D etection of Oncogene mrna Sequences in Cultured Cells by In Situ Hybridization*

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 17, No. 2 Copyright 1987, Institute for Clinical Science, Inc. D etection of Oncogene mrna Sequences in Cultured Cells by In Situ Hybridization* GARYD. STONER, PH.D.,f$ M ING YOU, M.D.,t JAN SKOUV, P h.d.j G. CO LIN BUDD, P h.d., BEN PANSKY, P h.d., M.D., and YIAN WANG, M.D.f Departments o f Pathology, f Physiology, and Anatomy, Medical College o f Ohio, Toledo, OH and Laboratory o f Environmental Carcinogenesis The Fibiger Institute, DK-2100, Copenhagen, Denmark ABSTRACT The present study was undertaken to determ ine the feasibility of using in situ hybridization techniques to identify oncogene transcription in cultu red cells. Following in situ hybridization with 32P-labeled v-src and v-ha-ra.v DNA probes, src and H a-ras related transcripts were identified in cell lines transfected with v-src and Ha-ras, respectively. In both the v-src and c-ha-ra.s- transfected cell lines, the num ber of silver grains over individual cells w ere significantly higher (p < 0.001, f-test) than in a nontransfected, non-tum origenic, rat esophageal epithelial cell line. There was a highly variable num ber of silver grains above individual cells. Significantly fewer silver grains were counted over cells that had been preincubated with either non-labeled v-src or v-ha-ras DNA or that w ere pretreated with RNase A. Both oncogene transfected cell lines contained approximately 10 times m ore oncogene related mrnas than non-transfected cells as judged by the num bers of silver grains over individual cells. Filter-hybridization analysis of the transfected and non-transfected cell lines confirm ed that the expression of src and H a-ras transcripts was higher in the transfected cell lines than in the non-transfected cell line. Therefore, the in situ hybridization technique would appear useful for the identification of oncogene transcripts in single cells and could potentially be applied to cytological preparations of hum an cells and to hum an tum or cells in culture. * This study was supported by U.S. N ational $ Gary D. Stoner, P h.d., Department of Pathol- Cancer Institute Grant No. CA Jan Skouv is ogy, Medical C ollege of Ohio, Health Education the recipient of a fellowship from the Danish Cancer Building, Rm. 202, Toledo, OH Society /87/ $01.50 Institute for Clinical Science, Inc.

2 Introduction ONCOGENE mrna DETECTION IN CULTURED CELLS 75 There is a growing body of evidence to suggest th at th e altered expression of cellular genes, the so-called oncogenes, is critical for the process of tum or formation.17,33,34 It is conceivable that the varied biological behavior of tum or cells, including their m alignant potential, is due to differences in the expression of one or m ore oncogenes. To examine this, it would be useful to develop a m ethod to identify oncogene expression in single cells. N ucleic acid hybrid izatio n offers a very specific and sensitive approach for the detection and quantitation of gene expression in cells.3,4,7,15 Semiquantitative in situ hybridization m ethods have b e e n d e v e lo p e d for the d etectio n of viral3,4,5,14 or other abundant RNAs2,8 in individual cells. In addition, quantitative m ethods for the detection of proteins, including oncogene products, in individual cells using histochemical or immunological approaches have b een d e v e l oped.27,35 At present, at least 30 different oncogenes originating from the cellular g en o m e have b e e n c h a ra c te riz e d.34 O ncogene specific D N A -probes for a majority of the oncogenes are available in the form