Simultaneous isolation of DNA, RNA, and antigenic protein exhibiting kinase activity from small tumor samples using guanidine isothiocyanate

ANALYTICAL

BIOCHEMISTRY

188,338-343

(1990)

Simultaneous Isolation of DNA, RNA, and Antigenic Protein Exhibiting Kinase Activity from Small Tumor Samples Using Guanidine Isothiocyanate Laurence M. Coombs,**t” Dennis Pigott,* Alison Jpachim Denner,* and Margaret A. Knowles*

Proctor,*

Marian

Eydmann,*

*Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 OTL, United Kingdom, and tInstitute of Urology, Middlesex Hospital, London, United Kingdom

Received

December

20,1989

Quantitative and qualitative abnormalities of genes and their expression as RNA and protein have been implicated in carcinogenesis. The ability to correlate gene copy number, structure, expression, and function with the disease process is fundamental to an understanding of the underlying mechanisms. Such studies in man are often hindered by the paucity of available tissue and the need to use various incompatible methods to extract DNA, RNA, and protein. Chirgwin (1) first suggested the use of guanidine isothiocyanate (GIT)* disruption of tissues to isolate intact

RNA, as both cation and anion are strongly chaotropic, capable of denaturing proteins and hence RNases faster than other chaotropes. Glison’s (2) method of separating RNA from the protein by ultracentrifugation using a cesium chloride cushion was incorporated, as it was already appreciated that dilution of denatured RNase resulted in renaturation of the active enzyme (3). Meselson’s (4) method of isolation of DNA in a cesium gradient has been incorporated by many contemporary technical manuals (5) into a combined method of extracting the DNA layer and RNA pellet from the same sample. In these methods the protein and lipid containing guanidine phase was aspirated and discarded prior to recovery of the DNA and RNA. Hager and Burgess (6) demonstrated that certain enzymes could be eluted from SDS-polyacrylamide gels and renatured to activity following further denaturation with guanidine hydrochloride. Tanford (7) in his review of protein denaturation and renaturation argued that denaturation was usually reversible and cited only theoretical cases where it might not be. He proposed that guanidine hydrochloride denatured proteins to random coils and that if disulfide bonds were also broken, correct reconformation would occur in dilute solution, with time, depending on the protein concerned and providing that the primary structure was intact. Tanford did not imply that this was technically easy but his arguments were further supported by Anfinsen’s work (8). This showed that the covalent structure of the primary sequence of ribonuclease was able to determine, without error, the complete native configuration. Therefore we investigated whether protein could be recovered quanti-

’ To whom correspondence should be addressed. * Abbreviations used: BSA, bovine serum albumin; threitol; EGFR, epidermal growth factor receptor; GIT,

thiocyanate; PBS, phosphate-buffered saline; RIPA, radioimmune precipitation buffer; SDS, sodium dodecyl sulfate; TE, Tris-EDTA; TEMED, N,N,N’,N’-tetramethyl-1,2-ethanediamine.

Correlative studies of genes and their expression in human tumors are often hampered by the small sample size and the need to use differing and incompatible techniques to obtain DNA, RNA, and protein. We describe an extension of the established guanidine isothiocyanate method for isolation of DNA and RNA which allows the simultaneous isolation of total cellular protein. The protein obtained by this method (from solid tumors and cell lines) was comparable to protein extracted by a standard detergent solubilization method. Antigenicity was retained as demonstrated by Western blotting for epidermal growth factor receptor and actin and by immunoprecipitation of ~53. Kinase activity was similar in proteins extracted by the two methods. It seems probable that most monomeric proteins can be obtained in a form suitable for Western analysis and immunoprecipitation and that. these may also retain some functional activity. 0 19VO Academic Press, Inc.

DTT, dithioguanidine iso-

338 All

Copyright 0 1990 rights of reproduction

0003-2697/90 $3.00 by Academic Press, Inc. in any form reserved.

