33, 39 and 45) and from two control synovial fibroblasts (patients `1 and °2) were applied . Total RNA from cell lines HBL-100, HL-60 and yeast rRNA (negative control) were hybridized with the Barn H1-fragment of the c-Ha-ras gene . Specific activity of the probe : 3 x 108 dpm gg - ' DNA; washing conditions : 0 . 5 x SSC, 42 ° C ; filter exposure at -80°C for 16 h . (b) Densitometric evaluation of the row containing 4lag of RNA . The absorption values correspond to the gene expression .

DISCUSSION Since the washing conditions after hybridization were done at relatively low temperatures (40-42 ° C) depending on the homology of the oncogenic probes the specificity of the hybridizations was checked for each oncogene by applying the denatured DNA and hybridizing it with itself (as described in Fig . 1) . This reaction was specific for each investigated oncogene . That the reactions were specific can also be seen in the case of the HL-60 cells which express a high level of c-myc due to the amplification of this gene but a very low level of c-fos ." No significant differences in expression between normal and rheumatoid arthritis patients' cultured

synovial fibroblasts were observed in the case of the following proto-oncogenes : c-ras, c-src, c-fos, and cmyb. Cellular oncogenes c-mil, c-erbB-2, c-int-2 and c-sis were not expressed in either of the cells . The highest differences in expression observed in all hybridization experiments between control fibroblasts and arthritic patients' fibroblasts were seen in the case of c-myc in patient 33 and control *1 . This difference could be due to different stages in the cell cycle at the time of harvesting . It is known that cmyc, c-myb, c-fos, and c-ras expression can be stimulated by serum factors . 18 Levels of c-myc mRNA are reported to vary by a factor of 2 in unsynchro-



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Fig. 3. (a) Autoradiography of the dot-blot filter hybridized with the v-myc gene. Weights, 8 zg and 2 gg of total RNA isolated from rheumatoid arthritic patients synovial fibroblasts as well as the same amounts of the control fibroblasts (1, "2) were applied . Weights, 8 and 2 µg of the HL-60 cell line RNA and 8 µg of yeast rRNA (negative control) and 200 pg v-myc DNA (positive control) were also added . The RNA dots were hybridized with the Sal 1-Barn Hi fragment of the v-myc gene . Specific activity of the probe : 1 x 108 dpm µg - ' DNA ; washing conditions : 0. 5 x SSC, 40 ° C ; filter exposure at -80 ° C for 24 h . (b) Densitometric evaluation of the row containing 8 µg RNA . The adsorption values correspond to the gene expression . Table 1 . Densitometric evaluation of the row of 8 µg of total RNA hybridized with v-myb (1 x 10 8 dpm µg - ' of DNA) Samples name

HL-60 '1 (control patient) '2 (control patient) 20 21 24 27 33 34 35 39 45

Absorption measured at 440 nm (in percent of total) 10 8 6 9 12 10 7 5 11 10 5 7

200 pg v-myb yielded in a very dark spot of a diameter of 7 mm .

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Fig. 4. (a) Autoradiography of the dot-blot filter hybridized with the v-src gene . Weights, 8 µg and 2 .tg of total RNA isolated from rheumatoid arthritic patients synovial fibroblasts as well as the same amounts of the control fibroblasts ("1, '2) and HL-60 cells were applied . A weight of 8 µg of rRNA served as a negative control, 200 pg of v-src DNA as a positive control . The filter-bound RNAs are hybridized with the Pvu 2-fragment of the v-src gene . Specific activity of the probe: 3 X 108 dpm .tg - ' DNA ; washing conditions : 0 . 5 x SSC, 40 ° C; filter exposure at - 80 ° C, 24 h . (b) Densitometric evaluation of the track corresponding to 8 µg RNA of (a) . The absorption values correspond to the gene expression .

nized fibroblasts ." Furthermore its concentration is dependent on cell density ." Thus it seems unlikely that these synovial fibroblasts of rheumatoid patients show any changed expression of proto-oncogenes at least when investigated as cultured synovial fibroblasts . It is probably more likely that this excessive proliferation of the synovial membrane in these patients is due to the permanent stimuli and/or the inflammatory mediators present .' Upon cultivation these factors would be removed and the rheumatoid patients' synovial fibroblasts would grow and proliferate like the ones of normal persons .