of bacterial plasmids or as ready-to-use preparations from various com m ercial sources. Therefore, in situ hybridization m ethods could be applied to d e te c t oncogene-specific mrnas in tissu e s an d cells in lab o ra to rie s not acquainted with all of the techniques of m odern m olecular biology. The present study was undertaken to determ ine the feasibility of using in situ h y b rid izatio n tech n iq u es to identify oncogene tran scrip tio n in individual cells in culture. In order to correlate the results obtained by in situ hybridization to those obtained by conventional filter hybridization oncogene expression in different cell lines was investigated in our laboratory. O ur results dem onstrate that in situ hybridization is specific for o ncogene mrnas and can pro v id e a sem iquantitative assessment of oncogene expression in single cells. M aterials and Methods C e l l L i n e s The cell lines used in this study w ere a R ous sarcom a v iru s-tra n sfo rm e d rat cereb ral endothelial cell lin e 13 designated P40+ ; a transform ed m urine fibroblast cell line (NIH RS 485) transfected by a hum an c-ha-ras gene containing an upstream long term inal rep eat (LTR) region from a retrovirus;9,10 and, a nontransfected, non-transform ed epithelial cell line (RE-149) derived in our laboratory from rat esophagus.1 The P40+ and N IH RS 485 cell lines were cultured at 37 C in D u lb e c c o s M E M * s u p p le m e n te d w ith 10 p e rc e n t fetal bovine serum f, as described.9,10,13 RE-149 cells w ere c u ltu re d in PFM R -4 m ed iu m 18 supplem ented with two percent dialysed fetal bovine se ru m t and seven growth factors as described previously.1 I n S it u H y b r id iz a t io n The procedure for in situ hybridization was essentially as d escribed 3,4,7,8 with m inor modifications. The following steps w ere used: preparation of radioactive probes, chemical treatm ent of glass slides, generating cytological specimens, nucleic acid denaturation, hybridization, autoradiography, and staining. The 0.7 Kb SacI-PstI fragm ent of the v-h a-ras gene12 and the 0.8 Kb P vull fragm ent of the v-src gene28,31 were used as probes. Roth probes w ere obtained as purified, nick translated DNA fragm ents * Grand Island Biological C o., Grand Island, NY. t Flow Laboratories, Rockville, MD.

3 76 STONER, YOU, SKOUV, BUDD, PANSKY, AND WANG labeled with 32P to a specific activity of ap p ro x im ate ly 5 X 108 cpm p e r fxg DNA.$ M icroscope slides were pre-treated by incubation at 70 C in 2X saline-sodium c itra te (2X SSC or 0.3M N ac l plus 0.03M trisodium citrate, p H 7.0) containing 0.02 percent (wt per vol) Ficoll, 0.02 percent polyvinylpyrrolidone, and 0.02 percent bovine serum albumin for two hrs. The slides w ere then fixed for 10 m inutes at room tem perature (22 C) in ethanol/acetic acid (3:1). This tre a t m ent prevents binding of DNA to glass.4 Sem i-confluent cultures of each cell line w ere suspended by treatm ent for 5 to 10 min with a H EPES buffered balanced salt solution (HBS) containing one percent polyvinylpyrrolidone, percent ethylenebis(oxyethylenenitrilo)tetracetic acid (EGTA) and 0.02 percent trypsin.1 The cells w ere washed once in HBS and deposited on treated m icroscope slides. Approxim ately 20,000 to 30,000 cells w ere deposited p er slide. After a few m inutes of drying, the cells w ere fixed for 10 m in at room tem peratu re in eth an o l/acetic acid, 3:1. The slides w ere then treated as follows: 20 min at room tem perature in 0.2 M HCL; five min in distilled water; five min in 2X SSC (2X); five min in distilled water; and 30 min at 37 C in 20 mm Tris-HCL (ph 7.4) per two mm CaCl2 