SIMULTANEOUS

ISOLATION

tatively from the guanidine phase and whether it could be used for studies of structure and function, thereby permitting a single extraction procedure to be used with small tissue samples. In this report we demonstrate that following isolation selected proteins are reactive on Western blots; they can be immunoprecipitated with specific antisera, and they display phosphorylating activity similar to that of proteins extracted by radioimmune precipitation buffer (RIPA) lysis. MATERIALS

AND

METHODS

The anti-peptide (2E) antiserum to EGFR (9), recognizing residues 429-440 of the receptor C terminal to the kinase domain, was kindly donated by Dr. W. Gullick, Imperial Cancer Research Fund, Hammersmith Hospital. Anti-actin antiserum (polyclonal) was purchased from Sigma Chemicals, and denaturation-insensitive anti-p53 monoclonal antibody 421 (10) and COS cells (expressing mutant human ~53) were gifts from Drs. Sturzbecher and Jenkins, Marie Curie Research Institute. Other cell lines used were COS (11) and A431 (12). Guanidine isothiocyanate, P-mercaptoethanol, N-lauroylsarcosine sodium salt, bovine serum albumin fraction V, bovine serum albumin (98%), trichloroacetic acid, Nonidet-P40, heparin sodium salt (Grade l), and antifoam A emulsion were purchased from Sigma. N,N-Methylenebis(acrylamide), ammonium persulfate, TEMED, and the silver staining kit were purchased from Bio-Rad. Acrylamide Electran was purchased from BDH. Coomassie blue G-250 and cesium chloride (optical grade) were from BRL. Collodion bags were from Sartorius. [T-~~P]ATP, 1251-labeledprotein A, and hyperfilm MP RPN6 were purchased from Amersham International. Protein A-Sepharose was from Pharmacia and fetal calf serum from Sera Labs U.K. The buffers used were as follows. Sample buffer: 1% SDS, 1% glycerol, 40 mM Tris-HCl (pH 6.7), 0.002% bromophenol blue, 0.05 M P-mercaptoethanol. Running buffer: 0.04 M Tris base, 0.19 M glycine, 0.1% SDS. Transfer buffer: 25 mM Tris-HCl (pH 8.3), 192 mM glytine, 20% (w/v) methanol. TE (1X): 10 mM Tris-HCl, 1 mM EDTA (pH 8.0). RIPA buffer without SDS: 150 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl (pH 8.0), 1% deoxycholic acid, 1% NP-40. Protein buffer following lyophilization: 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1% NP-40, 10% glycerol. The cesium chloride cushion was made from a 5.7 M solution containing 25 mM sodium acetate autoclaved prior to use. GIT solution (4 M) was made as follows: 50 g guanidine isothiocyanate, 0.5 g sodium lauroylsarcosine, and 2.5 ml 1 M sodium citrate, pH 7.0 (final concentration 25 mM), were made up to approximately 80 ml with doubly deionized water, adjusted to pH 7.0 using 10 mM sodium hy-