REFERENCES 1 . Harris, E . D . (1985) . Pathogenesis of rheumatoid arthritis . In Textbook of Rheumatology (Kelley,W . N ., Harris, E . D ., Ruddy, S . & Sledge, C. B ., eds) pp . 886-915 . Philadelphia: W . B . Saunders.

2 . Bishop, J . M. (1987). The molecular genetics of cancer . Science 235, 305-11 . 3 . Nishimura, S . & Sekiya, T . (1987) . Human cancer and cellular oncogenes . Biochemical Journal 243, 313-27 . 4. Yarden, Y . & Ullrich, A . (1988) . Growth factor receptor tyrosine kinases . Annual Review of Biochemistry 57, 443-78 . 5 . Barbacid, M. (1987) . ras genes . Annual Review of Biochemistry 56, 779-827 . 6 . Duesberg, P. (1985) . Activated proto-onc genes : Sufficient or necessary for cancer? Science 228, 669-77. 7 . Dayer, J . M ., Krane, S . M ., Graham, R., Russel, C . & Robinson, D . R . (1976) . Production of collagenase and prostaglandins by isolated adherent rheumatoid synovial cells . Proceedings of the National Academy of Science USA 73, 945-9 . 8 . Moscatelli, D. & Rifkin, D . B . (1988) . Membrane and matrix localization of proteinases : A common theme in tumor cell invasion and angiogenesis . Biochimica et Biophysica Acta 948, 67-85 . 9 . Muller, D ., Quantin, B ., Gesnel, M .-C ., Millon-Collard, R ., Abecassis, J . & Breathnach, R. (1988) . The collagenase gene family in humans consists of at least 4 members . Biochemical journal 253, 187-92 .

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Fig. 5. (a) Autoradiography of the dot-blot filter hybridized with the c-fos gene . A weight of 8 µg of total RNA isolated from rheumatoid arthritic patients synovial fibroblasts as well as the same amount of the control fibroblasts (°1, *2) was applied . A weight of 8 Vg of total RNA from the HL-60 cell line is also dotted . The RNAs were hybridized with the Pst 1-Sph 1-fragment of the c-fos gene . Specific activity of the probe : 3 x 10 8 dpm µg -1 DNA ; washing conditions : 0. 5 x SSC, 40° C ; filter exposure at -80 ° C for 16 h . (b) Densitometric evaluation of (a) . The absorption values correspond to the gene expression .

10. Liotta, L . A., Rao, C . N . & Wewer, U . M . (1986) . Biochemical interactions of tumor cells with the basement membrane . Annual Review of Biochemistry 55, 103757. 11 . Collins, S . J . (1987) . The HL-60 promyelotic leukemia cell line : Proliferation, differentiation, and cellular oncogene expression . Blood 70, 1223-44 . 12 . American type culture collection, 6th edn 1988 . Rockville, USA, Cell Lines and Hybridomas . 13 . Hanahan, D . (1983) . Studies on transformation of Escherichia coli with plasmids . Journal of Molecular Biology 166, 557-80. 14 . Maniatis, T ., Fritsch, E . F . & Sambrook, J . (1982) . Molecular Cloning: A Laboratory Manual . Cold Spring Harbor, New York . 15 . Meinkoth, J . & Wahl, G . M. (1987) . Nick translation . Methods of Enzymology 152, 91-4 . 16 . Chirgwin, J . M ., Przybyla, A . E ., MacDonald, R . J . &

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Rutter, W . J . (1979) . Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease . Biochemistry 18, 5294-9 . Thomas, P . (1983) . Hybridization of denatured RNA transferred or dotted to nitrocellulose paper . Methods of Enzymology 100, 255-66 . Heldin, C . H ., Betsholtz, C ., Claesson-Welsh, L . & Westermark, B . (1987) . Subversion of growth regulatory pathways in malignant transformation . Biochimica et Biophysica Acta 907, 219-44. Denhardt, D . T., Edwards, D . R . & Pafett, C . L . J . (1986) . Gene expression during the mammalian cell cycle . Biochimica et Biophysica Acta 865, 83-125 . Dean, M ., Levine, R. A ., Ran, W ., Kindy, M . S ., Sonenshein, G . E . & Campisi, J . (1986) . Regulation of c-myc transcription and mRNA abundance by serum growth factors and cell contact. Journal of Biological Chemistry 261, 9161-6 .