containing one julg of proteinase K per ml. The proteinase K digestion was followed by three washes in distilled w ater and dehydration in graded ethanol (30 percent, 75 percent, 95 percent; one min each). The slides w ere hybridized with one to two ng of [32P]DNA (3 to 6 X 105 cpm) p e r slide. T he h y b rid izatio n m ixture contained 50 percent deionized formamide, 0.02 percent (wt per vol) Ficoll, 0.02 p e rc e n t polyvinylpyrrolidone, 0.02 p e r cent bovine serum album in, 2X SSC, and 100 to 200 ng of labeled probe p er t Oncor, Inc., Gaithersburg, MD. ml. The hybridization mixture, including [32P]DNA, was heated at 100 C for 10 m in to denature the probes, and then quickly cooled to 0 C. Five microliters of the hybridization m ixture w ere placed on the cells and covered with a 12-mm glass coverslip. A few drops of m ineral oil w ere added to the area around the coverslip to prevent evaporation of the hybridization mixture. The slides were incubated in a hum idified cham ber for 48 hr at 25 C. At the end of the hybridizatio n p e rio d, th e coverslip s w ere rem oved by im m ersing the slides twice in chloroform (five min each time) and th en tw ice (five m in each tim e) in 50 percent formamide/tris-hcl buffer (10 mm Tris-HCL, ph 7.4; 1 mm ethylene diam ine tetraacetic acid [EDTA]; 600 mm NaCl). The slides w ere then washed in a large volum e of 2X SSC w ith constant stirring, followed by dehydration in graded ethanol (70 percent and 95 percent ethanol containing 300 mm am m onium acetate). All washes were done at room tem perature. Kodak NTB-2 nuclear track emulsion (one to two m icron in thickness) was m elted at 45 C and diluted 1:1 with 600 mm ammonium acetate. The slides were dipped in m elted em ulsion for one to two sec and allowed to dry in an upright position for one hr at room tem perature. T hey w ere tra n sfe rre d to light-proof boxes containing silica gel desiccant and exposed at 4 C. T he slides w ere developed in Kodak D-19 developer for three min at room tem perature followed by a brief wash in one percent acetic acid and fixation for th re e m in in Kodak fixer. After the slides w ere washed in distilled water, they w ere stained for 30 to 45 min with Giemsa stain diluted 1:50 in 10 mm phosphate buffer. A utoradiographic grains over individual cells w ere counted w ith an A rtek M odel 982 C ounter* fitted to a light * Artek Systems Corp., Farmingdale, NY.

4 ONCOGENE vnrna DETECTION IN CULTURED CELLS 77 m ic r o s c o p e. 1 A fte r e a c h c e ll w as counted, the grains in an equivalent area of th e slide adjacent to the cell w ere counted to determ ine the background. T he background grain count was automatically deducted from the grain count over the cells by the Artek Counter. The accuracy of the Artek counter for enum erating autoradiographic grains over cells was checked by com paring Artek counts of grains over 20 cells with those o f th e n a k e d e y e. T h e v a ria b ility betw een the two m ethods did not exceed 10 p e rc e n t after su b tractio n of background. F i l t e r H y b r id iz a t io n Total cellular ribonucleic acid (RNA) was p rep a re d by lysing exponentially growing cell monolayers in a lysis buffer (ph 7.0) containing: 4M guanidine thioc y a n ate,f 25 mm sodium citrate, 2.3 p e rc e n t (v/v) sodium N -lauroylsarcosinef, and seven percent (v/v) 2-mercaptoethanol in water. Following cell lysis, th e RNA was purified by ultracentrifugation through a 5.7 M CsCl cushion as described by Chirgwin et al.11 The integrity o f each sam ple was confirm ed by g el e le c tr o p h o r e s is o f g ly o x y la te d R N A.24 O nly RNA sam ples show ing intact 28S