OF DNA, RNA, AND PROTEIN

339

droxide, and filtered through a Nalgene (0.45 pm) filter. Antifoam A (0.33 ml) and P-mercaptoethanol (0.7 ml) were added, and the solution was made up to 100 ml with deionized water, aliquoted, and stored at -20°C. All preparation and work with this solution should be performed in a fume hood. Protein quantitation (13) was calculated with Coomassie blue by using a calibration curve of bovine serum albumin fraction V at 595 nm. Protein extracted with RIPA was first precipitated in trichloroacetic acid and dissolved in buffer to remove the detergent. Silver staining was achieved by following the manufacturer’s instructions. Preparation of Total Protein Cells were lysed in 4 M GIT after washing with PBS and then layered onto 5 ml cesium chloride in 14-ml Sorvall tubes. Tissues were taken from -7O”C, immersed in GIT, and chopped finely with a scalpel. The mixture was rotated at lo-20 rpm for 4-8 h to ensure dissolution and then layered onto cesium. (Maximum loading should not exceed 0.15 g tissue/ml as guanidine overloading interferes with RNA extraction (1,14).) Small samples (~80 mg) were layered onto 2-ml cesium chloride cushions in 4.4-ml Sorvall tubes. Samples were centrifuged at llO,OOO-150,OOOgat 20°C for 18 h to ensure sedimentation of RNA. The guanidine phase was aspirated to the level of birefringence in the cesium which marks the presence of DNA, placed in a collodion bag, and dialyzed at 4°C with four changes of 100 mM ammonium bicarbonate over 24 h. The minimum period of dialysis has not been assessed, as this time scale coincided with the method of extraction of DNA below. The samples were decanted, frozen at -7O”C, and lyophilized to complete dryness (24-48 h depending on sample size) before being dissolved in buffer or stored at -70°C. DNA and RNA Preparation The birefringent layer of cesium chloride was aspirated and dialyzed against 1X TE for 24-48 h at 4°C. The DNA was extracted with phenol, phenol:chloroform (l:l), and chloroform and precipitated with l/10 vol 3 M sodium acetate and 2.5 vol absolute ethanol. It was collected by centrifugation or spooling, washed in 70% ethanol, air-dried, and dissolved in 1X TE. The residual cesium chloride was aspirated and the RNA pellet allowed to air-dry. After being washed in 70% ethanol, it was dissolved in 300 ~10.3 M sodium acetate (pH 6.0) and precipitated with 1 ml absolute ethanol. Samples were stored for at least 1 h at -70°C and centrifuged at 1300 rpm and the RNA pellet was dried and dissolved in a 0.02% solution of heparin before quantitation and storage.

340 Standard

COOMBS

Protein

ET

AL.

Extraction

Where protein for comparison was required, RIPAextracted protein from cell lines was used. Dishes (g-cm diameter) of cells were washed three times in PBS and lysed in 3 ml of RIPA minus SDS. The solution was centrifuged at 100,OOOg at 4°C for 30 min and the supernatant recovered from the cell debris and stored at -70°C prior to use.

Western Blotting Proteins were run on SDS-polyacrylamide gels and transferred to nitrocellulose using a Bio-Rad transfer apparatus. The filters were blocked for 2 h in 10% BSA in PBS, rinsed in PBS, and incubated for 1 h in 1% BSA, 5% fetal calf serum, and antibody at 1:lOO dilution made up in PBS. Subsequently the filters were washed once in distilled water for 5 min and three times in PBS for 10 min each. Filters were then incubated in 1% BSA and 5% fetal calf serum in PBS with 1251-labeled protein A at a concentration of 0.2 &i/ml for 1 h. They were then washed consecutively for 10 min each time in PBS, 1% NP-40 in PBS, and PBS for a further three washes. All washes were done with vigorous shaking, and the filters were then air-dried, wrapped in Saran wrap, exposed to hyperfilm MP with intensifier screens at -70°C overnight, and developed the following day.

FIG. 1.

Guanidine (10 pg each) were stained.

run

isothiocyanateand RIPA-extracted proteins on 9% SDS-polyacrylamide gel and silver

Immunoprecipitation Protein solutions were precleared with 5 ~1 of normal rabbit serum and 35 ~1 protein A-Sepharose (in PBS) and incubated with rolling for 2 h at 4°C. After centrifugation for 1 min at 1300 rpm anti-p53 antibody and 35 ~1 protein A-Sepharose were added to the supernatant and incubated at 4°C for 4 h. The samples were centrifuged at 1300 rpm, the pellet was washed four times in 700 ~1 RIPA minus SDS, and the sample was boiled in 30 ~1 of loading buffer for 10 min before loading onto a gel.