and 18S ribosomal RNA bands w ith a 28S:18S ratio bigger than two w ere analyzed. The relative levels of oncogene related mrna w ere d e te r m ined by a variation of the dot-blot filter hybridization m eth o d using a M inifold II slot blot apparatus. $ H ybridization of serial dilutions (10, 5 and 2.5 (xg) of total RNA was perform ed at 42 C in 50 p e rc e n t form am ide using BA 85-SB nitrocellulose filters. $ After hybridization, the filters were washed three times (20 min each time) in 2X SSC and three tim es (20 min each) in 0. IX SSC, and 0.1 p e rc e n t sodium dodecy l sulfate. All washes w ere perform ed at 45 C using the same 32P-labeled deoxyribonucleic acid (DNA) p ro b es. A utoradiogram s w ere prepared using Kodak XAR-5 X-ray film and tungsten intensifying screens, and they w ere quantified by densitom e try. Results In s itu D N A -R N A h y b rid iz a tio n revealed that the majority of the P40 + cells showed src gene specific-labeling representing src gene mrnas, and most of the N IH RS 485 cells showed ras gene specific-labeling. In figure 1 is illustrated in situ DNA-mRNA hybridization of the P40+ and the RE-149 cell lines with the 32P-labeled w-src DNA probe. Eightyfive p e rc e n t of P cells had silver grains over th e cells, and th e m ean num ber of grains per cell (82.15) (SD = 29.87) was significantly (p < 0.001, t test) higher than in RE-149 cells (10.95) (SD = 17.06) (table I). The grains were d istrib u ted over the cell cytoplasm. Similar resu lts w ere found for ras gene mrna transcripts in N IH RS 485 and R E-149 cells (figure 2). U sing a 32Plabeled v-ras DNA probe, more than 95 percent of the N IH RS 485 cells contained silver grains over their cytoplasm and the m ean num ber of grains per cell (130.25) (SD = 30.91) was significantly (p < i-test) higher than in RE-149 cells (12.00) (SD = 21.96) (table I). To dem onstrate the specificity of the labeling for src and ras gene transcripts, P40+ and N IH RS 485 cell lines w ere pretreated with a 20-fold excess am ount of non-labeled src or ras DNA probes and then hybridized with 32P-labeled src or ras DNA probes. A utoradiographic preparations showed that there was no accum ulation of silver grains over the cells o ther than background w hen they t Fluka AG., Buchs, Switzerland. ± Schleicher and Schuell, Dassel, W. Germany. Kodak, Rochester, NY.

5 78 STONER, YOU, SKOUV, BUD D, PANSKY, AND WANG F ig u r e 1A. P 40+ cells reacted with 32P-labeled v-src D N A probe. N ote num erous autoradiographic grains over the cytoplasm. Original magnification X 11,000. F i g u r e IB. R E cells reacted with 32P-labeled v-src D N A probe. N ote fewer autoradiographic grains over the cytoplasm when compared to 1A. Original magnification X 11,000. Bars = 1.0 xm (A,B). w ere p retrea ted w ith non-labeled probe. In a d d itio n, follow ing tre a tm e n t w ith R N ase A, th e r e w e re no o b se rv a b le silver grains over the cytoplasm of P40 cells and N IH RS 485 cells. A slot blot filter hybridization was p er form ed on RNA isolated from mass cul tu re s of th e th re e cell lines: RS 485, P and RE-149 (figure 3). This analy sis show ed an approxim ately 50 tim es TABLE I Detection and Relative Abundance of src and ra s Oncogene mrnas in Cultured Cell Lines s r c - S p e c i f i c RN A Cell Line N u m b e r o f Cells Coun t e d P+40 RE r a s - S p e c i f i c RNA Autoradiographic G r a i n s p e r Cell ± ± 29.87* Cell Line N u m b e r o f Ce l l s Counted NIH RS 485 RE Autoradiographic G r a i n s p e r Cell ± ± Values are mean ± standard deviation and represent the number of grains p er cell after subtraction of the background counts.

6 O N C O G EN E mrna D ETECTIO N IN C U LTU R ED CELLS F i g u r e 2A. grains over the F ig u r e 2B. grains over the 79 RS 485 cells reacted with 32P-labeled v-ha-ras DN A probe. N ote numerous autoradiographic cytoplasm. Original magnification X 11,000. RE-149 cells reacted with 32P-labeled v-ha-ros D N A probe. N ote fewer autoradiographic cytoplasm w hen compared to 2A. Original magnification X 11,000. Bars = 1.0 Ji.m (A,B). overexpression of src in P 40+ cells when com pared to e ith e r R E -149 cells or RS 485 cells. T he expression of H a-ras in RS 485 cells exceeded that in P cells and RE-149 cells by factors of approxim ately 20 and 10, respectively. D iscussion In th e p re s e n t study, in situ DNAm RNA h y b rid iz atio n te c h n iq u e s w ere applied to th e detectio n of mrnas to two oncogenes, src and H a-ras, in trans f o rm e d c e lls. Q u a n tita tio n of s ilv e r grains over the cells indicated that the in situ hybridization technique is both sen sitive and specific in detecting oncogene mrna transcripts. However, a com pari son o f th e le v e l of o n c o g e n e re la te d m RNA by in situ h y b rid iz atio n using radioautography and by filter hybridiza tion suggested that the in situ hybridiza tion tec h n iq u e was not as sensitive as filter hybridization in revealing differ ences in oncogene mrna expression. This conclusion is m ade from the obser vation that in situ hybridization showed only a 10-fold d iffe re n c e in src g en e m RNA expression b e tw e e n P and RE-149 cells, w hereas filter hy b rid iza tion revealed a 50-fold difference. H ow ever, th e two m eth o d s w e re in close agreem ent w hen m easuring differences in H a-ras mrna expression betw een RS 485 and RE-149 cells. In situ hybridiza tio n sh o w e d th a t th e le v e l of H -ras related mrna was 10 tim es higher in RS

7 80 STONER, YOU, SKOUV, BUD D, PANSKY, AND WANG F i g u r e 3A. L evel of v-src and v-h a-ras related R N A in th e th r e e c e ll lines: RS 485, P and RE-149. RNA was extracted from five sepa rate culture flasks o f RS 485 and P 40+ cells (Nos. 1-5 ) and tw o s e p a r a te fla s k s o f R E c e l l s (Nos. 1,2). Total cellular RNA was sp otted onto a n itr o c e llu lo s e filte r in two-fold dilutions (10, 5, and 2.5 (xg) and hybrid iz e d w it h 32P - la b e l e d D N A probes sp ecific for src and Ha-ras. F ig u r e 3B. 125 x 10~3, 50 x 10_ 5 a n d 2 5 x jxg o f p u rified sr c and Ha-ras specific D N A w as s p o tte d o n to e a c h filter as a standard. Shown is the src D N A standard from the src-hybridization in figure 3A. B v-src DNA cells than in RE-149 cells; this differ ence was also obtained by filter hybrid ization. A possible explanation for this discrep a n c y is t h a t H a - r a s e x p r e s s io n in most cells is relatively abundant whereas t h a t o f t h e s r c g e n e is r e l a t i v e l y low,16 21,23,25,29 and the low level of src gene mrna expression may have been m asked by th e high background grain count observed in in situ preparations. It should b e em phasized that these data are prelim inary and a m ore extensive quan titative com parison of oncogene mrna expression in c u ltu re d cells by in situ hybridization versus filter hybridization w ould re q u ire studies utilizing several c o n c en tra tio n s o f rad io lab eled p ro b e, s u b stra te, etc. T h ese stu d ies are c u r rently underway. An im p o rta n t factor for the practical 25 5 a p p lic a tio n o f in s itu D N A - m R N A hybridization is the use of a radiolabeled probe that allows maximal labeling in a short period of tim e. It was found by us that silver grains could be observed in autoradiographic preparations w ithin 48 h r a fte r e x p o su re of th e cells to 32Plab eled DNA probes. In contrast, the tim e period for the appearance of silver grains after exposure of the same cells to DNAs labeled w ith eith er 35S or 3H was a p p ro x im a te ly 14 days a n d 28 days, resp ectiv ely (unpublished data). W hen using 32P labeled probes, the entire in situ D N A-m RNA hybridization p ro ce d ure to d etect oncogene specific mrnas can b e accom plished in th re e to four days. In situ hybridization is m ore sensitive than im m unof luorescence for the d etec tion of virus-infected cells. Haase et al14