Phosphorylation Protein solutions were adjusted to 100 mM Tris-HCl (pH 6.7) and 10 mM magnesium chloride and incubated for 10 min at 37°C with 10 PCi [T-~~P]ATP. The reaction was stopped by boiling in 10 ~1 sample buffer for 10 min. Samples boiled in SDS and DTT prior to incubating with [T-~~P]ATP were used as controls. Gels were stained with Coomassie blue to assess loading, dried, and exposed to hyperfilm MP with intensifier screens overnight at -70°C. RESULTS

most flexible. Other methods involving precipitation of proteins from the guanidine phase following dialysis by trichloroacetic acid or acetone led to problems with aggregation (and presumably intermolecular disulfide bonding). This also occurred after lyophilization when the protein content of the guanidine phase was too high. This problem was circumvented by addition of more GIT or buffer solutions prior to dialysis. Tris, NHlHC03, and an equimolar solution of NaCl and EDTA have been used as the dialysate. Concentrations of salt from 50 to 300 mM were tried in each case and all were equally effective. If ammonium salts are used as the dialysate, lyophilization must be complete because they cause distortion of the tracks on Western blotting. With small samples, i.e., those from g-cm dishes, there was loss of protein when GIT extraction was compared with RIPA extraction (as much as 50%); however, this loss was proportionally much less with larger samples. That intact DNA and RNA can be obtained by this method has been well documented and will not be considered further.

Method and Quantitation

Integrity of Guanidine Proteins

Isothiocyanate-Extracted

We have found the method of protein recovery by dialysis and lyophilization outlined above the simplest and

Proteins extracted by either GIT or RIPA methods, electrophoresed on 9% gels, and stained by silver (Fig. 1)

SIMULTANEOUS

ISOLATION

OF

DNA,

RNA,

AND

PROTEIN

341

with the actin antisera (Fig. 3) were due to nonspecific binding. Immunoprecipitation of ~53 (Fig. 4) from proteins extracted from g-cm dishes of cos cells expressing mutant human ~53 by the GIT (lane 5) or RIPA (lane 2) method were compared. In both samples the mutant human P53 and endogenous monkey P53 migrated to their correct positions at 42 and 53 kDa, respectively. There were marked quantitative differences, with approximately 50% less protein being recovered from the GIT extraction. Additionally, the ratio of human ~53 to the 35-kDa band was much less in the GIT-extracted protein lane 5 (only just visible in the RIPA extracted protein, lane 2). However, the ratio of human to endogenous monkey ~53 between the two was similar. Evidence for Functional Isothiocyanate-Extracted

Activity of Guanidine Proteins

To assess whether protein extracted with GIT could be “renatured” to regain functional activity, the phos-

FIG. 2. Western blot of GIT-extracted protein probed for EGFR. 100 or 50 pg of GIT-extracted protein from A431 cells was loaded on a 5% SDS-polyacrylamide gel and run at 35 mA, transferred to nitrocellulose, and probed with antiserum to peptide 2E. As a control 100 pg A431 protein probed with antisera was incubated for 1 h prior to use with peptide ZE.

or Coomassie blue (not illustrated) showed very similar profiles. The differences between bands were quantitative rather than qualitative. The integrity of the proteins obtained was demonstrated further by examining the Western blotting characteristics of a large glycosylated protein, EGFR from A431 cells (which express the protein at high levels (12)) (Fig. 2), and a smaller nonglycosylated protein, actin, from a human transitional cell tumor (Fig. 3). Both migrated to their anticipated positions (170 and 43 kDa, respectively) and were detected using appropriate antibodies. Incubating the EGFR antisera with the immunizing peptide (2E) prior to blotting confirmed the specificity of binding and indicated that the weaker signals occasionally obtained (data not shown) were due to nonspecific binding rather than to degradation products. Similarly, the additional bands seen at 36 and 70 kDa

92.5kD--!

46kD, 43kD-=’

30kD-

FIG. 3. Western blot of GIT-extracted tumor protein probed for actin. 100 pg of GIT-extracted protein from a human transitional cell carcinoma biopsy was run on a 9% SDS-poiyacrylamide gel at 35 mA, transferred to nitrocellulose, and probed with anti-actin antiserum.