8 ONCOGENE mrna DETECTION IN CULTURED CELLS 81 show ed th at in situ hybridization was m ore sensitive for the detection of viral nucleic acids in visna virus-infected cells than was im m unofluorescence for the detection of visna virus specific proteins. F urtherm ore, silver grain counts w ere converted to copy num bers by com paring the average num ber of grains per cell in cells infected with visna virus to the average copy n u m b e r as assessed by standard hybridization techniques using mrna extracted from th e cells. In spite of the intense research on oncogenes, th e re is relatively little information regarding the tem poral sequence of oncogene expression in cellular transform ation, and the potential role(s) of tu m o r in itia to r s a n d p ro m o te rs in influencing the expression of oncogenes in transform ed cells. In situ hybridization techniques provide an opportunity to investigate the expression of oncogene m essages in c u ltu red cells exposed to various carcinogens and prom oters, and to study the relationships betw een the expression of these genes and the acquisition of phenotypic alterations during the various stages of cell transformation. In addition, in combination with immunocytochem ical m ethods, in situ hybridization techniques can be used to comp a re in p a ra lle l, th e e x p re ssio n of o n cogene m RNAs and th e ir p ro te in products in single cells. E nhanced oncogene expression has b een dem o n strated in various hum an tum ors,19'20'29 as well as in certain norm al hum an tissu e s.26 In at least one instance, the expression of an oncogene (N -ras) in hum an neuroblastom as was shown to be of m ajor im portance for d eterm in in g the prognosis of the disease.6 How ever, w ith the exception of two recen t studies in which the expression of the H a -ras gene was examined in colonic mucosa during different stages of tum or progression,30'32 th ere is very little in f o r m a tio n on th e te m p o r a l sequen ce of oncogene expression in tu m o r initiation and progression. The adaptation of the in situ hybridization technique described in this report to tissue specim ens (frozen or fixed) could r e s u lt in c o m p a riso n s of o n co g en e expression in single cells in normal, prem alignant and m alignant tissues. Such comparisons could provide knowledge of p o ten tia l use in tu m o r diagnosis, in d eterm in in g the prognosis of the disease, and in designing m ore specific and effective m ethods of cancer treatm ent. A major advantage of the in situ hybridization tech n iq u e described h e re is that oncogene expression can be determ ined in single cells and, if adapted to tissue specim ens, w ould re q u ire relatively small am ounts of tissue. Acknowledgm ents Thanks are extended to Drs. Clement Diglio and D. James McCorquodale for providing the P40+ cell line, and to Dr. Esther Chang for the NIH RS 485 cell line. The secretarial assistance of Ms. Susan Hahn is greatly appreciated. References 1. B a b c o c k, M. S., M a r in o, M. R., G u n n in g III, W. T., and St o n e r, G. D.: Clonal growth and serial propagation of rat esophageal epithelial cells. In Vitro 79: , BERGER, C. N.: In situ hybridization of immunoglobulin-specific RNA in single cells o f the B lym phocyte lineage with radiolabelled DN A probes. The E M B O J. 5 : , BRAHIC, M. and H a a s e, A. T.: Detection of viral sequences of low reiteration frequency by in situ h yb rid ization. Proc. N atl. Acad. Sci. USA 75: , B r a h i c, M., H a a s e, A. T., and C a s h, E.: Simultaneous in situ detection of viral RNA and antigens. Proc. Natl. Acad. Sci. USA S I: , B r i g a t i, D. J., M y e r s o n, D., L e a r y, J. J., S p a l h o l z, B., T r a v is, S. Z., F o n g, C. K. Y., HSIUNG, G. D., and W a r d, D. C.: Detection of viral genom es in cultured cells and paraffinem bedded tissue sections using biotin-labeled hybridization probes. Virology 126: , B r o d u n, G. M., S e e g e r, R. C., S c h w a b, M., V a r m u s, H. E., and B is h o p, J. M.: A m p lifica tion o f N -m yc in un treated hum an neurob lastom as c o rrela tes w ith a d v a n ced d is e a se stage. S cien ce 224: , B u d d, G. C. and Pa n sk y, B.: In situ D N A- mrna hybridization as a microscopic cytoehem-

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