342

COOMBS

ET

AL.

samples, appropriate exposures revealed that the majority of protein species phosphorylated were similar. DISCUSSION

-96kD I ; -69kD -Monkey Anti-P53

P53

Ab:-Human

P53

Denaturation, irreversible denaturation, and renaturation of proteins have been the subjects of much controversy (7). In the presence of its ligand or substrate, a protein may partially reconform even in a strong denaturant (3). Whether a protein ever renatures to its “native” form outside its normal environment is questionable, and the conditions under which individual proteins renature will probably vary from protein to protein. We have demonstrated a simple method by which selected proteins can be recovered from small samples at the same time as DNA and RNA. The purified proteins appear to retain primary structure, as assessedby size and antigenicity on Western blotting and immunoprecipitation. That a degree of tertiary structure (and function) is regained is shown by the phosphorylating ability of

FIG. 4.

Immunoprecipitation of p53 from COS cells expressing human mutant ~53. Half the RIPA-extracted protein from a g-cm dish of cells was immunoprecipitated (lane 2) in parallel with the GIT-extracted protein from an identical dish (lane 5), 200 pg of which was run separately (lane 4), transferred to nitrocellulose, and probed with the same antibody used to precipitate it. Lanes 1 and 3 contain the anti-P53 antisera.

phorylating activity of RIPA-extracted protein was compared to that of GIT-extracted protein (Fig. 5). Initial results with GIT-extracted protein dissolved in RIPA following lyophilization (lane A) showed that activity was present but at levels much lower than those seen with RIPA-extracted proteins (lane D). However, when the protein was dissolved in water, phosphorylation was lo- to 2O-fold higher (lane B). To confirm that this activity depended on protein conformation, both GIT- and RIPA-extracted proteins were denatured by boiling with SDS and DTT prior to assay (lanes C and E). No phosphorylation occurred with denatured proteins. To compare the ratios of phosphorylated to nonphosphorylatedproteins obtained, equal counts of each phosphorylatedprotein mixture were loaded (lanes G and H). When the gel was stained with Coomassie blue, it was apparent that about twice as much GIT-extracted protein had been loaded, showing that there had been either a 50% loss of kinase activity or a 50% gain of inactive protein. In addition there were marked qualitative differences. Most noticeable were the higher amounts of phosphorylated proteins at 93 and 48 kDa and two illdefined bands at approximately 5 and 10 kDa which may represent degradation products in the GIT-extracted protein. Although there were marked variations in signal intensity between different protein bands in the two

m

-2OOkD

I -46kD

-3OkD

-14.3kD -1OkD -5kD FIG. 6.

Comparison of phosphorylating ability of GIT- and RIPAextracted proteins from COS cells. All samples were run on 5-15% gradient gels. GIT-extracted protein was taken up in water unless stated otherwise. Lanes A and B contain GIT-extracted protein taken up in RIPA (-SDS; lane A) or water (lane B) prior to phosphorylation. Lanes C and E contain RIPAand GIT-extracted controls, respectively (boiled in SDS and DTT prior to phosphorylation), compared to normally phosphorylated RIPA(lane D) and GIT-extracted protein (lane F). Lanes G and H contain equal counts of RIPAand GITextracted protein, respectively. Lane I contains size markers.

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ISOLATION

the proteins, although whether this is due to autophosphorylation of many proteins or to the reactivation of a specific kinase or both has not been determined. The apparent sensitivity of the kinase activity of GIT-extracted protein to RIPA buffer was confirmed by repetition. Equal volumes of the same solution of [r-32P]ATP were used and equal loading confirmed by Coomassie blue staining of the gel. This has not been investigated further but may be related to the detergent content (1% NP-40,1% deoxycholic acid). This could occur as a general effect, such that removal of detergent from RIPAextracted protein would enhance its activity. However, it may also be secondary to conformational changes induced by GIT leading to exposure of epitopes predisposing to micelle formation with detergents. The qualitative differences between the two methods of protein extraction shown by immunoprecipitation of p53 suggest that the GIT method of extraction either specifically enriches for the 35-kDa presumed internally initiated protein or perhaps more likely it is lost on centrifugation in RIPA-treated cells. If one assumes it is a degradation product, then the GIT method of extraction appears to cause more degradation. The qualitative changes above and those in the phosphorylation studies can be explained by fundamental differences in the methods. GIT causes dissolution of most of the protein present in the cell and its matrix, whereas RIPA dissolution followed by ultracentrifugation results in loss of many of the cytoskeletal and nuclear components. GIT extraction may result in increased degradation of proteins. However, it is also possible that the 5- and lo-kDa phosphorylated bands represent subunits of polymeric proteins following cleavage of disulfide bonds by /3-mercaptoethanol in the GIT solution. It is unlikely that in such a mixture of proteins the right subunits would realign, although in a pure solution of a single protein this may well be the case. While this work was in progress, Iadarola and Naranjo (14) emphasized the value of methods which allow the simultaneous isolation of active proteins and mRNA in correlative studies on human tissues. They demonstrated that active peptide hormones could be obtained from the guanidine phase of tissues treated with guanidine isothiocyanate and desalted using Sep Packs. Although it is unlikely that the proteins have been renatured to their native conforma-

OF

DNA,

RNA,

AND

343

PROTEIN

tion, we have demonstrated that selected proteins can be isolated intact and that activity which requires secondary and tertiary structure can be obtained. Denatured ribonucleases can be reactivated by dilution of the denaturant (3). E. coli RNA polymerase (a subunit), E. coli transcription termination factor p, P-galactosidase, alkaline phosphatase, wheat cr-amylase, and DNA topoisomerase have been extracted from SDS-polyacrylamide gels, further denatured with guanidine hydrochloride, and renatured to activity (6). Octapeptide hormones in an active form have been recovered from the guanidine phase of tissues dissolved in GIT (14). These observations suggest that it should be possible to isolate many proteins in an active form using this method. Minor modifications may be necessary depending on the class of proteins of interest. As such it should prove valuable especially in the analysis of human tissues. ACKNOWLEDGMENT I thank

Dr. Horst

Sturzbecher

for his friendly

advice

and help.

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A. E., Macdonald, 24,5294-5299.

R. J., and Rutter,

2. Glison, V., Crkvenjakov, R., Byus, C. (1974) Biochemistry 12, 2633-2637. 3. Sela, M., Anfinsen, C. B., and Harrington, W. F. (1956) Biochim. Biophys. Acta 26,502-512. 4. Meselson, M., Stahl, F. W., and Vinograd, Acad. Sci. USA 43,581-588.

J. (1957)

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5. Davis, L. G., Dibner, M. D., and Battey, J. F. (1986) Basic Methods in Molecular Biology, pp. 130-135, Elsevier, Amsterdam. 6. Hager, D. A., and Burgess, R. R. (1980) Anal. Biochem. 109, 7686. 7. Tanford, C. (1968) Adu. Protein Chem. 23,121-282. 8. Anfinsen, C. B. (1962) Brookhaven Symp. Biol. 15,184-198. 9. Gullick, W. J., Marsden, J. J., Downward, J., and Waterfield, M. D. (1985) J. Cell Sci. Suppl. 3,151-160. 10. Harlow, E. D., Crawford, L. V., Pim, C. D., and Williamson, N. M. (1981) J. Virol. 39,861-869. 11. Gluzman, Y. (1981) Cell 23,175-182. 12. Fabricant, R. N., DeLarco, J. E., and Todaro, Natl. Acad. Set USA 74,565-569. 13. Bradford, 14. Iadarola,

G. J. (1977)

M. M. (1976) Anal. Biochem. 72,248-254. M. J., and Naranjo, J. R. (1988) Peptides

9,669-671.

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